Advances in Experimental Medicine and Biology 1331

Cellular Neuroscience, Neural Circuits and Systems Neuroscience

Laura Calzà
Luigi Aloe
Luciana Giardino Editors

Recent Advances
in NGF and Related
Molecules
The Continuum of the NGF “Saga”
Advances in Experimental Medicine
and Biology

Cellular Neuroscience, Neural Circuits

and Systems Neuroscience

Volume 1331

Series Editors
Adalberto Merighi, Department of Veterinary Sciences, University of Turin,
Grugliasco, Italy
Laura Lossi, Department of Veterinary Science, University of Turin,
Grugliasco, Italy
Alberto Granato, Department of Psychology, Catholic University, Milan, Italy
This book subseries publishes reviews and original articles covering all
aspects of cellular and systems neuroscience, particularly neural circuits. It
is also open to contributions on neurological disorders and their molecular and
biochemical basis. A subseries of the highly successful Advances in Experi-
mental Medicine and Biology, it provides a publication vehicle for articles
focusing on the latest research, developments and methods, as well as
opinions and principles. Contributors are experienced neuroscientists and
researchers with extensive knowledge of the embryology, histology, anatomy,
physiology and pharmacology of central and peripheral neurons. It endeavors
to highlight the importance of the correct choice of animal models and the
implementation of the 3Rs principles to translate molecular and cellular
findings of basic research into clinical applications. This series is a valuable
source of state-of-art knowledge and inspiration for future research ideas for a
wide range of researchers and animal care professionals working in the field of
neuroscience.

More information about this subseries at http://www.springer.com/series/

16028
Laura Calzà • Luigi Aloe •
Luciana Giardino
Editors

Recent Advances in NGF

and Related Molecules
The Continuum of the NGF “Saga”
Editors
Laura Calzà Luigi Aloe
University of Bologna IRET Foundation
Ozzano dell’Emilia, Bologna, Italy Ozzano dell’Emilia, Bologna, Italy

Luciana Giardino
University of Bologna
Ozzano dell’Emilia, Bologna, Italy

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISSN 2524-6577 ISSN 2524-6585 (electronic)
Cellular Neuroscience, Neural Circuits and Systems Neuroscience
ISBN 978-3-030-74045-0 ISBN 978-3-030-74046-7 (eBook)
https://doi.org/10.1007/978-3-030-74046-7

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature
Switzerland AG 2021
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher,
whether the whole or part of the material is concerned, specifically the rights of translation,
reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any
other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are
exempt from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in
this book are believed to be true and accurate at the date of publication. Neither the publisher nor
the authors or the editors give a warranty, expressed or implied, with respect to the material
contained herein or for any errors or omissions that may have been made. The publisher remains
neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG.
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface: Rita Levi-Montalcini

Rita Levi-Montalcini received her medical degree and specialization in neu-

rology and psychiatry in 1936 from the Faculty of Medicine at the University
of Turin, Italy. She then entered the Institute of Anatomy as a postgraduate
student in the laboratory of Giuseppe Levi, a well-known anatomist and tutor
of two other future winners of the Nobel Prize in Physiology or Medicine—
Salvatore Luria (who won in 1969) and Renato Dulbecco (who won in 1975).
During her early postgraduate studies, Levi-Montalcini investigated the rela-
tionship between the developing central nervous system (CNS) and its periph-
eral targets, the results of which led her to suggest that the failure of neurons to
thrive in the absence of peripheral target tissues was due to a degenerative
process, rather than a failure of differentiation as had previously been
hypothesized by Victor Hamburger, a renowned embryologist and develop-
mental neuroscientist.
The NGF story began in 1949 when Hamburger showed Levi-Montalcini
the results achieved by one of his postgraduate students, Elmer Bueker.
Bueker had observed that implantation of a small fragment of malignant
mouse tumor into the body wall of 3-day chick embryos caused sensory fibers
to invade the tumor. Bueker hypothesized that this effect was due to the
rapidly expanding tumor allowing the sensory fibers to branch into the
much larger field offered by the neoplastic cells which replaced the embryonic
tissue, a hypothesis which did not convince Rita Levi-Montalcini, who
decided to study the effect more extensively. The results of her reinvestigation
led Levi-Montalcini to the hypothesis that the mouse sarcoma tissues 180 and
37, when transplanted into a chick embryo, produce and release a diffusible
agent which stimulates the growth and differentiation of developing nerve
cells. Using a variety of in vitro methodologies, she then demonstrated that
these tumor tissues produce and release a molecule able to stimulate neuritis
outgrowth from sensory and sympathetic nerve cells, and in 1952, she named
this molecule nerve growth factor (NGF).
In collaboration with Stanley Cohen, co-winner of the Nobel Prize in 1986,
she discovered that NGF is stored in snake venom and in the submaxillary
salivary gland of mice.
During the early 1950s, Levi-Montalcini—then Professor at the Depart-
ment of Zoology at the Washington University of St. Louis—intensified her
in vivo and in vitro studies of NGF on small rodents, while two other
scientists—first Stanley Cohen and later P. U. Angeletti—investigated the

v
vi Preface: Rita Levi-Montalcini

chemical and biochemical properties of NGF, both devising a methodology of

purifying large quantities of NGF from snake venom and from mouse salivary
glands. The availability of large amounts of NGF in these glands provided an
additional opportunity to carry out numerous and collaborative in vivo studies
and to investigate the structural, biochemical, and pharmacological action of
NGF on sympathetic and sensory nerve cells, as well as permitting the
production of anti-NGF antibodies, and to further characterize the effects of
NGF on the development of the peripheral nervous system by injecting this
antibody into newborn rodents. The immune deprivation of NGF via the
exogenous administration of NGF antibodies to these rodents, and the obser-
vation that inhibition of circulating NGF levels results in the death of
NGF-target cells, described in the late 1960s, became known as
immunosympathectomy.
In addition to this continued scientific activity, Levi-Montalcini wrote
several books. The first, In Praise of Imperfection, is an inspiring autobio-
graphical work where she tells of her success, but also of the delays and
distractions affecting researchers, the potential mistakes they can make, and
their tendency to underestimate difficulties. A second volume entitled Il tuo
futuro (“Your future”) is dedicated to young people who during their early
lives often face the need to make vital choices, while her third book, Senza
Olio e contro Vento (“Out of oil and against the wind”), contains ten true
stories of well-known and everyday people, united by the courage and honesty
they showed at very difficult times of their lives. In her most recent book
L’asso nella manica a brandelli (“The ace up a tattered sleeve”), she discusses
the skills and creativity of famous elderly people, and how to maintain and
improve cognitive abilities in later life. From a scientific point of view,
however, her most important book is perhaps the The NGF Saga.
More recently, basic preclinical studies have indicated that purified NGF
has potential clinical application in ocular disorders, such as corneal ulcers,
neurotrophic keratitis, maculopathy, glaucoma, and cutaneous ulcers such as
diabetic and rheumatoid ulcers and pressure sores.
The articles published in this issue are part of a scientific contribution
presented and discussed at the Second International NGF scientific meeting
dedicated to Rita Levi-Montalcini, held in Bologna on June 13–14, 2019, and
organized by Laura Calzà, Luigi Aloe, and Luciana Giardino. A major aim of
this international NGF meeting was to bring attendees up to date on the past,
recent, and ongoing studies in the field of the Italian Nobel Laureate in
Physiology or Medicine who discovered the first neurotrophic factor,
providing neurologists with a powerful tool for investigating the growth,
differentiation, and plasticity of nerve cells, as well as paving the way for
the identification of other specific growth factors for other cell types. As
progress is achieved by moving continuously forward toward further scientific
knowledge, we were happy that the invited speakers presented and discussed
the most recent findings in the field of NGF and other neurotrophic factors,
including the recently discovered recombinant human NGF with clinical
applications for certain human ocular diseases. It is a great pity that Rita
Levi-Montalcini is no longer with us, as she was at our first International NGF
meeting held in Modena in 2002. Finally, we owe a debt of gratitude to all the
Preface: Rita Levi-Montalcini vii

participants who contributed to the success of the NGF meeting conference in

Bologna, to the financial support provided by the IRET Foundation in
Ozzano, Bologna, and to the professional collaboration of all logistical
IRET staff. This meeting was also part of the “Italian Regenerative Medicine
Infrastructure—IRMI” project training program (MIUR,
CTN01_00177_888744 to LC).

Bologna, Italy Laura Calzà

Luigi Aloe
Luciana Giardino
Contents

Part I Basic Science

1 Nerve Growth Factor: The First Molecule of the
Neurotrophin Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Luca Lorenzini, Vito Antonio Baldassarro, Agnese Stanzani,
and Luciana Giardino
2 The First and Last Time I Met Rita Levi-Montalcini:
From the Insect Neurotrophic Factor to the Nerve Growth
Factor Clinical Application . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Luigi Aloe
3 NGF Signaling in Endosomes . . . . . . . . . . . . . . . . . . . . . . . . . 19
Naoya Yamashita
4 The NGF Metabolic Pathway: New Opportunities for
Biomarker Research and Drug Target Discovery . . . . . . . . . . 31
Rowan Pentz and M. Florencia Iulita

Part II Development and Regeneration

5 NGF and Endogenous Regeneration: From Embryology
Toward Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Vito Antonio Baldassarro, Luca Lorenzini, Andrea Bighinati,
Alessandro Giuliani, Giuseppe Alastra, Micaela Pannella,
Mercedes Fernandez, Luciana Giardino, and Laura Calzà
6 The Versatile Roles of Nerve Growth Factor in Neuronal
Attraction, Odontoblast Differentiation, and Mineral
Deposition in Human Teeth . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Thimios A. Mitsiadis and Pierfrancesco Pagella
7 An Overview of Adult Neurogenesis . . . . . . . . . . . . . . . . . . . . 77
Filipa F. Ribeiro and Sara Xapelli
8 Intervention of Brain-Derived Neurotrophic Factor
and Other Neurotrophins in Adult Neurogenesis . . . . . . . . . . 95
Filipa F. Ribeiro and Sara Xapelli

ix
x Contents

Part III Alzheimer and Central Nervous System

9 Rita Levi-Montalcini, NGF Metabolism in Health and in the
Alzheimer’s Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
A. Claudio Cuello
10 NGF and the Amyloid Precursor Protein in Alzheimer’s
Disease: From Molecular Players to Neuronal Circuits . . . . . 145
Viviana Triaca, Francesca Ruberti, and Nadia Canu
11 A Review of Techniques for Biodelivery of Nerve Growth
Factor (NGF) to the Brain in Relation to Alzheimer’s
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Sumonto Mitra, Ruchi Gera, Bengt Linderoth, Göran Lind,
Lars Wahlberg, Per Almqvist, Homira Behbahani,
and Maria Eriksdotter
12 NGF Released from Blood Cells or Collagen Hydrogels
as a Therapeutic Target in Alzheimer’s Disease? . . . . . . . . . . 193
Christian Humpel

Part IV Complex Behaviors

13 When Nerve Growth Factor Met Behavior . . . . . . . . . . . . . . 205
Daniela Santucci, Arianna Racca, and Enrico Alleva
14 Cross Talk of BDNF and GDNF in Spinal Substantia
Gelatinosa (Lamina II): Focus on Circuitry . . . . . . . . . . . . . . 215
Adalberto Merighi

Part V Clinical Medicine

15 BDNF Gene as a Precision Skill of Obesity Management . . . . 233
Helena Marcos-Pasero, Elena Aguilar-Aguilar,
Maria P. Ikonomopoulou, and Viviana Loria-Kohen
16 The Science of Love: State of the Art . . . . . . . . . . . . . . . . . . . 249
Donatella Marazziti, Stefania Palermo, and Federico Mucci
17 NGF and Retinitis Pigmentosa: Structural and Molecular
Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Maria Luisa Rocco, Laura Calzà, and Luigi Aloe
18 NGF in Inflammatory and Neurodegenerative Diseases
of the Eye: New Findings Supporting Neuroprotection and
Proper Tissue Remodeling in Vitreoretinal Disorders . . . . . . 265
Graziana Esposito, Bijorn Omar Balzamino, Luca Bruno,
Andrea Cacciamani, and Alessandra Micera
Contents xi

Part VI Veterinary Medicine

19 Nerve Growth Factor (NGF) and Animal Reproduction . . . . . 277
Margherita Maranesi, Cristiano Boiti, and Massimo Zerani
20 Neurotrophins in the Brain of Teleost Fish: The State
of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Paolo de Girolamo and Livia D’Angelo
 Part I
Basic Science
 Nerve Growth Factor: The First Molecule
of the Neurotrophin Family 1
Luca Lorenzini, Vito Antonio Baldassarro, Agnese Stanzani,
and Luciana Giardino

Abstract endocrine, and skeletal system, vascular

Neurotrophins (NTs) are molecules regulating
differentiation, maintenance, and functional
plasticity of vertebrate nervous systems.
Nerve growth factor (NGF) was the first to be
identified in the neurotrophin family. The long
scientific history of NTs provided not only
advancement in the neuroscience field but
opened new scenarios involving different
body districts in physiological and pathologi-
cal conditions, which include the immune,
districts, inflammation, etc. To date, many
biological aspects of NTs have been clarified,
but the new discoveries are still opening new
insights on molecular and cellular mechanisms
and systemic effects, also affecting the possi-
ble therapeutic application of NTs. This short
review summarizes the main aspects of NGF
biology and biochemistry, including the role
of the NGF precursor molecule, high- and
low-affinity receptors and related intracellular
pathways, and target cells.

L. Lorenzini
Department of Veterinary Medical Sciences, University of
Bologna, Bologna, Italy
Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
Bologna, Bologna, Italy
e-mail: luca.lorenzini8@unibo.it
V. A. Baldassarro · A. Stanzani
Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
Bologna, Bologna, Italy
e-mail: vito.baldassarro2@unibo.it; agnese.
stanzani2@unibo.it
L. Giardino (*)
Department of Veterinary Medical Sciences, University of
Bologna, Bologna, Italy
Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
Bologna, Bologna, Italy
IRET Foundation, Bologna, Italy
e-mail: luciana.giardino@unibo.it
1.1 Introduction
The pioneering work of Rita Levi-Montalcini in
the first half of the last century led to a revolu-
tionary view of neuroembryology, when she first
speculated (in collaboration with Viktor Ham-
burger) and then proved (in collaboration with
Stanley Cohen) that a target-derived soluble “fac-
tor” was responsible not only for the appropriate
development of sensory and sympathetic innerva-
tion of the chicken embryo but also for cell num-
ber control and, therefore, for the appropriate
shaping of neural pathways during development
(Levi-Montalcini 1997). This soluble factor was
then isolated in 1954 and initially named “nerve
growth-promoting factor.” Subsequently
abbreviated to nerve growth factor (NGF), it led

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 3

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_1
4 L. Lorenzini et al.
to the neurotrophic theory, postulating that NGF
(and subsequently all neurotrophins—NTs) is
responsible for regulating the balance between
neuronal survival and neuronal death, its absence
or withdrawal being the causal link to induce
neuronal death (Ichim et al. 2012).
NGF was the first molecule of the
“neurotrophin family” to be identified (Franco
et al. 2020): brain-derived neurotrophic factor
(BDNF) was discovered a few years later, with
neurotrophin-3 (NT-3) and neurotrophin-4
(NT-4) subsequently included in the family
(Hennigan et al. 2007).
NTs are synthesized in the endoplasmic retic-
ulum as precursor proteins of around 250 amino
acids (proNT, 35 kDa) which associate in a
homodimer. ProNGF is subsequently processed
by different proteases (e.g., furin), the presence of
a 103-residue N-terminal pro-part in proNGF
constituting the difference between the primary
structure of NGF and proNGF (Trabjerg et al.
2017). The mature forms (12–15 kDa) then end
up in two types of vesicles within the Golgi
network, belonging to the constitutive or
regulated secretory pathways (Costa et al. 2018).
In the former, vesicles release their cargos when
they reach the plasma membrane, whereas in the
regulated mode, release is calcium-dependent.
The cellular action of the neurotrophins, i.e.,
the biological effect of each NT, is mediated
through the activation of high- and low-affinity
receptors. High-affinity receptors belong to the
tropomyosin-related kinase (Trk) family of recep-
tor tyrosine kinases, NGF being the specific
ligand for TrkA, BDNF for TrkB, and NT-3/
NT-4 for TrkC. NGF specifically binds to TrkA,
while BDNF preferentially binds to TrkB. NT-3
preferentially binds to TrkC but can also activate
TrkA and TrkB, while NT-4 preferentially binds
to TrkB (Skaper 2012). TrkA has five extracellu-
lar binding domains that connect the extracellular
portion of the receptor to the single transmem-
brane domain and a juxtamembrane intracellular
region that is connected to the kinase domain. The
first (Trk-d1) is a leucine-rich region and is
flanked by two cysteine-rich domains (Trk-d2
and Trk-d3). The fourth and fifth domains
(Trk-d4 and Trk-d5) are immunoglobulin (Ig)-
like domains. The NGF binding domain is located
in the Trk-d5(Ig2) domain. According to a classi-
cal view, the ligand-receptor complex is
internalized in endosomes after binding to Trk
receptors and retrogradely transported to the cell
body regulating pro-apoptotic and anti-apoptotic
gene expression (Mok et al. 2009). Target-
derived NGF is also responsible for the trophic
support of selected neural populations in the cen-
tral nervous system (CNS) during adulthood, the
cholinergic basal forebrain neurons.
In addition, all NTs bind to the low-affinity
p75 receptor (p75NTR), a member of the TNF
receptor superfamily (Huang and Reichardt
2001). The p75NTR receptor is a monomer and
consists of three parts: a cysteine-rich extracellu-
lar domain involved in binding to NTs, a trans-
membrane region containing the cleavage site for
gamma-secretase, and an intracellular domain
containing what is known as the death domain,
responsible for the receptor’s pro-apoptotic activ-
ity. In some cells, NT binding to p75NTR triggers
a programmed cell death process via the c-Jun
N-terminal kinase (JNK) or caspase signaling
pathways (Chao 2003).
Over the years, many more molecular and
cellular aspects of NT biology and mechanisms
of action have been discovered and clarified,
including (1) the biological activity of proNTs,
(2) the multiple transmembrane pathways
activated by Trk receptors, and (3) the pleiotropic
nature of NTs, which act on many different cell
types also in adulthood. In this short review, we
will review these aspects with regard to NGF.
1.2 NGF and proNGF: Who Is Doing
What?
NGF is a product of a single gene found on
chromosome 1 and is produced according to the
aforementioned process of synthesis and release
from proNGF. In its mature form, NGF is assem-
bled in a non-covalent homodimer, denoted by a
ring structure formed by two disulfide bridges
penetrated by a third disulfide bridge (Trabjerg
et al. 2017). However, proNGF is not an inactive
precursor but takes active part in the biological
1 Nerve Growth Factor: The First Molecule of the Neurotrophin Family
activity of this NT, often producing opposite
effects to the mature NGF form.
Substantial contributions clarifying the
biological role of proNGF have come from the
study of the cholinergic neurons of the basal
forebrain in both human and animal brains.
Observations on these neurons have
demonstrated that degeneration in Alzheimer’s
disease, or in animal models such as the Down
syndrome mice Ts65Dn, is related to a defect in
the retrograde axonal transport of proNGF. This
is associated with a deficit in the NGF metabolic
pathway responsible for processing proNGF to
mature NGF (Iulita and Cuello 2014; Costa
et al. 2018; Fahnestock and Shekari 2019). The
ratio between pro- and mature forms of NGF,
responsible for determining neuronal fate, there-
fore derives from complex biochemical pathways
including not only intracellular synthesis and
release but also intracellular distribution, extracel-
lular proteases, and regulation of high- and
low-affinity receptor expression. For example,
the proNGF and NGF homodimers have a differ-
ent distribution within the neuron, with proNGF
localized in the cell body and mature NGF in the
cell body and neuronal processes. Moreover, the
cleavage site of proNGF, whether intracellular or
extracellular, is still disputed, and extracellular
matrix enzymes, including MMP7 and TIMP1,
seem to promote NGF biochemical maturation
(Costa et al. 2018).
In the meantime, proNT and proNGF receptors
continue to be a matter of debate. Since the origi-
nal demonstration that proNTs are secreted and
cleaved extracellularly and bind to receptors in
order to produce their biological effects (Lee et al.
2001), the p75NTR/sortilin high-affinity receptor
complex has been identified as responsible for
mediating proNGF-induced neuronal death by
apoptosis (Nykjaer et al. 2004; Teng et al.
2010). p75NTR expression is high during develop-
ment, while very few neural populations express
this receptor in the mature brain. However, recent
evidence indicates that p75NTR expression can be
induced in other neurons by lesion stimuli.
Sortilin is a member of the VPS10 domain trans-
membrane receptor family. It creates heterodimer
complexes with p75NTR, thus functioning as
5
co-receptor (Skeldal et al. 2012), and is a required
element for proNGF cell death signaling. More-
over, while most of the data excludes the possi-
bility of Trk activation by proNTs, recent studies
suggest that proNGF can bind TrkA receptors
with low affinity (5.4 times lower than mature
NGF). Thus, the biological effect of NGF must
be stated according to a number of concurrent
biochemical, cell-specific players.
1.3 TrkA- and p75NTR-Mediated
Intracellular Pathways
By interaction with TrkA, NGF regulates many
neuronal functions, including cell survival and
differentiation, axonal and dendritic growth, and
synaptic formation and plasticity (Fig. 1.1).
Ligand-receptor binding induces phosphorylation
of specific tyrosine residues, and the docking site
recruits pathway-activating proteins for Ras, Rac,
PI3 kinase, and PLC-γ1 and their downstream
effectors (Franco et al. 2020). These include
mitogen-activated protein (MAP) kinase cascades
(stimulated by Ras), Akt stimulation by PI3, and
PLC-γ1-dependent generation of IP3 and
diacylglycerol (DAG), which results in the mobi-
lization of Ca2+ stores and the activation of Ca2+
and DAG-regulated protein kinases (Reichardt
2006).
Transmembrane pathway activation does not
obscure gene transcription regulation through ret-
rograde transport of the NT-Trk endosomal com-
plex. This process is regulated by ubiquitination,
involves Shc, and is required for persistent acti-
vation of the MAP kinase cascade. Lastly, the
recruitment of distinct populations of endosomes,
such as the recruitment of the receptor in the
appropriate cell compartment, i.e., the plasma
membrane or the nucleus, may represent further
levels for control of NT biological activity.
The p75NTR receptor also mediates the activa-
tion of several signaling pathways, including
Traf6, NT receptor-interacting factor (NRIF),
melanoma-associated antigen (MAGE), p75NTR-
interacting MAGE homolog (NRAGE), Schwann
cell factor 1 (SC1), RhoGDI, and other proteins
(Fig. 1.2). A major pathway activated by p75NTR
6 L. Lorenzini et al.
Fig. 1.1 NGF/TrkA signaling transduction pathways.
TrkA mediates its action by phosphorylation, activating
MAPK, PI3K, and PLC-γ1 pathways, resulting in activa-
tion of gene expression, neuronal survival, and neurite
outgrowth. Activation of Ras activates MAP kinase sig-
naling cascade, promoting neuronal differentiation and
neurite outgrowth. Ras and Gab1 activate also PI3,
which promotes survival pathway activation and neuronal
growth. PLC-γ1 activation leads to Ca2+- and protein
kinase C-regulated pathway activation promoting synaptic
plasticity. TrkA acts also in endosomes moving through
the axon on microtubules by dynein binding. NGF remains
bound to TrkA keeping it activated, sustaining signals
facilitating the trafficking and leading to neuronal survival
and differentiation. Rabs are involved in biogenesis and
regulation of the transport of the endosomes.
Abbreviations: Akt protein kinase B, Bcl2 B-cell lym-
phoma 2, C1 cysteine cluster 1, C2 cysteine cluster
2, Erk extracellular signal-regulated kinases, Frs2 fibro-
blast growth factor receptor substrate 2, GAB1/2 growth
factor receptor-bound protein 2-associated protein 1, Gbr2
growth factor receptor-bound protein 2, IAPs inhibitors of
apoptosis, Ig1 immunoglobulin-like domain 1, Ig2
immunoglobulin-like domain 2, LRR1–3 extracellular
ligand-binding domain comprising leucine-rich motif,
MAPK mitogen-activated protein kinase, MEKK MAP
1 Nerve Growth Factor: The First Molecule of the Neurotrophin Family
is the c-Jun kinase signaling cascade, resulting in
p53/bax-mediated apoptosis. Notably, the recruit-
ment of these pathways seems to occur via the
p75NTR death domain (DD). This 10 kDa glob-
ular protein is present in many members of the
tumor necrosis factor receptor superfamily and
can recruit several interactor proteins which act
as pathway activators (Yuan et al. 2019).
The receptor p75NTR also promotes NF-κB-
dependent neuronal survival via p62 and the
kinase activity of IRAK. This mechanism, not
yet fully understood, is TrkA-independent and
has been demonstrated in several cell types
including Schwann cells and trigeminal and hip-
pocampal neurons (Franco et al. 2020). Other
pathways activated by NTs binding to p75NTR
include acidic sphingomyelinase, which results
in the generation of ceramide, and the Rho family
GTPase activity, which regulates cytoskeleton
assembly and stability and neurite outgrowth.
The role of the p75NTR intracellular domain
(ICD), produced by cleavage of the receptor by
gamma-secretase, is also unclear. The specific
29-amino acid peptide derived from the intracel-
lular cleavage of p75NTR interacts with and
potentiates the binding of NGF to TrkA-
expressing cells. Examples are the potentiation
of the effect of NGF on neurite outgrowth in
sympathetic neurons as a result of enhanced
Erk1/2 and Akt signaling (Matusica et al. 2013)
or the increase in the expression of vascular endo-
thelial growth factor, for example, after retinal
hypoxia via HIF1α (Le Moan et al. 2011).
Lastly, TrkA and p75NTR can signal synergis-
tically, antagonistically, or independently of each
other in different cellular contexts (Lu et al.
2005), meaning that the final fate produced by
NGF or proNGF exposure is cell-specific. In a
general sense, most studies suggest that in the
case of TrkA and p75NTR co-expression in the
same cell, the pro-apoptotic signals of p75NTR
ä
Fig. 1.1 (continued) kinase kinase kinase, NGF nerve
growth factor, PDKs pyruvate dehydrogenase kinases,
PI3K phosphatidylinositol 3 kinase, PLC-γ1 phospholi-
pase Cγ, Raf rapid accelerated fibrosarcoma, Ras reticular
7
are largely suppressed as a result of Trk
receptor-mediated signaling when mature NTs
bind to Trk receptors (Reichardt 2006).
1.4 NGF Target Cells
Currently, NGF remains the best-characterized
neurotrophic factor for its role in regulating sym-
pathetic and sensory neuron differentiation, in
phenotype maintenance and protection of target
neuronal populations, in the sensitivity of periph-
eral nerve fibers to heat and pain, and in
mediating physiological and abnormal pain per-
ception. In the CNS, NGF-producing and NGF
target cells are present in the basal forebrain,
striatum, thalamus, and several cortical regions
and in dentate granule cells in the hippocampus
as subpopulations of GABAergic interneurons.
NGF expression in the hippocampus is regulated
by neuronal activity, being increased by activa-
tion of glutamatergic and cholinergic neurotrans-
mission and decreased by GABAergic
transmission (Sofroniew et al. 2001).
NGF is also produced by glial cells, as
astrocytes and microglia, and NGF expression in
both cell types is markedly upregulated by local
tissue injury, inflammation, and cytokines. This
was observed in the periinfarct area in ischemic
cortex after transient stroke, after mechanical
injury in the hippocampus and in the cortex
surrounding the wound, and in Parkinson’s dis-
ease models at lesioned striatum (Pöyhönen et al.
2019).
These major physiological roles have also
prompted studies of the possible role of NGF
defects or signaling dysfunctions in pathological
conditions such as metabolic diseases—periph-
eral neuropathy in diabetes mellitus, for exam-
ple—or in infectious and inflammatory diseases,
due to the role of the neurotrophin in
activating system, Shc SH2 domain and collagen homolo-
gous region, SOS mammalian son of sevenless, TrkA
tropomyosin receptor kinase A
8 L. Lorenzini et al.
Fig. 1.2 NGF/p75NTR signaling transduction pathways.
P75NTR signaling may induce survival or apoptosis
response, depending on the cell type and activated cas-
cade. The receptor interacts with different proteins
(TRAF6, RhoA, NRAGE, SC-1, NRIF) regulating gene
expression, cell cycle, apoptosis, mitogenic responses, and
axonal growth. It activates three main pathways, NF-κB,
c-Jun, and Rho, which promotes, respectively, cell sur-
vival, apoptosis, and growth cone motility. The
pro-apoptotic c-Jun effect can be inhibited by Ras-PI3K
pathway activated by TrkA. Abbreviations: Ask1 apopto-
sis signal-regulating kinase 1, Cd42 glycoprotein lb,
CR1–4 complement receptor 1–4, IKK IκB kinase, IRAK
inflammatory and immune cells (Aarão et al.
2018). In fact, NGF production and biological
actions have been described in many
non-nervous cell types, such as immune
interleukin-1 receptor-associated kinase, JNK c-Jun N-ter-
minal kinase, NADE p75NTR-associated death executor,
NF-κB nuclear factor kappa-light-chain-enhancer of
activated B cells, NGF nerve growth factor, NIK
NF-kappa-B-inducing kinase, NRAGE neurotrophin
receptor-interacting MAGE homolog, NRIF neurotrophin
receptor-interacting factor, p62 sequestosome 1, p75NTR
neurotrophin low-affinity receptor, RhoA Ras homolog
family member A, RhoGDI Rho GDP-dissociation inhibi-
tor, RIP2 receptor-interacting-serine/threonine-protein
kinase 2, SC-1 Schwann cell factor 1, TRAF6 TNF
receptor-associated factor 6
inflammatory cells or epithelial cells, leading to
the general definition of NGF as a “pleiotropic”
molecule (Aloe and Calzà 2004; Micera et al.
2007).
1 Nerve Growth Factor: The First Molecule of the Neurotrophin Family
NGF high- and low-affinity receptors are pres-
ent in not only nerve cells and CNS tumors but in
immune cells such as macrophages, lymphocytes,
and mast cells. These cells synthesize, store, and
secrete NGF, suggesting a possible role for NGF
in the regulation of immune responses during
inflammatory, infectious, and autoimmune pro-
cesses (Aarão et al. 2018).
Other tissues and cell types expressing NGF
and related receptors include the skin (e.g.,
keratinocytes, fibroblasts, and melanocytes
(Gostynska et al. 2020)), the vascular system
(endothelial cells (Gostynska et al. 2020)), the
salivary (e.g., submandibular) glands, and various
endocrine organs/cells, such as the pituitary, the
thyroid, and the endocrine elements in the testes
and ovaries. Most of these cells continue to pro-
duce NGF during adulthood and modulate NGF
production in response to stimuli (Sofroniew et al.
2001).
This process also leads to local events, includ-
ing activation-induced histamine release, and an
increase in the number of mast cells and other
immune system cells (Aarão et al. 2018).
Several inflammatory, infectious, and autoim-
mune diseases also lead to an altered NGF physi-
ology, as indicated by altered plasma levels and
by the presence of anti-NGF antibody levels in
patients with rheumatoid arthritis, systemic lupus
erythematosus, and thyroiditis (Skaper 2017;
Aarão et al. 2018).
Finally, a special mention should be reserved
for the role of NGF, other neurotrophins, and
related receptors in cancer. The emerging concept
of “nerve dependency” in oncology has gained
consensus in recent years thanks to a large body
of data demonstrating that NGF and related
receptors can be expressed in human tumors,
including ovarian, breast, lung, pancreatic, skin,
liver, gastric, and thyroid malignancies. In these
context, neurotrophins can stimulate cancer cell
growth (Griffin et al. 2018), also leading to the
hypothesis of using the Trk receptor expression
level as a prognostic marker. A more recent
hypothesis, however, has suggested that nerves
infiltrated in the tumor microenvironment secrete
neurotransmitters, which can stimulate both
growth of tumor cells and angiogenesis, taking
9
part in a process known as “tumor neurogenesis”
(Griffin et al. 2018). This hypothesis refers to the
major role of peripheral innervation in the homeo-
static regulation of the microenvironment and its
adaptation in pathological conditions, suggesting
that NGF may play a role in cancer pain.
This evidence brings the research full circle to
the discoveries made by Rita Levi-Montalcini, a
mouse sarcoma being the tissue from which NGF
was originally isolated (Cohen et al. 1954).
References
Aarão TL d S, de Sousa JR, ASC F, LFM F, JAS Q (2018)
Nerve growth factor and pathogenesis of leprosy:
review and update. Front Immunol 9:939
Aloe L, Calzà L (2004) NGF and related molecules in
health and disease: preface. Prog Brain Res 146:1–544
Chao MV (2003) Neurotrophins and their receptors: a
convergence point for many signalling pathways. Nat
Rev Neurosci 4:299–309. https://doi.org/10.1038/
nrn1078
Cohen S, Levi-Montalcini R, Hamburger V (1954) A
nerve growth-stimulating factor isolated from
SARCOM as 37 and 180. Proc Natl Acad Sci
40:1014–1018. https://doi.org/10.1073/pnas.40.10.
1014
Costa RO, Perestrelo T, Almeida RD (2018) PROneuro-
trophins and CONSequences. Mol Neurobiol
55:2934–2951
Fahnestock M, Shekari A (2019) ProNGF and
neurodegeneration in Alzheimer’s disease. Front
Neurosci 13:129
Franco ML, Comaposada-Baró R, Vilar M (2020)
Neurotrophins and Neurotrophin receptors. In: Hor-
monal signaling in biology and medicine. Elsevier,
Amsterdam, pp 83–106
Gostynska N, Pannella M, Rocco ML, Giardino L, Aloe L,
Calzà L (2020) The pleiotropic molecule NGF
regulates the in vitro properties of fibroblasts,
keratinocytes, and endothelial cells: implications for
wound healing. Am J Physiol – Cell Physiol 318:
C360–C371. https://doi.org/10.1152/ajpcell.00180.
2019
Griffin N, Faulkner S, Jobling P, Hondermarck H (2018)
Targeting neurotrophin signaling in cancer: the renais-
sance. Pharmacol Res 135:12–17
Hennigan A, O’Callaghan RM, Kelly ÁM (2007)
Neurotrophins and their receptors: roles in plasticity,
neurodegeneration and neuroprotection. Biochem Soc
Trans 35(2):424–427. Portland Press
Huang EJ, Reichardt LF (2001) Neurotrophins: roles in
neuronal development and function. Annu Rev
Neurosci 24:677–736. https://doi.org/10.1146/
annurev.neuro.24.1.677
10
Ichim G, Tauszig-Delamasure S, Mehlen P (2012)
Neurotrophins and cell death. Exp Cell Res
318:1221–1228
Iulita MF, Cuello AC (2014) Nerve growth factor meta-
bolic dysfunction in Alzheimer’s disease and down
syndrome. Trends Pharmacol Sci 35:338–348. https://
doi.org/10.1016/j.tips.2014.04.010
Le Moan N, Houslay DM, Christian F, Houslay MD,
Akassoglou K (2011) Oxygen-dependent cleavage of
the p75 neurotrophin receptor triggers stabilization of
HIF-1α. Mol Cell 44:476–490. https://doi.org/10.
1016/j.molcel.2011.08.033
Lee R, Kermani P, Teng KK, Hempstead BL (2001) Reg-
ulation of cell survival by secreted proneurotrophins.
Science (80-) 294:1945–1948. https://doi.org/10.1126/
science.1065057
Levi-Montalcini R (1997) The Saga of the nerve growth
factor. World Scientific, Singapore
Lu B, Pang PT, Woo NH (2005) The yin and yang of
neurotrophin action. Nat Rev Neurosci 6:603–614
Matusica D, Skeldal S, Sykes AM, Palstra N, Sharma A,
Coulson EJ (2013) An intracellular domain fragment
of the p75 neurotrophin receptor (p75NTR) enhances
tropomyosin receptor kinase a(TrkA) receptor func-
tion. J Biol Chem 288:11144–11154. https://doi.org/
10.1074/jbc.M112.436469
Micera A, Lambiase A, Stampachiacchiere B, Bonini S,
Bonini S, Levi-Schaffer F (2007) Nerve growth factor
and tissue repair remodeling: trkANGFR and p75NTR,
two receptors one fate. Cytokine Growth Factor Rev
18:245–256
Mok SA, Lund K, Campenot RB (2009) A retrograde
apoptotic signal originating in NGF-deprived distal
axons of rat sympathetic neurons in compartmented
cultures. Cell Res 19:546–560. https://doi.org/10.
1038/cr.2009.11
Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen
MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow
TE, Hempstead BL, Petersen CM (2004) Sortilin is
essential for proNGF-induced neuronal cell death.
L. Lorenzini et al.
Nature 427:843–848. https://doi.org/10.1038/
nature02319
Pöyhönen S, Er S, Domanskyi A, Airavaara M (2019)
Effects of neurotrophic factors in glial cells in the
central nervous system: expression and properties in
neurodegeneration and injury. Front Physiol 10:486
Reichardt LF (2006) Neurotrophin-regulated signalling
pathways. Philos Trans R Soc B Biol Sci
361:1545–1564
Skaper SD (2012) The neurotrophin family of
neurotrophic factors: an overview. Methods Mol Biol
846:1–12
Skaper SD (2017) Nerve growth factor: a neuroimmune
crosstalk mediator for all seasons. Immunology
151:1–15
Skeldal S, Sykes AM, Glerup S, Matusica D, Palstra N,
Autio H, Boskovic Z, Madsen P, Castrén E, Nykjaer A,
Coulson EJ (2012) Mapping of the interaction site
between sortilin and the p75 neurotrophin receptor
reveals a regulatory role for the sortilin intracellular
domain in p75 neurotrophin receptor shedding and
apoptosis. J Biol Chem 287:43798–43809. https://doi.
org/10.1074/jbc.M112.374710
Sofroniew MV, Howe CL, Mobley WC (2001) Nerve
growth factor signaling, neuroprotection, and neural
repair. Annu Rev Neurosci 24:1217–1281. https://doi.
org/10.1146/annurev.neuro.24.1.1217
Teng KK, Felice S, Kim T, Hempstead BL (2010) Under-
standing proneurotrophin actions: recent advances and
challenges. Dev Neurobiol 70:350–359
Trabjerg E, Kartberg F, Christensen S, Rand KD (2017)
Conformational characterization of nerve growth fac-
tor- reveals that its regulatory pro-part domain
stabilizes three loop regions in its mature part. J Biol
Chem 292:16665–16676. https://doi.org/10.1074/jbc.
M117.803320
Yuan W, Ibáñez CF, Lin Z (2019) Death domain of p75
neurotrophin receptor: a structural perspective on an
intracellular signalling hub. Biol Rev 94:1282–1293.
https://doi.org/10.1111/brv.12502
 The First and Last Time I Met Rita
Levi-Montalcini: From the Insect 2
Neurotrophic Factor to the Nerve
Growth Factor Clinical Application
RLM and the Discovery of Two Neurotrophic Factors

Luigi Aloe

Abstract cardiometabolic diseases; and finally (4) NGF

The neurotrophic factor nerve growth factor
(NGF) has been discovered in the 1950s by
Rita Levi-Montalcini, first in a neoplastic tis-
sue and, later, in the mouse salivary gland (see
1A). Levi-Montalcini characterized its action
in the sensory and sympathetic neurons
(1B) and, a few years later, in central nervous,
endocrine, and immune systems. Nerve
growth factor plays its trophic role both during
development and in adulthood, ensuring the
maintenance of phenotypic and functional
characteristic of several populations of
neurons as well as immune cells. The aim of
the present overview is to describe my per-
sonal scientific and human experiences work-
ing with Rita Levi-Montalcini for over
45 years, first at Washington University in
St. Louis, Missouri, USA, searching (1) the
invertebrate neurotrophic factor in the
cockroaches and, later, at the Institute of Neu-
robiology of the National Research Council
(CNR) in Rome studying (2) the role of NGF
for various neuronal and non-neuronal
functions; (3) the potential involvement of
NGF in the pathobiology of human cutaneous,
ocular, neurodegenerative, and
L. Aloe (*)
IRET Foundation, Tecnopolo-Bologna “Rita Levi-
Montalcini”, Bologna, Italy
e-mail: luigi.aloe@cnr.it
potential clinical application.
2.1 Introduction
Rita Levi-Montalcini (RLM) received her medi-
cal degree in 1936 from the University of Turin
and entered in the Institute of Anatomy as a
postgraduate student in the laboratory of
Giuseppe Levi, a well-known anatomist and men-
tor of other two future Nobel Prize winners in
Physiology or Medicine, Salvatore Luria, in
1969, and Renato Dulbecco in 1975. During the
early postgraduate years, RLM studied the rela-
tionship between the developing nerve system
and peripheral targets and observed that many
sensory neurons died during normal development
and that the limb bud extirpation in chick
embryos caused an increase of cell death. These
observations led to the hypothesis that failure of
neurons to thrive in the absence of peripheral
target was because of a degenerative process
rather than a failure of differentiation. In 1946,
Viktor Hamburger, a well-known and famous
neuroembryologist working at the Department
of Zoology, Washington University in St. Louis,
Missouri, USA, invited RLM in his laboratory.
Working with Hamburger, she demonstrated that
nerve cells die during the course of normal devel-
opment and that limb bud extirpation causes an

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 11

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_2
12
increase of neuronal death, suggesting the
hypothesis that the developing neurons depend
on feedback signals from the peripheral tissues
and that neuronal target provides a specific mole-
cule required for neuronal survival, growth, and
differentiation. In 1950, she discovered the first
neurotrophic factor, the nerve growth factor
(NGF), in a mouse sarcoma tissue and later in
the snake venom and mouse submaxillary gland
(Fig. 2.1a). In collaboration with a young bio-
chemist, Stanley Cohen (who discovered the epi-
dermal growth factor, co-winner of the Nobel
Prize in 1986), RLM developed a series of exper-
imental approaches to purify the factor from the
mouse salivary gland that produces and stores
high amounts of NGF that was effective in
stimulating the growth and differentiation of neu-
ritis outgrowth from sensory and sympathetic
neurons, both in vivo and in vitro (Fig. 2.1b).
Using the NGF purified from this source and
antibodies against NGF, they then demonstrated
the functional biological significance of NGF on
mammals’ peripheral sensory and sympathetic
nerve cells (Cohen 1960; Levi-Montalcini 1952;
Levi-Montalcini 1964; Levi-Montalcini and
Booker 1960; Levi-Montalcini and Hamburger
1951).
2.2 How I Met Rita Levi-Montalcini
In the early June 1965, I received a letter from
Levi-Montalcini from St. Louis, MO, USA, for
fixing an interview in Rome during her next trip
to Rome. During the interview, she briefly told
me: “I was told that you are an expert of taking
care and raising Grasshoppers and I am sure that
you would be also able to take care and raise
cockroaches (Periplaneta Americana) in my Lab-
oratory in St. Louis.” She then continued: “I
would give you 400 dollars a month for six
months and I need an answer within two weeks.”
A week later, I decided to accept and traveled
with her to St. Louis 2 weeks later. The 6 months
became then nearly 45 years. Why did she decide
to leave the study of NGF? I was not told why she
decided stopping her studies on NGF, in spite of
she was already well-known among the
L. Aloe
international scientific community for her studies
on NGF. At that time, I personally did not know
almost anything about NGF studies, but only that
she was a famous international scientist. Only
later, I believe I understood her motivation. She
isolated and purified the molecule with her grad-
uate student Stanley Cohen and demonstrated that
it was able to promote growth and differentiation
of sensory and sympathetic neurons in vivo and
in vitro and that the administration of an antibody
against the purified NGF in developing rodents
caused immunosympathectomy, but no lethal
actions, nor an effect on their lifespan. Moreover,
after removing the largest mouse source of NGF,
the salivary gland, she observed neither adverse
nor lethal effect. Finally, she tested the action of
the purified molecule in invertebrates, particularly
in young and adult insects, and, again, found no
action on their nervous system. All these negative
observations led her to believe that NGF might
not have any potential clinical development and
future applications and hypothesized that insects
should produce their own neurotrophic factors, as
mammals. Convinced about this hypothesis,
RLM decided to take a sabbatical leave from the
NGF studies and to invest her time searching for
insect neurotropic factors.
2.3 Flight to St. Louis, MO, USA
In May 1966, I took the airplane for St. Louis,
USA, with RLM. To carry studies on insects’
nervous system, she selected adult developing
cockroaches because of their long lifespan and
the properties to lay bean-like eggs every week
containing about 12–14 embryos having all the
same stage of development. Once I raised a stable
colony of cockroaches and collected a sufficient
number of eggs and embryos, I started with RLM
to remove several different embryo tissues,
making tissue extracts and searching for the
unknown new insect neurotropic factor. Less
than 1 year later, we obtained the first positive
and novel result. We identified an NGF-like
neurotrophic factor in the digestive tract of
cockroaches (Fig. 2.2a) with properties of pro-
moting outgrowth and differentiation of insect
2 The First and Last Time I Met Rita Levi-Montalcini: From the Insect. . .
Fig. 2.1 (a) Photomicrograph of immunohistochemical-
stained section of adult mouse salivary gland storing and
releasing the nerve growth factor. Magnification: 95X. (b)
Photomicrograph of dissociated sympathetic neurons of
brain nerve cells in developing and adult
cockroaches (Fig. 2.2b). These findings were
published (Aloe and Levi-Montalcini 1972a, b).
Unfortunately, the amounts and release of the
factor in the digestive tract of this insect were
not sufficient for the molecule to be purified in
large amounts for further structural, biochemical,
Fig. 2.2 (a) Photomicrograph of adult cockroach intes-
tine producing and releasing insect neurotrophic factor.
Magnification: 25X. (b) Photomicrograph showing
13
developing rats cultured for 24 h in the presence of
10 nanograms of purified NGF showing neurite outgrowth
induced by NGF. Magnification: 45X
and molecular analyses, the commercial cost of
cockroaches became quite high, and the recombi-
nant methodologies were not yet available. There-
fore, she decided not to invest further time on
insect studies.
ganglia of developing cockroaches in culture in the pres-
ence of cockroach intestine extract inducing neurite
outgrowth after 48 h in vitro. Magnification: 200X
14
2.4 In 1972 RLM Decided
to Organize Her Return
in Rome
I remained in St. Louis to continue the studies of
the action of insect growth factor on the intestine
motility and to explore the role of NGF on rodent
peripheral neuropathies, behavioral response, and
potential pharmacotherapeutic properties. These
studies revealed that NGF was able to prevent the
peripheral neuropathies induced by chemical
drugs and by surgical axotomy. These findings
reinforced the hypothesis that NGF might have
indeed a potential clinical future. Furthermore,
other findings revealed that the NGF not only
prevented peripheral neuropathies but also pro-
moted the growth, differentiation, and degranula-
tion of mast cells. Moreover, we showed that mast
cells had the properties of synthesizing, storing,
and releasing NGF, which could exert profound
effects of autoimmune action, neuroimmune
interactions, tissue inflammation, and modulation
of lymphocytes’ memory abilities (Levi-
Montalcini et al. 1975). Furthermore, in vivo
administration of neutralizing anti-NGF
antibodies caused marked reduction in the titer
of specific IgGs in mice immunized with the
tetanus toxoid, nitrophen. Together these
observations indicated that NGF played a critical
role as an autocrine survival factor for memory B
lymphocytes.
In Rome, collaborating with Enrico Alleva
from the Italian National Institute of Health, we
found that the action of NGF was involved in
aggressive behavior, enhancing circulating NGF
levels, and promotes its synthesis in forebrain and
hypothalamic areas. These findings and other
numerous observations suggested that the
increase of NGF levels preceded the increase of
plasma cortisol and adrenocorticotropic hormone
and could be involved in the activation of cells of
the immune system, most probably associated
with homeostatic (homeodynamic, a term pro-
posed by RLM) mechanisms, as previously
shown for stressed rodents (Aloe and Levi-
Montalcini 1977; Torcia et al. 1996).
L. Aloe
2.5 NGF and the Central Nervous
System
In 1984, Siler and Schwab published the first
evidence that NGF was expressed in the central
nervous system of rodents. They demonstrated
that radiolabeled NGF, injected in very small
quantities into the frontal or dorsal anterior occip-
ital cortex of adult rats, was specifically taken up
and transported retrogradely to large, presumably
cholinergic neurons in the region of the nucleus
basalis (lateral preoptic nucleus, anterior lateral
hypothalamic nucleus, substantia innominata,
ventral globus pallidus, and internal capsule), as
revealed by light microscopic autoradiography.
They thus suggested that NGF might be
implicated in the protection of the brain choliner-
gic neurons that degenerated in aging and mem-
ory deficits (Aloe et al. 1986). Few years later, the
studies published by Hefti showed that NGF
receptors were localized in the rat forebrain and
that NGF promoted the survival of these neurons,
both in vivo and in vitro (Spillantini et al. 1989).
Other studies, published a few years later,
demonstrated that intraventricular injections of
NGF attenuated the degeneration of these
neurons, suggesting that NGF might be able to
attenuate and/or prevent the degeneration of the
forebrain cholinergic neurons involved in
Alzheimer’s disease (Hefti and Will 1987; Seiler
and Schwab 1984). The role of NGF in physiopa-
thology of brain nerve cells was subsequently
extended by the studies published by Laura
Calzà in collaboration with RLM (Calza et al.
2001; Calza et al. 2003; Fischer et al. 1987;
Giardino et al. 2002; Tuszynski et al. 1991).
In 1995, I was invited by Dr George
Chaldakov to present a lecture on NGF and mast
cells at the Biomedical Forum organized by the
Medical University of Varna, Bulgaria. Since
then, we had a fruitful scientific collaboration on
the possible significance of NGF and brain-
derived neurotrophic factor (BDNF) in multiple
biological phenomena, ranging from
neurotrophic, immunotrophic, to metabotropic
effects. We have for the first time reported data
that demonstrated significantly reduced local
2 The First and Last Time I Met Rita Levi-Montalcini: From the Insect. . .
and/or blood circulating levels of the two
neurotrophins in human cardiometabolic dis-
eases, including coronary atherosclerosis and
the metabolic syndrome and acute coronary
syndromes. We also suggested that TrkA and
TrkB agonists might have therapeutic benefit in
patients with these conditions. Further, we have
demonstrated that both NGF and BDNF were
secreted by adipose tissue cells.
2.6 Studies on NGF and Clinical
Potentiality
The first study to test the role of NGF on visual
cells was carried out with intravitreal administra-
tion of the factor on a mouse strain C3H, affected
by inherited retinitis pigmentosa, which is
characterized by a single gene mutation that
leads to photoreceptor degeneration. It was
found that the intraocular injection of NGF
induced a significant increase in the thickness of
the retinal compared to controls, indicating that
NGF administration protected and/or reduced the
degeneration of retinal cells (Lambiase and Aloe
1996).
2.7 NGF and Human Corneal
Neurotrophic Ulcer
Corneal neurotrophic ulcer is known to be
associated with impairment of sensory innerva-
tion that can lead to loss of vision. At that time,
we started to address the potential usefulness of
NGF in ameliorating the condition. Unfortu-
nately, no effective treatment was available. As
other available treatments were ineffective, based
on the results obtained with animal models
indicating that no undesired side effects occurred
after the topic administration of NGF to the eye,
after approval of an ethical committee and
informed consent of the patient, we topically
administered NGF to the eye of a patient affected
by the disorder. The treatment continued for
2 weeks, the ulcers healed, and the patients were
15
then followed up for up to 12 months. Corneal
integrity and sensitivity were maintained during
the follow-up period (Lambiase et al. 1998). Best-
corrected visual acuity increased progressively
during treatment and follow-up in all patients,
without systemic or local side effects of
treatment.
2.8 NGF and Glaucoma
One key event leading to glaucoma (GL) is the
elevated intraocular pressure. However, at that
time, whether the ocular application of NGF
could prevent or reduce the damage to retinal
ganglion cells (RGCs) was unknown. To test the
action of NGF, we induced GL in adult rats by the
injection of hypertonic saline into the episcleral
vein of the right eye and used the left eye as
control. The injection led to a progressive degen-
eration of the RGCs, with a loss of nearly 40% of
these cells after 7 weeks of treatment. This cellu-
lar loss was associated with the downregulation
of NGF and NGF receptor expression in the retina
and the glaucomatous eye, but ocular treatment
with NGF significantly reduced the deficit
induced by GL. These results unequivocally
revealed that NGF administration was protective
against RGC degeneration (Coassin et al. 2008;
Colafrancesco et al. 2011; Lambiase et al. 2009a;
Lambiase et al. 2009b). To further explore the
role of NGF on retinal degeneration, we used a
rodent model of intraocular hypertension. In this
study, we again investigated the effect of NGF
administration on RGCs. We found that both
animal models displayed a progressive degenera-
tion of RGCs and altered NGF and VEGF levels
in the retina and optic nerve. We then proved that
NGF eye drop administration exerted a protective
effect on the two models of retinal degeneration.
In brief, our findings indicated that NGF can play
a protective role against RGC degeneration
occurring in GL not only in rodents but also in
humans, suggesting that ocular topical NGF
administration could be a suitable pharmacologic
agent to counteract RGC degeneration in GL.
16
2.9 NGF and Macular Degeneration a powerful pharmacological tool for human cuta-
Age-related macular degeneration (ARMD) is a
severe disease affecting visual function in the
elderly. Currently available surgical and medical
options do not guarantee a protective outcome of
RGC degeneration, and visual acuity is progres-
sively worsening despite surgical and medical
treatments. RLM suffered from a maculopathy
for over 5 years, and the laser therapy and other
available therapies had no effect. Therefore, in
2002, RLM asked me to purify the NGF and to
the ophthalmologist, A. Lambiase, to monitor the
action of the factor on her macular degeneration.
After signing the informed consent, she was
treated with NGF eye drops for six consecutive
months. Daily comments from the patient and
weekly clinical examination from the ophthal-
mologist revealed the absence of NGF-related
undesired effects. Moreover, a clear improvement
of visual acuity and functional parameters were
consistently monitored 3 months after the begin-
ning of the treatment (Tuveri et al. 2000). In brief,
RLM discovered and used for her ocular disorder
the NGF treatment for nearly 6 years. Results
revealed that the ARMD was significantly
delayed with no undesired side effects. These
findings were published in 2009 (Lambiase et al.
2009a).
2.10 NGF and Cutaneous Wounds
Cutaneous wounds are known to elicit a series of
topical cellular responses that include clotting,
inflammatory infiltration, epithelialization, and
the formation of granulation tissue, including
new blood vessel, followed by tissue remodeling
and wound contraction. Based on the above and
other previous studies, we decided to investigate
the effect of the topical application of purified
NGF in human cutaneous ulcers induced by dia-
betes, vasculitis, and rheumatoid arthritis. The
results showed the efficacy of NGF was due to
its promoting activity on keratinocyte prolifera-
tion and vascular neoangiogenesis, suggesting
that topical application of NGF might represent
L. Aloe
neous ulcers, including in cutaneous ulcer healing
in “non-responder” transplanted skin (Aloe
2004b; Aloe and Chaldakov 2013; Chaldakov
2011; Chaldakov et al. 2001; Generini et al.
2004; Lambiase et al. 2009b; Landi et al. 2003;
Tuveri et al. 2000).
2.11 Concluding Remarks
During the last three decades, despite some doubt
by RLM regarding the effective development of
potential clinical applications of NGF, I provided
convincing demonstration, first in laboratory ani-
mal and later in human visual cells and cutaneous
ulcers, of the beneficial therapeutic effects of
NGF in clinical settings. Indeed, the Italian phar-
maceutical company Dompè has recently
registered the recombinant NGF as Cenegermin,
and the molecule has now been approved for
medical application in Italy, Switzerland,
Germany, and Canada.
Last Time I Meet RLM was less than 1 week
before her death in her house. Despite she was
rather tired, before leaving she asked me: “Luigi,
please remain a while more just to tell me some-
thing new about NGF.” Overall, for me, the sci-
entific and human experience working for nearly
45 years close to a Nobel winner, described as the
queen of modern neuroscience, brought a signifi-
cant contribution to both the basic and the clinical
saga of NGF.
References
Aloe L (2004a) Rita Levi-Montalcini: the discovery of
nerve growth factor and modern neurobiology. Trends
Cell Biol USA 14(7):395–399
Aloe L (2004b) Nerve growth factor, human skin ulcers
and vascularization. Our experience. Prog Brain Res
146:515–522. USA
Aloe L, Chaldakov GN (2013) Homage to Rita Levi-
Montalcini, the queen of modern neuroscience. Cell
Biol Int USA 37(8):761–765
Aloe L, Levi-Montalcini R (1972a) In vitro analysis of the
frontal and ingluvial ganglia from nymphal specimens
2 The First and Last Time I Met Rita Levi-Montalcini: From the Insect. . .
of the cockroach Periplaneta Americana. Brain Res
USA 44(1):147–163
Aloe L, Levi-Montalcini R (1972b) Interrelation and
dynamic activity of visceral muscle and nerve cells
from insect embryos in long term cultures. J Neurobiol
USA 3(1):3–23
Aloe L, Levi-Montalcini R (1977) Mast cells increase in
tissues of neonatal rats injected with NGF. Brain Res
USA 133(2):358–366
Aloe L, Alleva E, Böhm A, Levi-Montalcini R (1986)
Aggressive behavior induces release of nerve growth
factor from mouse salivary gland into the bloodstream.
Proc Natl Acad Sci USA 83(16):6184–6187
Calza L, Giardino L, Giuliani A, Aloe L, Levi-Montalcini
R (2001) Nerve growth factor control of neuronal
expression of angiogenetic and vasoactive factors.
Proc Natl Acad Sci USA 98(7):4160–4165
Calza L, Giuliani A, Fernandez M, Pirondi S, D’Intino G,
Aloe L, Giardino L (2003) Neural stem cells and cho-
linergic neurons: regulation by immunolesion and
treatment with mitogens, retinoic acid, and nerve
growth factor. Proc Natl Acad Sci USA 100
(12):7325–7330
Chaldakov G (2011) The metabotrophic NGF and BDNF:
an emerging concept. Arch Ital Biol Italy 149
(2):257–263
Chaldakov GN, Stankulov IS, Fiore M, Ghenev PI, Aloe L
(2001) Nerve growth factor levels and mast cell distri-
bution in human coronary atherosclerosis. Atheroscle-
rosis USA 159:57–66
Coassin M, Lambiase A, Sposato V, Micera A, Bonini S,
Aloe L (2008) Retinal p75 and bax overexpression is
associated with retinal ganglion cells apoptosis in a rat
model of glaucoma. Graefes Arch Clin Exp
Ophthalmol USA 246(12):1743–1749
Cohen S (1960) Purification of a nerve growth factor
promoting protein from a mouse salivary gland and
its neurocytoxic antiserum. Proc Natl Acad Sci USA
46(3):302–311
Colafrancesco V, Parisi V, Sposato V, Rossi S, Russo MA,
Coassin M, Lambiase A, Aloe L (2011) Ocular appli-
cation of nerve growth factor protects degenerating
retinal ganglion cells in a rat model of glaucoma. J
Glaucoma 20(2):100–108
Fischer W, Wictorin K, Björklund A, Williams LR,
Varon S, Gage FH (1987) Amelioration of cholinergic
neuron atrophy and spatial memory impairment in
aged rats by nerve growth factor. Nature USA 329
(6134):65–68
Generini S, Tuveri MA, Matucci Cerinic M, Mastinu F,
Manni L, Aloe L (2004) Topical application of nerve
growth factor in human diabetic foot ulcers. A study of
three cases. Exp Clin Endocrinol Diabetes USA 112
(9):542–544
17
Giardino L, Giuliani A, Battaglia A, Carfagna N, Aloe L,
Calza L (2002) Neuroprotection and aging of the cho-
linergic system: a role for the ergoline derivative
nicergoline (Sermion). Neuroscience USA 109
(3):487–497
Hefti F, Will B (1987) Nerve growth factor is a
neurotrophic factor for forebrain cholinergic neurons;
implications for Alzheimer’s disease. J Neural Transm
Suppl USA 24:309–315
Lambiase A, Aloe L (1996) Nerve growth factor delays
retinal degeneration in C3H mice. Graefes arch Clin
Exp Ophthalmol USA 234(1):96–100
Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L
(1998) Topical treatment with nerve growth factor for
corneal neurotrophic ulcers. N Engl J Med. USA 338
(17):1174–1180
Lambiase A, Aloe L, Centofanti M, Parisi V, Báo SN,
Mantelli F, Colafrancesco V, Manni GL, Bucci MG,
Bonini S, Levi-Montalcini R (2009a) Experimental
and clinical evidence of neuroprotection by nerve
growth factor eye drops: Implications for glaucoma.
Proc Natl Acad Sci USA 106(32):13469–13474
Lambiase A, Coassin M, And AL (2009b) Eye drops
improve visual acuity and electron functional activity
in age-related macular degeneration. Ann Ist super
Sanita’ Italy 45:439–442
Landi F, Aloe L, Russo A, Cesari M, Onder G, Bonini S,
Carbonin PU, Bernabei R (2003) Topical treatment of
pressure ulcers with nerve growth factor: a randomized
clinical trial. Ann Intern Med Italy 139(8):635–641
Levi-Montalcini R (1952) Effects of mouse tumor trans-
plantation on the nervous system. Ann New York Acad
Sci USA 55:330–343
Levi-Montalcini R (1964) Growth control of nerve cells by
a protein and its antiserum. Science USA 143
(3602):105–115
Levi-Montalcini R, Booker B (1960) Excessive growth of
the sympathetic ganglia evoked by a protein isolated
from the mouse salivary glands. Proc Natl Acad Sci
USA 46(3):373–384
Levi-Montalcini R, Hamburger V (1951) Selective growth
stimulating effects of mouse sarcoma on the sensory
and sympathetic nervous system of the chick embryos.
J Exp Zool USA 116(2):321–362
Levi-Montalcini R, Aloe L, Mugnaini E, Oesch F,
Thoenen H (1975) Nerve growth factor induces vol-
ume increase and enhances tyrosine hydroxylase syn-
thesis in chemically axotomized sympathetic ganglia
of newborn rats. Proc Natl Acad Sci USA 72
(2):595–599
Seiler M, Schwab ME (1984) Specific retrograde transport
of nerve growth factor (NGF) from neocortex to
nucleus basalis in the rat. Brain Res USA 300(1):33–39
Spillantini MG, Aloe L, Alleva E, De Simone R,
Goedert M, Levi-Montalcini R (1989) Nerve growth
18
factor mRNA and protein increase in hypothalamus in
a mouse model of aggression. Proc Natl Acad Sci USA
86(21):8555–8559
Torcia M, Bracci-Laudiero L, Lucibello M, Nencioni L,
Labardi D, Rubartelli A, Cozzolino F, Aloe L, Garaci E
(1996) Nerve growth factor is an autocrine survival
factor for memory b lymphocytes. Cell USA 85
(3):345–356
L. Aloe
Tuszynski MH, Sang H, Yoshida K, Gage FH (1991)
Recombinant human nerve growth factor infusions
prevent cholinergic neuronal degeneration in the adult
primate brain. Ann Neurol USA 30(5):625–636
Tuveri M, Generini S, Matucci-Cerinic M, Aloe L (2000)
NGF, a useful tool in the treatment of chronic
vasculitic ulcers in rheumatoid arthritis. Lancet USA
356(9243):1739–1740
 NGF Signaling in Endosomes
Naoya Yamashita
Abstract
During the development of the nervous sys-
tem, neurons respond to diffusible cues
secreted by target cells. Because such target-
derived factors regulate development, matura-
tion, and maintenance of axons as well as
somatodendritic compartments, signals
initiated at distal axons must be retrogradely
transmitted toward cell bodies. Neurotrophins,
including the nerve growth factor (NGF), pro-
vide one of the best-known examples of target-
derived growth factors. The cell biological
processes of endocytosis and retrograde traf-
ficking of their Trk receptors from growth
cones to cell bodies are key mechanisms by
which target-derived neurotrophins influence
neurons. Evidence accumulated over the past
several decades has begun to uncover the
molecular mechanisms of formation, transport,
N. Yamashita (*)
Department of Pharmacology, Juntendo University School
of Medicine, Tokyo, Japan
e-mail: ny-yamashita@juntendo.ac.jp
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_3
3
and biological functions of these specialized
endosomes called “signaling endosomes.”
3.1 Retrograde NGF Signaling
Through Endosomes
During development, newborn neurons migrate to
their proper locations and then extend their axons
and dendrites into correct target regions to form
synapses with suitable partners. The neurons that
fail to innervate these targets are eliminated by a
genetically coded mechanism. Target innervation
of axons influences axon development and matu-
ration as well as almost all aspects of the pro-
cesses involved in neuronal development.
Because neurons have long axons that extend
even more than several centimeters, the signals
from target-derived factors must be retrogradely
propagated to the cell body and/or dendrites.
Therefore, long-distance retrograde signaling
from distal axons is one of the fundamental
features for regulating the formation, mainte-
nance, and function of neuronal circuits
(Harrington and Ginty 2013; Cosker and Segal
2014; Scott-Solomon and Kuruvilla 2018). Yet,
the molecular mechanisms of this retrograde sig-
nal propagation remain to be elucidated in full.
Neurotrophins are often produced in postsyn-
aptic cells and act as target-derived factors to
initiate signals at axonal growth cones (Glebova
19
20
and Ginty 2005). Early studies using radiolabeled
125
I-NGF showed that in vivo injection into the
iris and salivary glands—both of which are target
organs for sympathetic neurons—resulted in the
accumulation of labeled NGF in the neuronal cell
bodies that provide innervation to these structures
(Hendry et al. 1974; Stoeckel et al. 1975). Simi-
larly, the retrograde accumulation of 125I-brain-
derived neurotrophic factor (BDNF),
125
I-neurotrophin 3 (NT-3), and 125I-NT-4 (also
known as NT-5) in the cell bodies of oculomotor
neurons were observed when those neurotrophins
were applied to the extraocular eye muscles
(Steljes et al. 1999), indicating that the target-
derived neurotrophins were retrogradely
transported along axons to mediate retrograde
signaling. Accumulating evidence further showed
that neurotrophins and their high-affinity Trk
receptor complexes were endocytosed into
growth cones and then retrogradely transported
to somatodendritic compartments for regulating
neuronal growth, differentiation, survival, and
synapse formation (Fig. 3.1). Because these
endosomes contain active neurotrophin signaling
components—including several downstream
molecules—they can transmit retrograde signal-
ing with high accuracy and persistency
(Harrington and Ginty 2013; Cosker and Segal
2014; Yamashita and Kuruvilla 2016).
The endosomes that provide retrograde signals
through this endosome-based transportation are
called “signaling endosomes,” a term that was
proposed by W.C. Mobley and colleagues
(Beattie et al. 1996). Initially, it was thought that
these specialized endosomes communicated with
the nucleus to regulate the gene expression. How-
ever, we now know that these endosomes also
influence neurons through transcription-
independent mechanisms. Over several decades,
the accumulating evidence—the majority of
which is discovered in NGF-TrkA signaling—
began to uncover the molecular mechanisms of
formation, transport, and biological functions of
signaling endosomes.
N. Yamashita
3.2 Endocytosis
of Neurotrophin-Receptor
Complexes in Growth Cones
Like other receptor tyrosine kinases, the Trk
receptors dimerize in response to a ligand (Jing
et al. 1992). The dimeric receptors phosphorylate
one another in trans to activate a downstream
cascade, including phosphatidylinositol 3-kinase
(PI3 kinase)-, Ras/MAPK-, and phospholipase
C-γ (PLCγ)-mediated pathways (Huang and
Reichardt 2003). In general, ligand-receptor
complexes are internalized by different endocytic
pathways, and, after internalization, some
receptors are recycled back to the plasma mem-
brane, while others are sorted together with their
ligands for degradation in late endosomes and
lysosomes. The internalization of ligand-receptor
signaling complexes by endocytosis was, there-
fore, considered for many years to be crucial for
the termination of signaling. However, it is now
clear that there is a high degree of compartmen-
talization of signaling needed to control and mod-
ulate the cell response. The NGF-bound TrkA
receptors are internalized into signaling
endosomes that have been found to include
components of their downstream molecules
(Grimes et al. 1996, 1997; Howe et al. 2001;
Wu et al. 2001). Pharmacological inhibition of
Trk kinase activity and ectopic expression of
kinase-dead Trk receptor mutant blocks ligand-
receptor endocytosis and subsequent retrograde
transport (Reynolds et al. 1998; Ye et al. 2003;
Heerssen et al. 2004). Inhibition of TrkA kinase
activity in distal axons, but not in middle axons,
of sympathetic neurons blocks retrograde trans-
port of 125I-NGF (Ye et al. 2003; Zweifel et al.
2005). Although the exact molecular mechanisms
are still unknown, it is therefore likely that kinase
activity of TrkA mediates the formation of signal-
ing endosomes, but it is not required for retro-
grade transport of these endosomes.
TrkA kinase activity is necessary for the asso-
ciation of clathrin with the plasma membrane in
3 NGF Signaling in Endosomes
Fig. 3.1 Nerve growth factor (NGF) secreted from target
organs activates TrkA receptors localized in distal axons.
In addition to activating the local signaling in growth
cones, the active NGF-TrkA complexes are endocytosed
and then undergo retrograde axonal transport to
somatodendritic compartments. TrkA-harboring
PC12 cells (Beattie et al. 2000). In addition,
clathrin-coated vesicles isolated from
NGF-treated PC12 cells contain NGF-TrkA
complexes and effectors of MAPK cascade, one
of the downstream pathways of NGF-TrkA sig-
naling (Howe et al. 2001). Therefore, clathrin-
dependent endocytosis may be at least one of
the major routes for the formation of signaling
endosomes. Several lines of evidence support the
idea that NGF-TrkA complexes are also
endocytosed by clathrin-independent
macropinocytosis. Pincher, an NGF-upregulated
GTPase, is involved in this process. The
overexpression of Pincher enhances NGF/TrkA
internalization both in PC12 cells and in sympa-
thetic neurons (Shao et al. 2002; Valdez et al.
2005). In contrast, inhibiting the activity of
Pincher suppresses internalization as well as
NGF retrograde signaling (Shao et al. 2002).
Thus, Pincher-mediated macropinocytosis is
21
endosomes, called “signaling endosomes,” transmit retro-
grade NGF signaling to regulate neuronal growth, differ-
entiation, survival, and synapse formation. Neurons that
fail to innervate their targets die because of the lack of
survival signals. P phosphate. All figures in this chapter
are created with Biorender.com
required for the formation of signaling
endosomes. At present, there is some debate as
to which endocytic route is the predominant mode
for the formation of signaling endosomes. How-
ever, because NGF signaling endosomes have
several biological functions and exist as several
distinct types of vesicles (see below), several
types of endocytosis may be involved, which is
probably related to the sorting of endosomes for
regulating diverse biological functions.
Both clathrin- and Pincher-mediated
endocytoses could be regulated by the endocytic
GTPase, dynamin, because NGF-TrkA signaling
adjusts the activity of neuron-specific splicing
isoform, dynamin 1. NGF stimulation induces
the tyrosine phosphorylation of TrkA at Tyr794
(in rats, Tyr784 in human TrkA), which activates
the calcium-responsive phosphatase, calcineurin,
by recruiting PLCγ to TrkA. Calcineurin
dephosphorylates PxIxIT motif-containing
22
dynamin 1 isoforms to promote TrkA endocyto-
sis, which is required for NGF retrograde signal-
ing through signaling endosomes (Bodmer et al.
2011). Once internalized, the NGF-TrkA
endosomes must disassemble an F-actin mesh-
work to promote their entry into the axon for
retrograde transport. This process is mediated by
the activation of Rac1, which leads to actin depo-
lymerization through the recruitment of cofilin to
the NGF-TrkA signaling endosomes (Fig. 3.2)
(Harrington et al. 2011). A recent study showed
that the Rac1 is locally translated in growth cones
in response to NGF followed by prenylation of
axonally translated Rac1. Because prenylation is
essential for retrograde transport of signaling
endosomes, prenylation coupled with the local
translation of Rac1 in growth cones may mediate
actin breakdown by neurotrophin signaling
endosomes (Scott-Solomon and Kuruvilla
2020). Although the exact contribution is
unknown, another TrkA effector, PI3 kinase, is
also involved in the NGF retrograde signaling
through signaling endosomes. The inhibition of
PI3 kinase prevents the accumulation of
NGF-TrkA endosomes in the cell bodies of sym-
pathetic neurons, but it does not block internali-
zation (Kuruvilla et al. 2000; Zweifel et al. 2005;
Yamashita and Kuruvilla 2016). Therefore, the
PI3 kinase pathway probably regulates post-
endocytic sorting in distal axons.
The retrograde transport of signaling
endosomes may not be the only fate of
neurotrophin-Trk receptor complexes that are
internalized into growth cones, because only a
minor population (approximately 2–2.5%) of
activated Trk receptors undergo retrograde trans-
port (Ure and Campenot 1997; Tsui-Pierchala and
Ginty 1999). The neurotrophin endosomes are
also recycled back to the growth cone membrane
or fused with lysosomes for degradation.
Although the sorting mechanisms are currently
unknown, molecular components of individual
endosomes may be a critical determinant. For
example, Nedd4-2, an E3 ubiquitin ligase, binds
to TrkA and promotes its ubiquitination, a step
that is involved in the sorting of neurotrophin
endosomes for degradation (Arevalo et al.
2006). Moreover, in developing sympathetic
N. Yamashita
neurons, NGF and NT-3 bind to and activate
TrkA, but only NGF supports survival because
it induces the TrkA internalization and retrograde
transport (Kuruvilla et al. 2004). NT-3 binds to
TrkA with low affinity compared with NGF and
is therefore thought to dissociate from TrkA
within endosomes at low pH, resulting in
endosomes that are incapable of recruiting and
activating Rac1 and cofilin, a prerequisite step
for retrograde transport (Harrington et al. 2011).
3.3 Retrograde Axonal Transport
of Signaling Endosomes
As with other types of transported vesicles, the
retrograde transport of neurotrophin-Trk
endosomes is driven by the dynein-microtubule
transport system (Fig. 3.2). Microtubule depo-
lymerization inhibits the retrograde appearance
of an active neurotrophin-Trk complex in the
cell bodies following neurotrophin stimulation
on distal axons (Watson et al. 1999). The disrup-
tion of the dynein-dynactin complex consistently
attenuates the retrograde transport of
neurotrophin endosomes (Wu et al. 2007). More-
over, the inhibition of dynein-dependent retro-
grade transport also blocks survival responses to
the target-derived neurotrophins (Heerssen et al.
2004). Thus, the dynein-dependent retrograde
transport of signaling endosomes is indeed
required for the neurotrophin retrograde
signaling.
The type of vesicle used by the NGF signaling
endosomes during retrograde transport is one of
the most important issues to be resolved. It has
long been argued that the neurotrophin signaling
endosomes are transported as a multivesicular
body (MVB), a Rab7-positive late endosome, or
a Rab5-positive early endosome (Bhattacharyya
et al. 2002; Delcroix et al. 2003; Deinhardt et al.
2006). Recent work by Ye et al. (2018) provided
new insight into this issue. By using FLAG-
tagged TrkA knock-in mice, they specifically
labeled endogenous FLAG-TrkA localized on
distal axons. They found that although TrkA+
endosomes in distal axons were mainly Rab5+,
the majority of distal axon-derived TrkA+
3 NGF Signaling in Endosomes 23

Fig. 3.2 The NGF-TrkA

complexes are endocytosed
into growth cones through a
PLCγ-calcineurin-dynamin
1 pathway. Once
internalized, the NGF-TrkA
endosomes disassemble an
F-actin meshwork through
Rac1 and cofilin to promote
entry into the axon for
retrograde transport. The
neurotrophin signaling
endosomes interact with the
dynein-dynactin complex,
thereby being transported
retrogradely to the
somatodendritic
compartments

endosomes observed in cell bodies were MVBs.
They further showed that the TrkA+ MVBs were
also Rab7-positive and these endosomes
associated the dynein complex with the dynein
adaptor, RILP, utilizing Rab7. MVBs were
shown to be attractive structures because they
were immune to lysosomal degradation and thus
provided persistent signaling during retrograde
transport. However, the axon-derived TrkA+
MVBs in cell bodies should be broken down to
permit signaling from individual endosomes as
the topology of TrkA in MVB is resistant to
relaying signals to cytoplasmic effectors. Interest-
ingly, once reaching the cell bodies, the TrkA+
MVBs evolved into single-membrane vesicles,
which may therefore be signaling competent
(Ye et al. 2018).
Whether or not an MVB is the sole organelle
responsible for the retrograde transport of
neurotrophin signaling endosomes is still
unknown. Trk receptors also interact with dynein
intermediate chains, suggesting a Rab7-
independent mode of interaction with a dynein
complex (Yano et al. 2001; Ha et al. 2008; Zhou
et al. 2012). In addition, other researchers showed
the retrograde transport of Rab11 (a marker for
recycling endosome)-positive signaling
endosomes derived from the axonal TrkA of
cultured sympathetic neurons from FLAG-TrkA
knock-in mice (Barford et al. 2018). As described
above, signaling endosomes are formed into
growth cones through several different types of
endocytosis. Ultrastructural analyses of the sciatic
nerve segment consistently showed that retro-
gradely transported TrkA+ endosomes were
observed as several structurally distinct types of
vesicles ranging from 50 to 200 nanometers
(nm) in diameter and included coated and
uncoated vesicles as well as multivesicular
structures (Bhattacharyya et al. 2002). Therefore,
signaling endosomes may be heterogenetic
organelles and thus may have many biological
functions.
24
3.4 Biological Functions
of Retrograde Signaling
Endosomes
During development, the neurons that compete
for limited amounts of target-derived factors can
survive and form a neuronal circuit (Fig. 3.1)
(Glebova and Ginty 2005). The neurotrophin sig-
naling endosomes surely contribute to this well-
accepted process. Upon arrival into the cell bod-
ies, signaling endosomes activate transcription
factors in the nucleus, including cyclic adenosine
monophosphate response-element binding pro-
tein (CREB), myocyte enhancer factor 2D
(MEF2D), and c-fos utilizing MAPK and PI3K
signaling pathways, thereby inducing the gene
expression required for neuronal survival (Riccio
et al. 1997; Watson et al. 1999; Pazyra-Murphy
et al. 2009). One gene responsible for a retrograde
neuronal survival response is Bcl-w, an anti-
apoptotic Bcl-2 family member, which is
upregulated by MEF2D through the MAPK path-
way (Fig. 3.3a) (Pazyra-Murphy et al. 2009). In
addition, NGF signaling endosomes also promote
neuronal survival by enhancing their own signal-
ing. In sympathetic neurons, NGF induces tran-
scriptional upregulation of TrkA, which enhances
NGF sensitivity and the duration of survival
signals in neurons responding to NGF
(Fig. 3.3a) (Deppmann et al. 2008). NGF further
induces the expression of Coronin-1 which
extends the duration of endosomal signaling.
Coronin-1 interacts with TrkA+ signaling
endosomes, which then mediate the local
recycling of TrkA+ signaling endosomes and pre-
vent them from fusing with lysosomes in cell
bodies (Fig. 3.3a and b) (Suo et al. 2014).
The target-derived signals from distal axons
also play a crucial role in dendrite development
and maturation (Fig. 3.3c). In sympathetic
neurons, the target size influences the size and
complexity of the dendritic arbors (Voyvodic
1989; Andrews et al. 1996). The administration
of NGF in vivo consistently enhances the den-
dritic arborization of sympathetic neurons (Snider
1988; Ruit et al. 1990). In addition, NGF is
required for the synaptic assembly of pre- and
N. Yamashita
post-ganglionic sympathetic neurons without
affecting pre-ganglionic axon innervation,
thereby suggesting the role of NGF in the matu-
ration of post-ganglionic sympathetic dendrites
(Sharma et al. 2010). Although how NGF signal-
ing endosomes influence dendritic arborization is
currently unknown, recent studies have begun to
uncover the role of NGF signaling endosomes in
postsynaptic maturation. NGF stimulation on
sympathetic axons enhances the formation of
postsynaptic receptor complexes, which are not
inhibited by de novo protein synthesis. Instead,
distal axon-derived TrkA+ endosomes that have
been transported to dendrites induce the cluster-
ing of postsynaptic components such as
membrane-associated guanylate kinases
(MAGUKs), Shank, guanylate kinase-associated
proteins (GKAPs), and nicotinic acetylcholine
receptors (nAChRs) in dendrites (Sharma et al.
2010). At least about 35% of the axon-derived
TrkA in dendrites is phosphorylated TrkA
(an active form of TrkA). Furthermore, local inhi-
bition of TrkA kinase activity in dendrites
suppresses NGF-dependent postsynaptic cluster-
ing (Lehigh et al. 2017). These data indicate that
the axon-derived TrkA signaling endosomes
found in dendrites are still signaling competent
and regulate synaptic assembly and maintenance
through a transcription-independent mechanism
(Fig. 3.3c).
The retrograde transport of signaling
endosomes further controls the specificity of
anterograde transport. In addition to a direct
secretory pathway, TrkA is also targeted to
growth cones through transcytosis; the naïve
TrkA receptors are initially localized on cell
body surfaces followed by endocytosis and anter-
ograde transport to the growth cone surface
(Ascano et al. 2009). This mode of TrkA axonal
targeting is enhanced by NGF signaling
endosomes. The retrogradely transported axon-
derived TrkA-harboring endosomes are
exocytosed on the soma surface, which then
induces the phosphorylation and endocytosis of
unliganded naïve TrkA receptors through the
interaction of somatic and axonal TrkA receptors
on the cell body membrane. Before anterograde
transport, phosphorylated naïve TrkA receptors
3 NGF Signaling in Endosomes
Fig. 3.3 Once reaching the cell bodies, the neurotrophin
signaling endosomes perform multiple biological
functions. The signaling endosomes activate several tran-
scription programs in the nucleus (a). Coronin-1, one of
the target genes for NGF signaling endosomes, interacts
with TrkA+ endosomes to extend the duration of
endosomal signaling by promoting local recycling of
TrkA+ signaling endosomes or preventing TrkA+
endosomes to fuse with lysosomes in cell bodies (b). The
active TrkA+ signaling endosomes are also transported to
the dendrite where they regulate the clustering of nAChR
are dephosphorylated by an endoplasmic
reticulum-resident protein tyrosine phosphatase
1B (PTP1B) (Fig. 3.3d). The inhibition of
PTP1B in cell bodies suppresses transcytosis
and induces the degradation of Trk receptors
that have been endocytosed from soma surfaces.
Thus, PTP1B acts as a gatekeeper that ensures the
targeting of inactive receptors (dephosphorylated
receptors) to growth cones to engage with the
ligand (Yamashita et al. 2017). Long-distance
axonal transport of endosomes takes an extended
25
and pre-existing postsynaptic density components, includ-
ing MAGUK, GKAP, and Shank (c). TrkA+ endosomes
further enhance their own trafficking. Axon-derived TrkA
interacts with naïve TrkA on the soma surface and subse-
quently elicits phosphorylation and endocytosis of naïve
unliganded TrkA. These naïve TrkA receptors are then
dephosphorylated by PTP1B followed by anterograde
transport to growth cones through the transcytotic pathway
(d). ER endoplasmic reticulum, CREB cyclic adenosine
monophosphate response-element binding protein,
MEF2D myocyte enhancer factor 2D
period of time and a tremendous amount of
energy (Chowdary et al. 2012). Therefore, the
NGF-enhanced transcytosis machinery would be
an economical way to deliver TrkA to growth
cones because it can deliver an appropriate
amount of TrkA to the neurons that compete for
a limited amount of target-derived NGF. Retro-
grade transport of signaling endosomes is an
attractive way of retrograde signaling because it
can specifically deliver the signaling components
themselves where they are needed. Because
26
retrograde signaling endosomes regulate the spec-
ificity of anterograde transport, it is possible that
the NGF-enhanced transcytosis further regulates
the trafficking of additional membrane proteins
necessary for NGF functioning on axons. Further
studies are needed to elucidate the molecular
identity of transcytosed vesicles.
3.5 Generality of Retrograde
Signaling Through Endosomes
Although some alternative models have been pro-
posed, the model of signaling endosomes that
transmit retrograde signaling is well accepted.
Several lines of evidence suggest that this model
may be a general mechanism that target-derived
growth factors utilize to transmit their retrograde
signals. Stimulation with ciliary neurotrophic fac-
tor (CNTF) or leukemia inhibitory factor (LIF) on
distal axons of sympathetic neurons activates
STAT3 in the cell bodies, which is microtubule
dependent (O’Brien and Nathanson 2007). Simi-
larly, the retrograde transport of distal axon-
derived bone morphogenetic protein (BMP) sig-
naling complexes is required for the activation of
SMAD in the cell bodies (Smith et al. 2012;
Walker et al. 2012). In addition, retrograde trans-
port of glial cell line-derived neurotrophic factor
(GDNF) in motor neurons is considered to be
involved in the activation of Pea3 (Leitner et al.
1999; Haase et al. 2002). Until now, however,
physiological functions and detailed molecular
mechanisms of these target-derived signals have
not been elucidated.
Recent studies have demonstrated the
functions and molecular mechanisms of the retro-
grade transport-dependent semaphorin 3A
(Sema3A) signal. Sema3A, a repulsive axon
guidance molecule, is also involved in the regula-
tion of dendritic development and maturation in
the cerebral cortex and hippocampus (Morita
et al. 2006; Yamashita et al. 2007; Nakamura
et al. 2009). In cultured hippocampal neurons,
the site of Sema3A activity is restricted to the
axonal growth cone. Sema3A and its receptor
complex, including PlexinA4, are internalized
into the growth cone and are then transported to
N. Yamashita
cell bodies by the dynein-dependent retrograde
transport. The axon-derived Sema3A signaling
complexes in cell bodies recruit the AMPA recep-
tor subunit GluA2 through interaction with
PlexinA4. These signaling complexes then
enhance the dendritic localization of GluA2,
thereby regulating dendritic development
(Yamashita et al. 2014). Sema3A and NGF sig-
naling endosomes share some signaling
components through the PlexinA4-TrkA interac-
tion. The kinase activity and dynein-interacting
activity of TrkA are required for the retrograde
transport of Sema3A signaling endosomes,
whereas retrograde transport of PlexinA4 is not
involved in retrograde NGF signaling (Yamashita
et al. 2016; Yamane et al. 2017). These findings
suggest that the molecular components of
Sema3A and NGF signaling endosomes do not
completely overlap, which indicates distinct sig-
naling pathways for Sema3A and NGF
(Yamashita 2019). The target-derived Sema3A
also induces cell death signaling in developing
sympathetic neurons through retrograde trans-
port. In this case, PlexinA3 (but not PlexinA4)
is involved in the effect (Wehner et al. 2016),
which may imply that the functions of signaling
endosomes are regulated by organizing their
molecular components.
3.6 Conclusions
From a classical view in which retrograde
neurotrophin signaling endosomes mediate
target-derived survival signals in neurons, the
scenario has broadened to incorporate many
other biological functions that depend on endocy-
tosis of neurotrophin-Trk complex from the
growth cone surface. Recent live cell imaging
experiments demonstrate that, although TrkA
endosomes in axons move exclusively in a retro-
grade manner, axon-derived TrkA endosomes
exhibit various behaviors in somatodendritic
compartments. Although the majority of somatic
TrkA endosomes are localized to the perinuclear
region, others are localized peripherally and then
undergo exo- and endocytosis and/or the move to
the dendrites (Lehigh et al. 2017; Ye et al. 2018).
3 NGF Signaling in Endosomes
In addition, some signaling endosomes undergo
fission and fusion events in somatodendritic
compartments (Barford et al. 2018). Thus, axon-
derived neurotrophin signaling endosomes are
well diversified in somatodendritic
compartments, and thus they may exert many
biological functions. However, we still do not
know when, where, or how the destination and
function of the individual signaling endosome are
determined. In this regard, future studies are
needed to clarify how the functions of signaling
endosomes are related to the type of endocytosis,
the mechanisms of dynein-dependent transport,
and the molecular components of individual
endosomes.
Neurons are unique cells with long axons, and
the long-distance communication between axons
and somatodendritic compartments is one of the
important cell biological activities that neurons
perform throughout life without regeneration.
The study of signaling endosomes helps clarify
the mechanism by which signals initiated at the
distal axon propagate within the cell. This mech-
anism is considered to be one of the foundations
for maintaining the neuronal survival and func-
tion. Because defects in signaling endosomes are
known to be associated with various neurological
disorders (BasuRay et al. 2010; Poon et al. 2011;
Poon et al. 2013; Patel et al. 2015), understanding
the formation, transport, and function of signaling
endosomes at the molecular level is necessary for
understanding the larger issues surrounding the
neuronal circuits in health and disease.
Acknowledgments The author’s work was supported by
the Naito Foundation, the Ichiro Kanehara Foundation, the
Daiichi Sankyo Foundation of Life Science, MEXT/JSPS
KAKENHI (20K07742), and the Institute for Diseases of
Old Age, Juntendo University Graduate School of
Medicine.
References
Andrews TJ, Thrasivoulou C, Nesbit W, Cowen T (1996)
Target-specific differences in the dendritic morphology
and neuropeptide content of neurons in the rat SCG
during development and aging. J Comp Neurol
368:33–44
27
Arevalo JC, Waite J, Rajagopal R, Beyna M, Chen ZY,
Lee FS, Chao MV (2006) Cell survival through Trk
neurotrophin receptors is differentially regulated by
ubiquitination. Neuron 50:549–559
Ascano M, Richmond A, Borden P, Kuruvilla R (2009)
Axonal targeting of Trk receptors via transcytosis
regulates sensitivity to neurotrophin responses. J
Neurosci 29:11674–11685
Barford K, Keeler A, McMahon L, McDaniel K, Yap CC,
Deppmann CD, Winckler B (2018) Transcytosis of
TrkA leads to diversification of dendritic signaling
endosomes. Sci Rep 8:4715
BasuRay S, Mukherjee S, Romero E, Wilson MC,
Wandinger-Ness A (2010) Rab7 mutants associated
with Charcot-Marie-tooth disease exhibit enhanced
NGF-stimulated signaling. PLoS One 5:e15351
Beattie EC, Zhou J, Grimes ML, Bunnett NW, Howe CL,
Mobley WC (1996) A signaling endosome hypothesis
to explain NGF actions: potential implications for
neurodegeneration. Cold Spring Harb Symp Quant
Biol 61:389–406
Beattie EC, Howe CL, Wilde A, Brodsky FM, Mobley
WC (2000) NGF signals through TrkA to increase
clathrin at the plasma membrane and enhance
clathrin-mediated membrane trafficking. J Neurosci
20:7325–7333
Bhattacharyya A, Watson FL, Pomeroy SL, Zhang YZ,
Stiles CD, Segal RA (2002) High-resolution imaging
demonstrates dynein-based vesicular transport of
activated Trk receptors. J Neurobiol 51:302–312
Bodmer D, Ascano M, Kuruvilla R (2011) Isoform-
specific dephosphorylation of dynamin1 by calcineurin
couples neurotrophin receptor endocytosis to axonal
growth. Neuron 70:1085–1099
Chowdary PD, Che DL, Cui B (2012) Neurotrophin sig-
naling via long-distance axonal transport. Annu Rev
Phys Chem 63:571–594
Cosker KE, Segal RA (2014) Neuronal signaling through
endocytosis. Cold Spring Harb Perspect Biol 6(2):
a020669
Deinhardt K, Salinas S, Verastegui C, Watson R, Worth D,
Hanrahan S, Bucci C, Schiavo G (2006) Rab5 and
Rab7 control endocytic sorting along the axonal retro-
grade transport pathway. Neuron 52:293–305
Delcroix JD, Valletta JS, Wu C, Hunt SJ, Kowal AS,
Mobley WC (2003) NGF signaling in sensory neurons:
evidence that early endosomes carry NGF retrograde
signals. Neuron 39:69–84
Deppmann CD, Mihalas S, Sharma N, Lonze BE,
Niebur E, Ginty DD (2008) A model for neuronal
competition during development. Science
320:369–373
Glebova NO, Ginty DD (2005) Growth and survival
signals controlling sympathetic nervous system devel-
opment. Annu Rev Neurosci 28:191–222
Grimes ML, Zhou J, Beattie EC, Yuen EC, Hall DE,
Valletta JS, Topp KS, LaVail JH, Bunnett NW,
Mobley WC (1996) Endocytosis of activated TrkA:
28
evidence that nerve growth factor induces formation of
signaling endosomes. J Neurosci 16:7950–7964
Grimes ML, Beattie E, Mobley WC (1997) A signaling
organelle containing the nerve growth factor-activated
receptor tyrosine kinase, TrkA. Proc Natl Acad Sci
USA 94:9909–9914
Ha J, Lo KW, Myers KR, Carr TM, Humsi MK, Rasoul
BA, Segal RA, Pfister KK (2008) A neuron-specific
cytoplasmic dynein isoform preferentially transports
TrkB signaling endosomes. J Cell Biol 181:1027–1039
Haase G, Dessaud E, Garces A, de Bovis B, Birling M,
Filippi P, Schmalbruch H, Arber S, deLapeyriere O
(2002) GDNF acts through PEA3 to regulate cell
body positioning and muscle innervation of specific
motor neuron pools. Neuron 35:893–905
Harrington AW, Ginty DD (2013) Long-distance retro-
grade neurotrophic factor signalling in neurons. Nat
Rev Neurosci 14:177–187
Harrington AW, St Hillaire C, Zweifel LS, Glebova NO,
Philippidou P, Halegoua S, Ginty DD (2011) Recruit-
ment of actin modifiers to TrkA endosomes governs
retrograde NGF signaling and survival. Cell
146:421–434
Heerssen HM, Pazyra MF, Segal RA (2004) Dynein
motors transport activated Trks to promote survival of
target-dependent neurons. Nat Neurosci 7:596–604
Hendry IA, Stockel K, Thoenen H, Iversen LL (1974) The
retrograde axonal transport of nerve growth factor.
Brain Res 68:103–121
Howe CL, Valletta JS, Rusnak AS, Mobley WC (2001)
NGF signaling from clathrin-coated vesicles: evidence
that signaling endosomes serve as a platform for the
Ras-MAPK pathway. Neuron 32:801–814
Huang EJ, Reichardt LF (2003) Trk receptors: roles in
neuronal signal transduction. Annu Rev Biochem
72:609–642
Jing S, Tapley P, Barbacid M (1992) Nerve growth factor
mediates signal transduction through trk homodimer
receptors. Neuron 9:1067–1079
Kuruvilla R, Ye H, Ginty DD (2000) Spatially and func-
tionally distinct roles of the PI3-K effector pathway
during NGF signaling in sympathetic neurons. Neuron
27:499–512
Kuruvilla R, Zweifel LS, Glebova NO, Lonze BE,
Valdez G, Ye H, Ginty DD (2004) A neurotrophin
signaling cascade coordinates sympathetic neuron
development through differential control of TrkA traf-
ficking and retrograde signaling. Cell 118:243–255
Lehigh KM, West KM, Ginty DD (2017) Retrogradely
transported TrkA endosomes signal locally within
dendrites to maintain sympathetic neuron synapses.
Cell Rep 19:86–100
Leitner ML, Molliver DC, Osborne PA, Vejsada R,
Golden JP, Lampe PA, Kato AC, Milbrandt J, Johnson
EM Jr (1999) Analysis of the retrograde transport of
glial cell line-derived neurotrophic factor (GDNF),
neurturin, and persephin suggests that in vivo signaling
for the GDNF family is GFRalpha coreceptor-specific.
J Neurosci 19:9322–9331
N. Yamashita
Morita A, Yamashita N, Sasaki Y, Uchida Y, Nakajima O,
Nakamura F, Yagi T, Taniguchi M, Usui H, Katoh-
Semba R, Takei K, Goshima Y (2006) Regulation of
dendritic branching and spine maturation by
semaphorin3A-Fyn signaling. J Neurosci
26:2971–2980
Nakamura F, Ugajin K, Yamashita N, Okada T, Uchida Y,
Taniguchi M, Ohshima T, Goshima Y (2009)
Increased proximal bifurcation of CA1 pyramidal api-
cal dendrites in sema3A mutant mice. J Comp Neurol
516:360–375
O’Brien JJ, Nathanson NM (2007) Retrograde activation
of STAT3 by leukemia inhibitory factor in sympathetic
neurons. J Neurochem 103:288–302
Patel A, Yamashita N, Ascano M, Bodmer D, Boehm E,
Bodkin-Clarke C, Ryu YK, Kuruvilla R (2015)
RCAN1 links impaired neurotrophin trafficking to
aberrant development of the sympathetic nervous sys-
tem in down syndrome. Nat Commun 6:10119
Pazyra-Murphy MF, Hans A, Courchesne SL, Karch C,
Cosker KE, Heerssen HM, Watson FL, Kim T,
Greenberg ME, Segal RA (2009) A retrograde neuro-
nal survival response: target-derived neurotrophins
regulate MEF2D and bcl-w. J Neurosci 29:6700–6709
Poon WW, Blurton-Jones M, Tu CH, Feinberg LM,
Chabrier MA, Harris JW, Jeon NL, Cotman CW
(2011) Beta-amyloid impairs axonal BDNF retrograde
trafficking. Neurobiol Aging 32:821–833
Poon WW, Carlos AJ, Aguilar BL, Berchtold NC,
Kawano CK, Zograbyan V, Yaopruke T,
Shelanski M, Cotman CW (2013) Beta-amyloid
(Abeta) oligomers impair brain-derived neurotrophic
factor retrograde trafficking by down-regulating
ubiquitin C-terminal hydrolase, UCH-L1. J Biol
Chem 288:16937–16948
Reynolds AJ, Bartlett SE, Hendry IA (1998) Signalling
events regulating the retrograde axonal transport of
125I-beta nerve growth factor in vivo. Brain Res
798:67–74
Riccio A, Pierchala BA, Ciarallo CL, Ginty DD (1997) An
NGF-TrkA-mediated retrograde signal to transcription
factor CREB in sympathetic neurons. Science
277:1097–1100
Ruit KG, Osborne PA, Schmidt RE, Johnson EM Jr,
Snider WD (1990) Nerve growth factor regulates sym-
pathetic ganglion cell morphology and survival in the
adult mouse. J Neurosci 10:2412–2419
Scott-Solomon E, Kuruvilla R (2018) Mechanisms of
neurotrophin trafficking via Trk receptors. Mol Cell
Neurosci 91:25–33
Scott-Solomon E, Kuruvilla R (2020) Prenylation of
axonally translated Rac1 controls NGF-dependent
axon growth. Dev Cell 53(691–705):e697
Shao Y, Akmentin W, Toledo-Aral JJ, Rosenbaum J,
Valdez G, Cabot JB, Hilbush BS, Halegoua S (2002)
Pincher, a pinocytic chaperone for nerve growth factor/
TrkA signaling endosomes. J Cell Biol 157:679–691
Sharma N, Deppmann CD, Harrington AW, St Hillaire C,
Chen ZY, Lee FS, Ginty DD (2010) Long-distance
3 NGF Signaling in Endosomes
control of synapse assembly by target-derived NGF.
Neuron 67:422–434
Smith RB, Machamer JB, Kim NC, Hays TS, Marques G
(2012) Relay of retrograde synaptogenic signals
through axonal transport of BMP receptors. J Cell Sci
125:3752–3764
Snider WD (1988) Nerve growth factor enhances dendritic
arborization of sympathetic ganglion cells in develop-
ing mammals. J Neurosci 8:2628–2634
Steljes TP, Kinoshita Y, Wheeler EF, Oppenheim RW,
von Bartheld CS (1999) Neurotrophic factor regulation
of developing avian oculomotor neurons: differential
effects of BDNF and GDNF. J Neurobiol 41:295–315
Stoeckel K, Schwab M, Thoenen H (1975) Specificity of
retrograde transport of nerve growth factor (NGF) in
sensory neurons: a biochemical and morphological
study. Brain Res 89:1–14
Suo D, Park J, Harrington AW, Zweifel LS, Mihalas S,
Deppmann CD (2014) Coronin-1 is a neurotrophin
endosomal effector that is required for developmental
competition for survival. Nat Neurosci 17:36–45
Tsui-Pierchala BA, Ginty DD (1999) Characterization of
an NGF-P-TrkA retrograde-signaling complex and
age-dependent regulation of TrkA phosphorylation in
sympathetic neurons. J Neurosci 19:8207–8218
Ure DR, Campenot RB (1997) Retrograde transport and
steady-state distribution of 125I-nerve growth factor in
rat sympathetic neurons in compartmented cultures. J
Neurosci 17:1282–1290
Valdez G, Akmentin W, Philippidou P, Kuruvilla R, Ginty
DD, Halegoua S (2005) Pincher-mediated
macroendocytosis underlies retrograde signaling by
neurotrophin receptors. J Neurosci 25:5236–5247
Voyvodic JT (1989) Peripheral target regulation of den-
dritic geometry in the rat superior cervical ganglion. J
Neurosci 9:1997–2010
Walker BA, Ji SJ, Jaffrey SR (2012) Intra-axonal transla-
tion of RhoA promotes axon growth inhibition by
CSPG. J Neurosci 32:14442–14447
Watson FL, Heerssen HM, Moheban DB, Lin MZ,
Sauvageot CM, Bhattacharyya A, Pomeroy SL, Segal
RA (1999) Rapid nuclear responses to target-derived
neurotrophins require retrograde transport of ligand-
receptor complex. J Neurosci 19:7889–7900
Wehner AB, Abdesselem H, Dickendesher TL, Imai F,
Yoshida Y, Giger RJ, Pierchala BA (2016)
Semaphorin 3A is a retrograde cell death signal in
developing sympathetic neurons. Development
143:1560–1570
Wu C, Lai CF, Mobley WC (2001) Nerve growth factor
activates persistent Rap1 signaling in endosomes. J
Neurosci 21:5406–5416
Wu C, Ramirez A, Cui B, Ding J, Delcroix JD, Valletta JS,
Liu JJ, Yang Y, Chu S, Mobley WC (2007) A
29
functional dynein-microtubule network is required for
NGF signaling through the Rap1/MAPK pathway.
Traffic 8:1503–1520
Yamane M, Yamashita N, Hida T, Kamiya Y,
Nakamura F, Kolattukudy P, Goshima Y (2017) A
functional coupling between CRMP1 and Nav1.7 for
retrograde propagation of semaphorin3A signaling. J
Cell Sci 130:1393–1403
Yamashita N (2019) Retrograde signaling via axonal
transport through signaling endosomes. J Pharmacol
Sci 141:91–96
Yamashita N, Kuruvilla R (2016) Neurotrophin signaling
endosomes: biogenesis, regulation, and functions. Curr
Opin Neurobiol 39:139–145
Yamashita N, Morita A, Uchida Y, Nakamura F, Usui H,
Ohshima T, Taniguchi M, Honnorat J, Thomasset N,
Takei K, Takahashi T, Kolattukudy P, Goshima Y
(2007) Regulation of spine development by
semaphorin3A through cyclin-dependent kinase
5 phosphorylation of collapsin response mediator pro-
tein 1. J Neurosci 27:12546–12554
Yamashita N, Usui H, Nakamura F, Chen S, Sasaki Y,
Hida T, Suto F, Taniguchi M, Takei K, Goshima Y
(2014) Plexin-A4-dependent retrograde semaphorin
3A signalling regulates the dendritic localization of
GluA2-containing AMPA receptors. Nat Commun
5:3424
Yamashita N, Yamane M, Suto F, Goshima Y (2016)
TrkA mediates retrograde semaphorin 3A signaling
through plexin A4 to regulate dendritic branching. J
Cell Sci 129:1802–1814
Yamashita N, Joshi R, Zhang S, Zhang ZY, Kuruvilla R
(2017) Phospho-regulation of soma-to-axon
transcytosis of neurotrophin receptors. Dev Cell
42:626–639.e625
Yano H, Lee FS, Kong H, Chuang J, Arevalo J, Perez P,
Sung C, Chao MV (2001) Association of Trk
neurotrophin receptors with components of the cyto-
plasmic dynein motor. J Neurosci 21:RC125
Ye H, Kuruvilla R, Zweifel LS, Ginty DD (2003) Evi-
dence in support of signaling endosome-based retro-
grade survival of sympathetic neurons. Neuron
39:57–68
Ye M, Lehigh KM, Ginty DD (2018) Multivesicular bod-
ies mediate long-range retrograde NGF-TrkA signal-
ing. elife 7:e33012
Zhou B, Cai Q, Xie Y, Sheng ZH (2012) Snapin recruits
dynein to BDNF-TrkB signaling endosomes for retro-
grade axonal transport and is essential for dendrite
growth of cortical neurons. Cell Rep 2:42–51
Zweifel LS, Kuruvilla R, Ginty DD (2005) Functions and
mechanisms of retrograde neurotrophin signalling. Nat
Rev Neurosci 6:615–625
 The NGF Metabolic Pathway: New
Opportunities for Biomarker Research 4
and Drug Target Discovery
NGF Pathway Biomarkers and Drug Targets

Rowan Pentz and M. Florencia Iulita

Abstract disease-modifying nature of the cholinergic

Recent research has demonstrated that degen-
eration of the basal forebrain cholinergic sys-
tem, far from being a mere downstream
mediator of Alzheimer’s disease
(AD) symptoms, may play a disease-
aggravating role in the continuum of AD
pathology. The search for novel biomarkers
of the cholinergic deficit in AD and novel
therapeutic targets for the sustenance of the
basal forebrain cholinergic system has there-
fore taken on more urgency. A novel model
that explains the preferential vulnerability of
basal forebrain cholinergic neurons in AD as
the result of pathological alterations to nerve
growth factor (NGF) metabolism offers an
integrated investigative platform for the devel-
opment of such biomarkers and therapeutics.
By positing a reciprocal trophic interaction
between the basal forebrain and its target
tissues, this model can also explain the
R. Pentz
Department of Neurology and Neurosurgery, McGill
University, Montreal, Canada
e-mail: rowan.pentz@mail.mcgill.ca
M. F. Iulita (*)
Sant Pau Memory Unit, Department of Neurology,
Hospital de la Santa Creu i Sant Pau, Biomedical Research
Institute Sant Pau, Universitat Autònoma de Barcelona,
Barcelona, Spain
Center of Biomedical Investigation Network for
Neurodegenerative Diseases (CIBERNED), Madrid, Spain
e-mail: miulita@santpau.cat
deficit in AD and can incorporate other key
factors in basal forebrain cholinergic degener-
ation, including NGF receptor changes and
retrograde transport deficits in AD. This chap-
ter will focus on the potential of NGF meta-
bolic pathway biomarkers in AD as well as
therapeutic targets to correct NGF metabolic
deficits, aiding the development of novel
pro-cholinergic therapeutics.
4.1 Introduction
It could be said that 2021 marks the beginning of
a new era for Alzheimer’s disease (AD) patients:
accurate blood-based biomarkers based on
phosphorylated tau isoforms (pTau-181 and
pTau-217) are at the forefront in diagnostics
(Janelidze et al. 2020; Karikari et al. 2020;
Palmqvist et al. 2020; Thijssen et al. 2020), neu-
roimaging tracers are being refined, and the anti-
amyloid monoclonal antibody aducanumab is
waiting for the FDA's decision on its application
by June 2021, which if approved, would be the
first disease-modifying treatment for AD. How-
ever, despite these promising developments, the
reality remains that the majority of AD patients
still get diagnosed when they develop cognitive
impairment and still receive anticholinesterase
medication, dying approximately after

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 31

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_4
32
8–10 years, just like they would have in the
previous century.
It is now widely recognized that Alzheimer’s
dementia and even mild cognitive impairment
(MCI) represent relatively late stages in the AD
pathological process and ones that may entail an
irreversible loss of the microstructural integrity of
the brain. Furthermore, it is increasingly
recognized that AD is a heterogenous disorder
for which no single biomarker or therapeutic
may be effective. Therefore, the effective man-
agement of AD will likely require a panel of
easily accessible and cost-effective biomarkers
that can be widely implemented, as well as
disease-modifying treatments that prevent,
reduce, or delay cognitive decline better than
current symptomatic medications (McDade and
Bateman 2017).
In this chapter, we will summarize the evi-
dence of a novel and promising source of poten-
tial biomarker candidates and drug targets
involving the nerve growth factor (NGF) meta-
bolic pathway. As discussed at length in Chap. 8
by A.C. Cuello, the discovery of the NGF meta-
bolic pathway has unified paradoxical findings to
account for the cholinergic deficit in AD (Cuello
et al. 2007; Iulita and Cuello 2014; Cuello et al.
2019), serving as a foundation and framework for
the discovery of novel therapeutic targets and
biomarker candidates.
4.2 Targeting the Cholinergic
System in AD: The Value
of Symptomatic Treatments
Cholinergic neurons that innervate the cerebral
cortex and hippocampus are essential for many
aspects of cognition and have their cell bodies in
the basal forebrain. Even though during develop-
ment all neurons depend on growth factors and
neurotrophins for survival, basal forebrain cholin-
ergic neurons (BFCNs) are the only ones that
retain this close dependency in the adult brain
(Hefti 1986; Hefti et al. 1986; Cuello 1996).
Specifically, these neurons depend on the contin-
uous supply of NGF for the maintenance of their
functional phenotype and synaptic integrity
R. Pentz and M. F. Iulita
(Debeir et al. 1999b; Allard et al. 2012b; Allard
et al. 2018b).
The relevance of the BFCN system in AD is
best evidenced by the fact that four of the five
licenced drugs to treat AD are acetylcholinester-
ase inhibitors (AChEIs), which prevent the break-
down of acetylcholine and thereby enhance
cholinergic neurotransmission. AChEIs are
widely applied drugs for AD and routinely pro-
vide a 6-month–2-year reprieve from cognitive
decline—though after these drugs cease to be
effective, patients return to their original patho-
logical trajectory (Giacobini and Becker 2007;
Pepeu and Giovannini 2010; Birks and Harvey
2018). Importantly, the efficacy of AChEIs
depends on some partial degree of cholinergic
integrity (Richter et al. 2018) and correlates to
inhibition of AChE (Giacobini et al. 2002),
demonstrating that the cholinergic system is a
validated therapeutic target in AD.
As AChEIs were considered to be “merely”
symptomatic drugs, interest in cholinergic
therapies waned. However, in the effort to priori-
tize the development of disease-modifying
treatments targeting core aspects of AD neuropa-
thology, perhaps an opportunity to build on
validated targets was missed. Indeed,
symptomatic treatments for diseases of old age
can substantially recapitulate the benefits of
disease-modifying therapies. It has been
estimated that a drug (e.g. a novel AChEI) that
could provide 3 rather than the existing 2 years of
symptomatic relief would produce an ~10%
reduction in the global prevalence of AD and a
cholinergic therapy that extended that symptom-
atic benefit to 5 years would produce an ~40%
reduction in prevalence (Zissimopoulos et al.
2015)—this is a viable notion, as L-DOPA
provides precisely such a 5-year reprieve for
patients with typical Parkinson’s disease
presentations. Indeed, projections in the context
of the Canadian healthcare system have indicated
that a drug offering 5 years of symptomatic relief
would cause cases of AD and associated
healthcare costs to increase only by 10% rather
than by 98% over the next 50 years (Manuel et al.
2016). Given that there are legitimate concerns
about the ability of modern healthcare systems to
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
meet the looming challenge of the AD epidemic
(El-Hayek et al. 2019), the value of better
treatments for AD becomes readily apparent.
Although AChEIs are considered symptomatic
drugs, there is evidence to suggest that they may
also have disease-modifying properties. From a
mechanistic standpoint, earlier reports had
indicated that physiological cholinergic signal-
ling (particularly through M1 and M3 muscarinic
receptors) reduces amyloidosis (Nitsch et al.
1992), lymphocyte proliferation, cytokine pro-
duction (Nizri et al. 2006), vasculopathy
(Claassen and Jansen 2006), and glutamate
excitotoxicity (Shen et al. 2010), leading to
neuroprotection (Fisher 2008). Thus, the loss of
this neuroprotective signalling, as a result of
BFCN degeneration and denervation of choliner-
gic input to target tissues (Bartus et al. 1982),
could help produce the neuropathology of AD
(Hampel et al. 2018).
Recent evidence has converged to support this
model (Risacher et al. 2016; Hampel et al. 2018).
Indeed, magnetic resonance imaging (MRI) stud-
ies have shown that AChEIs lead to reductions in
the rate of atrophy of the hippocampus, cerebral
cortex, and BFCN in patients with suspected pro-
dromal AD (Dubois et al. 2015; Cavedo et al.
2016; Cavedo et al. 2017). The validity of these
findings is further strengthened by preclinical
studies with novel pro-cholinergic drugs showing
lasting, disease-modifying effects of cholinergic
therapy in AD rodent models (Fig. 4.1) (Caccamo
et al. 2006; Hall et al. 2018). Given that M1
receptors are relatively spared in AD (Svensson
et al. 1992; Overk et al. 2010), finding novel ways
of activating these receptors (with minimal
peripheral side effects) remains an area of active
research.
4.3 NGF Dysmetabolism
and the Degeneration
of the Cholinergic System
in AD: A Self-Propagating
Feedback Loop?
Like the core AD pathological hallmarks, damage
to BFCNs accumulates silently over years or
33
decades as preclinical AD pathology advances.
Their atrophy is evident well prior to the onset
of cognitive impairments (Grothe et al. 2014),
and their degeneration predicts the atrophy of
the entorhinal cortex and cerebral cortex
according to the topography of cholinergic inner-
vation (Schmitz et al. 2016, 2018).
New insight into the causes of cholinergic
degeneration in AD has come from the discovery
of the NGF metabolic pathway, which is
described extensively in Chap. 8. Briefly, the
NGF precursor proNGF is secreted from neurons
in response to cholinergic stimulation, is
processed extracellularly to produce the mature
peptide, and is subsequently degraded; these steps
are accomplished by a set of co-released
zymogens and convertases (Bruno and Cuello
2006). Activated plasmin cleaves proNGF to
mature NGF (mNGF) in the synaptic space. This
plasmin is derived from cleavage of its inactive
zymogen, plasminogen, by tissue plasminogen
activator (tPA), which is regulated by
neuroserpin. The mNGF that is not taken up by
the presynaptic BFCN is degraded by matrix
metalloproteinase-9 (MMP-9) or MMP-3.
MMP-9 and MMP-3 are derived from cleavage
of their preprotein precursors, in a manner
regulated by tissue inhibitor of
metalloproteinase-1 (TIMP1) (Bruno and Cuello
2006).
The relevance of the NGF metabolic pathway
in modulating the availability of mNGF was
demonstrated by experiments showing that the
intracortical infusion of neuroserpin increased
proNGF and that the inhibition of MMP-9
increased mNGF in naïve rats (Bruno and Cuello
2006). These findings suggested that the NGF
pathway could indeed modulate cholinergic
tone, as the steady-state number of cortical cho-
linergic synapses is determined by NGF availabil-
ity (Debeir et al. 1998, 1999a). The link between
the NGF pathway and the status of the cholinergic
projection system was later demonstrated by
experiments in which NGF maturation was
disrupted with long-term intracortical infusions
of α2-antiplasmin in naïve rats, leading to
reduced density and integrity of cortical choliner-
gic synapses (Allard et al. 2012a) as well as

Fig. 4.1 A novel selective allosteric M1 muscarinic agonist (AF710B) attenuates
cognitive deficits and AD pathology in McGill-R-Thy1-APP transgenic rats. At
13 months of age (post-plaque stage), rats homozygous for mutant human APP bearing
the Swedish and Indiana mutations were treated with oral AF710B or vehicle for
5 months. After a 1-month washout, AF710B-treated rats demonstrated better
34
performance on the Novel Object Recognition Test and Morris Water Maze Test, as
well as reduced amyloid plaque density, normalized synaptophysin levels, and reduced
microgliosis. Reproduced with permission from John Wiley and Sons, Hall et al.,
Alzheimer’s & Dementia 2018
R. Pentz and M. F. Iulita
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . . 35

smaller soma size of BFCNs selectively (Allard (Mufson et al. 2019), it is worth noting that
et al. 2018a). These reductions in NGF metabo- TrkA mRNA is induced by mNGF signalling
lism/cholinergic tone also led to impaired (Holtzman et al. 1992; Gibbs and Pfaff 1994;
learning and memory on a variety of behavioural Venero et al. 1994; Figueiredo et al. 1995) and
tasks (Allard et al. 2012a). TrkA changes could be downstream of changes to
The inclusion of NGF metabolism into a NGF availability, possibility exacerbating these
model of cholinergic degeneration in AD can alterations. It is evident how this cycle could
further explain how such changes may become a become self-propagating in light of the
self-propagating feedback loop driving AD accumulating amyloid pathology, evolving
pathology (Fig. 4.2). Amyloidosis has been inflammation, and NGF dysmetabolism charac-
demonstrated to lead to NGF dysmetabolism, as teristic of AD, driving cholinergic and cognitive
the infusion of amyloid oligomers alone is suffi- deficits (Fig. 4.2).
cient to trigger AD-like NGF dysmetabolism in
naïve rats (Bruno et al. 2009a), as transgenic rats
expressing mutant human APP exhibit NGF
4.4 The NGF Pathway as a Platform
dysmetabolism (Iulita et al. 2017), and as brain
for the Search of New
amyloidosis correlates to various measures of
Biomarkers
NGF dysmetabolism in human AD (Bruno and
Cuello 2006; Bruno et al. 2009a; Pentz et al.
Biomarkers for the incipient stage of AD have
2020). Mediation of this effect by inflammation
advanced significantly over the past decades, and
is suggested by studies demonstrating that Aβ
it is now possible to identify people in early
oligomer-induced NGF dysmetabolism is
stages of the disease with relatively high cer-
restored to a trophic state by the application of
tainty, through imaging and CSF measures of
an anti-inflammatory (Bruno et al. 2009a), the
core AD pathological markers and, more recently,
well-established associations between Aβ pathol-
via ultrasensitive plasma assays (Blennow and
ogy and inflammation (Welikovitch et al. 2020),
Zetterberg 2018; Jack 2020). Despite these posi-
and the key role that NGF pathway proteins
tive advances, there is also an increasing recogni-
(including MMPs and the plasmin-activating sys-
tion that AD is a heterogenous condition
tem) play in a variety of inflammatory pathways
comprising several distinct clinical and patholog-
(Parks et al. 2004; Mehra et al. 2016).
ical subtypes (Murray et al. 2011), and meaning-
The resulting impairment of proNGF matura-
ful results may therefore not come from a single
tion and excessive mNGF degradation will then
biomarker.
lead to the atrophy of BFCNs and the loss of
In this regard, the NGF metabolic pathway
cortical cholinergic tone, as demonstrated causa-
offers an integrative exploratory platform to iden-
tively in experimental models (Allard et al.
tify new biomarkers of AD-related cholinergic
2012a, 2018a) and supported correlatively in
degeneration. Indeed, as cholinergic function/
human AD by a demonstration of the association
pathology is tightly tied to cognition (Drachman
between VAChT depletion and proNGF accumu-
and Leavitt 1974; Hasselmo and Sarter 2011) and
lation (Pentz et al. 2020). These deficits will then
differs across AD subtypes (Machado et al.
result in increased amyloidosis and/or inflamma-
2020), a biomarker that tracks this process could
tion by the mechanisms described above, and
have utility for sub-diagnosis and prognosis. As
excitotoxicity could play a role as well, as it is
the efficacy of AChEIs depends on partially intact
reduced by cholinergic signalling (Shen et al.
cholinergic tone (Richter et al. 2018), biomarkers
2010) and associated with acute increases in
associated with NGF metabolism could also be
proNGF (Le 2011).
used to inform about the efficacy of cholinergic
While a downregulation in TrkA levels has
therapies.
been implicated in AD cholinergic pathology
36 R. Pentz and M. F. Iulita

Fig. 4.2 Hypothetical basocortical feedback loop driving
Aβ pathology and NGF dysmetabolism in AD. Aβ pathol-
ogy is sufficient to unleash an early pro-inflammatory
process and to impair NGF metabolism by facilitating
proNGF accumulation and reduced mNGF availability.
A compromised NGF metabolism would lead to
downregulation of NGF receptor expression and dimin-
ished cholinergic trophic support resulting in reduced cho-
linergic tone. Because of reduced cholinergic
neurotransmission, the generation of toxic Aβ peptides
and inflammation will be favoured, contributing to the
self-propagation of this loop

alzheimer-down-unit/) (Fortea et al. 2018b,

4.5 Tracking NGF Metabolism
2020).
in AD Biofluids
The first study on the characterization of NGF
metabolic pathway proteins in plasma samples
Initial studies investigating NGF pathway
from a population-based cohort of individuals
markers in body fluids focused on individuals
with DS from Catania, Italy, found significant
with Down syndrome (DS). Because of the tri-
differences in DS with respect to euploid controls:
somy of chromosome 21, which harbours the
increased proNGF and increased MMP-9,
APP gene, individuals with DS are considered a
MMP-3, and MMP-1 in people with DS and
genetically determined high-risk population for
established dementia (DSAD), as well as in
AD. As they inevitably develop advanced AD
those who were asymptomatic (DS) (Iulita et al.
pathology by midlife (Head and Lott 2004;
2016). No changes were observed for tPA and
Head et al. 2012; Lott and Head 2019), studies
neuroserpin. A longitudinal assessment further
in DS represent a unique opportunity to investi-
showed that an increase in proNGF over a
gate biomarker trajectories particularly in the
1-year interval predicted prospective cognitive
early, silent stages of AD. This has been reflected
deterioration over 2 years (Iulita et al. 2016).
in the launch of several new projects assessing
A subsequent study, assessing a different
AD biomarkers, such as the Alzheimer’s
population-based cohort of people with DS from
Biomarkers Consortium-Down Syndrome
Barcelona, Spain, recapitulated these findings on
(ABC-DS, https://www.nia.nih.gov/research/
NGF markers in plasma and further extended
abc-ds) (Handen et al. 2020) and the Down
them to comparisons in CSF (Pentz et al.
Alzheimer Barcelona Neuroimaging Initiative
2021a). Highly similar profiles of NGF
(DABNI, https://santpaumemoryunit.com/
dysmetabolism were found in plasma, while
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
profiles in CSF bore yet more similarity to the
status of the NGF metabolic pathway in AD
(Pentz et al. 2020) and DSAD brains (Iulita
et al. 2014). Furthermore, CSF analysis permitted
the differentiation of different profiles in DS and
DSAD: DS was characterized by
increased proNGF (27 kDa and 50 kDa forms),
increased neuroserpin, increased MMP-3, and
increased MMP-1. DSAD in CSF recapitulated
these changes and was associated with signifi-
cantly further increased 50 kDa proNGF,
increased MMP-9, and normalized MMP-1.
ROC analysis showed that CSF MMP-9 and the
50 kDa form of proNGF differentiated DS from
DSAD with good diagnostic accuracy
(AUC ¼ 0.86–0.87). This study also revealed
that NGF pathway proteins correlated to core
biomarkers of AD, with particularly strong
associations observed between MMP-3 and total
tau as well as between proNGF (50 kDa) and the
Aβ42/40 ratio. Together, these results provide a
strong rationale for the idea that components of
the AD pathological process may be traceable by
signatures of NGF pathway proteins.
Below we will summarize and discuss previ-
ous studies assessing NGF pathway proteins indi-
vidually in biofluids as biomarkers of AD:
4.5.1 ProNGF
Other studies have examined NGF/proNGF in
body fluids independent from its metabolic path-
way. However, most proNGF and mNGF ELISA
systems do not effectively distinguish between
the two (Malerba et al. 2016)—a critical draw-
back in a condition where such markers are
regulated in opposite directions. Furthermore,
the independent and important contribution of
proNGF in the brain and in AD was not
recognized until 2001 (Fahnestock et al. 2001),
and as such, in biomarker studies prior to that
date, it was not independently assessed (Hock
et al. 2000).
In the sole other study to explicitly consider
CSF proNGF in the context of AD, CSF proNGF
levels were markedly higher in amnesic MCI and
AD patients. They were also elevated in persons
37
with clinical dementia ratings (CDRs) of 0.5 com-
pared to people with CDR 0 (Counts et al. 2016),
and the difference was higher when combined
with Aβ42 levels, indicating the changes in
proNGF precede the onset of dementia and are
associated with preclinical pathology. CSF
proNGF correlated to several cognitive test scores
(Counts et al. 2016), recapitulating similar
associations observed in brain tissue (Peng et al.
2015; Pentz et al. 2020). However, it must be
noted that this analysis was done in CSF taken
intracerebroventricularly, post-mortem, not spinal
CSF taken from living patients. In line with this
account, a previous study had identified increased
NGF in AD CSF using methods that did not
distinguish proNGF from mNGF (Massaro et al.
1994), and, given that that proNGF predominates
over mNGF in CSF, this could be counted as
confirmation.
4.5.2 tPA and Neuroserpin
Lower tPA levels have been reported in AD CSF,
where it correlates positively to cognitive scores
(Hanzel et al. 2014a)—a relationship that was
also demonstrated in human frontal cortex from
cognitively healthy aged individuals (Pentz et al.
2020). However, no differences in tPA levels in
CSF between MCI, AD, and controls were
reported in other studies (Martorana et al. 2012;
Marksteiner et al. 2014), in line with our findings
in a DS cohort (Pentz et al. 2021a). tPA may be
sensitive to storage conditions or to inter-assay
variance, or the results may be confounded by
another association—for example, tPA has been
suggested as a biomarker of geriatric depression
(Shi et al. 2010), and prodromal (and clinically
manifest) AD patients, as well as people with DS,
often manifest depression symptoms.
CSF neuroserpin has been reliably found to be
upregulated in AD CSF (Nielsen et al. 2007;
Hanzel et al. 2014a), in accordance with the
results in DSAD CSF from the Barcelona cohort.
Interestingly, one study found that neuroserpin
levels could identify people with AD from
controls and from patients with other neurodegen-
erative conditions, suggesting that a neuroserpin
38
upregulation might be a specific feature of AD
(Nielsen et al. 2007).
4.5.3 Matrix Metalloproteinase-3
and Metalloproteinase-9
CSF MMP-3 has been observed to be upregulated
in sporadic AD compared to MCI and healthy
controls (Hanzel et al. 2014b), in accordance
with the increase observed in DS/DSAD. CSF
MMP-3 was also shown to correlate to total tau
and p-Tau-181 levels, as well as cognitive decline
in the context of AD (Hanzel et al. 2014b).
Plasma MMP-3 was likewise demonstrated to be
upregulated in AD and to associate with cognitive
outcomes in a retrospective cohort analysis (Iulita
et al. 2019). Higher MMP-3 in AD plasma and
correlations to cognitive status were also
demonstrated by a second study (Lin et al.
2006), while a third, using both CSF and plasma,
showed that MMP-3 was increased in AD
plasma, and a trend was observable in AD CSF
(Horstmann et al. 2010).
MMP-3 is also higher in CSF from cognitively
healthy individuals classified as “at risk of AD”
by biomarker scores (Stomrud et al. 2010). This
study also confirmed the associations with
p-Tau-181 and total tau (Stomrud et al. 2010)
observed by Hanzel and colleagues (Hanzel
et al. 2014b) and in our work in DS (Pentz et al.
2021b). Diverging results were found in a study
that showed lower MMP-3 alongside reduced
CSF Aβ42 in people classified as having
neuroinflammation/neuroinfection while finding
no association between CSF tau and MMP-3
(Mlekusch and Humpel 2009). Although the
reasons behind these discrepant findings are not
clear, CSF was frozen 3 days after collection in
this study, which might have affected MMP-3
stability. Overall, there is general agreement that
plasma and CSF MMP-3 are increased in the AD
pathological process and that biofluid MMP-3 is
associated with p-Tau-181 and total tau rather
than other core biomarkers of AD.
Assessments of plasma MMP-9 in AD have
yielded divergent results. Several studies have
demonstrated no change in AD (Martín-Aragón
R. Pentz and M. F. Iulita
et al. 2009; Lim et al. 2011; Tuna et al. 2018;
Iulita et al. 2019), while others have described
increases (Lorenzl et al. 2003; Cai et al. 2007;
Lorenzl et al. 2008; Whelan et al. 2019), as in our
study in DS, or reductions (Horstmann et al.
2010). Similar discrepancies have been obtained
when MMP-9 was considered in CSF, with a
plurality of studies identifying increases (Lorenzl
et al. 2003; Cai et al. 2007; Lorenzl et al. 2008;
Stomrud et al. 2010; Whelan et al. 2019) but other
studies finding no change (Adair et al. 2004;
Hanzel et al. 2014a) or decreases (Mroczko
et al. 2014) with respect to controls. Several
factors could underlie these discrepancies.
MMP-9 is scarcely expressed in CSF (Horstmann
et al. 2010), and even temperatures of 80  C do
not fully protect it from degradation, rendering it
highly susceptible to differences in storage
conditions (Rouy et al. 2005). The preparation
of plasma, which commonly contains protease
inhibitors and chelators such as EDTA, may con-
found zymographic studies (which are used to
assess MMP activity), as MMPs are Zn+2-depen-
dent enzymes. Other storage conditions (as well
as inhibitors/peptidase levels) could also cause
activity-based and antigen-based MMP assays to
differ. Additionally, different comorbidities
between the study populations, some of which
are very common in DS syndrome, should also
be considered.
4.5.4 Correspondence Between NGF
Metabolism in Biofluids
and the CNS
How do the described biomarker results accord
with the status of the NGF metabolic pathway in
brains with AD pathology? ProNGF protein,
MMP-9 activity, and neuroserpin protein have
been demonstrated to be elevated in brains from
people with DSAD, concurrent with reduced tPA
mRNA (Iulita et al. 2014), as reviewed in (Iulita
and Cuello 2016). These changes, apart from tPA,
accord with findings in CSF in the DS population;
however, plasma did not reflect the changes to
MMP-9 or neuroserpin seen in the brain (Iulita
et al. 2014; Pentz et al. 2021a). Interestingly,
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . . 39

Table 4.1 Summary of changes in NGF pathway markers found in biofluids and the brain
Plasma CSF Brain Comment
proNGF " " " Diagnostic accuracy to distinguish AD in DS is superior in
CSF vs. plasma; 50 kDa form appears more sensitive to diagnosis
than 27 kDa form
tPA # or no # or no # tPA may be susceptible to storage conditions or to inter-assay
change change variance. Results may be confounded by another association,
e.g. depression
Neuroserpin No " " Results consistent across studies. Increased in AD vs. controls
change and vs. other dementias
MMP-3 " " " Consistent results across studies; biofluid MMP-3 is associated with
p-Tau-181 and total tau; MMP-3 levels differ by sex
MMP-9 "# or no "# or no " MMP-9 may be sensitive to degradation and susceptible to storage
change change conditions. EDTA tubes for plasma collection may impact MMP
activity as they are Zn+2-dependent enzymes
mNGF Data not Data not # ELISA cannot differentiate mNGF and proNGF. No accounts of
available available mNGF measured by Western blot in biofluids. With special
extraction protocols, it is possible to measure it in brain homogenates
AD Alzheimer’s disease, CSF cerebrospinal fluid, DS Down syndrome, mNGF mature NGF, MMP matrix
metalloproteinase, proNGF precursor of nerve growth factor, tPA tissue plasminogen activator

increased levels of proNGF, neuroserpin,
unchanged tPA, MMP-9, and MMP-3 also corre-
spond to the status of the NGF metabolic pathway
in sporadic AD brains, though in one study tPA
was found to be downregulated (Bruno et al.
2009a). These findings demonstrate that NGF
dysmetabolism is highly similar in DS and spo-
radic AD brains and that biofluids—particularly
CSF—reflect this cerebral NGF dysmetabolism
well (Pentz et al. 2021a). Table 4.1 offers a com-
parative summary between biofluids and brains.
4.6 Diagnostic Value of NGF
Pathway Biomarkers
Why does the AD field continue to fight for better
or additional biomarkers? Although AD can now
be diagnosed by biomarker expression at preclin-
ical stages (Dubois et al. 2014; McDade and
Bateman 2017; Jack et al. 2018), these measures
are imperfect as they fail to acknowledge the
heterogeneity inherent in AD (Murray et al.
2011) and offer limited prognostic value at such
early stages. Indeed, one recent model suggested
that 30% of Americans over the age of 50 would
be diagnosed with preclinical AD under interna-
tional diagnostic criteria (IWG-2/NIA-AA) but
that each of those people would have a 75%
chance of never manifesting the condition
(Brookmeyer and Abdalla 2018). As such, there
would be three unnecessary AD treatments for
every successful one—an extreme cost when
considering that these potentially expen-
sive treatments might have to be applied for
decades in a large swath of the elderly population.
These models suggest that biomarkers of preclin-
ical AD need to be refined by inclusion of other
markers beyond amyloid and tau that are also
surrogates of key neurodegenerative processes
in this complex disease. Given the strong associ-
ation between NGF metabolism, cholinergic tone,
and cognition, we suggest that the NGF metabolic
pathway could be a source of such biomarkers.
Could NGF pathway proteins assist in the
identifications of individuals with incipient
dementia? As described above, the ROC curve
values determined for CSF proNGF and CSF
MMP-9 in the DS population were good (0.86
and 0.87), though they were outperformed by the
diagnostic accuracy of Aβ42 or the Aβ42/40 ratio
(Fortea et al. 2018a). As such, NGF pathway
markers alone will not likely serve as a primary
means of diagnosing AD. However, NGF meta-
bolic pathway proteins could add value and fur-
ther specificity to traditional biomarkers (i.e. as
40
part of a panel), particularly to identify subtypes
of AD with prominent cholinergic lesions
(Machado et al. 2020). Indeed, a recent study
has demonstrated that neuronal density was low-
est and tau pathology was highest in the nucleus
basalis of Meynert (NbM)—part of the BFC sys-
tem—in the hippocampal-sparing subtype of spo-
radic AD, that typical AD was intermediate with
regard to both tau pathology and NbM cell count,
and that limbic-predominant AD showed the low-
est NbM tau pathology and the highest NbM
neuronal density, as well as the lowest NbM
plaque counts (Al-Shaikh et al. 2020). A second
study identified limbic-sparing AD as being
associated with the greatest longitudinal loss of
BFCN volume over several visits, while a “mini-
mal atrophy subtype” predictably displayed the
least BFCN volume loss (Machado et al. 2020).
When these patients were treated with the
encapsulated cell devices overexpressing NGF
(discussed in greater detail below), reduced atro-
phy of the precuneus and hippocampus was
observed only in the hippocampal-sparing and
typical AD subtypes (Machado et al. 2020).
While further work will be required, these initial
studies provide evidence that the cholinergic def-
icit may differ across subtypes of AD and
strongly suggest that NGF pathway-derived
biomarkers could identify certain of these
subtypes with different degree of cholinergic
degeneration. Follow-up studies should incorpo-
rate other novel biomarkers of cholinergic tone in
AD, including the proposed “cholinergic index”,
a ratio of ChAT to AChE levels in CSF, which
demonstrated the ability to measure treatment
response to AChEIs in a recent study (Karami
et al. 2019). Similar measures of treatment
response could provide a second potential signifi-
cant role for NGF pathway biomarkers in AD,
and, as AChEIs depend on some degree of cho-
linergic tone to exert their effect (Richter et al.
2018), identifying patients likely to respond to
AChEIs or other cholinergic therapies could pro-
vide a third.
R. Pentz and M. F. Iulita
4.7 NGF as a Therapeutic Target
in AD
AChEIs, while valuable, only transiently boost
the output of BFCNs. Could NGF be an effective
intervention to sustain BFCN output and cholin-
ergic tone more substantially in AD? Initial
results from a single patient who received intra-
ventricular NGF showed increased nicotinic
receptor expression and cerebral perfusion, with
positive indications on some—though not all—
cognitive tests and particularly good results in a
test of verbal memory (Olson et al. 1992). Similar
positive results (verbal fluency excepted) had pre-
viously been obtained with the direct infusion of
NGF into the brains of crab-eating macaques
(Tuszynski et al. 1990; Tuszynski et al. 1991).
However, further studies demonstrated that this
approach was nonviable in humans, as it led to
chronic pain and weight loss, presumably by the
cross-sprouting of spinal nerve afferents and
hypothalamic plasticity (Jönhagen et al. 1998;
McKelvey et al. 2013). The authors of these stud-
ies concluded they provided substantial proof of
concept for NGF therapy in AD but that direct
infusion would not be viable, indicating that other
methods of altering NGF-mediated trophic sup-
port would be required (Jönhagen 2000).
Gene therapy approaches can drive the
overexpression of mNGF in target tissues without
nociceptive or constitutional side effects
(Tuszynski et al. 1990; Hao et al. 2000). In a
successful phase I trial, this approach trended
towards reducing decline on the Mini-Mental Sta-
tus Examination and Alzheimer’s Disease
Assessment Scale-Cognitive subcomponent and
increased cortical glucose metabolism in eight
individuals with AD (Tuszynski et al. 2005).
Follow-up trials attempted to use advances in
adeno-associated virus technology to deliver
human mNGF directly to the NbM. This con-
struct, entitled CERE-110, enhanced cholinergic
trophism in rodent models (Bishop et al. 2008)
and AD patients (Rafii et al. 2014; Tuszynski
et al. 2015), with no signs of deleterious side
effects. Unfortunately, a larger clinical trial in
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
49 AD patients showed no effects on its
endpoints, which included cognitive testing,
FDG-PET, and longitudinal hippocampal
volumetry (Rafii et al. 2018). However, recent
results have suggested that this may have been
mediated by technical rather than conceptual
limitations, as a post-mortem analysis of the
brains of 15 patients revealed that not one of
their CERE-110 injections was correctly
localized to the basal forebrain (Castle et al.
2020). This, in addition to the fact that the study
was underpowered to detect efficacy and applied
only to patients with advanced AD (Rafii et al.
2018), suggests that this approach, while inevita-
bly challenging and invasive, may still hold
promise.
While the virus-mediated modification of
neurons to overexpress NGF has been abandoned,
a similar approach in which fibroblasts are col-
lected from a patient, modified to overexpress
mNGF, and implanted into the basal forebrain
within an encapsulation device is being actively
pursued (Eriksdotter-Jönhagen et al. 2012;
Eyjolfsdottir et al. 2016). There are positive
indications for the utility of this approach, as
small studies have demonstrated positive effects
on cognition, brain atrophy, and some (but not
all) fluid AD biomarkers (Ferreira et al. 2015;
Karami et al. 2015). Intriguingly, this treatment
also rectified CSF ChAT and AChE levels, which
are proposed biomarkers of cholinergic integrity.
Despite these promising findings, there are
reasons to be cautious. Many of the positive
changes were only observed in patients that
received a post hoc classification as “responders”
based on 2 point reduction on the Mini-Mental
State Examination (MMSE) (Ferreira et al. 2015;
Karami et al. 2015), and a recent report suggests
that CSF factors may impair the long-term effi-
cacy of the implants (Eriksdotter et al. 2018).
A third approach takes advantage of the dis-
covery that a condition called “type IV hereditary
sensory and autonomic neuropathy”, in which
patients have a significant nociceptive deficit,
results from a R100W point mutation in the
NGF gene (Capsoni et al. 2011). This mutant
NGF could permit the resurrection of direct
infusion approaches, as it retains full trophic
41
competence but does not induce a pain response
(Capsoni et al. 2011). This mutant NGF has been
primarily delivered intranasally, a protocol that
has been shown to alleviate plaque load and cog-
nitive decline in APP/PSEN1 mutant mice
(Capsoni et al. 2012). However, these effects do
not appear to be mediated by cholinergic
trophism, and the associated researchers now pro-
pose “painless” intranasal NGF as modulator of
inflammation (Capsoni et al. 2017; Cattaneo and
Capsoni 2019). Although more remains to be
learned about this protein, mutant NGF is an
exciting possibility in the future of pro-trophic
therapeutics.
The above approaches, though varied in many
ways, share not only their target but the fact that
the increased NGF they elicit is exogenously
derived and localized widely over a given area,
most of which lies outside cholinergic synapses.
This entails several disadvantages. Firstly, NGF
can have effects on off-target neurons and even
glia; in fact, many cells that are not usually
NGF-competent express the p75NTR in AD
(Mufson and Kordower 1992; McKelvey et al.
2013; Cattaneo and Capsoni 2019), so it is not
clear what the sum effect of exogenous NGF
would be on the AD pathological process. Sec-
ondly, these treatments would be applied in the
context of significantly elevated NGF-degrading
metalloproteinases (Bruno and Cuello 2006;
Iulita et al. 2014; Pentz et al. 2020)—therefore it
is not clear that the amount reaching cholinergic
synapses (if any) would suffice to overcome this
excessive degradation. Thirdly, many of these
approaches are highly invasive and technically
challenging, requiring injections or insertions
into the brain parenchyma or ventricles.
Targeting other proteins that participate in the
metabolism of NGF has the potential to overcome
all three of these following concerns. In the first,
NGF would still be solely produced at cholinergic
synapses; in the second, the excessive degrada-
tion of mNGF could itself be a target for therapy;
and in the third, if a target specific to cholinergic
synapses can be identified—as discussed
below—a drug could be applied systemically.
This type of therapy could have the potential to
produce a long-lasting rescue of BFCNs and
42
cortical cholinergic tone. Such a drug could also
be used adjunctly to enhance the effect of
AChEIs.
4.8 Targeting NGF Metabolism
A therapeutic that intervenes to restore trophic
support to BFCNs by normalizing NGF metabo-
lism could do so by two mechanisms: by restor-
ing/enhancing the normal conversion of proNGF
to mNGF and by diminishing the degradation of
mNGF. Both are valid approaches, as both
alterations occur in preclinical stages of AD and
correlate to AD neuropathology and cognitive
decline at these stages (Pentz et al. 2020), though
correlations to markers of cholinergic degenera-
tion have only been observed for the maturation
system. Below we will discuss various
approaches that could be used to target NGF
metabolism in the context of prior preclinical
studies.
4.8.1 Restoring Normal Conversion
of proNGF to mNGF
How could proNGF maturation be promoted?
tPA, which mediates the formation of plasmin,
is an already FDA-approved therapeutic for the
treatment of stroke (Group 1995) and has the
potential to enhance proNGF maturation.
Transgenesis experiments have demonstrated
that tg2576 mice deficient for the tPA gene
(PLAT /+) have increased cerebral amyloidosis
and mortality compared to PLAT +/+ littermates
(Oh et al. 2014). In addition, preclinical studies
showed that chronic systemic administration of
tPA to APPswe/PS1 transgenic mice reduced
amyloidosis and enhanced cognition (ElAli et al.
2016), which is also in line with its role in
facilitating neurovascular coupling (Park et al.
2008). Furthermore, studies in which central
tPA inhibitors such as neuroserpin have been
knocked down or inhibited with small molecules
also demonstrated positive effects on amyloidosis
and cognition (Fabbro et al. 2011; Liu et al. 2011;
Akhter et al. 2018). To further explore the
R. Pentz and M. F. Iulita
potential of tPA therapy in correcting NGF
dysmetabolism, several considerations should be
weighed. Some studies in cellular cultures have
suggested that tPA might induce or mediate AD
neuropathology, particularly amyloidosis and the
inflammatory activation of microglia (Siao and
Tsirka 2002; Medina et al. 2005), suggesting
that tPA is a complex target. It must further be
recalled that tPA therapy in cases of acute ische-
mic stroke is limited to a small window of 4.5 h
after the stroke occurs, as tPA applied later or for
longer increases the risk of intracerebral
haemorrhage and subsequent mortality. Therapies
that increase tPA activity in AD, while a
promising means of elevating proNGF matura-
tion, will therefore require careful dosing and
thorough analyses of potentially multifaceted
effects on wider AD pathology.
Neuroserpin, in contrast, is a tPA inhibitor that
has been demonstrated to be co-released with
proNGF in response to cholinergic stimulation
and which has been repeatedly linked to NGF
metabolism through correlational and pharmaco-
logical studies (Bruno and Cuello 2006; Pentz et
al. 2020). Neuroserpin has a restricted expression
pattern compared to other NGF pathway proteins,
with particularly high expression in the CNS,
particularly the isocortex and hippocampal for-
mation (Osterwalder et al. 1996; Krueger et al.
1997). As neuroserpin levels are elevated in
MCI/AD brains, their normalization at these
stages would presumably have even more limited
side effects as compared to a reduction in activity
from normal levels.
Preclinical evidence suggests that neuroserpin
inhibition could be efficacious in AD. The partial
knockdown of neuroserpin reduced amyloidosis
and enhanced cognition in the J20 mouse model
of AD (Fabbro et al. 2011), and indeed,
neuroserpin is known to have significant roles in
normal hippocampal plasticity (Hermann et al.
2020). However, in vitro aggregation studies
and in vivo studies in transgenic drosophila flies
have shown that neuroserpin has the capacity to
bind Aβ42, thereby reducing its toxicity to
cultured embryonic neurons (Kinghorn et al.
2006). Moreover, neuroserpin has unique phar-
macological properties that make it difficult to
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
target (Miranda and Lomas 2006), and there are
consequently no known inhibitors. Therefore,
novel drugs and careful analyses of side effects
will be required to effectively engage this target.
4.8.2 Restoring Normal mNGF
Degradation
While less mNGF is produced than usual in AD,
that which is produced is exposed to excessive
levels of the metalloproteinases that degrade it
(Bruno et al. 2009a, 2009b; Iulita et al. 2014;
Pentz et al. 2020), and inhibiting these
metalloproteinases is an equally viable approach
from pro-trophic, pro-cholinergic interventions in
AD. Additionally, it has recently been discovered
that MMP-3, in addition to MMP-9, is competent
to degrade mNGF both in vitro and in vivo and is
elevated at preclinical disease stages and that
these elevated MMP-3 levels correlate to early
decline on the MMSE in the AD continuum
(Pentz et al. 2021b). Furthermore, both MMP-9
and MMP-3 correlate to levels of mNGF in pre-
clinical stages of AD, suggesting that they may be
relevant for adjusting the steady state of NGF
metabolism.
Preclinical work has previously demonstrated
some efficacy for MMP-9 inhibition in AD. The
application of 100μg/kg of the MMP-9 inhibitor
GM6001, delivered intraparenchymally into the
cerebral cortices of young wild-type rats, caused
NGF levels to increase—in some cases, mature
NGF could only be detected in the brain when
MMP-9 was inhibited (Bruno and Cuello 2006).
Recently, a study showed that injections of the
MMP-3-specific inhibitor UK356618 into the
hippocampi of young wild-type rats provoked a
marked increase in mNGF levels. Since this drug
is highly selective (I.C. ¼ 5.9 nM), it could there-
fore be applied at very low doses (Pentz et al.
2021b). Additionally, minocycline, a broad-
spectrum MMP inhibitor and anti-inflammatory,
has previously been used to normalize the NGF
dysmetabolism induced by the intrahippocampal
injection of amyloid oligomers to young naïve
rats (Bruno et al. 2009a). Although the previously
discussed studies did not assess the effect of
43
MMP-3/MMP-9 inhibition on cholinergic integ-
rity, administration of an MMP-2/MMP-9 inhibi-
tor (CAS 193807-58-8) has been shown to
increase mNGF levels and cortical cholinergic
tone after 2 weeks of continuous infusion (Allard
et al. 2012a). However, it should also be consid-
ered that MMP-9 has a wide variety of targets
beyond NGF, including amyloid peptides
(Backstrom et al. 1996; Yan et al. 2006).
MMP-3, likewise, has a wide range of physiolog-
ical roles (Van Hove et al. 2012), and its levels
differ between men and women (Iulita et al. 2016;
Iulita et al. 2019; Pentz et al. 2020). Therefore,
while the preclinical evidence for MMP inhibi-
tion in AD is promising, its inhibition could have
a wide range of potential side and sex-specific
effects that should be carefully considered.
New interest in potential disease-modifying
roles for cholinergic therapeutics has
reinvigorated this field (Hampel et al. 2018;
Cuello et al. 2019). Of all the therapeutic avenues
under development, targeting NGF metabolism
appears to have the greatest potential to elevate
NGF levels at physiological sites with minimally
invasive procedures while addressing what is
likely to be the root cause of cholinergic degener-
ation in AD. However, identifying potential drug
candidates is challenging, based on the highly
varied localizations and activities that NGF path-
way proteins display, as well as the lack of spe-
cific inhibitors for certain proteins, thus
underscoring an opportunity to develop novel
drugs with the capability of restoring normal
NGF metabolism.
4.9 Conclusion
The discovery of the NGF metabolic pathway and
its essential role in the pathogenesis of choliner-
gic dysfunction in AD has opened new scientific
and therapeutic possibilities. ProNGF and NGF
pathway markers have demonstrated efficacy as
indicators of the AD pathological process and
interventions targeting NGF pathway proteins
have shown promise in preclinical studies.
While specific compounds and assays remain to
be developed, this new framework should offer
44
novel avenues for the clinical management
of AD.
Acknowledgements The authors wish to thank and dedi-
cate this chapter to Professor A. Claudio Cuello, for his
continued mentorship and support and for sharing with us
Rita Levi Montalcini’s inspirational legacy. Rowan Pentz
is the recipient of a Returning Student Fellowship from the
McGill Integrated Program in Neuroscience and CIHR
Doctoral Award no. 201610GSD-385496-2466860.
M. Florencia Iulita would like to acknowledge financial
support from a Postdoctoral Research Fellowship from the
Jérôme Lejeune and Sisley D’Ornano Foundations and
from a pilot exploratory grant (no. 1941) from the Jérôme
Lejeune Foundation.
References
Adair JC et al (2004) Measurement of gelatinase B
(MMP-9) in the cerebrospinal fluid of patients with
vascular dementia and Alzheimer disease. Stroke 35:
e159–e162
Akhter H et al (2018) A small molecule inhibitor of plas-
minogen activator inhibitor-1 reduces brain amyloid-β
load and improves memory in an animal model of
alzheimer’s disease. J Alzheimers Dis 64:447–457
Allard S et al (2012a) Impact of the NGF maturation and
degradation pathway on the cortical cholinergic system
phenotype. J Neurosci 32:2002–2012
Allard S et al (2012b) Impact of the NGF maturation and
degradation pathway on the cortical cholinergic system
phenotype. J Neurosci 32:2002–2012
Allard S et al (2018a) Compromise of cortical proNGF
maturation causes selective retrograde atrophy in cho-
linergic nucleus basalis neurons. Neurobiol Aging
67:10–20
Allard S et al (2018b) Compromise of cortical proNGF
maturation causes selective retrograde atrophy in cho-
linergic nucleus basalis neurons. Neurobiol Aging
67:10–20
Al-Shaikh FSH et al (2020) Selective vulnerability of the
nucleus basalis of Meynert among neuropathologic
subtypes of Alzheimer disease. JAMA Neurol
77:225–233
Backstrom JR et al (1996) Matrix metalloproteinase-9
(MMP-9) is synthesized in neurons of the human hip-
pocampus and is capable of degrading the amyloid-β
peptide (1–40). J Neurosci 16:7910–7919
Bartus RT et al (1982) The cholinergic hypothesis of
geriatric memory dysfunction. Science 217:408–414
Birks JS, Harvey RJ (2018) Donepezil for dementia due to
Alzheimer’s disease. Cochrane Database Syst Rev 6:
CD001190
Bishop KM et al (2008) Therapeutic potential of CERE-
110 (AAV2-NGF): targeted, stable, and sustained NGF
delivery and trophic activity on rodent basal forebrain
cholinergic neurons. Exp Neurol 211:574–584
R. Pentz and M. F. Iulita
Blennow K, Zetterberg H (2018) Biomarkers for
Alzheimer’s disease: current status and prospects for
the future. J Intern Med 284:643–663
Brookmeyer R, Abdalla N (2018) Estimation of lifetime
risks of Alzheimer’s disease dementia using
biomarkers for preclinical disease. Alzheimers Dement
14:981–988
Bruno MA, Cuello AC (2006) Activity-dependent release
of precursor nerve growth factor, conversion to mature
nerve growth factor, and its degradation by a protease
cascade. Proc Natl Acad Sci 103:6735–6740
Bruno MA et al (2009a) Amyloid β-induced nerve growth
factor dysmetabolism in Alzheimer disease. J
Neuropathol Exp Neurol 68:857–869
Bruno MA et al (2009b) Increased matrix
Metalloproteinase-9 activity in mild cognitive
impairment. J Neuropathol Exp Neurol 68:1309
Caccamo A et al (2006) M1 receptors play a central role in
modulating AD-like pathology in transgenic mice.
Neuron 49:671–682
Cai Z-Y et al (2007) Serum level of MMP-2, MMP-9 and
ox-LDL in Alzheimer’s disease with hyperlipoidemia.
J Med Coll PLA 22:352–356
Capsoni S et al (2011) Taking pain out of NGF: a “pain-
less” NGF mutant, linked to hereditary sensory auto-
nomic neuropathy type V, with full neurotrophic
activity. PloS One 6:e17321
Capsoni S et al (2012) Intranasal “painless” human nerve
growth factors slows amyloid neurodegeneration and
prevents memory deficits in app X PS1 mice. PLoS
One 7:e37555
Capsoni S et al (2017) The chemokine CXCL12 mediates
the anti-amyloidogenic action of painless human nerve
growth factor. Brain 140:201–217
Castle MJ et al (2020) Postmortem analysis in a clinical
trial of AAV2-NGF gene therapy for Alzheimer’s dis-
ease identifies a need for improved vector delivery.
Hum Gene Ther 31:415–422
Cattaneo A, Capsoni S (2019) Painless nerve growth fac-
tor: a TrkA biased agonist mediating a broad
neuroprotection via its actions on microglia cells.
Pharmacol Res 139:17–25
Cavedo E et al (2016) Reduced regional cortical thickness
rate of change in donepezil-treated subjects with
suspected prodromal Alzheimer’s disease. J Clin Psy-
chiatry 77(12):e1631–e1638
Cavedo E et al (2017) Reduced basal forebrain atrophy
progression in a randomized donepezil trial in prodro-
mal Alzheimer’s disease. Sci Rep 7:11706
Claassen JA, Jansen RW (2006) Cholinergically mediated
augmentation of cerebral perfusion in alzheimer’s dis-
ease and related cognitive disorders: the cholinergic–
vascular hypothesis. J Gerontol Ser A Biol Med Sci
61:267–271
Counts S et al (2016) Cerebrospinal fluid proNGF: a
putative biomarker for early Alzheimer’s disease.
Curr Alzheimer Res 13:800–808
Cuello AC (1996) Effects of trophic factors on the CNS
cholinergic phenotype. Prog Brain Res 109:347–358
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
Cuello A et al (2007) NGF-cholinergic dependency in
brain aging, MCI and Alzheimer’s disease. Curr
Alzheimer Res 4:351–358
Cuello AC et al (2019) The brain NGF metabolic pathway
in health and in Alzheimer’s pathology. Front Neurosci
13:62
Debeir T et al (1998) TrkA antagonists decrease
NGF-induced ChAT activity in vitro and modulate
cholinergic synaptic number in vivo. J Physiol Paris
92:205–208
Debeir T et al (1999a) A nerve growth factor mimetic
TrkA antagonist causes withdrawal of cortical cholin-
ergic boutons in the adult rat. Proc Natl Acad Sci
96:4067–4072
Debeir T et al (1999b) A nerve growth factor mimetic
TrkA antagonist causes withdrawal of cortical cholin-
ergic boutons in the adult rat. Proc Natl Acad Sci U S A
96:4067–4072
Drachman DA, Leavitt J (1974) Human memory and the
cholinergic system. A relationship to aging? Arch
Neurol 30:113–121
Dubois B et al (2014) Advancing research diagnostic
criteria for Alzheimer’s disease: the IWG-2 criteria.
Lancet Neurol 13:614–629
Dubois B et al (2015) Donepezil decreases annual rate of
hippocampal atrophy in suspected prodromal
Alzheimer’s disease. Alzheimers Dement
11:1041–1049
ElAli A et al (2016) Tissue-plasminogen activator
attenuates Alzheimer’s disease-related pathology
development in APPswe/PS1 mice. Neuropsychophar-
macology 41:1297
El-Hayek YH et al (2019) Tip of the iceberg: assessing the
global socioeconomic costs of Alzheimer’s disease and
related dementias and strategic implications for
stakeholders. J Alzheimers Dis 70:323–341
Eriksdotter M et al (2018) Cerebrospinal fluid from
Alzheimer patients affects cell-mediated nerve growth
factor production and cell survival in vitro. Exp Cell
Res 371:175–184
Eriksdotter-Jönhagen M et al (2012) Encapsulated cell
biodelivery of nerve growth factor to the basal fore-
brain in patients with Alzheimer’s disease. Dement
Geriatr Cogn Disord 33:18–28
Eyjolfsdottir H et al (2016) Targeted delivery of nerve
growth factor to the cholinergic basal forebrain of
Alzheimer’s disease patients: application of a second-
generation encapsulated cell biodelivery device.
Alzheimers Res Ther 8:30
Fabbro S et al (2011) Amyloid-beta levels are significantly
reduced and spatial memory defects are rescued in a
novel neuroserpin-deficient Alzheimer’s disease trans-
genic mouse model. J Neurochem 118:928–938
Fahnestock M et al (2001) The precursor pro-nerve growth
factor is the predominant form of nerve growth factor
in brain and is increased in Alzheimer’s disease. Mol
Cell Neurosci 18:210–220
Ferreira D et al (2015) Brain changes in Alzheimer’s
disease patients with implanted encapsulated cells
45
releasing nerve growth factor. J Alzheimers Dis
43:1059–1072
Figueiredo B et al (1995) Differential expression of
p140trk, p75NGFR and growth-associated phospho-
protein-43 genes in nucleus basalis magnocellularis,
thalamus and adjacent cortex following neocortical
infarction and nerve growth factor treatment. Neuro-
science 68:29–45
Fisher A (2008) Cholinergic treatments with emphasis on
m1 muscarinic agonists as potential disease-modifying
agents for Alzheimer’s disease. Neurotherapeutics
5:433–442
Fortea J et al (2018a) Plasma and CSF biomarkers for the
diagnosis of Alzheimer’s disease in adults with down
syndrome: a cross-sectional study. Lancet Neurol
17:860–869
Fortea J et al (2018b) Plasma and CSF biomarkers for the
diagnosis of Alzheimer’s disease in adults with down
syndrome: a cross-sectional study. Lancet Neurol
17:860–869
Fortea J et al (2020) Clinical and biomarker changes of
Alzheimer’s disease in adults with down syndrome: a
cross-sectional study. Lancet 395:1988–1997
Giacobini E, Becker RE (2007) One hundred years after
the discovery of Alzheimer’s disease. A turning point
for therapy? J Alzheimers Dis 12:37–52
Giacobini E et al (2002) Inhibition of acetyl- and butyryl-
cholinesterase in the cerebrospinal fluid of patients
with Alzheimer’s disease by rivastigmine: correlation
with cognitive benefit. J Neural Transm
109:1053–1065
Gibbs RB, Pfaff D (1994) In situ hybridization detection of
trkA mRNA in brain: distribution, colocalization with
p75NGFR and up-regulation by nerve growth factor. J
Comp Neurol 341:324–339
Grothe MJ et al (2014) Basal forebrain atrophy and corti-
cal amyloid deposition in nondemented elderly
subjects. Alzheimers Dement 10:S344–S353
Group, N. I. o. N. D. S. r.-P. S. S (1995) Tissue plasmino-
gen activator for acute ischemic stroke. N Engl J Med
333:1581–1588
Hall H et al (2018) AF710B, an M1/sigma-1 receptor
agonist with long-lasting disease-modifying properties
in a transgenic rat model of Alzheimer’s disease.
Alzheimers Dement 14:811–823
Hampel H et al (2018) The cholinergic system in the
pathophysiology and treatment of Alzheimer’s disease.
Brain 141:1917–1933
Handen BL et al (2020) The Alzheimer’s biomarker
consortium-down syndrome: rationale and methodol-
ogy. Alzheimers Dement (Amst) 12:e12065
Hanzel CE et al (2014a) Analysis of matrix metallo-
proteases and the plasminogen system in mild cogni-
tive impairment and Alzheimer’s disease cerebrospinal
fluid. J Alzheimers Dis 40:667–678
Hanzel CE et al (2014b) Neuronal driven pre-plaque
inflammation in a transgenic rat model of Alzheimer’s
disease. Neurobiol Aging 35:2249–2262
46
Hao J-X et al (2000) Intracerebroventricular infusion of
nerve growth factor induces pain-like response in rats.
Neurosci Lett 286:208–212
Hasselmo ME, Sarter M (2011) Modes and models of
forebrain cholinergic neuromodulation of cognition.
Neuropsychopharmacology 36:52–73
Head E, Lott IT (2004) Down syndrome and beta-amyloid
deposition. Curr Opin Neurol 17:95–100
Head E et al (2012) Aging and down syndrome. Curr
Gerontol Geriatr Res 2012:412536
Hefti F (1986) Nerve growth factor promotes survival of
septal cholinergic neurons after fimbrial transections. J
Neurosci 6:2155–2162
Hefti F et al (1986) Localization of nerve growth factor
receptors in cholinergic neurons of the human basal
forebrain. Neurosci Lett 69:37–41
Hermann M et al (2020) Deficits in developmental
neurogenesis and dendritic spine maturation in mice
lacking the serine protease inhibitor neuroserpin. Mol
Cell Neurosci 102:103420
Hock C et al (2000) Increased CSF levels of nerve growth
factor in patients with Alzheimer’s disease. Neurology
54:2009–2011
Holtzman DM et al (1992) p140trk mRNA marks
NGF-responsive forebrain neurons: evidence that trk
gene expression is induced by NGF. Neuron
9:465–478
Horstmann S et al (2010) Matrix metalloproteinases in
peripheral blood and cerebrospinal fluid in patients
with Alzheimer’s disease. Int Psychogeriatr
22:966–972
Iulita MF, Cuello AC (2014) Nerve growth factor meta-
bolic dysfunction in Alzheimer’s disease and down
syndrome. Trends Pharmacol Sci 35:338–348
Iulita MF, Cuello AC (2016) The NGF metabolic pathway
in the CNS and its Dysregulation in down syndrome
and Alzheimer’s disease. Curr Alzheimer Res
13:53–67
Iulita MF et al (2014) Nerve growth factor metabolic
dysfunction in Down’s syndrome brains. Brain 137
(Pt 3):860–872. https://doi.org/10.1093/brain/awt372
Iulita MF et al (2016) An inflammatory and trophic dis-
connect biomarker profile revealed in down syndrome
plasma: relation to cognitive decline and longitudinal
evaluation. Alzheimers Dement 12(11):1132–1148
Iulita MF et al (2017) Differential deregulation of NGF
and BDNF neurotrophins in a transgenic rat model of
Alzheimer’s disease. Neurobiol Dis 108:307–323
Iulita MF et al (2019) Identification and preliminary vali-
dation of a plasma profile associated with cognitive
decline in dementia and at-risk individuals: a retrospec-
tive cohort analysis. J Alzheimers Dis 67:327–341
Jack CR Jr et al (2018) NIA-AA research framework:
toward a biological definition of Alzheimer’s disease.
Alzheimers Dement 14:535–562
Jack CR Jr (2020) The transformative potential of plasma
phosphorylated tau. Lancet Neurol 19:373–374
Janelidze S et al (2020) Plasma P-tau181 in Alzheimer’s
disease: relationship to other biomarkers, differential
R. Pentz and M. F. Iulita
diagnosis, neuropathology and longitudinal progres-
sion to Alzheimer’s dementia. Nat Med 26:379–386
Jönhagen ME (2000) Nerve growth factor treatment in
dementia. Alzheimer Dis Assoc Disord 14:S31–S38
Jönhagen ME et al (1998) Intracerebroventricular infusion
of nerve growth factor in three patients with
Alzheimer’s disease. Dement Geriatr Cogn Disord
9:246–257
Karami A et al (2015) Changes in CSF cholinergic
biomarkers in response to cell therapy with NGF in
patients with Alzheimer’s disease. Alzheimers Dement
11:1316–1328
Karami A et al (2019) CSF cholinergic index, a new
biomeasure of treatment effect in patients with
Alzheimer’s disease. Frontiers in molecular. Neurosci-
ence 12:239
Karikari TK et al (2020) Blood phosphorylated tau 181 as
a biomarker for Alzheimer’s disease: a diagnostic per-
formance and prediction modelling study using data
from four prospective cohorts. Lancet Neurol
19:422–433
Kinghorn KJ et al (2006) Neuroserpin binds Aβ and is a
neuroprotective component of amyloid plaques in
Alzheimer disease. J Biol Chem 281:29268–29277
Krueger SR et al (1997) Expression of neuroserpin, an
inhibitor of tissue plasminogen activator, in the devel-
oping and adult nervous system of the mouse. J
Neurosci 17:8984–8996
Le AP (2011) Regulation of proNGF processing and its
effects on p75NTR-mediated cell death following sei-
zure. Rutgers University-Graduate School-Newark
Lim NK-H et al (2011) Investigation of matrix metallopro-
teinases, MMP-2 and MMP-9, in plasma reveals a
decrease of MMP-2 in Alzheimer’s disease. J
Alzheimers Dis 26:779–786
Lin Z et al (2006) The plasma matrix metalloproteinase-3
increased in patients with Alzheimer’s disease. Chin J
Psychiatry 39:209
Liu R-M et al (2011) Knockout of plasminogen activator
inhibitor 1 gene reduces amyloid beta peptide burden
in a mouse model of Alzheimer’s disease. Neurobiol
Aging 32:1079–1089
Lorenzl S et al (2003) Increased plasma levels of matrix
metalloproteinase-9 in patients with Alzheimer’s dis-
ease. Neurochem Int 43:191–196
Lorenzl S et al (2008) Profiles of matrix metallopro-
teinases and their inhibitors in plasma of patients with
dementia. Int Psychogeriatr 20:67–76
Lott IT, Head E (2019) Dementia in down syndrome:
unique insights for Alzheimer disease research. Nat
Rev Neurol 15:135–147
Machado A et al (2020) The cholinergic system in
subtypes of Alzheimer’s disease: an in vivo longitudi-
nal MRI study. Alzheimers Res Ther 12:1–11
Malerba F et al (2016) NGF and proNGF reciprocal inter-
ference in immunoassays: open questions, criticalities,
and ways forward. Front Mol Neurosci 9:63
Manuel DG et al (2016) Alzheimer’s and other dementias
in Canada, 2011 to 2031: a microsimulation population
4 The NGF Metabolic Pathway: New Opportunities for Biomarker Research and. . .
health modeling (POHEM) study of projected preva-
lence, health burden, health services, and caregiving
use. Popul Health Metr 14:37
Marksteiner J et al (2014) Analysis of 27 vascular-related
proteins reveals that NT-proBNP is a potential bio-
marker for Alzheimer’s disease and mild cognitive
impairment: a pilot-study. Exp Gerontol 50:114–121
Martín-Aragón S et al (2009) Metalloproteinase’s activity
and oxidative stress in mild cognitive impairment and
Alzheimer’s disease. Neurochem Res 34:373
Martorana A et al (2012) Plasmin system of Alzheimer’s
disease patients: CSF analysis. J Neural Transm
119:763–769
Massaro A et al (1994) Nerve growth factor (NGF) in
cerebrospinal fluid (CSF) from patients with various
neurological disorders. Ital J Neurol Sci 15:105–108
McDade E, Bateman RJ (2017) Stop Alzheimer’s before it
starts. Nature 547:153
McKelvey L et al (2013) Nerve growth factor-mediated
regulation of pain signalling and proposed new inter-
vention strategies in clinical pain management. J
Neurochem 124:276–289
Medina MG et al (2005) Tissue plasminogen activator
mediates amyloid-induced neurotoxicity via Erk1/
2 activation. EMBO J 24:1706–1716
Mehra A et al (2016) The plasminogen activation system
in neuroinflammation. Biochim Biophys Acta (BBA)-
molecular basis of disease 1862:395–402
Miranda E, Lomas D (2006) Neuroserpin: a serpin to think
about. Cell Mol Life Sci CMLS 63:709–722
Mlekusch R, Humpel C (2009) Matrix metalloproteinases-
2 and-3 are reduced in cerebrospinal fluid with low
beta-amyloid1–42 levels. Neurosci Lett 466:135–138
Mroczko B et al (2014) Concentrations of matrix
metalloproteinases and their tissue inhibitors in the
cerebrospinal fluid of patients with Alzheimer’s dis-
ease. J Alzheimers Dis 40:351–357
Mufson EJ, Kordower JH (1992) Cortical neurons express
nerve growth factor receptors in advanced age and
Alzheimer disease. Proc Natl Acad Sci 89:569–573
Mufson EJ et al (2019) Nerve growth factor pathobiology
during the progression of Alzheimer’s disease. Front
Neurosci 13:533
Murray ME et al (2011) Neuropathologically defined
subtypes of Alzheimer’s disease with distinct clinical
characteristics: a retrospective study. Lancet Neurol
10:785–796
Nielsen HM et al (2007) Plasma and CSF serpins in
Alzheimer disease and dementia with Lewy bodies.
Neurology 69:1569–1579
Nitsch RM et al (1992) Release of Alzheimer amyloid
precursor derivatives stimulated by activation of mus-
carinic acetylcholine receptors. Science 258:304–307
Nizri E et al (2006) Anti-inflammatory properties of cho-
linergic up-regulation: a new role for acetylcholinester-
ase inhibitors. Neuropharmacology 50:540–547
Oh SB et al (2014) Tissue plasminogen activator arrests
Alzheimer’s disease pathogenesis. Neurobiol Aging
35:511–519
47
Olson L et al (1992) Nerve growth factor affects
11 C-nicotine binding, blood flow, EEG, and verbal
episodic memory in an Alzheimer patient (case report).
J Neural Transm–Parkinson’s disease and dementia
section 4:79–95
Osterwalder T et al (1996) Neuroserpin, an axonally
secreted serine protease inhibitor. EMBO J
15:2944–2953
Overk CR et al (2010) Cortical M1 receptor concentration
increases without a concomitant change in function in
Alzheimer’s disease. J Chem Neuroanat 40:63–70
Palmqvist S et al (2020) Discriminative accuracy of
plasma Phospho-tau217 for Alzheimer disease vs
other neurodegenerative disorders. JAMA
324:772–781
Park L et al (2008) Key role of tissue plasminogen activa-
tor in neurovascular coupling. Proc Natl Acad Sci
105:1073–1078
Parks WC et al (2004) Matrix metalloproteinases as
modulators of inflammation and innate immunity. Nat
Rev Immunol 4:617
Peng M et al (2015) Plasma gelsolin and matrix
metalloproteinase 3 as potential biomarkers for
Alzheimer disease. Neurosci Lett 595:116–121
Pentz R et al (2020) The human brain NGF metabolic
pathway is impaired in the pre-clinical and clinical
continuum of Alzheimers disease. Mol Psych 1–15
Pentz R et al (2021a) Nerve growth factor (NGF) pathway
biomarkers in Down syndrome prior to and after the
onset of clinical Alzheimer’s disease: a paired CSF and
plasma study. Alzheimers Dement 17(4):605–617
Pentz R et al (2021b) A new role for matrix
metalloproteinase-3 in the NGF metabolic pathway:
proteolysis of mature NGF and sex-specific differences
in the continuum of Alzheimer’s pathology. Neurobiol
Dis 148:105150
Pepeu G, Giovannini MG (2010) Cholinesterase inhibitors
and memory. Chem Biol Interact 187:403–408
Rafii MS et al (2014) A phase 1 study of stereotactic gene
delivery of AAV2-NGF for Alzheimer’s disease.
Alzheimers Dement 10:571–581
Rafii MS et al (2018) Adeno-associated viral vector (sero-
type 2)–nerve growth factor for patients with alzheimer
disease: a randomized clinical trial. JAMA Neurol
75:834–841
Richter N et al (2018) Effect of cholinergic treatment
depends on cholinergic integrity in early Alzheimer’s
disease. Brain 141:903–915
Risacher SL et al (2016) Association between anticholin-
ergic medication use and cognition, brain metabolism,
and brain atrophy in cognitively normal older adults.
JAMA Neurol 73:721–732
Rouy D et al (2005) Plasma storage at 80 C does not
protect matrix metalloproteinase-9 from degradation.
Anal Biochem 338:294–298
Schmitz TW et al (2016) Basal forebrain degeneration
precedes and predicts the cortical spread of
Alzheimer’s pathology. Nat Commun 7:13249
48
Schmitz TW et al (2018) Longitudinal Alzheimer’s degen-
eration reflects the spatial topography of cholinergic
basal forebrain projections. Cell Rep 24:38–46
Shen H et al (2010) Neuroprotection by donepezil against
glutamate excitotoxicity involves stimulation of α7
nicotinic receptors and internalization of NMDA
receptors. Br J Pharmacol 161:127–139
Shi Y et al (2010) Plasma BDNF and tPA are associated
with late-onset geriatric depression. Psychiatry Clin
Neurosci 64:249–254
Siao C-J, Tsirka SE (2002) Tissue plasminogen activator
mediates microglial activation via its finger domain
through annexin II. J Neurosci 22:3352–3358
Stomrud E et al (2010) Alterations of matrix metallopro-
teinases in the healthy elderly with increased risk of
prodromal Alzheimer’s disease. Alzheimer’s Res Ther
2:20
Svensson AL et al (1992) Characterization of muscarinic
receptor subtypes in Alzheimer and control brain corti-
ces by selective muscarinic antagonists. Brain Res
596:142–148
Thijssen EH et al (2020) Diagnostic value of plasma
phosphorylated tau181 in Alzheimer’s disease and
frontotemporal lobar degeneration. Nat Med
26:387–397
Tuna G et al (2018) Evaluation of matrix
Metalloproteinase-2 (MMP-2) and-9 (MMP-9) and
their tissue inhibitors (TIMP-1 and TIMP-2) in plasma
from patients with neurodegenerative dementia. J
Alzheimers Dis 66:1265–1273
Tuszynski MH et al (1990) Nerve growth factor infusion in
the primate brain reduces lesion-induced cholinergic
neuronal degeneration. J Neurosci 10:3604–3614
R. Pentz and M. F. Iulita
Tuszynski MH et al (1991) Recombinant human nerve
growth factor infusions prevent cholinergic neuronal
degeneration in the adult primate brain. Ann Neurol:
Official Journal of the American Neurological Associ-
ation and the Child Neurology Society 30:625–636
Tuszynski MH et al (2005) A phase 1 clinical trial of nerve
growth factor gene therapy for Alzheimer disease. Nat
Med 11:551–555
Tuszynski MH et al (2015) Nerve growth factor gene
therapy: activation of neuronal responses in Alzheimer
disease. JAMA Neurol 72:1139–1147
Van Hove I et al (2012) Matrix metalloproteinase-3 in the
central nervous system: a look on the bright side. J
Neurochem 123:203–216
Venero J et al (1994) Expression of neurotrophin and trk
receptor genes in adult rats with fimbria transections:
effect of intraventricular nerve growth factor and brain-
derived neurotrophic factor administration. Neurosci-
ence 59:797–815
Welikovitch LA et al (2020) Early intraneuronal amyloid
triggers neuron-derived inflammatory signaling in APP
transgenic rats and human brain. Proc Natl Acad Sci
117:6844–6854
Whelan CD et al (2019) Multiplex proteomics identifies
novel CSF and plasma biomarkers of early
Alzheimer’s disease. Acta Neuropathol Commun
7:1–14
Yan P et al (2006) Matrix metalloproteinase-9 degrades
amyloid-β fibrils in vitro and compact plaques in situ. J
Biol Chem 281:24566–24574
Zissimopoulos J et al (2015) The value of delaying
Alzheimer’s disease onset. Forum Health Econ Policy
18:25–39. De Gruyter
 Part II
Development and Regeneration
 NGF and Endogenous Regeneration:
From Embryology Toward Therapies 5
Vito Antonio Baldassarro, Luca Lorenzini, Andrea Bighinati,
Alessandro Giuliani, Giuseppe Alastra, Micaela Pannella,
Mercedes Fernandez, Luciana Giardino, and Laura Calzà

Abstract through the regulation of the stem cell niches.

The self-repair ability of tissues and organs in
case of injury and disease is a fundamental
biological mechanism and an important thera-
peutic target. The tissue plasticity and the pres-
ence of adult stem cell niches open a new path
in the development of pharmacological and
non-pharmacological treatments finalized to
improve the intrinsic regeneration.
In this context, nerve growth factor (NGF)
is widely studied for its capability of driving
endogenous regeneration of ectoderm-derived
tissues, directly acting on the cell targets and
In fact, this growth factor is very promising for
its key role in the development and multiplic-
ity of the cellular targets.
In this chapter, we have traveled across the
recent history of NGF pleiotropic role in ecto-
dermal tissue generation and repair, from
embryonic development to skin wound
healing, axonal regrowth, and remyelination.
The better understanding of both the
biological mechanisms underlying regenera-
tion and the physiological role of NGF in
development and injury response will open
new therapeutic strategies, driven by the

V. A. Baldassarro · G. Alastra
Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
L. Giardino
Bologna, Ozzano Emilia, Bologna, Italy
Department of Veterinary Medical Sciences, University of
e-mail: vito.baldassarro2@unibo.it; giuseppe.
Bologna, Bologna, Italy
alastra2@unibo.it
Health Science and Technologies Interdepartmental
L. Lorenzini
Center for Industrial Research (HST-ICIR), University of
Health Science and Technologies Interdepartmental
Bologna, Bologna, Italy
Center for Industrial Research (HST-ICIR), University of
Bologna, Ozzano Emilia, Bologna, Italy IRET Foundation, Bologna, Italy
e-mail: luciana.giardino@unibo.it
Department of Veterinary Medical Sciences, University of
Bologna, Ozzano Emilia, Bologna, Italy L. Calzà (*)
e-mail: luca.lorenzini8@unibo.it Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
A. Bighinati · A. Giuliani · M. Fernandez
Bologna, Ozzano Emilia, Bologna, Italy
Department of Veterinary Medical Sciences, University of
Bologna, Ozzano Emilia, Bologna, Italy Department of Pharmacy and Biotechnology, University
e-mail: andrea.bighinati@unibo.it; a.giuliani@unibo.it; of Bologna, Bologna, Italy
mercedes.fernandez@unibo.it
Montecatone Rehabilitation Institute, Montecatone,
M. Pannella Bologna, Italy
IRET Foundation, Ozzano Emilia, Bologna, Italy e-mail: laura.calza@unibo.it

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 51

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_5
52
potential applications of this growth factor as
an agent for improving endogenous regenera-
tion processes.
5.1 Introduction
“Endogenous regeneration” refers to the ability of
tissues and organs to self-repair in the event of
injury or disease. This capability varies signifi-
cantly between the different animal species and
districts of the body; tailed amphibians, for exam-
ple, can completely regenerate parts of the body,
such as limbs, tails, jaws, eyes, and a variety of
internal structures. In mammals, tissues with a
high cell turnover such as the skin may effectively
regenerate in the event of extensive lesions, while
the nervous tissue, such as that of the central
nervous system (CNS), has a very limited regen-
erative capability, confined to the myelin sheath.
In both cases, this capability may be severely
impaired by disease or excessive lesion area.
Since the discovery of stem cell niches in most
adult mammalian tissues and organs, which con-
tribute to the respective and tissue-specific cell
turnover rate (Bagnara 2020), the self-repair capa-
bility of adult tissue has been the focus of increas-
ing interest. In some tissues, such as the skin, the
proliferation rate of niche stem and precursor
cells increases in the event of injury, while asym-
metric division provides precursors, thus increas-
ing cell turnover, and ultimately leading to tissue
repair. In other tissues, such as that of the CNS,
stem cells in the neurogenic niches react weakly
to injuries, an activation which has no substantial
effects on lesion or pathology evolution (Sun
2016). On the contrary, oligodendrocyte precur-
sor cells disseminated throughout the white and
gray matter, or newly generated from the
glycogenic niches, may efficiently repair the mye-
lin, restoring appropriate axonal function (Bruce
et al. 2010).
The subsequent hypothesis was that organ and
tissue plasticity, and endogenous regeneration as a
result, could be improved by pharmacological and
non-pharmacological treatments able to direct the
stem cell niches (Wells and Watt 2018). This area
V. A. Baldassarro et al.
is also defined “in vivo regenerative medicine” or
“autotherapies” (Lumelsky et al. 2018).
The role of growth and neurotrophic factors
during development makes these biomolecules
natural candidates for improving endogenous
regeneration. NGF in particular is very attractive
from this point of view, being a pleiotropic
molecule acting on many different cell types,
both during development and in adulthood
(Aloe and Calzà 2004). In this short review,
we will focus on data supporting the potential
applications of NGF as an agent for improving
endogenous regeneration of ectoderm-derived
tissues, summarizing the basic and translational
findings supporting the development of NGF as
a drug.
5.2 NGF and Embryonic
Development
NGF and its receptors appear very early during
development (reviewed by Bracci-Laudiero and
De Stefano 2016). The NGF mRNA is found in
oocytes at different maturation stages and is
involved in follicle and oocyte maturation.
p75NTR and TrkA appear in mouse embryos at
the blastocyst stage, being confined to the inner
cell mass and absent in the trophoblast. NGF
receptor expression then differentiates into spe-
cific areas during gastrulation and neurulation,
suggesting that NGF plays a role in germinal
layer differentiation and early body shape specifi-
cation. NGF neutralization experiments in
chickens indicate that NGF absence alters body
axis rotation as well as notochord and somite
formation and evolution.
During neurulation, p75NTR expression in neu-
ral crest cells correlates with the beginning of cell
migration (Wislet et al. 2018). The different
isoforms of Trk receptors then appear during neu-
ral crest cell migration and differentiation
(Martin-Zanca et al. 1990).
Both mouse (Moscatelli et al. 2009) and
human embryonic stem cells (Pyle et al. 2006)
express NGF receptors, and differentiation stud-
ies of 3D embryonic stem cell cultures indicate
that exposure to NGF plus retinoic acid
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies 53

(RA) favors the ectodermal and mesodermal toward differentiation, gradually losing their
lineages (Inanç et al. 2008). pluripotency to become specific germinative
We studied the expression of NGF and its layer committed cells.
receptors in embryonic stem cells derived from Figure 5.1 shows the mRNA expression pro-
rat blastocytes (RESCs), using a cell line isolated file of NGF, trkA, and p75NTR under these differ-
in our laboratory (Fernández et al. 2011) ent in vitro conditions. We observed that all
(Fig. 5.1). These cells can be maintained in sev- mRNAs are expressed in the aforementioned cul-
eral ways: first as clusters (CL), growing in the ture conditions, with a significant increase of
presence of LIF (leukemia inhibitory factor) and p75NTR expression level in EBs, around
bFGF (basic-fibroblast growth factor) in ultra-low 10 times higher than in CL (**p ¼ 0.0077) and
attachment plates; second as a mixed population all the other studied conditions (SC, CL mixed
of single cells (SC) and CL, growing in the pres- population, **p ¼ 0.0093, p ¼ 0.0091). Interest-
ence of LIF and bFGF, in attaching and floating ingly, the trkA expression was significantly lower
conditions, respectively; third as embryoid bodies (around 0.5-fold) in single cells only compared to
(EB), whose formation can be induced by grow- the reference group (**p ¼ 0.0084).
ing the cells obtained from cluster splitting NGF also regulates organogenesis and tissue
(RESC-CL) in ultra-low attachment plates with- maturation in several districts, acting through
out LIF but in the presence of bFGF (Pannella both sensory innervation and angiogenesis (Kim
et al. 2018). These EBs recapitulate the first steps et al. 2013), as in the case of endochondral bone,

A B C D

E NGF mRNA F TrkA mRNA G P75 mRNA

2 2 15 **
R e la tiv e e x p re s s io n

**
n o rm a liz e d to C L

**

**
10

1 1
5

0 0 0
c l s c l s L c l s
c B
L

s
L

s c B s c B C
C

E
C

E E
L

mixed population mixed population mixed population

Floating + - + +

LIF + + + -
bFGF + + + +

Fig. 5.1 NGF, trkA, and p75 gene expression in RESCs. independent experiments performed in duplicate are
Representative images of RESCs grown as CLs (a), mixed included in the graphs. Statistical analysis was performed
population of SCs and CLs (b, c), and EBs (d) in the using one-way ANOVA and Tukey’s multiple comparison
specified culture conditions and mediums containing the test. Significant results were fixed at p < 0.05.
mitogens indicated. Graphs show relative mRNA expres- Abbreviations: bFGF, basic fibroblast growth factor; CL,
sion of NGF (e) and its receptors, trkA (f) and p75 (g), clusters; EBs, embryoid bodies; LIF, leukemia inhibitory
normalized to CL. Mean values SEM from three factor; SC, single cells
54
but also acts directly on non-neural cells, such as
during odontogenesis (Mitsiadis and Pagella
2016).
Finally, classical neutralization experiments
have indicated that several neural populations
and pathways in the peripheral nervous system
(PNS), including the sensory and sympathetic
neurons, and selective neural populations in the
CNS, such as the cholinergic neurons of the basal
forebrain, require NGF for appropriate axonal
growth, cell differentiation, cell migration, and
promotion and control of nerve regeneration
(Levi-Montalcini 1997). The sympathetic
neurons of the superior cervical ganglion are the
best-studied example of the role of NGF in
controlling pro-survival vs. pro-apoptotic
pathways during development. These cells
require NGF at the time of target innervation,
and in vitro experiments indicate that NGF with-
drawal activates the mitochondrial (intrinsic)
pathway of apoptosis involving caspases, Bcl-2
(B-cell lymphoma 2) family proteins, and XIAP
(X-linked inhibitor of apoptosis protein)
(Kristiansen and Ham 2014).
However, the role of NGF in mature neural
phenotype maintenance within the adult nervous
system, in cell reaction to lesion as either survival
or repair, and as a pro-apoptotic factor, is still
disputed, being dependent on: (1) the involved
receptor subtype, (2) the synthesis pathway from
the pro-NGF, and (3) the target cell type.
5.3 NGF and Epithelial Wound
Healing
The skin is the largest organ in the body, covering
its entire external surface, and forms the physical
barrier between the host and the external environ-
ment. The skin protects the entire body against
numerous insults, prevents excessive body water
loss, and plays a role in immune surveillance and
sensory detection.
The epithelial component of the skin derives
from the embryonic surface ectoderm, whereas
the underlying derma derives from the mesoderm.
During development, NGF plays a major role in
the development of selective skin components: in
V. A. Baldassarro et al.
particular, the development of thinly myelinated
Aδ or unmyelinated C-fibers that innervate the
skin is NGF-dependent (Indo 2012), and half of
the nociceptive sensory neurons innervating the
mature skin are dependent upon an appropriate
NGF supply (Snider and McMahon 1998).
Neurotrophins, including NGF, play a complex
role in the hair follicle cycle (Botchkarev et al.
2004), and the low-affinity neurotrophin receptor
is a key regulator of the melanocytic cell lineage
(Kasemeier-Kulesa and Kulesa 2018). During
development, cell types other than
keratinocytes—for example, melanocytes—also
require NGF, which derives from the neural
crest: p75NTR is in fact a marker for neural crest
cells and is thus used for cell sorting experiments.
Recently, p75NTR - (CD271) positivity has been
associated with an aggressive clinical subtype of
melanoma which metastasizes to the brain, being
indicated as a mediator of phenotype switching
which suppresses melanoma cell proliferation,
while concomitantly promoting metastasis forma-
tion in vivo (Kasemeier-Kulesa and Kulesa
2018).
The breakdown of skin integrity, when it is
incorrectly and rapidly repaired, results in a sub-
stantial physiological imbalance and renders a
patient vulnerable to a number of pathological
conditions, such as infection, fluid loss, and elec-
trolyte imbalance (Reinke and Sorg 2012),
underlining the critical importance of a complete
and efficient healing of cutaneous wounds. The
wound healing process is characterized by four
sequential and overlapping phases: hemostasis
and early inflammation (0–several hours after
injury), inflammation (1–3 days), proliferation
(4–21 days), and maturation and remodeling
(21 days–1 year) (Reinke and Sorg 2012). Dereg-
ulation of any of these steps results in impaired
healing, potentially leading to chronic ulcers (Sen
et al. 2009).
Several in vitro and in vivo studies have
shown that NGF affects several cell types
involved in skin wound healing. TrkA and/or
p75NTR expression is not limited to sensory
nerve endings but also includes inflammatory
cells (neutrophils, macrophages, mast cells, and
lymphocytes), keratinocytes, fibroblasts, and
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies
endothelial cells. NGF is also secreted by
fibroblasts (Matsuda et al. 1991), keratinocytes
(Tron et al. 1990), mast cells (Leon et al. 1994),
and T cells (Ehrhard et al. 1993), and has angio-
genic properties, via nitric oxide synthase (NOS)
and vascular endothelial growth factor (VEGF)
(Calzà et al. 2001).
We recently demonstrated that human
fibroblasts (BJ cells) and keratinocytes (HEK
cells), as well as human endothelial cells
(HUVECs), express both high- (TrkA) and
low-affinity (p75NTR) receptors for NGF, and
key physiological properties of these cells, such
as proliferation, migration, and angio-tube forma-
tion, are affected by NGF exposure in both phys-
iological and pathological (hyperglycemia)
conditions (Gostynska et al. 2019).
The underlying molecular mechanism,
activated by TrkA, includes the PI3K/Akt and
ERK/MAPK signaling pathways and the down-
stream mTOR. Notably, several NGF-mediated
effects during development, such as endothelial
cell invasion and cord formation (Park et al.
2007), angiogenesis in bone (Yu et al. 2019), as
well as neuroprotection (Elsherbiny et al. 2019),
activate these pathways. Moreover, an
impairment of the AKT-mTOR pathway has
been indicated as a possible cause of wound
healing impairment in diabetic mice (Jere et al.
2019).
A role of endogenous NGF in wound healing
has been suggested by several studies in animal
models, human pathologies, and in vitro models.
In particular, the diabetic skin is characterized by
a reduced epidermal innervation, considered to be
a main cause of diabetic “small-fiber neuropathy”
(Ebenezer and Polydefkis 2014), leading to
chronic ulcers (Laverdet et al. 2015). Notably, a
reduction in neurotrophic support to PNS neurons
has been indicated as responsible for peripheral
neuropathy (Sima 2003).
Topical application of NGF has therefore been
considered as a possible therapy for skin wound
healing in chronic ulcers (Tuveri et al. 2000;
Generini et al. 2004), and several in vivo studies
report the positive effect on fibroblast migration,
re-epithelialization and capillary lumen forma-
tion, and an increase in wound contraction in the
55
full-thickness excision model. One limiting side
effect of the therapeutic use of NGF is local
hyperalgesia to mechanical and electrical stimuli
lasting for weeks, due to nociceptor sensitization
(Obreja et al. 2018).
The use of NGF as therapeutic agent has been
approved for lesions of the corneal epithelium
(Sacchetti et al. 2020), and recombinant human
NGF has been an approved drug (Oxervate®)
since 2017 as eye drops for neurotrophic keratitis.
5.4 NGF and Axonal Regrowth
NGF exerts multiple effects on NGF-sensitive
neural pathway formation, including axonal elon-
gation and guidance (Guthrie 2007). Moreover,
NGF binding at TrkA or p75NTR receptors
activates either pro-survival or pro-apoptotic
pathways. During development, nervous system
post-synaptic cells are responsible for the major-
ity of NGF release, thus directing the appropriate
target innervation. The binding of NGF to TrkA
on sympathetic and sensory axon terminals, for
example, leads to receptor dimerization and inter-
nalization; then the complex NGF/TkA is retro-
gradely transported to the neuronal cell body,
where it activates molecular pathways for neuro-
nal survival and differentiation (Johnson et al.
1980). Neuronal cell survival is mediated by the
phosphorylation and activation of phosphatidy-
linositol 3-kinase (PI-3K) and MAPK which
leads to Akt activation, the final result of which
is the inhibition of pro-apoptotic genes (Bim and
FasL) in the developing neuron (Reichardt 2006).
The role of p75NTR in nervous system devel-
opment is complex, promoting either survival and
growth or apoptosis and degeneration, depending
on the expression of its coreceptors and ligands.
p75NTR-TrkA complexes elicit pro-survival and
axonal growth signaling in response to NGF,
while neurotrophin binding to p75NTR alone, or
the binding of proNGF, can trigger cell death and
axonal degeneration. Moreover, p75NTR intracel-
lular trafficking is not yet fully understood
(Edward Hickman et al. 2018).
In addition to the neurotrophic effect mediated
by the action on pro-survival genes, NGF
56
promotes neurite and axonal elongation, the
biological effect from which this neurotrophin
takes its name, as derived from the historical
experiments by Levi-Montalcini and Hamburger.
The original experiments described the sympa-
thetic innervation of solid tumors secreting this
molecule, when ectopically implanted in chick
embryos (Levi-Montalcini and Hamburger
1951). In this system, NGF acts as
chemoattractant for neurites, and it has been
demonstrated that the elongation of neuron pro-
cesses mediated by NGF depends on neurite
arborization rather than retrograde axonal trans-
port for its action. In fact, as demonstrated in an
in vitro model of sympathetic neuron cultures,
NGF application to the cell body compartment
does not promote neurite extension retrogradely
or anterogradely, while NGF application to the
neurite compartment promotes neurite elonga-
tion, depending on concentration gradient
(Campenot 1994).
At molecular level, TrkA activation in neuro-
nal cells leads to an increase in β-actin concentra-
tion and its polymerization in the growth cones,
ultimately resulting in axonal elongation via
PI-3K and PLC-γ activation and subsequent sta-
bilization by de-phosphorylation of the actin-
stabilizing complex ADF/Cofilin (Meberg et al.
1998). The activation of p75NTR in the develop-
ing nervous system also leads to growth cone
stabilization and axon elongation through inacti-
vation of RhoA, which normally phosphorylates
and destabilizes ADF/Cofilin via the ROCK cas-
cade (Yamashita et al. 1999).
The role of NGF on the target neural popula-
tion in the mature nervous system is much more
disputed, and differs in the CNS and PNS. It is
noteworthy that p75NTR levels are reduced in
neuronal cells during adulthood, and activation
of this receptor by NGF has been shown to
cause neuronal death in motor neurons, in partic-
ular through activation of the JNK and NF-κB
pathway (Wiese et al. 1999), suggesting a switch
between pro- and anti-neurotrophic NGF signal-
ing via p75NTR in adulthood. In addition, NGF
levels are reduced in adult neurons once nervous
system development is complete (Terenghi
1999).
V. A. Baldassarro et al.
The PNS and the CNS differ substantially in
their ability to respond and regenerate axons after
damage. In both districts, the “sprouting” of a
stump occurs following axon transection, but
this sprouting is followed by axonal elongation
in the PNS only, while this attempt aborts in the
CNS (Cafferty et al. 2010). The main reason for
this different behavior, as proven by Aguayo and
collaborators’ pioneering work in bridging spinal
cord lesions with peripheral nerve grafts, seems to
be the non-permissive microenvironment of the
CNS compared to the PNS (Richardson et al.
1980; David and Aguayo 1981; Benfey and
Aguayo 1982; Richardson and Riopelle 1984).
This microenvironment is determined by several
factors, including inflammatory stimuli (glial acti-
vation and scarring in the CNS versus Wallerian
degeneration in the PNS), myelin and cellular
debris from the core of the injury, extracellular
matrix composition, availability of growth-
promoting proteins, and different remyelinating
cells (oligodendrocytes and astrocytes in the CNS
versus Schwann cells and macrophages in the
PNS) (Mietto et al. 2015).
In the CNS, maintenance of the basal cholin-
ergic system phenotype is NGF-dependent, but
spontaneous axonal regrowth does not occur
even in the event of extreme axonal degeneration,
as in the case of experimental lesions or in
Alzheimer’s disease (Mesulam et al. 2019). In
the PNS, damage to the adult nerves induces a
rapid and robust increase in the synthesis of
neurotrophins by the surrounding milieu near
the lesion. NGF synthesis, for example, increases
in the sensory neurons in the dorsal root ganglia
(DRG), thus reactivating the cellular mechanisms
active during development, and switching their
phenotype from “transmitting mode” to
“regenerating/regrowth mode” (Richner et al.
2014).
Due to the pleiotropic nature of this molecule,
however, NGF synthesis also increases in
non-neuronal cells, such as the Schwann cells
and perineural fibroblasts surrounding the stumps
following axon lesion (Matsuoka et al. 1991), and
acts on axons of the afferent sensory neurons in
the proximal stump via TrkA. NGF is retro-
gradely transported to the cell body, where it
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies
mediates a trophic effect, enhancing the levels of
the pro-survival gene GAP-43 (Melville et al.
1989; Wiese et al. 1992; Verge 1996). NGF also
stimulates Schwann cell migration and recruit-
ment in the distal stump of the transected nerve
(Taniuchi et al. 1988).
On the contrary, deafferentation of adult sym-
pathetic neurons does not modify the expression
level of NGF or TrkA (DeCouto et al. 2003),
while axotomy of the cervical vagus nerve results
in increased expression of mRNAs for NGF in
non-neuronal cells at both the proximal and distal
segments of the transected nerve segment (Lee
et al. 2001).
The indirect effects of NGF on axonal
regrowth support the pharmacological use of
this neurotrophin in non-NGF-dependent
pathways, including the use of innovative
approaches based on biomaterials. Taking the
results obtained from conventional grafts loaded
with NGF, such as nerve bridges (Derby et al.
1993), or vein grafts (Gravvanis et al. 2004) as a
starting point, more recent approaches have
extended the potential use of NGF to treating
spinal cord injury. For example, the incorporation
of NGF into an electrospun scaffold (Colello et al.
2016) or nanoparticles (Xu et al. 2019) has led to
improved axonal regrowth in spinal cord injury,
possibly regulating the inflammation and angio-
genesis associated with contusion damage.
5.5 NGF and Remyelination
Myelination is the process whereby the axons are
wrapped in a multilayer lipid-enriched membrane
known as myelin sheath, the thickness of which
depends upon axon diameter, to guarantee the
appropriate axonal potential conduction.
Myelination occurs during development in both
CNS and PNS during late gestation and the early
post-natal period and is carried out by two distinct
cell types, oligodendrocyte precursor cells
(OPCs) and Schwann cells precursors (SCP).
In the CNS, three different sequential waves
(E12.5, E15.5, E18.5) in mice generate the OPCs,
which spread throughout all brain and spinal cord
regions, with a single mature oligodendrocyte
57
(OL) wrapping multiple axons, in a process
which reaches its peak at 2–4 week postnatal
age. During the development of the PNS, SCPs
are generated from the neural crest and then
migrate along with growing axons to form the
peripheral nerves, where the whole cell body
wraps around the nude axon. Notably, the tran-
scriptional networks that drive OPC and SCP
differentiation, such as central and peripheral
myelination and remyelination, are substantially
different (Sock and Wegner 2019).
A role of the neurotrophins in regulating
myelination has been suggested and
demonstrated by a number of studies, but still
remains a matter of investigation. It has, in fact,
been suggested that NGF acts as a potent regula-
tor of the axonal signals controlling the
myelination of TrkA-expressing DRG neurons.
Unexpectedly, these NGF-regulated axonal
signals have opposite effects on the myelination
of the peripheral and central branch of the
pseudounipolar neurons in DRGs, promoting
peripheral myelination by Schwann cells (SCs)
but reducing central myelination by OLs (Chan
et al. 2004).
This discrepant effect is still a topic of debate,
since little is known about the role of NGF and
other neurotrophins in the regulation of CNS
myelination, with most of the data deriving from
in vitro models (Xiao et al. 2009). Whereas NGF
is ineffective in promoting myelination in axon-
OL DRG co-cultures, while promoting
myelination in axon-SC DRG (Chan et al.
2004), the neurotrophin increases the expression
of the genes encoding for myelin-related proteins
in OL by acting on both p75NTR and TrkA
receptors (Du et al. 2003, 2006). Moreover,
since NGF is also able to interact directly with
the myelin oligodendrocyte glycoprotein (MOG),
modulating axon growth and survival, this novel
mechanism may mediate communication
between growing axons and myelination (von
Büdingen et al. 2015).
In the PNS, converging results indicate that
NGF regulates both the migration and differenti-
ation of SCPs. The study of Ntrk1 (the gene
encoding for TrkA) expression in the sciatic
nerve during development shows its high
58
expression at E17, before decreasing at E19 and
disappearing postnatally. p75NTR also increases at
E19 and decreases at the postnatal stages (Piirsoo
et al. 2010). This dynamic expression is also
involved in SCP/SC differentiation and
myelination, supporting the bi-directional signal-
ing occurring between developing axons and SCs.
In vitro data also support the role of NGF in SC
differentiation, the Ngf gene being constantly
over-expressed at all stages compared to
differentiated cells. Ntrk1 expression is also
higher than during proliferation and the growth-
arrest state, and p75NTR expression is induced by
differentiation (Bentley and Lee 2000).
In both the CNS and the PNS, numerous cell
characteristics and signaling molecules, deriving
from the demyelination process, or from glial and
neuronal cell signals, orchestrate the complex
machinery of myelin repair in adulthood. In the
CNS, a significant number of the OPCs generated
during development do not differentiate but
remain quiescent, producing the pool of adult
precursors identified by the expression of specific
markers (e.g., NG2 and PDGFRα). Resident and
newly formed OPCs, derived from neural stem
cells (NSCs), are activated by demyelinating
insults, and by signals produced by demyelinated
axons, resident astrocytes, and microglia, and
may efficiently repair the myelin, thus restoring
saltatory conduction. Indeed, remyelination offers
a unique example of endogenous regeneration in
a system with poor regeneration capabilities, such
as the CNS (Crawford et al. 2013). This
remyelination process shares some aspects and
mechanisms with developmental myelination, as
supported by the recapitulation hypothesis (Fancy
et al. 2011). However, it is now clear that devel-
opmental myelination and remyelination present
important differences (Miron et al. 2011;
Baldassarro et al. 2019a, b); there are also impor-
tant regional differences in the remyelination
capabilities of the adult CNS (Horiuchi et al.
2017; Kuhn et al. 2019). A large body of data
on the possible role of NGF in myelin protection
and/or repair in the CNS comes from animal
models of inflammatory/demyelinating diseases.
In experimental allergic encephalomyelitis, the
most widely used animal model for multiple
V. A. Baldassarro et al.
sclerosis (MS), an increase in tissue NGF concen-
tration has been described (Calzà et al. 1997;
Micera et al. 1998; Acosta et al. 2013), such as
an increase of p75NTR positive cells in the
subventricular zone (SVZ) of the lateral ventricles
(Calzà et al. 1997). These cells, recognized as
OPCs (Oderfeld-Nowak et al. 2009), migrate to
the corpus callosum (Calzà et al. 1998), p75NTR
being a main regulator of the migration process
during remyelination also (Anton et al. 1994;
Bentley and Lee 2000). In addition, OLs
expressing p75NTR have also been found in MS
patients (Chang et al. 2000). The importance of
the NGF low-affinity receptor in OPC biology is
also confirmed by the exacerbation of symptoms
in MS animal models lacking p75NTR (Copray
et al. 2004), in which an altered composition of
the inflammatory cellular infiltrates is described
(Küst et al. 2006). On the other hand, a protective
effect on OLs has been shown by a synthetic
ligand for TrkA in the cuprizone demyelination
model (Bonetto et al. 2017).
The general view of the possible role of NGF
in OPC/OL cell fate, as derived from in vitro
experiments, has been proposed by Casaccia-
Bonefill and Chao (Casaccia-Bonnefil et al.
2000). In brief, the binding of neurotrophins to
Trk receptor tyrosine kinases initiates signaling
cascades that promote cell survival and differen-
tiation. In contrast, p75NTR modulates the suscep-
tibility to death of fully differentiated, but not
OPCs, in specific conditions. NGF has no toxic
effects on trkA or p75NTR pig-derived OPCs
(Althaus and Klöppner 2006), such as OL
isolated from the rat cerebrum and expressing
p75NTR (Starkey et al. 2001); in fact NGF
overexpression in human NSCs increases OL dif-
ferentiation (Marei et al. 2013). Lastly, NGF
exposure protects OLs from TNFa-induced toxic-
ity, not only in resident precursors, but also in
OPCs generated in the SVZ (Takano et al. 2000;
Petratos et al. 2004). Overall, the final picture of
p75NTR and Trks expression throughout OPC dif-
ferentiation is still disputed, depending on the
animal species, cell system, and animal model
used for in vitro and in vivo experiments (Althaus
et al. 2008; Guo et al. 2013).
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies
SCs, like OLs, are postmitotic and fully
differentiated cells. Peripheral demyelination,
however, leads to a rapid response of these cells,
which de-differentiate, re-enter the cell cycle, and
activate the repair process (Jessen et al. 2015). In
addition, SC lineage is characterized by
differences throughout the different phases of
development and differentiation, but heterogene-
ity is lower compared to OLs (Jessen et al. 2015).
There is much evidence to suggest that NGF
and other neurotrophins play different roles in
these complex contexts (Althaus et al. 2008;
Webber and Zochodne 2010). p75NTR plays a
role in the regulation of SCP migration to the
PNS (Anton et al. 1994). Throughout p75NTR
activation, NGF treatment following peripheral
nerve lesions promotes SC autophagy, enhancing
the clearance of myelin debris and accelerating
recovery from injury (Li et al. 2020). NGF also
acts as chemo-attractant and chemo-kinetic cue
for both SCs and SCPs (Maniwa et al. 2003;
Cornejo et al. 2010).
The data briefly summarized above show a
clear portrait of the pro-myelinating role of NGF
in PNS, while still uncertain is the effect on
OPCs/OLs, and, therefore, on central
myelination. In both districts, moreover, it is
still unclear whether NGF acts directly on
myelinating cells, or indirectly through axons or
the satellite cells (fibroblasts, inflammatory cells,
glial cells other than myelinating, etc.).
References
Acosta CMR, Cortes C, MacPhee H, Namaka MP (2013)
Exploring the role of nerve growth factor in multiple
sclerosis: implications in myelin repair. CNS Neurol
Disord Drug Targets 12:1242–1256
Aloe L, Calzà L (2004) NGF and related molecules in
health and disease: preface. In: Progress in brain
research, p 544
Althaus HH, Klöppner S (2006) Mature pig oligoden-
drocytes rapidly process human recombinant
pro-nerve growth factor and do not undergo cell
death. J Neurochem 98:506–517. https://doi.org/10.
1111/j.1471-4159.2006.03891.x
Althaus HH, Klöppner S, Klopfleisch S, Schmitz M (2008)
Oligodendroglial cells and neurotrophins: a poly-
phonic cantata in major and minor. J Mol Neurosci
35:65–79. https://doi.org/10.1007/s12031-008-9053-y
59
Anton ES, Weskamp G, Reichardt LF, Matthew WD
(1994) Nerve growth factor and its low-affinity recep-
tor promote Schwann cell migration. Proc Natl Acad
Sci U S A 91:2795–2799. https://doi.org/10.1073/
pnas.91.7.2795
Bagnara GP (2020) Stem cells. Societa Editrice Esculapi
Baldassarro VA, Krężel W, Fernández M, Schuhbaur B,
Giardino L, Calzà L (2019a) The role of nuclear
receptors in the differentiation of oligodendrocyte pre-
cursor cells derived from fetal and adult neural stem
cells. Stem Cell Res 37:101443. https://doi.org/10.
1016/j.scr.2019.101443
Baldassarro VA, Marchesini A, Giardino L, Calzà L
(2019b) Differential effects of glucose deprivation on
the survival of fetal versus adult neural stem cells-
derived oligodendrocyte precursor cells. Glia. https://
doi.org/10.1002/glia.23750
Benfey M, Aguayo AJ (1982) Extensive elongation of
axons from rat brain into peripheral nerve grafts.
Nature 296:150–152. https://doi.org/10.1038/
296150a0
Bentley CA, Lee KF (2000) p75 Is important for axon
growth and Schwann cell migration during develop-
ment. J Neurosci 20:7706–7715. https://doi.org/10.
1523/jneurosci.20-20-07706.2000
Bonetto G, Charalampopoulos I, Gravanis A, Karagogeos
D (2017) The novel synthetic microneurotrophin
BNN27 protects mature oligodendrocytes against
cuprizone-induced death, through the NGF receptor
TrkA. Glia 65:1376–1394. https://doi.org/10.1002/
glia.23170
Botchkarev VA, Botchkareva N V., Peters EMJ, Paus R
(2004) Epithelial growth control by neurotrophins:
leads and lessons from the hair follicle. In: Progress
in brain research. Elsevier, Amsterdam, pp 493–513
Bracci-Laudiero L, De Stefano ME (2016) NGF in early
embryogenesis, differentiation, and pathology in the
nervous and immune systems. In: Current topics in
behavioral neurosciences. Springer, Cham, pp
125–152
Bruce CC, Zhao C, Franklin RJM (2010) Remyelination—
An effective means of neuroprotection. Horm Behav
57:56–62. https://doi.org/10.1016/j.yhbeh.2009.06.
004
Cafferty WBJ, Duffy P, Huebner E, Strittmatter SM
(2010) MAG and OMgp synergize with Nogo-A to
restrict axonal growth and neurological recovery after
spinal cord trauma. J Neurosci 30:6825–6837. https://
doi.org/10.1523/JNEUROSCI.6239-09.2010
Calzà L, Giardino L, Pozza M, Micera A, Aloe L (1997)
Time-course changes of nerve growth factor,
corticotropin-releasing hormone, and nitric oxide
synthase isoforms and their possible role in the devel-
opment of inflammatory response in experimental
allergic encephalomyelitis. Proc Natl Acad Sci U S A
94:3368–3373. https://doi.org/10.1073/pnas.94.7.3368
Calzà L, Giardino L, Pozza M, Bettelli C, Micera A, Aloe
L (1998) Proliferation and phenotype regulation in the
subventricular zone during experimental allergic
60
encephalomyelitis: in vivo evidence of a role for nerve
growth factor. Proc Natl Acad Sci U S A
95:3209–3214. https://doi.org/10.1073/pnas.95.6.3209
Calzà L, Giardino L, Giuliani A, Aloe L, Levi-Montalcini
R (2001) Nerve growth factor control of neuronal
expression of angiogenetic and vasoactive factors.
Proc Natl Acad Sci U S A 98:4160–4165. https://doi.
org/10.1073/pnas.051626998
Campenot RB (1994) NGF and the local control of nerve
terminal growth. J Neurobiol 25:599–611. https://doi.
org/10.1002/neu.480250603
Casaccia-Bonnefil P, Gu C, Chao MV (2000)
Neurotrophins in cell survival/death decisions. Adv
Exp Med Biol 468:275–282. https://doi.org/10.1007/
978-1-4615-4685-6_22
Chan JR, Watkins TA, Cosgaya JM, Zhang C, Chen L,
Reichardt LF, Shooter EM, Barres BA (2004) NGF
controls axonal receptivity to myelination by Schwann
cells or oligodendrocytes. Neuron 43:183–191. https://
doi.org/10.1016/j.neuron.2004.06.024
Chang A, Nishiyama A, Peterson J, Prineas J, Trapp BD
(2000) NG2-positive oligodendrocyte progenitor cells
in adult human brain and multiple sclerosis lesions. J
Neurosci 20:6404–6412. https://doi.org/10.1523/
jneurosci.20-17-06404.2000
Colello RJ, Chow WN, Bigbee JW, Lin C, Dalton D,
Brown D, Jha BS, Mathern BE, Lee KD, Simpson
DG (2016) The incorporation of growth factor and
chondroitinase ABC into an electrospun scaffold to
promote axon regrowth following spinal cord injury.
J Tissue Eng Regen Med 10:656–668. https://doi.org/
10.1002/term.1805
Copray S, Küst B, Emmer B, Lin MY, Liem R, Amor S,
De Vries H, Floris S, Boddeke E (2004) Deficient p75
low-affinity neurotrophin receptor expression
exacerbates experimental allergic encephalomyelitis
in C57/BL6 mice. J Neuroimmunol 148:41–53.
https://doi.org/10.1016/j.jneuroim.2003.11.008
Cornejo M, Nambi D, Walheim C, Somerville M,
Walker J, Kim L, Ollison L, Diamante G,
Vyawahare S, De Bellard ME (2010) Effect of
NRG1, GDNF, EGF and NGF in the migration of a
Schwann cell precursor line. Neurochem Res
35:1643–1651. https://doi.org/10.1007/s11064-010-
0225-0
Crawford AH, Chambers C, Franklin RJM (2013)
Remyelination: the true regeneration of the central
nervous system. J Comp Pathol 149:242–254. https://
doi.org/10.1016/j.jcpa.2013.05.004
David S, Aguayo A (1981) Axonal elongation into periph-
eral nervous system “bridges” after central nervous
system injury in adult rats. Science 214:931–933.
https://doi.org/10.1126/science.6171034
DeCouto SA, Jones EE, Kudwa AE, Shoemaker SE,
Shafer AJ, Brieschke MA, James PF, Vaughn JC,
Isaacson LG (2003) The effects of deafferentation
and exogenous NGF on neurotrophins and
neurotrophin receptor mRNA expression in the adult
superior cervical ganglion. Brain Res Mol Brain Res
V. A. Baldassarro et al.
119:73–82. https://doi.org/10.1016/j.molbrainres.
2003.08.015
Derby A, Engleman VW, Frierdich GE, Neises G, Rapp
SR, Roufa DG (1993) Nerve growth factor facilitates
regeneration across nerve gaps: morphological and
behavioral studies in rat sciatic nerve. Exp Neurol
119:176–191. https://doi.org/10.1006/exnr.1993.1019
Du Y, Fischer TZ, Lee LN, Lercher LD, Dreyfus CF
(2003) Regionally specific effects of BDNF on
oligodendrocytes. Dev Neurosci 25:116–126. https://
doi.org/10.1159/000072261
Du Y, Fischer TZ, Clinton-Luke P, Lercher LD, Dreyfus
CF (2006) Distinct effects of p75 in mediating actions
of neurotrophins on basal forebrain oligodendrocytes.
Mol Cell Neurosci 31:366–375. https://doi.org/10.
1016/j.mcn.2005.11.001
Ebenezer G, Polydefkis M (2014) Epidermal innervation
in diabetes. In: Handbook of clinical neurology.
Elsevier, pp 261–274
Edward Hickman F, Stanley EM, Carter BD (2018)
Neurotrophin responsiveness of sympathetic neurons
is regulated by rapid mobilization of the p75 receptor
to the cell surface through trka activation of arf6. J
Neurosci 38:5606–5619. https://doi.org/10.1523/
JNEUROSCI.0788-16.2018
Ehrhard PB, Erb P, Graumann U, Otten U (1993) Expression
of nerve growth factor and nerve growth factor receptor
tyrosine kinase Trk in activated CD4-positive T-cell
clones. Proc Natl Acad Sci U S A 90:10984–10988.
https://doi.org/10.1073/pnas.90.23.10984
Elsherbiny NM, Abdel-Mottaleb Y, Elkazaz AY, Atef H,
Lashine RM, Youssef AM, Ezzat W, El-Ghaiesh SH,
Elshaer RE, El-Shafey M, Zaitone SA (2019) Carba-
mazepine alleviates retinal and optic nerve neural
degeneration in diabetic mice via nerve growth
factor-induced PI3K/Akt/mTOR activation. Front
Neurosci 13. https://doi.org/10.3389/fnins.2019.01089
Fancy SPJ, Chan JR, Baranzini SE, Franklin RJM,
Rowitch DH (2011) Myelin regeneration: a recapitula-
tion of development? Annu Rev Neurosci 34:21–43.
https://doi.org/10.1146/annurev-neuro-061010-
113629
Fernández M, Pirondi S, Chen BL, Del Vecchio G,
Alessandri M, Farnedi A, Pession A, Feki A, Jaconi
MEE, Calzà L (2011) Isolation of rat embryonic stem-
like cells: a tool for stem cell research and drug discov-
ery. Dev Dyn 240:2482–2494. https://doi.org/10.1002/
dvdy.22761
Generini S, Tuveri MA, Matucci Cerinic M, Mastinu F,
Manni L, Aloe L (2004) Topical application of nerve
growth factor in human diabetic foot ulcers. A study of
three cases. Exp Clin Endocrinol Diabetes
112:542–544. https://doi.org/10.1055/s-2004-821313
Gostynska N, Pannella M, Rocco ML, Giardino L, Aloe L,
Calzà L (2019) The pleiotropic molecule NGF
regulates the in vitro properties of fibroblasts,
keratinocytes and endothelial cells: implications for
wound healing. Am J Physiol Physiol. https://doi.org/
10.1152/ajpcell.00180.2019
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies
Gravvanis AI, Tsoutsos DA, Tagaris GA, Papalois AE,
Patralexis CG, Iconomou TG, Panayotou PN,
Ioannovich JD (2004) Beneficial effect of nerve
growth factor-7S on peripheral nerve regeneration
through inside-out vein grafts: an experimental study.
Microsurgery 24:408–415. https://doi.org/10.1002/
micr.20055
Guo J et al (2013) proNGF inhibits proliferation and
oligodendrogenesis of postnatal hippocampal neural
stem/progenitor cells through p75NTR in vitro. Stem
Cell Res 11:874–887. https://doi.org/10.1016/j.scr.
2013.05.004
Guthrie S (2007) Neurotrophic factors: are they axon
guidance molecules? Adv Exp Med Biol 621:81–94
Horiuchi M, Suzuki-Horiuchi Y, Akiyama T, Itoh A,
Pleasure D, Carstens E, Itoh T (2017) Differing intrin-
sic biological properties between forebrain and spinal
oligodendroglial lineage cells. J Neurochem
142:378–391. https://doi.org/10.1111/jnc.14074
Inanç B, Elçin AE, Elçin YM (2008) Human embryonic
stem cell differentiation on tissue engineering
scaffolds: effects of NGF and retinoic acid induction.
Tissue Eng Part A. https://doi.org/10.1089/tea.2007.
0213
Indo Y (2012) Nerve growth factor and the physiology of
pain: lessons from congenital insensitivity to pain with
anhidrosis. Clin Genet 82:341–350
Jere SW, Houreld NN, Abrahamse H (2019) Role of the
PI3K/AKT (mTOR and GSK3β) signalling pathway
and photobiomodulation in diabetic wound healing.
Cytokine Growth Factor Rev. 50:52–59
Jessen KR, Mirsky R, Lloyd AC (2015) Schwann cells:
development and role in nerve repair. Cold Spring
Harb Perspect Biol 7:1–15. https://doi.org/10.1101/
cshperspect.a020487
Johnson EM, Gorin PD, Brandeis LD, Pearson J (1980)
Dorsal root ganglion neurons are destroyed by expo-
sure in utero to maternal antibody to nerve growth
factor. Science 210:916–918. https://doi.org/10.1126/
science.7192014
Kasemeier-Kulesa JC, Kulesa PM (2018) The convergent
roles of CD271/p75 in neural crest-derived melanoma
plasticity. Dev Biol 444:S352–S355. https://doi.org/
10.1016/j.ydbio.2018.04.008
Kim YS, Jo DH, Lee H, Kim JH, Kim KW, Kim JH (2013)
Nerve growth factor-mediated vascular endothelial
growth factor expression of astrocyte in retinal vascu-
lar development. Biochem Biophys Res Commun
431:740–745. https://doi.org/10.1016/j.bbrc.2013.01.
045
Kristiansen M, Ham J (2014) Programmed cell death dur-
ing neuronal development: the sympathetic neuron
model. Cell Death Differ 21:1025–1035
Kuhn S, Gritti L, Crooks D, Dombrowski Y (2019)
Oligodendrocytes in development, myelin generation
and beyond. Cells 8:1424. https://doi.org/10.3390/
cells8111424
Küst B, Mantingh-Otter I, Boddeke E, Copray S (2006)
Deficient p75 low-affinity neurotrophin receptor
61
expression does alter the composition of cellular infil-
trate in experimental autoimmune encephalomyelitis in
C57BL/6 mice. J Neuroimmunol 174:92–100. https://
doi.org/10.1016/j.jneuroim.2006.01.020
Laverdet B, Danigo A, Girard D, Magy L, Demiot C,
Desmoulière A (2015) Skin innervation: important
roles during normal and pathological cutaneous repair.
Histol Histopathol 30:875–892
Lee P, Zhuo H, Helke CJ (2001) Axotomy alters
neurotrophin and neurotrophin receptor mRNAs in
the vagus nerve and nodose ganglion of the rat. Brain
Res Mol Brain Res 87:31–41. https://doi.org/10.1016/
s0169-328x(00)00277-1
Leon A, Buriani A, Dal Toso R, Fabris M, Romanello S,
Aloe L, Levi-Montalcini R (1994) Mast cells synthe-
size, store, and release nerve growth factor. Proc Natl
Acad Sci U S A 91:3739–3743. https://doi.org/10.
1073/pnas.91.9.3739
Levi-Montalcini R (1997) The saga of the nerve growth
factor. World Scientific, River Edge, NJ
Levi-Montalcini R, Hamburger V (1951) Selective growth
stimulating effects of mouse sarcoma on the sensory
and sympathetic nervous system of the chick embryo. J
Exp Zool 116:321–361. https://doi.org/10.1002/jez.
1401160206
Li R, Li D, Wu C, Ye L, Wu Y, Yuan Y, Yang S, Xie L,
Mao Y, Jiang T, Li Y, Wang J, Zhang H, Li X, Xiao J
(2020) Nerve growth factor activates autophagy in
Schwann cells to enhance myelin debris clearance
and to expedite nerve regeneration. Theranostics
10:1649–1677. https://doi.org/10.7150/thno.40919
Lumelsky N, O’Hayre M, Chander P, Shum L, Somerman
MJ (2018) Autotherapies: enhancing endogenous
healing and regeneration. Trends Mol Med
24:919–930
Maniwa S, Iwata A, Hirata H, Ochi M (2003) Effects of
neurotrophic factors on chemokinesis of Schwann cells
in culture. Scand J Plast Reconstr Surg Hand Surg
37:14–17. https://doi.org/10.1080/alp.37.1.14.17
Marei HES, Althani A, Afifi N, Abd-Elmaksoud A,
Bernardini C, Michetti F, Barba M, Pescatori M,
Maira G, Paldino E, Manni L, Casalbore P, Cenciarelli
C (2013) Over-expression of hNGF in adult human
olfactory bulb neural stem cells promotes cell growth
and oligodendrocytic differentiation. PLoS One 8:
e82206. https://doi.org/10.1371/journal.pone.0082206
Martin-Zanca D, Barbacid M, Parada LF (1990) Expres-
sion of the trk proto-oncogene is restricted to the sen-
sory cranial and spinal ganglia of neural crest origin in
mouse development. Genes Dev 4:683–694. https://
doi.org/10.1101/gad.4.5.683
Matsuda H, Kannan Y, Ushio H, Kiso Y, Kanemoto T,
Suzuki H, Kitamura Y (1991) Nerve growth factor
induces development of connective tissue-type mast
cells in vitro from murine bone marrow cells. J Exp
Med 174:7–14. https://doi.org/10.1084/jem.174.1.7
Matsuoka I, Meyer M, Thoenen H (1991) Cell-type-spe-
cific regulation of nerve growth factor (NGF) synthesis
in non-neuronal cells: comparison of Schwann cells
62
with other cell types. J Neurosci 11:3165–3177. https://
doi.org/10.1523/jneurosci.11-10-03165.1991
Meberg PJ, Ono S, Minamide LS, Takahashi M, Bamburg
JR (1998) Actin depolymerizing factor and cofilin
phosphorylation dynamics: response to signals that
regulate neurite extension. Cell Motil Cytoskeleton
39:172–190. https://doi.org/10.1002/(SICI)1097-0169
(1998)39:2<172::AID-CM8>3.0.CO;2-8
Melville S, Sherburn TE, Coggeshall RE (1989) Preserva-
tion of sensory cells by placing stumps of transected
nerve in an impermeable tube. Exp Neurol
105:311–315. https://doi.org/10.1016/0014-4886(89)
90135-0
Mesulam MM, Lalehzari N, Rahmani F, Ohm D,
Shahidehpour R, Kim G, Gefen T, Weintraub S,
Bigio E, Geula C (2019) Cortical cholinergic denerva-
tion in primary progressive aphasia with Alzheimer
pathology. Neurology 92:E1580–E1588. https://doi.
org/10.1212/WNL.0000000000007247
Micera A, Vigneti E, Aloe L (1998) Changes of NGF
presence in nonneuronal cells in response to experi-
mental allergic encephalomyelitis in Lewis rats. Exp
Neurol 154:41–46. https://doi.org/10.1006/exnr.1998.
6864
Mietto BS, Mostacada K, Martinez AMB (2015)
Neurotrauma and inflammation: CNS and PNS
responses. Mediators Inflamm. https://doi.org/10.
1155/2015/251204
Miron VE, Kuhlmann T, Antel JP (2011) Cells of the
oligodendroglial lineage, myelination, and
remyelination. BBA Mol Basis Dis 1812:184–193.
https://doi.org/10.1016/j.bbadis.2010.09.010
Mitsiadis TA, Pagella P (2016) Expression of nerve
growth factor (NGF), TrkA, and p75NTR in develop-
ing human fetal teeth. Front Physiol 7. https://doi.org/
10.3389/fphys.2016.00338
Moscatelli I, Pierantozzi E, Camaioni A, Siracusa G,
Campagnolo L (2009) p75 neurotrophin receptor is
involved in proliferation of undifferentiated mouse
embryonic stem cells. Exp Cell Res 315:3220–3232.
https://doi.org/10.1016/j.yexcr.2009.08.014
Obreja O, Rukwied R, Nagler L, Schmidt M, Schmelz M,
Namer B (2018) Nerve growth factor locally sensitizes
nociceptors in human skin. Pain 159:416–426. https://
doi.org/10.1097/j.pain.0000000000001108
Oderfeld-Nowak B, Zaremba M, Kwiatkowska-Patzer B,
Lipkowski AW, Kurkowska-Jastrzȩbska I, Triaca V,
Aloe L (2009) NG2 positive cells of rat spinal cord
activated during experimental autoimmune encephalo-
myelitis are spatially associated with radially oriented
astroglia and express p75 receptor: a role for nerve
growth factor in oligodendrocyte progenitor migra-
tion? Arch Ital Biol 147:105–115. https://doi.org/10.
4449/aib.v147i4.871
Pannella M, Giardino L, Calzà L, Fernández M (2018)
Growth and neurotrophic factors in embryonic stem
cells. In: Methods in molecular biology. Humana,
Totowa, NJ, pp 275–294
V. A. Baldassarro et al.
Park MJ, Kwak HJ, Lee HC, Yoo DH, Park IC, Kim MS,
Lee SH, Chang HR, Hong S Il (2007) Nerve growth
factor induces endothelial cell invasion and cord for-
mation by promoting matrix metalloproteinase-2-
expression through the phosphatidylinositol 3-kinase/
Akt signaling pathway and AP-2 transcription factor. J
Biol Chem 282:30485–30496. https://doi.org/10.1074/
jbc.M701081200
Petratos S, Gonzales MF, Azari MF, Marriott M,
Minichiello RA, Shipham KA, Profyris C,
Nicolaou A, Boyle K, Cheema SS, Kilpatrick TJ
(2004) Expression of the low-affinity neurotrophin
receptor, p75NTR, is upregulated by oligodendroglial
progenitors adjacent to the subventricular zone in
response to demyelination. Glia 48:64–75. https://doi.
org/10.1002/glia.20056
Piirsoo M, Kaljas A, Tamm K, Timmusk T (2010) Expres-
sion of NGF and GDNF family members and their
receptors during peripheral nerve development and
differentiation of Schwann cells in vitro. Neurosci
Lett 469:135–140. https://doi.org/10.1016/j.neulet.
2009.11.060
Pyle AD, Lock LF, Donovan PJ (2006) Neurotrophins
mediate human embryonic stem cell survival. Nat
Biotechnol 24:344–350. https://doi.org/10.1038/
nbt1189
Reichardt LF (2006) Neurotrophin-regulated signalling
pathways. Philos Trans R Soc B Biol Sci
361:1545–1564
Reinke JM, Sorg H (2012) Wound repair and regeneration.
Eur Surg Res 49:35–43
Richardson PM, Riopelle RJ (1984) Uptake of nerve
growth factor along peripheral and spinal axons of
primary sensory neurons. J Neurosci 4:1683–1689.
https://doi.org/10.1523/jneurosci.04-07-01683.1984
Richardson PM, McGuinness UM, Aguayo AJ (1980)
Axons from CNS neurones regenerate into PNS grafts.
Nature 284:264–265. https://doi.org/10.1038/
284264a0
Richner M, Ulrichsen M, Elmegaard SL, Dieu R, Pallesen
LT, Vaegter CB (2014) Peripheral nerve injury
modulates neurotrophin signaling in the peripheral and
central nervous system. Mol Neurobiol 50:945–970
Sacchetti M, Lambiase A, Schmidl D, Schmetterer L,
Ferrari M, Mantelli F, Allegretti M, Garhoefer G
(2020) Effect of recombinant human nerve growth
factor eye drops in patients with dry eye: a phase IIa,
open label, multiple-dose study. Br J Ophthalmol
104:127–135. https://doi.org/10.1136/bjophthalmol-
2018-312470
Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt
TK, Gottrup F, Gurtner GC, Longaker MT (2009)
Human skin wounds: a major and snowballing threat
to public health and the economy: perspective article.
Wound Repair Regen 17:763–771
Sima AAF (2003) New insights into the metabolic and
molecular basis for diabetic neuropathy. Cell Mol Life
Sci 60:2445–2464
5 NGF and Endogenous Regeneration: From Embryology Toward Therapies
Snider WD, McMahon SB (1998) Tackling pain at the
source: new ideas about nociceptors. Neuron
20:629–632
Sock E, Wegner M (2019) Transcriptional control of
myelination and remyelination. Glia 67
(11):2153–2165
Starkey GD, Petratos S, Shipham KA, Butzkueven H,
Bucci T, Lowry K, Cheema SS, Kilpatrick TJ (2001)
Neurotrophin receptor expression and responsiveness
by postnatal cerebral oligodendroglia. Neuroreport
12:4081–4086. https://doi.org/10.1097/00001756-
200112210-00044
Sun D (2016) Endogenous neurogenic cell response in the
mature mammalian brain following traumatic injury.
Exp Neurol 275:405–410
Takano R, Hisahara S, Namikawa K, Kiyama H, Okano H,
Miura M (2000) Nerve growth factor protects
oligodendrocytes from tumor necrosis factor-alpha-
induced injury through Akt-mediated signaling
mechanisms. J Biol Chem 275:16360–16365. https://
doi.org/10.1074/jbc.M910419199
Taniuchi M, Clark HB, Schweitzer JB, Johnson EM
(1988) Expression of nerve growth factor receptors
by Schwann cells of axotomized peripheral nerves:
ultrastructural location, suppression by axonal contact,
and binding properties. J Neurosci 8:664–681
Terenghi G (1999) Peripheral nerve regeneration and
neurotrophic factors. J Anat 194:1–14. https://doi.org/
10.1046/j.1469-7580.1999.19410001.x
Tron VA, Coughlin MD, Jang DE, Stanisz J, Sauder DN
(1990) Expression and modulation of nerve growth
factor in murine keratinocytes (PAM 212). J Clin
Invest 85:1085–1089. https://doi.org/10.1172/
JCI114539
Tuveri M, Generini S, Matucci-Cerinic M, Aloe L (2000)
NGF, a useful tool in the treatment of chronic
vasculitic ulcers in rheumatoid arthritis. Lancet
356:1739–1740. https://doi.org/10.1016/S0140-6736
(00)03212-8
Verge VMK (1996) Neurotrophins and nerve injury in the
adult. Philos Trans R Soc B Biol Sci 351:423–430.
https://doi.org/10.1098/rstb.1996.0038
von Büdingen H-C, Mei F, Greenfield A, Jahn S, Shen
Y-AA, Reid HH, McKemy DD, Chan JR (2015) The
myelin oligodendrocyte glycoprotein directly binds
63
nerve growth factor to modulate central axon circuitry.
J Cell Biol 210:891–898. https://doi.org/10.1083/jcb.
201504106
Webber C, Zochodne D (2010) The nerve regenerative
microenvironment: early behavior and partnership of
axons and Schwann cells. Exp Neurol 223:51–59
Wells JM, Watt FM (2018) Diverse mechanisms for
endogenous regeneration and repair in mammalian
organs. Nature 557:322–328
Wiese UH, Ruth JL, Emson PC (1992) Differential expres-
sion of growth-associated protein (GAP-43) mRNA in
rat primary sensory neurons after peripheral nerve
lesion: a non-radioactive in situ hybridisation study.
Brain Res 592:141–156. https://doi.org/10.1016/0006-
8993(92)91669-6
Wiese S, Metzger F, Holtmann B, Sendtner M (1999) The
role of p75NTR in modulating neurotrophin survival
effects in developing motoneurons. Eur J Neurosci
11:1668–1676. https://doi.org/10.1046/j.1460-9568.
1999.00585.x
Wislet S, Vandervelden G, Rogister B (2018) From neural
crest development to cancer and vice versa: how
p75NTR and (Pro)neurotrophins could act on cell
migration and invasion? Front Mol Neurosci 11:244
Xiao J, Kilpatrick TJ, Murray SS (2009) The role of
neurotrophins in the regulation of myelin development.
Neurosignals 17:265–276. https://doi.org/10.1159/
000231893
Xu D, Wu D, Qin M, Nih LR, Liu C, Cao Z, Ren J,
Chen X, He Z, Yu W, Guan J, Duan S, Liu F, Liu X,
Li J, Harley D, Xu B, Hou L, Chen ISY, Wen J,
Chen W, Pourtaheri S, Lu Y (2019) Efficient delivery
of nerve growth factors to the central nervous system
for neural regeneration. Adv Mater 31:e1900727.
https://doi.org/10.1002/adma.201900727
Yamashita T, Tucker KL, Barde YA (1999) Neurotrophin
binding to the p75 receptor modulates Rho activity and
axonal outgrowth. Neuron 24:585–593. https://doi.org/
10.1016/S0896-6273(00)81114-9
Yu X, Qi Y, Zhao T, Fang J, Liu X, Xu T, Yang Q, Dai X
(2019) NGF increases FGF2 expression and promotes
endothelial cell migration and tube formation through
PI3K/Akt and ERK/MAPK pathways in human
chondrocytes. Osteoarthr Cartil 27:526–534. https://
doi.org/10.1016/j.joca.2018.12.007
 The Versatile Roles of Nerve Growth
Factor in Neuronal Attraction, 6
Odontoblast Differentiation,
and Mineral Deposition in Human Teeth

Thimios A. Mitsiadis and Pierfrancesco Pagella

Abstract the NGF secreting cells. These results show

Nerve growth factor (NGF) is an important
molecule for the development and differentia-
tion of neuronal and non-neuronal cells. Here
we analyze by immunohistochemistry the dis-
tribution of NGF in the dental pulp mesen-
chyme of embryonic and functional human
teeth. In the dental pulp of both embryonic
and healthy functional teeth, NGF is mainly
expressed in the odontoblasts that are respon-
sible for dentine formation, while in functional
teeth NGF is also expressed in nerve fibers
innervating the dental pulp. In injured teeth,
NGF is expressed in the newly formed
odontoblastic-like cells, which replace the
dying odontoblasts. In these teeth, NGF
expression is also upregulated in the intact
odontoblasts, suggesting a role for this mole-
cule in dental tissue repair. Similarly, in
cultures of human dental pulp cells, NGF
expression is strongly upregulated during
their differentiation into odontoblasts as well
as during the mineralization process. In
microfluidic devices, release of NGF from
cultured human dental pulp cells induced neu-
ronal growth from trigeminal ganglia toward
T. A. Mitsiadis (*) · P. Pagella
Orofacial Development and Regeneration, Institute of Oral
Biology, Centre for Dental Medicine, University of
Zurich, Zurich, Switzerland
e-mail: thimios.mitsiadis@zzm.uzh.ch; pierfrancesco.
pagella@zzm.uzh.ch
that NGF is closely linked to the various
functions of odontoblasts, including secretory
and neuronal attraction processes.
6.1 Introduction
Nerve growth factor (NGF) is the first identified
member of a family of neurotrophic molecules
called neurortophins, which have essential roles
in the fate, development, survival, outgrowth, and
maintenance of selected group of neurons
(Tomellini et al. 2014; Reichardt 2006; Levi-
Montalcini 1987; Lewin and Carter 2014). NGF
binds with low affinity to the transmembrane
glycoprotein receptor p75NTR that is expressed
on both neurons and non-neuronal target cells,
and its activation can induce either cell survival
or apoptosis (Lee et al. 2001; Ibanez and Simi
2012; Lewin and Carter 2014). The 140 kDa
tyrosine kinase TrkA glycoprotein acts as the
high-affinity receptor for NGF (Tomellini et al.
2014; Reichardt 2006; Levi-Montalcini 1987;
Lewin and Carter 2014), and its function is
influenced by the concomitant presence of
p75NTR (Reichardt 2006; Lewin and Carter
2014; Benedetti et al. 1993; Bibel et al. 1999).
In teeth, apart from the known functions of
NGF in the nervous system, additional roles
have been suggested for this molecule during

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 65

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_6
66
odontogenesis, dental tissue healing upon injury,
and differentiation of ameloblasts and
odontoblasts (Mitsiadis et al. 1992, 1993, 2017;
Mitsiadis and Luukko 1995; Mitsiadis and
Pagella 2016). Indeed, our previous studies and
work from others have shown that NGF and the
p75NTR and TrkA receptors are expressed in the
developing human teeth during embryogenesis,
thus indicating that NGF regulates dental physio-
logical processes (Mitsiadis et al. 1992, 1993;
Byers et al. 1990; Byers 1990; Luukko et al.
1997; Christensen et al. 1993; Nosrat et al.
2002; Mitsiadis and Pagella 2016). Moreover,
our recent findings have shown that NGF is
involved in dental tissue regeneration and the
behavior of dental pulp cells (Mitsiadis et al.
2017; Pagella et al. 2020b). Indeed, NGF pro-
duced by dental pulp cells cultured in
microfluidic devices acts as a neuroattractive
agent that allows neurons to establish direct
contacts with these cells (Pagella et al. 2020b).
Therefore, these results, in combination with pre-
viously reported findings in dental pulp
fibroblasts (Nosrat et al. 2001), indicate that
NGF plays a key role in the attraction and ramifi-
cation of neurons within the dental pulp during
tooth development and regeneration. Previous
studies in rodents, dogs, and humans have
shown that expression of NGF and p75NTR
increased after dental injury (Byers et al. 1990;
Byers 1990; Mitsiadis et al. 2017; Asaumi et al.
2000; Byun et al. 2008). Taken together, these
findings illustrate the importance of NGF in a
variety of biological processes linked to tooth
development, homeostasis, and repair.
In this study, we analyze the in vivo distribution
of NGF in the odontoblasts of developing, func-
tional healthy and injured human teeth, as well as
in cultured human dental pulp cells either alone or
in the presence of trigeminal neurons in vitro.
6.2 Materials and Methods
6.2.1 Materials
Antibodies Preparation, purification, and charac-
terization of the polyclonal anti-NGF antibody
T. A. Mitsiadis and P. Pagella
(Mitsiadis et al. 1992, 1993) and the mouse anti-
human p75NTR monoclonal antibody 20.4 (Grob
et al. 1985; Ross et al. 1984; Chesa et al. 1988)
have already been described. The p75NTR mono-
clonal antibody was a kind gift of Dr
E.M. Johnson Jr. and Dr C. Osborn (St. Louis,
USA). Mouse anti-neurofilament (NFP, Zymed
labs, San Francisco, CA, USA), rabbit
anti-β-III-tubulin (ab18207, Abcam, Cambridge,
UK), mouse anti-NuMA (GT3611; GeneTex,
Irvine, CA, U.S.A.), alexa-488-conjugated anti-
rabbit IgG (A-32731), and alexa-568-conjugated
anti-mouse IgG (A-11004; Thermo Fisher, Basel,
Switzerland) antibodies were also used.
Chemicals Vector Vectastain ABC kit was pur-
chased from Biosys (Compiègne, France). For the
preparation of the culture media, all materials
were purchased from Gibco BRL (Life
Technologies Inc., NY, USA). Other chemicals
were obtained from Sigma (St. Louis, MO, USA).
For cultures, Minimum Essential Medium
(MEM) was supplemented with 10% fetal bovine
serum, 2 mM glutamine, 100 UI/mL penicillin,
100 μg/mL streptomycin (Biowhittaker, Gagny,
France), and 0.25 μg/mL amphotericin B
(Fungizone®). Trigeminal ganglia were cultured
in Neurobasal Medium (Gibco) supplemented
with B27 (17504-044, Gibco), GlutaMAX, peni-
cillin/streptomycin (10 U/mL), 50 ng/mL Nerve
Growth Factor (NGF, R&D Systems), and
0.25 pM Arabinose Cytoside.
6.2.2 Embryonic Human Tissues
Tissues Human fetal tissues were obtained from
legal abortions. The material comprises teeth from
three fetuses (23 gestational week). The gestation
age was estimated from the fetal foot length and
from the last menstruation of the mother. Embryos
were non-infected, and all tissues were both mac-
roscopically and microscopically normal. The
fetuses were immediately fixed in 10% buffered
formalin for 5 days and then decalcified for
3 weeks in formic acid/10% formalin prior to
embedding in Paraplast. 4 to 6 μm thick sections
6 The Versatile Roles of Nerve Growth Factor in Neuronal Attraction. . . 67

were used for immunohistochemistry. This study healthy patients as previously described
was carried out in compliance with the French (Woloszyk et al. 2016). The dental pulps were
legislation, after the approval of the Regional gently removed with forceps, minced with
Ethics Committee of Development and Reproduc- scalpels, rinsed with PBS, and finally enzymati-
tion of the U.F.R. of Medicine of Reims-France cally digested for one hour at 37  C in a solution
(INSERM 314 Reims). of collagenase (3 mg/mL; Life Technologies BV,
Zug ZG, Switzerland) and Dispase (4 mg/mL;
Sigma-Aldrich Chemie GmbH, Buchs SG,
Switzerland). Dental pulp cells were cultured in
6.2.3 Adult Human Tissues
100-μm diameter culture dishes (Becton
Dickinson Labware, Lincoln Park, NJ, USA) in
All procedures were performed in accordance
MEM supplemented with 2 mM
with the current guidelines. The Cantonal Ethic
β-glycerophosphate (Sigma Chemical Co., St
Commission of Zurich approved experimental
Louis, USA). The cells were cultured at 37  C
procedures involving the use of human specimens
in a humidified atmosphere of 5% CO2, 95% air,
(reference number 2012-0588). All procedures
and the culture medium changed every other day.
were performed after obtaining the informed writ-
Confluent cultures were collected by
ten consent from all patients. Professional dentists
trypsinization (0.2% trypsin and 0.02% EDTA).
performed cavity preparations and tooth
The cells were plated at 3  103/cm2 on tissue
extractions.
culture treated 8-chambers glass slides (Becton
For studies on injured teeth, cavities were
Dickinson Labware, Lincoln Park, NJ, USA).
performed in healthy first premolars scheduled
After 6 weeks of culture, cells were fixed with
for extraction following the orthodontic treatment
70% ethanol for one hour at 4  C and processed
plan. Cavities 2–3 mm wide and 1.3–1.8 mm
for immunohistochemistry.
deep were prepared in the flank of the crown
(Fig. 6.3a). The pulp chambers were not exposed
during the preparation of the cavities. The cavities
6.2.5 Immunohistochemistry
were restored with the calcium hydroxide product
Dycal (Dentsplay, USA). Injured teeth were
Immunoperoxidase staining on sections was
extracted the ninth post-operation week.
performed as previously described (Mitsiadis
Extracted teeth were fixed in 10% neutral-
et al. 1992, 1993, 2003). Briefly, the sections
buffered formalin for 24 h, demineralized in
were deparaffinized, treated with 0.4% pepsin,
sodium formiate for 21 days, and then embedded
exposed to a 0.3% solution of hydrogen peroxide
in paraffin wax. Thereafter teeth were serially
in methanol, and then incubated overnight at 4  C
sectioned (6 μm thick sections) and processed
in a humid atmosphere with the polyclonal anti-
for immunohistochemistry. For the detection of
NGF antibody diluted 1:300 in PBS/0.2% BSA,
NGF in intact functional teeth, we proceeded with
the monoclonal anti-p75NTR antibody 20.4 (dilu-
immediate pulp extraction, followed by 10%
tion 1:1000), and the mouse anti-neurofilament
neutral-buffered formalin fixation for 24 h and
antibody (NFP, dilution 1:50). Peroxidase was
embedding in paraffin without prior
revealed by incubation with either 3.3-
decalcification.
diaminobenzidine tetrahydrochloride (DAB) or
3-amino-9-ethylcarbazole (AEC) reaction
solutions. After staining, the sections were
6.2.4 Cultures of Human Dental mounted with Eukit. In control sections the
Pulp Cells antibodies were omitted.
For immunohistochemistry on cultured dental
Human dental pulp cells were isolated from the pulp cells, the incubation with the NGF antibody
dental pulp of extracted impacted wisdom teeth of was performed overnight at 4  C after
68
permeabilization of cells for 15 min with 0.5%
Triton X-100 in PBS. Upon staining, the slides
were mounted with Aquamount (BDH, Gurr,
England). Controls were performed by
incubations with unrelated primary antibodies.
For immunofluorescence identification of
β-III-tubulin and NuMA in microfluidic
chambers, cells were incubated with the primary
antibodies diluted 1:100 in PBS/0.2% BSA and
then with a 1:250 dilution of Alexa-488-conju-
gated anti-rabbit IgG and Alexa-568-conjugated
anti-mouse IgG. Cells were then counterstained
with DAPI and mounted with Fluorescence
Mounting Medium (S302380-2, DAKO,
Agilent, Basel, Switzerland) before examination
with a Leica DM6000 widefield fluorescent
microscope.
6.2.6 Preparation of Microfluidic
Devices
Microfluidic devices were prepared as previously
described (Pagella and Mitsiadis 2020; Pagella
et al. 2015, Pagella et al. 2020b). Glass coverslips
were coated overnight at 37  C with 0.1 mg/mL
poly-D-lysine and stored in 70% of ethanol at
4  C. Polydimethylsiloxane (PDMS) microfluidic
devices (Millipore A150, Switzerland, 2  2 cm)
were punched with a 1-mm-diameter biopsy
punch on the neuronal side to enable the insertion
of the trigeminal ganglion and then sterilized
with 70% of ethanol. Both glass coverslips
and microfluidic devices were then let dry
completely under the laminar flow hood for 2 h.
The microfluidic devices were then mounted
onto the glass coverslips and pressed gently to
ensure proper adhesion. After mounting, the
microfluidic devices were coated with laminin
(5 μg/mL in Neurobasal Medium) overnight at
37  C. To prevent the persistence of air bubbles
in the culture chambers, the coated microfluidic
devices were placed under vacuum. After coat-
ing, the laminin solution was removed and the
culture chambers filled with the appropriate cul-
ture medium.
T. A. Mitsiadis and P. Pagella
6.2.7 Embryonic Mouse Tissues
All mice were maintained and handled according
to the Swiss Animal Welfare Law and in compli-
ance with the regulations of the Cantonal Veteri-
nary Office, Zurich (License number 151/2014).
The animal facility provided standardized hous-
ing conditions, with a mean room temperature of
21  C, relative humidity of 50%  5%, and
15 complete changes of filtered air per hour
(HEPA H 14 filter); air pressure was controlled
at 50 Pa. The light/dark cycle in the animal rooms
was set to a 12 h/12 h cycle (lights on at 07:00,
lights off at 19:00) with artificial light of approxi-
mately 40 Lux in the cage. The animals had
unrestricted access to sterilized drinking water,
and ad libitum access to a pelleted and extruded
mouse diet in the food hopper (Kliba No. 3436;
Provimi Kliba/Granovit AG, Kaiseraugst,
Switzerland). Mice were housed in a barrier-
protected specific pathogen-free unit and were
kept in groups of maximum five adult mice per
cage in standard IVC cages (Allentown Mouse
500; 194 mm  181 mm  398 mm, floor area
500 cm2; Allentown, New Jersey, USA) with
autoclaved dust-free poplar bedding (JRS GmbH
+ Co KG, Rosenberg, Germany). A standard
cardboard house (Ketchum Manufacturing,
Brockville, Canada) served as a shelter, and tissue
papers were provided as nesting material. Addi-
tionally, crinklets (SAFE® crinklets natural, JRS
GmbH + Co KG, Rosenberg, Germany) were
provided as enrichment and further nesting mate-
rial. The specific pathogen-free status of the
animals was monitored frequently and confirmed
according to FELASA guidelines by a sentinel
program. The mice were free of all viral, bacterial,
and parasitic pathogens listed in FELASA
recommendations (FELASA working group on
revision of guidelines for health monitoring of
rodents and rabbits et al. 2014).
C57/BL6J mice were time-mated. Successful
mating was assessed by vaginal plug check, and
the day of plug was considered as the day of
embryonic development 0.5 (E0.5). Trigeminal
ganglia were dissected from embryonic day 14.5
6 The Versatile Roles of Nerve Growth Factor in Neuronal Attraction. . . 69

Fig. 6.1 Distribution of NGF protein in dental tissues at
the bell stage of developing human teeth. (a–c)
Immunostaining against NGF (red color). In c, higher
magnification of the cusp area of the developing tooth
showing NGF expression in odontoblasts. Abbreviations:
d, dentine; df, dental follicle; dp, dental pulp; ide, inner
dental epithelium; o, odontoblasts; ode, outer dental epi-
thelium; pa, preameloblasts; sr, stellate reticulum. Scale
bars: (a, b) 100 μm; (c) 25 μm

(E14.5) mouse embryos. Dissections were

6.3 Results
performed in cold Dulbecco’s PBS. Dissected
ganglia were preserved in PBS, on ice, until cul-
6.3.1 NGF Distribution
ture. Trigeminal ganglia were then cultured in
in Odontoblasts of Human
Neurobasal Medium (Gibco) supplemented with
Embryonic Teeth
B27 (Gibco 17504-044), GlutaMAX, penicillin/
streptomycin (10 U/mL), 50 ng/mL nerve growth
Odontoblasts start to differentiate and secrete the
factor (NGF, R&D Systems), and 0.25 pM Arab-
dentine matrix during the late bell stage of human
inose Cytoside.
tooth development (23rd gestation week). At the
same time, inner dental epithelial cells stop divid-
ing and differentiate into the enamel-forming
6.2.8 Co-culture of Trigeminal
preameloblasts/ameloblasts. During this stage,
Ganglia and Human Dental
NGF immunoreactrivity was observed in all den-
Pulp Cells
tal epithelial cells, whereas labeling was absent
from the dental pulp (Fig. 6.1a, b). Strong NGF
Trigeminal ganglia were placed through the
staining was observed in differentiated functional
punched hole into the neuronal chamber of the
odontoblasts at the tip of the cusp (Fig. 6.1b, c),
microfluidic device. Ganglia were cultured alone
while immunoreactivity was also detected in the
for 5–7 days, until axons projected into the adja-
dental follicle (Fig. 6.1b).
cent chamber. Cells were then added to the
co-culture system. 20 μl of human dental pulp
cells in suspension were added directly in the
chamber at a density of 1  104 cells/chamber. 6.3.2 NGF and p75NTR Expression
Cells were allowed to be attached for 1 h and in Intact Functional
30 min, then 300 μl of the appropriate medium Human Teeth
were added to all chambers. Co-cultures were
maintained for 4 days. At the end of the culture In healthy functional human teeth (Fig. 6.2a),
period, the medium was removed and the samples NGF staining was observed in odontoblasts
were fixed in paraformaldehyde 4% (PFA 4%; (Fig. 6.2c, d), neurons (Fig. 6.2e), and some of
300 μl/chamber) for 15 min. the pulp fibroblasts (Fig. 6.2c, d) of freshly
70
Fig. 6.2 Expression of NGF and p75NTR in adult intact
and injured human teeth. (a) Histology of an adult intact
human tooth. (b) Immunostaining against p75NTR on
intact tooth. (c–e) Immunostaining against NGF on the
dental pulp of intact teeth. (c, d) show NGF expression
in odontoblasts. (e) shows NGF expression in nerve fibers
localized within the dental pulp. (f) Histology of adult
human tooth after cavity preparation. (g–i)
Immunostaining against NGF on the dental pulp of teeth
extracted dental pulps, while strong p75NTR
staining was confined to nerve fibers (Fig. 6.2b).
In the dental pulp, NGF immunoreactivity was
sporadically observed in few fibroblasts
(Fig. 6.2c–e). Immunostaining for NGF and
p75NTR was not detected in control sections
where the antibodies were omitted (data not
shown).
6.3.3 NGF Distribution in Functional
Human Teeth After Cavity
Preparation
Tooth cavity preparation leads to the apoptosis of
the odontoblasts beneath the injury site (Mitsiadis
et al. 2008). Nine weeks post-operation, the
apoptotic odontoblasts were replaced by
T. A. Mitsiadis and P. Pagella
subjected to cavity preparation. (g) shows NGF distribu-
tion far from the injury site. (h) shows NGF expression in
newly formed odontoblasts under the injured area. (i)
shows NGF expression in nerve fibers within the dental
pulp. Abbreviations: d, dentine; dp, dental pulp; nf, nerve
fibers; no, newly formed odontoblasts; o, odontoblasts; td,
tertiary dentine; v, vessel. Scale bars: (a, f) 5 mm; (b, c)
80 μm; (d, e) 50 μm; (g) 1 mm; (h, i) 20 μm
odontoblast-like cells, which produced a layer
of tertiary dentine beneath the injury site
(Fig. 6.2f, h). Strong NGF immunoreactivity
was detected in the odontoblast-like cells
(Fig. 6.2h), but also in odontoblasts located at a
distance from the cavity preparation site
(Fig. 6.2g). NGF staining was also observed in
few pulp fibroblasts (Fig. 6.2g) and neurons
(Fig. 6.2i). NGF staining was not detected in
control sections (data not shown).
6.3.4 NGF Expression in Human
Dental Pulp Cells In Vitro
In order to understand better the role of NGF in
tooth repair, we have analyzed NGF expression in
dental pulp cells freshly extracted from human
6 The Versatile Roles of Nerve Growth Factor in Neuronal Attraction. . .
Fig. 6.3 NGF protein distribution in cultured human
dental pulp cells. (a) Freshly extracted human intact
teeth, which are used for the collection of dental pulp
cells. (b–d) Expression of NGF in dental pulp cells
cultured under mineralizing conditions for 2 days (not
yet confluent cells, b), 1 week (confluent cells, c), and
4 weeks (formation of mineralization nodules, d). (e)
Schematic representation of the microfluidic “organ-on-
a-chip” device that is used for the co-culture of the trigem-
inal ganglion with the dental pulp cells. (f) Immunofluo-
rescent staining against βIII-tubulin showing neuronal
healthy teeth (Fig. 6.3a). Cells were cultured in
mineralizing conditions. All dental pulp cells
cultured in the presence of ß-glycerophosphate
for different periods of time up to 4 weeks
exhibited NGF immunoreactivity (Fig. 6.3b–d).
The strongest NGF staining was observed in con-
fluent cells (Fig. 6.3c) and in cells forming
mineralized nodules (Fig. 6.3d).
6.3.5 Co-culture of Trigeminal
Ganglia and Human Dental
Pulp Cells in Microfluidic
Devices
Trigeminal ganglia from embryonic day 14.5
C57/BL6J mouse embryos were isolated and
cultured alone at the neuronal compartment of
the microfluidic device (Fig. 6.3e). Upon the ini-
tiation of axonal outgrowths (4 days of culture)
71
growth from the chamber containing the trigeminal gan-
glion toward the chamber containing the dental pulp cells.
(g) Higher magnification of f, showing the passage of
axons through the microgrooves. (h) Immunofluorescent
staining against βIII-tubulin (green color) showing trigem-
inal neurons establishing contacts (white arrowheads) with
dental pulp cells (marked by NuMA nuclear staining, red
color). DAPI (blue color) marks the nuclei of the cells.
Abbreviations: cc1, culture chamber 1; cc2, culture cham-
ber 2; mg, microgrooves; tgg, trigeminal ganglion. Scale
bars: (b–d, i) 25 μm; (f) 500 μm; (g) 75 μm
(Fig. 6.3f, g), human dental pulp cells were added
to the other side of the microfluidic device. We
then analyzed the outgrowth of trigeminal
neurons co-cultured with dental pulp cells. Tri-
geminal ganglia and dental pulp cells were
co-cultured for 4 days. Staining with βIII-tubulin
and analysis using a wide-field fluorescent
microscopy showed many trigeminal axonal
projections toward the dental pulp cell compart-
ment (Fig. 6.3b). Rich axonal networks were
observed in the dental cell compartment, where
neuronal projections established direct contacts
with the dental pulp cells (Fig. 6.3h).
6.4 Discussion
A complex network of molecules ensures dental
tissue integrity and function during tooth devel-
opment, homeostasis, and regeneration. Among
72
these molecules, NGF emerged as an important
modulator of innervation, cytodifferentiation, and
mineralization processes in human and rodent
teeth (Mitsiadis et al. 1992, 1993, 2017; Mitsiadis
and Pagella 2016; Mitsiadis and Luukko 1995),
thus indicating that the functions of NGF in dental
tissues are highly conserved among different spe-
cies (Mitsiadis et al. 1992, 1993; Mitsiadis and
Pagella 2016). In our previous studies, we have
demonstrated that NGF and its high and low
affinity receptors, TrkA and p75NTR respectively,
are expressed in specific cell types during the
various developmental stages of human teeth
(Mitsiadis and Pagella 2016). In the present
study, we highlight the roles of NGF in the dif-
ferentiation of odontoblasts, formation of dentine,
and establishment of dental pulp innervation. Our
findings confirm the previous studies and show
that NGF expression in odontoblasts precedes the
dental pulp innervation, thus indicating that this
molecule is involved in odontoblasts differentia-
tion, dentine matrix production, and mineraliza-
tion events during odontogenesis. In contrast, the
low levels of NGF expression by odontoblasts of
functional intact adult teeth are correlated to the
slow, throughout life deposition of secondary
dentine. However, the synthesis of dentine by
odontoblasts is accelerated in carious or injured
teeth (Mitsiadis and Rahiotis 2004; About and
Mitsiadis 2001), a process associated with NGF
upregulation (Mitsiadis et al. 2017). Deep cavity
preparations are more drastic and lead to exten-
sive apoptosis of the odontoblasts beneath the
injured site (Mitsiadis et al. 2008). In this case,
dental pulp cells are recruited at the injured site;
they differentiate into odontoblast-like cells and
form a reparative dentin (Mitsiadis and Rahiotis
2004). Our present findings show that in these
conditions, NGF expression is upregulated not
only in odontoblast-like cells but also in
odontoblasts located far from the injury site, in
pulp fibroblasts, and neurons, thus clearly
demonstrating the involvement of this molecule
in a complex dental tissue healing process.
Indeed, our present and previous in vitro
T. A. Mitsiadis and P. Pagella
experiments showed that NGF is upregulated dur-
ing the mineralization of dental pulp fibroblasts
and suggested an active role for this molecule in
hard tissue formation (Mitsiadis et al. 2017), a
function similar to that recently reported during
osteogenesis (Tomlinson et al. 2017).
The role of innervation in the tooth repair
events is not yet well elucidated. It is well
established from previous pioneer studies that
NGF plays a pivotal role in tissue innervation,
acting as a neurotrophic and neuroattractive factor
(Levi-Montalcini 1987; Levi-Montalcini and
Aloe 1980; Ruit et al. 1992; Davies et al. 1981;
Cohen et al. 1954). Human tooth is one of the
most innervated organs of the body (Mitsiadis
and Luukko 1995; Mitsiadis et al. 2017), and
therefore NGF could play an important role in
pain perception (Ugolini et al. 2007; Kumar and
Mahal 2012). A dense neuronal plexus is com-
posed around the NGF-secreting odontoblasts
(Mitsiadis et al. 2017; Mitsiadis and Pagella
2016). NGF upregulation upon tooth injury
promotes reorganization of the neuronal network
within the dental pulp (Mitsiadis et al. 2017). This
reorganization and the neuroattractive properties
of dental pulp cells on trigeminal ganglia could be
studied in vitro using microfluidic “organ-on-a-
chip” devices (Pagella et al. 2021). These
miniaturized platforms have already been used
to emulate the neurotrophic properties of dental
pulp stem cells, as well as dental epithelial cancer
stem cells (ameloblastoma) (Pagella and
Mitsiadis 2020; Pagella et al. 2020a, b). These
two stem cell populations produce and secrete
NGF, which attracts the neurons toward them
(Pagella et al. 2020a, b). Here we show that, in
the microfluidic devices, neurons are also
attracted by dental pulp fibroblasts and establish
complex and abundant contacts with them. In
addition to this function, NGF can have stimula-
tory roles in regenerative processes by
modulating the secretion of neuropeptides and
other bioactive molecules by neurons (Pagella
et al. 2014). Stem cells are involved in dental
pulp regeneration, and therefore nerve-derived
6 The Versatile Roles of Nerve Growth Factor in Neuronal Attraction. . . 73

Fig. 6.4 Schematic

representation of the
functions of NGF in
odontoblasts. NGF plays
significant roles in the
differentiation of
odontoblasts, the
mineralization of the dentin
matrix, and the
establishment of a rich
network of neurons in the
dental pulp

molecules affect the behavior of stem cells in the

healing processes of many organs, including teeth
(Kumar and Brockes 2012; Orsini et al. 2018;
Mitsiadis et al. 2015).
In conclusion, our present and previous
results in human teeth (Mitsiadis et al. 2017;
Mitsiadis and Pagella 2016) clearly show that
NGF is associated with the differentiation of
odontoblasts, the formation of dentine, and the
organization of the neuronal network within the
dental pulp during development, pathology, and
regeneration (Fig. 6.4).
Ackowledgments This work was supported by institu-
tional grants from the University of Zurich.
Competing Financial Interests The authors declare
that there are no conflicts of interest associated with
this work.
References
About I, Mitsiadis TA (2001) Molecular aspects of tooth
pathogenesis and repair: in vivo and in vitro models.
Adv Dent Res 15:59–62
Asaumi K, Nakanishi T, Asahara H, Inoue H, Takigawa M
(2000) Expression of neurotrophins and their receptors
(TRK) during fracture healing. Bone 26:625–633
Benedetti M, Levi A, Chao MV (1993) Differential
expression of nerve growth factor receptors leads to
altered binding affinity and neurotrophin
responsiveness. Proc Natl Acad Sci U S A
90:7859–7863
Bibel M, Hoppe E, Barde YA (1999) Biochemical and
functional interactions between the neurotrophin
receptors trk and p75NTR. EMBO J 18:616–622
Byers MR (1990) Segregation of NGF receptor in sensory
receptors, nerves and local cells of teeth and
periodontium demonstrated by EM immunocytochem-
istry. J Neurocytol 19:765–775
Byers MR, Schatteman GC, Bothwell M (1990) Multiple
functions for NGF receptor in developing, aging and
74
injured rat teeth are suggested by epithelial, mesenchy-
mal and neural immunoreactivity. Development
109:461–471
Byun JH, Lee JH, Choi YJ, Kim JR, Park BW (2008)
Co-expression of nerve growth factor and p75NGFR
in the inferior alveolar nerve after mandibular distrac-
tion osteogenesis. Int J Oral Maxillofac Surg
37:467–472
Chesa PG, Rettig WJ, Thomson TM, Old LJ, Melamed
MR (1988) Immunohistochemical analysis of nerve
growth factor receptor expression in normal and malig-
nant human tissues. J Histochem Cytochem
36:383–389
Christensen LR, Mollgard K, Kjaer I, Janas MS (1993)
Immunocytochemical demonstration of nerve growth
factor receptor (NGF-R) in developing human fetal
teeth. Anat Embryol (Berl) 188:247–255
Cohen S, Levi-Montalcini R, Hamburger V (1954) A
nerve growth-stimulating factor isolated from sarcom
as 37 and 180. Proc Natl Acad Sci U S A
40:1014–1018
Davies AM, Lumsden AG, Slavkin HC, Burnstock G
(1981) Influence of nerve growth factor on the embry-
onic mouse trigeminal ganglion in culture. Dev
Neurosci 4:150–156
Felasa Working Group on Revision of Guidelines for
Health Monitoring of Rodents and Rabbits, Mahler
Convenor M, Berard M, Feinstein R, Gallagher A,
Illgen-Wilcke B, Pritchett-Corning K, Raspa M
(2014) FELASA recommendations for the health mon-
itoring of mouse, rat, hamster, guinea pig and rabbit
colonies in breeding and experimental units. Lab Anim
48:178–192
Grob PM, Ross AH, Koprowski H, Bothwell M (1985)
Characterization of the human melanoma nerve growth
factor receptor. J Biol Chem 260:8044–8049
Ibanez CF, Simi A (2012) p75 neurotrophin receptor sig-
naling in nervous system injury and degeneration: par-
adox and opportunity. Trends Neurosci 35:431–440
Kumar A, Brockes JP (2012) Nerve dependence in tissue,
organ, and appendage regeneration. Trends Neurosci
35:691–699
Kumar V, Mahal BA (2012) NGF—the TrkA to successful
pain treatment. J Pain Res 5:279–287
Lee R, Kermani P, Teng KK, Hempstead BL (2001) Reg-
ulation of cell survival by secreted proneurotrophins.
Science 294:1945–1948
Levi-Montalcini R (1987) The nerve growth factor
35 years later. Science 237:1154–1162
Levi-Montalcini R, Aloe L (1980) Tropic, trophic, and
transforming effects of nerve growth factor. Adv
Biochem Psychopharmacol 25:3–15
Lewin G, Carter BD (2014) Neurotrophic factors.
Springer, Berlin
Luukko K, Arumae U, Karavanov A, Moshnyakov M,
Sainio K, Sariola H, Saarma M, Thesleff I (1997)
Neurotrophin mRNA expression in the developing
tooth suggests multiple roles in innervation and organ-
ogenesis. Dev Dyn 210:117–129
T. A. Mitsiadis and P. Pagella
Mitsiadis TA, Luukko K (1995) Neurotrophins in
odontogenesis. Int J Dev Biol 39:195–202
Mitsiadis TA, Pagella P (2016) Expression of nerve
growth factor (NGF), TrkA, and p75(NTR) in devel-
oping human fetal teeth. Front Physiol 7:338
Mitsiadis TA, Rahiotis C (2004) Parallels between tooth
development and repair: conserved molecular
mechanisms following carious and dental injury. J
Dent Res 83:896–902
Mitsiadis TA, Dicou E, Joffre A, Magloire H (1992)
Immunohistochemical localization of nerve growth
factor (NGF) and NGF receptor (NGF-R) in the devel-
oping first molar tooth of the rat. Differentiation
49:47–61
Mitsiadis TA, Couble P, Dicou E, Rudkin BB, Magloire H
(1993) Patterns of nerve growth factor (NGF),
proNGF, and p75 NGF receptor expression in the rat
incisor: comparison with expression in the molar. Dif-
ferentiation 54:161–175
Mitsiadis TA, Romeas A, Lendahl U, Sharpe PT, Farges
JC (2003) Notch2 protein distribution in human teeth
under normal and pathological conditions. Exp Cell
Res 282:101–109
Mitsiadis TA, De Bari C, About I (2008) Apoptosis in
developmental and repair-related human tooth
remodeling: a view from the inside. Exp Cell Res
314:869–877
Mitsiadis TA, Orsini G, Jimenez-Rojo L (2015) Stem cell-
based approaches in dentistry. Eur Cell Mater
30:248–257
Mitsiadis TA, Magloire H, Pagella P (2017) Nerve growth
factor signalling in pathology and regeneration of
human teeth. Sci Rep 7:1327
Nosrat IV, Widenfalk J, Olson L, Nosrat CA (2001) Dental
pulp cells produce neurotrophic factors, interact with
trigeminal neurons in vitro, and rescue motoneurons
after spinal cord injury. Dev Biol 238:120–132
Nosrat I, Seiger A, Olson L, Nosrat CA (2002) Expression
patterns of neurotrophic factor mRNAs in developing
human teeth. Cell Tissue Res 310:177–187
Orsini G, Pagella P, Mitsiadis TA (2018) Modern trends in
dental medicine: an update for internists. Am J Med
131:1425–1430
Pagella P, Mitsiadis TA (2020) Analysis of tooth innerva-
tion in microfluidic coculture devices. Methods Mol
Biol 2155:99–106
Pagella P, Jimenez-Rojo L, Mitsiadis TA (2014) Roles of
innervation in developing and regenerating orofacial
tissues. Cell Mol Life Sci 71:2241–2251
Pagella P, Miran S, Mitsiadis T (2015) Analysis of devel-
oping tooth germ innervation using microfluidic
co-culture devices. J Vis Exp e53114
Pagella P, Caton J, Meisel CT, Mitsiadis TA (2020a)
Ameloblastomas exhibit stem cell potential, possess
neurotrophic properties, and establish connections
with trigeminal neurons. Cells 9
Pagella P, Miran S, Neto E, Martin I, Lamghari M,
Mitsiadis TA (2020b) Human dental pulp stem cells
exhibit enhanced properties in comparison to human
6 The Versatile Roles of Nerve Growth Factor in Neuronal Attraction. . .
bone marrow stem cells on neurites outgrowth. FASEB
J 34:5499–5511
Pagella P, Cordiale A, Marconi GD, Trubiani O, Rasponi
M, Mitsiadis TA (2021) Bioengineered tooth emula-
tion systems for regenerative and pharmacological
purposes. Eur Cell Mater 41:502–516
Reichardt LF (2006) Neurotrophin-regulated signalling
pathways. Philos Trans R Soc Lond B Biol Sci
361:1545–1564
Ross AH, Grob P, Bothwell M, Elder DE, Ernst CS,
Marano N, Ghrist BF, Slemp CC, Herlyn M, Atkinson
B et al (1984) Characterization of nerve growth factor
receptor in neural crest tumors using monoclonal
antibodies. Proc Natl Acad Sci U S A 81:6681–6685
Ruit KG, Elliott JL, Osborne PA, Yan Q, Snider WD
(1992) Selective dependence of mammalian dorsal
root ganglion neurons on nerve growth factor during
embryonic development. Neuron 8:573–587
75
Tomellini E, Lagadec C, Polakowska R, Le Bourhis X
(2014) Role of p75 neurotrophin receptor in stem cell
biology: more than just a marker. Cell Mol Life Sci
71:2467–2481
Tomlinson RE, Li Z, Li Z, Minichiello L, Riddle RC,
Venkatesan A, Clemens TL (2017) NGF-TrkA signal-
ing in sensory nerves is required for skeletal adaptation
to mechanical loads in mice. Proc Natl Acad Sci U S A
114:E3632–E3E41
Ugolini G, Marinelli S, Covaceuszach S, Cattaneo A,
Pavone F (2007) The function neutralizing anti-TrkA
antibody MNAC13 reduces inflammatory and neuro-
pathic pain. Proc Natl Acad Sci U S A 104:2985–2990
Woloszyk A, Buschmann J, Waschkies C, Stadlinger B,
Mitsiadis TA (2016) Human dental pulp stem cells and
gingival fibroblasts seeded into silk fibroin scaffolds
have the same ability in attracting vessels. Front
Physiol 7:140
 An Overview of Adult Neurogenesis
7
Filipa F. Ribeiro and Sara Xapelli

Abstract review the main properties of the adult neuro-

Neurogenesis is maintained in the mammalian
brain throughout adulthood in two main
regions: the subventricular zone (SVZ) of the
lateral ventricles and the subgranular zone
(SGZ) of the hippocampal dentate gyrus.
Adult neurogenesis is a process composed of
multiple steps by which neurons are generated
from dividing adult neural stem cells and
migrate to be integrated into existing neuronal
circuits. Alterations in any of these steps
impair neurogenesis and may compromise
brain function, leading to cognitive
impairment and neurodegenerative diseases.
Therefore, understanding the cellular and
molecular mechanisms that modulate adult
neurogenesis is the centre of attention of
regenerative research. In this chapter, we
F. F. Ribeiro · S. Xapelli (*)
Instituto de Farmacologia e Neurociências, Faculdade de
Medicina, Universidade de Lisboa, Lisbon, Portugal
Instituto de Medicina Molecular João Lobo Antunes,
Faculdade de Medicina, Universidade de Lisboa, Lisbon,
Portugal
e-mail: anaribeiro@medicina.ulisboa.pt;
sxapelli@medicina.ulisboa.pt
genic niches.
7.1 Adult Neurogenesis: An
Historical Perspective
For over 100 years, a central dogma in the field of
neuroscience was that new neurons are not added
to the adult mammalian brain. Indeed, until
recently, it was assumed that neurogenesis, the
formation of new neurons, in the mammalian
brain was thought to occur only during develop-
ment, stopping soon after birth. Then, the brain
would remain a fixed structure, incapable of pro-
ducing new neurons during adulthood. This cen-
tral dogma in neuroscience was defended by
influential figures, such as the Spanish Nobel
Prize Winner, the neuroanatomist Santiago
Ramón y Cajal (Ramón y Cajal and May 1928),
who contributed for the scepticism of the scien-
tific community.
In 1962, with the introduction of [3H]-thymi-
dine autoradiography, a technique where [3H]-
thymidine is incorporated into the DNA of divid-
ing cells, Joseph Altman reported the first evi-
dence of [3H]-thymidine presence in neurons
(Altman 1962). He also demonstrated that new
neurons are present in different areas of the post-
natal brain, such as in the neocortex, the dentate
gyrus (DG) of the hippocampus, the olfactory

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 77

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_7
78
bulb (OB) and the cerebellar cortex of different
mammals (Altman 1963; Altman and Das 1965,
1966, 1967).
Despite Altman’s discoveries, the evidence of
postnatal neurogenesis was ignored for two more
decades. It was then that, with electron micros-
copy, Kaplan and his colleagues confirmed
Altman’s results by showing the existence of
[3H]-thymidine-labelled neurons in the DG, OB
and visual cortex, as well as mitotic cells in the
SVZ of adult macaque monkeys (Kaplan and
Hinds 1977; Kaplan 1981, 1983; Kaplan and
Bell 1984). Simultaneously, Nottebohm and
colleagues demonstrated that neurogenesis occurs
during adulthood in the brain of songbirds,
namely in the SVZ and in the forebrain, including
in the hippocampus. These SVZ new neurons
migrated to the vocal control nucleus to replace
pre-existent neurons in a process dependent on
synaptic inputs (Goldman and Nottebohm 1983;
Paton and Nottebohm 1984; Nottebohm 1985;
Burd and Nottebohm 1985). Later, Reynolds
and Weiss isolated proliferating cells from the
adult mouse striatum, demonstrating that these
cells have the potential to originate neurons and
astrocytes (Reynolds and Weiss 1992).
By the nineties, the phenotypic identification
of new generated cells was facilitated by the
emergence of new techniques, namely the syn-
thetic thymidine analogue BrdU (5-bromo-2-
0
-deoxyuridine) which is incorporated during the
S-phase of the cell cycle and cell-type specific
markers associated with immunohistochemistry
and confocal microscopy. These techniques
contributed to demonstrate in vivo adult
neurogenesis in mammals, such as in rodents
(Kuhn et al. 1996) and primates (Gould et al.
1998, 1999a; Kornack and Rakic 1999). This
kind of prospective labelling of newborn cells
with BrdU was a huge improvement for adult
mammalian neurogenesis studies, showing its
conservation in almost all mammals, including
in humans, by demonstrating the presence of
newborn neurons in the adult human DG
(Eriksson et al. 1998).
Together, this evidence allowed concluding
that the brain does not have an immutable neuro-
nal composition, with the concept moving
F. F. Ribeiro and S. Xapelli
towards a more plastic brain, where new neurons
are continuously being formed throughout adult-
hood in specific areas.
7.2 The Doubts Behind Adult
Human Neurogenesis
The existence of adult human neurogenesis was
controversial, with great scepticism among the
scientific community. In 2013, Jonas Friesen’s
lab used the retrospective birth dating of cells
with 14C, which constituted an innovative strat-
egy to study neurogenesis in the human adult
brain. This carbon isotype, which resulted from
nuclear bomb tests of the Cold War, was spread in
the atmosphere, and incorporated in the food
through plant photosynthesis and then in animals
eating plants, and finally into human body. In the
human body, mitotic cells integrated 14C into
DNA, thus allowing to determinate cell birth dat-
ing by measuring the concentration of 14C in the
DNA of post-mortem brain. Carbon dating has
demonstrated that neuronal turnover mainly
occurs in hippocampus and striatum, with
700 neurons being added each day into the
human DG. Moreover, this method failed to
reveal neuronal turnover in the OB or in the
cerebral neocortex of the adult human brain
(Bhardwaj et al. 2006; Bergmann et al. 2012;
Spalding et al. 2013; Ernst et al. 2014). However,
the use of carbon dating was also pointed to be
prone to contamination, providing inaccurate
results. Consequently, the occurrence of adult
neurogenesis in the human brain was still not
consensual amongst the scientific community.
In the past 5 years, several studies have
focused in resolving the unsolved mystery of
whether new neurons are formed or not in the
human adult brain. In 2016, Dennis and
colleagues using immunohistochemical methods
failed to demonstrate any continued neurogenesis
in the adult human. They examined cell prolifera-
tion and neurogenesis in 23 individuals aged
0.2–59 years and observed a marked decline in
neurogenesis in both SVZ and SGZ regions in
early childhood. Moreover, microglia were the
only proliferating cells found in both niches and
7 An Overview of Adult Neurogenesis
adjacent parenchyma after 3 years of age (Dennis
et al. 2016). In 2018, Sorrells and colleagues
collected post-mortem tissue from healthy adults
and postoperative hippocampal tissue from
humans with epilepsy. The subjects ranged from
foetuses with 14 gestational weeks to adults with
77 years old. Samples were stained with the fluo-
rescent marker antibodies Ki-67 (nuclear protein
associated with cellular proliferation), SOX1 (Sex
determining region Y-box 1) and SOX2 (Sex
determining region Y-box 2) to identify neural
progenitor cells, and DCX (Doublecortin) and
PSA-NCAM (Polysialylated-neural cell adhesion
molecule) to identify immature neurons. Results
showed that the density of proliferating cells and
young neurons decreased with age, with the latter
reducing from around 1618 cells/mm2 at birth to
12 cells/mm2 by age 7, to 2 cells/mm2 by age
13, and to almost none in the samples of
individuals with more than 18 years old. With
this study, the authors concluded that the recruit-
ment of new neurons to the primate hippocampus
decreases rapidly during the first years of life,
with neurogenesis in the DG being extremely
rare, or extinct, in adult humans (Sorrells et al.
2018).
On the other hand, at the same time, other
studies emerged supporting the existence of
adult human hippocampal neurogenesis. Using
similar immunohistochemical methods, Boldrini
and colleagues studied the DG from post-mortem
healthy humans aged 14–79 years and found that
the total numbers of intermediate neural
progenitors and immature neurons, which were
thousands, were stable across ages. Moreover,
they observed angiogenesis and changes in the
volume of DG with aging. They employed unbi-
ased stereology, which is the gold standard for
counting the number of neurons, and they
concluded that while DG neurogenesis and vol-
ume is unchanged with aging, there is a decline in
quiescent stem cell pools, angiogenesis, and
neuroplasticity (Boldrini et al. 2018). More
recently, Moreno-Jiménez and colleagues
identified thousands of immature neurons
exhibiting variable degrees of maturation along
the differentiation stages of adult hippocampal
neurogenesis in neurologically healthy humans
79
up to the ninth decade of life. In contrast,
neurogenesis declined in the DG of patients with
Alzheimer’s disease with disease progression
(Moreno-Jiménez et al. 2019). Tobin and
colleagues not only reported that hippocampal
neurogenesis is persistent in aged human brains,
through the tenth decade of life, but also that
hippocampal neurogenesis is still present,
although not as significantly, in patients with
mild cognitive impairments and Alzheimer’s dis-
ease (Tobin et al. 2019).
Bioinformatic methods have also been used to
analyse the differential expression of
neurogenesis signature markers in the human hip-
pocampal transcriptome, from prenatal, through
infancy and adolescence, to adulthood. Results
found persistent but minimal hippocampal
neurogenesis in adult humans, suggesting that a
significant number of newborn hippocampal cells
could be glial cells and not neurons (Kumar et al.
2019).
Apart from the DG, Ernst and colleagues
observed that new neurons integrate in the adult
human striatum. They showed that DCX-positive
and PSA-NCAM-positive neuroblasts are present
both in the SVZ and in the adjacent striatum of
control human brains, with these cells being
responsible for the turnover of striatal
interneurons (Ernst et al. 2014). While in rodents,
new neurons generated in the SVZ migrate to the
OB to become interneurons, only entering the
striatum under pathological conditions, such as
in ischemia, in humans SVZ-derived cells mainly
migrate to the adjacent striatum to become
interneurons, and rarely to the OB (Kempermann
2014). Apart from the physiological role, which is
yet unknown, it is thought that striatal adult
neurogenesis may be important in pathological
conditions, such as in cerebral ischemia, where
upregulation of neurogenesis could reduce dam-
age, and psychiatric disorders, where
neurogenesis could enhance susceptibility to ill-
ness or support its treatment (Inta et al. 2015).
Whether and/or how much adult neurogenesis
really occurs in humans is a question of great
importance to understand its functional role in
our species (Ghosh 2019). Importantly, adult
neurogenesis has been widely studied in other
80 F. F. Ribeiro and S. Xapelli

organisms. These findings paved the way for a Weiss 1992; Kilpatrick and Bartlett 1993; Palmer
new research field that, in the last two decades, et al. 1997; Gage et al. 1998).
has been extensively explored and has provided In contrast to what is observed in vitro, in vivo
several insights on the identity of neural stem clonal studies have shown limited NSC self-
cells (NSCs), on the properties and cellular com- renewal capacity with a fast progeny expansion
position of neurogenic niches, the cellular dynam- (Encinas et al. 2011; Calzolari et al. 2015), with a
ics and the migratory behaviour of newly NSC being mostly unilineage, generating
generated cells, as well as the survival, integration neurons, and, sometimes, having the capacity to
and functional relevance of these adult born cells. generate either a neuron or a glial cell (Encinas
et al. 2011; Bonaguidi et al. 2011; Calzolari et al.
2015). Although an adult NSC might be intrinsi-
cally multipotent, displaying the capacity to gen-
7.3 Neural Stem Cells
erate cells from the neuronal and glial lineages,
the niche environment seems to limit, in some
One of the things that was against the acceptance
way, its potential and long-term self-renewal
of adult neurogenesis was the unconceivable idea
capacity. The extracellular environment, such as
that mature neurons, considered to be terminally
the different types of neighbouring cells that
differentiated and unable to re-enter the cell-
secrete or present distinct signals on their plasma
division cycle, could be able to divide to originate
membrane, as well as molecules of the extracellu-
new neurons. The concept of NSC (Kempermann
lar matrix, is essential to establish a difference
and Gage 1999), being both proliferative cells,
between lineage and potential (Götz et al. 2015).
with the capacity to self-renew, and multipotent
Therefore, this will have an impact in cell genesis,
cells, meaning that after exiting the cell cycle
turning this process region-specific. For instance,
initiate differentiation towards a specialized neu-
in the SVZ, the higher number of neuronal line-
ral cell type, was introduced more recently (Hall
age progenitors is located in the lateral wall, with
and Watt 1989; Gage 2000; Temple 2001)
NSCs being more prone for neurogenesis, while
(Fig. 7.1).
oligodendrocyte progenitors are enriched in the
NSCs were first identified (Reynolds and
septal wall, with gliogenesis being more substan-
Weiss 1992) from isolated cells from the striatum
tial in this area (Mizrak et al. 2019). While the
of the adult mouse brain. When cultured in the
lineage of a cell is what a single NSC originates
presence of epidermal growth factor (EGF), these
in vivo, in a conditioned environment, the poten-
isolated cells were able to extensively proliferate
tial corresponds to what a single NSC can do
as non-adherent spherical structures, the
when it is exposed to different environments
neurospheres, which were composed of clonally
(Götz et al. 2015).
derived precursors. These cells were able to give
rise to more neurospheres with the same pheno-
type, thus showing self-renewal capacity. In con-
7.4 Adult Neurogenic Niches
trast, when removing the growth factors from the
medium and providing an adhesion substrate of
In the adult mammalian brain, NSCs are mainly
poly-L-ornithine, neurosphere cells formed a
restricted to specific brain regions, the SVZ in the
pseudomonolayer culture which differentiated
lateral ventricles and the subgranular zone (SGZ)
into astrocytes and neurons, thus exhibiting
in the DG of the hippocampus where constitutive
multipotency (Reynolds and Weiss 1992).
neurogenesis takes place throughout adulthood
The two main properties that are attributed to
(Fig. 7.2). These regions have a specialized and
NSCs are the capacity to self-renew through cell
diverse microenvironment that embeds NSCs, the
division and to generate a differentiated progeny
reason why it is called a niche. In these niches,
of the three neural lineages, namely neurons,
NSCs generate neural progenitor cells (NPCs), or
astrocytes and oligodendrocytes (Reynolds and
transit amplifying progenitors, which rapidly
7 An Overview of Adult Neurogenesis
Fig. 7.1 Neural stem cells
are self-renewing and
multipotent cells with the
capacity to generate
neurons, astrocytes and
oligodendrocytes (from
Ribeiro 2017)
divide and form migrating neuroblasts.
Neuroblasts differentiate into neurons that inte-
grate into the pre-existing circuits. While SGZ
newborn neurons migrate short distances to inte-
grate into the circuits of the DG granule cell layer,
SVZ newborn neurons migrate long distances
towards the OB (Alvarez-Buylla and Lim 2004).
Fig. 7.2 The main neurogenic niches in the adult mam-
malian brain are the subventricular zone (SVZ), in the
lateral ventricles (LV), and the subgranular zone (SGZ),
in the dentate gyrus (DG) of the hippocampus. These
neurogenic niches contain neural stem cells (NSCs) with
the capacity to divide and generate new neurons. Newborn
neurons from SGZ migrate short distances to be integrated
into the circuits of the DG, whereas SVZ newborn neurons
migrate long distances, through the rostral migratory
stream (RMS), towards the olfactory bulb (OB) (from
Ribeiro 2017)
81
The generation of new neurons from the SVZ
and SGZ is possible because adult NSCs are
embedded in a specialized and diverse microen-
vironment that is the niche and that is permissive
for neurogenesis to occur. Apart from NSCs and
NPCs, the neurogenic niche is also composed of
glial, endothelial and immune (as microglia and
macrophages) cells. Giving all this cell diversity,
the neurogenic niche provides an unique milieu
consisting of extracellular matrix, short and long-
ranged signalling factors, cell-to-cell contacts,
which is responsible for tightly regulating NSC
self-renewal and differentiation (Palmer et al.
2000; Alvarez-Buylla and Lim 2004; Ma et al.
2005; Merkle and Alvarez-Buylla 2006).
Vasculature and systemic factors also have a
great impact on adult neurogenesis. The impor-
tance of this relationship has been demonstrated
by heterochronic parabiosis experiments. In this
parabiosis experiments, the blood circulations of
a young and old mice were surgically connected
through the attachment of their skins on the lateral
torso. Heterochronic parabiosis experiments
showed that the presence of young mouse blood
in the circulatory system of an older animal
resulted in increased proliferation of NPCs and
increased adult neurogenesis in the neurogenic
82
niches. Moreover, old heterochronic parabionts
displayed increased vessel volume and cerebral
blood flow comparable to young mice. In con-
trast, adult neurogenesis in the younger animal
was reduced (Villeda et al. 2011; Katsimpardi
et al. 2014).
SVZ NSCs are in contact with the cerebrospi-
nal fluid (CSF) which is produced inside the
ventricles by a highly vascular structure, the cho-
roid plexus (CP). The CP acts as an interface
between the brain and the periphery. Moreover,
many altered factors in the blood have been
shown to influence SVZ neurogenesis by affect-
ing the activated NSCs. Besides not being in
contact with the CSF, DG NSCs have an ample
interaction with the DG vasculature, which starts
from the arterioles of the hippocampal fissure,
passing as capillaries through the granule cell
(GC) layer along the rostro-caudal axis (Ghosh
2019).
Due to differences in the microenvironment of
SGZ and SVZ, self-renewal and differentiation of
NSCs are differently regulated. On the one hand,
self-renewal through symmetric division
represents the fate of a minority of DG NSCs,
with most of them undergoing asymmetric cell
division resulting in one NSCs and one
differentiated cell, either an astrocyte or neuron,
but never an oligodendrocyte under normal
conditions (Encinas et al. 2011; Bonaguidi et al.
2011). On the other hand, self-renewal and differ-
entiation of SVZ NSCs occur through symmetric
cell division. About 20–30% of SVZ NSCs divide
symmetrically to maintain their pool, whereas
most NSCs undergo differentiative symmetric
divisions, undergoing neuronal lineage progres-
sion (Obernier et al. 2018). In addition to this,
adult DG NSCs possess limited self-renewal, pro-
liferation and migration capacities (Seaberg and
van der Kooy 2002; Bull and Bartlett 2005),
when compared to SVZ NSCs. In pathological
conditions, such as in multiple sclerosis, SVZ
NSCs can also give raise to oligodendrocyte pre-
cursor cells (OPCs), thus generating oligoden-
drocytes (Menn et al. 2006).
Apart from these two niches in the adult mam-
malian brain, non-typical NSC niches have also
been postulated where neurogenesis can occur.
F. F. Ribeiro and S. Xapelli
Although more limited, this process has been
observed in neocortex (Gould et al. 1999b;
Dayer et al. 2005; Ohira et al. 2010), amygdala
(Bernier et al. 2002), substantia nigra (Lie et al.
2002; Zhao et al. 2003; Zhao and Janson Lang
2009), hypothalamus (Kokoeva et al. 2005, 2007)
and striatum (Bédard et al. 2006). This evidence
contributes to the idea that the potential of the
brain to generate new neurons is greater than
initially thought. Nevertheless, the neurogenic
process in these non-classic neurogenic areas is
still in the beginning of its characterisation, with
some evidence still controversial (Breunig et al.
2007). Progenitor cells residing in non-classic
neurogenic locations, such as the substantia
nigra, can give rise to neurons when transplanted
into neurogenic niches, but the contrary is not
possible. In in vitro conditions, these restricted
progenitors can give rise to both neurons and glia.
These experiments suggest that the niches have
the capacity to provide both ‘restrictive’ and
‘instructive’ cues to a neural progenitor for a
certain fate, depending on its location and
requirements (Ghosh 2019).
7.5 The Subventricular Zone (SVZ)
Neurogenic Niche
The SVZ is the major neurogenic niche of the
adult mammalian brain, and it is localised along
the ependymal cell layer that lines the walls of the
lateral ventricles (Fig. 7.3) (Doetsch et al. 1997).
SVZ contains a subpopulation of quiescent cells
with astrocyte-like characteristics, the type B
cells, which function as NSCs (Morshead et al.
1994; Nam and Benezra 2009).
In the SVZ niche, the type B cells are slow
dividing cells. These cells are in direct contact
with the CSF, which fills the lumen of the lateral
ventricles, through a specialized apical process
surrounded by ependymal cells (Shen et al.
2008; Falcão et al. 2012; Fuentealba et al.
2012). The adult CP, present in the ventricle
lumen, expresses and secretes to the CSF not
only numerous trophic factors but also cytokines,
which can influence the behaviour of SVZ NSCs
and NPCs, by modulating their self-renewal
7 An Overview of Adult Neurogenesis 83

Fig. 7.3 Schematic

representation of the adult
subventricular zone (SVZ)
neurogenic niche. SVZ
lines the lateral walls of the
lateral ventricles and is
comprised of three main
cell types: the multipotent
type B NSCs that give rise
to type C cells (fast dividing
transient amplifying cells)
that, in turn, generate type
A neuroblasts. Type B cells
interact basally with blood
vessels and apically with
the cerebrospinal fluid
(CSF). The composition of
the CSF is modified by the
choroid plexus, a thin
vascularized membrane
mainly composed by
epithelial cells, which
secretes several cytokines
and trophic factors to the
CSF (from Armada-
Moreira et al. 2015)

capacity, proliferation and differentiation (Falcão neuroblasts commonly migrate in a saltatory

et al. 2012; Fuentealba et al. 2012). On the basal
side, they are also in contact with blood vessels
through the extension of a basal process
(Mirzadeh et al. 2008). Type B NSCs give rise
to intermediate progenitors (IPCs), or fast divid-
ing transient amplifying progenitors, known as
type C cells (Fig. 7.4) (Doetsch et al. 1999).
In rodents, type C cells mostly generate
neuroblasts, or type A cells, which migrate
along the rostral migratory stream (RMS) towards
the OB where they differentiate into olfactory
interneurons and are integrated into the neuronal
circuitry involved in olfaction (Altman 1969;
Luskin 1993; Lois and Alvarez-Buylla 1994;
Carleton et al. 2003; Belluzzi et al. 2003).
Neuroblasts migrate tangentially along the
RMS forming chains surrounded by a meshwork
of specialized GFAP-positive astrocytes that form
a tubular structure longitudinally linking the SVZ
to the OB (Lois et al. 1996; Peretto et al. 1997).
Migrating neuroblasts have a bipolar morphol-
ogy, displaying a long leading process and a
short trailing process. Individual bipolar-shaped
manner which involves three steps: leading-
process extension; swelling formation and
centrosomal migration; and somal translocation
(Kaneko et al. 2017). Neuroblasts express DCX,
as well as PSA-NCAM and N-cadherin, which
enable them to migrate along one another through
homophilic interaction, thus forming elongated
clusters called chains (Rousselot et al. 1995;
Lois et al. 1996; Yagita et al. 2009). After a
brain injury, apart from individual migration, sim-
ilar chain formations are observed for neuroblasts
migrating towards injured areas (Zhang et al.
2009).
Externally to the chains of neuroblasts, there
are astrocytic tunnels called glial tubes that sur-
round and separate the chains of neuroblasts from
the nearby brain parenchyma, which helps to
maintain the long-distance migration route
(Kaneko et al. 2010). This astroglial matrix in
the RMS is composed by specialized astrocytes
that overexpress non-soluble factors such as
extracellular matrix and cell adhesion molecules,
and promote neuronal migration in a contact-
84
Fig. 7.4 Adult neurogenesis from the subventricular zone
of the lateral ventricle to the olfactory bulb in rodents.
Schematic representation of adult subventricular zone
(SVZ) neurogenesis in five developmental stages: (1) acti-
vation of radial glia-like cells in the SVZ in the lateral
ventricle (LV) and proliferation of transient amplifying
progenitors; (2) fate specification with the generation of
neuroblasts/type A cells; (3) chain tangential migration of
neuroblasts within the rostral migratory stream (RMS);
dependent manner (García-Marqués et al. 2010).
These astrocytes also modulate neuroblast migra-
tion by secreting or trapping soluble factors
(Mason et al. 2001; Bolteus and Bordey 2004;
Snapyan et al. 2009). Apart from this, blood
F. F. Ribeiro and S. Xapelli
(4) radial migration of immature neurons in the olfactory
bulb (OB); (5) synaptic integration and maturation of
granule cells and periglomerular neurons (PG) in the olfac-
tory bulb. Also shown is the expression of stage-specific
markers during SVZ-OB adult neurogenesis. GFAP, glial
fibrillary acidic protein; Dlx2, distal-less homeobox 2;
DCX, doublecortin; PSA-NCAM, polysialylated neural
cell adhesion molecule; NeuN, neuronal nuclei (from
Ribeiro 2017)
vessels have a very important function in rostral
migration by retaining neuroblasts in the migra-
tory stream, by serving as a physical substrate
towards the OB, or even to an injured area, as
well as by providing and regulating molecular
7 An Overview of Adult Neurogenesis
cues (Snapyan et al. 2009; Whitman et al. 2009;
Grade et al. 2013).
The OB is a complex and multi-layered struc-
ture. The main OB is composed of the glomerular
layer, external plexiform layer, mitral cell layer,
internal plexiform layer and granule cell layer
(Lazarini and Lledo 2011). Although yet not
clear, it is thought that OB neurogenesis may be
used for plasticity, olfactory discrimination, and
olfactory learning and memory (Lledo and Valley
2016). In the OB, there is a continuous cell turn-
over, with newly generated granule cells thought
to replace older ones. Importantly, cell death
occurs through the whole SVZ-OB pathway,
being more marked in the OB (Biebl et al. 2000;
Petreanu and Alvarez-Buylla 2002).
Directional migration of neuroblasts towards
the OB is regulated by several signalling
molecules that mediate not only the interaction
between type A cells, astrocytes and blood
vessels, but also the interaction of these cells
with the surrounding tissues, which make possi-
ble this long-way migration process. Among
them are attractive cues, such as netrin-1 and
prokineticin 2, that are produced by the OB (Liu
and Rao 2003; Prosser et al. 2007); repulsive
cues, like Slit proteins, that are expressed in the
septum (Hu and Rutishauser 1996; Hu 1999) and
CP (Wu et al. 1999; Nguyen-Ba-Charvet et al.
2004). Other signalling molecules known to con-
trol neuroblast migration are ephrins (Conover
et al. 2000), integrins (Murase and Horwitz
2002), growth factors such as glial cell line-
derived neurotrophic factor (GDNF) (Paratcha
et al. 2006), brain-derived neurotrophic factor
(BDNF) (Chiaramello et al. 2007; Snapyan et al.
2009) and vascular endothelial growth factor
(VEGF) (Wittko et al. 2009), and gamma-
aminobutyric acid (GABA) (Bolteus and Bordey
2004; Snapyan et al. 2009).
Once arrived to the OB, neuroblasts detach
from rostral migratory chains and radially migrate
into the OB, with the majority of them using
blood vessels in a process called vasophilic neu-
ronal migration (Bovetti et al. 2007). SVZ adult-
born cells differentiate into olfactory
interneurons, with the majority migrating towards
85
the granule cell layer to become granule cells
(Winner et al. 2002; Carleton et al. 2003) and
the others to the glomerular layer to become
periglomerular interneurons (Belluzzi et al.
2003). These newborn cells differentiate more
slowly into periglomerular interneurons, with a
delay of more than 1 month when compared to
the granule cells (Winner et al. 2002).
Neuroblasts differentiate into GABAergic
interneurons, such as calretinin+ or calbindin+,
into dopaminergic tyrosine hydroxylase+ neurons
(Batista-Brito et al. 2008), or into glutamatergic
neurons (Brill et al. 2009). These neurons estab-
lish dendro-dendritic synapses with the tufted
cells (Abrous et al. 2005) and begin a maturation
process by receiving GABAergic and
glutamatergic synaptic inputs (Belluzzi et al.
2003). A large amount of SVZ-derived cells that
arrive to the OB firstly differentiate into GABA-
containing granule cells, followed by the forma-
tion of glutamatergic synaptic contacts (Carleton
et al. 2003; Belluzzi et al. 2003). At earlier stages
of differentiation in OB, newborn neurons remain
unable to fire action potentials (Carleton et al.
2003). This delay in excitability may be important
to protect circuits from uncontrolled neurotrans-
mitter release and neural network disruption
(Lledo et al. 2006; Pignatelli and Belluzzi
2010). The majority of cells that disperse
throughout the granule cell layer develop into
GABAergic granule cells, while a small percent-
age that move into the periglomerular region
develop mainly into interneurons of either
GABAergic or dopaminergic phenotypes (Kuhn
et al. 2005b).
Within the SVZ, thousands of cells are born
each day (Winner et al. 2002). The majority of
new generated cells, whether periglomerular or
granule cells, that reach the OB die, with around
40% of newly born cells surviving up to 19 months
(Petreanu and Alvarez-Buylla 2002; Winner et al.
2002). These data suggest that OB neurogenesis
promotes an overproduction and turnover of
young neurons, rather than replacing the old
ones. Per month, only around 6–10% of the
existing cellular population within the OB granule
cell layer is replaced (Winner et al. 2002). The
86
survival of neurons is largely influenced by the
odour environment (Rochefort et al. 2002;
Petreanu and Alvarez-Buylla 2002; Yamaguchi
and Mori 2005). Enriched odour exposure
increases the number of newborn neurons in the
adult OB and improves odour memory (Rochefort
et al. 2002). Therefore, the system may function
by overproducing potential neurons which will be
selected through an experience-dependent
process.
The route newborn SVZ neurons take in direc-
tion to OB is not fixed. In fact, although
neuroblasts migrate to the OB in healthy
conditions, they can also be called upon demand.
For instance, when a lesion occurs in the cerebral
cortex, corpus callosum or striatum, newborn
neuroblasts can migrate from the SVZ to the
injured areas and form functional neurons
(Bernier et al. 2002; Arvidsson et al. 2002; Parent
et al. 2002; Bédard et al. 2006; Yamashita et al.
2006). Interestingly, neuroblasts migrating in an
ischemic striatum display higher exploratory
behaviour and longer stationary periods than
cells migrating in the RMS (Grade et al. 2013).
Some reports show that, in addition to the germi-
nal zone located in the walls of the lateral
ventricles facing the striatum (lateral walls),
other areas also have cells that in culture possess
stem cell-like properties, as it is the case of parts
of the lateral ventricular wall facing the septum,
as well as the whole rostral migratory stream
(RMS) (Doetsch et al. 1999; Gritti et al. 2002).
These stem cell-like properties were also
observed in vivo (Merkle et al. 2007), showing
to possess neurogenic potential (Gritti et al. 2002;
Aguirre and Gallo 2004). Moreover, several lines
of evidence also suggest that the adult OB is a
source of local NSCs (Pagano et al. 2000; Liu and
Martin 2003; Giachino and Taylor 2009;
Moreno-Estellés et al. 2012).
Finally, a minority of type C cells enter in the
oligodendroglial lineage. They produce oligoden-
drocyte progenitor cells (OPCs), which migrate
radially out of the SVZ into the surrounding cor-
tex and white matter where they differentiate into
oligodendrocytes (Suzuki and Goldman 2003;
Menn et al. 2006).
F. F. Ribeiro and S. Xapelli
7.6 The Subgranular Zone (SGZ)
Neurogenic Niche
The other neurogenic niche in the adult mamma-
lian brain where NSCs reside is the SGZ of the
hippocampal DG. The DG is a V-shaped structure
that is composed of three layers (Fig. 7.5): the
molecular layer, which is mainly occupied by the
dendrites of the dentate granule cells and the
fibres of the perforant path coming from the ento-
rhinal cortex; the granule cell layer, which is
mainly made by densely packed granule cells
with a 6–8 neuron-thickness and encloses the
third layer; and the hilus or the polymorphic cell
layer, which is a region composed of a variety of
cell types, including the mossy cells (Amaral
et al. 2007). The SGZ is a thin band of cells that
is located at the border between the inner granule
cell layer and the hilus of the DG, which contains
NSCs and NPCs, as well as the bodies of
parvalbumin-expressing interneurons, i.e. the
GABAergic basket cells (Toni and Schinder
2016).
NSCs that reside in the SGZ are called type
1 cells and are radial glial-like precursor cells
(Fig. 7.6). They display astrocytic electrophysio-
logical properties and express the astrocytic
marker GFAP, as well as other markers such as
the intermediate filament nestin and Sox2 (Seri
et al. 2001; Filippov et al. 2003; Fukuda et al.
2003; Steiner et al. 2006). With the cell body in
the SGZ, these early precursor cells have pro-
cesses that allow them to be in permanent contact
not only with the molecular layer, through an
extending apical process, but also with blood
vessels (Filippov et al. 2003; Fukuda et al.
2003). Type 1 cells are early progenitor cells
that rarely divide. Responsive to tonic GABA
from local interneurons, these cells are
maintained in quiescence by the GABA released
from GABAergic basket cells (Song et al. 2012).
When type 1 cells exit the quiescent state, they
generate intermediate progenitor cells, the type
2 cells (Fig. 7.6). Unlike the former, type 2 cells
display a high proliferative activity that allows the
SGZ pool of precursor cells to expand. Moreover,
they have a simpler morphology, without radial
7 An Overview of Adult Neurogenesis
Fig. 7.5 Structure and circuits of the hippocampus. Sche-
matic representation of a transversal slice of the hippocam-
pus depicting the dentate gyrus (DG), cornus ammonis
(CA)3 and CA1. The DG is composed of three layers:
the molecular layer, which is mainly occupied by the
dendrites of the dentate granule cells and the fibres of the
perforant path; the granule cell layer, made by densely
packed granule cells and the hilus, mostly composed of
basket and mossy cells, apart from other cell types. In the
87
border between the inner granule cell layer and the hilus of
the DG is the subgranular zone (SGZ), containing NSCs
and NPCs. Granule cells are synaptically connected via the
so-called trisynaptic circuit. The main circuit input is the
performant path, composed of axons coming from the
lateral and the medial entorhinal cortex. Then, granule
neurons project mossy fibres to CA3 pyramidal neurons,
which then project Schaffer collaterals to CA1 pyramidal
neurons (from Ribeiro 2017)
88
Fig. 7.6 Cell types and the main markers expressed dur-
ing the process of adult neurogenesis in the dentate gyrus
of the hippocampus. Schematic representation of adult
hippocampal neurogenesis in five developmental stages:
(1) activation of quiescent radial glia-like cell in the
subgranular zone (SGZ) and proliferation of non-radial
precursor and intermediate progenitors; (2) fate specifica-
tion with the generation of neuronal precursors/type 2b
cells; (3) short-distance radial migration of neuroblasts/
but horizontally oriented short processes (Fukuda
et al. 2003). Two distinct subsets of type 2 cells
can be identified, the type 2a and the type 2b.
Similarly to type 1 cells, type 2a cells still express
glial markers and give rise to a subpopulation of
type 2b lacking GFAP and expressing the neuro-
nal markers DCX and PSA-NCAM (Fukuda et al.
2003; Steiner et al. 2006). Type 3 cells are then
generated from type 2b cells and have little
F. F. Ribeiro and S. Xapelli
type 3 cells to the granule cell layer; (4) axons and dendrite
development of immature neurons and (5) adult-born den-
tate granule cell synaptic integration. The expressed cell
stage-specific markers are also represented during adult
hippocampal neurogenesis. GFAP, glial fibrillary acidic
protein; DCX, doublecortin; PSA-NCAM, polysialylated
neural cell adhesion molecule; NeuN, neuronal nuclei
(from Ribeiro 2017)
proliferative activity. They lack nestin and glial
markers and express neuronal lineage markers,
including DCX and PSA-NCAM. With a great
diversity of morphologies, type 3 cells can have
either the radial or tangential process. Further-
more, these cells migrate radially short distances
to the granule cell layer where they exit cell cycle
to become granule neurons that express the
postmitotic neuronal markers neuronal nuclei
7 An Overview of Adult Neurogenesis
(NeuN) and the transient calcium-binding protein
calretinin, which is later replaced by the mature
neuronal marker calbindin (Brandt et al. 2003).
Once they arrive to their destination in the granule
cell layer, newborn neurons continue to maturate
their processes. Initially they project the axon
through the mossy fibre tract in the hilar region
to contact hippocampal CA3 and then extend
their dendrites towards the molecular layer,
establishing synaptic contacts with axons from
the entorhinal cortex (Fig. 7.5) (Hastings and
Gould 1999; Zhao et al. 2006; Sun et al. 2013).
Hippocampal activity regulates neurogenesis
in the adult DG. Type 2 cells, as type 1 cells,
also respond to GABA. However, while GABA
maintains type 1 cells quiescent, type 2 cells start
to be responsive to active GABAergic excitatory
inputs from the hippocampal circuitry, which are
responsible for promoting neuronal differentia-
tion (Tozuka et al. 2005) and to promote dendritic
development and synaptic integration of young
newborn neurons (Ge et al. 2006). In rodents, by
the time glutamatergic synapses are formed, new
neurons present a very high synaptic plasticity
during the “critical period” of 4–6 weeks of the
cell age, with immature neurons displaying a
lower threshold than mature neurons to induce
long-term potentiation (LTP) (Wang et al. 2000;
Schmidt-Hieber et al. 2004; Ge et al. 2007).
These adult-born neurons might be preferentially
recruited into hippocampal neural circuits to
mediate novelty recognition, contextual fear con-
ditioning, spatial information processing and
memory formation (Gu et al. 2013). The critical
period finishes when new neurons start to be
inhibited by the surrounding interneurons, like
mature neurons, when GABAergic contacts
switch to inhibitory (Espósito at al. 2005; Ge
et al. 2006).
During the postmitotic phase, there is a dra-
matic decrease in the number of new neurons,
through apoptosis (Biebl et al. 2000; Kuhn et al.
2005a). In fact, most of the cells are eliminated
before establishing synaptic contacts with the tar-
get area in CA3 or receiving correct dendritic
input from the entorhinal cortex in the molecular
layer of the DG. From the 10,000 cells that are
produced every day, just a small percentage
89
survives and becomes permanently integrated in
the granule cell layer.
Adult hippocampal neurogenesis is important
for several hippocampal-dependent functions.
Numerous studies over the past years have
associated hippocampal neurogenesis with con-
text learning and spatial memory, as well as emo-
tional behaviours related to stress, anxiety and
depression, and also with memory formation,
consolidation and decay, pattern separation and
discrimination behaviours, addiction, and atten-
tion (Snyder and Drew 2020).
7.7 Conclusion
Over the last years, there has been a growing
increase in the number of reports highlighting
the existence of adult neurogenesis both in animal
models and in humans. In fact, adult-generated
neurons have important functional contributions
to neural plasticity and cognition across human
lifespan. Importantly, the prospect of using NSC
as regenerative therapies is very promising.
Therefore, the potential actions of pharmacologi-
cal agents such as neurotrophins used to modulate
NSC activity are highly relevant and will be
discussed in the second part of this chapter.
Acknowledgements This work was supported by
IF/01227/2015 project funded by Fundação para a Ciência
e a Tecnologia (FCT). F.F.R. (IMM/CT/35-2018) received
a fellowship from FCT.
References
Abrous DN, Koehl M, Le Moal M (2005) Adult
neurogenesis: from precursors to network and physiol-
ogy. Physiol Rev 85:523–569. https://doi.org/10.1152/
physrev.00055.2003
Aguirre A, Gallo V (2004) Postnatal neurogenesis and
gliogenesis in the olfactory bulb from
NG2-expressing progenitors of the subventricular
zone. J Neurosci 24:10530–10541. https://doi.org/10.
1523/JNEUROSCI.3572-04.2004
Altman J (1962) Are new neurons formed in the brains of
adult mammals? Science 135:1127–1128
Altman J (1963) Autoradiographic investigation of cell
proliferation in the brains of rats and cats. Anat Rec
145:573–591
90
Altman J (1969) Autoradiographic and histological studies
of postnatal neurogenesis. IV. Cell proliferation and
migration in the anterior forebrain, with special refer-
ence to persisting neurogenesis in the olfactory bulb. J
Comp Neurol 137:433–457. https://doi.org/10.1002/
cne.901370404
Altman J, Das GD (1965) Autoradiographic and histologi-
cal evidence of postnatal hippocampal neurogenesis in
rats. J Comp Neurol 124:319–335
Altman J, Das GD (1966) Autoradiographic and histologi-
cal studies of postnatal neurogenesis. I. A longitudinal
investigation of the kinetics, migration and transforma-
tion of cells incorporating tritiated thymidine in neo-
nate rats, with special reference to postnatal
neurogenesis in some brain regions. J Comp Neurol
126:337–389. https://doi.org/10.1002/cne.901260302
Altman J, Das GD (1967) Postnatal neurogenesis in the
guinea-pig. Nature 214:1098–1101
Alvarez-Buylla A, Lim DA (2004) For the long run:
maintaining germinal niches in the adult brain. Neuron
41:683–686
Amaral DG, Scharfman HE, Lavenex P (2007) The den-
tate gyrus: fundamental neuroanatomical organization
(dentate gyrus for dummies). Prog Brain Res
163:3–22. https://doi.org/10.1016/S0079-6123(07)
63001-5
Armada-Moreira A, Ribeiro F, Sebastião A, Xapelli S
(2015) Neuroinflammatory modulators of oligoden-
drogenesis. Neuroimmunol Neuroinflamm 2:263.
https://doi.org/10.4103/2347-8659.167311
Arvidsson A, Collin T, Kirik D et al (2002) Neuronal
replacement from endogenous precursors in the adult
brain after stroke. Nat Med 8:963–970. https://doi.org/
10.1038/nm747
Batista-Brito R, Close J, Machold R, Fishell G (2008) The
distinct temporal origins of olfactory bulb interneuron
subtypes. J Neurosci 28:3966–3975. https://doi.org/10.
1523/JNEUROSCI.5625-07.2008
Bédard A, Gravel C, Parent A (2006) Chemical character-
ization of newly generated neurons in the striatum of
adult primates. Exp Brain Res 170:501–512. https://
doi.org/10.1007/s00221-005-0233-5
Belluzzi O, Benedusi M, Ackman J, LoTurco JJ (2003)
Electrophysiological differentiation of new neurons in
the olfactory bulb. J Neurosci 23:10411–10418
Bergmann O, Liebl J, Bernard S et al (2012) The age of
olfactory bulb neurons in humans. Neuron
74:634–639. https://doi.org/10.1016/j.neuron.2012.
03.030
Bernier PJ, Bedard A, Vinet J et al (2002) Newly
generated neurons in the amygdala and adjoining cor-
tex of adult primates. Proc Natl Acad Sci USA
99:11464–11469. https://doi.org/10.1073/pnas.
172403999
Bhardwaj RD, Curtis MA, Spalding KL et al (2006) Neo-
cortical neurogenesis in humans is restricted to devel-
opment. Proc Natl Acad Sci USA 103:12564–12568.
https://doi.org/10.1073/pnas.0605177103
F. F. Ribeiro and S. Xapelli
Biebl M, Cooper CM, Winkler J, Kuhn HG (2000) Analy-
sis of neurogenesis and programmed cell death reveals
a self-renewing capacity in the adult rat brain. Neurosci
Lett 291:17–20
Boldrini M, Fulmore CA, Tartt AN, et al (2018) Human
hippocampal neurogenesis persists throughout aging.
Cell Stem Cell 22:589–599.e5. https://doi.org/10.
1016/j.stem.2018.03.015
Bolteus AJ, Bordey A (2004) GABA release and uptake
regulate neuronal precursor migration in the postnatal
subventricular zone. J Neurosci 24:7623–7631. https://
doi.org/10.1523/JNEUROSCI.1999-04.2004
Bonaguidi MA, Wheeler MA, Shapiro JS et al (2011) In
vivo clonal analysis reveals self-renewing and
multipotent adult neural stem cell characteristics. Cell
145:1142–1155. https://doi.org/10.1016/j.cell.2011.
05.024
Bovetti S, Hsieh Y-C, Bovolin P et al (2007) Blood vessels
form a scaffold for neuroblast migration in the adult
olfactory bulb. J Neurosci 27:5976–5980. https://doi.
org/10.1523/JNEUROSCI.0678-07.2007
Brandt MD, Jessberger S, Steiner B et al (2003) Transient
calretinin expression defines early postmitotic step of
neuronal differentiation in adult hippocampal
neurogenesis of mice. Mol Cell Neurosci 24:603–613
Breunig JJ, Arellano JI, Macklis JD, Rakic P (2007)
Everything that glitters isn’t gold: a critical review of
postnatal neural precursor analyses. Cell Stem Cell
1:612–627. https://doi.org/10.1016/j.stem.2007.11.
008
Brill MS, Ninkovic J, Winpenny E et al (2009) Adult
generation of glutamatergic olfactory bulb
interneurons. Nat Neurosci 12:1524–1533. https://doi.
org/10.1038/nn.2416
Bull ND, Bartlett PF (2005) The adult mouse hippocampal
progenitor is neurogenic but not a stem cell. J Neurosci
25:10815–10821. https://doi.org/10.1523/
JNEUROSCI.3249-05.2005
Burd GD, Nottebohm F (1985) Ultrastructural characteri-
zation of synaptic terminals formed on newly
generated neurons in a song control nucleus of the
adult canary forebrain. J Comp Neurol 240:143–152.
https://doi.org/10.1002/cne.902400204
Calzolari F, Michel J, Baumgart EV et al (2015) Fast
clonal expansion and limited neural stem cell self-
renewal in the adult subependymal zone. Nat Neurosci
18:490–492. https://doi.org/10.1038/nn.3963
Carleton A, Petreanu LT, Lansford R et al (2003) Becom-
ing a new neuron in the adult olfactory bulb. Nat
Neurosci 6:507–518. https://doi.org/10.1038/nn1048
Chiaramello S, Dalmasso G, Bezin L et al (2007) BDNF/
TrkB interaction regulates migration of SVZ precursor
cells via PI3-K and MAP-K signalling pathways. Eur J
Neurosci 26:1780–1790. https://doi.org/10.1111/j.
1460-9568.2007.05818.x
Conover JC, Doetsch F, Garcia-Verdugo JM et al (2000)
Disruption of Eph/ephrin signaling affects migration
and proliferation in the adult subventricular zone. Nat
Neurosci 3:1091–1097. https://doi.org/10.1038/80606
7 An Overview of Adult Neurogenesis
Dayer AG, Cleaver KM, Abouantoun T, Cameron HA
(2005) New GABAergic interneurons in the adult neo-
cortex and striatum are generated from different
precursors. J Cell Biol 168:415–427. https://doi.org/
10.1083/jcb.200407053
Dennis CV, Suh LS, Rodriguez ML et al (2016) Human
adult neurogenesis across the ages: an immunohisto-
chemical study. Neuropathol Appl Neurobiol
42:621–638. https://doi.org/10.1111/nan.12337
Doetsch F, García-Verdugo JM, Alvarez-Buylla A (1997)
Cellular composition and three-dimensional organiza-
tion of the subventricular germinal zone in the adult
mammalian brain. J Neurosci 17:5046–5061
Doetsch F, Caillé I, Lim DA et al (1999) Subventricular
zone astrocytes are neural stem cells in the adult mam-
malian brain. Cell 97:703–716
Encinas JM, Michurina TV, Peunova N et al (2011)
Division-coupled astrocytic differentiation and
age-related depletion of neural stem cells in the adult
hippocampus. Cell Stem Cell 8:566–579. https://doi.
org/10.1016/j.stem.2011.03.010
Eriksson PS, Perfilieva E, Björk-Eriksson T et al (1998)
Neurogenesis in the adult human hippocampus. Nat
Med 4:1313–1317. https://doi.org/10.1038/3305
Ernst A, Alkass K, Bernard S et al (2014) Neurogenesis in
the striatum of the adult human brain. Cell
156:1072–1083. https://doi.org/10.1016/j.cell.2014.
01.044
Falcão AM, Marques F, Novais A et al (2012) The path
from the choroid plexus to the subventricular zone: go
with the flow! Front Cell Neurosci 6:34. https://doi.
org/10.3389/fncel.2012.00034
Filippov V, Kronenberg G, Pivneva T et al (2003) Sub-
population of nestin-expressing progenitor cells in the
adult murine hippocampus shows electrophysiological
and morphological characteristics of astrocytes. Mol
Cell Neurosci 23:373–382
Fuentealba LC, Obernier K, Alvarez-Buylla A (2012)
Adult neural stem cells bridge their niche. Cell Stem
Cell 10:698–708. https://doi.org/10.1016/j.stem.2012.
05.012
Fukuda S, Kato F, Tozuka Y et al (2003) Two distinct
subpopulations of nestin-positive cells in adult mouse
dentate gyrus. J Neurosci 23:9357–9366
Gage FH (2000) Mammalian neural stem cells. Science
287:1433–1438
Gage FH, Kempermann G, Palmer TD et al (1998)
Multipotent progenitor cells in the adult dentate
gyrus. J Neurobiol 36:249–266
García-Marqués J, Carlos JAD, Greer CA, López-
Mascaraque L (2010) Different astroglia permissivity
controls the migration of olfactory bulb interneuron
precursors. Glia 58:218–230. https://doi.org/10.1002/
glia.20918
Ge S, Goh ELK, Sailor KA et al (2006) GABA regulates
synaptic integration of newly generated neurons in the
adult brain. Nature 439:589–593. https://doi.org/10.
1038/nature04404
91
Ge S, Yang C-H, Hsu K-S et al (2007) A critical period for
enhanced synaptic plasticity in newly generated
neurons of the adult brain. Neuron 54:559–566.
https://doi.org/10.1016/j.neuron.2007.05.002
Ghosh HS (2019) Adult neurogenesis and the promise of
adult neural stem cells. J Exp Neurosci 13. https://doi.
org/10.1177/1179069519856876
Giachino C, Taylor V (2009) Lineage analysis of quiescent
regenerative stem cells in the adult brain by genetic
labelling reveals spatially restricted neurogenic niches
in the olfactory bulb. Eur J Neurosci 30:9–24. https://
doi.org/10.1111/j.1460-9568.2009.06798.x
Goldman SA, Nottebohm F (1983) Neuronal production,
migration, and differentiation in a vocal control
nucleus of the adult female canary brain. Proc Natl
Acad Sci USA 80:2390–2394
Götz M, Sirko S, Beckers J, Irmler M (2015) Reactive
astrocytes as neural stem or progenitor cells: in vivo
lineage, In vitro potential, and genome-wide expres-
sion analysis. Glia 63:1452–1468. https://doi.org/10.
1002/glia.22850
Gould E, Tanapat P, McEwen BS et al (1998) Proliferation
of granule cell precursors in the dentate gyrus of adult
monkeys is diminished by stress. Proc Natl Acad Sci
USA 95:3168–3171
Gould E, Reeves AJ, Fallah M et al (1999a) Hippocampal
neurogenesis in adult Old World primates. Proc Natl
Acad Sci USA 96:5263–5267
Gould E, Reeves AJ, Graziano MS, Gross CG (1999b)
Neurogenesis in the neocortex of adult primates. Sci-
ence 286:548–552
Grade S, Weng YC, Snapyan M et al (2013) Brain-derived
neurotrophic factor promotes vasculature-associated
migration of neuronal precursors toward the ischemic
striatum. PLoS One 8:e55039. https://doi.org/10.1371/
journal.pone.0055039
Gritti A, Bonfanti L, Doetsch F et al (2002) Multipotent
neural stem cells reside into the rostral extension and
olfactory bulb of adult rodents. J Neurosci 22:437–445
Gu Y, Janoschka S, Ge S (2013) Neurogenesis and hippo-
campal plasticity in adult brain. Curr Top Behav
Neurosci 15:31–48. https://doi.org/10.1007/7854_
2012_217
Hall PA, Watt FM (1989) Stem cells: the generation and
maintenance of cellular diversity. Development
106:619–633
Hastings NB, Gould E (1999) Rapid extension of axons
into the CA3 region by adult-generated granule cells. J
Comp Neurol 413:146–154
Hu H (1999) Chemorepulsion of neuronal migration by
Slit2 in the developing mammalian forebrain. Neuron
23:703–711
Hu H, Rutishauser U (1996) A septum-derived
chemorepulsive factor for migrating olfactory interneu-
ron precursors. Neuron 16:933–940
Inta D, Cameron HA, Gass P (2015) New neurons in the
adult striatum: from rodents to humans. Trends
Neurosci 38:517–523. https://doi.org/10.1016/j.tins.
2015.07.005
92
Kaneko N, Marín O, Koike M et al (2010) New neurons
clear the path of astrocytic processes for their rapid
migration in the adult brain. Neuron 67:213–223.
https://doi.org/10.1016/j.neuron.2010.06.018
Kaneko N, Sawada M, Sawamoto K (2017) Mechanisms
of neuronal migration in the adult brain. J Neurochem
141:835–847. https://doi.org/10.1111/jnc.14002
Kaplan MS (1981) Neurogenesis in the 3-month-old rat
visual cortex. J Comp Neurol 195:323–338. https://doi.
org/10.1002/cne.901950211
Kaplan MS (1983) Proliferation of subependymal cells in
the adult primate CNS: differential uptake of DNA
labelled precursors. J Hirnforsch 24:23–33
Kaplan MS, Bell DH (1984) Mitotic neuroblasts in the
9-day-old and 11-month-old rodent hippocampus. J
Neurosci 4:1429–1441
Kaplan MS, Hinds JW (1977) Neurogenesis in the adult
rat: electron microscopic analysis of light
radioautographs. Science 197:1092–1094
Katsimpardi L, Litterman NK, Schein PA et al (2014)
Vascular and neurogenic rejuvenation of the aging
mouse brain by young systemic factors. Science
344:630–634. https://doi.org/10.1126/science.
1251141
Kempermann G (2014) Off the beaten track: new neurons
in the adult human striatum. Cell 156:870–871. https://
doi.org/10.1016/j.cell.2014.02.027
Kempermann G, Gage FH (1999) New nerve cells for the
adult brain. Sci Am 280:48–53
Kilpatrick TJ, Bartlett PF (1993) Cloning and growth of
multipotential neural precursors: requirements for pro-
liferation and differentiation. Neuron 10:255–265
Kokoeva MV, Yin H, Flier JS (2005) Neurogenesis in the
hypothalamus of adult mice: potential role in energy
balance. Science 310:679–683. https://doi.org/10.
1126/science.1115360
Kokoeva MV, Yin H, Flier JS (2007) Evidence for consti-
tutive neural cell proliferation in the adult murine
hypothalamus. J Comp Neurol 505:209–220. https://
doi.org/10.1002/cne.21492
Kornack DR, Rakic P (1999) Continuation of
neurogenesis in the hippocampus of the adult macaque
monkey. Proc Natl Acad Sci USA 96:5768–5773
Kuhn HG, Dickinson-Anson H, Gage FH (1996)
Neurogenesis in the dentate gyrus of the adult rat:
age-related decrease of neuronal progenitor prolifera-
tion. J Neurosci 16:2027–2033
Kuhn HG, Biebl M, Wilhelm D et al (2005a) Increased
generation of granule cells in adult Bcl-2-
overexpressing mice: a role for cell death during
continued hippocampal neurogenesis. Eur J Neurosci
22:1907–1915. https://doi.org/10.1111/j.1460-9568.
2005.04377.x
Kuhn HG, Cooper-Kuhn C, Eriksson P, Nilsson M
(2005b) Signals regulating neurogenesis in the adult
olfactory bulb. Chem Senses 30:i109–i110. https://doi.
org/10.1093/chemse/bjh138
Kumar A, Pareek V, Faiq MA et al (2019) Transcriptomic
analysis of the neurogenesis signature suggests
F. F. Ribeiro and S. Xapelli
continued but minimal neurogenesis in the adult
human hippocampus. bioRxiv. https://doi.org/10.
1101/664995
Lazarini F, Lledo P-M (2011) Is adult neurogenesis essen-
tial for olfaction? Trends Neurosci 34:20–30. https://
doi.org/10.1016/j.tins.2010.09.006
Lie DC, Dziewczapolski G, Willhoite AR et al (2002) The
adult substantia nigra contains progenitor cells with
neurogenic potential. J Neurosci 22:6639–6649.
https://doi.org/10.1523/JNEUROSCI.22-15-06639.
2002
Liu Z, Martin LJ (2003) Olfactory bulb core is a rich
source of neural progenitor and stem cells in adult
rodent and human. J Comp Neurol 459:368–391.
https://doi.org/10.1002/cne.10664
Liu G, Rao Y (2003) Neuronal migration from the fore-
brain to the olfactory bulb requires a new attractant
persistent in the olfactory bulb. J Neurosci
23:6651–6659
Lledo P-M, Valley M (2016) Adult olfactory bulb
neurogenesis. Cold Spring Harb Perspect Biol 8:
a018945. https://doi.org/10.1101/cshperspect.a018945
Lledo P-M, Alonso M, Grubb MS (2006) Adult
neurogenesis and functional plasticity in neuronal
circuits. Nat Rev Neurosci 7:179–193. https://doi.org/
10.1038/nrn1867
Lois C, Alvarez-Buylla A (1994) Long-distance neuronal
migration in the adult mammalian brain. Science
264:1145–1148
Lois C, García-Verdugo JM, Alvarez-Buylla A (1996)
Chain migration of neuronal precursors. Science
271:978–981
Luskin MB (1993) Restricted proliferation and migration
of postnatally generated neurons derived from the fore-
brain subventricular zone. Neuron 11:173–189
Ma DK, Ming G-L, Song H (2005) Glial influences on
neural stem cell development: cellular niches for adult
neurogenesis. Curr Opin Neurobiol 15:514–520.
https://doi.org/10.1016/j.conb.2005.08.003
Mason HA, Ito S, Corfas G (2001) Extracellular signals
that regulate the tangential migration of olfactory bulb
neuronal precursors: inducers, inhibitors, and
repellents. J Neurosci 21:7654–7663
Menn B, Garcia-Verdugo JM, Yaschine C et al (2006)
Origin of oligodendrocytes in the subventricular zone
of the adult brain. J Neurosci 26:7907–7918. https://
doi.org/10.1523/JNEUROSCI.1299-06.2006
Merkle FT, Alvarez-Buylla A (2006) Neural stem cells in
mammalian development. Curr Opin Cell Biol
18:704–709. https://doi.org/10.1016/j.ceb.2006.09.
008
Merkle FT, Mirzadeh Z, Alvarez-Buylla A (2007) Mosaic
organization of neural stem cells in the adult brain.
Science 317:381–384. https://doi.org/10.1126/sci
ence.1144914
Mirzadeh Z, Merkle FT, Soriano-Navarro M et al (2008)
Neural stem cells confer unique pinwheel architecture
to the ventricular surface in neurogenic regions of the
7 An Overview of Adult Neurogenesis
adult brain. Cell Stem Cell 3:265–278. https://doi.org/
10.1016/j.stem.2008.07.004
Mizrak D, Levitin HM, Delgado AC et al (2019) Single-
cell analysis of regional differences in adult V-SVZ
neural stem cell lineages. Cell Rep 26:394–406.e5.
https://doi.org/10.1016/j.celrep.2018.12.044
Moreno-Estellés M, González-Gómez P, Hortigüela R
et al (2012) Symmetric expansion of neural stem cells
from the adult olfactory bulb is driven by astrocytes via
WNT7A. Stem Cells 30:2796–2809. https://doi.org/
10.1002/stem.1243
Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J
et al (2019) Adult hippocampal neurogenesis is abun-
dant in neurologically healthy subjects and drops
sharply in patients with Alzheimer’s disease. Nat
Med 25:554–560. https://doi.org/10.1038/s41591-
019-0375-9
Morshead CM, Reynolds BA, Craig CG et al (1994)
Neural stem cells in the adult mammalian forebrain: a
relatively quiescent subpopulation of subependymal
cells. Neuron 13:1071–1082
Murase S, Horwitz AF (2002) Deleted in colorectal carci-
noma and differentially expressed integrins mediate the
directional migration of neural precursors in the rostral
migratory stream. J Neurosci 22:3568–3579. https://
doi.org/10.1523/JNEUROSCI.22-09-03568.2002
Nam H, Benezra R (2009) High levels of Id1 expression
define B1 type adult neural stem cells. Cell Stem Cell
5:515–526. https://doi.org/10.1016/j.stem.2009.08.
017
Nguyen-Ba-Charvet KT, Picard-Riera N, Tessier-Lavigne
M et al (2004) Multiple roles for slits in the control of
cell migration in the rostral migratory stream. J
Neurosci 24:1497–1506. https://doi.org/10.1523/
JNEUROSCI.4729-03.2004
Nottebohm F (1985) Neuronal replacement in adulthood.
Ann N Y Acad Sci 457:143–161
Obernier K, Cebrian-Silla A, Thomson M, et al (2018)
Adult neurogenesis is sustained by symmetric self-
renewal and differentiation. Cell Stem Cell
22:221–234.e8. https://doi.org/10.1016/j.stem.2018.
01.003
Ohira K, Furuta T, Hioki H et al (2010) Ischemia-induced
neurogenesis of neocortical layer 1 progenitor cells.
Nat Neurosci 13:173–179. https://doi.org/10.1038/nn.
2473
Pagano SF, Impagnatiello F, Girelli M et al (2000) Isola-
tion and characterization of neural stem cells from the
adult human olfactory bulb. Stem Cells 18:295–300.
https://doi.org/10.1634/stemcells.18-4-295
Palmer TD, Takahashi J, Gage FH (1997) The adult rat
hippocampus contains primordial neural stem cells.
Mol Cell Neurosci 8:389–404. https://doi.org/10.
1006/mcne.1996.0595
Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche
for adult hippocampal neurogenesis. J Comp Neurol
425:479–494
Paratcha G, Ibáñez CF, Ledda F (2006) GDNF is a
chemoattractant factor for neuronal precursor cells in
93
the rostral migratory stream. Mol Cell Neurosci
31:505–514. https://doi.org/10.1016/j.mcn.2005.11.
007
Parent JM, Vexler ZS, Gong C et al (2002) Rat forebrain
neurogenesis and striatal neuron replacement after
focal stroke. Ann Neurol 52:802–813. https://doi.org/
10.1002/ana.10393
Paton JA, Nottebohm FN (1984) Neurons generated in the
adult brain are recruited into functional circuits. Sci-
ence 225:1046–1048
Peretto P, Merighi A, Fasolo A, Bonfanti L (1997) Glial
tubes in the rostral migratory stream of the adult rat.
Brain Res Bull 42:9–21
Petreanu L, Alvarez-Buylla A (2002) Maturation and
death of adult-born olfactory bulb granule neurons:
role of olfaction. J Neurosci 22:6106–6113
Pignatelli A, Belluzzi O (2010) Neurogenesis in the adult
olfactory bulb. In: Menini A (ed) The neurobiology of
olfaction. CRC/Taylor & Francis, Boca Raton, FL
Prosser HM, Bradley A, Caldwell MA (2007) Olfactory
bulb hypoplasia in Prokr2 null mice stems from defec-
tive neuronal progenitor migration and differentiation.
Eur J Neurosci 26:3339–3344. https://doi.org/10.1111/
j.1460-9568.2007.05958.x
Ramón y Cajal S, May RM (1928) Degeneration & regen-
eration of the nervous system. Oxford University
Press, Humphrey Milford, London
Reynolds BA, Weiss S (1992) Generation of neurons and
astrocytes from isolated cells of the adult mammalian
central nervous system. Science 255:1707–1710
Ribeiro FF (2017) Unveiling the trophic actions of adeno-
sine A2A receptors in neurite outgrowth and postnatal
neurogenesis: interaction with brain-derived
neurotrophic factor. PhD thesis. Universidade de
Lisboa, Lisbon, Portugal
Rochefort C, Gheusi G, Vincent J-D, Lledo P-M (2002)
Enriched odor exposure increases the number of new-
born neurons in the adult olfactory bulb and improves
odor memory. J Neurosci 22:2679–2689. https://doi.
org/10.1523/JNEUROSCI.22-07-02679.2002
Rousselot P, Lois C, Alvarez-Buylla A (1995) Embryonic
(PSA) N-CAM reveals chains of migrating neuroblasts
between the lateral ventricle and the olfactory bulb of
adult mice. J Comp Neurol 351:51–61. https://doi.org/
10.1002/cne.903510106
Schmidt-Hieber C, Jonas P, Bischofberger J (2004)
Enhanced synaptic plasticity in newly generated gran-
ule cells of the adult hippocampus. Nature
429:184–187. https://doi.org/10.1038/nature02553
Seaberg RM, van der Kooy D (2002) Adult rodent neuro-
genic regions: the ventricular subependyma contains
neural stem cells, but the dentate gyrus contains
restricted progenitors. J Neurosci 22:1784–1793
Seri B, García-Verdugo JM, McEwen BS, Alvarez-Buylla
A (2001) Astrocytes give rise to new neurons in the
adult mammalian hippocampus. J Neurosci
21:7153–7160
Shen Q, Wang Y, Kokovay E et al (2008) Adult SVZ stem
cells lie in a vascular niche: a quantitative analysis of
94
niche cell-cell interactions. Cell Stem Cell 3:289–300.
https://doi.org/10.1016/j.stem.2008.07.026
Snapyan M, Lemasson M, Brill MS et al (2009) Vascula-
ture guides migrating neuronal precursors in the adult
mammalian forebrain via brain-derived neurotrophic
factor signaling. J Neurosci 29:4172–4188. https://
doi.org/10.1523/JNEUROSCI.4956-08.2009
Snyder JS, Drew MR (2020) Functional neurogenesis over
the years. Behav Brain Res 382:112470. https://doi.
org/10.1016/j.bbr.2020.112470
Song J, Zhong C, Bonaguidi MA et al (2012) Neuronal
circuitry mechanism regulating adult quiescent neural
stem-cell fate decision. Nature 489:150–154. https://
doi.org/10.1038/nature11306
Sorrells SF, Paredes MF, Cebrian-Silla A et al (2018)
Human hippocampal neurogenesis drops sharply in
children to undetectable levels in adults. Nature
555:377–381. https://doi.org/10.1038/nature25975
Spalding KL, Bergmann O, Alkass K et al (2013) Dynam-
ics of hippocampal neurogenesis in adult humans. Cell
153:1219–1227. https://doi.org/10.1016/j.cell.2013.
05.002
Steiner B, Klempin F, Wang L et al (2006) Type-2 cells as
link between glial and neuronal lineage in adult hippo-
campal neurogenesis. Glia 54:805–814. https://doi.org/
10.1002/glia.20407
Sun GJ, Sailor KA, Mahmood QA et al (2013) Seamless
reconstruction of intact adult-born neurons by serial
end-block imaging reveals complex axonal guidance
and development in the adult hippocampus. J Neurosci
33:11400–11411. https://doi.org/10.1523/
JNEUROSCI.1374-13.2013
Suzuki SO, Goldman JE (2003) Multiple cell populations
in the early postnatal subventricular zone take distinct
migratory pathways: a dynamic study of glial and
neuronal progenitor migration. J Neurosci
23:4240–4250
Temple S (2001) The development of neural stem cells.
Nature 414:112–117. https://doi.org/10.1038/
35102174
Tobin MK, Musaraca K, Disouky A et al (2019) Human
hippocampal neurogenesis persists in aged adults and
Alzheimer’s disease patients. Cell Stem Cell
24:974–982.e3. https://doi.org/10.1016/j.stem.2019.
05.003
Toni N, Schinder AF (2016) Maturation and functional
integration of new granule cells into the adult hippo-
campus. Cold Spring Harb Perspect Biol 8:a018903.
https://doi.org/10.1101/cshperspect.a018903
Tozuka Y, Fukuda S, Namba T et al (2005) GABAergic
excitation promotes neuronal differentiation in adult
hippocampal progenitor cells. Neuron 47:803–815.
https://doi.org/10.1016/j.neuron.2005.08.023
Villeda SA, Luo J, Mosher KI et al (2011) The ageing
systemic milieu negatively regulates neurogenesis and
cognitive function. Nature 477:90–94. https://doi.org/
10.1038/nature10357
F. F. Ribeiro and S. Xapelli
Wang S, Scott BW, Wojtowicz JM (2000) Heterogenous
properties of dentate granule neurons in the adult rat. J
Neurobiol 42:248–257
Whitman MC, Fan W, Rela L et al (2009) Blood vessels
form a migratory scaffold in the rostral migratory
stream. J Comp Neurol 516:94–104. https://doi.org/
10.1002/cne.22093
Winner B, Cooper-Kuhn CM, Aigner R et al (2002) Long-
term survival and cell death of newly generated
neurons in the adult rat olfactory bulb. Eur J Neurosci
16:1681–1689. https://doi.org/10.1046/j.1460-9568.
2002.02238.x
Wittko IM, Schänzer A, Kuzmichev A et al (2009)
VEGFR-1 regulates adult olfactory bulb neurogenesis
and migration of neural progenitors in the rostral
migratory stream in vivo. J Neurosci 29:8704–8714.
https://doi.org/10.1523/JNEUROSCI.5527-08.2009
Wu W, Wong K, Chen J et al (1999) Directional guidance
of neuronal migration in the olfactory system by the
protein Slit. Nature 400:331–336. https://doi.org/10.
1038/22477
Yagita Y, Sakurai T, Tanaka H et al (2009) N-cadherin
mediates interaction between precursor cells in the
subventricular zone and regulates further differentia-
tion. J Neurosci Res 87:3331–3342. https://doi.org/10.
1002/jnr.22044
Yamaguchi M, Mori K (2005) Critical period for sensory
experience-dependent survival of newly generated
granule cells in the adult mouse olfactory bulb. Proc
Natl Acad Sci USA 102:9697–9702. https://doi.org/10.
1073/pnas.0406082102
Yamashita T, Ninomiya M, Acosta PH et al (2006)
Subventricular zone-derived neuroblasts migrate and
differentiate into mature neurons in the post-stroke
adult striatum. J Neurosci 26:6627–6636. https://doi.
org/10.1523/JNEUROSCI.0149-06.2006
Zhang RL, Chopp M, Gregg SR et al (2009) Patterns and
dynamics of subventricular zone neuroblast migration
in the ischemic striatum of the adult mouse. J Cereb
Blood Flow Metab 29:1240–1250. https://doi.org/10.
1038/jcbfm.2009.55
Zhao M, Janson Lang AM (2009) Bromodeoxyuridine
infused into the cerebral ventricle of adult mice labels
nigral neurons under physiological conditions--a
method to detect newborn nerve cells in regions with
a low rate of neurogenesis. J Neurosci Methods
184:327–331. https://doi.org/10.1016/j.jneumeth.
2009.08.007
Zhao M, Momma S, Delfani K et al (2003) Evidence for
neurogenesis in the adult mammalian substantia nigra.
Proc Natl Acad Sci USA 100:7925–7930. https://doi.
org/10.1073/pnas.1131955100
Zhao C, Teng EM, Summers RG et al (2006) Distinct
morphological stages of dentate granule neuron matu-
ration in the adult mouse hippocampus. J Neurosci
26:3–11. https://doi.org/10.1523/JNEUROSCI.3648-
05.2006
 Intervention of Brain-Derived
Neurotrophic Factor and Other 8
Neurotrophins in Adult Neurogenesis

Filipa F. Ribeiro and Sara Xapelli

Abstract the most studied neurotrophin. In this chapter,

Cell survival during adult neurogenesis and
the modulation of each step, namely, prolifer-
ation, lineage differentiation, migration, matu-
ration, and functional integration of the
newborn cells into the existing circuitry, is
regulated by intrinsic and extrinsic factors.
Transduction of extracellular niche signals
triggers the activation of intracellular
mechanisms that regulate adult neurogenesis
by affecting gene expression. While the intrin-
sic factors include transcription factors and
epigenetic regulators, the extrinsic factors are
molecular signals that are present in the neuro-
genic niche microenvironment. These include
morphogens, growth factors,
neurotransmitters, and signaling molecules
secreted as soluble factors or associated to
the extracellular matrix. Among these molecu-
lar mechanisms are neurotrophins and
neurotrophin receptors which have been
implicated in the regulation of adult
neurogenesis at different levels, with brain-
derived neurotrophic factor (BDNF) being
F. F. Ribeiro · S. Xapelli (*)
Instituto de Farmacologia e Neurociências, Faculdade de
Medicina, Universidade de Lisboa, Lisbon, Portugal
Instituto de Medicina Molecular João Lobo Antunes,
Faculdade de Medicina, Universidade de Lisboa, Lisbon,
Portugal
e-mail: anaribeiro@medicina.ulisboa.pt;
sxapelli@medicina.ulisboa.pt
we review the current knowledge about the
role of neurotrophins in the regulation of
adult neurogenesis in both the subventricular
zone (SVZ) and the hippocampal subgranular
zone (SGZ).
8.1 Neurotrophins
Neurotrophins are pleiotropic molecules that not
only play important roles in regulating cell sur-
vival but also exert a wide variety of other
biological functions, including the regulation of
neuronal differentiation, axonal and dendritic
growth, and synaptic transmission and plasticity.
Hence, neurotrophins are essential for the devel-
opment of the architecture of the embryonic
brain, and also for its maintenance during adult-
hood, having important roles in learning and
memory processes (Bothwell 2014). Among the
family of neurotrophins, BDNF has been the most
studied neurotrophin in the regulation of postnatal
neurogenesis, with NGF, neurotrophin-3 (NT-3),
and neurotrophin-4 (NT-4) being less studied
(Huang and Reichardt 2001; Chao 2003; Vilar
and Mira 2016).
All neurotrophins are initially synthesized as
precursors or proneurotrophins containing signal
peptides followed by the pro-region at the
N-terminal. Proneurotrophins may be secreted

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 95

L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_8
96
from cells or undergo intracellular proteolytic
cleavage to yield mature neurotrophins, which
dimerize after translation (Kolbeck et al. 1994;
Foltran and Diaz 2016). Proneurotrophins and
neurotrophins have distinct biological functions.
8.2 Brain-Derived Neurotrophic
Factor (BDNF)
BDNF, a small basic protein with 252 amino acid
residues, is abundantly expressed in the nervous
system (Katoh-Semba et al. 1997). BDNF synthe-
sis is highly regulated in space and time at several
levels. The BDNF gene comprises one common
encoding region and multiple promoters that reg-
ulate the transcription of the BDNF gene. In
rodents, the main encoding region is spliced to
one out of eleven upstream different 50 UTR alter-
native exons. In addition, BDNF transcripts can
be subjected to polyadenylation in two different
sites, generating two different transcripts, one
with a long and other with a short 30 UTR
(untranslated region), raising the resultant
transcripts to a total of 22 variants (Aid et al.
2007; Pruunsild et al. 2007). This structural dif-
ference determines the subcellular localization of
the BDNF mRNAs: BDNF mRNAs with a short
30 UTR are restricted to the soma, where they are
constitutively translated to maintain the basal
levels of BDNF protein, whilst long 30 UTR
mRNAs are targeted to dendritic spines, where
they are locally translated in response to neuronal
activation (An et al. 2008; Lau et al. 2010). There
is also evidence that BDNF 50 UTR mRNA splice
variants have differences in the expression and
localization of individual BDNF transcripts
between cell types, creating a spatial code that
facilitates local structural and functional plasticity
in specific synaptic connections between hippo-
campal neurons (Maynard et al. 2017). In
neurons, BDNF mRNA variants have a distinct
subcellular distribution, constituting a “spatial
code,” with exon 1, 3, 5, 7, and 8 located in
neuronal soma, exon 4 extending into proximal
dendrites, and exon 2 and 6 reaching distal
dendrites (Aliaga et al. 2009; Baj et al. 2011;
Colliva and Tongiorgi 2020). In a similar way to
F. F. Ribeiro and S. Xapelli
other neurotrophins, BDNF is not translated from
the mRNA as a mature protein, but it is initially
synthesized as a precursor protein, the pre-pro-
BDNF. The presence of a signal peptide directs
this pre-pro-neurotrophin to the endoplasmic
reticulum where the signal peptide is cleaved to
form a 32 kDa pro-BDNF (Halban and Irminger
1994). Pro-BDNF is then transported to the Golgi
for sorting into two different secretory pathways
(Fig. 8.1): the constitutive pathway, which
comprises small vesicles that are transported to
the cell periphery to fuse with the plasma mem-
brane, in an intracellular Ca2+-independent man-
ner; and the regulated pathway, which comprises
large vesicles that fuse with the plasma membrane
in a Ca2+-dependent manner (Lessmann et al.
2003). While the constitutive secretory pathway
for BDNF secretion occurs in both non-neuronal
and neuronal cells, the regulated secretory path-
way exclusively occurs in neurons (Seidah et al.
1996; Mowla et al. 2001). Through these pro-
cesses, pro-BDNF is secreted to the extracellular
space. Either pro-BDNF can be simply secreted
without further digestion, thus serving as an
endogenous ligand by itself, or pro-BDNF can
be secreted and extracellularly processed to
mature BDNF by the endopeptidases tissue plas-
minogen activator (tPA)/plasmin system (Pang
et al. 2004) and matrix-metalloproteinases (Lee
et al. 2001; Hwang et al. 2005; Mizoguchi et al.
2011).
Alternatively, pro-BDNF can suffer intracellu-
larly proteolytic cleavage in the trans-Golgi
through the action of furin, to be constitutively
released, or within secretory granules by
convertases in the regulated pathway (Seidah
et al. 1996; Mowla et al. 1999). In these
situations, pro-BDNF is intracellularly converted
and secreted as a 14 kDa mature BDNF. As a
result, there are two secreted BDNF forms, the
pro-BDNF and the mature BDNF, which increase
the complexity of biological effects.
Pro-BDNF seems not only to represent a pre-
cursor for the biologically active BDNF but also
to play biologically active functions, such as
modulation of synaptic plasticity, neuronal mor-
phology, or cell survival (Lee et al. 2001;
Nagappan et al. 2009; Yang et al. 2009; Sun
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
Fig. 8.1 Production and processing of BDNF. BDNF
mRNAs with a short 30 UTR are restricted to the soma,
while long 30 UTR mRNAs are targeted to dendritic spines.
Pro-BDNF may be processed to mature BDNF by several
cellular mechanisms. Pro-BDNF can be cleaved in the
trans-Golgi by furin and in regulated secretory vesicles
et al. 2012; Dieni et al. 2012). Indeed, pro-BDNF
was shown to be predominantly secreted in
response to low frequency stimulation, being
responsible for facilitating long-term depression
(LTD), while mature BDNF was mostly secreted
following high frequency stimulation, promot-
ing long-term potentiation (LTP) (LTP and LTD
are cellular mechanisms of long-term synaptic
plasticity underlying learning and memory forma-
tion, maintenance, and decay). In fact, as a conse-
quence of high frequency stimulation of cultured
97
by convertase enzymes. If pro-BDNF reaches the extracel-
lular milieu, it can be processed by the endopeptidases
tPA/plasmin system and matrix-metalloproteinases, and
the resulting mature BDNF then activates cell surface
TrkB receptors. Alternatively, extracellular pro-BDNF
can bind to p75NTR (from Ribeiro 2017)
hippocampal neurons or electroconvulsive stimu-
lation of rat brains, extracellular mature BDNF
levels were highly increased mainly due to
co-secretion of pro-BDNF and tPA that extracel-
lularly produce mature BDNF, and probably also
to increased levels of intracellular prohormone
convertase 1, which intracellularly converts
pro-BDNF into mature BDNF (Nagappan et al.
2009; Segawa et al. 2013). Apparently,
pro-BDNF is the main form secreted in vivo and
the ratio pro-BDNF/mature BDNF is crucial for
98
the regulation of long-term synaptic plasticity, as
well as pathological conditions (Foltran and Diaz
2016).
Moreover, BDNF can be anterogradely
transported from the cell soma to axon terminals
(Smith et al. 1997; Adachi et al. 2005; Lu et al.
2014). For instance, in hippocampal mossy fibers,
BDNF is transcribed and translated at the soma of
granule neurons and then transported
anterogradely to provide trophic support of CA3
pyramidal neurons (Smith et al. 1997). For that,
BDNF and pro-BDNF have been suggested to be
stored in presynaptic large dense core vesicles
(Dieni et al. 2012).
As mentioned above, pro-BDNF is enzymati-
cally cleaved to generate mature BDNF. Apart
from mature BDNF, as a result of this proteolytic
processing, it is generated a N-terminal fragment
from the pro-BDNF, which called the
pro-peptide. Interestingly, this pro-peptide also
has biological activity. Both BDNF and its
pro-peptide have been found to be stored in pre-
synaptic dense core vesicles in hippocampal
neurons, and the BDNF pro-peptide also seems
to be involved in neuronal plasticity, such as in
facilitating LTD (Mizui et al. 2017; Dieni et al.
2012).
8.3 Neurotrophin Receptors
Neurotrophins interact with two types of trans-
membrane cell surface receptors: the high-affinity
tropomyosin-related kinase receptor (Trk), which
belongs to the tyrosine kinase family of receptors;
and the low-affinity pan-neurotrophin receptor
p75NTR, which belongs to the tumor necrosis
factor (TNF) receptor superfamily. The mature
neurotrophins bind to the class of the Trk family
of tyrosine protein kinase receptors. These are the
major membrane signal-transducing receptors
that include the TrkA, TrkB, and TrkC receptors,
for which the main ligands are, respectively,
NGF, BDNF/NT-4, and NT-3 (Chao 2003). In
addition, all neurotrophins can also bind to
p75NTR with similar low affinity (approximately
100-fold less than to Trk receptors). On the other
hand, pro-neurotrophins, the precursors of the
F. F. Ribeiro and S. Xapelli
above mature neurotrophins, bind with high affin-
ity to this second class of receptors (Lee et al.
2001), also signaling through a specific
co-receptor, sortilin (Teng et al. 2005).
The mature BDNF elicits its trophic actions
through high-affinity binding to TrkB, which
triggers receptor dimerization and subsequent
autophosphorylation of multiple tyrosine residues
in the cytoplasmic receptor domain. These
phosphorylated tyrosine residues promote the
activation of three main intracellular signaling
pathways (Fig. 8.2), by the recruitment of various
adaptor proteins, such as Src homologous and
collagen-like (Shc) adaptor protein, or phospholi-
pase C (PLC)γ. Shc adaptor protein links the
activated TrkB receptor to two separated intracel-
lular signaling cascades, namely, phosphatidy-
linositol 3-kinase (PI3K)/Akt pathway and Ras
pathway which leads to the activation of
mitogen-activated protein kinases/extracellular
signal-regulated kinases (MAPK/ERK) cascade.
In addition, PLCγ binds to activated Trk receptors
and initiates an intracellular signaling cascade
resulting in the release of inositol phosphates,
with consequent release of intracellular calcium
(Ca2+), and activation of protein kinase C (PKC).
There are several isoforms of TrkB receptors.
The afore described isoform is called the TrkB
full-length (TrkB-FL). Other isoforms contain an
extracellular ligand binding domain and a single
transmembrane domain, lacking, however, the
intracellular tyrosine kinase domain. These are
the truncated TrkB isoforms that are generated
from alternative splicing (Klein et al. 1990;
Middlemas et al. 1991; Stoilov et al. 2002;
Luberg et al. 2010). There are two most known
C-terminal truncated TrkB isoforms, TrkB-T1
and TrkB-T2 (Klein et al. 1990; Middlemas
et al. 1991). In humans, however, three isoforms
have been described, the C-terminal truncated
receptor (TrkB-T1), the C-terminal truncated
receptor containing a Shc-binding site (TrkB-T-
Shc), and a C-terminal truncated receptor
retaining tyrosine kinase activity (TrkB-T-TK)
(Stoilov et al. 2002; Luberg et al. 2010). TrkB-
T1 and TrkB-T2 are similar to TrkB-FL except at
the intracellular domain, where they only have,
respectively, 23 and 21 amino acid residues and
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
Fig. 8.2 Signaling
cascades activated by the
neurotrophin receptor
TrkB. BDNF binding to
TrkB triggers the activation
of signaling cascades, such
as Ras,
phosphatidylinositol
3-kinase (PI3K), and
phospholipase C (PLC)γ
pathways, resulting in gene
expression, neuronal
survival, and neurite
outgrowth (from Ribeiro
2017)
lack the kinase domain (Klein et al. 1990;
Middlemas et al. 1991). The first 12 amino acid
residues of the intracellular domain of both
truncated TrkB isoforms are common with those
of TrkB-FL, and the remaining residues of the
intracellular domain are isoform-specific of each
truncated receptor (Baxter et al. 1997). TrkB-FL
is abundantly expressed in neuronal cells, while
truncated receptors are mainly found in glial cells,
being widely expressed during development and
in the adult brain (Reichardt 2003). TrkB-T-Shc
has been identified only in human neurons
(Stoilov et al. 2002). In addition to these truncated
receptors originated from alternative splicing,
TrkB-FL protein can be cleaved at the cytosolic
domain by calpains to produce the TrkB truncated
form TrkB-T0 and the TrkB-ICD fragment of
32 kDa containing the protein kinase domain
(Jerónimo-Santos et al. 2015; Tanqueiro et al.
2018; Fonseca-Gomes et al. 2019).
There is no clear answer about the function or
signaling pathways activated by truncated
receptors. Some findings suggest that truncated
TrkB isoforms function as selective scavengers
that prevent extracellular BDNF diffusion since,
when BDNF binds to these receptors, it is
99
internalized (Biffo et al. 1995). The truncated
receptors may also function as dominant negative
receptors through the formation of non-functional
heterodimers with TrkB-FL, thus inhibiting BDNF
signaling (Eide et al. 1996; Haapasalo et al. 2002).
However, other studies suggest that TrkB-T1 and
TrkB-T2 are able to induce signaling transduction
in response to BDNF binding, through their short
cytoplasmic domain (Baxter et al. 1997). Also,
TrkB-T1 has been shown to evoke rapid calcium
transients in astrocytes (Rose et al. 2003) and to
alter astrocytic morphology via Rho GTPase
(Ohira et al. 2005) in response to BDNF binding,
as well as to allow glycine (Aroeira et al. 2015) and
GABA (Vaz et al. 2011) transport. In fact,
astrocytes were shown to predominantly express
TrkB-T1, which is a TrkB isoform that was
demonstrated to increase astrocyte morphological
complexity in response to BDNF, during the astro-
cytic maturation process (Holt et al. 2019), as well
as to be important in cell migration and prolifera-
tion (Matyas et al. 2017).
Regarding p75NTR, it is composed of an
extracellular cysteine-rich domain, a single trans-
membrane domain and a cytoplasmic domain that
contains a “death” domain. Activation of
100
p75NTR triggers nuclear factor-kB (NF-kB) and
Jun kinase-signaling pathways, thus, respec-
tively, regulating cell survival and death
(Reichardt 2006). Besides lacking an intracellular
kinase domain, p75NTR can cooperate with other
proteins to enhance biological responses, forming
functional receptor complexes with Trk receptors
to increase binding affinity to their preferred
neurotrophins (Barker 2004), and with sortilin,
forming a p75NTR-sortilin complex to which
pro-neurotrophins bind, like pro-BDNF (Teng
et al. 2005). Evidence suggests that sortilin also
interacts with TrkA, TrkB, and TrkC receptors,
enabling their anterograde axonal transport,
thereby enhancing neurotrophin signaling
(Vaegter et al. 2011). Moreover, there are two
known p75NTR isoforms: a short (s-p75NTR)
and a full-length isoform. The full-length isoform
is able to bind to neurotrophins, while the short
isoform, whose functions are largely unknown,
lacks the neurotrophin binding site (Fujii and
Kunugi 2009).
These receptors have shown to mediate utterly
opposite effects. For instance, studies have shown
that pro-BDNF binds to p75NTR to facilitate
LTD in hippocampal neurons, initiates
programmed neuronal death, collapses neurite
outgrowth, or negatively regulates dendritic com-
plexity and spine density (Lee et al. 2001; Woo
et al. 2005; Nagappan et al. 2009; Sun et al. 2012;
Yang et al. 2014). In opposition, when activated
by BDNF, TrkB enhances LTP, cell survival, and
dendritic complexity (Pang et al. 2004; Nagappan
et al. 2009). Hence, a bidirectional regulation
seems to exist between both receptors and pro-
and mature BDNF.
8.4 BDNF Role in Adult
Hippocampal Neurogenesis
The role of BDNF in adult neurogenesis has been
the subject of several studies. This neurotrophin
has been implicated in the regulation of adult
neurogenesis at different levels, in both dentate
gyrus (DG) and SVZ neurogenic niches. Despite
F. F. Ribeiro and S. Xapelli
contradictory findings, BDNF constitutes a key
regulatory player in the neurogenic process. Both
BDNF and its high-affinity TrkB receptor are
expressed in the adult mouse hippocampus in
neurons and astrocytes (Ivanova and Beyer
2001; Li et al. 2008). Within the hippocampus,
TrkB is strongly expressed in the granule layer of
the DG, as well as in the stratum pyramidale of
areas CA1–CA3 and within the subiculum, and in
moderate levels in the stratum radiatum, stratum
lacunosum moleculare, and the molecular layer of
the DG (Yan et al. 1997). TrkB expression levels
have been shown to increase with the progression
toward neuronal maturity. However, stem-like
cells, which rarely divide, express the TrkB pro-
tein, in contrast to proliferating progenitors
(Donovan et al. 2008). Using primary murine
hippocampal cultures, it has been shown that
BDNF levels increased stepwise from embryonic
day 15 until the second postnatal week, whereas
TrkB expression was stable throughout develop-
ment. BDNF was shown to be strongly expressed
in the granule layer of the DG (Li et al. 2008), and
particularly in mossy fiber axons of the dentate
granule neurons (Conner et al. 1997). However,
in humans, the levels of BDNF mRNA did not
change significantly in the hippocampus with
aging, whereas the levels of TrkB mRNA,
whether of the TrkB-T isoform or the TrkB-FL
isoform, decreased (Webster et al. 2006). More-
over, post-mortem findings from patients with
bipolar disorder, major depressive disorder,
schizophrenia, and Alzheimer’s disease showed
that BDNF levels are decreased in hippocampus
(Hock et al. 2000; Reinhart et al. 2015).
On the other hand, the lower-affinity receptor
for BDNF, p75NTR, can also be found within the
hippocampal formation. In young mice, both
granule cells of the DG and pyramidal neurons
of CA1-CA3 express high levels of p75NTR
mRNA (Zagrebelsky et al. 2005). Within the
DG, p75NTR is mainly localized presynaptically,
but can also be found in astrocytes and in the
dendrites and cell bodies of some granule cells
(Dougherty and Milner 1999). p75NTR has been
shown to localize in dendritic spines, in addition
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
to afferent terminals, of CA1 neurons (Woo et al.
2005), and in cell bodies within the DG (Barrett
et al. 2005). Neural progenitor cell (NPC)-specific
knockout of p75NTR did not affect proliferation
of NPCs nor altered neurogenesis in the adult
hippocampus (Groves et al. 2019). Regarding
the co-receptor sortilin, it is highly expressed in
the hippocampus, and particularly in the soma
and dendrites of neurons located in CA1-CA3
and within the DG (Sarret et al. 2003).
BDNF has been described to be an important
player in DG neurogenesis (Scharfman et al.
2005; Taliaz et al. 2010; Ferreira et al. 2018).
Using antibodies to scavenge BDNF in vitro led
to decreased neuronal differentiation of progeni-
tor cells (Babu et al. 2009). However, there are
contradictory reports on the role of BDNF upon
proliferation, with some showing an increase
(Li et al. 2008; Ferreira et al. 2018), while others
no changes in progenitor cell proliferation in the
presence of BDNF (Lee et al. 2002; Choi et al.
2009), and others demonstrating that BDNF
depletion induces SGZ progenitor cell prolifera-
tion, concluding that BDNF is required for the
terminal differentiation of new neurons in the
adult hippocampus (Chan et al. 2008). Similarly,
there are also conflicting data regarding the
BDNF role upon survival of new neurons in the
adult DG (Sairanen et al. 2005; Chan et al. 2008;
Choi et al. 2009), with reports showing that
BDNF signaling is required for the survival of
newborn neurons in mouse hippocampus
(Sairanen et al. 2005; Choi et al. 2009), and others
that it is not necessary for cell survival (Chan
et al. 2008).
The high-affinity BDNF receptor TrkB seems
to play a role in these BDNF-mediated actions on
adult hippocampal neurogenesis. Deletion of the
BDNF or NTRK2 (TrkB gene) in progenitor cells
blocked BDNF-induced neurosphere growth
in vitro, and impaired proliferation and
neurogenesis in vivo, together with a reduction
of DG thickness (Lee et al. 2002; Li et al. 2008).
The generation of new neurons during adulthood
involves local precursor cell migration and termi-
nal differentiation in the DG, which is influenced
by the hippocampal microenvironment. BDNF,
101
through TrkB cascade, favored cell survival and
migration of murine adult hippocampal precursor
cells during differentiation to glial and neuronal
lineages, thus increasing these cell types. (Ortiz-
López et al. 2017). Moreover, BDNF, through
TrkB signaling, has been consistently shown to
have an essential regulatory role in dendritic com-
plexity, as well as in synaptic formation, matura-
tion, and plasticity of newborn SGZ neurons
(Chan et al. 2008; Gao et al. 2009; Wang et al.
2015).
BDNF is an important factor that is involved in
the regulation of hippocampal spine densities and
spine shape, and these effects seemed to be
mediated by TrkB receptors, since receptor dele-
tion in adult neural progenitors reduced the den-
dritic complexity and spine density in adult-born
hippocampal neurons (Bergami et al. 2008), thus
suggesting the relevance of BDNF/TrkB signal-
ing for dendrite morphogenesis of adult-born
neurons. In in vivo, during adult neurogenesis in
the DG, BDNF has been described to be locally
synthesized and released from the dendrites of
mature granule cells which through stimulation
of TrkB receptors on parvalbumin (PV)-
expressing GABAergic interneurons leads to mat-
uration of adult-born neurons through GABA
inputs (Waterhouse et al. 2012). In fact, ErbB4
ablation in PV neurons inhibited adult hippocam-
pal neurogenesis in mice through down-
regulation of the BDNF–TrkB pathway (Zhang
et al. 2018). Rather than a paracrine action, den-
drite morphogenesis of adult-born granule cells is
mainly regulated by an autocrine action of BDNF
in these neurons, possibly as a result of
GABAergic or glutamatergic inputs (Wang et al.
2015), whereas BDNF released from adult-born
granule cells also proved to have an impact, even
though minor, in adult-born neuronal dendrite
growth and formation of the dendritic branches
(Wang et al. 2015). Mice, in which long 30 UTR
BDNF mRNA is not generated, displayed
decreased differentiation, maturation, and inte-
gration of neuronal precursors in the dentate
gyrus (Waterhouse et al. 2012). Using specific
knockout mice lacking BDNF production from
exons 1, 2, 4, or 6 splice variants, it was found
102
that loss of BDNF from different BDNF mRNA
variants differentially affected dendritic complex-
ity and spine morphology in the hippocampus
(Maynard et al. 2017). These data demonstrate
that the diverse actions of BDNF rely on different
splice variants.
Not only TrkB activation promotes adult
neurogenesis enhancement, but also p75NTR
(Bernabeu and Longo 2010). p75NTRs are
expressed by adult dentate progenitor persisting
until the early stages of neuronal and glial cell
maturation. These receptors are important for pro-
moting proliferation and/or early maturation of
not only neural, but also glial cells, being impor-
tant for regulating neuronal and non-neuronal cell
genesis. In fact, mice lacking the p75NTR recep-
tor gene (p75(NTR-/-)) have significantly reduced
hippocampal neurogenesis (Bernabeu and Longo
2010). P75NTR is also important for axon speci-
fication. In cultured hippocampal neurons, local
exposure to neurotrophins leads to p75NTR
asymmetrical localization in the differentiating
neuron, accumulating into that undifferentiated
neurite. This process leads to specification of the
future axon. Moreover, knockout or knockdown
of p75NTR results in axon initiation failure dur-
ing in vivo adult hippocampal neurogenesis
(Zuccaro et al. 2014). As a p75NTR ligand,
BDNF induces the specification of axons in
cultured developing hippocampal neurons (Shelly
et al. 2007; Cheng et al. 2011). These beneficial
effects of p75NTR may be possible through
partnering with TrkB and binding to mature
BDNF. Contrary to the beneficial effects of
mature BDNF on adult neurogenesis, pro-BDNF
seems to attenuate neurogenesis in the hippocam-
pus (Chen et al. 2016), possibly through binding
to the complex p75NTR/sortilin. Moreover, loss-
of-function experiments show that mutant lines of
p75NTR have a higher spine density and greater
dendritic complexity than wild-type (WT) mice.
On the contrary, p75NTR overexpression signifi-
cantly reduced dendritic complexity and spine
density in all dendritic compartments. These
results show that p75NTR negatively modulates
dendritic morphology in hippocampal neurons, in
opposition to TrkB (Zagrebelsky et al. 2005).
F. F. Ribeiro and S. Xapelli
8.5 Role of Environmental Factors
on BDNF and Adult
Hippocampal Neurogenesis
Environmental factors can also stimulate the pro-
duction of BDNF in the hippocampus and thus
regulate adult hippocampal neurogenesis, such as
physical exercise (Cotman and Berchtold 2002;
Berchtold et al. 2005; Marlatt et al. 2012), and
enriched environment (Kuzumaki et al. 2011).
Adult hippocampal neurogenesis can be
enhanced by voluntary exercise. Indeed, running
increases cell proliferation and neurogenesis in
the adult mouse DG (van Praag et al. 1999).
Rats subjected to forced run on a treadmill for
30 min 5 times a week for 4 weeks showed
improvements in short-term memory which was
impaired by maternal lipopolysaccharide (LPS)
exposure. Moreover, increased cell proliferation
and neurogenesis in the DG and increased BDNF
and TrkB expression were observed in the hippo-
campus of rats with treadmill exercise born from
LPS-exposed maternal rats (Kim et al. 2015). On
the other hand, in mice allowed to voluntarily
exercise, with free access to a running wheel,
only a few days of exercise was enough to induce
an increase in BDNF mRNA levels in the DG
(Neeper et al. 1995). By either inhibiting BDNF
action or blocking TrkB, the benefit induced by
exercise on cognitive function, such as learning
and recall abilities, was reduced to levels similar
to sedentary control (Vaynman et al. 2004, 2006).
Other studies have shown beneficial effects of
exercise on hippocampal neurogenesis through-
out life span. For example, aerobic exercise train-
ing has shown to increase hippocampal volume in
adult humans by 2%, thus reversing age-related
loss in volume by 1–2 years. Also, increased
hippocampal volume was associated with greater
serum levels of BDNF and spatial memory
improvement (Erickson et al. 2011). Another
work studied the effect of a 30-min running
once a day for 6 weeks in hippocampal
neurogenesis of young and adult rats. Results
showed an increase in hippocampal neurogenesis
in both ages, being more active in young than in
adult rats. BDNF and TrkB expression in the
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
hippocampus were also enhanced in both ages,
but they were greater in the adult rats. Results
showed that adults have much better spatial
learning abilities than young rats in the absence
of exercise. But the improvement of exercise-
induced spatial learning ability through
neurogenesis was better in younger age (Jin
et al. 2017). Exercise was also reported to provide
cognitive benefit to a mouse model of
Alzheimer’s disease, 5FAD, by inducing adult
hippocampal neurogenesis and elevating the
levels of BDNF. In fact, combining adult
neurogenesis with BDNF mimicked exercise
effects on cognition in an Alzheimer’s mouse
model (Choi et al. 2018). Long-term exercise
not only elevates the rate of adult neurogenesis
and BDNF levels (Marlatt et al. 2012), but also
pro-BDNF levels in the hippocampus. In addi-
tion, voluntary exercise increases the activity of
the serine protease tPA that facilitates pro-BDNF
cleavage into mature BDNF. This suggests that
the beneficial effects on adult neurogenesis
depend on cleavage of pro-BDNF to mature
BDNF, which, in turn, can signal through TrkB
(Ding et al. 2011).
Exposure to an enriched environment
increases neurogenesis in the DG of adult rodents.
BDNF is required for the enhancement of hippo-
campal neurogenesis following environmental
enrichment (Rossi et al. 2006). Indeed, environ-
mental enrichment increases BDNF mRNA
expression via sustained epigenetic modification
in the mouse hippocampus (Kuzumaki et al.
2011). However, environmental enrichment
consists of many components, such as expanded
learning opportunities, increased social interac-
tion, more physical activity, and larger housing.
A study assigned adult mice to a separate envi-
ronmental enrichment component: water-maze
learning (learner), swim-time-yoked control
(swimmer), voluntary wheel running (runner),
and enriched (enriched) and standard housing
(control) groups and evaluated the number of
BrdU+ cells. They observed that neither maze
training nor yoked swimming had any effect on
BrdU+ cell number, but running doubled the
number of surviving newborn cells, in amounts
like enriched conditions, which included running
103
wheels. These data demonstrate that voluntary
exercise is sufficient to enhance neurogenesis in
the adult mouse DG (van Praag et al. 1999), also
suggesting that the benefits from environmental
enrichment are highly dependent on the access to
running wheels (Russo-Neustadt et al. 2000;
Kobilo et al. 2011), as voluntary exercise in run-
ning wheels was, by itself, able to increase BDNF
levels and promote adult hippocampal
neurogenesis (Neeper et al. 1995). Mice lacking
expression of BDNF through promoter IV
(BDNF-KIV mice) exhibit a depression-like phe-
notype (Sakata et al. 2009). Three weeks in an
enriched environment, including running wheels,
rescued depression-like behavior of BDNF-KIV
mice and increased other BDNF transcripts,
restoring BDNF protein levels in the hippocam-
pus. An enriched environment also restored the
reduction in cell survival and dendritic length and
increased cell proliferation in BDNF-KIV mice.
These results suggest that other BDNF promoters
compensate the loss of a specific BDNF
promotor, rescuing BDNF levels and BDNF-
mediated function associated to environmental
enrichment (Jha et al. 2011).
Nutrition is also an environmental factor that
can influence BDNF levels and adult hippocam-
pal neurogenesis. For instance, rodents fasted
intermittently exhibit enhanced hippocampal
neurogenesis and LTP at hippocampal synapses
compared with sedentary animals fed an ad
libitum diet. Curiously, an increase in BDNF
was also observed in animals subjected to inter-
mittent fasting (Baik et al. 2020). Conversely,
obesity and psychosocial stress combined in the
same individual, as it happens frequently in the
Western society, augmented anxiety-like behav-
ior, and impaired spatial memory of adult mice.
These changes were associated with reduced hip-
pocampal volume, neurogenesis, local BDNF and
TrkB levels, and amplified astrogliosis (Agrimi
et al. 2019).
Neurogenesis in the SGZ of the DG is also
negatively regulated by stressful experiences,
leading to a depression condition, and positively
by treatment with antidepressant drugs (Hanson
et al. 2011). In fact, chronic antidepressant
administration also promotes an increase in
104
BDNF expression (Nibuya et al. 1995; Russo-
Neustadt et al. 2000; Sairanen et al. 2005), while
BDNF infusion mimics the antidepressant behav-
ioral effects (Shirayama et al. 2002). More
recently, TrkB-dependent adult hippocampal
neurogenesis was shown to mediate sustained
ketamine antidepressant response (Ma et al.
2017).
8.6 BDNF Role in SVZ-Derived
Neurogenesis
The SVZ expresses low levels of BDNF (Galvão
et al. 2008), with levels not changing throughout
the adult lifespan (Werry et al. 2010), and with
SVZ astrocytes and ependymal cells being the
possible source of BDNF (Tonchev 2011).
Proliferating cells in the SVZ have been reported
to express both the full-length and the truncated
form of the TrkB receptors, but not p75NTR
(Tervonen et al. 2006; Galvão et al. 2008).
p75NTRs are expressed in SVZ intermediate
progenitors (type C cells) and migrating
neuroblasts (type A cells) (Hosomi et al. 2003;
Galvão et al. 2008).
BDNF has been shown to increase the number
of new neurons in the olfactory bulb (OB) (Zigova
et al. 1998; Benraiss et al. 2001; Henry et al.
2007). However, studies on the role of BDNF in
regulating SVZ-neuronal survival have provided
conflicting results. First studies, in rat cell
cultures, showed BDNF as a mediator of neuronal
survival (Kirschenbaum and Goldman 1995). In
agreement, later in vivo studies showed increased
cell death in animals where BDNF is genetically
knocked out or mutated (Linnarsson et al. 2000;
Bath et al. 2008), or enhanced survival of new
neurons in BDNF overexpressing animals
(Benraiss et al. 2001). In contradiction, others
have shown that BDNF does not affect the sur-
vival of SVZ-derived neurons (Galvão et al.
2008). These different responses to BDNF may
rely on BDNF receptor expression in the different
cells throughout neurogenic process.
Galvão and colleagues found that TrkB knock-
out mice decreased cell proliferation and survival
in the SVZ (Galvão et al. 2008). Accordingly, we
F. F. Ribeiro and S. Xapelli
have demonstrated that in vitro BDNF enhances
cell proliferation and increases the number of
neurospheres, favoring symmetric divisions
toward self-renewal (Ferreira et al. 2018). How-
ever, Tervonen and colleagues demonstrated that
the number of neurosphere-forming progenitors,
obtained from SVZ cells from mice
overexpressing TrkB-T1, was reduced, and apo-
ptosis increased, when compared with WT
neurospheres. Interestingly, these overexpressing
TrkB-T1 cells showed a higher cell proliferation
rate, measured by the increase in the diameter of
postnatal mice SVZ-derived neurospheres
(Tervonen et al. 2006). These results reveal a
role for truncated TrkB receptors in modulating
postnatal neurogenesis, namely, the capacity of
neural progenitors to proliferate, and suggest that
these receptors may be modifying the action
of BDNF.
Work aimed at testing the role of BDNF in the
migration of SVZ-derived cells has been most
consensual. Along the rostral migratory stream
(RMS), BDNF is synthesized and secreted by
the endothelial cells of the blood vessels that
outline the migratory stream (Snapyan et al.
2009), being a key player in the regulation of
neuroblast migration (Snapyan et al. 2009;
Bagley and Belluscio 2010). Indeed, BDNF scav-
enging using TrkB-Fc (a fusion protein compris-
ing the Fc domain of human IgG and the
ectodomain of TrkB) or genetical ablation of
BDNF from vascular endothelial cells decreased
neuroblast migration in vitro and in vivo
(Snapyan et al. 2009). However, the type of
receptors through which BDNF signals, either
TrkB-FL, TrkB-T or p75NTR, to regulate
neuroblast migration is not yet established.
Some studies have shown that TrkB-FL is not
essential for RMS migration (Galvão et al. 2008;
Bergami et al. 2013) since it is not expressed in
neuroblasts until they arrive to the OB, but only in
astrocytes and ependymal cells (Galvão et al.
2008; Bergami et al. 2013). In fact, TrkB-T was
shown to be abundantly expressed by nestin+
cells within the neurosphere, in differentiated
glia cells, and in immature neurons within adher-
ent differentiated neurospheres, whereas TrkB-
FL was expressed in fully differentiated post-
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
mitotic neurons and in immature astrocytes
(Frisén et al. 1993; Rudge et al. 1994; Climent
et al. 2000; Galvão et al. 2008; Islam et al. 2009).
Instead, migrating neuroblasts express TrkB-T1
and p75NTR (Galvão et al. 2008; Snapyan et al.
2009), with p75NTR being pointed as a good
candidate (Snapyan et al. 2009). It is suggested
that BDNF promotes neuroblast migration via
p75NTR. Then, GABA released from neuroblasts
induces Ca2+-dependent insertion of high-affinity
TrkB receptors on the plasma membrane of
astrocytes that surround neuronal precursors. As
a result, astrocytes trap extracellular BDNF,
reducing the amount of BDNF that binds to the
low-affinity receptor p75NTR on neuroblast,
hence regulating cell migration (Snapyan et al.
2009; Grade et al. 2013). In accordance, in
p75NTR null animals, a significant reduction in
OB volume was observed, concomitant with a
decrease in the number of proliferating and
migrating neuroblasts (Young et al. 2007). How-
ever, complete ablation of p75NTR gene in mice
did not affect newly born cell density in the OB
(Bath et al. 2008).
In a similar way to the RMS, BDNF is highly
expressed in the OB (Bath et al. 2008), being
suggested to be released by local glutamatergic
neurons and long projecting neurons from the
anterior olfactory nucleus and the piriform cortex
(Bergami et al. 2013). Only when they arrive to
the OB, immature neurons start to express TrkB-
FL. Through TrkB-FL, BDNF has a physiologi-
cal role during the integration of newborn
neurons, regulating the degree of connectivity in
integrating neurons, particularly in
periglomerular cells, by shaping their dendritic
complexity and spine density, through, respec-
tively, Shc/PI3K and PLCγ activation (Bergami
et al. 2013). Indeed, TrkB-deficient neurons
displayed a marked impairment in dendritic
arborization and spine growth. Moreover, BDNF
has also a critical role in balancing the turnover of
adult-born periglomerular cells, as TrkB deletion
compromised the survival of the new dopaminer-
gic neurons, but not of the granule cells (Bergami
et al. 2013). In accordance, when SVZ cells from
TrkB knockout mice (TrkB-KO) were grafted
into the SVZ of WT, grafted progenitors
105
produced neuroblasts that migrated to the OB in
the absence of TrkB. The survival and differenti-
ation of granule cells and calbindin+
periglomerular interneurons seemed unaffected
by the loss of TrkB, but dopaminergic
periglomerular neurons were reduced (Galvão
et al. 2008). On the other hand, overexpressing
BDNF in the granule cell layer did not affect
survival, as well as the numbers of new granule
cells when mice were sensory deprived by unilat-
eral naris occlusion (McDole et al. 2020).
Although not affecting the turnover of granule
cell layer in vivo, sustained BDNF over-
expression produces a marked increase in granule
cell spine density, leading to spine maturation on
their apical dendrites (McDole et al. 2015). In
fact, BDNF has been previously demonstrated to
promote dendritic growth of SVZ-derived neuro-
nal precursors during the initial 3 days in culture
(Gascon et al. 2005), and to selectively regulate
dendritogenesis of PV-expressing interneurons in
the main OB in vivo (Berghuis et al. 2006). Mor-
phometric analysis demonstrated that changes in
spine density in apical dendrites of granule cell
neurons were most observed in the distal and
proximal domains, suggesting that multiple excit-
atory inputs are regulated by BDNF (McDole
et al. 2015).
In addition to neurogenic regions, intraventric-
ular administration of BDNF for 12 days has been
shown to increase the number of BrdU+ cells in
specific parenchymal structures lining the lateral
and third ventricles, including the striatum, sep-
tum, thalamus, and hypothalamus, thus showing
an association between BDNF and a positive
regulation of adult neurogenesis in these noncon-
ventional neurogenic areas (Pencea et al. 2001).
8.7 Regulation of Adult
Neurogenesis by NGF
The role of pro-NGF in the biology of adult
neural stem cells (NSCs) is still not clear. How-
ever, there are several reports that study the inter-
vention of this proneurotrophin in the adult
hippocampal neurogenesis. Interestingly,
pro-NGF has been reported to be highly
106
expressed in aging brains and in the brains of
patients with mild cognitive impairment and
with Alzheimer’s disease (Peng et al. 2004; Guo
et al. 2013). Indeed pro-NGF accumulation in the
adult hippocampus has been correlated with
memory deficits and loss of cognitive function
(Peng et al. 2004; Guo et al. 2013). Pro-NGF
infusion into adult mouse hippocampus signifi-
cantly reduced the density of BrdU+ cells and the
density of BrdU/DCX-double positive cells in the
SGZ, indicating an inhibitory effect of pro-NGF
on hippocampal neurogenesis. Moreover,
pro-NGF infusion induced prominent cell apopto-
sis and activated resident astrocyte and microglia,
which might further contribute to an impairment
in the hippocampal neurogenesis (Guo et al.
2013). With the high-affinity receptor of
pro-NGF, p75NTR, being expressed on both
proliferating cells and postmitotic mature cells
in the adult mouse hippocampus (Guo et al.
2013), the pro-NGF role in inhibiting
neurogenesis in this region could be possible
through the creation of a toxic signaling complex
by simultaneously binding to p75NTR and
sortilin, given that sortilin is expressed in the
adult hippocampus and is essential for pro-NGF-
induced neuronal cell death (Nykjaer et al. 2004;
Xu et al. 2019). In aging and related
neurodegeneration, studies have shown that the
prefrontal cortex and hippocampus have around
two-fold increase in the levels of the
proneurotrophin, pro-NGF, p75NTR receptors,
and sortilin, and decreased levels of mature
NGF and phospho-TrkA receptors (Jansen et al.
2007; Al-Shawi et al. 2008; Terry et al. 2011;
Meeker and Williams 2014). These data support
the argument that the sortilin/TrkA ratio is
responsible for NGF-altered signaling in the
aging brain, and that such changes may contribute
to an age-related decline in cognitive function. On
the other hand, pro-NGF has been shown to act as
mitogen on hippocampal neurogenesis. Using
AD11 transgenic mice, in which the constitutive
expression of anti-NGF antibody leads to an
imbalance of pro-NGF over mature NGF, an
increase in the proliferation of progenitor cells
F. F. Ribeiro and S. Xapelli
with a reduction in hippocampal neurogenesis
was reported. In accordance, AD11 hippocampal
NSCs proliferated more in vitro but were unable
to differentiate into morphologically mature
neurons. Moreover, radial glial-like cell
expressed high levels of p75NTR and responded
to chronic pro-NGF by reactivating their prolifer-
ation, leading to the formation of neurospheres
(Corvaglia et al. 2019). Altogether, these studies
are consensual in showing that pro-NGF impairs
hippocampal neurogenesis.
Regarding the mature form, NGF is highly
expressed in the adult hippocampus
(Maisonpierre et al. 1990). Chronic intracerebro-
ventricular infusion of NGF for 6 weeks
enhanced recognition memory and increased the
expression of TrkA, synaptogenesis, and cell pro-
liferation in the DG (Birch and Kelly 2013).
However, other data suggest that treating adult
and aged male rats intracerebroventricularly
with NGF for 6 or 20 days increased hippocampal
cholinergic activity, without affecting the prolif-
eration of progenitor cells in the granule cell layer
of the DG. Nevertheless, continuous NGF infu-
sion improved survival of new neurons in the
granule cell layer of adult, but not of aged rats
(Frielingsdorf et al. 2007).
In the absence of exercise, short-term environ-
mental enrichment improves memory, and
increases NGF concentration, early neuronal sur-
vival, and synaptogenesis in the DG in a time-
dependent manner (Birch et al. 2013). A six-week
period of housing in an enriched environment,
without access to exercise equipment, improved
recognition memory. The improvement in cogni-
tive function was coincident with increased
expression of NGF, but not BDNF in the rat
hippocampus, and increased expression of synap-
tic vesicle proteins and cell proliferation in the
DG (Birch et al. 2013).
Very little is known regarding the role of NGF
upon SVZ-derived neurogenesis in physiological
conditions. It was demonstrated that swimming
exercise in adult rats for 5 days/week over a
period of 8 weeks increases neurogenesis, neuro-
nal survival, and neuronal maintenance in the
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
SVZ. At the same time, swimming exercise was
associated to an increase in the levels of NGF in
the OB (Chae et al. 2014). Therefore, it is possible
that NGF may have a role in regulating OB
neurogenesis. Importantly, experiments
performed with adult male Sprague-Dawley rats
subjected to middle cerebral artery occlusion for
2 h showed that treatment with intranasal NGF
failed to increase cell proliferation but improved
survival of newly generated cells in ipsilateral
striatum and SVZ (Zhu et al. 2011). In fact, B
cells in the SVZ do not express TrkA (Delgado
et al. 2014), which is in accordance with the
absence of an effect in cell proliferation. More-
over, cells double labeled with BrdU and the
mature neuronal marker NeuN were increased in
striatum and SVZ in rats treated with intranasal
NGF. Therefore, these results demonstrate that
intranasal NGF promotes neurogenesis and sur-
vival of newly generated cells, an action that was
accompanied by functional recovery after cere-
bral ischemia. (Zhu et al. 2011).
8.8 Regulation of Adult
Neurogenesis by NT-3
Contrary to NGF and BDNF, NT-3 is by far the
most highly expressed neurotrophin in the imma-
ture regions of the CNS in which proliferation,
migration, and differentiation of neuronal
precursors is ongoing. NT-3 expression dramati-
cally decreases with the maturation of these
regions. High levels of NT-3 are synthesized by
discrete populations of neurons and astrocytes in
the nervous system; however, it can be also pro-
duced, at lower levels, by endothelial cells and by
the microvasculature in the choroid plexus.
Therefore, the NT-3 protein is also found in
the CSF.
In the adult hippocampus, in a similar way to
BDNF and NGF, NT-3 is highly expressed
(Maisonpierre et al. 1990) as well as TrkC
receptors (Ip et al. 1993). Indeed, in the adult
brain, the expression of NT-3 is largely confined
to the hippocampal DG (Maisonpierre et al.
107
1990). In fact, the differentiation of NPCs in DG
lacking NT-3 is compromised, rather than cell
proliferation. Particularly, NT-3 affects the num-
ber of newly differentiated neurons, but not glia.
Moreover, NT-3 conditional knockout mice
displayed impaired LTP in the lateral, but not
medial perforant path-granule neuron synapses,
and exhibited deficits in spatial memory tasks,
indicating that NT-3 facilitates hippocampal plas-
ticity and learning and memory by regulating
hippocampal neurogenesis (Shimazu et al. 2006).
Conditional deletion of NT-3 in endothelial
cells leads to the loss of NT-3 protein in both
SVZ and CSF, showing that these cells are a
major source of NT-3 in SVZ stem cell niche.
Type B cells and ependymal cells do not synthe-
size NT-3, but type B cells express TrkC
receptors. In young adult (60-day-old) heterozy-
gous NT-3 mice, the proportion of dividing
BrdU-labeled retaining cells over GFAP+ cells
was increased, which was followed by higher
numbers of OB neurons and significant changes
in olfactory behavior. Moreover, NT-3 promotes
an increase in the number of neurospheres
through a mechanism dependent on TrkC. Inter-
estingly, while heterozygous NT-3 young adult
mice exhibited an increase in cell proliferation, in
middle-aged mice there was a decrease in
neurosphere formation, with B1 stem cells
becoming depleted (Delgado et al. 2014).
8.9 Regulation of Adult
Neurogenesis by NT-4
NT-4 is also known as neurotrophin-5 (NT-5) and
is the least well-understood member of the
neurotrophin family. Together with BDNF,
NT-4 is a ligand for TrkB receptor. Therefore, it
would be expected for them to have similar
effects. In fact, both ligands have been reported
to promote survival and differentiation of hippo-
campal neurons (Ip et al. 1993), as well as neurite
extension and survival of differentiated cerebellar
granule cells, through TrkB (Gao et al. 1995),
among other findings. However, evidence
108
suggests that TrkB receptor can also be differen-
tially stimulated by BDNF and NT-4, and conse-
quently, depending on the nature of the
stimulation, each neurotrophin can trigger differ-
ent synaptic signals, and thus mediate different
effects (Hernandez-Echeagaray 2020). For
instance, both BDNF and NT-4 are known to
modulate corticostriatal synaptic transmission.
When corticostriatal synapses were exposed to
BDNF and NT-4 separately, they increased the
phosphorylated levels of TrkB receptors, thus
demonstrating no differences in the regulation of
the receptor. However, sequential exposure to
both neurotrophins results in different modulatory
effects on corticostriatal transmission, depending
on the exposure order. When NT-4 is added after
BDNF, it elicits an antagonistic effect, with
phosphorylated levels of TrkB being reduced.
On the other hand, if BDNF follows NT-4, it
elicits a synergistic effect, with activated levels
of TrkB being enhanced. Each effect was
demonstrated to be respectively dependent on
TrkB-T and TrkB-FL (Torres-Cruz et al. 2019).
Moreover, exposure to an enriched environment
is known to enhance the number of newly
generated neurons in the DG. WT mice and
NT-4–/– mice placed in an enriched environment
for 8 weeks showed a two-fold increase in the
mitotic marker BrdU in the DG, as well as in the
number of cells double-stained for BrdU and the
neuronal marker NeuN. This enhancement in hip-
pocampal neurogenesis was, however, not
observed in BDNF+/– mice under enriched
conditions. As a consequence, these data demon-
strate that NT-4 is not required for the environ-
mental induction of neurogenesis, although
BDNF is (Rossi et al. 2006).
Consequently, besides NT-4 acting on the
same receptor as BDNF, the role of the two
neurotrophins upon adult neurogenesis may be
different. These different actions could be depen-
dent on the different availability of each TrkB
isoform on the membrane surface, as well as in
the extracellular concentration of each ligand and
their different affinity for each receptor. This is an
area that is very poorly explored and whose
knowledge will contribute to further understand
the role of TrkB signaling.
F. F. Ribeiro and S. Xapelli
8.10 Conclusion
BDNF is the neurotrophin that has been the center
of most attention over the last two decades, in
terms of regulating adult neurogenesis. Alto-
gether, this chapter strongly suggests that BDNF
is an important regulator at different levels of the
process, whether through binding as a
pro-neurotrophin or as a mature neurotrophin to
TrkB and/or p75NTR and sortilin. All these dif-
ferent mechanisms may result in distinct actions
of BDNF, demonstrating the richness and fine-
tune regulation of adult neurogenesis exerted by
this molecule. Hence, this field needs and
deserves to be further explored to contribute to a
better and deeper knowledge about the actions of
BDNF and its receptors in physiological and
pathological conditions.
Although being less explored, the few studies
that focus on the actions of NGF, NT-3, and NT-4
also strongly suggest an important role for these
neurotrophins in modulating adult neurogenesis.
Alterations in the expression levels of some of
these mature and pro-neurotrophins are observed
with aging and in neurodegenerative diseases.
Therefore, future research should pay attention
to these ligands to further understand the intricate
modulatory mechanisms of adult neurogenesis
that involve all the neurotrophins, and therefore
contribute to develop new strategies for brain
regeneration.
Acknowledgments This work was supported by
IF/01227/2015 project funded by Fundação para a Ciência
e a Tecnologia (FCT). F.F.R. (IMM/CT/35-2018) received
a fellowship from FCT.
References
Adachi N, Kohara K, Tsumoto T (2005) Difference in
trafficking of brain-derived neurotrophic factor
between axons and dendrites of cortical neurons,
revealed by live-cell imaging. BMC Neurosci 6:42.
https://doi.org/10.1186/1471-2202-6-42
Agrimi J, Spalletti C, Baroni C et al (2019) Obese mice
exposed to psychosocial stress display cardiac and
hippocampal dysfunction associated with local brain-
derived neurotrophic factor depletion. EBioMedicine
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
47:384–401. https://doi.org/10.1016/j.ebiom.2019.08.
042
Aid T, Kazantseva A, Piirsoo M et al (2007) Mouse and rat
BDNF gene structure and expression revisited. J
Neurosci Res 85:525–535. https://doi.org/10.1002/jnr.
21139
Aliaga EE, Mendoza I, Tapia-Arancibia L (2009) Distinct
subcellular localization of BDNF transcripts in
cultured hypothalamic neurons and modification by
neuronal activation. J Neural Transm (Vienna)
116:23–32. https://doi.org/10.1007/s00702-008-0159-
8 Berchtold NC, Chinn G, Chou M et al (2005) Exercise
Al-Shawi R, Hafner A, Olsen J et al (2008) Neurotoxic and
neurotrophic roles of proNGF and the receptor sortilin
in the adult and ageing nervous system. Eur J Neurosci
27:2103–2114. https://doi.org/10.1111/j.1460-9568.
2008.06152.x
An JJ, Gharami K, Liao G-Y et al (2008) Distinct role of
long 30 UTR BDNF mRNA in spine morphology and
synaptic plasticity in hippocampal neurons. Cell
134:175–187. https://doi.org/10.1016/j.cell.2008.05.
045
Aroeira RI, Sebastião AM, Valente CA (2015) BDNF, via
truncated TrkB receptor, modulates GlyT1 and GlyT2
in astrocytes. Glia 63:2181–2197. https://doi.org/10.
1002/glia.22884
Babu H, Ramirez-Rodriguez G, Fabel K et al (2009)
Synaptic network activity induces neuronal differenti-
ation of adult hippocampal precursor cells through
BDNF signaling. Front Neurosci 3:49. https://doi.org/
10.3389/neuro.22.001.2009
Bagley JA, Belluscio L (2010) Dynamic imaging reveals
that brain-derived neurotrophic factor can indepen-
dently regulate motility and direction of neuroblasts
within the rostral migratory stream. Neuroscience
169:1449–1461. https://doi.org/10.1016/j.neurosci
ence.2010.05.075
Baik S-H, Rajeev V, Fann DY-W et al (2020) Intermittent
fasting increases adult hippocampal neurogenesis.
Brain Behav 10:e01444. https://doi.org/10.1002/brb3.
1444
Baj G, Leone E, Chao MV, Tongiorgi E (2011) Spatial
segregation of BDNF transcripts enables BDNF to
differentially shape distinct dendritic compartments.
PNAS 108:16813–16818. https://doi.org/10.1073/
pnas.1014168108
Barker PA (2004) p75NTR is positively promiscuous:
novel partners and new insights. Neuron 42:529–533.
https://doi.org/10.1016/j.neuron.2004.04.001
Barrett GL, Greferath U, Barker PA et al (2005)
Co-expression of the P75 neurotrophin receptor and
neurotrophin receptor-interacting melanoma antigen
homolog in the mature rat brain. Neuroscience
133:381–392. https://doi.org/10.1016/j.neuroscience.
2005.01.067
Bath KG, Mandairon N, Jing D et al (2008) Variant brain-
derived neurotrophic factor (Val66Met) alters adult
olfactory bulb neurogenesis and spontaneous olfactory
109
discrimination. J Neurosci 28:2383–2393. https://doi.
org/10.1523/JNEUROSCI.4387-07.2008
Baxter GT, Radeke MJ, Kuo RC et al (1997) Signal
transduction mediated by the truncated trkB receptor
isoforms, trkB.T1 and trkB.T2. J Neurosci
17:2683–2690
Benraiss A, Chmielnicki E, Lerner K et al (2001) Adeno-
viral brain-derived neurotrophic factor induces both
neostriatal and olfactory neuronal recruitment from
endogenous progenitor cells in the adult forebrain. J
Neurosci 21:6718–6731
primes a molecular memory for brain-derived
neurotrophic factor protein induction in the rat hippo-
campus. Neuroscience 133:853–861. https://doi.org/
10.1016/j.neuroscience.2005.03.026
Bergami M, Rimondini R, Santi S et al (2008) Deletion of
TrkB in adult progenitors alters newborn neuron inte-
gration into hippocampal circuits and increases
anxiety-like behavior. Proc Natl Acad Sci U S A
105:15570–15575. https://doi.org/10.1073/pnas.
0803702105
Bergami M, Vignoli B, Motori E et al (2013) TrkB signal-
ing directs the incorporation of newly generated
periglomerular cells in the adult olfactory bulb. J
Neurosci 33:11464–11478. https://doi.org/10.1523/
JNEUROSCI.4812-12.2013
Berghuis P, Agerman K, Dobszay MB et al (2006) Brain-
derived neurotrophic factor selectively regulates
dendritogenesis of parvalbumin-containing
interneurons in the main olfactory bulb through the
PLCgamma pathway. J Neurobiol 66:1437–1451.
https://doi.org/10.1002/neu.20319
Bernabeu RO, Longo FM (2010) The p75 neurotrophin
receptor is expressed by adult mouse dentate progeni-
tor cells and regulates neuronal and non-neuronal cell
genesis. BMC Neurosci 11:136. https://doi.org/10.
1186/1471-2202-11-136
Biffo S, Offenhäuser N, Carter BD, Barde YA (1995)
Selective binding and internalisation by truncated
receptors restrict the availability of BDNF during
development. Development 121:2461–2470
Birch AM, Kelly ÁM (2013) Chronic intracerebroven-
tricular infusion of nerve growth factor improves rec-
ognition memory in the rat. Neuropharmacology
75:255–261. https://doi.org/10.1016/j.neuropharm.
2013.07.023
Birch AM, McGarry NB, Kelly ÁM (2013) Short-term
environmental enrichment, in the absence of exercise,
improves memory, and increases NGF concentration,
early neuronal survival, and synaptogenesis in the den-
tate gyrus in a time-dependent manner. Hippocampus
23:437–450. https://doi.org/10.1002/hipo.22103
Bothwell M (2014) NGF, BDNF, NT3, and NT4. Handb
Exp Pharmacol 220:3–15. https://doi.org/10.1007/978-
3-642-45106-5_1
Chae C-H, Jung S-L, An S-H et al (2014) Swimming
exercise stimulates neuro-genesis in the subventricular
zone via increase in synapsin I and nerve growth factor
110
levels. Biol Sport 31:309–314. https://doi.org/10.5604/
20831862.1132130
Chan JP, Cordeira J, Calderon GA et al (2008) Depletion
of central BDNF in mice impedes terminal differentia-
tion of new granule neurons in the adult hippocampus.
Mol Cell Neurosci 39:372–383. https://doi.org/10.
1016/j.mcn.2008.07.017
Chao MV (2003) Neurotrophins and their receptors: a
convergence point for many signalling pathways. Nat
Rev Neurosci 4:299–309. https://doi.org/10.1038/
nrn1078
Chen J, Li C-R, Yang H et al (2016) proBDNF Attenuates
Hippocampal Neurogenesis and Induces Learning and
Memory Deficits in Aged Mice. Neurotox Res
29:47–53. https://doi.org/10.1007/s12640-015-9568-2
Cheng P-L, Song A-H, Wong Y-H et al (2011) Self-
amplifying autocrine actions of BDNF in axon devel-
opment. Proc Natl Acad Sci U S A 108:18430–18435.
https://doi.org/10.1073/pnas.1115907108
Choi SH, Li Y, Parada LF, Sisodia SS (2009) Regulation
of hippocampal progenitor cell survival, proliferation
and dendritic development by BDNF. Mol
Neurodegener 4:52. https://doi.org/10.1186/1750-
1326-4-52
Choi SH, Bylykbashi E, Chatila ZK et al (2018) Combined
adult neurogenesis and BDNF mimic exercise effects
on cognition in an Alzheimer’s mouse model. Science
361(6406):eaan8821. https://doi.org/10.1126/science.
aan8821
Climent E, Sancho-Tello M, Miñana R et al (2000)
Astrocytes in culture express the full-length Trk-B
receptor and respond to brain derived neurotrophic
factor by changing intracellular calcium levels: effect
of ethanol exposure in rats. Neurosci Lett 288:53–56
Colliva A, Tongiorgi E (2020) Distinct role of 50 UTR
sequences in dendritic trafficking of BDNF mRNA:
additional mechanisms for the BDNF splice variants
spatial code. Mol Brain. https://doi.org/10.1186/
s13041-020-00680-8
Conner JM, Lauterborn JC, Yan Q et al (1997) Distribu-
tion of brain-derived neurotrophic factor (BDNF) pro-
tein and mRNA in the normal adult rat CNS: evidence
for anterograde axonal transport. J Neurosci
17:2295–2313
Corvaglia V, Cilli D, Scopa C et al (2019) ProNGF is a
cell-type-specific mitogen for adult hippocampal and
for induced neural stem cells. Stem Cells
37:1223–1237. https://doi.org/10.1002/stem.3037
Cotman CW, Berchtold NC (2002) Exercise: a behavioral
intervention to enhance brain health and plasticity.
Trends Neurosci 25:295–301
Delgado AC, Ferrón SR, Vicente D et al (2014) Endothe-
lial NT-3 delivered by vasculature and CSF promotes
quiescence of subependymal neural stem cells through
nitric oxide induction. Neuron 83:572–585. https://doi.
org/10.1016/j.neuron.2014.06.015
Dieni S, Matsumoto T, Dekkers M et al (2012) BDNF and
its pro-peptide are stored in presynaptic dense core
F. F. Ribeiro and S. Xapelli
vesicles in brain neurons. J Cell Biol 196:775–788.
https://doi.org/10.1083/jcb.201201038
Ding Q, Ying Z, Gómez-Pinilla F (2011) Exercise
influences hippocampal plasticity by modulating
BDNF processing. Neuroscience 192:773–780.
https://doi.org/10.1016/j.neuroscience.2011.06.032
Donovan MH, Yamaguchi M, Eisch AJ (2008) Dynamic
expression of TrkB receptor protein on proliferating
and maturing cells in the adult mouse dentate gyrus.
Hippocampus 18:435–439. https://doi.org/10.1002/
hipo.20410
Dougherty KD, Milner TA (1999) p75NTR immunoreac-
tivity in the rat dentate gyrus is mostly within presyn-
aptic profiles but is also found in some astrocytic and
postsynaptic profiles. J Comp Neurol 407:77–91.
https://doi.org/10.1002/(sici)1096-9861(19990428)
407:1<77::aid-cne6>3.0.co;2-s
Eide FF, Vining ER, Eide BL et al (1996) Naturally
occurring truncated trkB receptors have dominant
inhibitory effects on brain-derived neurotrophic factor
signaling. J Neurosci 16:3123–3129
Erickson KI, Voss MW, Prakash RS et al (2011) Exercise
training increases size of hippocampus and improves
memory. Proc Natl Acad Sci USA 108:3017–3022.
https://doi.org/10.1073/pnas.1015950108
Ferreira FF, Ribeiro FF, Rodrigues RS et al (2018) Brain-
derived neurotrophic factor (BDNF) role in
cannabinoid-mediated neurogenesis. Front Cell
Neurosci 12:441. https://doi.org/10.3389/fncel.2018.
00441
Foltran RB, Diaz SL (2016) BDNF isoforms: a round trip
ticket between neurogenesis and serotonin? J
Neurochem 138:204–221. https://doi.org/10.1111/jnc.
13658
Fonseca-Gomes J, Jerónimo-Santos A, Lesnikova A et al
(2019) TrkB-ICD fragment, originating from BDNF
receptor cleavage, is translocated to cell nucleus and
phosphorylates nuclear and axonal proteins. Front Mol
Neurosci 12:4. https://doi.org/10.3389/fnmol.2019.
00004
Frielingsdorf H, Simpson DR, Thal LJ, Pizzo DP (2007)
Nerve growth factor promotes survival of new neurons
in the adult hippocampus. Neurobiol Dis 26:47–55.
https://doi.org/10.1016/j.nbd.2006.11.015
Frisén J, Verge VM, Fried K et al (1993) Characterization
of glial trkB receptors: differential response to injury in
the central and peripheral nervous systems. Proc Natl
Acad Sci USA 90:4971–4975
Fujii T, Kunugi H (2009) p75NTR as a therapeutic target
for neuropsychiatric diseases. Curr Mol Pharmacol
2:70–76. https://doi.org/10.2174/
1874467210902010070
Galvão RP, Garcia-Verdugo JM, Alvarez-Buylla A (2008)
Brain-derived neurotrophic factor signaling does not
stimulate subventricular zone neurogenesis in adult
mice and rats. J Neurosci 28:13368–13383. https://
doi.org/10.1523/JNEUROSCI.2918-08.2008
Gao WQ, Zheng JL, Karihaloo M (1995) Neurotrophin-4/
5 (NT-4/5) and brain-derived neurotrophic factor
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
(BDNF) act at later stages of cerebellar granule cell
differentiation. J Neurosci 15(4):2656–2667. https://
pubmed.ncbi.nlm.nih.gov/7722620/. Accessed 10 Sept
2020
Gao X, Smith GM, Chen J (2009) Impaired dendritic
development and synaptic formation of postnatal-
born dentate gyrus granular neurons in the absence of
brain-derived neurotrophic factor signaling. Exp
Neurol 215:178–190. https://doi.org/10.1016/j.
expneurol.2008.10.009
Gascon E, Vutskits L, Zhang H et al (2005) Sequential
activation of p75 and TrkB is involved in dendritic
development of subventricular zone-derived neuronal
progenitors in vitro. Eur J Neurosci 21:69–80. https://
doi.org/10.1111/j.1460-9568.2004.03849.x
Grade S, Weng YC, Snapyan M et al (2013) Brain-derived
neurotrophic factor promotes vasculature-associated
migration of neuronal precursors toward the ischemic
striatum. PLoS One 8:e55039. https://doi.org/10.1371/
journal.pone.0055039
Groves N, O’Keeffe I, Lee W et al (2019) Blockade of
TrkB but not p75NTR activates a subpopulation of
quiescent neural precursor cells and enhances
neurogenesis in the adult mouse hippocampus. Dev
Neurobiol 79:868–879. https://doi.org/10.1002/dneu.
22729
Guo J, Wang J, Zhang Z et al (2013) proNGF inhibits
neurogenesis and induces glial activation in adult
mouse dentate gyrus. Neurochem Res 38:1695–1703.
https://doi.org/10.1007/s11064-013-1071-7
Haapasalo A, Sipola I, Larsson K et al (2002) Regulation
of TRKB surface expression by brain-derived
neurotrophic factor and truncated TRKB isoforms. J
Biol Chem 277:43160–43167. https://doi.org/10.1074/
jbc.M205202200
Halban PA, Irminger JC (1994) Sorting and processing of
secretory proteins. Biochem J 299(Pt 1):1–18
Hanson ND, Owens MJ, Nemeroff CB (2011) Depression,
antidepressants, and neurogenesis: a critical reap-
praisal. Neuropsychopharmacology 36:2589–2602.
https://doi.org/10.1038/npp.2011.220
Henry RA, Hughes SM, Connor B (2007) AAV-mediated
delivery of BDNF augments neurogenesis in the nor-
mal and quinolinic acid-lesioned adult rat brain. Eur J
Neurosci 25:3513–3525. https://doi.org/10.1111/j.
1460-9568.2007.05625.x
Hernandez-Echeagaray E (2020) The role of the TrkB-T1
receptor in the neurotrophin-4/5 antagonism of brain-
derived neurotrophic factor on corticostriatal synaptic
transmission. Neural Regen Res 15:1973–1976.
https://doi.org/10.4103/1673-5374.282224
Hock C, Heese K, Hulette C et al (2000) Region-specific
neurotrophin imbalances in Alzheimer disease:
decreased levels of brain-derived neurotrophic factor
and increased levels of nerve growth factor in hippo-
campus and cortical areas. Arch Neurol 57:846–851.
https://doi.org/10.1001/archneur.57.6.846
Holt LM, Hernandez RD, Pacheco NL et al (2019) Astro-
cyte morphogenesis is dependent on BDNF signaling
111
via astrocytic TrkB.T1. eLife 8:e44667. https://doi.org/
10.7554/eLife.44667
Hosomi S, Yamashita T, Aoki M, Tohyama M (2003) The
p75 receptor is required for BDNF-induced differenti-
ation of neural precursor cells. Biochem Biophys Res
Commun 301:1011–1015
Huang EJ, Reichardt LF (2001) Neurotrophins: roles in
neuronal development and function. Annu Rev
Neurosci 24:677–736. https://doi.org/10.1146/
annurev.neuro.24.1.677
Hwang JJ, Park M-H, Choi S-Y, Koh J-Y (2005) Activa-
tion of the Trk signaling pathway by extracellular zinc.
Role of metalloproteinases. J Biol Chem
280:11995–12001. https://doi.org/10.1074/jbc.
M403172200
Ip N, Li Y, Yancopoulos G, Lindsay R (1993) Cultured
hippocampal neurons show responses to BDNF, NT-3,
and NT-4, but not NGF. J Neurosci 13:3394–3405.
https://doi.org/10.1523/JNEUROSCI.13-08-03394.
1993
Islam O, Loo TX, Heese K (2009) Brain-derived
neurotrophic factor (BDNF) has proliferative effects
on neural stem cells through the truncated TRK-B
receptor, MAP kinase, AKT, and STAT-3 signaling
pathways. Curr Neurovasc Res 6:42–53
Ivanova T, Beyer C (2001) Pre- and postnatal expression
of brain-derived neurotrophic factor mRNA/protein
and tyrosine protein kinase receptor B mRNA in the
mouse hippocampus. Neurosci Lett 307:21–24. https://
doi.org/10.1016/s0304-3940(01)01905-x
Jansen P, Giehl K, Nyengaard JR et al (2007) Roles for the
pro-neurotrophin receptor sortilin in neuronal develop-
ment, aging and brain injury. Nat Neurosci
10:1449–1457. https://doi.org/10.1038/nn2000
Jerónimo-Santos A, Vaz SH, Parreira S et al (2015)
Dysregulation of TrkB receptors and BDNF function
by amyloid-β peptide is mediated by calpain. Cereb
Cortex 25:3107–3121. https://doi.org/10.1093/cercor/
bhu105
Jha S, Dong B, Sakata K (2011) Enriched environment
treatment reverses depression-like behavior and
restores reduced hippocampal neurogenesis and pro-
tein levels of brain-derived neurotrophic factor in mice
lacking its expression through promoter IV. Transl
Psychiatry 1:e40. https://doi.org/10.1038/tp.2011.33
Jin J-J, Ko I-G, Kim S-E et al (2017) Age-dependent
differences of treadmill exercise on spatial learning
ability between young- and adult-age rats. J Exerc
Rehabil 13:381–386. https://doi.org/10.12965/jer.
1735070.535
Katoh-Semba R, Takeuchi IK, Semba R, Kato K (1997)
Distribution of brain-derived neurotrophic factor in rats
and its changes with development in the brain. J
Neurochem 69:34–42
Kim K, Sung Y-H, Seo J-H et al (2015) Effects of tread-
mill exercise-intensity on short-term memory in the
rats born of the lipopolysaccharide-exposed maternal
rats. J Exerc Rehabil 11:296–302. https://doi.org/10.
12965/jer.150264
112
Kirschenbaum B, Goldman SA (1995) Brain-derived
neurotrophic factor promotes the survival of neurons
arising from the adult rat forebrain subependymal
zone. Proc Natl Acad Sci USA 92:210–214
Klein R, Conway D, Parada LF, Barbacid M (1990) The
trkB tyrosine protein kinase gene codes for a second
neurogenic receptor that lacks the catalytic kinase
domain. Cell 61:647–656. https://doi.org/10.1016/
0092-8674(90)90476-U
Kobilo T, Liu Q-R, Gandhi K et al (2011) Running is the
neurogenic and neurotrophic stimulus in environmen-
tal enrichment. Learn Mem 18:605–609. https://doi.
org/10.1101/lm.2283011
Kolbeck R, Jungbluth S, Barde Y-A (1994)
Characterisation of neurotrophin dimers and
monomers. Eur J Biochem 225:995–1003. https://doi.
org/10.1111/j.1432-1033.1994.0995b.x
Kuzumaki N, Ikegami D, Tamura R et al (2011) Hippo-
campal epigenetic modification at the brain-derived
neurotrophic factor gene induced by an enriched envi-
ronment. Hippocampus 21:127–132. https://doi.org/
10.1002/hipo.20775
Lau AG, Irier HA, Gu J et al (2010) Distinct 30 UTRs
differentially regulate activity-dependent translation
of brain-derived neurotrophic factor (BDNF). Proc
Natl Acad Sci USA 107:15945–15950. https://doi.
org/10.1073/pnas.1002929107
Lee R, Kermani P, Teng KK, Hempstead BL (2001) Reg-
ulation of cell survival by secreted proneurotrophins.
Science 294:1945–1948. https://doi.org/10.1126/sci
ence.1065057
Lee J, Seroogy KB, Mattson MP (2002) Dietary restriction
enhances neurotrophin expression and neurogenesis in
the hippocampus of adult mice. J Neurochem
80:539–547
Lessmann V, Gottmann K, Malcangio M (2003)
Neurotrophin secretion: current facts and future
prospects. Prog Neurobiol 69:341–374
Li Y, Luikart BW, Birnbaum S et al (2008) TrkB regulates
hippocampal neurogenesis and governs sensitivity to
antidepressive treatment. Neuron 59:399–412. https://
doi.org/10.1016/j.neuron.2008.06.023
Linnarsson S, Willson CA, Ernfors P (2000) Cell death in
regenerating populations of neurons in BDNF mutant
mice. Brain Res Mol Brain Res 75:61–69
Lu J-J, Yang M, Sun Y, Zhou X-F (2014) Synthesis,
trafficking and release of BDNF. In: Kostrzewa RM
(ed) Handbook of neurotoxicity. Springer, New York,
pp 1955–1971
Luberg K, Wong J, Weickert CS, Timmusk T (2010)
Human TrkB gene: novel alternative transcripts, pro-
tein isoforms and expression pattern in the prefrontal
cerebral cortex during postnatal development. J
Neurochem 113:952–964. https://doi.org/10.1111/j.
1471-4159.2010.06662.x
Ma Z, Zang T, Birnbaum SG et al (2017) TrkB dependent
adult hippocampal progenitor differentiation mediates
sustained ketamine antidepressant response. Nat
F. F. Ribeiro and S. Xapelli
Commun 8:1668. https://doi.org/10.1038/s41467-
017-01709-8
Maisonpierre PC, Belluscio L, Friedman B et al (1990)
NT-3, BDNF, and NGF in the developing rat nervous
system: parallel as well as reciprocal patterns of
expression. Neuron 5:501–509. https://doi.org/10.
1016/0896-6273(90)90089-x
Marlatt MW, Potter MC, Lucassen PJ, van Praag H (2012)
Running throughout middle-age improves memory
function, hippocampal neurogenesis, and BDNF levels
in female C57BL/6J mice. Dev Neurobiol 72:943–952.
https://doi.org/10.1002/dneu.22009
Matyas JJ, O’Driscoll CM, Yu L et al (2017) Truncated
TrkB.T1-mediated astrocyte dysfunction contributes to
impaired motor function and neuropathic pain after
spinal cord injury. J Neurosci 37:3956–3971. https://
doi.org/10.1523/JNEUROSCI.3353-16.2017
Maynard KR, Hobbs JW, Sukumar M et al (2017) Bdnf
mRNA splice variants differentially impact CA1 and
CA3 dendrite complexity and spine morphology in the
hippocampus. Brain Struct Funct 222:3295–3307.
https://doi.org/10.1007/s00429-017-1405-3
McDole B, Isgor C, Pare C, Guthrie K (2015) BDNF over-
expression increases olfactory bulb granule cell den-
dritic spine density in vivo. Neuroscience
304:146–160. https://doi.org/10.1016/j.neuroscience.
2015.07.056
McDole B, Berger R, Guthrie K (2020) Genetic increases
in olfactory Bulb BDNF do not enhance survival of
adult-born granule cells. Chem Senses 45:3–13. https://
doi.org/10.1093/chemse/bjz058
Meeker R, Williams K (2014) Dynamic nature of the p75
neurotrophin receptor in response to injury and disease.
J Neuroimmune Pharmacol 9:615–628. https://doi.org/
10.1007/s11481-014-9566-9
Middlemas DS, Lindberg RA, Hunter T (1991) trkB, a
neural receptor protein-tyrosine kinase: evidence for a
full-length and two truncated receptors. Mol Cell Biol
11:143–153. https://doi.org/10.1128/MCB.11.1.143
Mizoguchi H, Nakade J, Tachibana M et al (2011) Matrix
metalloproteinase-9 contributes to kindled seizure
development in pentylenetetrazole-treated mice by
converting pro-BDNF to mature BDNF in the hippo-
campus. J Neurosci 31:12963–12971. https://doi.org/
10.1523/JNEUROSCI.3118-11.2011
Mizui T et al (2017) BDNF pro-peptide: a novel synaptic
modulator generated as an N-terminal fragment from
the BDNF precursor by proteolytic processing. Neural
Regen Res 12(7):1024–1027. http://www.nrronline.
o r g / a r t i c l e . a s p ? i s s n ¼1 6 7 3 - 5 3 7 4 ; y e a r ¼2 0 1 7 ;
volume¼12;issue¼7;spage¼1024;epage¼1027;
aulast¼Mizui. Accessed 9 Sept 2020
Mowla SJ, Pareek S, Farhadi HF et al (1999) Differential
sorting of nerve growth factor and brain-derived
neurotrophic factor in hippocampal neurons. J
Neurosci 19:2069–2080
Mowla SJ, Farhadi HF, Pareek S et al (2001) Biosynthesis
and post-translational processing of the precursor to
brain-derived neurotrophic factor. J Biol Chem
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
276:12660–12666. https://doi.org/10.1074/jbc.
M008104200
Nagappan G, Zaitsev E, Senatorov VV et al (2009) Con-
trol of extracellular cleavage of ProBDNF by high
frequency neuronal activity. Proc Natl Acad Sci USA
106:1267–1272. https://doi.org/10.1073/pnas.
0807322106
Neeper SA, Gómez-Pinilla F, Choi J, Cotman C (1995)
Exercise and brain neurotrophins. Nature 373:109.
https://doi.org/10.1038/373109a0
Nibuya M, Morinobu S, Duman RS (1995) Regulation of
BDNF and trkB mRNA in rat brain by chronic electro-
convulsive seizure and antidepressant drug treatments.
J Neurosci 15:7539–7547
Nykjaer A, Lee R, Teng KK et al (2004) Sortilin is essen-
tial for proNGF-induced neuronal cell death. Nature
427:843–848. https://doi.org/10.1038/nature02319
Ohira K, Kumanogoh H, Sahara Y et al (2005) A truncated
tropomyosin-related kinase B receptor, T1, regulates
glial cell morphology via Rho GDP dissociation inhib-
itor 1. J Neurosci 25:1343–1353. https://doi.org/10.
1523/JNEUROSCI.4436-04.2005
Ortiz-López L, Vega-Rivera NM, Babu H, Ramírez-
Rodríguez GB (2017) Brain-derived neurotrophic fac-
tor induces cell survival and the migration of murine
adult hippocampal precursor cells during differentia-
tion in vitro. Neurotox Res 31:122–135. https://doi.
org/10.1007/s12640-016-9673-x
Pang PT, Teng HK, Zaitsev E et al (2004) Cleavage of
proBDNF by tPA/plasmin is essential for long-term
hippocampal plasticity. Science 306:487–491. https://
doi.org/10.1126/science.1100135
Pencea V, Bingaman KD, Wiegand SJ, Luskin MB (2001)
Infusion of brain-derived neurotrophic factor into the
lateral ventricle of the adult rat leads to new neurons in
the parenchyma of the striatum, septum, thalamus, and
hypothalamus. J Neurosci 21:6706–6717
Peng S, Wuu J, Mufson EJ, Fahnestock M (2004)
Increased proNGF levels in subjects with mild cogni-
tive impairment and mild Alzheimer disease. J
Neuropathol Exp Neurol 63:641–649. https://doi.org/
10.1093/jnen/63.6.641
Pruunsild P, Kazantseva A, Aid T et al (2007) Dissecting
the human BDNF locus: bidirectional transcription,
complex splicing, and multiple promoters. Genomics
90:397–406. https://doi.org/10.1016/j.ygeno.2007.05.
004
Reichardt LF (2003) Neurobiology: signals that make
waves. Nature 426:25–26. https://doi.org/10.1038/
426025a
Reichardt LF (2006) Neurotrophin-regulated signalling
pathways. Philos Trans R Soc Lond, B, Biol Sci
361:1545–1564. https://doi.org/10.1098/rstb.2006.
1894
Reinhart V, Bove SE, Volfson D et al (2015) Evaluation of
TrkB and BDNF transcripts in prefrontal cortex, hip-
pocampus, and striatum from subjects with schizophre-
nia, bipolar disorder, and major depressive disorder.
113
Neurobiol Dis 77:220–227. https://doi.org/10.1016/j.
nbd.2015.03.011
Ribeiro FF (2017) Unveiling the trophic actions of adeno-
sine A2A receptors in neurite outgrowth and postnatal
neurogenesis: interaction with brain-derived
neurotrophic factor. PhD thesis. Universidade de
Lisboa, Lisbon, Portugal
Rose CR, Blum R, Pichler B et al (2003) Truncated TrkB-
T1 mediates neurotrophin-evoked calcium signalling
in glia cells. Nature 426:74–78. https://doi.org/10.
1038/nature01983
Rossi C, Angelucci A, Costantin L et al (2006) Brain-
derived neurotrophic factor (BDNF) is required for
the enhancement of hippocampal neurogenesis follow-
ing environmental enrichment. Eur J Neurosci
24:1850–1856. https://doi.org/10.1111/j.1460-9568.
2006.05059.x
Rudge JS, Li Y, Pasnikowski EM et al (1994)
Neurotrophic factor receptors and their signal transduc-
tion capabilities in rat astrocytes. Eur J Neurosci
6:693–705
Russo-Neustadt AA, Beard RC, Huang YM, Cotman CW
(2000) Physical activity and antidepressant treatment
potentiate the expression of specific brain-derived
neurotrophic factor transcripts in the rat hippocampus.
Neuroscience 101:305–312
Sairanen M, Lucas G, Ernfors P et al (2005) Brain-derived
neurotrophic factor and antidepressant drugs have dif-
ferent but coordinated effects on neuronal turnover,
proliferation, and survival in the adult dentate gyrus.
J Neurosci 25:1089–1094. https://doi.org/10.1523/
JNEUROSCI.3741-04.2005
Sakata K, Woo NH, Martinowich K et al (2009) Critical
role of promoter IV-driven BDNF transcription in
GABAergic transmission and synaptic plasticity in
the prefrontal cortex. Proc Natl Acad Sci U S A
106:5942–5947. https://doi.org/10.1073/pnas.
0811431106
Sarret P, Krzywkowski P, Segal L et al (2003) Distribution
of NTS3 receptor/sortilin mRNA and protein in the rat
central nervous system. J Comp Neurol 461:483–505.
https://doi.org/10.1002/cne.10708
Scharfman H, Goodman J, Macleod A et al (2005)
Increased neurogenesis and the ectopic granule cells
after intrahippocampal BDNF infusion in adult rats.
Exp Neurol 192:348–356. https://doi.org/10.1016/j.
expneurol.2004.11.016
Segawa M, Morinobu S, Matsumoto T et al (2013) Elec-
troconvulsive seizure, but not imipramine, rapidly
up-regulates pro-BDNF and t-PA, leading to mature
BDNF production, in the rat hippocampus. Int J
Neuropsychopharmacol 16:339–350. https://doi.org/
10.1017/S1461145712000053
Seidah NG, Benjannet S, Pareek S et al (1996) Cellular
processing of the nerve growth factor precursor by the
mammalian pro-protein convertases. Biochem J 314
(Pt 3):951–960
Shelly M, Cancedda L, Heilshorn S et al (2007) LKB1/
STRAD promotes axon initiation during neuronal
114
polarization. Cell 129:565–577. https://doi.org/10.
1016/j.cell.2007.04.012
Shimazu K, Zhao M, Sakata K et al (2006) NT-3 facilitates
hippocampal plasticity and learning and memory by
regulating neurogenesis. Learn Mem 13:307–315.
https://doi.org/10.1101/lm.76006
Shirayama Y, Chen AC-H, Nakagawa S et al (2002)
Brain-derived neurotrophic factor produces antidepres-
sant effects in behavioral models of depression. J
Neurosci 22:3251–3261. https://doi.org/10.1523/
JNEUROSCI.22-08-03251.2002
Smith MA, Zhang LX, Lyons WE, Mamounas LA (1997)
Anterograde transport of endogenous brain-derived
neurotrophic factor in hippocampal mossy fibers.
Neuroreport 8:1829–1834. https://doi.org/10.1097/
00001756-199705260-00008
Snapyan M, Lemasson M, Brill MS et al (2009) Vascula-
ture guides migrating neuronal precursors in the adult
mammalian forebrain via brain-derived neurotrophic
factor signaling. J Neurosci 29:4172–4188. https://
doi.org/10.1523/JNEUROSCI.4956-08.2009
Stoilov P, Castren E, Stamm S (2002) Analysis of the
human TrkB gene genomic organization reveals
novel TrkB isoforms, unusual gene length, and splic-
ing mechanism. Biochem Biophys Res Commun
290:1054–1065. https://doi.org/10.1006/bbrc.2001.
6301
Sun Y, Lim Y, Li F et al (2012) ProBDNF collapses
neurite outgrowth of primary neurons by activating
RhoA. PLoS One 7:e35883. https://doi.org/10.1371/
journal.pone.0035883
Taliaz D, Stall N, Dar DE, Zangen A (2010) Knockdown
of brain-derived neurotrophic factor in specific brain
sites precipitates behaviors associated with depression
and reduces neurogenesis. Mol Psychiatry 15:80–92.
https://doi.org/10.1038/mp.2009.67
Tanqueiro SR, Ramalho RM, Rodrigues TM et al (2018)
Inhibition of NMDA receptors prevents the loss of
BDNF function induced by amyloid β. Front
Pharmacol 9:237. https://doi.org/10.3389/fphar.2018.
00237
Teng HK, Teng KK, Lee R et al (2005) ProBDNF induces
neuronal apoptosis via activation of a receptor complex
of p75NTR and sortilin. J Neurosci 25:5455–5463.
https://doi.org/10.1523/JNEUROSCI.5123-04.2005
Terry AV, Kutiyanawalla A, Pillai A (2011)
Age-dependent alterations in nerve growth factor
(NGF)-related proteins, sortilin, and learning and
memory in rats. Physiol Behav 102:149–157. https://
doi.org/10.1016/j.physbeh.2010.11.005
Tervonen TA, Ajamian F, Wit JD et al (2006)
Overexpression of a truncated TrkB isoform increases
the proliferation of neural progenitors. Eur J Neurosci
24:1277–1285. https://doi.org/10.1111/j.1460-9568.
2006.05010.x
Tonchev AB (2011) Brain ischemia, neurogenesis, and
neurotrophic receptor expression in primates. Arch
Ital Biol 149:225–231
F. F. Ribeiro and S. Xapelli
Torres-Cruz FM, Mendoza E, Vivar-Cortés IC et al (2019)
Do BDNF and NT-4/5 exert synergistic or occlusive
effects on corticostriatal transmission in a male mouse
model of Huntington’s disease? J Neurosci Res
97:1665–1677. https://doi.org/10.1002/jnr.24507
Vaegter CB, Jansen P, Fjorback AW et al (2011) Sortilin
associates with Trk receptors to enhance anterograde
transport and signaling by neurotrophins. Nat Neurosci
14. https://doi.org/10.1038/nn.2689
van Praag H, Kempermann G, Gage FH (1999) Running
increases cell proliferation and neurogenesis in the
adult mouse dentate gyrus. Nat Neurosci 2:266–270.
https://doi.org/10.1038/6368
Vaynman S, Ying Z, Gomez-Pinilla F (2004) Hippocam-
pal BDNF mediates the efficacy of exercise on synaptic
plasticity and cognition. Eur J Neurosci 20:2580–2590.
https://doi.org/10.1111/j.1460-9568.2004.03720.x
Vaynman SS, Ying Z, Yin D, Gomez-Pinilla F (2006)
Exercise differentially regulates synaptic proteins
associated to the function of BDNF. Brain Res
1070:124–130. https://doi.org/10.1016/j.brainres.
2005.11.062
Vaz SH, Jørgensen TN, Cristóvão-Ferreira S et al (2011)
Brain-derived neurotrophic factor (BDNF) enhances
GABA transport by modulating the trafficking of
GABA transporter-1 (GAT-1) from the plasma mem-
brane of rat cortical astrocytes. J Biol Chem
286:40464–40476. https://doi.org/10.1074/jbc.M111.
232009
Vilar M, Mira H (2016) Regulation of neurogenesis by
neurotrophins during adulthood: expected and unex-
pected roles. Front Neurosci 10:26. https://doi.org/10.
3389/fnins.2016.00026
Wang L, Chang X, She L et al (2015) Autocrine action of
BDNF on dendrite development of adult-born hippo-
campal neurons. J Neurosci 35:8384–8393. https://doi.
org/10.1523/JNEUROSCI.4682-14.2015
Waterhouse EG, An JJ, Orefice LL et al (2012) BDNF
promotes differentiation and maturation of adult-born
neurons through GABAergic transmission. J Neurosci
32:14318–14330. https://doi.org/10.1523/
JNEUROSCI.0709-12.2012
Webster MJ, Herman MM, Kleinman JE, Shannon
Weickert C (2006) BDNF and trkB mRNA expression
in the hippocampus and temporal cortex during the
human lifespan. Gene Expr Patterns 6:941–951.
https://doi.org/10.1016/j.modgep.2006.03.009
Werry EL, Enjeti S, Halliday GM et al (2010) Effect of age
on proliferation-regulating factors in human adult neu-
rogenic regions. J Neurochem 115:956–964. https://
doi.org/10.1111/j.1471-4159.2010.06992.x
Woo NH, Teng HK, Siao C-J et al (2005) Activation of
p75NTR by proBDNF facilitates hippocampal long-
term depression. Nat Neurosci 8:1069–1077. https://
doi.org/10.1038/nn1510
Xu S-Y, Zhang Q-L, Zhang Q et al (2019) Regional and
cellular mapping of sortilin immunoreactivity in adult
human brain. Front Neuroanat 13:31. https://doi.org/
10.3389/fnana.2019.00031
8 Intervention of Brain-Derived Neurotrophic Factor and Other Neurotrophins. . .
Yan Q, Radeke MJ, Matheson CR et al (1997) Immunocy-
tochemical localization of TrkB in the central nervous
system of the adult rat. J Comp Neurol 378:135–157
Yang J, Siao C-J, Nagappan G et al (2009) Neuronal
release of proBDNF. Nat Neurosci 12:113–115.
https://doi.org/10.1038/nn.2244
Yang J, Harte-Hargrove LC, Siao C-J et al (2014)
proBDNF negatively regulates neuronal remodeling,
synaptic transmission, and synaptic plasticity in hippo-
campus. Cell Rep 7:796–806. https://doi.org/10.1016/
j.celrep.2014.03.040
Young KM, Merson TD, Sotthibundhu A et al (2007) p75
neurotrophin receptor expression defines a population
of BDNF-responsive neurogenic precursor cells. J
Neurosci 27:5146–5155. https://doi.org/10.1523/
JNEUROSCI.0654-07.2007
Zagrebelsky M, Holz A, Dechant G et al (2005) The p75
neurotrophin receptor negatively modulates dendrite
complexity and spine density in hippocampal neurons.
J Neurosci 25:9989–9999. https://doi.org/10.1523/
JNEUROSCI.2492-05.2005
115
Zhang H, He X, Mei Y, Ling Q (2018) Ablation of ErbB4
in parvalbumin-positive interneurons inhibits adult
hippocampal neurogenesis through down-regulating
BDNF/TrkB expression. J Comp Neurol
526:2482–2492. https://doi.org/10.1002/cne.24506
Zhu W, Cheng S, Xu G et al (2011) Intranasal nerve
growth factor enhances striatal neurogenesis in adult
rats with focal cerebral ischemia. Drug Deliv
18:338–343. https://doi.org/10.3109/10717544.2011.
557785
Zigova T, Pencea V, Wiegand SJ, Luskin MB (1998)
Intraventricular administration of BDNF increases the
number of newly generated neurons in the adult olfac-
tory bulb. Mol Cell Neurosci 11:234–245. https://doi.
org/10.1006/mcne.1998.0684
Zuccaro E, Bergami M, Vignoli B et al (2014) Polarized
expression of p75(NTR) specifies axons during devel-
opment and adult neurogenesis. Cell Rep 7:138–152.
https://doi.org/10.1016/j.celrep.2014.02.039
 Part III
Alzheimer and Central Nervous System
 Rita Levi-Montalcini, NGF Metabolism
in Health and in the Alzheimer’s 9
Pathology

A. Claudio Cuello

My life has been enriched by excellent human relations, work, and interests. I have never
felt lonely.
Rita Levi-Montalcini

Abstract found enhanced protein levels and enzymatic

activity of the proteases responsible for the
This chapter relates biographic personal and
proteolytic degradation of mNGF. A biochem-
scientific interactions with Rita Levi-
ical prospect explaining the tropic factor vul-
Montalcini. It highlights research from our
nerability of the NGF-dependent basal
laboratory inspired by Rita’s fundamental dis-
forebrain cholinergic neurons and of their syn-
covery. This work from studies on potentially
aptic terminals. The NGF deregulation of this
neuro-reparative gangliosides, their
metabolic pathway is evident at preclinical
interactions with NGF, the role of exogenous
stages and reflected in body fluid particularly
NGF in the recovery of degenerating choliner-
in the cerebrospinal fluid (CSF). The findings
gic neurons of the basal forebrain to the evi-
of a deregulation of the NGF metabolic path-
dence that endogenous NGF maintains the
way and its reflection in plasma and CSF are
“day-to-day” cortical synaptic phenotype and
opening doors for the development of novel
the discovery of a novel CNS “NGF metabolic
biomarkers for preclinical detection of AD
pathway.” This brain pathway’s conceptual
pathology both in Alzheimer’s and in Down
platform allowed the investigation of its status
syndrome (DS) with “silent” AD pathology.
during the Alzheimer’s disease
(AD) pathology. This revealed a major com-
promise of the conversion of the NGF precur-
9.1 Preface
sor molecule (proNGF) into the most
biologically active molecule, mature NGF
I wish to begin this chapter by congratulating my
(mNGF). Furthermore, in this pathology, we
colleagues Luigi Aloe and Laura Calza for
keeping Rita’s legacy alive, by promoting excit-
A. C. Cuello (*) ing and enriching NGF-related meetings and by
Department of Pharmacology and Therapeutics, McGill allowing us to write reviews of our work with
University, Montreal, QC, Canada
commentaries in the first person regarding our
e-mail: claudio.cuello@mcgill.ca

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 119
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_9
120
stories and interactions with Rita, stories that
inspired our work for many subsequent years.
The excellence in human relations, sharing
research interests, as signaled by Rita’s quotation,
is reflected in this account as are the
investigations on trophic responses in health and
disease.
9.2 What I Knew About Rita During
My Early Immersion
in the Neurosciences
Rita’s quotation has guided my narrative; it is
largely in the first person and it brings to forum
the significance of our friendship and the impact
of her discovery of NGF to Neurosciences. This
discovery led to the finding of a family of
neurotrophins and of their corresponding
receptors. Besides the neurotrophins, other tro-
phic factors were found consequently, such as
the epidermal, fibroblast, and the ciliary trophic
factors, among others.
Before getting to know Rita, I knew that she
was from the laboratory of Giuseppe Levi at
Torino University, a lab which trained scientists
like Rita, Salvador Luria, and Renato Dulbecco—
all of whom received the Nobel Prize. My first
mentor in Argentina, Eduardo De Robertis, dis-
coverer of the synaptic vesicles, was an admirer
of Giuseppe Levi and his histology book was
prominent in the library of our department at the
Buenos Aires University Medical School.
During my initial lab training at medical
school and in later years, I transited through for-
mal research in neuroanatomy, electron micros-
copy, neuroendocrinology, catecholamines, novel
neuroactive peptides, the CNS cholinergic sys-
tem, and ultimately Alzheimer’s pathology.
Throughout that route, I became aware of Rita’s
discovery of NGF and of the saga of Torino,
Belgium, Rio de Janeiro, and St Louis (USA).
My first awareness of the NGF neurobiology
came when I was still a medical student and a
TA at the “De Robertis Institute” of the Univer-
sity of Buenos Aires. By then, we all knew about
her spectacular 1964 paper in Science revealing
the classical “halo” of growing neuronal
A. C. Cuello
processes induced by “a protein factor” (Levi-
Montalcini 1964).
Eduardo De Robertis was the son of Italian
immigrants with humble origins. He made signif-
icant contributions to cell and neurobiology
starting in the United States and became the
Chair of the Department and Director of a Cell
Biology Research Institute at the Buenos Aires
University, where in 1962 the first isolation of
synaptic vesicles took place (De Robertis et al.
1962). As selected medical undergraduates, and
as Departmental TAs, we had the privilege of
having access to labs, making microscope
preparations and samples for histology and neu-
roanatomy teachings, and assisting with access to
advanced techniques in the pioneering years of
electron microscopy. Near the completion of my
medical studies, in 1965, Maria Teresa Sabatini
from the “De Robertis” Institute collaborated at a
distance with Pietro (“Piero”) Angeletti, from
Rita’s research group, on the effects of NGF
immunoneutralization, a work which was
published the following year in a non-very visible
Italian journal (Sabatini et al. 1966).
It was only afterwards during my research
years in Cambridge and Oxford that I learnt
more details about the fascinating life Odyssey
of Rita and her further contributions. During my
Cambridge period, first as a post-doctoral fellow,
and later as an MRC scientific staff at Les
Iversen’s Unit, I followed Les’ pioneering
collaborations with Hans Thoenen and Ian
Hendry on the retrograde transport of NGF
(Hendry et al. 1974).
Like many neuroscience colleagues, I knew
about the evolution of Rita’s seminal work with
Viktor Hamburger on the development of the
chick embryo (Hamburger and Levi-Montalcini
1949) which led to the discovery of NGF,
initially identified as a “proteinaceous factor,” a
saga well narrated by Luigi Aloe in 2004 (Aloe
2004). In later years, well into my “auto-exile”
to England and through the long-lasting and
inspiring friendship with Giancarlo Pepeu
(Firenze) and with Ezio Giacobini (Illinois,
USA and Geneva, Switzerland), I gained the
full picture of Rita Levi-Montalcini, the persona
and the scientist.
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
During my chats with Ezio, Giancarlo, and
many Italian colleagues, I realized just how
“heroic” and unusual Rita’s story was. I came to
know about Rita’s clandestine work in Italy, the
paper contradicting Hamburger’s interpretations
on the influence of the limbs on the development
of the nervous system, “the axonal bridges” in
particular. That paper was published with her past
mentor, Giuseppe Levi, “en francais,” in a rather
obscure Belgian journal, in the middle of WWII
(Levi-Montalcini 1997)!
The above publication, which could have eas-
ily been lost . . . provoked—after the war years—
Viktor Hamburger to invite Rita to come to his
lab in St. Louis to study the problem together to
resolve their differences. . . This was quite an
extraordinary gesture, a gesture that nowadays
would be rare to find. The move to St Louis
offered Rita an excellent platform for applying
her talents to the outmost, establishing very pro-
ductive and transformational collaborations with
Viktor Hamburger and Stanley Cohen. These
studies built an entire new conceptual platform
in the neurosciences.
Knowing all the above, I never suspected that
Rita Levi-Montalcini, a person of my continued
admiration, would ever become one of my close
and personal friends.
9.3 My Encounters with Rita
My first encounter with Rita was most unusual. I
was, by then, in my Oxford lab, doing stereotactic
surgery, when I heard a voice from behind me
saying: “Are you Dr Cuello?” Concentrated on
my task, I simple replied, “Yes,” without turning
around. The voice behind me continued, “I am
Rita Levi-Montalcini I wanted to consult with
you about a research project.” It goes without
saying that I interrupted my work, took my lab
coat out, and invited Rita to continue the conver-
sation in my office. There, I indicated to her how
pleased I was in meeting her. I then asked Rita
about her interests. She told me that she wanted to
study the effects of NGF on the phenotypic
changes during the development of the nervous
system in the Xenopus and expressed that she
121
wanted to collaborate with us given—in her
words—our well-recognized expertise in
immunohistochemistry.
After she had explained the project in detail, I
told Rita that I found the project straightforward
and that I would be pleased to welcome to my lab
one of her collaborators, to perform the work
under my guidance at our Oxford laboratory.
She agreed and arranged for her close collabora-
tor Luigi Aloe to come to our lab. I then had the
pleasure of having daily interactions with Luigi
and practicing with him mi povero italiano. Luigi
spent some time in Oxford doing his first hands-
on experience in immunohistochemistry. He
returned to Rome with a new expertise and with
a provision of reagents as well as recently devel-
oped monoclonal antibodies. A few months
elapsed when I received by mail a draft of a
paper intended for PNAS authored by Levi-
Montalcini, Luigi Aloe, and A. Claudio Cuello.
I made my corrections and edits to the paper,
crossing out my name in the authorship list and
mailed it back. When the paper arrived back to
Rome, I received a call from Rita thanking me for
the corrections and she asked me why I had
crossed out my name from the authorship list.
Somewhat irritated she asked if I did not like the
work (no te è piaciuto il lavoro). I responded that
it was a nice piece of work but that I felt I did not
have any intellectual participation in it, that my
contribution was simply to instruct Luigi in
immunohistochemical procedures, and that it
was for me a privilege to assist her. A long silence
followed broken by the sentence: Nothing like
this has happened to me before.
The study with my modest “ghost” participa-
tion was ultimately published in PNAS 1985
(Levi-Montalcini and Aloe 1985). The above epi-
sode was the beginning of long and close friend-
ship. Rita opened her heart to us and likewise my
wife, Martha, and I shared with her all our dreams
and expectations. She narrated to us the most
significant aspects of her very rich, fascinating,
and inspiring life. We visited each other’s homes
in Rome and from our side her visitation in
Oxford and later in Montreal. She knew our
daughters Paula and Karina well and we became
close to her sister Paola. Paola was an artist. She
122
Fig. 9.1 Paola and Rita
posing for us in the balcony
of their apartment in Rome,
which overlooked
“Mussolini’s villa”
in Rome
was a very gentle and petite person who looked
and dressed like a fashionable character from the
late 1800s . On one occasion, she let us see her
work. In the expectation of seeing delicate, mini-
ature, paintings, we were most surprised to see
modern and forceful paintings and sculptures.
Much later, I met her cousin Piera Levi-
Montalcini, and while visiting Rita in Rome, we
had the pleasure of meeting a more distant cousin
of hers, Dr. Sacerdote de Lustig, from Argentina.
Through many “at home” lunch and dinner
occasions, besides science, we discussed with
Rita art, literature, history, and the state of the
world. It was a well-known fact that the
Mussolini regime blunted her career in Torino; a
lesser-known irony was that the balcony of Rita’s
apartment in Rome overlooked the park, which
was part of Mussolini’s villa (see Fig. 9.1).
The irony resides in the fact that Mussolini’s
regime established the “Racial Laws” in Italy due
to which she was banned from Torino University
and it could have ended her career as well as her
life. This dramatic period of Rita’s life is well
reflected in her inspiring book In Praise of Imper-
fection (Levi-Montalcini 1988).
Rita was a true “Renacentist” individual. Very
few were the subjects on which Rita did not have
an opinion. She was a humanist and a defender of
minority rights. I remember that over her desk
there was a portrait of Dr. Martin Luther King.
Besides science, during our encounters, we often
A. C. Cuello
discussed literature, history, and politics. She
wrote several non-scientific books in Italian and
I have the entire collection at home with her
signature (Tempo di mutanti, Abbi il coraggio di
conoscere, Tempo di azioni, and La Galassia
mente). I learnt that she was a life-long close
friend of Primo Levi, an author I admire and
from whom I have read and reread If This Is a
Man and The Periodic Table. Rita was Primo
Levi’s confidant. In 1987, I received an unex-
pected call at home from Rita in tremendous
anguish and she was nearly crying. This was to
let me know that Primo Levi died by suicide. He
was one of her closest friends with whom she
shared transcendental moments while hiding
from and resisting the fascistic Italian regime.
She felt partially responsible for his death because
she missed one of his calls asking for help. It was
a long talk. I found myself in the estranged pre-
dicament of comforting Rita; it was an intense
emotional experience, which fortified our friend-
ship. Our encounters were always en famille with
my wife Martha and while in Oxford also with my
daughters Paula and Karina. We also often visited
our mutual labs (see Fig. 9.2).
Rita followed with interest my career evolu-
tion. She thought that I should not leave the
historically rich Oxford environment, my univer-
sity position, and my fellowship with a Lincoln
College, with late medieval traditions. Her initial
resistance was converted in her unreserved
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Fig. 9.2 I am visiting
Rita’s lab in Rome with the
slides for my talk at hand
and in the company of
Pietro Calissano, circa 1986
support when she understood about my enthusi-
asm of taking a larger responsibility, across the
Atlantic, at McGill University. While at McGill,
she visited our lab and interacted for several days,
in the most youthful fashion, with my new lab
trainees. Also, I had the satisfaction that my prop-
osition of inviting Rita to give one of the first
lectures for the Faculty of Medicine Series on
Foundations of Medical Sciences at McGill Uni-
versity was enthusiastically accepted. This was an
initiative organized by the very inspiring dean of
medicine, Richard Cruess, and a number of
McGill notables. These lectures included a good
list of Nobel Laureates and other outstanding
trailblazer scientists that were celebrated with a
formal black-tie dinner. . .a tradition that I helped
to “transport” from Oxford to McGill.
In a 1984 meeting in Italy, though I had no
idea she had already been nominated, I forecasted
to Rita that she would win the Nobel Prize, to
which she responded: “That will never happen,
Claudio, but if it happens, you will be the first to
be invited to the Nobel ceremony.” And so she
did. I received a phone call and a formal invita-
tion. Unfortunately, the 1986 Nobel ceremony
coincided with the celebration in Argentina of
the 50th wedding anniversary of my parents. I
123
still regret that I missed this great occasion and
most of all I regret not being able to share the
moment in which Rita received the highest inter-
national recognition for her scientific
contributions.
Another memorable encounter with Rita was
when I was invited by her to give one of the first
lectures following the opening of the EBRI in
Rome and other lectures and interactions with
Rita and EBRI colleagues thereafter.
With the passing of the years, I had the privi-
lege of participating in the symposium organized
by Luigi Aloe and Italian colleagues in 2009, for
celebrating Rita’s hundredth anniversary and her
enormous legacy to science. I also attended the
more formal ceremony held in the Roman
Palazzo Senatorial Capitol hill, where Rita gave
a magnificent speech next to the statue of Julius
Cesar. Again, a most ironic distinction for a “non-
Arian” person once persecuted by the Race Laws
of fascistic Italy to give a speech next to the
imposing statue of Julius Cesar.
While I was leading the McGill University
“Brain@McGill” initiative, I proposed that the
university recognize the exceptional
contributions to medicine and to society made
by Rita by awarding her an honorary doctor of
124 A. C. Cuello

Fig. 9.3 Having the honor

of “hooding” Rita with the
gown representing the
McGill Science Doctorate,
with the participation of the
Rector of The Sapienza
University of Rome, on
February 23rd, 2011.
McGill Provost Anthony
Massi led the ceremony
with the endorsement of
McGill Principal Heather
Monroe-Bloom and with
the approval of the McGill
Senate

science degree. This was done with the collabora-
tion of the Rector of the Sapienza Università di
Roma in 2011 and the McGill Provost Anthony
C. Masi. Rita was delighted with the distinction. I
“hooded” Rita with the McGill robe upon com-
pletion of the ceremony (see Fig. 9.3). This was a
very special and exceptional occasion, as it was
the first time in McGill’s 190-year history that the
University conferred an honorary doctorate on
foreign soil and the second time that such a
degree was awarded off campus. The first was in
1944, at the end of WWII, when Winston
Churchill and Franklin Delano Roosevelt were
honored by McGill University in Quebec City.
That was my last encounter with Rita.
9.4 My First Interest in Neural
Repair Leading to NGF
Research
My involvement in NGF research has the rooting
in the development of an excellent model for the
study of the retrograde atrophy of cholinergic
neurons of the nucleus basalis in the rat following
a stroke-like type of cortical lesions (Sofroniew
et al. 1983), a study led by Michael Sofroniew, by
then my Oxford graduate student. We
demonstrated that this retrograde atrophy of the
n. basalis neurons was accompanied by a marked
loss of its biochemical cholinergic phenotype,
i.e., loss of choline acetyltransferase activity
(Stephens et al. 1985). During our initial studies
with that model, we demonstrated that the
shrunken n. basalis neurons could be salvaged
with the parenteral application of
monogangliosides (Cuello et al. 1986; Sofroniew
et al. 1986). The ganglioside GM1 was a com-
pound reported to have neuroprotective properties
and at the time applied clinically. Its
manufacturing process was proprietary of an Ital-
ian pharmaceutical company: Fidia. The funda-
mental biochemical aspects of ganglioside were
already revealed in the notable and extensive
works of Tettamanti in Italy (Tettamanti 1988),
Svennerholm in Sweden (Svennerholm 1980),
and Ledeen in the United States (Ledeen and Yu
1982; Ledeen 1984).
My interest in the ganglioside GM1 was
aroused by a number of studies in the peripheral
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
nervous system, such as the demonstration of a
GM1-induced improvement in the rate of recov-
ery of the contractile responses of sympathetically
denervated nictitating membranes (Ceccarelli
et al. 1976), and the effects of brain gangliosides
on functional recovery in experimental regenera-
tion and re-innervation. The demonstration of the
neurorepair properties of GM1 on denervated
skeletal muscle was reported by Caccia and
coworkers (Caccia et al. 1979) followed by com-
prehensive histological and functional studies.
Gorio et al. (1980, 1983a, b) provided further
support to ganglioside-induced axonal-synaptic
sprouting in models of compromised motor
nerve innervation.
Impressed by the above evidence, I had
discussions with colleagues at Fidia laboratory,
and during my last years in Oxford and my initial
years at McGill, we tested the hypothesis that
systemic administration of the GM1 ganglioside
could have a reparative function in the CNS,
taking advantage of our model of retrograde atro-
phy of nucleus basalis neurons. To our amaze-
ment, we found that the daily intraperitoneal
application of 30 mg of GM1 for 30 days
provided a remarkable protection of the morphol-
ogy and neurochemistry of the forebrain n. basalis
cholinergic neurons (Cuello et al. 1986;
Sofroniew et al. 1986; Stephens et al. 1987). We
later observed that these neurotrophic-like effects
of GM1 gangliosides included improved in vivo
release of acetyl choline from the cerebral cortex
after extensive (Maysinger et al. 1988, 1990a, b),
studies which were led by Dr. Maysinger with our
collaborators Herrera-Marschitz and Ungerstedt
at the Karolinska Institute. These investigations
included the evidence that the monogangliosides
have reparative effects on GM1 in diminishing
the ensuing deficits in memory-related tasks in
animals bearing neocortical devascularizing
lesions (Elliott et al. 1989; Garofalo and Cuello
1994).
That period allowed me to have extensive
discussions with Rita about neural repair. It also
facilitated interactions with leading scientists in
ganglioside research and forming a long-lasting
friendship with Richard Ledeen, in honor of
125
whom I wrote more recently a testimonial honor-
ing his long career in the field (Cuello 2012).
Much of the interest in gangliosides research
plunged when the clinically applied GM1 was
suspected of provoking Guillain-Barre syndrome.
One publication in 1994, based on a small patient
cohort, stated that the clinically applied GM1
could incite the Guillain-Barré syndrome (Landi
et al. 1994) and provoked much media coverage,
which, regrettably, resulted in the suspension of
the GM1 therapy for peripheral neuropathies and
in the suspension of the endorsement of ganglio-
side research by granting institutions. As such,
the fundamental neuropharmacology of
gangliosides and their potential as reparative
molecules, per se, or combined with trophic
factors were never fully developed. With the
passing of the years, it became quite clear that
there was no solid evidence of causality between
GM1 administration and the development of the
Guillain-Barre syndrome, but rather there is solid
evidence to the contrary (Granieri et al. 1991;
Samson and Fiori 1994). The original paper of
Landi and coworkers was shown to be based on
faulty epidemiological premises. However, the
concern remained regarding the presence in the
Guillain Barre syndrome of a diversity of anti-
ganglioside antibodies (including anti-GM1).
Such concern has been currently nullified through
numerous publications which demonstrated that
antibodies against microbial glycolipids and
glycoproteins present in the Guillain-Barre neu-
ropathy do cross-react with gangliosides. This
aspect is nowadays interpreted as a cross-reaction
(“mimicry”) of likely causative anti-microbial
antibodies detected in the syndrome
(Wanleenuwat et al. 2020).
In the last period of our investigations of the
neurotrophic-neuroreparative effects of GM1 in
experimental models of CNS cholinergic degen-
eration and of the potential interactions with
mature NGF, I was invited by Advances in Phar-
macology to write a review on the neuropharma-
cology of glycosphingolipids toward nerve
growth and repair (Cuello 1990) where I
highlighted the state of the field and the possible
application of GM1 alone and/or in combination
126
with NGF. Ideas which were often discussed with
Rita and which led to our lab entering fully
fledged NGF research.
9.5 NGF Functions in the Adult
Central Nervous System
and Reparative Properties
for Basal Forebrain Cholinergic
Neurons
The investigations on the reparative actions of
gangliosides in halting the atrophy of basal fore-
brain cholinergic neurons brought us to study the
likely reparative effects of NGF in our lesion
model of retrograde degeneration of basal fore-
brain cholinergic neurons (BFCN). At the time, it
was already well established that NGF played a
significant role in the survival and differentiation
of primary sensory and autonomic neurons
through the pioneering work or Rita. A similar
NGF role on the maturation of the basal forebrain
cholinergic neurons and their terminations in the
cerebral cortex and the hippocampus was
established (Mobley et al. 1986; Molnar et al.
1998).
By then, it was assumed that BFCNs were
completely dependent on NGF not only for their
developmental differentiation but as well for their
survival in the adult CNS. Such a concept was
reinforced by the apparent death of the BFCN of
the medial septum, when deprived of “target-
derived” NGF by the interruption of their connec-
tion with the hippocampus, by the axotomy of the
fimbria-fornix projection, and their subsequent
rescue of such neuronal cholinergic set by the
timely application of exogenous mature NGF in
the ventricular space (Hefti 1986; Kromer 1987;
Williams et al. 1986; Gage et al. 1988).
The above studies were significant in
establishing the “proof of principle” of NGF
reparative properties in the CNS. They generated
a great deal of anticipation in the field, since it had
already been established that this cholinergic sys-
tem appeared most vulnerable in Alzheimer’s
disease (AD) (Bowen et al. 1976; Davies and
Maloney 1976). This was of great interest as it
was shown in humans and primates that the CNS
cholinergic system played a substantive role in
A. C. Cuello
higher CNS functions and in memory in particu-
lar (Drachman and Leavitt 1974; Bartus et al.
1982).
Further to it, the histopathology of the nucleus
basalis with Nissl staining demonstrated the loss
of “magnocellular” neurons, the cells responsible
for the sole source of cholinergic innervation for
the human cerebral cortex (Whitehouse et al.
1982). This was a most influential scientific
report. However, the study overestimated the
loss of the nucleus basalis “magnocellular” cho-
linergic neurons; because the measurements were
based on cell size, neuronal atrophy was
incorrectly counted as neuronal loss. The above
studies and a Science publication of Coyle and
collaborators (Coyle et al. 1983) promoted “the
cholinergic hypothesis of AD,” with the implied
concept of a primary involvement of the basal
forebrain in the development of the pathology.
The resulting collective “cholinergic” studies
had the merit of provoking the development of
therapeutic anticholinesterases, with the objective
of improving the brain’s “cholinergic tone” by
blocking the extracellular degradation of acetyl
choline. This approach remains, even nowadays,
the most successful symptomatic treatment in the
AD pathology and its effects are most surprising,
given the advanced brain pathology at the time of
clinical presentation. Since then, the field has
evolved considerably. In a review by the “Cho-
linergic Working Group” (Hampel et al. 2018),
more contemporaneous ideas have been advanced
regarding the significance of BFCN in AAD.
In the early years of the above saga, with
Michael Sofroniew, we proposed that the
involvement of BFCN in the AD pathology was
a not a “primary” as initially thought but rather a
“secondary” consequence to a primary cortical/
hippocampal pathology—a hypothesis which was
advanced in an invited TINS review (Cuello and
Sofroniew 1984). Nowadays, it is largely
accepted that the presence of endogenous NGF
is not essential for BFCN survival, but rather for
the maintenance of the cholinergic phenotype at
the cell body and axonal terminations. The first
and most unequivocal case was made by
Sofroniew and collaborators (Sofroniew et al.
1990). In that report the case was made (quite
convincingly) that, even after the excitotoxicity
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
elimination of most hippocampal neurons supply-
ing “target-derived” NGF, the medial septum
cholinergic neurons survive in a shrunken state.
This was in direct contradiction of the prevalent
concept of neuronal death following a drastic
axotomy of the fimbria-fornix: an axotomy that
was too close to the cell body of BFCN, thus
leading to neuronal loss. In other words, the
reported cell death of the NGF-dependent cholin-
ergic neurons in the investigations applying the
interruption of the septal-hippocampal axonal
connection was not due to the deprivation of
NGF supply but rather by the classical concept
of neuronal cell death provoked by proximal
axotomy (Ramón y Cajal and May 1928). Subse-
quently, we and others demonstrated that, in the
human AD pathology, the predominant conse-
quence is the cellular atrophy, more than cell
death of BFCN of the nucleus basalis (Pearson
et al. 1983; Kordower et al. 1989).
With the guiding hypothesis that cholinergic
neurons of the nucleus basalis suffer a secondary,
retrograde, atrophy to a primary cortical lesion,
we investigated the potential of exogenous NGF
for the recovery of degenerating NGF-dependent
cholinergic neurons of the nucleus basalis. Our
initial studies on the retrograde atrophy of BFCN
cell bodies and nerve terminals following cortical
stroke-type lesions revealed the ganglioside
potentiation of the NGF biochemical and struc-
tural reparative effects (Cuello et al. 1989). With
Lorella Garofalo we did an exhaustive analysis
comparing the therapeutic impact of NGF or the
ganglioside GM1 alone or their therapeutic com-
bination on cholinergic high-affinity choline
uptake, receptor binding and choline acetyl trans-
ferase enzymatic activity in the cortical synaptic
area, and the micro-dissected n. basalis after
stroke-like cortical lesions on the retrograde cho-
linergic atrophy model in the rat (Garofalo and
Cuello 1995). The study confirmed the coopera-
tive actions of NGF and GM1 in re-establishing
biochemical cholinergic markers after CNS
lesions and illustrated narrower “pseudotrophic”
actions of GM1 (less potency and narrower thera-
peutic window (Garofalo and Cuello 1995)). We
further interpreted the GM1 potentiation of NGF
effects might be due as a facilitation of the NGF
binding/activation of the TrkA receptor (Garofalo
127
et al. 1993). Such interpretation was later con-
firmed experimentally by Rabin and Mochetti by
providing evidence of a direct GM1 activation of
the TrkA receptors (Rabin and Mocchetti 1995),
by Mutoh and collaborators showing that GM1
tightly associates with the high-affinity tyrosine
kinase TrkA facilitating it (Mutoh et al. 1995),
and by Farooqui and collaborators supporting a
GM1-induced dimerization of TrkA receptors
(Farooqui et al. 1997), both molecular alterations
signifying increased responses to NGF receptor
activation.
This line of work brought us to demonstrate
that exogenous NGF alone and/or potentiated by
gangliosides, besides preventing the atrophy of
nucleus basalis neurons NGF, had the capacity
of generating de novo cholinergic synaptogenesis
in the non-lesioned areas of the cerebral cortex of
adult rats, following stroke-like lesions (Garofalo
et al. 1992) (Fig. 9.4).
The ensuing comprehensive neurochemical
and pharmacological analysis of the NGF
neurotrophic effects in the CNS cholinergic sys-
tem demonstrated correlative improvements in
the choline uptake mechanisms and on the
in vivo microdialysis release of acetyl choline
(Garofalo and Cuello 1995; Maysinger et al.
1992), observations which are consistent with an
NGF-induced generation of cortical cholinergic
synapses in the adult and fully differentiated ner-
vous system. Such studies in our view fulfill the
predictions of Ramon y Cajal in that “...if experi-
mental neurology is some day to supply artifi-
cially the deficiencies in question ... it must give
to the sprouts (axonal sprouts), by means of ade-
quate alimentation, a vigorous capacity for
growth...” (Ramón y Cajal and May 1928).
A further demonstration of the fine-tuning
exerted by NGF on the maintenance of the den-
sity of basal forebrain cholinergic synapses was
provided by experiments in which the cortical
endogenous NGF was mopped-out with the appli-
cation of anti-NGF monoclonal antibodies or,
alternatively, with the injection of TrkA peptide
mimetic blockers. Thus, negating the NGF occur-
rence or blocking its TrkA receptor activation was
sufficient to provoke the loss of pre-existing cor-
tical cholinergic synapses, in proportion to the
proximity of the site of injection (Debeir et al.
 128

Fig. 9.4 Upper left-hand panel, the gray-shaded area represents the neocortical sites of such brain level. The aggrupation of cholinergic neurons in the inner portions of the
basal forebrain nerve terminals and site of the generation of NGF; the red area globus pallidus represents the nucleus basalis. On the right side the cholinergic (ChAT-
illustrates the unilateral experimental cortical stroke-like lesion. Upper right-hand immunoreactive) n. basalis neurons of control animals, middle micrograph
A. C. Cuello

panel on the left, the black dots represent the localization of cholinergic neurons at representing the retrograde atrophy of n. basalis cholinergic neurons following a

ä
Fig. 9.4 (continued) cortical lesion. The lower micrograph represents the recovery of
these n. basalis cholinergic neurons with the administration of exogenous NGF. Lower
left-hand panel illustrates micrographs and histograms of the density of the varicosities
(presynaptic sites) of cholinergic terminals in the remaining, non-lesioned, cerebral
cortex in control rats, control plus NGF, adjacent stroke-type lesions without NGF and
after NGF treatment, respectively. Note the above average density of cholinergic
9
presynaptic boutons in the NGF-treated cortically lesioned rats, indicative of
synaptogenesis. Lower right-hand panel illustrates the representative ultrastructural
images of the shrinkage of cortical cholinergic presynaptic in the lesioned rats and
the hypertrophy and large area of contact of cholinergic varicosities in the lesioned
NGF-treated rats. As per Garofalo et al., PNAS, 1992
Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
129
130 A. C. Cuello

Fig. 9.5 The upper

drawing represents
schematically the
experimental approach
utilized to investigate the
impact of the cerebral
cortex endogenous mature
NGF in the maintenance of
the NGF-dependent
cholinergic synapses
originated from nerve
terminals of the n. basalis of
the rat. The occurring
mature NGF was
immunoneutralized either
by the application of anti-
NGF monoclonal
antibodies or by blocking
their activation of the TrkA
receptor by applying a
mimetic peptide. As
represented in the
histogram at the bottom,
both procedures resulted in
the significant loss of the
density of pre-existing
cholinergic presynaptic
boutons (VAChT-IR:
Vesicular Acetyl Choline
Transporter-
Immunoreactive sites), as in
Debeir et al., PNAS, 1999

1999). This was a finding indicating that the “day-
to-day” density of cortical cholinergic neurons is
entirely dependent on the continued supply of
minute amounts of endogenous NGF. This con-
cept is well in line with the classical Hebbian
principle “of synaptic growth following neuronal
activity” (Hebb 1949) (Fig. 9.5).
9.6 A Novel NGF Metabolic
Pathway
9.6.1 The Background
We came across the evidence that proNGF and
not mature NGF (mNGF) was the molecular form
of this neurotrophin being released in an activity-
dependent manner. A failure in a release mecha-
nism could have provided an explanation for tro-
phic disconnection of basal forebrain cholinergic
neurons in aging or neurodegenerative
conditions. The situation of the NGF sustenance
of NGF-dependent neurons in the case of AD was
particularly challenging. In early years, authorita-
tive publications suggested a causative role of
NGF failure for the retrograde atrophy death of
FBCN (Stewart and Appel 1988; Hefti and
Weiner 1986). However, such assumptions of an
NGF trophic disconnect explaining the atrophy of
basal cholinergic neurons and the loss of cortical
and hippocampal synapses in AD was not
supported by the neurochemical evidence. It was
shown that in AD brains the NGF synthesis
remained unchanged (Goedert et al. 1986; Jette
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
et al. 1994; Fahnestock et al. 1996) and that the
offer of the NGF precursor molecule, proNGF,
contrary to expectations, was not diminished but
supranormally elevated (Fahnestock et al. 2001,
2004; Peng et al. 2005; Pedraza et al. 2005).
Such a situation created an AD NGF paradox
regarding the apparent healthy status of the tro-
phic support in the occurrence of a progressive
atrophy of BFCN. The first explanation has been
the diminished TrkA-immunoreactive sites in AD
brains (Mufson et al. 1997; Hock et al. 1998).
Another explanation has been that in the AD
pathology there is an Aβ amyloid-induced
impairment in the axonal retrograde transport
responsible for NGF mobilization from nerve
terminals to neuronal soma to affect the trophic
functions (Salehi et al. 2006). Both the
impairment of axonal transport and the losses of
the cognate high-affinity receptor could be plau-
sible scenarios. On the other hand, the presence of
mature NGF is fundamentally responsible for
regulating the expression of all housekeeping
genes, including those related to axonal transport,
transmitter-specific proteins, i.e., NGF-dependent
neurons. This is also the case for the expression of
TrkA protein, the high-affinity NGF receptor,
since NGF has been shown in in vivo conditions
where the TrkA receptor expression is controlled
by its own ligand: NGF (Figueiredo et al. 1995;
Venero et al. 1994).
The more challenging aspect is to resolve
whether the reported tau and Aβ intraneuronal
pathologies reported in the AD pathology are
the cause of the atrophy of the BFCN or if it is a
pre-existing NGF trophic disconnect which
makes these neurons vulnerable to the propaga-
tion of toxic tau and Aβ amyloid proteins. We
propose that in AD there is progressive loss of
mature NGF, which is best explained by the exis-
tence of a NGF metabolic pathway, as described
below.
9.6.2 The Discovery of the NGF
Metabolic Pathway
Keeping in mind the above background regarding
the pathophysiology of NGF, we were interested
131
with Martin Bruno, by then my graduate student,
in deciphering how NGF was actually released in
an activity-dependent fashion in the fully matured
central nervous system. The preceding work, as
stimulating it was at the time, had some signifi-
cant drawback.
The preceding studies, in fact, supported the
decades-long prevalent idea that the molecule
released in an activity-dependent manner was
mature NGF. This assumption was based on
in vitro studies on dissociated hippocampal cells
or tissue slices transfected with either proNGF or
mature NGF cDNA constructs and analyzing the
supernatants resulting from the application of
high molarity of KCl (Blochl and Thoenen
1996; Hoener 2000; Mowla et al. 1999). In
these historical studies, the resulting supernatants
were assayed with the available anti-NGF
antibodies in an ELISA format in the assumption
that the signals thus obtained represented the
actual mature NGF. However, it is nowadays
clear that these antibodies, and current ones at
large, do not differentiate mature NGF from its
precursor, the proNGF molecule.
To avoid the above experimental drawbacks,
we decided to go back to “first principles.” We
resorted to use ex vivo rat cerebral cortex tissue
blocks, i.e., neurons preserving their contacts to a
great extent and in a conventional milieu, as
opposed to dissociated neurons or tissue slices,
genetically manipulated to express mature NGF
nor proNGF. The cerebral cortex blocks were in
contrast taken from intact tissue. In consequence,
the experiments reflected the activity-dependent
release of endogenous neurotrophins in physio-
logical conditions. These cortical tissue blocks
were continuously superfused with a balanced
and oxygenated buffer, obtaining time-dependent
fractions of superfusates after neuronal stimula-
tion with high KCl, carbachol, or glutamate. An
approach not very far from the Otto Loewi’s
classical “vagusstoff” experiments leading to the
discovery of chemical nerve transmission and
eventually the identification of acetyl choline
(Loewi 1921). For that, Martin Bruno was able
to reproduce the tissue superfusion system which
I learnt at the MRC Cambridge Iversen’s Unit in
Neurochemical Pharmacology. In the knowledge
132
Fig. 9.6 On top, a schematic representation of ex vivo
system utilized to investigate the nature of endogenous
neurotrophins released by neurotransmission or high
molarity K+ stimulation. Small rat cortical tissue blocks
were placed into a chamber and constantly superfused with
a modified Hanks’ buffer equilibrated with 95% O2 and
5% CO2 with a slow flow rate. After 30 min of equilibra-
tion of 0.25 ml/min at 37
C, the tissue was subjected to
stimulations with carbachol (100 nM), glutamate (60 μM),
or KCl (50 mM) applied over a 5-min period. (a) A typical
Western blot analysis of the detected neurotrophins,
that ELISA methods do not properly distinguish
mature NGF from ProNGF, to investigate the
superfusate fractions following tissue stimulation,
we resorted to apply a Western blot approach. To
our great surprise, the superfusate fractions
emerging after stimulation with either KCl, car-
bachol, or glutamate inexorably revealed that the
activity-dependent released neurotrophin in phys-
iological conditions was not mNGF, as the dogma
had established, but rather its precursor, proNGF
(Fig. 9.6).
A. C. Cuello
indicating that only proNGF was readily found in fractions
collected immediately after any type of tissue-stimulation.
NGF indicates a control band from loading with 5 ng of
mNGF. (b) and (c) illustrate the superfusate fractions
collected at 5-min intervals. In (b), the peak in the graph
represents the increase in proNGF released after carbachol.
A second carbachol stimulation was blocked by the Ca++
intracellular chelator AM BAPTA, while in (c) the block-
ade of extracellular Ca++ with BAPTA failed to prevent
the transmitter-stimulated release
The finding of a stimulus-dependent proNGF
release created a problem. How was the mature
NGF generated and how was it going to reach its
cognate receptors to exert its neurotrophic
actions? It took us about 2 years to discover the
occurrence of a complex metabolic cascade
responsible for the conversion of proNGF to
mNGF and of the degradation in the extracellular
space of the remnants of mNGF which were not
internalized and transported retrogradely, i.e., fol-
lowing their binding TrkA/p75 receptor
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Fig. 9.7 The top panels demonstrate the immunoreactive
sites within large pyramidal neurons of layer V of the
cerebral cortex of the rat. Scale bar-xxx (from Bruno and
Cuello, PNAS, 2006). The other panel illustrates the
cohort of activity-dependent molecules released
complexes. These proteins responsible for the
NGF metabolism were plasminogen, tPA (tissue
plasminogen activator), neuroserpin, and matrix
metalloproteinases. We found this mix of zymo-
gen, convertases, and endogenous inhibitors to
co-release along with proNGF. We found that
the conversion of proNGF to mNGF is made by
the plasmin, which is generated by the action of
(tPA over the zymogen plasminogen. The actions
of tPA are, in turn, regulated by neuroserpin
inhibiting the extent of tPA activity (Bruno and
Cuello 2006). The newly formed mNGF is there-
fore readily available to bound the cognate recep-
tor, internalized and retrogradely transported
axonally, to the cell soma of the NGF-dependent
neurons to activate the generation of all proteins
required for the maintenance of the anatomical
133
simultaneously in the extracellular space upon transmitter
or membrane-depolarizing KCl stimuli (image from video
animation of the NGF metabolic pathway: https://
meetings.ami.org/2018/project/ngf-metabolism-in-
alzheimers-disease/)
and biochemical phenotype: a process well
illustrated by Mobley, Schwab, and others (Seiler
and Schwab 1984; Grimes et al. 1996). The
remaining mNGF in the extracellular space is
ultimately degraded by activated Matrix Mettallo-
proteases (MMP9) and also, more recently
identified also by the actions of MMP3 (Pentz
et al. 2020). The activity of the MMPs is also
regulated by the endogenous inhibitor Timp-1.
The key proteins encompassing the NGF meta-
bolic pathway have been co-localized along
proNGF in cortical pyramidal neurons, more
prominently in lamina V (Bruno and Cuello
2006), as illustrated in Fig. 9.7.
The description of this metabolic pathway was
published in PNAS in 2006 (Bruno and Cuello
2006) with the approval of the elected Editor,
134 A. C. Cuello

Fig. 9.8 Schematic

representations of events
leading to proNGF
conversion into mNGF and
its degradation. Neuronally
stored proNGF,
plasminogen, tPA,
neuroserpin, proMMP-9,
andTIMP-1would be
released into the
extracellular space upon
neuronal stimulation.
Released tPA would induce
the conversion of
plasminogen into plasmin.
tPA activity will be tightly
regulated by the released
neuroserpin. The generated
plasmin would convert
proNGF into mNGF.
mNGF would interact with
its cognate receptors (TrkA
and p75 neurotrophin
receptor) and internalized
by synapses of
NGF-dependent neurons.
The remaining mNGF will
be ultimately degraded by
active MMP-9. From Bruno
and Cuello, PNAS, 2006

Hans Thoenen, who made towering contributions
to our understanding of fundamental aspects of
the biochemistry and physiology of
neurotrophins. Sending the submission for review
and accepting its publication was an exemplary
gesture of scientific gentlemanship as the path-
way contradicted his original ideas of intracellular
conversion of proNGF to mNGF and its release as
the mature neurotrophin. A schematic summary
of the pathway is represented in Fig. 9.8.
This pathway was validated pharmaco-
logically in the rat brain initially by the applica-
tion of exogenous neuroserpin blocking tPA
activity and, therefore, inhibiting the plasmin for-
mation and leading to splicing the proNGF, thus
preventing the formation of mNGF. The result is
a spectacular buildup of proNGF. Contrary to it,
the application of an MMP inhibitor protects
mNGF from its proteolytic degradation by
MM9, with the consequential elevation of brain
levels of mNGF, which otherwise are close to
undetectable at physiological conditions
(Fahnestock et al. 2001) (Fig. 9.9).
Further to the pharmacological validation of
the NGF pathway, with Simon Allard further we
demonstrated that this pathway is downstream
responsible for the maintenance of the cholinergic
synaptic phenotype at the cerebral cortex level.
By blocking the proNGF conversion to mNGF
with local administration of α2-antiplasmin in the
rat prefrontal cortex, we observed, as expected, a
diminution of the local cortical levels of
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Fig. 9.9 Histogragrams of pharmacological interventions
on the NGF metabolic pathway. On the left, note the
remarkable increase in the cortical levels of proNGF in
neuroserpin-treated animals after 72 h post-injection
(mean +/– SEM; P  0.001; t test). The right-hand histo-
gram illustrates that the cortical application of the MMP
inhibitor GM6001 results in an over tenfold increment of
endogenous NGF and elevation of proNGF. Such
a scenario resulted in the significant loss or
pre-existing NGF-dependent cholinergic
pre-synaptic boutons, without affecting the den-
sity of glutamatergic, GABAergic, or catechol-
aminergic synapses (Allard et al. 2012). In
opposition, applying in the prefrontal cortex an
MMP inhibitor with the same experimental
approach resulted in an increment in the density
of cholinergic synaptic boutons.
At a later stage, we investigated with Simon
whether the phenotypic regulatory influence of
the NGF metabolic pathway was extensive to
the status of the cell bodies of the n. basalis
BFCN. For those studies we used a combination
of marking the n. basalis neurons receiving the
plasmin inhibitor at cortical synaptic level and
investigating the pharmacological effects on neu-
ronal size and classical transmitter and receptor
markers.
To achieve that, we applied α2-antiplasmin in
a restricted area of the neocortex injecting simul-
taneously FluoroGold, a classical axonal retro-
grade transport marker. In that manner we could
identify in the n. basalis which choline acetylase
(ChAT)-immunoreactive neurons were, or were
not, exposed to α2-antiplasmin. As such we found
that the cortical blockade of the proNGF matura-
tion into mNGF resulted in an atrophy of the cell
135
mNGF as compared with the control levels from cortical
tissues receiving the inactive isomer GM(-) and thus dra-
matically inverted the proNGF to mNGF physiological
ratio. The mNGF cortical levels were raised highly signif-
icantly (P  0.001) having a consequential decrease of
proNGF (P  0.01) as compared to basal levels
bodies of n. basalis BFCN. This was
accompanied by a marked downregulation of the
NGF p75 neurotrophin receptor and of TrkA
receptor as well as a reduction in choline
acetyltransferase protein expression. These
changes were restricted to the cholinergic
n. basalis which were marked with the retro-
gradely transported FluoroGold (Allard et al.
2018)—see Fig. 9.10.
This study provided strong evidence
demonstrating the cortical compromise of the
extracellular maturation of proNGF sufficient to
cause a somatodendritic retrograde degeneration
of the cell bodies of the NGF-dependent BFCN of
the n. basalis. Such experimental evidence would
also give credence to the postulated idea that the
cholinergic basal forebrain atrophy observed in
AD and in DS is due to a deregulation of the NGF
metabolic pathway, as discussed below.
9.7 The Deregulation of the NGF
Metabolic Pathway
in Alzheimer’s Disease
and in Down Syndrome
The discovery of the NGF metabolic pathway
allowed us to test the hypothesis of its possible
deregulation in the AD pathology as a plausible
136 A. C. Cuello

Fig. 9.10 Schematic

representation illustrating
the experimental models
and indicating that only the
n. basalis neurons exposed
to α2-antiplasmin (white-
brown ovals) at their
cerebral cortex suffered
retrograde degeneration
(cell body atrophy)
resulting from the inhibition
of plasmin activity
converting proNGF into
mNGF, while neurons
retrogradely transported
fluorogold particles (
yellow spots) were not
affected. The histograms
illustrate the degree of
neuronal cell shrinkage of
NGF-dependent cholinergic
BCFN after 2 or 4 weeks of
cortical application of α2-
antiplasmin and the rapid
loss of the expression of the
TrkA receptors in those
cells at the same time
intervals. This is an
observation reinforcing the
concept that in the
cholinergic neurons the
expression of TrkA receptor
proteins, as well as that of
all the neuronal
housekeeping proteins, is
dependent on the
continuous supply of
endogenous NGF. From
Allard et al, Neurobiology
of Aging, 2018, with the
publisher’s permission

outcome. That hypothesis, if proven, could (previously unexplained) elevation of proNGF

resolve the prevalent “NGF-cholinergic paradox”
of cholinergic atrophy in the apparent trophic
factor supply. This we did initially with Martin
Bruno. We applied the biochemical paradigm of
the NGF metabolic pathway by the investigation
of the status of the pathway in control and AD
postmortem brain samples from the Netherlands
brain bank. This revealed the existence of a com-
promise in the conversion of proNGF to the bio-
logically active mNGF, with the consequential
content. This compromise in the proNGF conver-
sion was accompanied by increased levels of the
mNGF-degrading MMP9 protein (Bruno et al.
2009b). In other words, we found in AD brains
a scenario of “double jeopardy,” i.e., diminished
mNGF production and augmented mNGF prote-
olysis. Further to it, in collaboration with Elliott
Mufson, in cortical postmortem samples from the
brain bank of the Religious Order Study, we
found a notable accumulation of both the protein
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
and the zymogenic activity of MMP9 in samples
of individual identified as MCI (mild cognitive
impaired) (Bruno et al. 2009a). More importantly
that study demonstrated a correlation between the
degree of MMP9 content and the degree of cog-
nitive losses (Bruno et al. 2009b). These findings
were highly suggestive that the NGF deregulation
could start at preclinical levels of the AD
pathology.
With the arrival of Florencia Iulita to our lab,
we embarked on a study of the status of the NGF
metabolic pathway in Down syndrome (DS). We
hypothesized that DS individuals should have an
equally deregulated NGF metabolic pathway as
they develop inexorably an AD pathology, given
that the trisomy of the chromosome 21 results in
triplication of the gene expressing APP (amyloid
precursor protein), which is provoking the pathol-
ogy (Lott 1982; Wisniewski and Wrzolek 1988;
Hardy et al. 1989; Head et al. 2016; Lott and
Head 2019). As such, DS individuals should
also display a deregulated NGF brain metabolism,
an aspect which would be consistent with the
known atrophy of the basal forebrain cholinergic
system.
The investigation on the status of the NGF
metabolic pathway in DS was led by Florencia;
and it was facilitated through an excellent collab-
oration with Sonia Do Carmo, Lotta Granholm,
Jorge Busciglio, and Thomas Wisniewski. As
such we confirmed the expected high expression
Fig. 9.11 Scattergrams illustrating a positive correlation
between proNGF and amyloid-β42 in (a) the temporal
(r ¼ 0.626, P ¼ 0.017) and in (b) the frontal (r ¼ 0.629,
137
of APP and Aβ proteins in cerebral cortex post-
mortem samples of DS individuals. More signifi-
cantly, these studies eloquently illustrated that
there was indeed a compromise of the NGF met-
abolic pathway responsible for the conversion of
proNGF to mNGF in DS brains bearing AD
pathology. This was shown by lower levels of
plasminogen and a reduced tPA mRNA as well
as an increment in neuroserpin expression and
also an increase in brain proNGF levels (Iulita
et al. 2014). These conditions eloquently signaled
a marked impairment in the maturation of
proNGF to mNGF, providing a rationale for the
known DS cholinergic atrophy (Yates et al.
1980). As is in the AD brains, we observed in
DS brains elevated zymogenic activity of MMP9,
which would exacerbate the degradation of the
already diminished levels of the biologically
active mNGF. Interestingly the genetic model of
DS and the cultures of human fetal cortical cells
also demonstrated an age-related increment of
proNGF. Importantly, the levels of proNGF in
DS brains correlated with the Aβ42 amyloid con-
tent; see Fig. 9.11 (Iulita et al. 2014).
As the AD in DS individuals develops silently
over decades, we thought that the analysis of body
fluid samples in adults at stages prior to the presen-
tation of clinical AD could provide clues or poten-
tial biomarkers signaling the evolving, silent, AD
brain pathology in DS. For our first study we had
the good fortune of interacting with Filippo Caraci
P ¼ 0.001) cortices of post-mortem brain samples of DS
individuals. From Iulita et al, Brain 2014 with the
Publisher’s permission
138
who, with his collaborators, was following longitu-
dinally a mature DS cohort performing cognitive
assessments and extracting blood samples.
Through a collaborative work led by Florencia
Iulita, we investigated the plasma levels of Aß
amyloid peptide, proNGF, tPA, neuroserpin,
metalloproteases in 31 individuals with DS (with
and without dementia) and in 31 healthy controls.
The examination of associations between
biomarkers and cognitive decline demonstrated
foremost that declining plasma Aß peptides and
increasing proNGF levels correlated with cognitive
decline (Iulita et al. 2016b). These findings support
the notion that incremental levels of proNGF could
be of value in identifying individuals who are at
preclinical AD stages. This is being an unmet need
in the field. Figure 9.12 illustrates the changes in
proNGF levels combined with “self-to-self” longi-
tudinal analysis that are strategies of great promise
(Fig. 9.13).
Fig. 9.12 Illustration of time-dependent relationship
between changes in blood proNGF with cognitive decline
in DS individuals. Graphical representation of cognitive
score drop in the Test of Severe Impairment (TSI) (left
graph) and in the Modified Mini Mental (3-MS) (right
hand graph) scores associated with longitudinal change
(d) in proNGF as analyzed by linear regression analysis.
The upward triangles represent non-demented DS
individuals whose plasma levels of proNGF increased
over 12 months and downward triangles depict DS
individuals whose levels of proNGF decreased. Black
A. C. Cuello
9.8 The NGF Metabolic Pathway
in Health and Disease
and Future Challenges
It is of interest that the Alzheimer’s-like amyloid
pathology in a rat transgenic model
overexpressing the human app gene bearing AD
mutations is sufficient to deregulate the brain
NGF metabolic pathway. The obvious conclusion
is that the sole occurrence of just one of hallmarks
of the AD pathology like the abnormal brain
accumulation of Aβ amyloid peptides can elicit
alterations of this pathway similar to those
observed in human brains’ postmortem samples
from individuals with sporadic AD. In the
McGill-APP transgenic rat the brain neurochemi-
cal analysis revealed, as in AD, a differential
dysregulation of NGF and BDNF transcripts and
protein expression as the amyloid pathology
squares represent individuals whose plasma proNGF
levels did not change significantly. Regression lines are
modeling those individuals whose proNGF levels
increased over 12 months. From Iulita et al. 2016
reproduced with the Publisher’s permission. The deregu-
lation of the NGF metabolic pathway has been discussed
in extent in a number of reviews (Iulita and Cuello 2014,
2016; Iulita et al. 2016a; Cuello et al. 2019) and the overall
ideas discussed above are schematically represented in
Fig. 9.13
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Fig. 9.13 Left, schematic representation of the biochem-
ical alterations of the NGF metabolic pathway in AD and
DS with AD pathology. Deficits in tPA diminish plasmin-
ogen conversion into plasmin, therefore leading to
impaired proNGF conversion with consequent proNGF
accumulation. The low amounts of mNGF are further
compromised by higher MMP-9 activity.
Pro-inflammatory mediators as well as other metallo-
proteases like MMP-3 would increase MMP-9 activation
in the AD pathology. From Iulita and Cuello (2016).
Right, scheme indicating that the abnormal brain accumu-
lation of toxic Aβ peptides per se or conjointly with the
early AD inflammation will provoke the impaired NGF
metabolism as represented on the left. The reduced mNGF
progresses. Briefly, BDNF mRNA levels start to
diminish significantly at very early stages of the
amyloid pathology, even before the occurrence of
the amyloid plaques (Iulita et al. 2017). Loss of
BDNF expression is characteristic of AD pathol-
ogy (Holsinger et al. 2000). Contrary to that, no
changes were observed in NGF mRNA expres-
sion at any stage of the amyloid pathology in the
transgenic rats, while the levels of proNGF were
increasingly elevated paralleling the progress of
the amyloid pathology (Iulita et al. 2017) simi-
larly to the case in AD pathology, as discussed in
the preceding sections. As in the human condi-
tion, the transgenic rat exhibiting progressive
amyloid pathology displayed the same AD-like
compromise of the conversion of proNGF into
mature NGF resulting in a brain build-up of
139
production will diminish the generation of all proteins in
NGF-dependent BFCN, including the expression of TrkA
receptors. That will result in the progressive atrophy of
these neurons and therefore in the expression of the ace-
tylcholine (Ach) synthesizing enzyme (choline acetyl
transferase: (ChAT) and the reduced release of its neuro-
transmitter Ach. That will diminish the so-called “cholin-
ergic tone” at their cortical presynaptic sites. A diminished
cholinergic tone would cease to facilitate the activation of
the “non-amyloidogenic” APP pathway (Nitsch et al.
1992, 1993) in the cerebral cortex, i.e., facilitating instead
the APP “amyloidogenic” pathway generating more Aβ
amyloid peptides and, consequently, creating a pathologi-
cal vicious cycle
proNGF molecules accompanied at late stages
with an increased expression of MMP9, the
NGF-degrading protease. Again, this experimen-
tal evidence would favor a direct or indirect caus-
ative role of Aβ amyloid on the AD pathology
NGF metabolic deregulation. Whether amyloid
per se or indirectly remains to be established;
however, an amyloid-induced CNS inflammation
could contribute to the NGF metabolic deregula-
tion. This has been insinuated by the
demonstrating that blocking in vivo with
minocycline the inflammatory effects elicited Aβ
amyloid oligomers also blunted the Aβ peptide
oligomers rise of proNGF levels (Bruno et al.
2009b)
While we have strong experimental evidence
regarding an Aβ amyloid causality for the NGF
140
metabolic deregulation of the NGF metabolic
pathway in AD, the likely added participation of
tau pathology to this metabolic deregulation has
not yet been explored.
One of the questions often made to us is
“whether the pharmacological manipulation of
this pathway could reestablish the NGF ‘trophic
tone’ in AD and therefore reestablish the dimin-
ished ‘cholinergic tone,’” which has a definitive
role in cognitive functions. The assumption being
that a timely and effective therapeutic strategy
reestablishing the normality of the endogenous
NGF supply, at its physiological sites, would be
of undeniable value. This would be a great prom-
ise as even in AD brains, as Eriksdotter and
collaborators have shown, the application of
exogenous NGF elicits a reparative improvement
in the brain’s glucose metabolism (Ferreira et al.
2015) along with a beneficial effect on CSF cho-
linergic markers (Karami et al. 2015).
The pharmacological reestablishment of a
“healthy” NGF metabolism is a desirable thera-
peutic objective. It is also a most challenging
endeavor, given that the molecular members of
the protease-convertase cascade are also involved
in other fundamental aspects of the CNS bio-
chemistry and physiology. Notwithstanding such
obstacles, our lab is investigating a potential,
demanding, experimental avenue.
A most immediately promising route would be
to further explore the likelihood that the
pathology-induced deregulation of the NGF met-
abolic pathway could render predictable
biomarkers of the preclinical AD pathology.
This is a very likely scenario. As discussed
above, progressive raise of blood proNGF levels
are observed in DS in their transition to DS plus
clinical AD. Preliminary work of our lab with the
lab of Juan Fortea (Sant Pau Memory Unit,
Barcelona would support that an even better
reflection of the NGF deregulation in DS is
found in CSF material (Pentz et al. 2019).
Another encouraging information is emerging
from an ongoing study in our lab, led by Rowan
Pentz in collaboration with David Bennett. In
these advanced studies there is a clear indication
that in postmortem brain samples of NCI
A. C. Cuello
individuals already show AD-like alterations in
the tissue levels of key molecules participants on
the NGF pathway. In consequence, it would be
reasonable to assume that body fluids, in particu-
lar CSF, should reflect some of these brain molec-
ular changes.
Acknowledgments The work narrated above was made
historically possible by several UK, Canada, and US
research-granting institutions, notably the UK MRC and
Wellcome Trust, from Canada the MRC, CIHR, and the
Alzheimer Society of Canada, and from the United States
the Alzheimer Association and the NIH. Most importantly,
the research narrated here would not have been possible
without the creativity and dedication of my outstanding lab
members or without the contributions from my outstand-
ing external collaborators.
Dedication I would like to dedicate these reflections
about trophic responses not only to the memory of Rita
Levi Montalcini and her stellar career, but also as a tribute
to Mario Bunge’s legacy. He was one of the world’s
greatest philosophers of his generation. A philosopher
with formal and deep knowledge of the physical and
biological sciences. A philosopher who understood synap-
tic plasticity and who strongly made the case that mind,
consciousness, and all psychological responses were
strictly governed by neuronal connections and biochemi-
cal brain events. I had the privilege of sharing with Mario
many interminable scientific discussions and with him and
his family many memorable events. I will sorely miss his
loss as a dear friend and as an inspiring McGill colleague.
References
Allard S, Leon WC, Pakavathkumar P, Bruno MA,
Ribeiro-Da-Silva A, Cuello AC (2012) Impact of the
NGF maturation and degradation pathway on the cor-
tical cholinergic system phenotype. J Neurosci
32:2002–2012
Allard S, Jacobs ML, Do Carmo S, Cuello AC (2018)
Compromise of cortical proNGF maturation causes
selective retrograde atrophy in cholinergic nucleus
basalis neurons. Neurobiol Aging 67:10–20
Aloe L (2004) Rita Levi-Montalcini: the discovery of
nerve growth factor and modern neurobiology. Trends
Cell Biol 14:395–399
Bartus RT, Dean RL 3rd, Beer B, Lippa AS (1982) The
cholinergic hypothesis of geriatric memory dysfunc-
tion. Science 217:408–414
Blochl A, Thoenen H (1996) Localization of cellular stor-
age compartments and sites of constitutive and
activity-dependent release of nerve growth factor
(NGF) in primary cultures of hippocampal neurons.
Mol Cell Neurosci 7:173–190
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Bowen DM, Smith CB, White P, Davison AN (1976)
Neurotransmitter-related enzymes and indices of hyp-
oxia in senile dementia and other abiotrophies. Brain
99:459–496
Bruno MA, Cuello AC (2006) Activity-dependent release
of precursor nerve growth factor, conversion to mature
nerve growth factor, and its degradation by a protease
cascade. Proc Natl Acad Sci U S A 103:6735–6740
Bruno MA, Leon WC, Fragoso G, Mushynski WE,
Almazan G, Cuello AC (2009a) Amyloid beta-induced
nerve growth factor dysmetabolism in Alzheimer dis-
ease. J Neuropathol Exp Neurol 68:857–869
Bruno MA, Mufson EJ, Wuu J, Cuello AC (2009b)
Increased matrix metalloproteinase 9 activity in mild
cognitive impairment. J Neuropathol Exp Neurol
68:1309–1318
Caccia M, Meola G, Cerri C, Frattola L, Scarlato G, Aporti
FJM (1979) Treatment of denervated muscle by
gangliosides. Muscle Nerve 2:382–389
Ceccarelli B, Aporti F, Finesso M (1976) Effects of brain
gangliosides on functional recovery in experimental
regeneration and reinnervation. Adv Exp Med Biol
71:275–293
Coyle JT, Price DL, Delong MR (1983) Alzheimer’s dis-
ease: a disorder of cortical cholinergic innervation.
Science 219:1184–1190
Cuello AC (1990) Glycosphingolipids that can regulate
nerve growth and repair. Adv Pharmacol 21:1–50
Cuello AC (2012) Gangliosides, NGF, brain aging and
disease: a mini-review with personal reflections.
Neurochem Res 37:1256–1260
Cuello AC, Sofroniew MV (1984) The anatomy of the
CNS cholinergic neurons. Trends Neurosci 7:74–78
Cuello AC, Stephens PH, Tagari PC, Sofroniew MV,
Pearson RC (1986) Retrograde changes in the nucleus
basalis of the rat, caused by cortical damage, are
prevented by exogenous ganglioside GM1. Brain Res
376:373–377
Cuello AC, Garofalo L, Kenigsberg RL, Maysinger D
(1989) Gangliosides potentiate in vivo and in vitro
effects of nerve growth factor on central cholinergic
neurons. Proc Natl Acad Sci U S A 86:2056–2060
Cuello AC, Pentz R, Hall H (2019) The brain NGF meta-
bolic pathway in health and in Alzheimer’s pathology.
Front Neurosci 13:62
Davies P, Maloney AJ (1976) Selective loss of central
cholinergic neurons in Alzheimer’s disease. Lancet
2:1403
Debeir T, Saragovi HU, Cuello AC (1999) A nerve growth
factor mimetic TrkA antagonist causes withdrawal of
cortical cholinergic boutons in the adult rat. Proc Natl
Acad Sci U S A 96:4067–4072
De Robertis E, De Lores Arnaiz GR, De Iraldi AP (1962)
Isolation of synaptic vesicles from nerve endings of the
rat brain. Nature 194:794–795
Drachman DA, Leavitt J (1974) Human memory and the
cholinergic system. A relationship to aging? Arch
Neurol 30:113–121
141
Elliott PJ, Garofalo L, Cuello AC (1989) Limited neocor-
tical devascularizing lesions causing deficits in mem-
ory retention and choline acetyltransferase activity—
effects of the monosialoganglioside GM1. Neurosci-
ence 31:63–76
Fahnestock M, Scott SA, Jette N, Weingartner JA,
Crutcher KA (1996) Nerve growth factor mRNA and
protein levels measured in the same tissue from normal
and Alzheimer’s disease parietal cortex. Brain Res Mol
Brain Res 42:175–178
Fahnestock M, Michalski B, Xu B, Coughlin MD (2001)
The precursor pro-nerve growth factor is the predomi-
nant form of nerve growth factor in brain and is
increased in Alzheimer’s disease. Mol Cell Neurosci
18:210–220
Fahnestock M, Yu G, Michalski B, Mathew S,
Colquhoun A, Ross GM, Coughlin MD (2004) The
nerve growth factor precursor proNGF exhibits
neurotrophic activity but is less active than mature
nerve growth factor. J Neurochem 89:581–592
Farooqui T, Franklin T, Pearl DK, Yates AJ (1997) Gan-
glioside GM1 enhances induction by nerve growth
factor of a putative dimer of TrkA. J Neurochem
68:2348–2355
Ferreira D, Westman E, Eyjolfsdottir H, Almqvist P,
Lind G, Linderoth B, Seiger A, Blennow K,
Karami A, Darreh-Shori T, Wiberg M, Simmons A,
Wahlund LO, Wahlberg L, Eriksdotter M (2015) Brain
changes in Alzheimer’s disease patients with
implanted encapsulated cells releasing nerve growth
factor. J Alzheimers Dis 43:1059–1072
Figueiredo BC, Skup M, Bedard AM, Tetzlaff W, Cuello
AC (1995) Differential expression of p140trk,
p75NGFR and growth-associated phosphoprotein-43
genes in nucleus basalis magnocellularis, thalamus
and adjacent cortex following neocortical infarction
and nerve growth factor treatment. Neuroscience
68:29–45
Gage FH, Armstrong DM, Williams LR, Varon S (1988)
Morphological response of axotomized septal neurons
to nerve growth factor. J Comp Neurol 269:147–155
Garofalo L, Cuello AC (1994) Nerve growth factor and the
monosialoganglioside GM1: analogous and different
in vivo effects on biochemical, morphological, and
behavioral parameters of adult cortically lesioned rats.
Exp Neurol 125:195–217
Garofalo L, Cuello AC (1995) Pharmacological character-
ization of nerve growth factor and/or monosialogan-
glioside GM1 effects on cholinergic markers in the
adult lesioned brain. J Pharmacol Exp Ther
272:527–545
Garofalo L, Ribeiro-Da-Silva A, Cuello AC (1992) Nerve
growth factor-induced synaptogenesis and hypertrophy
of cortical cholinergic terminals. Proc Natl Acad Sci U
S A 89:2639–2643
Garofalo L, Ribeiro-Da-Silva A, Cuello AC (1993) Poten-
tiation of nerve growth factor-induced alterations in
cholinergic fibre length and presynaptic terminal size
142
in cortex of lesioned rats by the monosialoganglioside
GM1. Neuroscience 57:21–40
Goedert M, Fine A, Hunt SP, Ullrich A (1986) Nerve
growth factor mRNA in peripheral and central rat
tissues and in the human central nervous system: lesion
effects in the rat brain and levels in Alzheimer’s dis-
ease. Brain Res 387:85–92
Gorio A, Carmignoto G, Facci L, Finesso M (1980) Motor
nerve sprouting induced by ganglioside treatment. Pos-
sible implications for gangliosides on neuronal growth.
Brain Res 197:236–241
Gorio A, Carmignoto G, Finesso M, Polato P, Nunzi MG
(1983a) Muscle reinnervation—II. Sprouting, synapse
formation and repression. Neuroscience 8:403–IN1
Gorio A, Marini P, Zanoni R (1983b) Muscle reinnerva-
tion—III. Motoneuron sprouting capacity, enhance-
ment by exogenous gangliosides. Neuroscience
8:417–429
Granieri E, Casetta I, Govoni V, Tola MR, Paolino E,
Rocca WA (1991) Ganglioside therapy and Guillain-
Barre syndrome. A historical cohort study in Ferrara,
Italy, fails to demonstrate an association. Neuroepi-
demiology 10:161–169
Grimes ML, Zhou J, Beattie EC, Yuen EC, Hall DE,
Valletta JS, Topp KS, Lavail JH, Bunnett NW, Mobley
WC (1996) Endocytosis of activated TrkA: evidence
that nerve growth factor induces formation of signaling
endosomes. J Neurosci 16:7950–7964
Hamburger V, Levi-Montalcini R (1949) Proliferation,
differentiation and degeneration in the spinal ganglia
of the chick embryo under normal and experimental
conditions. J Exp Zool 111:457–501
Hampel H, Mesulam MM, Cuello AC, Farlow MR,
Giacobini E, Grossberg GT, Khachaturian AS,
Vergallo A, Cavedo E, Snyder PJ, Khachaturian ZS
(2018) The cholinergic system in the pathophysiology
and treatment of Alzheimer’s disease. Brain
141:1917–1933
Hardy JA, Owen MJ, Goate AM, James LA, Haynes AR,
Rossor MN, Roques P, Mullan MJ (1989) Molecular
genetics of Alzheimer’s disease. Biochem Soc Trans
17:75–76
Head E, Lott IT, Wilcock DM, Lemere CA (2016) Aging
in Down syndrome and the development of
Alzheimer’s disease neuropathology. Curr Alzheimer
Res 13:18–29
Hebb DO (1949) The organization of behavior: a neuro-
psychological theory. Wiley, Chapman & Hall,
New York
Hefti F (1986) Nerve growth factor promotes survival of
septal cholinergic neurons after fimbrial transections. J
Neurosci 6:2155–2162
Hefti F, Weiner WJ (1986) Nerve growth factor and
Alzheimer’s disease. Ann Neurol 20:275–281
Hendry IA, Stöckel K, Thoenen H, Iversen LL (1974) The
retrograde axonal transport of nerve growth factor.
Brain Res 68:103–121
Hock C, Heese K, Müller-Spahn F, Hulette C, Rosenberg
C, Otten U (1998) Decreased trkA neurotrophin
A. C. Cuello
receptor expression in the parietal cortex of patients
with Alzheimer’s disease. Neurosci Lett 241(2):151–
154. https://doi.org/10.1016/S0304-3940(98)00019-6
Hoener MC (2000) Role played by sodium in activity-
dependent secretion of neurotrophins—revisited. Eur
J Neurosci 12:3096–3106
Holsinger RM, Schnarr J, Henry P, Castelo VT,
Fahnestock M (2000) Quantitation of BDNF mRNA
in human parietal cortex by competitive reverse
transcription-polymerase chain reaction: decreased
levels in Alzheimer’s disease. Brain Res Mol Brain
Res 76:347–354
Iulita MF, Cuello AC (2014) Nerve growth factor meta-
bolic dysfunction in Alzheimer’s disease and Down
syndrome. Trends Pharmacol Sci 35:338–348
Iulita MF, Cuello AC (2016) The NGF metabolic pathway
in the CNS and its dysregulation in down syndrome
and Alzheimer’s disease. Curr Alzheimer Res
13:53–67
Iulita MF, Do Carmo S, Ower AK, Fortress AM, Flores
Aguilar L, Hanna M, Wisniewski T, Granholm A-C,
Buhusi M, Busciglio J, Cuello AC (2014) Nerve
growth factor metabolic dysfunction in Down’s syn-
drome brains. Brain 137:860–872
Iulita MF, Caraci F, Cuello AC (2016a) A link between
nerve growth factor metabolic deregulation and
amyloid-beta-driven inflammation in Down syndrome.
CNS Neurol Disord Drug Targets 15:434–447
Iulita MF, Ower A, Barone C, Pentz R, Gubert P,
Romano C, Cantarella RA, Elia F, Buono S,
Recupero M, Romano C, Castellano S, Bosco P, DI
Nuovo S, Drago F, Caraci F, Cuello AC (2016b) An
inflammatory and trophic disconnect biomarker profile
revealed in Down syndrome plasma: relation to cogni-
tive decline and longitudinal evaluation. Alzheimers
Dement 12:1132–1148
Iulita MF, Bistue Millon MB, Pentz R, Aguilar LF, Do
Carmo S, Allard S, Michalski B, Wilson EN,
Ducatenzeiler A, Bruno MA, Fahnestock M, Cuello
AC (2017) Differential deregulation of NGF and
BDNF neurotrophins in a transgenic rat model of
Alzheimer’s disease. Neurobiol Dis 108:307–323
Jette N, Cole MS, Fahnestock M (1994) NGF mRNA is
not decreased in frontal cortex from Alzheimer’s dis-
ease patients. Brain Res Mol Brain Res 25:242–250
Karami A, Eyjolfsdottir H, Vijayaraghavan S, Lind G,
Almqvist P, Kadir A, Linderoth B, Andreasen N,
Blennow K, Wall A, Westman E, Ferreira D,
Kristoffersen Wiberg M, Wahlund LO, Seiger A,
Nordberg A, Wahlberg L, DARREH-Shori T,
Eriksdotter M (2015) Changes in CSF cholinergic
biomarkers in response to cell therapy with NGF in
patients with Alzheimer’s disease. Alzheimers Dement
11:1316–1328
Kordower JH, Gash DM, Bothwell M, Hersh L, Mufson
EJ (1989) Nerve growth factor receptor and choline
acetyltransferase remain colocalized in the nucleus
basalis (Ch4) of Alzheimer’s patients. Neurobiol
Aging 10:67–74
9 Rita Levi-Montalcini, NGF Metabolism in Health and in the Alzheimer’s Pathology
Kromer LF (1987) Nerve growth factor treatment after
brain injury prevents neuronal death. Science
235:214–216
Landi G, Ciccone A, D’alessandro R, Dossi BC, Ricci S,
Simone IL (1994) Gangliosides and the Guillain-Barre
syndrome. BMJ 308:1638
Ledeen RW (1984) Biology of gangliosides: neuritogenic
and neuronotrophic properties. J Neurosci Res
12:147–159
Ledeen RW, Yu RK (1982) Gangliosides: structure, isola-
tion, and analysis. Methods Enzymol 83:139–191
Levi-Montalcini R (1964) Growth control of nerve cells by
a protein factor and its antiserum: discovery of this
factor may provide new leads to understanding of
some neurogenetic processes. Science 143:105–110
Levi-Montalcini R (1988) In praise of imperfection : my
life and work. Basic Books, New York
Levi-Montalcini R (1997) Les conséquences de la destruc-
tion d’un territoire d’innervation périphérique sur le
développement des centres nerveux correspondants
dans l’embryon de poulet. In: The Saga of the nerve
growth factor: preliminary studies, discovery, further
development. World Scientific
Levi-Montalcini R, Aloe L (1985) Differentiating effects
of murine nerve growth factor in the peripheral and
central nervous systems of Xenopus laevis tadpoles.
Proc Natl Acad Sci U S A 82:7111–7115
Loewi O (1921) Über humorale übertragbarkeit der
Herznervenwirkung. Pflüger’s Archiv für die gesamte
Physiologie des Menschen und der Tiere 189:239–242
Lott IT (1982) Down’s syndrome, aging, and Alzheimer’s
disease: a clinical review. Ann N Y Acad Sci
396:15–27
Lott IT, Head E (2019) Dementia in Down syndrome:
unique insights for Alzheimer disease research. Nat
Rev Neurol 15:135–147
Maysinger D, Herrera-Marschitz M, Carlsson A,
Garofalo L, Cuello AC, Ungerstedt U (1988) Striatal
and cortical acetylcholine release in vivo in rats with
unilateral decortication: effects of treatment with
monosialoganglioside GM1. Brain Res 461:355–360
Maysinger D, Herrera-Marschitz M, Ungerstedt U, Cuello
AC (1990a) Acetylcholine release in vivo: effects of
chronic treatment with monosialoganglioside GM1.
Neuropharmacology 29:151–159
Maysinger D, Herrera-Marschitz M, Ungerstedt U, Cuello
AC (1990b) Effects of treatment with
microencapsulated monosialoganglioside GM1 on cor-
tical and striatal acetylcholine release in rats with cor-
tical devascularizing lesions. Neurosci Lett
118:252–256
Maysinger D, Herrera-Marschitz M, Goiny M,
Ungerstedt U, Claudio Cuello, A. (1992) Effects of
nerve growth factor on cortical and striatal acetylcho-
line and dopamine release in rats with cortical
devascularizing lesions. Brain Research 577:300–305
Mobley WC, Rutkowski JL, Tennekoon GI, Gemski J,
Buchanan K, Johnston MV (1986) Nerve growth factor
143
increases choline acetyltransferase activity in develop-
ing basal forebrain neurons. Mol Brain Res 1:53–62
Molnar M, Tongiorgi E, Avignone E, Gonfloni S,
Ruberti F, Domenici L, Cattaneo A (1998) The effects
of anti-nerve growth factor monoclonal antibodies on
developing basal forebrain neurons are transient and
reversible. Eur J Neurosci 10:3127–3140
Mowla SJ, Pareek S, Farhadi HF, Petrecca K, Fawcett JP,
Seidah NG, Morris SJ, Sossin WS, Murphy RA (1999)
Differential sorting of nerve growth factor and brain-
derived neurotrophic factor in hippocampal neurons. J
Neurosci 19:2069–2080
Mufson EJ, Lavine N, Jaffar S, Kordower JH, Quirion R,
Saragovi HU (1997) Reduction in p140-TrkA receptor
protein within the nucleus basalis and cortex in
Alzheimer’s Disease. Exp Neurol 146(1):91–103.
https://doi.org/10.1006/EXNR.1997.6504
Mutoh T, Tokuda A, Miyadai T, Hamaguchi M, Fujiki N
(1995) Ganglioside GM1 binds to the Trk protein and
regulates receptor function. Proc Natl Acad Sci U S A
92:5087–5091
Nitsch RM, Slack BE, Wurtman RJ, Growdon JH (1992)
Release of Alzheimer amyloid precursor derivatives
stimulated by activation of muscarinic acetylcholine
receptors. Science 258:304–307
Nitsch RM, Farber SA, Growdon JH, Wurtman RJ (1993)
Release of amyloid beta-protein precursor derivatives
by electrical depolarization of rat hippocampal slices.
Proc Natl Acad Sci U S A 90:5191–5193
Pearson RC, Sofroniew MV, Cuello AC, Powell TP,
Eckenstein F, Esiri MM, Wilcock GK (1983) Persis-
tence of cholinergic neurons in the basal nucleus in a
brain with senile dementia of the Alzheimer’s type
demonstrated by immunohistochemical staining for
choline acetyltransferase. Brain Res 289:375–379
Pedraza CE, Podlesniy P, Vidal N, Arevalo JC, Lee R,
Hempstead B, Ferrer I, Iglesias M, Espinet C (2005)
Pro-NGF isolated from the human brain affected by
Alzheimer’s disease induces neuronal apoptosis
mediated by p75NTR. Am J Pathol 166:533–543
Peng S, Wuu J, Mufson EJ, Fahnestock M (2005) Precur-
sor form of brain-derived neurotrophic factor and
mature brain-derived neurotrophic factor are decreased
in the pre-clinical stages of Alzheimer’s disease. J
Neurochem 93:1412–1421
Pentz R, Iulita MF, Juan Fortea A, Cuello C (2019)
Identifying Alzheimer’s disease in Down syndrome
with NGF metabolism: hope for better treatment and
diagnosis? T21RS Sci Soc Bull 1–3
Pentz R, Iulita MF, Ducatenzeiler A, Bennett DA, Cuello
AC (2020) The human brain NGF metabolic pathway
is impaired in the pre-clinical and clinical continuum of
Alzheimers disease. Mol Psychiatry:1–15. https://doi.
org/10.1038/S41380-020-0797-2
Rabin SJ, Mocchetti I (1995) GM1 ganglioside activates
the high-affinity nerve growth factor receptor trkA. J
Neurochem 65:347–354
144
Ramón y Cajal S, May RM (1928) Degeneration & regen-
eration of the nervous system. Oxford University
Press, Humphrey Milford, London
Sabatini MT, Levi-Montalcini R, Angeletti PU (1966)
[Early effects of anti-nerve growth factor serum on
the nerve cell]. Ann Ist Super Sanita 2:349–355
Salehi A, Delcroix JD, Belichenko PV, Zhan K, Wu C,
Valletta JS, Takimoto-Kimura R, Kleschevnikov AM,
Sambamurti K, Chung PP, Xia W, Villar A, Campbell
WA, Kulnane LS, Nixon RA, Lamb BT, Epstein CJ,
Stokin GB, Goldstein LS, Mobley WC (2006)
Increased App expression in a mouse model of
Down’s syndrome disrupts NGF transport and causes
cholinergic neuron degeneration. Neuron 51:29–42
Samson J, Fiori MJB (1994) Gangliosides and the
Guillain-Barre syndrome. BMJ Clin Res 308
(6944):1638
Seiler M, Schwab ME (1984) Specific retrograde transport
of nerve growth factor (NGF) from neocortex to
nucleus basalis in the rat. Brain Res 300:33–39
Sofroniew MV, Pearson RC, Eckenstein F, Cuello AC,
Powell TP (1983) Retrograde changes in cholinergic
neurons in the basal forebrain of the rat following
cortical damage. Brain Res 289:370–374
Sofroniew MV, Pearson RC, Cuello AC, Tagari PC,
Stephens PH (1986) Parenterally administered GM1
ganglioside prevents retrograde degeneration of cho-
linergic cells of the rat basal forebrain. Brain Res
398:393–396
Sofroniew MV, Galletly NP, Isacson O, Svendsen CN
(1990) Survival of adult basal forebrain cholinergic
neurons after loss of target neurons. Science
247:338–342
Stephens PH, Cuello AC, Sofroniew MV, Pearson RC,
Tagari P (1985) Effect of unilateral decortication on
choline acetyltransferase activity in the nucleus basalis
A. C. Cuello
and other areas of the rat brain. J Neurochem
45:1021–1026
Stephens PH, Tagari PC, Garofalo L, Maysinger D,
Piotte M, Cuello AC (1987) Neural plasticity of basal
forebrain cholinergic neurons: effects of gangliosides.
Neurosci Lett 80:80–84
Stewart SS, Appel SH (1988) Trophic factors in neuro-
logic disease. Annu Rev Med 39:193–201
Svennerholm L (1980) Ganglioside designation. In: Struc-
ture and function of gangliosides. Springer
Tettamanti G (1988) Towards the understanding of the
physiological role of gangliosides. J New Trends Gan-
glioside Res Neurochem Neuroregenerative Aspects
14:625–646
Venero JL, Knusel B, Beck KD, Hefti F (1994) Expression
of neurotrophin and trk receptor genes in adult rats
with fimbria transections: effect of intraventricular
nerve growth factor and brain-derived neurotrophic
factor administration. Neuroscience 59:797–815
Wanleenuwat P, Iwanowski P, Kozubski W (2020)
Antiganglioside antibodies in neurological diseases. J
Neurol Sci 408:116576
Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle
JT, Delon MR (1982) Alzheimer’s disease and senile
dementia: loss of neurons in the basal forebrain. Sci-
ence 215:1237–1239
Williams LR, Varon S, Peterson GM, Wictorin K,
Fischer W, Bjorklund A, Gage FH (1986) Continuous
infusion of nerve growth factor prevents basal fore-
brain neuronal death after fimbria fornix transection.
Proc Natl Acad Sci U S A 83:9231–9235
Wisniewski HM, Wrzolek M (1988) Pathogenesis of amy-
loid formation in Alzheimer’s disease, Down’s syn-
drome and scrapie. Ciba Found Symp 135:224–238
Yates CM, Simpson J, Maloney AF, Gordon A, Reid AH
(1980) Alzheimer-like cholinergic deficiency in Down
syndrome. Lancet 2:979
 NGF and the Amyloid Precursor Protein
in Alzheimer’s Disease: From Molecular 10
Players to Neuronal Circuits

Viviana Triaca, Francesca Ruberti, and Nadia Canu

Abstract (MRI) and positron emission tomography

Alzheimer’s disease (AD), one of the most
common causes of dementia in elderly people,
is characterized by progressive impairment in
cognitive function, early degeneration of basal
forebrain cholinergic neurons (BFCNs),
abnormal metabolism of the amyloid precursor
protein (APP), amyloid beta-peptide (Aβ)
depositions, and neurofibrillary tangles.
According to the cholinergic hypothesis, dys-
function of acetylcholine-containing neurons
in the basal forebrain contributes markedly to
the cognitive decline observed in AD. In addi-
tion, the neurotrophic factor hypothesis posits
that the loss nerve growth factor (NGF) signal-
ling in AD may account for the vulnerability to
atrophy of BFCNs and consequent impairment
of cholinergic functions. Though acetylcholin-
esterase inhibitors provide only partial and
symptomatic relief to AD patients, emerging
data from in vivo magnetic resonance imaging
V. Triaca · F. Ruberti
Institute of Biochemistry and Cell Biology (IBBC),
National Research Council (CNR), Campus A. Buzzati-
Traverso, Monterotondo, RM, Italy
e-mail: viviana.triaca@cnr.it; francesca.ruberti@cnr.it
N. Canu (*)
Institute of Biochemistry and Cell Biology (IBBC),
National Research Council (CNR), Campus A. Buzzati-
Traverso, Monterotondo, RM, Italy
Department of System Medicine, Section of Physiology,
University of Rome “Tor Vergata”, Rome, Italy
e-mail: nadia.canu@uniroma2.it
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_10
(PET) studies in mild cognitive impairment
(MCI) and AD patients highlight the early
involvement of BFCNs in MCI and the early
phase of AD. These data support the choliner-
gic and neurotrophic hypotheses of AD and
suggest new targets for AD therapy.
Different mechanisms account for selective
vulnerability of BFCNs to AD pathology, with
regard to altered metabolism of APP and tau.
In this review, we provide a general overview
of the current knowledge of NGF and APP
interplay, focusing on the role of APP in
regulating NGF receptors trafficking/signal-
ling and on the involvement of NGF in
modulating phosphorylation of APP, which
in turn controls APP intracellular trafficking
and processing. Moreover, we highlight the
consequences of APP interaction with
p75NTR and TrkA receptor, which share the
same binding site within the APP juxta-
membrane domain. We underline the impor-
tance of insulin dysmetabolism in AD pathol-
ogy, in the light of our recent data showing that
overlapping intracellular signalling pathways
stimulated by NGF or insulin can be compen-
satory. In particular, NGF-based signalling is
able to ameliorates deficiencies in insulin sig-
nalling in the medial septum of 3
Tg-AD
mice. Finally, we present an overview of
NGF-regulated microRNAs (miRNAs).
These small non-coding RNAs are involved
in post-transcriptional regulation of gene
145
146
expression, and we focus on a subset that are
specifically deregulated in AD and thus poten-
tially contribute to its pathology.
10.1 Alzheimer’s Disease Pathology
In 1906, the German clinical psychiatrist and
neuroanatomist Alois Alzheimer first identified
and described a ‘peculiar severe disease process
of the cerebral cortex’ in Auguste Deter, a
51-year-old woman, who rapidly developed
dementia typical of older individuals. This dis-
ease process, which would become known as
Alzheimer’s disease (AD), is today the most com-
mon cause of dementia among elderly people.
AD can be either sporadic or familial. Sporadic
AD (SAD) usually occurs after age 65 and is the
most common form of AD. Familial AD (FAD),
which typically presents in middle age, is a very
rare genetic condition caused by a mutation in the
type 1 transmembrane protein APP, Presenilin
1 (PS1) and 2 (PS2) genes, whose protein
products regulate APP processing (Dorszewska
et al. 2016), and increase copy number for APP
gene, as in Down syndrome (DS).
Clinical manifestations of AD include spatial
navigation, memory, thinking, comprehension,
learning capacity loss, as well as significant dete-
rioration of cognitive function as compared to
normal aging-related changes. The weakening of
cognitive function is generally associated with,
and sometimes precedes, deterioration in emo-
tional control, social behaviour, or motivation.
Thus, patients progress from normal brain ageing
to mild cognitive impairment (MCI) followed by
increasing dementia severity (i.e., mild, moderate,
and severe) in SAD.
Anatomopathological Hallmarks: APP, Tau,
Glial Response, Neuronal
and Synaptic Loss The main neuropathological
features of AD include amyloid plaques, neurofi-
brillary tangles (NFT), glial responses, and neu-
ronal and synaptic loss (Serrano-Pozo et al.
2011). The amyloid plaques result from the
V. Triaca et al.
abnormal extracellular accumulation and deposi-
tion in the brain and the cerebral vessel [cerebral
amyloid angiopathy] of the amyloid-β peptide
(Aβ) with 40 or 42 amino acids (Aβ 40 and Aβ
42). These peptides are the products of the physi-
ological metabolism of the APP after its sequen-
tial cleavage by the aspartyl protease β-site APP
cleaving enzyme 1 (BACE1, also termed
β-secretase) and the γ-secretase complex (includ-
ing PS1 and PS2) in neurons. While Aβ 40 is the
most abundant form, neurotoxicity is mainly
mediated by Aβ 42. NFTs are essentially made
of paired helical filaments (PHFs), which are
fibrils of different morphology mainly composed
of the abnormal misfolded and hyperpho-
sphorylated microtubule-associated protein tau.
Invariably accompanying NFTs are the neuropil
threads (i.e., abnormal neurites containing, con-
trary to normal neurites, straight and paired heli-
cal filaments composed of tau and ubiquitin;
Serrano-Pozo et al. 2011).
Glial responses have come to assume a pivotal
role in both clinical expression and progression of
cognitive decline in AD. Indeed, reactive
astrocytes and activated microglial cells normally
decorate amyloid plaques, suggesting that Aβ is a
major trigger. However, glial responses show a
positive relationship also with NFT and contrib-
ute to the ongoing neurodegeneration (Serrano-
Pozo et al. 2011).
Neuronal loss is the cause of pathological cor-
tical atrophy as revealed by Nissl or NeuN
staining of brain sections, computed tomography
(CT) scan and MRI analysis in vivo. Its regional
and laminar distribution meets that of NFTs. Not-
withstanding, within the same region neuronal
loss overcomes the numbers of NFTs, and
neurons bearing NFT may be still alive. These
findings led to considering the neuronal loss a
better correlate of cognitive deficits than the num-
ber of NFTs. Synapse loss is another causative
agent of cortical atrophy in AD. Synapse degen-
eration is responsible for the loss of structural
plasticity and inefficiency of the neuronal net-
work in AD, as shown post mortem by
immunohistochemistry using antibodies against
pre- or post-synaptic proteins, by electron
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . . 147

microscopy, and by 18F-fluorodeoxyglucose pos- Neurotrophic Hypothesis Intimately connected

itron emission tomography ([18F] FDG PET) in to the cholinergic hypothesis is the neurotrophic
human subjects (Kashyap et al. 2019). Interest- hypothesis (Appel 1981; Chen et al. 2018).
ingly, network activity seems to be disproportion- According to this, loss of a neurotrophic factor
ately weakened in the early stages of AD, [in particular NGF (although today we refer to the
possibly due to the regional specific effects of loss of NGF-signalling)] in the AD brain may
altered metabolism of APP and tau on synapses account for the dysfunction of cholinergic system.
(Spires-Jones and Hyman 2014). This hypothesis has been recently reinforced by
data from single-cell neuron-specific gene expres-
sion profiles and functional network analysis that
have predicted a strong association of the
10.2 Past and Current
neurotrophin signalling pathway with neuronal
Aetiopathological Hypotheses
vulnerability to AD (Roussarie et al. 2020).
in Sporadic Alzheimer’s
Disease (SAD)
Aβ and Aβ-Oligomer (AβO) Cascade
Hypothesis In 1992, it was proposed that the
There are several hypotheses regarding the causes
accumulation of fibrillar Aβ peptides in the
of SAD.
brain—as result of an imbalance between Aβ
production and Aβ clearance—is a crucial step
Cholinergic Hypothesis In the late 1970s,
in AD pathogenesis and that NFT, cell loss, vas-
researchers enunciated the ‘cholinergic hypothe-
cular damage, and dementia all result from Aβ
sis of age-associated memory dysfunction’ that
deposition (Hardy and Higgings 1992). This
was extended also to AD. According to this
hypothesis relied on evidence that people carry-
hypothesis, AD dementia was attributed to a fail-
ing mutations in three genes involved in Aβ pro-
ure of the cholinergic system (Davies and
duction (APP, PS1, PS2) and patients with DS
Maloney 1976). One class of symptom-
(overexpressing APP due to a triplication the
alleviating medications, such as the cholinester-
gene coding for APP, which is located on chro-
ase inhibitors, are in common use today. The
mosome 21) experienced memory loss and neu-
cholinergic hypothesis implied that the altered
rological changes characteristic of
physiology of basal forebrain cholinergic neurons
AD. Moreover, the generation of different APP
(BFCNs) contributed to AD pathology. A sugges-
or APP/PS1 transgenic mouse models developing
tion later confirmed by finding that a massive
some of the main features of FAD (Puzzo et al.
neurons reduction in the nucleus basalis of
2014) reinforced the Aβ hypothesis. However, the
Meynert (NBM) (Whitehouse et al. 1981, 1982)
evidence that Aβ causes SAD is complex and
preceded by years the cholinergic impaiments
hazy, thus prompting criticisms about the central
characteristic of AD (Cuello et al. 2007; Schliebs
role of the Aβ cascade in AD pathogenesis.
and Arendt 2011).
Among these are the weak temporal and
Recently this concept has been questioned by
anatomical correlation between Aβ deposition,
the only modest and transient cognitive relief
neuronal death, and clinical symptoms in SAD,
provided by cholinesterase inhibitors, possibly
and the finding that all attempts to develop A-
because of late administration to severe AD
β-targeting drugs to treat AD have been mostly
patients. On the other hand, novel drugs targeting
unsuccessful so far.
the cholinergic system, like muscarinic receptor
In 1998, the Aβ cascade evolved into the AβO
agonists (Fisher 2012; Verma et al. 2018), as well
hypothesis. According to this hypothesis, soluble
as deep brain stimulation (DBS) of basal fore-
and oligomeric forms of Aβ trigger the cascade of
brain nuclei (Mankin and Fried 2020), are
events leading to AD (Cline et al. 2018). The
among the most promising approaches for early
main issue related to the AβO hypothesis is the
phase of AD therapy.
148
time of appearance and exact identity of the more
toxic AβO species. This aspect deserves further
investigation to develop Aβ-oriented therapeutics
against the correct target at the desired time.
Moreover, Aβ monomer homeostasis is essential
for the synaptic function and it should be taken
into consideration since the Aβ ‘loss-of-function’
in neurons is a key determinant in AD pathophys-
iology (Giuffrida et al. 2009).
Tau Hypothesis In contrast to plaque deposition,
tau pathology correlates more closely with neuro-
nal loss and with the severity, and duration of
cognitive deficits (Arriagada et al. 1992; Bierer
et al. 1995; Giannakopoulos et al. 2003; Bejanin
et al. 2017). Tau pathology [due to an imbalance
of tau isoforms for splicing deficits and/or aber-
rant post-translational modifications (hyperpho-
sphorylation, glycosylation, and truncation)]
disrupts neuronal homeostasis by affecting the
transport system (of the neurotrophic receptors,
APP, etc.), the cytoskeletal system, signalling
system, and mitochondrial integrity, and
culminates in aberrant cell death even in the
absence of NFTs (Iqbal et al. 2016). Moreover,
tau pathology usually first appears in discrete and
specific areas to later spread trans-synaptically, to
other brain regions, eventually affecting circuit
functional connectivity and physiology (Frost
et al. 2009). For several years, Aβ and tau deposi-
tion have been seen as two distinct pathogno-
monic signs and causative agents of
AD. However, a huge amount of experimental
evidence has shown a mutual control between
Aβ and tau (Selkoe 2011). Indeed, the high affin-
ity between Aβ and tau induces the assembly of a
‘soluble complex that may promote self-
aggregation of both into the insoluble forms’
(Guo et al. 2006). Moreover, AβO seeds the for-
mation of highly toxic tau oligomers, redistributes
tau to dendrites, and drives the synaptic spread of
tau (Bilousova et al. 2016). Also, the extracellular
region of APP, with or without FAD mutations,
forms a complex with tau fibrils. This complex
promotes the uptake of tau fibrils into the cells
and causes the seed-dependent intracellular tau
aggregation. This latter, in turn, favours the cell-
V. Triaca et al.
to-cell spreading of tau pathology (Takahashi
et al. 2015).
An Integrated Hypothesis for AD An emerging
point of view sustains that AD pathogenesis ‘is
not simply a neuron-centric, linear cascade
initiated by Aβ/AβO and leading to dementia,
but rather a long, complex cellular phase
consisting of feedback and feedforward responses
of astrocytes, microglia, and vasculature’
(De Strooper and Karran 2016). A series of events
elicit reactive gliosis and neuroinflammation.
Among these are ROS activation, impaired astro-
cytic clearance of Aβ [associated or not to induc-
tion of microglial phagocytosis (Batarseh et al.
2016; Nielsen et al. 2010)], AβO-mediated switch
in microglial phenotype to a pro-inflammatory
phenotype leading to improper tumour necrosis
factor secretion (El-Shimy et al. 2015). In turn,
reactive microglia and astrocytes secrete numer-
ous pro-inflammatory cytokines, disturb the sig-
nalling of insulin, alter astrocytic insulin
secretion, contribute to glucose hypometabolism,
impair neurotrophic supply, impact the expres-
sion of excitatory amino acid transporters 1 and
2, thus impairing synaptic plasticity. This point of
view has flowed into a more integrated hypothesis
for SAD (De Felice 2013). According to this
hypothesis, SAD results from the accumulation
over the time of the insults to the brain and
peripheral organs that cause an increase of AβO
levels.
10.3 BFCNs’ Vulnerability in AD
Target Circuits
Aβ and tau pathologies impair the circuits
involved in spatial orientation, learning, and
memory. Nevertheless showing different spatial/
temporal distributions. Aβ deposition begins
within the cortex (frontal, temporal, and occipital)
and later attacks the hippocampus, the EC, insular
and cingulate cortices, and amygdala. Then, it
colonizes several subcortical regions, including
BFCNs, and later the substantia nigra, brainstem
nuclei, and also the molecular layer of the
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
cerebellum (Thal et al. 2002). NFT deposition is
quite different from that for Aβ plaques. NFT
deposition exhibits the identical regional pattern
as neurodegeneration and occurs in an exceed-
ingly predictable, stereotypical spatiotemporal
sequence of six stages (Stages I–VI) (Braak and
Braak 1991).
In line with the foremost recent clinical
findings, current experimental approaches focus
on specific neuronal vulnerability and network
deficit as the main driver of MCI to AD progres-
sion and AD pathology occurrence, replacing the
old view of AD as a single-molecule single-cell
neurodegenerative disease. Recently,
neurodegeneration in AD has been demonstrated
to specifically affect the circuits of the basal fore-
brain in a predictable manner (Schmitz et al.
2018).
BFCNs include the medial septum (Ch1), the
vertical and horizontal diagonal Bands of Broca
(VDB or Ch2 and HDB or Ch3, respectively), the
substantia innominata, and the NBM (Ch4). As
described by anterograde, retrograde, and trans-
synaptic labellings, primates’ cholinergic inputs
to the hippocampus arise from the medial septum
and vertical limb of the diagonal band of Broca
(DbB) innervates the hippocampal target neurons
within the CA1 and dentate gyrus, both through
the fornix and a non-fornical route (reviewed in
Ballinger et al. 2016).
Due to their intrinsic anatomy, biochemistry,
and neurotransmitter release, BFCNs have higher
propensity to accumulate disease-related proteins
(Aβ and tau specifically). In particular, early
accumulation of intraneuronal Aβ in adult life
and Aβ oligomerization during the aging process
makes BFCNs more prone to AD degeneration
(Baker-Nigh et al. 2015).
Alteration of cortical cholinergic neurons and
tau accumulation in the NBM are observed in
patients > 60 years and found increased in elderly
people, particularly those affected by AD (Geula
et al. 2008; Sassin et al. 2000). Accordingly,
in vivo MRI findings confirmed the reduced
BFCNs’ volume in MCI and AD (Kilimann
et al. 2014; Teipel et al. 2005, 2011; Grothe
et al. 2010).
149
In line with the cholinergic and neurotrophic
hypotheses, BFCNs are selectively vulnerable to
Aβ and tau oligomers due to their dependence on
NGF, though the underlying mechanism is not
well known. If the availability of NGF is ham-
pered due to an imbalance in maturation/degrada-
tion of NGF, and/or a concomitant increase in
precursor pro-NGF, BFCNs undergo progressive
atrophy (Cuello et al. 2007). Further they may
become more vulnerable (Allen et al. 2011) or
even contribute to the emergence of Aβ and tau
pathology (Calissano et al. 2010; Capsoni et al.
2000), finally leading to dementia exacerbation
and/or anticipation. Recently, BFCN vulnerabil-
ity has been demonstrated to occur also because
of the higher metabolic activity required by their
recruitment in reinforced feedback, during rapid
reconfiguration of cortical state and plasticity
(Hangya et al. 2015). Moreover, their requirement
of acetyl-CoA for the biosynthesis of both ATP
and acetylcholine may concur to their higher sus-
ceptibility to AD pathology (Szutowicz et al.
2013; Wurtman 1992).
BFCN atrophy was also reported by the AD
neuroimaging initiative (ADNI), demonstrating
that BF abnormalities precede and predict cortical
degeneration in AD subjects, as also
demonstrated by cortical thinning as detected by
PET in MCI patients, but not in cognitively nor-
mal people (Schmitz et al. 2018) and by increased
Aβ burden in the prefrontal cortex (Kerbler et al.
2015). Interestingly, DbB and frontal cortex dam-
age correlate well with cognitive decline, with
Meynert’s nucleus deficits being more associated
with impaired delayed recall in MCI subjects
(Grothe et al. 2010), particularly in women
(Cantero et al. 2016). Also, secondary BFCN
atrophy occurs in a late stage as a result of neuron
shrinkage in their cortical target circuits (Pearson
et al. 1983; Vogels et al. 1990). Of note, the
global cognitive deterioration rate can be
predicted based on the volume of basal choliner-
gic nuclei, with higher sensitivity than that
observed in hippocampal neurons (Teipel et al.
2005).
Moreover, the medial septum is also able to
reconfigure circuit function restoring the residual
150
memory in case of a fornical deficit (Zatorre et al.
2012), further pinpointing BFCNs as a promising
target for AD therapy. The medial septum net-
work has been shown to modulate hippocampal
memory via regulated theta oscillations by low
firing neurons (Petersen and Buzsáki 2020;
Tsanov 2017; Patel et al. 2012) and gamma
oscillations (Leung and Ma 2018), and to inhibit
the occurrence of an abnormal excitatory status
(Colom et al. 2006). In line, wave induction by
DBS resulted in ameliorated cognition (Jeong
et al. 2014; Stypulkowski et al. 2017), and distur-
bance in the theta and gamma network is pro-
posed as an early AD biomarker (Kitchigina
2018). Taken together, these results support the
hypothesis that BFCNs dysfunction is an early
event, significantly contributing to the AD con-
tinuum, and that re-activation of BF circuits may
lead to a significant improvement of synaptic
activity and cognition.
10.4 APP and NGF Interplay
As already mentioned, the viability and physiol-
ogy of BFCNs rely on NGF, which is initially
produced as precursor pro-NGF by BFCN targets
in the hippocampus and cortical tissues. NGF,
here produced, binds to its high-affinity TrkA
and the low-affinity (p75NTR) receptors present
at the distal end of BFCNs axons and, once
internalized, it is retrogradely transported to the
cell body. Internalization and transport of the
NGF-receptors complex to the cell body are
required for acetylcholine neurotransmitter
release to initiate responses in the BFCN targets.
In AD cholinergic neurons, the NGF/NGF recep-
tor complex is affected at many levels including
the following: (1) unbalanced NGF maturation
with an increase in the pro-NGF; (2) reduced
TrkA/p75NTR ratio; (3) increased pro-apoptotic
activity of p75NTR via its interaction with
pro-NGF; (4) stimulation of p75NTR by A-
β-peptide binding; (5) loss of TrkA receptor that
increases the affinity of pro-NGF for p75NTR,
thus favouring the pro-apoptotic activity of
p75NTR in cooperation with sortilin (reviewed
by Mufson et al. 2019; Ioannou and Fahnestock
2017).
V. Triaca et al.
APP is a type-I transmembrane protein
concentrated in the synapse, synthesized in the
endoplasmic reticulum (ER), transported to the
Golgi/trans-Golgi network (TGN), and inserted
in the plasma membrane (Caporaso et al. 1994).
Cell surface APP can be internalized for
endosomal recycling to the cell surface, for late
endosomal/lysosomal degradation, or for trans-
port from endosomes back to the TGN (reviewed
in Eggert et al. 2018). By regulating APP phos-
phorylation at specific sites, NGF may dictate
APP trafficking and processing in neurons
(Matrone et al. 2011; Triaca et al. 2016). It is
well known that APP is cleaved in the
amyloidogenic pathway by BACE1 at an extra-
cellular site adjacent to the plasma membrane to
yield the βCTF fragment (transmembrane
β-carboxyterminal), which is further cleaved by
γ-secretase complex [a complex comprising
presenilin, nicastrin, anterior pharynx defective
1 (APH-1), and presenilin enhancer 2 (PEN-2)]
to release the soluble APP intracellular cytoplas-
mic domain (AICD) fragment, Aβ 40, and Aβ
42 peptides. APP can also be cleaved though the
non-amyloidogenic pathway by cell surface
α-secretase (remarkably ADAM10). Cleavage
by α-secretase results in the release of the
ectodomain of APP-soluble fragment (sAPPα),
and the simultaneous generation of a membrane
retained C-terminal fragment (APP-CTF). This
latter can be further processed by γ-secretase
(reviewed by Zhang et al. 2011). Recent studies
have indicated that, while APP α-secretases
cleavage occurs at the plasma membrane, proteo-
lytic processing by BACE1, and then Aβ produc-
tion, requires APP internalization from the cell
surface to intracellular, acidic, endocytic
compartments (Koo and Squazzo 1994; Selkoe
et al. 1996; Haass et al. 2012).
10.4.1 APP Control of the NGF
Receptor Trafficking
and Signalling
Several pieces of evidence demonstrated a mutual
influence between trafficking and signalling of
NGF receptors with the trafficking and signalling
of APP. Besides being the precursor of Aβ
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
peptides, APP is a protein whose physiological
functions are likely lost in AD; thus, the loss-
function of APP contributes to AD pathogenesis.
APP has been implicated in neuronal plasticity,
synapse formation, iron and calcium metabolism,
cell adhesion, and also the regulation of traffick-
ing of BACE1, PS1 (Kamal et al. 2001; Liu et al.
2009), and choline transporter (Wang et al. 2007).
Beyond its own metabolism, APP somehow
interacts with the retrograde axonal transport
mechanism in the BFCNs that are among the
earliest neurons to degenerate in
AD. Particularly it has been reported that
increased APP gene dosage [triplicated in Down
syndrome (DS)] and overexpression of APP strik-
ingly affect NGF retrograde transport and cause
degeneration of BFCNs in a DS mouse model.
Also other transmembrane APP_CTF may likely
negatively impact NGF retrograde transport
(Salehi et al. 2006) by perturbing the endocytic-
lysosomal pathway. Further investigations to
identify the mechanism by which APP and its
various products impair the endocytic-lysosomal
pathway were performed in PC12 cells, cultured
rat BFCNs, and primary BFCNs fromTs65Dn
mice, a DS mouse model. It has been found that
Rab5 (major regulator of endosome biogenesis)
positive endosomes were enlarged in cultured
BFCNs from Ts65Dn mice, and endosomal size
was restored by APP RNAi, indicating that APP
is directly involved in such event. Moreover,
overexpression of full-length wild-type and
mutant forms of APP, as well as of the β-CTF
fragment, induced the activation of Rab5 and
enlargement of Rab5 endosomes in cultured
PC12 and rat BFCNs (Xu et al. 2016). Recently,
super-resolution structured illumination micros-
copy (SR-SIM) and transmission electron micros-
copy (TEM) have shown that endosome had a
normal size but appeared clustered in fibroblasts
and IPCS-derived neurons from individual DS,
and BFCN cultures from DS human subjects,
suggesting that APP overexpression causes a
‘traffic jam’ in the endosomal compartment
(Botté et al. 2020).
Altogether, these findings suggest that
overexpression of APP perturbs the Rab5 positive
151
endosomal compartment and that Rab5 increased
activation plays a paramount role in perturbing
NGF trafficking and signalling in AD and DS,
thus leading to BFCN atrophy. Since APP
interacts with both p75NTR and TrkA
(Fombonne et al. 2009; Matrone et al. 2011),
Zang and collaborators asked whether the mecha-
nism of APP-mediated impairment of NGF retro-
grade transport relied on altered trafficking of its
TrkA and p75NTR receptors. Thus, they
investigated the effects of APP on the p75NTR
and TrkA trafficking and the downstream signal-
ling under NGF stimulation. Interestingly, they
found that APP regulates cell surface levels of
the two NGF receptors. In particular, down-
regulation and over-expression of APP resulted
in increased or decreased NGF/NGF receptors’
endocytosis, respectively (Zhang et al. 2013).
Moreover, they showed that APP deficiency, by
enhancing the NGF-mediated PI3K/Akt and
MAPK, pathways, resulted in an increased neu-
ronal differentiation and survival in response to
NGF. It follows that the down-regulation of these
NGF stimulated pathways, as occurring when
APP is overexpressed, may result in early BFCN
suffering and eventually cell death.
10.4.2 Bad and Good Interactions
of APP with NGF Receptors
As already mentioned, the APP can interact with
p75NTR and TrkA NGF-receptors. However, the
effect of such different interactions regarding sur-
vival and APP metabolism is different.
Fombonne et al. (2009) showed that APP and
p75NTR interact directly, using different
techniques (including the yeast two-hybrid
assay, bioluminescence, resonance, energy trans-
fer, confocal microscopy, and coimmunopreci-
pitation in vitro and in vivo). By deletion
studies, they found that β-CTF (C99) (APP597-
695) but not C83 fragment (APP613-695) is suf-
ficient for this interaction, indicating that the Aβ
region of APP is required for APP/p75NTR inter-
action. Moreover, they found that the
APP/p75NTR complex is negatively modulated
152
by NGF and Aβ peptide, both in vitro and in vivo.
Indeed, colocalization of APP and p75NTR was
reduced in a mouse model of AD expressing a
high level of Aβ (PDAPP, J20 line). The authors
suggested that the APP/p75NTR complex is
blocked in AD as a consequence of the likely
more favourite binding of Aβ to APP. Interest-
ingly, they demonstrated that NGF and Aβ pep-
tide by preventing this association block its
downstream effects. In detail, they demonstrated
that the interaction between APP and p75NTR
‘shifts the effect of APP from a trophic one,
with the production of sAPPα and the Fe65-
dependent transcriptional effects, to an anti-
trophic one, with the reduction in sAPPα and
the increase in Aβ peptide production’
(Fombonne et al. 2009). The negative impact of
the APP/p75NTR interaction was further
supported by the finding that co-expression of
APP and p75NTR in neuroblastoma cells line
‘enhanced caspase-3 dependent cells death over
that induced by either APP or p75NTR alone’
(Fombonne et al. 2009). Equally, the APP may
also disturb p75NTR processing and signalling
by jamming the endocytic pathway (see above).
Regarding APP/TrkA interaction (Matrone
et al. 2011; Zhang et al. 2013; Triaca et al.
2016), we recently investigated this issue by
bimolecular fluorescence complementation
(BiFC), proximity ligation assay (PLA), and
co-immunoprecipitation of full-length and
selected APP and TrkA fragments in transfected
HEK293 cells and rat primary septal cultures
(Canu et al. 2017a, b). Interestingly, we found
that, as for APP/p75NTR interaction (Fombonne
et al. 2009), the APP juxta-membrane region
encompassing β- and α-secretase cleavage sites
is sufficient for its interaction with TrkA. On the
other side, TrkA binds APP using its own juxta-
transmembrane, as well as its intracellular
domain. The latter TrkA domain is likely crucial
for modulating the phosphorylation state of APP,
known to affect in turn the interaction with TrkA
(Triaca et al. 2016). Although BiFC and PLA are
able to detect the interaction among proteins in
close proximity (distance range 7–15 nm), they
do not exclude the involvement of a third partner
in favouring the assembly of a multimolecular
V. Triaca et al.
complex. We have ruled out strong candidates
such as p75NTR, ShcC, and Mint-2 in bridging
APP and TrkA, since their co-expression with
APP and TrkA BIFC plasmids did not lead to an
increased BIFC signal (Canu et al. 2017a, b).
Likewise, we suggested that a potential partner
may be sortilin. Sortilin is a member of the
Vps10p-domain receptor family that acts as a
receptor of neurotrophic factors, mediates traf-
ficking from the secretory pathway to endosomes,
and retrograde transport to the TGN (Nielsen
et al. 2001). Particularly, sortilin binds TrkA,
enables TrkA anterograde axonal transport,
enhances neurotrophin signalling (Vaegter et al.
2011), and interacts with APP to influence its
production and cellular uptake (Gustafsen et al.
2013). Despite this physiological role, sortilin
acts as a co-receptor with p75NTR to mediate
Pro-NGF-induced pro-death signalling (Nykjaer
et al. 2004). APP/TrkA complex is confined to the
plasma membrane, ER, Golgi, and endocytic
vesicles, as evidenced by the colocalization of
the APP/TrkA complex with compartment-
specific markers by both BiFC analysis
(in HEK-293 cells expressing exogenous APP
and TrkA) and PLA assay in rat septal neuronal
culture. Interestingly, drugs that perturb cellular
trafficking block this interaction consistently with
the known physiological transport of APP and
TrkA in these organelles. Our findings suggest
that the reciprocal physiological and pathological
interplay between APP and TrkA might occur
within these compartments where both proteins
form homodimers in the ER/Golgi before
travelling to the cell surface.
Moreover, we found that in primary septal
neurons, NGF increases the number of TrkA/
APP complexes in every compartment, including
the ER and Golgi apparatus within 1 h of NGF
treatment. This effect is likely dependent on
NGF-mediated changes in APP phosphorylation
(Triaca et al. 2016; Matrone et al. 2011) since
TrkA and APP levels did not change under NGF
stimulation (Canu et al. 2017a). Conversely,
TrkA/APP complexes decrease in number with-
out any apparent decrease of TrkA or APP and
before the loss of cell viability following pro-
death-inducing stimuli like NGF deprivation, Aβ
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
peptide, staurosporine, and rapamycin treatments
(Canu et al. 2017a). Such agents, via different
mechanisms, activate the amyloidogenic APP
processing [as observed in NGF-deprived pri-
mary hippocampal neurons (Matrone et al.
2008)] by disentangling the physiological TrkA/
APP complex. Since APP/APP oligomerization
promotes the amyloidogenic APP processing,
we hypothesize that TrkA/APP reduces the num-
ber of APP homodimers in physiological
conditions and under NGF stimulation and that,
during cell death (Canu et al. 2017a) and AD
neurodegeneration (Triaca et al. 2016), the disso-
ciation of APP/TrkA complex allows the assem-
bly of APP homodimers, which are more
susceptible to processing by β- and γ-secretase.
10.4.3 NGF Regulates APP Trafficking
and Processing in BFCNs
Regardless of the disease aetiology (sporadic or
familial), protein toxicity seems to be a main
hallmark of several neurodegenerative diseases
(including AD, Parkinson’s disease, Huntington’s
disease, frontotemporal dementia, and
amyotrophic lateral sclerosis) and involves accu-
mulation, mislocalization, and/or aggregation of
the disease-specific proteins. In neurodegenera-
tive diseases, protein toxicity is often
phosphorylation-dependent and leads to cellular
defects such as transcriptional alteration, mito-
chondrial dysfunction, and an impaired protein/
RNA quality control system. Each of these events
critically contributes to the initiation and progres-
sion of cognitive/motor deficits (Chung et al.
2018). Protein phosphorylation of APP in AD,
of tau in AD and PD, and of alpha-synuclein in
Lewy bodies of PD and synucleinopathies, is
responsible for initiating and sustaining the
degenerative process in vulnerable BFCNs,
substantia nigra, and neurons, respectively
(Oueslati 2016; Tenreiro et al. 2014; Spillantini
et al. 1998; Goedert et al. 1992). In the case of
APP, phosphorylation at specific tyrosine (tyr) or
serine/threonine (ser/thr) residues at its intracellu-
lar tail (C-terminal) is key to alternative cleavage
pathways by α or β secretases (ADAM10/17 and
153
BACE, respectively; Lee et al. 2003) and to its
subsequent trafficking and processing. Particu-
larly, the phosphorylation of APP at Thr668
(T6889 in the YENPT668Y motif of the APP
C-tail has been described to control its intracellu-
lar trafficking to abnormal Rab5 positive early
endosomes, favouring Aβ fragments generation
by BACE1, thus contributing to
neurodegeneration (Barbagallo et al. 2010;
Chang et al. 2006; Cataldo et al. 2004). In line,
hyperphosphorylation of APP is observed in
human AD brain (Lee et al. 2003; Schettini
et al. 2010) and it is mediated by a number of
ser/thr kinases, including JNKs 1-3, GSK3β,
Cdk5, and ROCK1, all recognized key patholog-
ical players in Aβ-driven synaptic dysfunctions,
axonal swelling, and neuronal death (Hu et al.
2019). Accordingly, specifically targeting APP
phosphorylation was hypothesized as a promising
AD therapy (Lee et al. 2003).
Recently, it has been demonstrated that NGF
modulates APP phosphorylation at T668 by
down-regulating the JNK kinase in BFCNs
in vitro, as well as in intact septo-hippocampal
slices ex vivo, and in AD animal models. The
NGF-TrkA signalling pathway stimulates ShcC-
mediated PI3K inhibition of JNK, in particular of
the 54kDa isoform JNK(p54), thus reducing
APPpT668 level in adult BFCNs (Triaca et al.
2016). Activation of ShcC, the adult brain iso-
form of the early TrkA adaptor Shc, represents a
key signalling event in NGF-driven
neuroprotection of BFCNs and related cognition.
Indeed, the ShcC genetic ablation is able per se to
increase JNK activation (Triaca et al. 2016, 2018)
by releasing the Shc-PI3K control over the initia-
tion of the apoptotic route (Molton et al. 2003),
thus specifically fostering β40 and βCTF genera-
tion in the hippocampus of shcC-KO mice, and
affecting related cognitive performance in the
visual object recognition test (vORT; Triaca
et al. 2018). In line, the aberrant activation of
the alternative TrkA-PLCγ pathway has been
suggested to promote p75NTR-dependent
amyloidogenesis and apoptosis in hippocampal
neurons (Matrone et al. 2008, 2009).
Upon NGF-elicited autophosphorylation at
Tyr490 (Y490), TrkApY490 binds APP and
154
moves to the Golgi system, where APP exposure
to BACE1 is irrelevant, possibly because of the
prevailing TrkA competitive binding to APP and
of reduced physiological BACE1 level in the
compartment. The reduced APP/BACE1 interac-
tion directly results in the down-regulation of
amyloidogenic APP cleavage, and βCTF and Aβ
levels (Triaca et al. 2016).
Following T668 phosphorylation, APPpT668
is not able to bind TrkA, as observed by
co-immunoprecipitation and colocalization stud-
ies. Released by TrkA, APP is now free to be
trafficked to the BACE1-enriched endo-lyso-
somal compartment, where more Aβ is produced
(Triaca et al. 2016). Similarly, the T668 phos-
phorylation is known to cause the detachment of
another well-known APP interactor, Fe65, lead-
ing to neuronal death (Chang et al. 2006). On the
other hand, the neuroprotective interaction of
APP with TrkA is affected in the cortex and
hippocampus of AD patients and of the 3xTg-
AD mouse model, where the APPpT668 level is
augmented. Thus, in addition to favouring the
amyloidogenic APP-APP interaction by competi-
tion (Canu et al. 2017a, b), the lack of APP-TrkA
binding directly promotes Aβ generation via the
BACE1 cleavage of APP (Triaca et al. 2016).
Noteworthy, the role of TrkA as APP shuttling
protein was hypothesized a decade ago (Heese
et al. 2004). Further evidence of a role for the
NGF pathway in the modulation of APP phos-
phorylation came from the observation that
transactivated TrkA induces Y682 phosphoryla-
tion at the Y682NPTY motif of the APP C-tail
in vitro and subsequent binding of the APP intra-
cellular domain (AID) with Shc (Tarr et al. 2002).
Opposite, APP-TrkA interaction is lost in APPYG
transgenic mice lacking the Y682 residue
(Matrone et al. 2011), resulting in reduced APP
degradation and cholinergic demise (La Rosa
et al. 2015; Matrone et al. 2012).
Conclusively, these findings show the signal-
ling and molecular substrates, and the intracellu-
lar mechanisms related to the anti-amyloidogenic
NGF action in BFCNs of the septo-hippocampal
pathway in physio-pathological conditions.
Promoting APP-TrkA interaction and/or pro-
moting the ShcC-PI3K pathway are envisaged as
V. Triaca et al.
promising tools to restore the physiological
NGF-TrkA system control over APP
processing—the final goal being the restoration
of normal cholinergic activity in the basal fore-
brain of early AD patients and possibly MCI.
10.5 BFCN Insulin Resistance
as a Risk Factor for AD.
Improvement by NGF
The brain has been long time thought to be
insulin-independent. Instead, recent work has
demonstrated the expression of the insulin recep-
tor (IR), insulin receptor substrate (IRS), and
glucose transporters 1-8 (Glut1-8) in the BF, and
highlighted the key insulin’s action on brain glu-
cose homeostasis, memory, and mood (reviewed
in de la Monte 2012). In particular, insulin has
been reported to affect the expression of N-
methyl-D-aspartate (NMDA) receptors (Skeberdis
et al. 2001), modulate levels of the
neurotransmitters, like acetylcholine (Fishwick
and Rylett 2015), and influence long-term poten-
tiation (LTP; Zhao and Alkon 2001). In turn, the
deficiency/lack of insulin signalling in the brain,
also known as neuronal insulin resistance, leads
to changes in PI3K, Akt, and GSK3β activities, as
well as Tau hyperphosphorylation, and Aβ pro-
duction in Neuronal Insulin Receptor KO mice
(NIRKO; Schubert et al. 2004).
Chronic exposure to an excess of insulin
determines insulin resistance via feedback inhibi-
tion of the IR by the Ser306 phosphorylation of
the insulin receptor substrate 1 (IRS1), the insulin
signalling hub, hampering subsequent metabolic
stimulation of the insulin pathway (De Felice
et al. 2014). Age-related hyperinsulinemia,
which is know to induce a decrease in cell sensi-
tivity to a subsequent glucose challange,
quadriplicates the risk of developing AD (Chow
et al., 2019).
In line, systemic hyperinsulinemia, type 2 dia-
betes mellitus (T2D), obesity, and metabolic
conditions may trigger tissue ‘desensitization’ to
insulin, increasing the risk to develop AD (Kellar
and Craft 2020; Ferreira et al. 2018). Detection of
cerebral Aβ with 18-F fluorbetan amyloid PET,
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
brain FDG-PET, and blood HOMA-IR index
(homeostasis model assessment of insulin resis-
tance (HOMA-IR) has correlated central
hypoglycaemia with peripheral insulin resistance
in AD patients, and associated central
dysmetabolism with cortical memory deficits
(Willette et al. 2015). Consistently, a diabetes
diagnosis is a good predictor of cognitive decline
and mortality over 10 years in the elderly popula-
tion (Exalto et al. 2013; Tilvis et al. 2004). Now-
adays, AD is considered now as type 3 diabetes,
because of the well-characterized status reminis-
cent of insulin resistance in the brain promoting
amyloidogenesis and neurodegeneration (Arnold
et al. 2018; De Felice et al. 2014). Central meta-
bolic dysfunction is considered a well-established
feature of AD, and brain glucose hypometabolism
is associated with AD dementia (Whitmer et al.
2007; Craft 2007) even before early AD manifes-
tation (Sperling et al. 2011).
Accordingly, metabolic derangement and con-
current early synaptic failure in BFCNs have been
reported to parallel memory deficits in MCI and
AD, and proposed as a good predictor of MCI
progression towards AD (Chen and Mobley
2019), although the underlying mechanisms are
still under debate.
To investigate these mechanisms, we devel-
oped an in vitro model of insulin resistance in
rat cholinergic neurons by means of
hyperinsulinemic culture conditions. BFCNs
were shown to develop insulin resistance as
shown by reduced activation of both insulin
receptor (IR) and insulin substrate receptor
1 (IRS1). Further, insulin-resistant neurons
expressed a higher level of serine phosphorylated
IRS1, a well-known hallmark of insulin resis-
tance, and showed reduced glucose transporter
2 (Glut2) translocation to the plasma membrane
resulting in lower glucose uptake. Also, neuronal
activity was repressed, as demonstrated by
decreased nuclear c-Fos expression (Sposato
et al. 2019).
Significantly, NGF was shown to improve
insulin resistance by TrkA-driven tyrosine phos-
phorylation and activation of IRS1, both in the
in vitro model and in 3xTg-AD mice. Interest-
ingly enough, nasal administration of NGF to
155
3xTg-AD mice rescued insulin resistance in the
medial septum at the pre-symptomatic stage, sev-
eral months before its detection in the neocortex
and hippocampus (Sposato et al. 2019).
Since insulin resistance correlates with Aβ
burden, even before the AD onset of memory
decline (Wang et al. 2019a, b), the ability of
NGF to reactivate the insulin pathway and control
Aβ production and related cognition further
pinpoints its use for insulin resistance, glucose
dysmetabolism, and synaptic derangement in
early AD.
10.6 Interplay Between MicroRNAs,
APP, and NGF in Alzheimer’s
Disease
MicroRNAs and AD MicroRNAs (miRNAs)
are evolutionarily conserved small non-coding
RNAs around 22 nucleotides in length. They
bind messenger RNA (mRNA) of target genes
mainly within the 30 untranslated region (UTR)
and regulate, at the post-transcriptional level,
gene expression through transcript destabilization
or translation inhibition. In humans and
mammals, each miRNA can potentially modulate
the expression of several target genes and in turn
regulate many cellular processes. Brain-
expressed miRNAs play a role in neuronal devel-
opment, synaptic transmission, synaptic plastic-
ity, and behaviour. Furthermore, specific
miRNAs are highly enriched in several neuronal
cell types (reviewed in Kiltschewskij and Cairns
2019). Alteration of miRNA expression profiles
has been described in AD human brain tissues.
Dysregulation of miRNAs is implicated in APP
expression, Aβ production/clearance, and tau
phosphorylation, as well as in insulin signalling,
and neuroinflammation; and it has been shown to
contribute to AD pathology (reviewed in Wang
et al. 2019a; Improta-Caria et al. 2020).
The interplay between APP and NGF
metabolisms could also be modulated by
miRNAs. MiRNAs regulating APP expression
and, in turn, amyloidogenesis (i.e. miR-16,
miR-20 family, miR-101, miR-153 miR-346,
etc) are down-regulated in AD mouse models
156
and AD human brain tissues (reviewed in
Amakiri et al. 2019). It has been shown that the
modulation of these microRNAs has functional
effects on relevant hallmarks of AD pathology.
For example, the brain delivery of miR-16 in
mice reduces APP, BACE1, and tau in a region-
specific manner (Parsi et al. 2015). Conversely,
inhibition of miR-101 in mouse hippocampus not
only up-regulates AD-related genes but also neg-
atively affects learning and memory (Barbato
et al. 2020). It might be relevant to investigate
whether the down-regulation of these miRNAs
leading to APP over-expression could also inter-
fere with NGF trafficking, as observed in BFCNs
of the DS mouse model (see Sect. 10.4.1).
NGF-Modulated miRNAs and Their
Dysregulation in AD Few data are available
on the miRNA-mediated regulation of NGF and
NGF receptors in the nervous system. Interest-
ingly, it has been shown that Let-7 miRNAs
induce peripheral nerve regeneration by
regulating NGF expression (Li et al. 2015). Evi-
dence of NGF receptors modulation by miRNAs
has been observed in an in vivo mouse model of
brain focal ischemia, where TrkA and p75NTR
receptors exert protective and deleterious effects,
respectively. After cerebral ischemia, a decrease
in the level of miR-592 induces an increase of
p75NTR that is associated also to an increase of
pro-NGF and, in turn, cell death (Irmady et al.
2014). However, at this time there is no evidence
that miRNA-mediated regulation of NGF and/or
TrkA/p75NTR genes may occur in AD vulnera-
ble neuronal circuitry.
Instead, the effects of NGF on miRNAs
expression have been more extensively
investigated. Several expression profiles have
been mainly performed in NGF-differentiated
PC12 cells to identify NGF-regulated miRNAs
(Terasawa et al. 2009; Hamada et al. 2012;
Montalban et al. 2014; Pandey et al. 2015).
These studies described miRNAs involved in
NGF-related cell survival and differentiation, but
also neuronal degeneration due to NGF depriva-
tion. As different experimental conditions, such
V. Triaca et al.
as times and doses of NGF treatment, were used,
the NGF-regulated miRNAs identified were often
different in the various studies, and not always
associated with the same function. For example,
up-regulation of miR-221 in PC12 cells has been
found after 48 h of NGF treatment and it has been
associated with cell survival and activation of
extracellular signal-regulated kinase (ERK)
(Terasawa et al. 2009). In another study
up-regulation of miR-221 after 48 h and 8 days
of NGF exposure was associated with cell differ-
entiation (Hamada et al. 2012; Pandey et al.
2015).
Montalban et al. (2014) profiled miRNA
expression in PC12 cells not only upon NGF
treatment but also upon NGF deprivation of ter-
minally differentiated cells. The expression of a
small number of miRNAs was up-regulated by
NGF, and their induction was lost upon 24 h NGF
deprivation. Among the regulated miRNAs, they
found the miR-212/132 cluster, already known to
be modulated by BDNF, and including miR-21,
miR-29c, miR-30c miR-93, miR-103, miR-207,
miR-691, and miR-709. Interestingly, a few of
these miRNAs have also been associated with
AD. Among them, noteworthy is miR-132 that
is involved in several CNS functions and it is one
of the most down-regulated miRNAs in the inter-
mediate and late Braak stages of AD. Several
in vitro and in vivo studies have shown that
miR-132 depletion may aggravate AD pathology
acting on both Aβ and tau pathways, altering
several mechanisms of cellular homeostasis,
inducing dysfunction in synaptic plasticity and
memory processes (reviewed by Salta and De
Strooper 2017). To correlate miRNAs deregula-
tion to failure of NGF signalling is relevant to
highlight that the expression of miR-132 has been
studied over the course of AD in relation to Aβ,
tau, and ChAT expression in post-mortem NBM
human samples. It was shown that miR-132
expression levels were quite stable during the
early stage of AD and decreased significantly
during the late stages, showing a positive correla-
tion with ChAT (Zhu et al. 2016). Thus, it is
tempting to speculate that loss of NGF supply,
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
in the course of AD, may contribute to miR-132
down-regulation and lead to the vulnerability of
NBM in the late phase of AD.
Another NGF-regulated miRNA in PC12 cells
potentially associated with AD is miR-21.
Montalban et al. (2014) showed that miR-21 is
highly up-regulated from 6 h to 10 days of NGF
treatment and participates in NGF signalling. It
enhances MAPK and Akt signalling and induces
neuronal differentiation by increasing the neurite
length. Notably, over-expression of this miRNA
protects differentiated-PC12 cells from degenera-
tion upon NGF deprivation. More recently, it has
been shown that the level of miR-21 in cortical
and hippocampal neurons decreases significantly
after Aβ exposure (Chatterjee et al. 2016). More-
over, miR-21 over-expression in the hippocam-
pus of a mouse model of AD rescued memory
deficits and decreased both Aβ deposition and
proinflammatory factors (Cui et al. 2018).
Whether miR-21 down-regulation in AD is
strictly associated with the loss of NGF signalling
needs to be further investigated.
Altered expression of few miRNAs in AD
might also be part of a protective/adaptative
response to the pathology. A recent study showed
that miR-200c, a miRNA gradually induced in
NGF treated PC12 cells, is also up-regulated in
a mouse model of AD- and Aβ-treated neuronal
cells (Wu et al. 2016). The authors found that
miR-200c increase supports the survival and dif-
ferentiation of cultured neurons by PTEN down-
regulation. Moreover, they also suggested that
only TrkA positive cholinergic neurons are sensi-
tive to miR-200c-mediated neurite outgrowth.
Thus, the Aβ-miR-200c-PTEN pathway could
protect cholinergic neurons at the early stage of
AD. Besides changes of miRNA expression,
NGF also induces PC12 neuronal differentiation
by a reduction of let-7a miRNA activity. In par-
ticular, the uncoupling of miRNA from Ago2
protein (Patranabis and Bhattacharyya 2016), the
main component of the RNA-induced silencing
complex implicated in miRNA-mediated post-
transcriptional regulation of mRNA targets, has
been observed. Likewise, it has been shown that
NGF signalling in dorsal root ganglia may
157
regulate axonal elongation without changing
miRNA expression. Indeed, Map1b and Calm1
mRNA were released from the Fragile X Mental
retardation protein (FMRP) and miR-181d-
repressing granules (Wang et al. 2015).
Overall, a deep investigation of the role of
NGF on miRNAs regulation and their effects on
specific targets in TrkA/p75NTR cells of the CNS
is required. These studies in control and AD dis-
ease models may lead to the discovery of
NGF-regulated miRNAs potentially associated
with disease progression.
10.7 Conclusions
The current view of AD aetiopathology posits
that Aβ and tau depositions display a well-defined
spatial and temporal pattern of appearance. One
take-home lesson from unsuccessful clinical trials
centred on Aβ is that once Aβ has been deposited
in vulnerable BF circuits, the application of A-
β-targeting antibodies and/or BACE inhibitors
fails to improve the neuronal deficits or to induce
even partial recovery of cognition. The most plau-
sible reasons for the failure of these therapeutical
approaches for AD include insufficiently early
intervention, but also inaccurate choice of treat-
ment targets, improper drug dosages, loss of the
physiological function of Aβ peptides, and an
inadequate understanding of the complex patho-
physiology of AD.
Aβ-centred clinical trials have stimulated the
search for a more integrated view of AD patho-
genesis, which has helped to improve AD diag-
nosis, development of new disease-specific
tracking markers, and prediction of disease pro-
gression. In line with this, AD aetiopathology has
been re-conceptualized as a neurodegenerative
continuum, with three different phases reflecting
the progressive cognitive decline (Jack et al.
2018). In addition to considering the clinically
defined syndromal phases (MCI, early AD, severe
AD), AD staging takes into account the unique
combination of biological features within the A
(Aβ) - T (tau) - N (Neurodegeneration) system,
where N is a measure of FDG-PET
158
Fig. 10.1 A schematic model illustrating the control of
APP metabolism by the NGF/TrkA signalling, the early
insulin pathway and miR-132 level in healthy (up) and AD
(bottom) BFCNs. Healthy brain: The NGF pathway and
the anti-amyloidogenic route are shown. Upon binding to
its ligand NGF, TrkA autophosphorylates at the Y490
residue, thus acting as a docking site for ShcC. Once
bound and activated, ShcC hinders the JNK-mediated
phosphorylation of APP at the T668 site. Since TrkA
does not bind APP if phosphorylated at T668, the reduc-
tion of NGF-mediated APPpT668 promotes: (1) the
APP-TrkA binding, (2) the subsequent TrkA-mediated
V. Triaca et al.
trafficking of APP to the Golgi compartment and to the
plasma-membrane, and (3) preferential physiological
cleavage by ADAM10/17, and sAPPα level increase.
Upon NGF stimulation, pTrkAY490 is also able to induce
the insulin/IR pathway by tyrosine transphosphorylation
of the IRS1, promoting glucose uptake mainly through
Glut4 and sustaining cholinergic neurons activity, and
metabolism. In healthy brain, NGF signalling in choliner-
gic neurons may modulate the expression of miR-132
implicated in the homeostasis of Aβ and tau phosphoryla-
tion. Tau, not within the focus of this review, is not
hyperphosphorylated, and is in equilibrium between
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
hypometabolism and MRI-detected atrophy
(Ebenau et al. 2020).
As reported in AD Drug Development Pipe-
line 2020, several new drugs are in clinical trials
(phases 1–3) for the treatment of AD. Among
these are drugs for improving synaptic plasticity/
neuroprotection (24%) and drugs targeting
metabolism (12%), such as insulin sensitizers.
The neurotrophin NGF, with its clear
neuroprotective activity over BFCNs, has been
demonstrated to exert double-edged effects. It
acts as an Aβ-modulating molecule under
physio-pathological conditions (Triaca et al.
2016; Matrone et al. 2008; Canu et al.
2017a, b), but also as an insulin sensitizer by
activating IRS1, a central metabolic hub for neu-
ronal metabolism and synaptic activity in the
normal and AD brain (Sposato et al. 2019).
In light of recent findings, effective AD treat-
ment may require a combination of treatments
rather than a monotherapy. Target-specific non--
invasive delivery of a combination of classical
APP/tau-targeted molecules, together with novel
compounds that sustain neuronal trophism,
recover neuronal metabolism and improve synap-
tic plasticity and are likely candidates as thera-
peutic interventions to AD dementia. Useful tools
may come from NGF-based and/or miRNA-
based approaches (Fig. 10.1).
Acknowledgements This work was supported by
Finanziamento delle Attività Base di Ricerca FFABR to
NC. Graphic elaboration of the illustration by VT was
partially realized with vectorial images from ‘FreeVector.
com’, under Creative Common Attribution 4.0
ä
Fig. 10.1 (continued) binding and detaching from
microtubles. Alzheimer’s brain: The impairment of
NGF/TrkA signalling and the amyloidogenic route.
Reduced availability of mature NGF, and impaired expres-
sion levels or activation of TrkA affects APP metabolism
in BFCN. In fact, disturbances in the NGF/TrkA-ShcC
signalling pathway and the alternative assembly of the
pro-apototic p75NTR/sortilin complex grant the activation
of JNK. This latter results in augmented APPpT668 levels
and disentanglement of the APP/TrkA complex. APPpT668
is then trafficked to the endosomal-lysosomal
compartments where it is cleaved by BACE1, to generate
159
International (CC BY 4.0). A special thanks to
Dr. V. Sposato for her contribution to the
experimental work.
The authors apologize for the scientific publications not
acknowledged in this manuscript because of space
restrictions.
The authors sincerely apologize to all those colleagues
whose important work was not cited in this paper owing
to space limitations.
Conflict of Interest Statement The authors declare that
they have no actual or potential conflicts of interest and
that these data are not published elsewhere. In addition all
authors approve the study described in this report.
References
Allen SJ, Watson JJ, Dawbarn D (2011) The neurotrophins
and their role in Alzheimer’s disease. Curr
Neuropharmacol 9:559–573
Amakiri N, Kubosumi A, Tran J, Reddy PH (2019) Amy-
loid beta and microRNAs in Alzheimer’s disease.
Front Neurosci 13:430
Appel SH (1981) A unifying hypothesis for the cause of
amyotrophic lateral sclerosis, parkinsonism, and
Alzheimer disease. Ann Neurol 10:499–505
Arnold SE, Arvanitakis Z, Macauley-Rambach SL et al
(2018) Brain insulin resistance in type 2 diabetes and
Alzheimer disease: concepts and conundrums. Nat Rev
Neurol 14:168–181
Arriagada PV, Growdon JH, Hedley-Whyte ET, Hyman
BT (1992) Neurofibrillary tangles but not senile
plaques parallel duration and severity of Alzheimer’s
disease. Neurology 42(3 Pt 1):631–639
Baker-Nigh A, Vahedi S, Davis EG, Weintraub S, Bigio
EH, Klein WL, Geula C (2015) Neuronal amyloid-β
accumulation within cholinergic basal forebrain in age-
ing and Alzheimer’s disease. Brain 138:1722–1737
Ballinger EC, Ananth M, Talmage DA, Role LW (2016)
Basal forebrain cholinergic circuits and signaling in
cognition and cognitive decline. Neuron
91:1199–1218
CTFβ and Aβ. In AD, miR-132 expression is reduced and
contributes to the increased level of Aβ and tau phosphor-
ylation in AD. Abnormal tau phosphorylation causes its
aggregation. Concomitantly, ser/thr kinases, like JNK,
phosphorylate IRS1 at key serine residues, hampering its
further activation and leading to neuronal insulin resis-
tance, a condition characterized by insulin insensitivity,
reduced glucose uptake, and deficits in synaptic activity
and memory. Grey and white syringes indicate the classi-
cal and suggested AD targets, respectively, for a combined
therapeutical approach to this devastating pathology
160
Barbagallo AP, Weldon R, Tamayev R, Zhou D,
Giliberto L, Foreman O, D’Adamio L (2010) Tyr
(682) in the intracellular domain of APP regulates
amyloidogenic APP processing in vivo. PLoS One 5
(11):e15503
Barbato C, Giacovazzo G, Albiero F, Scardigli R,
Scopa C, Ciotti MT, Strimpakos G, Coccurello R,
Ruberti F (2020) Cognitive decline and modulation of
Alzheimer’s disease-related genes after inhibition of
microRNA-101 in mouse hippocampal neurons. Mol
Neurobiol 57(7):3183–3194
Batarseh YS, Duong QV, Mousa YM, Al Rihani SB,
Elfakhri K, Kaddoumi A (2016) Amyloid-beta and
astrocytes interplay in amyloid-beta related disorders.
Int J Mol Sci 17:338
Bejanin A, Schonhaut DR, La Joie R, Krame JH, Baker
SL, Sosa N et al (2017) Tau pathology and
neurodegeneration contribute to cognitive impairment
in Alzheimer’s disease. Brain 140:3286–3300
Bierer LM, Haroutunian V, Gabriel S, Knott PJ, Carlin LS,
Purohit DP, Perl DP, Schmeidler J, Kanof P, Davis KL
(1995) Neurochemical correlates of dementia severity
in Alzheimer’s disease: relative importance of the cho-
linergic deficits. J Neurochem 64:749–760
Bilousova T, Miller CA, Poon WW, Vinters HV,
Corrada M, Kawas C, Hayden EY, Teplow DB,
Glabe C, Albay R 3rd, Cole GM, Teng E, Gylys KH
(2016) Synaptic amyloid-beta oligomers precede
p-Tau and differentiate high pathology control cases.
Am J Pathol 186:185–198
Botté A, Lainé J, Xicota L, Heiligenstein X, Fontaine G,
Kasri A, Rivals I, Goh P, Faklaris O, Cossec JC,
Morel E, Rebillat AS, Nizetic D, Raposo G, Potier
MC (2020) Ultrastructural and dynamic studies of the
endosomal compartment in Down syndrome. Acta
Neuropathol Commun 8(1):89
Braak H, Braak E (1991) Neuropathological stageing of
Alzheimer-related changes. Acta Neuropathol
82:239–259
Calissano P, Amadoro G, Matrone C, Ciaffrè S,
Corsetti V, Marolda R, Ciotti MT, DiLuzio A,
Severini C, Mercanti D, Provenzano C, Canu N
(2010) Does the term “NGF” actually means anti-
amyloidogenic ? The case of NGF. Cell Death Diff
17(7):1126–1133
Cantero JL, Zaborszky L, Atienza M (2016) Volume loss
of the nucleus basalis of meynert is associated with
atrophy of innervated regions in mild cognitive
impairment. Cereb Cortex 27(8):3881–3889
Canu N, Pagano I, LaRosa LR, Pellegrino M, Ciotti MT,
Mercanti D, Moretti F, Sposato V, Triaca V, Petrella C,
Maruyama IN, Levi A, Calissano P (2017a) Associa-
tion of TrkA and APP is promoted by NGF and
reduced by cell death-promoting agents. Front Mol
Neurosci 10:15
Canu N, Amadoro G, Triaca V, Latina V, Sposato V,
Corsetti V, Severini C, Ciotti MT, Calissano P
(2017b) The intersection of NGF/TrkA signaling and
amyloid precursor protein processing in Alzheimer’s
disease neuropathology. Int J Mol Sci 18(6):1319
V. Triaca et al.
Caporaso GL, Takei K, Gandy SE, Matteoli M,
Mundigl O, Greengard P, De Camilli P (1994) Mor-
phologic and biochemical analysis of the intracellular
trafficking of the Alzheimer beta/A4 amyloid precursor
protein. Neurosci 14:3122–3138
Capsoni S, Ugolini G, Comparini A, Ruberti F, Berardi N,
Cattaneo A (2000) Alzheimer-like neurodegeneration
in aged antinerve growth factor transgenic mice. Proc
Natl Acad Sci USA 97:6826–6831
Cataldo AM, Petanceska S, Terio NB, Peterhoff CM,
Durham R, Mercken M, Mehta PD, Buxbaum J,
Haroutunian V, Nixon RA (2004) Abeta localization
in abnormal endosomes: association with earliest
Abeta elevations in AD and Down syndrome.
Neurobiol Aging 25:1263–1272
Chang KA, Kim HS, Ha TY, Ha JW, Shin KY, Jeong YH,
Lee JP, Park CH, Kim S, Baik TK, Suh YH (2006)
Phosphorylation of amyloid precursor protein (APP) at
Thr668 regulates the nuclear translocation of the APP
intracellular domain and induces neurodegeneration.
Mol Cell Biol 26:4327–4338
Chatterjee N, Sanphui P, Kemeny S, Greene LA, Biswas
SC (2016) Role and regulation of Cdc25A phosphatase
in neuron death induced by NGF deprivation or
β-amyloid. Cell Death Discov 2:16083
Chen X-Q, Mobley WC (2019) Exploring the pathogene-
sis of Alzheimer disease in basal forebrain cholinergic
neurons: converging insights from alternative
hypotheses. Front Neurosci 13:446
Chen Q, Sawa M, Mobley WC (2018) Dysregulation of
neurotrophin signaling in the pathogenesis of
Alzheimer disease and of Alzheimer disease in Down
syndrome. Free Radic Biol Med 114:52–61
Chow H-M, Shi M, Cheng A, Gao Y, Chen G, Song X, So
RWL, Zhang J, Herrup K (2019) Age-related
hyperinsulinemia leads to insulin resistance in neurons
and cell-cycle-induced senescence. Nat Neurosci
22:1806–1819
Chung CG, Lee H, Lee SB (2018) Mechanisms of protein
toxicity in neurodegenerative diseases. Cell Mol Life
Sci 75:3159–3180
Cline EN, Bicca MA, Viola KL, Klein WL (2018) The
amyloid-β oligomer hypothesis: beginning of the third
decade. J Alzheimer Dis 64(S1):S567–S610
Colom LV, García-Hernández A, Castañeda MT, Perez-
Cordova MG, Garrido-Sanabria ER (2006) Septo-
hippocampal networks in chronically epileptic rats:
potential antiepileptic effects of theta rhythm genera-
tion. J Neurophysiol 95:3645–3653
Craft S (2007) Insulin resistance and Alzheimer’s disease
pathogenesis: potential mechanisms and implications
for treatment. Curr Alzheimer Res 4(2):147–152
Cuello AC, Bruno MA, Bell KFS (2007) NGF-cholinergic
dependency in brain aging, MCI and Alzheimer’s dis-
ease. Curr Alzheimer Res 4:351–358
Cui GH, Wu J, Mou FF, Xie WH, Wang FB, Wang QL,
Fang J, Xu YW, Dong YR, Liu JR, Guo HD (2018)
Exosomes derived from hypoxia-preconditioned mes-
enchymal stromal cells ameliorate cognitive decline by
rescuing synaptic dysfunction and regulating
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
inflammatory responses in APP/PS1 mice. FASEB J
32(2):654–668
Davies P, Maloney AJ (1976) Selective loss of central
cholinergic neurons in Alzheimer’s disease. Lancet
2:1403
De Felice FG (2013) Alzheimer’s disease and insulin
resistance: translating basic science into clinical
applications. J Clin Invest 123:531–539
De Felice FG, Lourenco MV, Ferreira ST (2014) How
does brain insulin resistance develop in Alzheimer’s
disease? Alzheimer Dement 10:S26–S32
de la Monte SM (2012) Brain insulin resistance and defi-
ciency as therapeutic targets in Alzheimers disease.
Curr Alzheimer Res 9(1):35–66
De Strooper B, Karran E (2016) The cellular phase of
Alzheimer’s disease. Cell 164:603–615
Dorszewska J, Prendecki M, Oczkowska A, Dezor M,
Kozubski W (2016) Molecular basis of familial and
sporadic Alzheimer’s disease. Curr Alzheimer Res
13:952–963
Ebenau JL, Timmers T, Wesselman LMP et al (2020)
ATN classification and clinical progression in subjec-
tive cognitive decline. Neurology 95:e46–e58
Eggert S, Thomas C, Kins S, Hermey G (2018) Trafficking
in Alzheimer’s disease: modulation of APP transport
and processing by the transmembrane proteins LRP1,
SorLA, SorCS1c, Sortilin, and Calsyntenin. Mol
Neurobiol 55(7):5809–5829
El-Shimy IA, Heikal OA, Hamdi N (2015) Minocycline
attenuates Abeta oligomers-induced pro-inflammatory
phenotype in primary microglia while enhancing Abeta
fibrils phagocytosis. Neurosci Lett 609:36–41
Exalto LG, Biessels GJ, Karter AJ, Huang ES, Katon WJ,
Minkoff JR, Whitmer RA (2013) Risk score for pre-
diction of 10 year dementia risk in individuals with
type 2 diabetes: a cohort study. Lancet Diabetes
Endocrinol 1:183–190
Ferreira LSS, Fernandes CS, Vieira MNN, de Felice FG
(2018) Insulin resistance in Alzheimer’s disease. Front
Neurosci 12:830
Fisher A (2012) Cholinergic modulation of amyloid pre-
cursor protein processing with emphasis on M1 mus-
carinic receptor: perspectives and challenges in
treatment of Alzheimer’s disease. J Neurochem 120
(Suppl 1):22–33
Fishwick KJ, Rylett RJ (2015) Insulin regulates the activ-
ity of the high-affinity choline transporter CHT. PLoS
One 10:e0132934
Fombonne J, Rabizadeh S, Banwait S, Mehlen P,
Bredesen DE (2009) Selective vulnerability in
Alzheimer’s disease: amyloid precursor protein and
p75(NTR) interaction. Ann Neurol 65(3):294–303
Frost B, Jacks RL, Diamond MI (2009) Propagation of tau
misfolding from the outside to the inside of a cell. J
Biol Chem 284:12845–12852
Geula C, Nagykery N, Nicholas A, Wu C-K (2008) Cho-
linergic neuronal and axonal abnormalities are present
early in aging and in Alzheimer disease. J Neuropathol
Exp Neurol 67:309–318
161
Giannakopoulos P, Herrmann FR, Bussière T, Bouras C,
Kövari E, Perl DP, Morrison JH, Gold G, Hof PR
(2003) Tangle and neuron numbers, but not amyloid
load, predict cognitive status in Alzheimer’s disease.
Neurology 60:1495–1500
Giuffrida ML, Caraci F, Pignataro B, Cataldo S, De
Bona P, Bruno V, Molinaro G, Pappalardo G,
Messina A, Palmigiano A, Garozzo D, Nicoletti F,
Rizzarelli E, Copani A (2009) Beta-amyloid
monomers are neuroprotective. J Neurosci
29:10582–10587
Goedert M, Spillantini MG, Cairns NJ, Crowther RA
(1992) Tau proteins of alzheimer paired helical
filaments: abnormal phosphorylation of all six brain
isoforms. Neuron 8:159–168
Grothe M, Zaborszky L, Atienza M, Gil-Neciga E,
Rodriguez-Romero R, Teipel SJ, Amunts K, Suarez-
Gonzalez A, Cantero JL (2010) Reduction of basal
forebrain cholinergic system parallels cognitive
impairment in patients at high risk of developing
Alzheimer’s disease. Cereb Cortex 20:1685–1695
Guo JP, Arai T, Miklossy J, McGeer PL (2006) Aβ and tau
form soluble complexes that may promote self aggre-
gation of both into the insoluble forms observed in
Alzheimer’s disease. Proc Natl Acad Sci USA
103:1953–1958
Gustafsen C, Glerup S, Pallesen LT, Olsen D, Andersen
OM, Nykjær A, Madsen P, Petersen CM (2013)
Sortilin and SorLA display distinct roles in processing
and trafficking of amyloid precursor protein. J
Neurosci 33:64–71
Haass C, Kaether C, Thinakaran G, Sisodia S (2012)
Trafficking and proteolytic processing of APP. Cold
Spring Harb Perspect Med 2(5):a006270
Hamada N, Fujita Y, Kojima T, Kitamoto A, Akao Y,
Nozawa Y, Ito M (2012) MicroRNA expression
profiling of NGF-treated PC12 cells revealed a critical
role for miR-221 in neuronal differentiation.
Neurochem Int 60:743–750
Hangya B, Ranade SP, Lorenc M, Kepecs A (2015) Cen-
tral cholinergic neurons are rapidly recruited by rein-
forcement feedback. Cell 162:1155–1168
Hardy JA, Higgings GA (1992) Alzheimer’s disease: the
amyloid cascade hypothesis. Science 256:184–185
Heese K, Inoue N, Sawada T (2004) APP, NGF & the
‘Sunday-driver’ in a Trolley on the Road. Resto Neurol
Neurosci 22(2):131–136
Hu YB, Ren RJ, Zhang YF, Huang Y, Cui HL, Ma C, Qiu
WY, Wang H, Cui PJ, Chen HZ, Wang G (2019)
Rho-associated coiled-coil kinase 1 activation
mediates amyloid precursor protein site-specific
Ser655 phosphorylation and triggers amyloid pathol-
ogy. Aging Cell 18(5):e13001
Improta-Caria AC, Nonaka CKV, Cavalcante BRR, De
Sousa RAL, Aras Júnior R, Souza BSF (2020) Modu-
lation of microRNAs as a potential molecular mecha-
nism involved in the beneficial actions of physical
exercise in Alzheimer disease. Int J Mol Sci 21
(14):4977
162
Ioannou MS, Fahnestock M (2017) ProNGF, but Not
NGF, switches from neurotrophic to apoptotic activity
in response to reductions in TrkA receptor levels. Int J
Mol Sci 18:599
Iqbal K, Liu F, Gong CX (2016) Tau and neurodegenera-
tive disease: the story so far. Nat Rev Neurol 12
(1):15–27
Irmady K, Jackman KA, Padow VA, Shahani N, Martin
LA, Cerchietti L, Unsicker K, Iadecola C, Hempstead
BL (2014) Mir-592 regulates the induction and cell
death-promoting activity of p75NTR in neuronal ische-
mic injury. J Neurosci 34:3419–3428
Jack CR Jr, Bennett DA, Blennow K, Carrillo MC,
Dunn B, Haeberlein SB, Holtzman DM, Jagust W,
Jessen F, Karlawish J, Liu E, Molinuevo JL,
Montine T, Phelps C, Rankin KP, Rowe CC,
Scheltens P, Siemers E, Snyder HM, Sperling R,
Contributors (2018) NIA-AA research framework:
toward a biological definition of Alzheimer’s disease.
Alzheimerd Dement 14(4):535–562
Jeong DU, Lee JE, Lee SE, Chang WS, Kim SJ, Chang JW
(2014) Improvements in memory after medial septum
stimulation are associated with changes in hippocam-
pal cholinergic activity and neurogenesis. BioMed Res
Int 2014:1–10
Kamal A, Almenar-Queralt A, LeBlanc JF, Roberts EA,
Goldstein LS (2001) Kinesin-mediated axonal trans-
port of a membrane compartment containing beta-
secretase and presenilin-1 requires APP. Nature 414
(6864):643–648
Kashyap G, Bapat D, Das D, Gowaikar R, Amritkar RE,
Rangarajan G, Ravindranath V, Ambika G (2019)
Synapse loss and progress of Alzheimer’s disease—a
network model. Sci Rep 9:6555
Kellar D, Craft S (2020) Brain insulin resistance in
Alzheimer’s disease and related disorders: mechanisms
and therapeutic approaches. Lancet Neurol
19:758–766
Kerbler GM, Fripp J, Rowe CC, Villemagne VL,
Salvado O, Rose S, Coulson EJ (2015) Basal forebrain
atrophy correlates with amyloid β burden in
Alzheimer’s disease. NeuroImage Clin 7:105–113
Kilimann I, Grothe M, Heinsen H, Alho EJ, Grinberg L,
Amaro E Jr, Dos Santos GA, da Silva RE, Mitchell AJ,
Frisoni GB, Bokde AL, Fellgiebel A, Filippi M,
Hampel H, Klöppel S, Teipel SJ (2014) Subregional
basal forebrain atrophy in Alzheimer’s disease: a mul-
ticenter study. J Alzheimers Dis 40:687–700
Kiltschewskij D, Cairns MJ (2019) Temporospatial guid-
ance of activity-dependent gene expression by
microRNA: mechanisms and functional implications
for neural plasticity. Nucleic Acids Res 47:533–545
Kitchigina VF (2018) Alterations of coherent theta and
gamma network oscillations as an early biomarker of
temporal lobe epilepsy and Alzheimer’s disease. Front
Integr Neurosci 12:36
Koo EH, Squazzo SL (1994) Evidence that production and
release of amyloid beta-protein involves the endocytic
pathway. J Biol Chem 269:17386–17389
La Rosa LR, Perrone L, Nielsen MS, Calissano P,
Andersen OM, Matrone C (2015) Y682G mutation of
V. Triaca et al.
amyloid precursor protein promotes endo-lysosomal
dysfunction by disrupting APP-SorLA interaction.
Front Cell Neurosci 9:109
Lee MS, Kao SC, Lemere CA, Xia W, Tseng HC, Zhou Y,
Neve R, Ahlijanian MK, Tsai LH (2003) APP
processing is regulated by cytoplasmic phosphoryla-
tion. J Cell Biol 163:83–95
Leung LS, Ma J (2018) Medial septum modulates hippo-
campal gamma activity and prepulse inhibition in an
N-methyl-d-aspartate receptor antagonist model of
schizophrenia. Schizoph Res 198:36–44
Li S, Wang X, Gu Y, Chen C, Wang Y, Liu J, Hu W,
Yu B, Wang Y, Ding F, Liu Y, Gu X (2015) Let-7
microRNAs regenerate peripheral nerve regeneration
by targeting nerve growth factor. Mol Ther 23
(3):423–433
Liu Y, Zhang YW, Wang X, Zhang H, You X, Liao FF,
Xu HJ (2009) Intracellular trafficking of presenilin 1 is
regulated by beta-amyloid precursor protein and phos-
pholipase D1. Biol Chem 284:12145–12152
Mankin EA, Fried I (2020) Modulation of human memory
by deep brain stimulation of the entorhinal-
hippocampal circuitry. Neuron 106(2):218–235
Matrone C, Ciotti MT, Mercanti D, Marolda R, Calissano
P (2008) NGF and BDNF signaling control
amyloidogenic route and a production in hippocampal
neurons. Proc Natl Acad Sci USA 105:13139–13144
Matrone C, Marolda R, Ciafrè S, Ciotti MT, Mercanti D,
Calissano P (2009) Tyrosine kinase nerve growth fac-
tor receptor switches from prosurvival to proapoptotic
activity via Abeta-mediated phosphorylation. Proc
Natl Acad Sci U S A 106(27):11358–11363
Matrone C, Barbagallo AP, La Rosa LR, Florenzano F,
Ciotti MT, Mercanti D, Chao MV, Calissano P,
D’Adamio L (2011) APP is phosphorylated by TrkA
and regulates NGF/TrkA signaling. J Neurosci
31:11756–11761
Matrone C, Luvisetto S, La Rosa LR, Tamayev R,
Pignataro A, Canu N, Yang L, Barbagallo AP,
Biundo F, Lombino F, Zheng H, Ammassari-Teule M,
D’Adamio L (2012) Tyr682 in the Aβ-precursor pro-
tein intracellular domain regulates synaptic connectiv-
ity, cholinergic function, and cognitive performance.
Aging Cell 6:1084–1093
Molton SA, Todd DE, Cook SJ (2003) Selective activation
of the c-Jun N-terminal kinase (JNK) pathway fails to
elicit Bax activation or apoptosis unless the phosphoi-
nositide 30 ‐kinase (PI3K) pathway is inhibited. Onco-
gene 22:4690–4701
Montalban E, Mattugini N, Ciarapica R, Provenzano C,
Savino M, Scagnoli F, Prosperini G, Carissimi C,
Fulci V, Matrone C, Calissano P, Nasi S (2014)
MiR-21 is an NGF-modulated microRNA that
supports NGF signaling and regulates neuronal degen-
eration in PC12 cells. Neuromolecular Med
16:415–430
Mufson EJ, Counts SE, Ginsberg SD, Mahady L, Perez
SE, Massa SM, Longo FM, Ikonomovic MD (2019)
Nerve growth factor pathobiology during the progres-
sion of Alzheimer’s disease. Front Neurosci 13:533
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . .
Nielsen MS, Madsen P, Christensen EI, Nykjær A,
Gliemann J, Kasper D, Pohlmann R, Petersen CM
(2001) The sortilin cytoplasmic tail conveys Golgi-
endosome transport and binds the VHS domain of the
GGA2 sorting protein. EMBO J 20:2180–2190
Nielsen HM, Mulder SD, Belien JA, Musters RJ,
Eikelenboom P, Veerhuis R (2010) Astrocytic A beta
1-42 uptake is determined by A beta-aggregation state
and the presence of amyloid-associated proteins. Glia
58:1235–1246
Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen
MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow
TE, Hempstead BL, Petersen CM (2004) Sortilin is
essential for proNGF-induced neuronal cell death.
Nature 427(6977):843–848
Oueslati AJ (2016) Implication of alpha-synuclein phos-
phorylation at S129 in synucleinopathies: what have
we learned in the last decade? Parkinsons Dis 26
(1):39–51
Pandey A, Singh P, Jauhari A, Singh T, Khan F, Pant AB,
Parmar D, Yadav S (2015) Critical role of the miR-200
family in regulating differentiation and proliferation of
neurons. J Neurochem 133:640–652
Parsi S, Smith PY, Goupil C, Dorval V, Hébert SS (2015)
Preclinical evaluation of miR-15/107 family members
as multifactorial drug targets for Alzheimer’s disease.
Mol Ther Nucleic Acids 4(10):e256
Patel J, Fujisawa S, Berényi A, Royer S, Buzsáki G (2012)
Traveling theta waves along the entire septotemporal
axis of the hippocampus. Neuron 75:410–417
Patranabis S, Bhattacharyya SN (2016) Phosphorylation of
Ago2 and subsequent inactivation of let-7a
RNP-specific microRNAs control differentiation of
mammalian sympathetic neurons. Mol Cell Biol
36:1260–1271
Pearson RCA, Gatter KC, Powell TPS (1983) The cortical
relationships of certain basal ganglia and the choliner-
gic basal forebrain nuclei. Brain Res 261:327–330
Petersen PC, Buzsáki G (2020) Cooling of medial septum
reveals theta phase lag coordination of hippocampal
cell assemblies. Neuron 107:731–744.e3
Puzzo D, Lee L, Palmeri A, Calabrese G, Arancio O
(2014) Behavioral assays with mouse models of
Alzheimer’s disease: practical considerations and
guidelines. Biochem Pharmacol 88:450–467
Roussarie JP, Yao V, Rodriguez-Rodriguez P,
Oughtred R, Rust J, Plautz Z, Kasturia S,
Albornoz C, Wang W, Schmidt EF, Dannenfelser R,
Tadych A, Brichta L, Barnea-Cramer A, Heintz N, Hof
PR, Heiman M, Dolinski K, Flajolet M, Troyanskaya
OG, Greengard P (2020) Selective neuronal vulnera-
bility in Alzheimer’s disease: a network-based analy-
sis. Neuron 107:1–15
Salehi A, Delcroix JD, Belichenko PV, Zhan K, Wu C,
Valletta JS, Takimoto-Kimura R, Kleschevnikov AM,
Sambamurti K, Chung PP, Xia W, Villar A, Campbell
WA, Kulnane LS, Nixon RA, Lamb BT, Epstein CJ,
Stokin GB, Goldstein LS, Mobley WC (2006)
Increased App expression in a mouse model of
Down’s syndrome disrupts NGF transport and causes
cholinergic neuron degeneration. Neuron 51:29–42
163
Salta E, De Strooper B (2017) microRNA-132: a key
noncoding RNA operating in the cellular phase of
Alzheimer’s disease. FASEB J 31:424–433
Sassin I, Schultz C, Thal DR, Rüb U, Arai K, Braak E,
Braak H (2000) Evolution of Alzheimer’s disease-
related cytoskeletal changes in the basal nucleus of
Meynert. Acta Neuropathol 100:259–269
Schettini G, Govoni S, Racchi M, Rodriguez G (2010)
Phosphorylation of APP-CTF-AICD domains and
interaction with adaptor proteins: signal transduction
and/or transcriptional role—relevance for Alzheimer
pathology. J Neurochem 115:1299–1308
Schliebs R, Arendt T (2011) The cholinergic system in
aging and neuronal degeneration. Behav Brain Res
221:555–563
Schmitz TW, Mur M, Aghourian M, Bedard M-A, Spreng
RN, Alzheimer’s Disease Neuroimaging Initiative
(2018) Longitudinal Alzheimer’s degeneration reflects
the spatial topography of cholinergic basal forebrain
projections. Cell Rep 24:38–46
Schubert M, Gautam D, Surjo D et al (2004) Role for
neuronal insulin resistance in neurodegenerative
diseases. Proc Natl Acad Sci U S A 101:3100–3105
Selkoe DJ (2011) Resolving controversies on the path to
Alzheimer’s therapeutics. Nat Med 17:1060–1065
Selkoe DJ, Yamazaki T, Citron M, Podlisny MB, Koo EH,
Teplow DB, Haass C (1996) The role of APP
processing and trafficking pathways in the formation
of amyloid beta-protein. Ann N Y Acad Sci 777:57–64
Serrano-Pozo A, Frosch MP, Masliah E, Hyman BT
(2011) Neuropathological alterations in Alzheimer dis-
ease. Cold Spring Harb Perspect Med 1:a006189
Skeberdis VA, Lan J, Zheng X, Zukin RS, Bennett MVL
(2001) Insulin promotes rapid delivery of N-methyl-D-
aspartate receptors to the cell surface by exocytosis.
Proc Natl Acad Sci U S A 98:3561–3566
Sperling RA, Aisen PS, Beckett LA et al (2011) Toward
defining the preclinical stages of Alzheimer’s disease:
recommendations from the National Institute on
Aging-Alzheimer’s Association workgroups on diag-
nostic guidelines for Alzheimer’s disease. Alzheimers
Dement 7:280–292
Spillantini MG, Crowther RA, Jakes R, Hasegawa M,
Goedert M (1998) α-Synuclein in filamentous
inclusions of Lewy bodies from Parkinson’s disease
and dementia with Lewy bodies. Proc Natl Acad Sci
USA 95:6469–6473
Spires-Jones TL, Hyman BT (2014) The intersection of
amyloid beta and tau at synapses in Alzheimer’s dis-
ease. Neuron 82(4):756–771
Sposato V, Canu N, Fico E, Fusco S, Bolasco G, Ciotti
MT, Spinelli M, Mercanti D, Grassi C, Triaca V,
Calissano P (2019) The medial septum is insulin resis-
tant in the AD presymptomatic phase: rescue by nerve
growth factor-driven IRS1 activation. Mol Neurobiol
56:535–552
Stypulkowski PH, Stanslaski SR, Giftakis JE (2017) Mod-
ulation of hippocampal activity with fornix deep brain
stimulation. Brain Stimul 10:1125–1132
Szutowicz A, Bielarczyk H, Jankowska-Kulawy A,
Pawełczyk T, Ronowska A (2013) Acetyl-CoA the
164
key factor for survival or death of cholinergic neurons
in course of neurodegenerative diseases. Neurochem
Res 38:1523–1542
Takahashi M Kametani F, Nonaka T, Akiyama H,
Hisanaga S, Hasegawa M (2015) Extracellular associ-
ation of APP and tau fibrils induces intracellular aggre-
gate formation of tau. Acta Neuropathol 129
(6):895–907
Tarr PE, Contursi C, Roncarati R, Noviello C, Ghersi E,
Scheinfeld MH, Zambrano N, Russo T, D’Adamio L
(2002) Evidence for a role of the nerve growth factor
receptor TrkA in tyrosine phosphorylation and
processing of beta-APP. Biochem Biophys Res
Commun 295(2):324–329
Teipel SJ, Flatz WH, Heinsen H, Bokde AL, Schoenberg
SO, Stockel S, Dietrich O, Reiser MF, Moller HJ,
Hampel H (2005) Measurement of basal forebrain
atrophy in Alzheimer’s disease using MRI. Brain 128
(Part 11):2626–2644
Teipel SJ, Meindl T, Grinberg L, Grothe M, Cantero JL,
Reiser MF, Möller H-J, Heinsen H, Hampel H (2011)
The cholinergic system in mild cognitive impairment
and Alzheimer’s disease: an in vivo MRI and DTI
study. Hum Brain Mapp 32:1349–1362
Tenreiro S, Eckermann K, Outeiro TF (2014) Protein
phosphorylation in neurodegeneration: friend or foe?
Front Mol Neurosci 7:42
Terasawa K, Ichimura A, Sato F, Shimizu K, Tsujimoto G
(2009) Sustained activation of ERK1/2 by NGF
induces microRNA-221 and 222 in PC12 cells. FEBS
J. 276:3269–3276
Thal DR, Rüb U, Orantes M, Braak H (2002) Phases of A
beta-deposition in the human brain and its relevance
for the development of AD. Neurology 58:1791–1800
Tilvis RS, Kahonen-Vare MH, Jolkkonen J, Valvanne J,
Pitkala KH, Strandberg TE (2004) Predictors of cogni-
tive decline and mortality of aged people over a
10-year period. J Gerontol Ser A Biol Sci Med Sci
59:M268–M274
Triaca V, Sposato V, Bolasco G, Ciotti MT, Pelicci P,
Bruni AC, Cupidi C, Maletta R, Feligioni M,
Nisticò R, Canu N, Calissano P (2016) NGF controls
APP cleavage by downregulating APP phosphoryla-
tion at Thr668: relevance for Alzheimer’s disease.
Aging Cell 15:661–672
Triaca V, Coccurello R, Giacovazzo G (2018) The neuro-
nal Shc adaptor in Alzheimer’s disease. Aging 10
(1):5–6
Tsanov M (2017) Speed and oscillations: medial septum
integration of attention and navigation. Front Syst
Neurosci 11:67
Vaegter CB, Jansen P, Fjorback AW, Glerup S, Skeldal S,
Kjolby M, Richner M, Erdmann B, Nyengaard JR,
Tessarollo L, Lewin GR, Willnow TE, Chao MV,
Nykjaer A (2011) Sortilin associates with Trk receptors
to enhance anterograde transport and neurotrophin sig-
naling. Nat Neurosci 14:54–61
Verma S, Kumar A, Tripathi T, Kumar A (2018) Musca-
rinic and nicotinic acetylcholine receptor agonists:
V. Triaca et al.
current scenario in Alzheimer’s disease therapy. J
Pharm Pharmacol 70(8):985–993
Vogels OJM, Broere CAJ, ter Laak HJ, ten Donkelaar HJ,
Nieuwenhuys R, Schulte BPM (1990) Cell loss and
shrinkage in the nucleus basalis Meynert complex in
Alzheimer’s disease. Neurobiol Aging 11:3–13
Wang B, Yang L, Wang Z, Zheng H (2007) Amyolid
precursor protein mediates presynaptic localization
and activity of the high-affinity choline transporter.
Proc Natl Acad Sci U S A 104:14140–14145
Wang B, Pan L, Wei M, Wang Q, Liu WW, Wang N,
Jiang XY, Zhang X, Bao L (2015) FMRP-mediated
axonal delivery of miR-181d regulates axon elongation
by locally targeting Map1b and Calm1. Cell Rep
13:2794–2807
Wang M, Qin L, Tang B (2019a) MicroRNAs in
Alzheimer’s disease. Front Genet 10:153
Wang W, Tanokashira D, Fukui Y, Maruyama M,
Kuroiwa C, Saito T, Saido TC, Taguchi A (2019b)
Serine phosphorylation of IRS1 correlates with A-
β-unrelated memory deficits and elevation in Aβ level
prior to the onset of memory decline in AD. Nutrients
11:1942
Whitehouse PJ, Price DL, Clark AW, Coyle JT, Delong
MR (1981) Alzheimer disease: evidence for selective
loss of cholinergic neurons in the nucleus basalis. Ann
Neurol 10:122–126
Whitehouse PJ, PriceDL StrubleRG, Clark AW, Coyle JT,
De-lon MR (1982) Alzheimer’s disease and senile
dementia: loss of neurons in the basal forebrain. Sci-
ence 215:1237–1239
Whitmer RA (2007) Type 2 diabetes and risk of cognitive
impairment and dementia. Curr Neurol Neurosci Rep
7:373–380
Willette AA, Bendlin BB, Starks EJ et al (2015) Associa-
tion of insulin resistance with cerebral glucose uptake
in late middle–aged adults at risk for Alzheimer dis-
ease. JAMA Neurol 72:1013
Wu Q, Ye X, Xiong Y, Zhu H, Miao J, Zhang W, Wan J
(2016) The protective role of microRNA-200c in
Alzheimer’s disease pathologies is induced by beta
amyloid-triggered endoplasmic reticulum stress. Front
Mol Neurosci 9:140
Wurtman RJ (1992) Choline metabolism as a basis for the
selective vulnerability of cholinergic neurons. Trends
Neurosci 15:117–122
Xu W, Weissmiller AM, White JA 2nd, Fang F, Wang X,
Wu Y, Pearn ML, Zhao X, Sawa M, Chen S,
Gunawardena S, Ding J, Mobley WC, Wu C (2016)
Amyloid precursor protein-mediated endocytic path-
way disruption induces axonal dysfunction and
neurodegeneration. J Clin Invest 126(5):1815–1833
Zatorre RJ, Fields RD, Johansen-Berg H (2012) Plasticity
in gray and white: neuroimaging changes in brain
structure during learning. Nat Neurosci 15(4):528–536
Zhang YW, Thomposn R, Zang H, Xu H (2011) APP
processing in Alzheimer’s disease. Mol Brain 4:3
Zhang YW, Chen Y, Liu Y, Zhao Y, Liao FF, Xu H (2013)
APP regulates NGF receptor trafficking and
10 NGF and the Amyloid Precursor Protein in Alzheimer’s Disease: From. . . 165

NGF-mediated neuronal differentiation and survival.
PLoS One 8(11):e80571
Zhao W-Q, Alkon DL (2001) Role of insulin and insulin
receptor in learning and memory. Mol Cell Endocrinol
177:125–134
Zhu QB, Unmehopa U, Bossers K, Hu YT, Verwer R,
Balesar R, Zhao J, Bao AM, Swaab D (2016)
MicroRNA-132 and early growth response-1 in
nucleus basalis of Meynert during the course of
Alzheimer’s disease. Brain 139:908–921
 A Review of Techniques for Biodelivery
of Nerve Growth Factor (NGF) 11
to the Brain in Relation to Alzheimer’s
Disease

Sumonto Mitra, Ruchi Gera, Bengt Linderoth, Göran Lind,

Lars Wahlberg, Per Almqvist, Homira Behbahani,
and Maria Eriksdotter

Abstract affected individual and associated family.

Age-dependent progressive neurodegeneration
and associated cognitive dysfunction represent
a serious concern worldwide. Currently,
dementia accounts for the fifth highest cause
of death, among which Alzheimer’s disease
(AD) represents more than 60% of the cases.
AD is associated with progressive cognitive
dysfunction which affects daily life of the
S. Mitra (*) · R. Gera
Division of Clinical Geriatrics, NVS Department,
Karolinska Institutet, Stockholm, Sweden
e-mail: sumonto.mitra@ki.se; ruchi.gera@ki.se
B. Linderoth · G. Lind · P. Almqvist
Section of Neurosurgery, Department of Clinical
Neuroscience, Karolinska Institutet, Stockholm, Sweden
e-mail: bengt.linderoth@ki.se; goran.lind@sll.se; per.
almqvist@ki.se
L. Wahlberg
Gloriana Therapeutics Inc., Warren, RI, USA
e-mail: luw@glorianatx.com
H. Behbahani
Division of Clinical Geriatrics, NVS Department,
Karolinska Institutet, Stockholm, Sweden
Karolinska Universitets laboratoriet (LNP5), Karolinska
University Hospital, Stockholm, Sweden
e-mail: homira.behbahani@ki.se
M. Eriksdotter
Division of Clinical Geriatrics, NVS Department,
Karolinska Institutet, Stockholm, Sweden
Theme Aging, Karolinska University Hospital, Huddinge,
Sweden
e-mail: maria.eriksdotter@ki.se
The cognitive dysfunctions are at least par-
tially due to the degeneration of a specific set
of neurons (cholinergic neurons) whose cell
bodies are situated in the basal forebrain
region (basal forebrain cholinergic neurons,
BFCNs) but innervate wide areas of the
brain. It has been explicitly shown that the
delivery of the neurotrophic protein nerve
growth factor (NGF) can rescue BFCNs and
restore cognitive dysfunction, making NGF
interesting as a potential therapeutic substance
for AD. Unfortunately, NGF cannot pass
through the blood-brain barrier (BBB) and
thus peripheral administration of NGF protein
is not viable therapeutically. NGF must be
delivered in a way which will allow its brain
penetration and availability to the BFCNs to
modulate BFCN activity and viability. Over
the past few decades, various methodologies
have been developed to deliver NGF to the
brain tissue. In this chapter, NGF delivery
methods are discussed in the context of AD.
Abbreviations
Aβ Amyloid-beta
AD Alzheimer’s disease
APP Amyloid precursor protein
BBB Blood-brain barrier

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 167
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_11
168 S. Mitra et al.

BFCN Basal forebrain cholinergic neuron dysfunction and Alzheimer’s disease (AD). The
CSF Cerebrospinal fluid currently available drugs against AD targeting the
ECB Encapsulated cell biodelivery cholinergic system, the cholinesterase inhibitors,
EEG Electroencephalography have a modest albeit persistent effect on cognition
FTD Frontotemporal dementia (Birks and Harvey 2018; Zhu et al. 2013; Secnik
HIBI Hypoxic-ischemic brain injuries et al. 2020; Dou et al. 2018). Among many other
ICD Intracerebral delivery targets, several clinical trials have attempted the
ICVD Intracerebroventricular delivery reduction of amyloid-beta (Aβ) peptides or
MMP3/ Matrix metalloproteinase 3/9 plaques, although a majority of the trials have
9 not produced convincing result so far in improv-
mNGF Mature-NGF ing cognition (Cummings et al. 2019, 2020;
MSCs Mesenchymal stem cells Scheltens et al. 2016; Liu et al. 2019). However,
nbM Nucleus basalis of Meynert in the last year, modest positive results on cogni-
NGF Nerve growth factor tion have been attributed to aducanumab, a mono-
NGFR NGF receptor clonal antibody against Aβ (Schneider 2020), and
NSCs Neural stem cells approval by the American FDA is now pending. It
p75 Low-affinity NGF receptor or is important, however, to widen the search for
LNGFR, or p75 neurotrophin therapies for AD, and therefore, delivery of
receptor, or p75NTR NGF as one therapeutic approach is of great
PLGA Poly(lactic-co-glycolic acid) interest.
proNGF Precursor-NGF Under normal physiological conditions, NGF
rh Recombinant human is secreted from cells in a precursor form
TBI Traumatic brain injury (proNGF) which needs to be converted into
tPA Tissue plasminogen activator mature form (mNGF) (Fahnestock and Shekari
TrkA Tropomyosin receptor kinase A 2019; Cuello et al. 2019). This conversion of
uPA Urokinase plasminogen activation proNGF to mNGF is done by the enzyme plas-
min, which itself is generated from the proteolytic
cleavage of plasminogen by tissue plasminogen
activator (tPA) or urokinase plasminogen activa-
11.1 Introduction and Background
tion (uPA), respectively (Bruno and Cuello
2006). Once formed, mNGF can initiate its down-
NGF was the first neurotrophic factor discovered
stream signaling through its receptor interaction
in the 1950s by Rita Levi-Montalcini and Viktor
with TrkA (tropomyosin receptor kinase A) or
Hamburger, which established the family of
p75 (known as low-affinity NGF receptor,
neurotrophic factors and further led to the discov-
LNGFR; or p75 neurotrophin receptor, or
ery of other related proteins (Cohen and Levi-
p75NTR), both collectively called
Montalcini 1956; Cohen et al. 1954; Bradshaw
NGF-receptors (NGFR). Alternatively, mNGF
et al. 2017). Due to its involvement in various
can be further degraded and cleared from the
physiological processes, NGF is a highly
system by the matrix metalloproteinase 3/9
researched molecule (Aloe et al. 2012, 2015,
(MMP3 and MMP9) enzyme. Depending on this
2016; Lambiase et al. 2011; Bracci-Laudiero
metabolic dynamics-controlled availability of dif-
and Manni 2014; Mitra et al. 2019; Simone
ferent NGF forms and expression of the receptor,
et al. 1999; Rocco et al. 2018). A quick search
the final biological effects are initiated (as shown
in PubMed (https://pubmed.ncbi.nlm.nih.gov/)
in Fig. 11.1). If NGF maturation is hampered,
using the keywords “nerve growth factor”
proNGF levels may get accumulated and affect
retrieved more than 74,190 results (as of 29th
neuronal survival through the cooperative signal-
December, 2020). Interestingly, hampered NGF
ing of p75NTR and sortilin receptor (Capsoni
metabolism has also been linked to cognitive
et al. 2013; Skeldal et al. 2012).
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Plasminogen
tPA/uPA
proNGF
Plasmin
Pro-domain
p75 Sortilin p75
JNK
NF-κB
Caspases
c-Jun
NF-κB
Apoptosis
Fig. 11.1 NGF signaling in the brain. NGF is initially
released as proNGF from cells, which is then metabolized
to mNGF by plasmin, and mNGF is ultimately degraded
by MMP3/9 enzyme. NGF (pro or mature) can induce
different downstream signaling based on their interaction
with various NGF receptor combinations (i.e., TrkA-p75
or p75-sortilin). Based on specific NGF and receptor type
interaction, cell signaling cascades for apoptosis (death),
survival, or differentiation take place via specific signaling
pathways. Abbreviations: NGF (nerve growth factor),
Under AD condition, the level of mNGF is
reduced while proNGF is elevated (Fahnestock
and Shekari 2019), implying hampered NGF
metabolism. This could be due to decreased tPA
levels (reduced mNGF production), upregulated
MMP9 activity (enhanced clearance of mNGF),
altered TrkA/p75 mediated signaling, or a combi-
nation of all of them (Mitra et al. 2019). Simulta-
neously, hampered retrograde transport of NGF
from hippocampus and cortical tissue has also
169
NGF NGF degradation
MMP3/9
TrkA
P P
PKB/AKT MAPK PKC
TFs Cell Survival &
differentiation
Cell Survival
proNGF (precursor-NGF), mNGF (mature-NGF), TrkA
(tropomyosin receptor kinase A), p75 (p75 neurotrophin
receptor), MMP3/9 (matrix metalloproteinase 3 or 9), tPA
(tissue plasminogen activator), uPA (urokinase plasmino-
gen activation), JNK (c-Jun N-terminal kinases), NK-κB
(nuclear factor kappa-light-chain-enhancer of activated B
cells), PKB/AKT (protein kinase B), MAPK (mitogen-
activated protein kinase (MAPK), PKC (protein kinase
C), and TFs (transcription factors). ‘P’ denotes phosphor-
ylation sites
been reported in AD (Mufson et al. 1995). Recent
studies have confirmed the hampered activity of
tPA and elevated levels of MMP3/9 in AD,
indicating reduced production and enhanced
clearance of mNGF from the brain tissue (Hanzel
et al. 2014; Pentz et al. 2020). Reduced mNGF
levels hinder cholinergic signaling (production of
the neurotransmitter—acetylcholine) resulting in
inflammation due to glial activation. Thus, restor-
ing the mNGF levels in the brain for recovery of
170 S. Mitra et al.

NGF delivery methods

Peripheral Intracerebroventricular Intracerebral

Intranasal and Stem Cell Intracerebral

Parenteral Tissue graft
Intraocular mediated infusion

Encapsulated
Gene therapy
biodelivery
Genetically Focused Small
Carrier
modified cell ultrasound molecule
mediated
mediated guided mediators
Genetically Viral vector Non-
•Nanoparticles modified cell direct Biodegradable
Biodegradable
•Microspheres mediated delivery
•Conjugates
•Conduits

Fig. 11.2 Various methods for NGF delivery. NGF is broadly classified as peripheral, intracerebroventricular,
delivered using various strategies targeting the brain tis- and intracerebral, respectively. Further classification is
sue. Based on the initial administration route, methods are based on the technical method employed to deliver NGF

the cholinergic signaling in various clinical and must diffuse wide enough to reach the target cells
pre-clinical studies has attracted significant atten- for an efficacious outcome, and (3) the drug must
tion (Hampel et al. 2018). be delivered in a stable manner over long term to
Although the incidence of dementia has been enhance the probability of a clinically relevant
reported to be declining in high-income countries outcome. Although various delivery methods
(Satizabal et al. 2016), recent results are have been utilized for several types of
conflicting (Binder et al. 2019). Thus, the effort neurotrophic molecules, few newer options
to combat AD-related neuronal dysfunction showing increased clinical efficacy have emerged
remains a primary concern. In the last four in the last few decades (Olson 1993; Ruozi et al.
decades, various research groups have tried 2012; Yi et al. 2014), as further described.
to deliver mNGF to the brain tissue in order to
revive the cholinergic signaling with an aim to
recover cognition. Although one of the major
11.2 Different Methods of NGF
issues with brain delivery of NGF is the intrinsic
Delivery
complexity of delivering drugs continuously for
extended periods of time to achieve a clinical
Based on the tissue targeting ability, the various
output, one major problem is the biological
methods utilized for NGF delivery can be broadly
off-target impact of mNGF on nociception and
classified into three major classes: (1) peripheral
pain induction (Denk et al. 2017; Chang et al.
delivery (systemic administration), (2) intracere-
2016). To achieve an efficacious therapy, some
bral delivery (ICD) (within brain parenchyma),
of the following conditions needs to be fulfilled—
and (3) intracerebroventricular delivery (ICVD)
(1) the drug has to be delivered precisely to the
(to the cerebrospinal fluid—CSF). These classes
requisite target region of the brain at appropriate
are subdivided into different methods depending
quantity to stimulate intended outcome while
on the technical approach as shown in Fig. 11.2.
reducing off-target effects, (2) the delivered drug
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
11.2.1 Intracerebroventricular
Delivery (ICVD)
Early studies during 1970s–1980s demonstrated
the correlation between loss of BFCNs and pro-
gressive memory deficits in AD patients, which
led to the proposal of the cholinergic theory of
geriatric memory dysfunction in 1982 (Bartus
et al. 1982). Before and after the proposal of the
cholinergic hypothesis, several reports have
demonstrated the consequence of declining NGF
levels and the potential therapeutic effects of
exogenous NGF delivery with respect to memory
function (Cuello et al. 2019; Hampel et al. 2018;
Chen and Mobley 2019). One of the earliest
methods for delivery of NGF to the brain tissue
was by direct injection of NGF to the CSF using
the ICVD method (Hefti and Schneider 1989).
Starting in 1986, different groups reported the
administration of purified NGF protein to adult
rats where ICVD NGF infusion over 4 weeks
resulted in enhanced survival of cholinergic
neurons post fimbria transection (Williams et al.
1986; Hefti 1986). Subsequently, Fischer et al.
demonstrated that NGF infusion in old rats for
similar duration recovered spatial memory and
reduced neuron atrophy (Fischer et al. 1987).
Further studies showed that NGF not only was
able to recover damaged neurons, but NGF could
also dramatically restore the cholinergic pheno-
type from a point when no cholinergic cells were
apparent by immunostaining of the brain tissue,
indicating that NGF is needed for maintaining the
cholinergic phenotype (Hagg et al. 1988).
Subsequent rodent studies demonstrated the pres-
ence of NGFRs on the BFCNs, and the impor-
tance of NGF for regulation of its receptors
(Montero and Hefti 1988; Montero and Hefti
1989). These findings were replicated in higher
mammals (i.e. monkeys) using mouse-derived
NGF (Koliatsos et al. 1990; Tuszynski et al.
1990) showing the conservation and efficacy of
NGF cross-species. Several other reports showed
the beneficial impact of NGF infusion on the
cerebral vasculature, cellular regeneration, and
proliferation (Winkler et al. 1997; Saffran et al.
1989; Isaacson et al. 1990, 1992; Schlachetzki
171
et al. 2014) but also identified hypophagic effect
of NGF in rats leading to weight loss (Williams
1991). It is at this juncture in 1990 that possible
NGF therapy for AD condition was proposed,
based on the ability of NGF to recover lesioned
cholinergic neurons.
In the following years, Koliatsos et al.
(1991a, b) prepared the recombinant human
NGF (rhNGF) protein and showed its efficacy
on rats to revive lesioned BFCNs. For the first
time, Olson and coworkers from Karolinska
Institutet showed that mouse NGF (75 μg/day
for 3 months) could be therapeutically delivered
to the lateral cerebral ventricle of one AD patient
and reported improved 11C-nicotine binding,
blood flow, electroencephalography (EEG), and
verbal episodic memory (Olson et al. 1992;
Seiger et al. 1993). These encouraging results
prompted the group to perform another trial on
3 AD patients which apart from replicating simi-
lar beneficial effects on 11C-nicotine binding and
improved blood-flow unfortunately demonstrated
side effects which included back pain and weight
loss (Eriksdotter Jonhagen et al. 1998). These
side effects subsided when the NGF infusion
was stopped, but the patients continued to show
improved cerebral blood flow for several months
after the infusion was terminated (Jonhagen
2000). Follow-up experiments in rats receiving
7 or 0.7 μg/day NGF intracerebroventricularly
for 2 weeks reproduced the pain-inducing effects
of NGF while delivery to the brain parenchyma
did not elicit pain (Hao et al. 2000). The ICVD
method has been shown to allow minimal diffu-
sion of delivered neurotrophic molecule into the
brain parenchyma, thereby reducing the
biological effectiveness (Yan et al. 1994). Due
to these off-target effects, NGF delivery by
ICVD was not recommended at higher concentra-
tion, but a later study used a combination of NGF
with other neurotrophic factors to enhance cho-
linergic signaling in rats (Tirassa et al. 2003).
Interestingly, years after the NGF infusion studies
were stopped for AD, NGF ICVD infusion was
shown to be capable of offering neuroprotective
effects in two new-born children suffering from
hypoxic-ischemic brain injuries (HIBI) with
172
prolonged comatose state, wherein NGF infusion
led to normalization of EEG and cerebral perfu-
sion (Fantacci et al. 2013; Chiaretti et al. 2005).
These studies do not report the impact of NGF on
pain induction in these children, which otherwise
would be an interesting data to evaluate. This
difference may be explained by a different
response to NGF in young versus old brains,
indicating that the restorative capabilities of
NGF are quite strong, especially when
administered in younger individuals.
11.2.2 Intracerebral Delivery
To circumvent the off-target effects of NGF (pain
and weight loss), targeted delivery within the
brain parenchyma is considered a preferred
method of choice. Due to the different dynamics
of drug distribution and clearance pathways
within the brain tissue (when compared to CSF),
NGF delivery has been attempted by using vari-
ous technical methods described further.
11.2.2.1 Implantation of Tissue Grafts
Due to the dependence of BFCN’s on cortical and
hippocampal regions for retrograde NGF supply
(Mitra et al. 2019), early studies have
demonstrated the possibility of using fetal tissue
grafts in the cortical regions of the brain to restore
the BFCNs (Fine et al. 1985; Springer et al.
1988a). Briefly, a cortical lesion is introduced
(by chemical agents, e.g., Ibotenic acid) which
has been shown to reduce the cholinergic function
of the basal forebrain in rats, which is then res-
cued by implanted fetal ventral forebrain tissue
rich in NGF producing cells. These grafts have
been shown to induce reversal of memory deficits
introduced by the cortical lesion. Due to extensive
production of NGF by the submaxillary glands in
rodents, these tissues have also been used as
grafts in the lateral ventricle of rats, resulting in
increased cell survival after fimbria-fornix tran-
section (Springer et al. 1988b). Subsequently,
grafts of hippocampal tissue isolated from
embryos (day 18) transplanted in fimbria-fornix
transection models along with simultaneous
ICVD infusion for NGF for 9 weeks provided
S. Mitra et al.
long-lasting protection of cholinergic cells until
after 6 months post NGF therapy (Tuszynski and
Gage 1995). These models provide exciting data
about the possible use of embryonic grafts to
revive cholinergic tissue in injury models, but
these studies do not allow the possibility to eval-
uate the consequences of grafting in a relevant
AD setting. Moreover, issues regarding transplant
rejection by the host immune system could be a
serious setback. Attempts have been made to use
autologous adrenal tissues to revive motor deficits
in Parkinson’s patients wherein NGF infusion for
a month was performed to support the adrenal
grafts (Sydow et al. 1995), but need for regular
NGF infusion and engraftment of non-nervous
tissue in the brain makes the delivery less
feasible.
11.2.2.2 Intracerebral Infusion
As described earlier, several groups have
experimented with direct delivery of NGF infu-
sion into the cerebral tissue, in order to avoid the
side effects of ICVD delivery of NGF. This
method was established by Williams et al.
(1987) when they improved the accuracy of ste-
reotaxic implantation of a cannula within the
brain tissue and combined it with the capability
of Alzet-pumps for continuous infusion of thera-
peutic substances. Further support for intracere-
bral infusion came from the work of Venero et al.
(1996), who demonstrated that 100 times lower
NGF concentration is capable of inducing similar
biological effects when NGF is administered
intracerebrally as compared to ICVD. This pre-
ferred method of NGF delivery was later utilized
to rescue BFCNs in a model of fimbria-fornix
transection (Tuszynski 2000). This approach of
NGF delivery was not utilized in AD-related
models, possibly due to the enhanced degradation
of NGF in the AD brain environment which is due
to increased activity of MMP3/9 enzyme activity
(Pentz et al. 2020; Allard et al. 2012; Hanzel et al.
2014). Interestingly, NGF infusion to the brain
tissue has been shown to reduce the impact of
viral infection and protect cells from lysis post
infection (Pakzaban et al. 1994), which is
regarded as one of the latest discovered etiologies
of AD (Sochocka et al. 2017; Cairns et al. 2020;
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Balin and Hudson 2018). A robotically assisted
placement of a catheter system has been recently
developed to intermittently deliver infusions to
brain parenchyma, although this has not been
tested to deliver NGF in an AD setting (https://
www.renishaw.com) (Killick-Cole et al. 2019).
11.2.2.3 Stem Cell Mediated
Stem cells have been used for several decades in
the field of regenerative medicine, as an inex-
haustible source of self-replicative cells which
can replace damaged tissue and cells (Polak and
Mantalaris 2008; De Luca et al. 2019). Certain
type of stem cells, neural stem cells (NSCs) and
mesenchymal stem cells (MSCs), has been shown
to produce NGF and other neurotrophic factors
(Kamei et al. 2007; Paradisi et al. 2014). Con-
versely, various neurotrophic molecules have
been shown to influence stem cell behavior; for
example, proNGF has been shown to induce pro-
liferation in NSCs while mNGF is needed for its
differentiation, indicating the importance of NGF
metabolism in maintaining neuron generation
from NSCs (Corvaglia et al. 2019; Pramanik
et al. 2017). Several studies have reported the
neuroprotective ability of different stem cell
populations on cell regeneration,
neuroinflammation, and neurobehavior when
implanted within the brain tissue under different
pathological conditions including AD (Kim et al.
2010, 2020; Gu et al. 2015; Lee et al. 2012a; Ager
et al. 2015; McGinley et al. 2018; Zhang et al.
2014). Unfortunately, recent reports indicate that
amyloid precursor protein (APP) expression and
Aβ peptides can modulate the stem cell prolifera-
tion and differentiation, thereby affecting their
therapeutic efficiency and survival (Kwak et al.
2006; Sugaya 2008; Bernabeu-Zornoza et al.
2019). Stem cell therapy is also complicated by
the involvement of immune rejection wherein the
host immune system attacks the implanted stem
cells which reduces the therapeutic efficacy and
aggravates inflammatory responses (Liu et al.
2017). Thus, the efficiency of stem cell therapy
especially in the case of AD needs to be evaluated
carefully (Hayashi et al. 2020; Wang et al. 2019).
Significant improvements in this field has been
made recently, wherein instead of using stem
173
cells themselves, some studies reported the use
of stem cell-derived extracellular vesicles as a
therapeutic option when injected intracerebrally
or delivered through intranasal passage in animal
models of AD (Elia et al. 2019; Losurdo et al.
2020).
11.2.2.4 Gene Therapy
To enhance delivery, without complication from
NGF-associated side effects and dysregulation of
stem cell behavior (aberrant differentiation, sur-
vival, immune response), different approaches
have been undertaken by utilizing gene therapy.
Either cells are transfected with NGF-producing
vectors and then injected, or viral particles are
directly injected into the brain areas which are
targeted to express and be revived by NGF. One
advantage over allogeneic grafting is the use of
autologous cells which are immune-compatible
with the host, while the viral particles can target
multiple cell types at the injection site, transduce
them with NGF coding vectors and induce NGF
secretion locally. But the disadvantage with both
approaches is that once injected, these cells or
viral particles cannot be recovered. These
approaches are further elaborated.
Use of Genetically Modified Cells To secrete
enhanced levels of NGF in an AD context, the
use of genetically modified cells was initiated in
the late 1980s when a mouse fibroblast cell line
was transfected with rat-NGF-secreting vectors
and implanted into rat brains (Ernfors et al.
1989). Improved survival of fetal cholinergic neu-
ronal grafts and increased sprouting of adult
neurons/interneurons were observed when
mixed with genetically modified NGF delivering
fibroblast during implantation to cortical tissue in
a model of unilateral fimbria fornix dissection.
The same group later showed that continuous
NGF delivery can be successfully achieved over
several months by implanting genetically
modified NSCs to aged rat brains, leading to
functional recovery in spatial memory
(Martinez-Serrano et al. 1996; Martinez-Serrano
and Bjorklund 1998). Similar studies were
reported by other groups where autologous
fibroblasts were used to harbor NGF-expressing
174
vectors and were implanted in the brain of adult
primates long term (Tuszynski et al. 1996).
Follow-up work in rats demonstrated the capabil-
ity of genetically identical but modified
fibroblasts to provide partial recovery from spinal
cord injury even when administered several
months post-injury induction (Grill et al. 1997).
These promising effects of NGF delivery using
ex-vivo gene manipulation led to the first-in-
human trial of ex-vivo gene therapy and were
reported in 2005 by Tuzynski et al. (2005). A
10-year follow-up (until brain collection from
those patients’ post-mortem) showed response to
the therapy by neuronal sprouting toward the
implanted fibroblasts (Tuszynski et al. 2015),
indicating long-lasting effect of NGF administra-
tion. NGF delivering human NSCs implanted in
the brain tissue has also been shown to improve
cognitive functions in ibotenic acid-induced hip-
pocampal lesion in rats and aging mice, respec-
tively (Lee et al. 2012b; Park et al. 2013).
Viral Vector Direct Delivery With the advent of
methodologies which offered greater control on
gene expression (reducing uncontrolled
non-specific viral infection in tissues), hybrid
viral vectors allowed the revival of virus-
mediated direct gene delivery, which had been
associated with several technical problems
(Thomas et al. 2003; Goswami et al. 2019). One
of the first reports of NGF delivery using viral
vectors was performed using a hybrid viral vector
containing the AAV2 genome with AAV sero-
type 5 (AAV2/5) in a rat model of fimbria fornix
transection (Wu et al. 2005). As compared to
AAV2 vector, AAV2/5 vectors consistently
showed higher transduction efficiency, while the
duration of gene expression in both cases was
similar (17 weeks). Simultaneously, other groups
using AAV2 vectors expressing NGF in various
pre-clinical studies could show stable gene
expression for long term (1 year), paving the
way for trials in AD patients (Bishop et al.
2008; Nagahara et al. 2009).
A pilot phase 1 clinical trial on ten AD
patients, eight patients receiving ex-vivo geneti-
cally modified fibroblasts (described under
S. Mitra et al.
genetically modified cell section) and two
patients receiving in-vivo AAV2 injection
showed enhanced NGF signaling and protein
levels in the brain tissue post-mortem (Tuszynski
et al. 2015). This encouraging data using AAV2-
NGF vectors was further translated to a dose-
escalating phase 1 study on ten AD patients
which showed that the delivery was feasible in
humans, well tolerated, safe, and resulted in long-
term NGF expression (Rafii et al. 2014). Further
analysis of post-mortem brains from the phase
1 dose escalation study revealed that although
AAV2-NGF vectors were capable of inducing
NGF signaling for almost 7 years in the injected
sites, those sites were not within the cholinergic
nuclei (nucleus basalis of Meynert, nbM) which
were intended to be activated by the therapy. Due
to the limited spread of the injected AAV2-NGF
vectors coupled with incorrect stereotactic
targeting, the clinical trial was not able to induce
the cholinergic pathways which were needed for
the improvement of cognition in AD patients
(Castle et al. 2020). A follow-up multicenter pla-
cebo controlled phase 2 clinical trial on 49 AD
patients replicated the observation from the phase
I study that AAV2-NGF delivery was safe but
failed to show any considerable improvement in
cognitive score at 2-year follow-up (Rafii et al.
2018), in part because the vector targeting of the
brain was unsatisfactory.
11.2.2.5 Encapsulated Biodelivery
With the prospect of developing new tools for
targeted delivery of neurotrophins to the brain,
several important aspects had to be considered,
including the complexity and format of delivery
(stereotactic injection/implantation, ICVD,
peripheral delivery) targeting the correct tissue
or cell type, potential side effects with uncon-
trolled delivery (viral vectors), immune rejection
(stem cells), and many others. The necessity of
delivering NGF to the correct anatomical struc-
ture within the brain tissue highlights the need for
a delivery system which can deliver NGF in pre-
cise location for an extended period of time in
order to elicit a relevant biologic response trans-
lating into a positive clinical outcome (Mahoney
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
and Saltzman 1999). One of the delivery methods
that was developed to achieve these goals was the
encapsulated cell biodelivery (ECB, using cells
for encapsulation) or encapsulated biodelivery
(EB, using protein encapsulation). This technique
essentially contains two parts: (1) a protein of
interest to be delivered or a genetically modified
cell which produces a therapeutic protein and
(2) a semi-permeable membrane for encapsula-
tion. Similarly, the ECB/EB technique can be
considered as a hybrid method which possesses
the qualities of targeted in-situ delivery, long-
term release, with a biologically (immunologi-
cally) safe method. Thus, the fate of the ECB
cells/EB-proteins can be verified. Methods
utilizing encapsulated delivery can be
differentiated based on the material used to pre-
pare the outer membrane, and its contents within
(cells or pure proteins). Broadly, they are classi-
fied as biodegradable and non-biodegradable as
further discussed.
Non-biodegradable This type of encapsulation
devices contains an outer semi-permeable mem-
brane which is non-biodegradable but biocompat-
ible, allowing the influx of nutrients and oxygen
necessary for cell survival with simultaneous
out-flow of the therapeutic molecule
(s) (applicable for ECB). Initially, polymer-
coated pellets of purified NGF proteins were
used to deliver NGF over a limited time period
of time (Powell et al. 1990; Hoffman et al. 1990;
Saltzman et al. 1999). However, remarkable
innovations allowed the use of live genetically
modified cells encapsulated within a biocompati-
ble semi-permeable membrane (Tresco et al.
1992). Using encapsulated NGF-transfected
fibroblast cells, neuroprotective effects were
observed in an ibotenic acid rodent model of
striatal degeneration (Emerich et al. 1994a). Sim-
ilarly, NGF delivery using transfected baby ham-
ster kidney cells in a primate model of fornix
transection showed enhanced sprouting of septal
cholinergic neurons (Emerich et al. 1994b;
Kordower et al. 1994). With the advantage of
potential long life and regeneration capacity of
cell lines which can produce the intended
175
therapeutic protein over a longer time, and due
to the possibility to use different types of cells
within the device and its implantation to any
desired location, this kind of ECB systems were
preferred to deliver NGF in-vivo/in-situ (Date
et al. 1996; Orive et al. 2015; Emerich et al.
2014).
Tremendous efforts and significant optimiza-
tion of cell types, gene expression vectors, and
ECB engineering led to the first successful open-
label human phase1 clinical trial using ECB-NGF
in 6 AD patients by our group (Eriksdotter-
Jonhagen et al. 2012; Wahlberg et al. 2012).
This study showed that ECB-NGF therapy in
AD patients is feasible, safe, and well tolerated
for up to 1 year (experimental endpoint of the
study). Improvement in cognition, EEG, and nic-
otinic receptor binding were reported, along with
improved cholinergic markers in CSF and
reduced basal forebrain atrophy in three patients
(Karami et al. 2015; Machado et al. 2020; Ferreira
et al. 2015). An improved version (second gener-
ation) of the ECB-NGF devices was developed
which released ten times more NGF and was
utilized in a follow-up dose escalation study in
four AD patients for 6 months, confirming the
observations of beneficial effects of first trial
(Fjord-Larsen et al. 2012; Eyjolfsdottir et al.
2016; Machado et al. 2020). The NGF releasing
ability of the ECB-NGF devices was found to be
affected post-explantation after 12 months and
after 6 months, respectively, and further in-vitro
studies identified the impact of inflammatory
cytokines on the NGF releasing cells (Eriksdotter
et al. 2018).
Further identification of “factors” which can
affect ECB-NGF cells and by modifying the cells
to become resistant against these “factors” may
further improve our ECB-NGF therapy to attain
long-term (several years) clinical efficacy. One
major drawback of using non-biodegradable
ECBs is the invasive procedure which is needed
for its implantation. On the contrary, the major
advantage is that once ECB is placed in its target
region, no further drugs or modifications are
required for an extended period (up to 1 year
currently). The ECB can be removed from the
176
brain if needed to terminate the therapy giving
full control over the procedure and treatment
duration. Additionally, a “reloading system”
may be applied in the future to enable the replace-
ment of old with new ECB device, thereby
extending clinical therapy duration without the
need of stereotactic procedures during follow-up
procedures. Similarly, due to the presence of
active protein-releasing cells, ECBs overcome
the problem of protein storage (like in Alzet
pumps) which would otherwise affect protein
quality, aggregation, and biological activity due
to storage conditions of pure proteins. Presently,
many other types of cell encapsulation procedures
are being evaluated in tandem with different
encapsulation materials which may lead to further
advancements in the field (Orive et al. 2019; Oh
et al. 2020; Son et al. 2017).
Biodegradable This class of EBs uses encapsu-
lation materials which are usually biological
molecules which could interact with the
surrounding tissue upon implantation and would
be degraded over time to allow slow and
sustained release of materials encapsulated within
them. Usually these EBs deliver therapeutics over
a short period of time (days to weeks), due to their
dissolution by the host tissue enzymes and limited
amount of storage. The advantage is its biocom-
patibility and ability to release therapeutics in-situ
which allows its implantation within the tissue.
However, one of the drawbacks would be the
inability to control their activity (protein release
and degradation over time), or explantation
(if needed) along with their alteration in shape
due to tissue dynamics and sensitivity towards
enzymes, pH or temperature. Although the use
of biodegradable encapsulation strategies was
utilized to deliver various biological materials
from the early 1980s (Lim and Sun 1980), deliv-
ery of NGF using in-vitro and in-vivo rodent
models was developed from 1990s and onward
(Camarata et al. 1992; Xu et al. 2002; de Boer
et al. 2010). Optimization of the materials used in
the encapsulation of NGF led to the production of
biodegradable microspheres which can slowly
S. Mitra et al.
release NGF over extended time periods and
demonstrated enhanced properties to induce
NGF-mediated signaling (Sakiyama-Elbert and
Hubbell 2000) and protect from neurotoxicity
(Menei et al. 2000). Further optimization of
encapsulation materials produced
thermosensitive and chemosensitive materials
which allow activity-based release of NGF from
the encapsulation matrix (Lee et al. 2003; Zhao
et al. 2016; Hu et al. 2020). In a few applications,
NGF protein was modified to form polypeptides
to control its release and diffusion (Sakiyama-
Elbert et al. 2001; Song et al. 2012). Currently,
nanostructured carriers are being tested for its
efficiency to deliver NGF in models of spinal
cord injury and AD (Zhu et al. 2016; Zilony-
Hanin et al. 2019), although various optimization
in the delivery vehicles have been proposed for
the future (Wang et al. 2017).
11.2.3 Peripheral Delivery
Due to the invasiveness of procedures mentioned
in previous sections (Sects. 11.2.1 and 11.2.2),
other less-invasive options have been proposed to
reduce risk and complications associated with the
more invasive NGF delivery methods described
earlier. Peripheral delivery methods have the pos-
sibility to consider different approaches which are
not bound to NGF protein or gene delivery (small
molecules, coated vesicles, etc.) as mentioned in
the following and has the advantage of continu-
ous multiple administration, which is not possible
with invasive approaches. Conversely, one of the
major disadvantages is the inability to control the
tissue-targeted delivery of the delivered molecule
and associated side effects on other body organs.
Aberrantly increasing NGF signaling has been
shown to be associated with different forms of
cancer in vulnerable organs (Aloe et al. 2016;
Hayakawa et al. 2017; Blondy et al. 2019; Griffin
et al. 2018). Several approaches have been
undertaken to achieve NGF delivery to the brain
through parenteral administration by overcoming
these limitations, as further described.
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
11.2.3.1 Intranasal and Intraocular
Delivery
Administration of therapeutics has been tried for
various drugs through the ocular and nasal
passages owing to their direct connection to the
olfactory and frontal brain regions (Ganger and
Schindowski 2018; Mandal et al. 2018; Pardridge
2019). Large molecules like NGF, which is also
very sensitive to enzymatic degradation, need a
direct access to brain tissue to generate a compre-
hensive clinically relevant effect, which is actu-
ally offered by the ocular and nasal passages since
NGF delivered through these routes bypasses the
BBB without reaching systemic circulation.
Although NGF has been used to treat various
eye disease conditions and NGF expression has
been found to be increased in optic-nerve tissue
(Lambiase et al. 1998; Bonini et al. 2000;
Lambiase et al. 2005; Guo et al. 2020), the
mechanisms of delivery to the brain tissue
through this route are not thoroughly understood.
Moreover, as mentioned previously, NGF deliv-
ery may induce side effects including pain and
weight loss and using NGF in its natural full-
length form intranasally or intraocularly may
elicit adverse side effects. Discovery of a mutated
form of NGF, termed painless NGF, owing to its
cholinergic signaling inducing properties without
inducing pain (Minde et al. 2004; Cattaneo and
Capsoni 2019), led to studies on this mutated
form for NGF delivery (mentioned below in this
section). Nevertheless, there are a few studies
indicating the possibility to stimulate brain
regions through ocular delivery of NGF without
generating anti-NGF antibody response from the
host (Lambiase et al. 2007a, b; Tirassa et al.
2018), but whether the effects can enhance cogni-
tive abilities in the context of AD has not yet been
explored.
In comparison to ocular delivery, intranasal
delivery of NGF has already been a topic of
thorough research during the early days of NGF
therapy development in 1990s where it was
shown to efficiently reach the brain tissue to
induce NGF signaling (Altar and Bakhit 1991;
Chen et al. 1998). Intranasal administration of
NGF was found to show neuroprotection and
177
rescue cognitive deficits in animal models of
AD (Capsoni et al. 2002,2012; De Rosa et al.
2005). Using a mouse model of AD which intrin-
sically produces anti-NGF antibodies, thereby
depriving the brain tissue of autologously pro-
duced NGF (Capsoni et al. 2011), a direct com-
parison between ocular and nasal administration
of recombinant NGF demonstrated enhanced dis-
tribution and efficacy of intranasally delivered
NGF to rescue the phenotypic changes of these
mice (Capsoni et al. 2009; Covaceuszach et al.
2009). Moreover, intranasal delivery of therapeu-
tics has also been shown to be more effective than
intraperitoneal administration in reaching the
brain tissue, in part by bypassing the BBB
(Chauhan and Chauhan 2015). Thus, the utility
of the intranasal approach to deliver NGF seems
more promising than other non-invasive
approaches (considering the delivery of NGF pro-
tein). The efficiency of intranasal NGF delivery-
mediated neuroprotection has been demonstrated
in two female patients with frontotemporal
dementia (FTD) who received NGF therapy
only (for 12–16 months) and were compared to
another two female patients who were treated
with memantine and idebenone therapy (de Bellis
et al. 2018). Another study on a 4-year-old trau-
matic brain injury (TBI) patient showed a benefi-
cial impact of intranasal NGF therapy, resulting
in improved voluntary responses and clinical con-
dition (Chiaretti et al. 2017). As discussed previ-
ously, although intranasal NGF delivery shows
great promise as a translational candidate for
future human use, the complex anatomy of the
nasal cavity and its interaction with other physio-
logical functions needs to be evaluated closely
prior to the development of a clinically relevant
therapy (Erdo et al. 2018; Bourganis et al. 2018;
Gomez et al. 2012).
11.2.3.2 Parenteral Delivery
Apart from the delivery methods discussed Sect.
11.2.3.1, most of the peripheral delivery methods
utilize parenteral delivery, i.e., injections of vari-
ous molecules in different forms delivered intra-
venously, to be further discussed.
178
Small Molecule Mediators Due to the realiza-
tion that stimulation of the TrkA receptor is
needed for cell survival, or conversely possible
inhibition of p75 may reduce cell death, and that
both of these biological effects can be activated
without the need for an actual neurotrophin mol-
ecule, neurotrophin mimicking compounds
(peptides and chemical analogues) were devel-
oped from the late 1990s (Spiegel et al. 1995;
Estenne-Bouhtou et al. 1996; Longo et al. 1997;
LeSauteur et al. 1995). With the advent of time
and technology, various kinds and classes of
small-molecule agonists and antagonists have
also been developed toward TrkA and p75
receptors (Mitra et al. 2019; Covaceuszach et al.
2009; Cattaneo et al. 2008; Longo and Massa
2013). Among the various molecules developed
till date, a small-molecule p75 modulator
(LM11A-31) has been found to reduce basal fore-
brain cholinergic degeneration and rescue
neurons from death in various rodent models of
AD when administered orally (Yang et al. 2020a;
Xie et al. 2019; Nguyen et al. 2014). The com-
pound is currently tested in an ongoing clinical
trial in mild-to-moderate AD patients
(ClinicalTrials.gov Identifier: NCT03069014).
Genetically Modified Cell-Mediated
Delivery Another approach for NGF delivery to
the brain was undertaken using ex-vivo autolo-
gous genetically modified monocytes, which
showed neuroprotection when injected directly
ICVD (Zassler and Humpel 2006) or peripherally
via the blood (Hohsfield et al. 2014). When
injected into the cerebral ventricles, the
monocytes were found to survive for 7 days and
resulted in restored basal forebrain function. Sim-
ilarly, when genetically modified
NGF-expressing monocytes were injected intra-
venously, they were found to reach the brain
tissue and elevate cognitive outcome. In both
intraventricular and parenteral deliveries, geneti-
cally modified monocytes were found to deliver
NGF without inducing inflammatory reactions.
Carrier-Mediated Delivery Due to the sensitiv-
ity of the NGF protein by rapid degradation by
S. Mitra et al.
proteolytic enzymes when present in the blood,
and since NGF cannot cross the BBB, it is neces-
sary to conjugate it with various carrier
molecules. Diverse methods of conjugation have
been applied to different kinds of carrier
molecules, as further discussed.
Protein Conjugated Direct conjugation of NGF
to other proteins has been tried to achieve
receptor-mediated uptake through BBB. Initial
reports in 1990s showed the efficacy of the anti-
transferrin receptor antibody-NGF conjugates
injected intravenously in reviving septal choliner-
gic and non-cholinergic grafted neurons
implanted in the anterior chamber of the eye
(Friden et al. 1993; Granholm et al. 1994). Intra-
venous NGF-conjugated delivery was found to be
effective in rescuing BFCNs from excitotoxic
injury, reversed age-related cognitive dysfunc-
tion, and restored electrophysiological currents
(Charles et al. 1996; Backman et al. 1996; Albeck
et al. 1999). Follow-up studies showed that by
using transferrin-avidin conjugation to bind
NGF-biotin conjugates, this could increase the
NGF binding capacity of the transferrin protein
and delivered higher amounts of NGF to the brain
tissue when compared to NGF-anti-transferrin
receptor antibody conjugates (Liao et al. 2001;
Li et al. 2000).
Nanoparticle Conjugated Nanomaterial-
mediated drug delivery to the brain has been an
attractive option under various experimental
conditions due to its flexibility and tissue
targeting capabilities (Singh et al. 2019). NGF
protein can be delivered to the brain when
adsorbed on various nanoparticle materials and
has been reported to enhance cognition and mem-
ory in pharmacological models of injury in-vivo
(Kurakhmaeva et al. 2009). Recently nanocapsule
structures have been developed which can deliver
NGF to the rodent and primate brain when
injected intravenously (Xu et al. 2019). Further-
more, advanced nanocarriers which carry
NGF-secreting plasmids entrapped within a lipo-
some vehicle passing the BBB by incorporating a
transferrin ligand on the surface of the nanocarrier
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
have been developed. These liposome
nanocarriers have been shown to reduce Aβ levels
and revive synaptic proteins when injected intra-
venously in an AD mice model (Rodrigues et al.
2020). Several other types of nanocarriers have
been tested in-vitro and showed improved reten-
tion and slower degradation of NGF when conju-
gated to nanomaterials (Marcus et al. 2015;
Razavi et al. 2019), but they are yet to be tested
in-vivo.
Microspheres Mediated Microspheres are
entrapment procedures (much like nanoparticles)
but with better biocompatibility, mechanical flex-
ibility and control of drug release kinetics (Varde
and Pack 2004). Initial work in the early 1990s
showed the ability of intracerebrally injected
NGF containing microspheres to constantly
release its contents for more than 4 weeks
(Camarata et al. 1992). Similar studies in later
work demonstrated that although microspheres
can be used for continuous controlled delivery
of NGF, the biological activity may abate due to
the preparation step or due to protein denaturation
and degradation over time (Hadlock et al. 2003).
Newer versions of biodegradable microspheres
using fibrin were recently reported to deliver
NGF when injected to the brain (Samal et al.
2015). In-vivo studies in rat showed that
peripherally administered liposomes can deliver
NGF to different brain regions including the stri-
atum, cortex, and basal forebrain (Xie et al. 2005;
Gu et al. 2007), and can recover spatial learning
ability when implanted in fimbria-fornix transec-
tion models (Gu et al. 2009). Furthermore, an
animal model of sciatic nerve injury demonstrated
that poly lactic-co-glycolic acid (PLGA)
microspheres containing NGF injected to the
injury site can increase nerve recovery (Zhang
et al. 2017). A recent study using exosomes to
simultaneously pack NGF protein and NGF
expressing vectors together and inject them sys-
temically in a stroke model showed beneficial
effects on neuronal cell survival and inflamma-
tory reactions (Yang et al. 2020b). With new
technologies at the horizon, the development of
targeted viable in-vivo delivery of NGF is eagerly
179
expected for studies in AD-relevant animal
models (Qu et al. 2020).
Incorporated Within Nerve Guidance
Conduits Although conduits have yet not been
used in studies of treatment for dementia or AD,
this technology has been demonstrated to provide
support and guidance for axonal growth in spinal
cord injury and peripheral nerve injury cases
(Piotrowicz and Shoichet 2006). Conduits are
3D structures which are coated internally with
molecules (biological or chemical) which
enhance the axonal growth. NGF has been used
as the chemical que in several studies and shown
to enhance guided axonal growth and improved
nerve health (Liu et al. 2013; Xia and Lv 2018;
Hsu et al. 2019). Injectable hydrogels intended
for stem cell applications in nerve regeneration
have also been reported to enhance cell survival
and proliferation when used in conjunction with
NGF (Li et al. 2020).
Ultrasound-Guided Brain Delivery Although
this method is yet not used in assisting an
NGF-carrying device or anything similar, ultra-
sound waves have been used in conjunction with
other NGF delivery methods to enhance the brain
penetration of NGF, when delivered peripherally.
Since peripherally injected neurotrophins cannot
cross the BBB, guided focused ultrasounds were
used to momentarily break the BBB in specific
brain regions, thus facilitating drug uptake. This
approach of using ultrasound-guided delivery of
nano-/micro-particles is termed “sonochem-
otherapy” (Lammertink et al. 2015). In a spinal
cord injury model, a NGF plasmid contained in
PLGA nanobubbles was intravenously injected
and focally disintegrated within the injured tissue
to release the plasmid using guided ultrasound
which led to neuroprotection (Song et al. 2017).
Safety and tolerability of focused ultrasound-
guided BBB opening and drug delivery have
recently been discussed as a promising technol-
ogy for delivering peripherally administered
drugs to specific brain regions in AD patients
(Rezai et al. 2020; Lipsman et al. 2018). Efficacy
of an intravenously administered TrkA agonist
180
Parenteral
ICVD
Stereotactic injection
Encapsulated
Genetically modified
Viral vectors
Stem cell
Intracerebral
Fig. 11.3 Brain targeting capabilities of NGF delivery
methods: Although NGF can be administered by various
techniques into different regions of the body, but the brain
availability of NGF, post-administration, defines the effi-
cacy of these methods. This figure highlights the initial
(D3) was delivered to the basal forebrain using
focused ultrasound and shown to enhance cholin-
ergic signaling and neurotransmission in a mice
model of AD (Xhima et al. 2020).
11.3 Conclusion and Future
Developments
Over the last three decades, several different
methods have been applied to deliver NGF to
the brain, as illustrated in Fig. 11.3. The various
methods have evolved over time to meet the
medical needs of clinical efficacy, tolerability,
and safety, and to minimize off-target effects.
Nonetheless, every delivery method has its pros
and cons, which should be taken into consider-
ation when assessing their potential for clinical
application.
Invasive techniques have been shown to have
potential for clinically relevant effects in AD
patients (ECB-NGF, viral vectors, genetically
modified cells) but need high expertise for
S. Mitra et al.
Delivery to brain tissue
Delivery to CSF
Parenteral
Parenteral with targeted
possibilities
Intraocular
&
Intranasal
Focused
guided
ultrasound
+
Parenteral
drug
administration route of each technique and provides an
overall view of invasiveness of the technique and its
brain targeting capability. Different administration routes
are color coded. Abbreviations: ICVD (intracerebroven-
tricular), CSF (cerebrospinal fluid)
administration, whereas data on long-term safety
and efficacy of non-invasive procedures are still
awaiting clinical testing. Painless NGF
equivalents and novel small molecule modulators
are promising for future therapies but need further
evaluation for off-target effects and safety, along
with the possibility to use multiple neurotrophin
molecules in close connection or simultaneously.
References
Ager RR, Davis JL, Agazaryan A, Benavente F, Poon
WW, LaFerla FM, Blurton-Jones M (2015) Human
neural stem cells improve cognition and promote syn-
aptic growth in two complementary transgenic models
of Alzheimer’s disease and neuronal loss. Hippocam-
pus 25(7):813–826. https://doi.org/10.1002/hipo.
22405
Albeck DS, Backman C, Veng L, Friden P, Rose GM,
Granholm A (1999) Acute application of NGF
increases the firing rate of aged rat basal forebrain
neurons. Eur J Neurosci 11(7):2291–2304. https://doi.
org/10.1046/j.1460-9568.1999.00644.x
Allard S, Leon WC, Pakavathkumar P, Bruno MA,
Ribeiro-da-Silva A, Cuello AC (2012) Impact of the
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
NGF maturation and degradation pathway on the cor-
tical cholinergic system phenotype. J Neurosci 32
(6):2002–2012. https://doi.org/10.1523/JNEUROSCI.
1144-11.2012
Aloe L, Rocco ML, Bianchi P, Manni L (2012) Nerve
growth factor: from the early discoveries to the poten-
tial clinical use. J Transl Med 10:239. https://doi.org/
10.1186/1479-5876-10-239
Aloe L, Rocco ML, Balzamino BO, Micera A (2015)
Nerve growth factor: a focus on neuroscience and
therapy. Curr Neuropharmacol 13(3):294–303.
h t t p s : / / d o i . o r g / 1 0 . 2 1 7 4 /
1570159x13666150403231920
Aloe L, Rocco ML, Balzamino BO, Micera A (2016)
Nerve growth factor: role in growth, differentiation
and controlling cancer cell development. J Exp Clin
Cancer Res 35(1):116. https://doi.org/10.1186/s13046-
016-0395-y
Altar CA, Bakhit C (1991) Receptor-mediated transport of
human recombinant nerve growth factor from olfactory
bulb to forebrain cholinergic nuclei. Brain Res 541
(1):82–88. https://doi.org/10.1016/0006-8993(91)
91077-e
Backman C, Rose GM, Hoffer BJ, Henry MA, Bartus RT,
Friden P, Granholm AC (1996) Systemic administra-
tion of a nerve growth factor conjugate reverses
age-related cognitive dysfunction and prevents cholin-
ergic neuron atrophy. J Neurosci 16(17):5437–5442
Balin BJ, Hudson AP (2018) Herpes viruses and
Alzheimer’s disease: new evidence in the debate. Lan-
cet Neurol 17(10):839–841. https://doi.org/10.1016/
S1474-4422(18)30316-8
Bartus RT, Dean RL 3rd, Beer B, Lippa AS (1982) The
cholinergic hypothesis of geriatric memory dysfunc-
tion. Science 217(4558):408–414
Bernabeu-Zornoza A, Coronel R, Palmer C,
Monteagudo M, Zambrano A, Liste I (2019) Physio-
logical and pathological effects of amyloid-beta spe-
cies in neural stem cell biology. Neural Regen Res 14
(12):2035–2042. https://doi.org/10.4103/1673-5374.
262571
Binder N, Balmford J, Schumacher M (2019) A multi-state
model based reanalysis of the Framingham heart study:
is dementia incidence really declining? Eur J
Epidemiol 34(11):1075–1083. https://doi.org/10.
1007/s10654-019-00567-6
Birks JS, Harvey RJ (2018) Donepezil for dementia due to
Alzheimer’s disease. Cochrane Database Syst Rev 6:
CD001190. https://doi.org/10.1002/14651858.
CD001190.pub3
Bishop KM, Hofer EK, Mehta A, Ramirez A, Sun L,
Tuszynski M, Bartus RT (2008) Therapeutic potential
of CERE-110 (AAV2-NGF): targeted, stable, and
sustained NGF delivery and trophic activity on rodent
basal forebrain cholinergic neurons. Exp Neurol 211
(2):574–584. https://doi.org/10.1016/j.expneurol.
2008.03.004
Blondy S, Christou N, David V, Verdier M, Jauberteau
MO, Mathonnet M, Perraud A (2019) Neurotrophins
181
and their involvement in digestive cancers. Cell Death
Dis 10(2):123. https://doi.org/10.1038/s41419-019-
1385-8
Bonini S, Lambiase A, Rama P, Caprioglio G, Aloe L
(2000) Topical treatment with nerve growth factor for
neurotrophic keratitis. Ophthalmology 107
(7):1347–1351.; Discussion 1351–1342. https://doi.
org/10.1016/s0161-6420(00)00163-9
Bourganis V, Kammona O, Alexopoulos A, Kiparissides
C (2018) Recent advances in carrier mediated nose-to-
brain delivery of pharmaceutics. Eur J Pharm
Biopharm 128:337–362. https://doi.org/10.1016/j.
ejpb.2018.05.009
Bracci-Laudiero L, Manni L (2014) NGF and immune
regulation. In: Kostrzewa RM (ed) Handbook of neu-
rotoxicity. Springer, New York
Bradshaw RA, Mobley W, Rush RA (2017) Nerve growth
factor and related substances: a brief history and an
introduction to the international NGF meeting series.
Int J Mol Sci 18(6):1143. https://doi.org/10.3390/
ijms18061143
Bruno MA, Cuello AC (2006) Activity-dependent release
of precursor nerve growth factor, conversion to mature
nerve growth factor, and its degradation by a protease
cascade. Proc Natl Acad Sci U S A 103
(17):6735–6740. https://doi.org/10.1073/pnas.
0510645103
Cairns DM, Rouleau N, Parker RN, Walsh KG, Gehrke L,
Kaplan DL (2020) A 3D human brain-like tissue model
of herpes-induced Alzheimer’s disease. Sci Adv 6(19):
eaay8828. https://doi.org/10.1126/sciadv.aay8828
Camarata PJ, Suryanarayanan R, Turner DA, Parker RG,
Ebner TJ (1992) Sustained release of nerve growth
factor from biodegradable polymer microspheres. Neu-
rosurgery 30(3):313–319. https://doi.org/10.1227/
00006123-199203000-00001
Capsoni S, Giannotta S, Cattaneo A (2002) Nerve growth
factor and galantamine ameliorate early signs of
neurodegeneration in anti-nerve growth factor mice.
Proc Natl Acad Sci U S A 99(19):12432–12437.
https://doi.org/10.1073/pnas.192442999
Capsoni S, Covaceuszach S, Ugolini G, Spirito F,
Vignone D, Stefanini B, Amato G, Cattaneo A (2009)
Delivery of NGF to the brain: intranasal versus ocular
administration in anti-NGF transgenic mice. J
Alzheimers Dis 16(2):371–388. https://doi.org/10.
3233/JAD-2009-0953
Capsoni S, Brandi R, Arisi I, D’Onofrio M, Cattaneo A
(2011) A dual mechanism linking NGF/proNGF
imbalance and early inflammation to Alzheimer’s dis-
ease neurodegeneration in the AD11 anti-NGF mouse
model. CNS Neurol Disord Drug Targets 10
(5):635–647
Capsoni S, Marinelli S, Ceci M, Vignone D, Amato G,
Malerba F, Paoletti F, Meli G, Viegi A, Pavone F,
Cattaneo A (2012) Intranasal “painless” human nerve
growth factor [corrected] slows amyloid
neurodegeneration and prevents memory deficits in
182
App X PS1 mice. PLoS One 7(5):e37555. https://doi.
org/10.1371/journal.pone.0037555
Capsoni S, Amato G, Vignone D, Criscuolo C, Nykjaer A,
Cattaneo A (2013) Dissecting the role of sortilin recep-
tor signaling in neurodegeneration induced by NGF
deprivation. Biochem Biophys Res Commun 431
(3):579–585. https://doi.org/10.1016/j.bbrc.2013.01.
007
Castle MJ, Baltanas FC, Kovacs I, Nagahara AH, Barba D,
Tuszynski MH (2020) Postmortem analysis in a clini-
cal trial of AAV2-NGF gene therapy for Alzheimer’s
disease identifies a need for improved vector delivery.
Hum Gene Ther 31(7-8):415–422. https://doi.org/10.
1089/hum.2019.367
Cattaneo A, Capsoni S (2019) Painless nerve growth fac-
tor: a TrkA biased agonist mediating a broad
neuroprotection via its actions on microglia cells.
Pharmacol Res 139:17–25. https://doi.org/10.1016/j.
phrs.2018.10.028
Cattaneo A, Capsoni S, Paoletti F (2008) Towards non
invasive nerve growth factor therapies for Alzheimer’s
disease. J Alzheimers Dis 15(2):255–283. https://doi.
org/10.3233/jad-2008-15210
Chang DS, Hsu E, Hottinger DG, Cohen SP (2016) Anti-
nerve growth factor in pain management: current evi-
dence. J Pain Res 9:373–383. https://doi.org/10.2147/
JPR.S89061
Charles V, Mufson EJ, Friden PM, Bartus RT, Kordower
JH (1996) Atrophy of cholinergic basal forebrain
neurons following excitotoxic cortical lesions is
reversed by intravenous administration of an NGF
conjugate. Brain Res 728(2):193–203. https://doi.org/
10.1016/0006-8993(96)00398-8
Chauhan MB, Chauhan NB (2015) Brain uptake of
Neurotherapeutics after intranasal versus intraperito-
neal delivery in mice. J Neurol Neurosurg 2:1–9
Chen XQ, Mobley WC (2019) Exploring the pathogenesis
of Alzheimer disease in basal forebrain cholinergic
neurons: converging insights from alternative
hypotheses. Front Neurosci 13:446. https://doi.org/10.
3389/fnins.2019.00446
Chen XQ, Fawcett JR, Rahman YE, Ala TA, Frey IW
(1998) Delivery of nerve growth factor to the brain
via the olfactory pathway. J Alzheimers Dis 1
(1):35–44. https://doi.org/10.3233/jad-1998-1102
Chiaretti A, Genovese O, Riccardi R, Di Rocco C, Di
Giuda D, Mariotti P, Pulitano S, Piastra M,
Polidori G, Colafati GS, Aloe L (2005) Intraventricular
nerve growth factor infusion: a possible treatment for
neurological deficits following hypoxic-ischemic brain
injury in infants. Neurol Res 27(7):741–746. https://
doi.org/10.1179/016164105X35611
Chiaretti A, Conti G, Falsini B, Buonsenso D, Crasti M,
Manni L, Soligo M, Fantacci C, Genovese O, Calcagni
ML, Di Giuda D, Mattoli MV, Cocciolillo F, Ferrara P,
Ruggiero A, Staccioli S, Colafati GS, Riccardi R
(2017) Intranasal nerve growth factor administration
improves cerebral functions in a child with severe
traumatic brain injury: a case report. Brain Inj 31
S. Mitra et al.
(11):1538–1547. https://doi.org/10.1080/02699052.
2017.1376760
Cohen S, Levi-Montalcini R (1956) A nerve growth-
stimulating factor isolated from snake venom. Proc
Natl Acad Sci U S A 42(9):571–574. https://doi.org/
10.1073/pnas.42.9.571
Cohen S, Levi-Montalcini R, Hamburger V (1954) A
nerve growth-stimulating factor isolated from sarcom
as 37 and 180. Proc Natl Acad Sci U S A 40
(10):1014–1018. https://doi.org/10.1073/pnas.40.10.
1014
Corvaglia V, Cilli D, Scopa C, Brandi R, Arisi I,
Malerba F, La Regina F, Scardigli R, Cattaneo A
(2019) ProNGF is a cell-type-specific mitogen for
adult hippocampal and for induced neural stem cells.
Stem Cells 37(9):1223–1237. https://doi.org/10.1002/
stem.3037
Covaceuszach S, Capsoni S, Ugolini G, Spirito F,
Vignone D, Cattaneo A (2009) Development of a non
invasive NGF-based therapy for Alzheimer’s disease.
Curr Alzheimer Res 6(2):158–170. https://doi.org/10.
2174/156720509787602870
Cuello AC, Pentz R, Hall H (2019) The brain NGF meta-
bolic pathway in health and in Alzheimer’s pathology.
Front Neurosci 13:62. https://doi.org/10.3389/fnins.
2019.00062
Cummings J, Feldman HH, Scheltens P (2019) The
“rights” of precision drug development for
Alzheimer’s disease. Alzheimers Res Ther 11(1):76.
https://doi.org/10.1186/s13195-019-0529-5
Cummings J, Lee G, Ritter A, Sabbagh M, Zhong K
(2020) Alzheimer’s disease drug development pipe-
line: 2020. Alzheimers Dement (N Y) 6(1):e12050.
https://doi.org/10.1002/trc2.12050
Date I, Ohmoto T, Imaoka T, Ono T, Hammang JP,
Francis J, Greco C, Emerich DF (1996) Cografting
with polymer-encapsulated human nerve growth
factor-secreting cells and chromaffin cell survival and
behavioral recovery in hemiparkinsonian rats. J
Neurosurg 84(6):1006–1012. https://doi.org/10.3171/
jns.1996.84.6.1006
de Bellis A, de Bellis M, Aloe L (2018) Long-term non--
invasive treatment via intranasal administration of
nerve growth factor protects the human brain in
frontotemporal dementia associated with corticobasal
syndrome: a pilot study. J Alzheimers Dis Rep 2
(1):67–77. https://doi.org/10.3233/ADR-180055
de Boer R, Knight AM, Spinner RJ, Malessy MJ,
Yaszemski MJ, Windebank AJ (2010) In vitro and
in vivo release of nerve growth factor from biodegrad-
able poly-lactic-co-glycolic-acid microspheres. J
Biomed Mater Res A 95(4):1067–1073. https://doi.
org/10.1002/jbm.a.32900
De Luca M, Aiuti A, Cossu G, Parmar M, Pellegrini G,
Robey PG (2019) Advances in stem cell research and
therapeutic development. Nat Cell Biol 21
(7):801–811. https://doi.org/10.1038/s41556-019-
0344-z
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Denk F, Bennett DL, McMahon SB (2017) Nerve growth
factor and pain mechanisms. Annu Rev Neurosci
40:307–325. https://doi.org/10.1146/annurev-neuro-
072116-031121
De Rosa R, Garcia AA, Braschi C, Capsoni S, Maffei L,
Berardi N, Cattaneo A (2005) Intranasal administration
of nerve growth factor (NGF) rescues recognition
memory deficits in AD11 anti-NGF transgenic mice.
Proc Natl Acad Sci U S A 102(10):3811–3816. https://
doi.org/10.1073/pnas.0500195102
Dou KX, Tan MS, Tan CC, Cao XP, Hou XH, Guo QH,
Tan L, Mok V, Yu JT (2018) Comparative safety and
effectiveness of cholinesterase inhibitors and
memantine for Alzheimer’s disease: a network meta-
analysis of 41 randomized controlled trials. Alzheimers
Res Ther 10(1):126. https://doi.org/10.1186/s13195-
018-0457-9
Elia CA, Tamborini M, Rasile M, Desiato G, Marchetti S,
Swuec P, Mazzitelli S, Clemente F, Anselmo A,
Matteoli M, Malosio ML, Coco S (2019) Intracerebral
injection of extracellular vesicles from mesenchymal
stem cells exerts reduced Abeta plaque burden in early
stages of a preclinical model of Alzheimer’s disease.
Cells 8(9):1059. https://doi.org/10.3390/cells8091059
Emerich DF, Hammang JP, Baetge EE, Winn SR (1994a)
Implantation of polymer-encapsulated human nerve
growth factor-secreting fibroblasts attenuates the
behavioral and neuropathological consequences of
quinolinic acid injections into rodent striatum. Exp
Neurol 130(1):141–150. https://doi.org/10.1006/exnr.
1994.1193
Emerich DF, Winn SR, Harper J, Hammang JP, Baetge
EE, Kordower JH (1994b) Implants of polymer-
encapsulated human NGF-secreting cells in the nonhu-
man primate: rescue and sprouting of degenerating
cholinergic basal forebrain neurons. J Comp Neurol
349(1):148–164. https://doi.org/10.1002/cne.
903490110
Emerich DF, Orive G, Thanos C, Tornoe J, Wahlberg LU
(2014) Encapsulated cell therapy for neurodegenera-
tive diseases: from promise to product. Adv Drug
Deliv Rev 67–68:131–141. https://doi.org/10.1016/j.
addr.2013.07.008
Erdo F, Bors LA, Farkas D, Bajza A, Gizurarson S (2018)
Evaluation of intranasal delivery route of drug admin-
istration for brain targeting. Brain Res Bull
143:155–170. https://doi.org/10.1016/j.brainresbull.
2018.10.009
Eriksdotter Jonhagen M, Nordberg A, Amberla K,
Backman L, Ebendal T, Meyerson B, Olson L, Seiger,
Shigeta M, Theodorsson E, Viitanen M, Winblad B,
Wahlund LO (1998) Intracerebroventricular infusion
of nerve growth factor in three patients with
Alzheimer’s disease. Dement Geriatr Cogn Disord
9 (5):246–257. https://doi.org/10.1159/000017069
Eriksdotter M, Navarro-Oviedo M, Mitra S, Wahlberg L,
Linderoth B, Tjernberg LO, Behbahani H (2018) Cere-
brospinal fluid from Alzheimer patients affects cell-
mediated nerve growth factor production and cell
183
survival in vitro. Exp Cell Res 371(1):175–184.
https://doi.org/10.1016/j.yexcr.2018.08.007
Eriksdotter-Jonhagen M, Linderoth B, Lind G,
Aladellie L, Almkvist O, Andreasen N, Blennow K,
Bogdanovic N, Jelic V, Kadir A, Nordberg A,
Sundstrom E, Wahlund LO, Wall A, Wiberg M,
Winblad B, Seiger A, Almqvist P, Wahlberg L
(2012) Encapsulated cell biodelivery of nerve growth
factor to the Basal forebrain in patients with
Alzheimer’s disease. Dement Geriatr Cogn Disord 33
(1):18–28. https://doi.org/10.1159/000336051
Ernfors P, Ebendal T, Olson L, Mouton P, Stromberg I,
Persson H (1989) A cell line producing recombinant
nerve growth factor evokes growth responses in intrin-
sic and grafted central cholinergic neurons. Proc Natl
Acad Sci U S A 86(12):4756–4760. https://doi.org/10.
1073/pnas.86.12.4756
Estenne-Bouhtou G, Kullander K, Karlsson M, Ebendal T,
Hacksell U, Luthman K (1996) Design, synthesis, tan-
dem mass spectrometric sequencing and biological
activity of NGF mimetics. Int J Pept Protein Res 48
(4):337–346. https://doi.org/10.1111/j.1399-3011.
1996.tb00850.x
Eyjolfsdottir H, Eriksdotter M, Linderoth B, Lind G,
Juliusson B, Kusk P, Almkvist O, Andreasen N,
Blennow K, Ferreira D, Westman E, Nennesmo I,
Karami A, Darreh-Shori T, Kadir A, Nordberg A,
Sundstrom E, Wahlund LO, Wall A, Wiberg M,
Winblad B, Seiger A, Wahlberg L, Almqvist P
(2016) Targeted delivery of nerve growth factor to
the cholinergic basal forebrain of Alzheimer’s disease
patients: application of a second-generation
encapsulated cell biodelivery device. Alzheimers Res
Ther 8(1):30. https://doi.org/10.1186/s13195-016-
0195-9
Fahnestock M, Shekari A (2019) ProNGF and
neurodegeneration in Alzheimer’s disease. Front
Neurosci 13:129. https://doi.org/10.3389/fnins.2019.
00129
Fantacci C, Capozzi D, Ferrara P, Chiaretti A (2013)
Neuroprotective role of nerve growth factor in
hypoxic-ischemic brain injury. Brain Sci 3
(3):1013–1022. https://doi.org/10.3390/
brainsci3031013
Ferreira D, Westman E, Eyjolfsdottir H, Almqvist P,
Lind G, Linderoth B, Seiger A, Blennow K,
Karami A, Darreh-Shori T, Wiberg M, Simmons A,
Wahlund LO, Wahlberg L, Eriksdotter M (2015) Brain
changes in Alzheimer’s disease patients with
implanted encapsulated cells releasing nerve growth
factor. J Alzheimers Dis 43(3):1059–1072. https://doi.
org/10.3233/JAD-141068
Fine A, Dunnett SB, Bjorklund A, Iversen SD (1985)
Cholinergic ventral forebrain grafts into the neocortex
improve passive avoidance memory in a rat model of
Alzheimer disease. Proc Natl Acad Sci U S A 82
(15):5227–5230. https://doi.org/10.1073/pnas.82.15.
5227
184
Fischer W, Wictorin K, Bjorklund A, Williams LR,
Varon S, Gage FH (1987) Amelioration of cholinergic
neuron atrophy and spatial memory impairment in
aged rats by nerve growth factor. Nature 329
(6134):65–68. https://doi.org/10.1038/329065a0
Fjord-Larsen L, Kusk P, Emerich DF, Thanos C, Torp M,
Bintz B, Tornoe J, Johnsen AH, Wahlberg LU (2012)
Increased encapsulated cell biodelivery of nerve
growth factor in the brain by transposon-mediated
gene transfer. Gene Ther 19(10):1010–1017. https://
doi.org/10.1038/gt.2011.178
Friden PM, Walus LR, Watson P, Doctrow SR, Kozarich
JW, Backman C, Bergman H, Hoffer B, Bloom F,
Granholm AC (1993) Blood-brain barrier penetration
and in vivo activity of an NGF conjugate. Science 259
(5093):373–377. https://doi.org/10.1126/science.
8420006
Ganger S, Schindowski K (2018) Tailoring formulations
for intranasal nose-to-brain delivery: a review on archi-
tecture, physico-chemical characteristics and
mucociliary clearance of the nasal olfactory mucosa.
Pharmaceutics 10(3):116. https://doi.org/10.3390/
pharmaceutics10030116
Gomez D, Martinez JA, Hanson LR, Frey WH 2nd, Toth
CC (2012) Intranasal treatment of neurodegenerative
diseases and stroke. Front Biosci (Schol Ed) 4:74–89.
https://doi.org/10.2741/252
Goswami R, Subramanian G, Silayeva L, Newkirk I,
Doctor D, Chawla K, Chattopadhyay S, Chandra D,
Chilukuri N, Betapudi V (2019) Gene therapy leaves a
vicious cycle. Front Oncol 9:297. https://doi.org/10.
3389/fonc.2019.00297
Granholm AC, Backman C, Bloom F, Ebendal T, Gerhardt
GA, Hoffer B, Mackerlova L, Olson L, Soderstrom S,
Walus LR et al (1994) NGF and anti-transferrin recep-
tor antibody conjugate: short and long-term effects on
survival of cholinergic neurons in intraocular septal
transplants. J Pharmacol Exp Ther 268(1):448–459
Griffin N, Faulkner S, Jobling P, Hondermarck H (2018)
Targeting neurotrophin signaling in cancer: the renais-
sance. Pharmacol Res 135:12–17. https://doi.org/10.
1016/j.phrs.2018.07.019
Grill RJ, Blesch A, Tuszynski MH (1997) Robust growth
of chronically injured spinal cord axons induced by
grafts of genetically modified NGF-secreting cells.
Exp Neurol 148(2):444–452. https://doi.org/10.1006/
exnr.1997.6704
Gu H, Song C, Long D, Mei L, Sun H (2007) Controlled
release of recombinant human nerve growth factor
(rhNGF) from poly[(lactic acid)-co-(glycolic acid)]
microspheres for the treatment of neurodegenerative
disorders. Polym Int 56(10):1272–1280. https://doi.
org/10.1002/pi.2272
Gu H, Long D, Song C, Li X (2009) Recombinant human
NGF-loaded microspheres promote survival of basal
forebrain cholinergic neurons and improve memory
impairments of spatial learning in the rat model of
Alzheimer’s disease with fimbria-fornix lesion.
S. Mitra et al.
Neurosci Lett 453(3):204–209. https://doi.org/10.
1016/j.neulet.2009.02.027
Gu G, Zhang W, Li M, Ni J, Wang P (2015) Transplanta-
tion of NSC-derived cholinergic neuron-like cells
improves cognitive function in APP/PS1 transgenic
mice. Neuroscience 291:81–92. https://doi.org/10.
1016/j.neuroscience.2015.01.073
Guo L, Davis BM, Ravindran N, Galvao J, Kapoor N,
Haamedi N, Shamsher E, Luong V, Fico E, Cordeiro
MF (2020) Topical recombinant human Nerve growth
factor (rh-NGF) is neuroprotective to retinal ganglion
cells by targeting secondary degeneration. Sci Rep 10
(1):3375. https://doi.org/10.1038/s41598-020-60427-2
Hadlock TA, Sheahan T, Cheney ML, Vacanti JP,
Sundback CA (2003) Biologic activity of nerve growth
factor slowly released from microspheres. J Reconstr
Microsurg 19(3):179–184.; Discussion 185–176.
https://doi.org/10.1055/s-2003-39831
Hagg T, Manthorpe M, Vahlsing HL, Varon S (1988)
Delayed treatment with nerve growth factor reverses
the apparent loss of cholinergic neurons after acute
brain damage. Exp Neurol 101(2):303–312. https://
doi.org/10.1016/0014-4886(88)90013-1
Hampel H, Mesulam MM, Cuello AC, Farlow MR,
Giacobini E, Grossberg GT, Khachaturian AS,
Vergallo A, Cavedo E, Snyder PJ, Khachaturian ZS
(2018) The cholinergic system in the pathophysiology
and treatment of Alzheimer’s disease. Brain 141
(7):1917–1933. https://doi.org/10.1093/brain/awy132
Hanzel CE, Iulita MF, Eyjolfsdottir H, Hjorth E,
Schultzberg M, Eriksdotter M, Cuello AC (2014)
Analysis of matrix metallo-proteases and the plasmin-
ogen system in mild cognitive impairment and
Alzheimer’s disease cerebrospinal fluid. J Alzheimers
Dis 40(3):667–678. https://doi.org/10.3233/JAD-
132282
Hao J, Ebendal T, Xu X, Wiesenfeld-Hallin Z, Eriksdotter
Jonhagen M (2000) Intracerebroventricular infusion of
nerve growth factor induces pain-like response in rats.
Neurosci Lett 286(3):208–212. https://doi.org/10.
1016/s0304-3940(00)01107-1
Hayakawa Y, Sakitani K, Konishi M, Asfaha S, Niikura R,
Tomita H, Renz BW, Tailor Y, Macchini M,
Middelhoff M, Jiang Z, Tanaka T, Dubeykovskaya
ZA, Kim W, Chen X, Urbanska AM, Nagar K,
Westphalen CB, Quante M, Lin CS, Gershon MD,
Hara A, Zhao CM, Chen D, Worthley DL, Koike K,
Wang TC (2017) Nerve growth factor promotes gastric
tumorigenesis through aberrant cholinergic signaling.
Cancer Cell 31(1):21–34. https://doi.org/10.1016/j.
ccell.2016.11.005
Hayashi Y, Lin HT, Lee CC, Tsai KJ (2020) Effects of
neural stem cell transplantation in Alzheimer’s disease
models. J Biomed Sci 27(1):29. https://doi.org/10.
1186/s12929-020-0622-x
Hefti F (1986) Nerve growth factor promotes survival of
septal cholinergic neurons after fimbrial transections. J
Neurosci 6(8):2155–2162
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Hefti F, Schneider LS (1989) Rationale for the planned
clinical trials with nerve growth factor in Alzheimer’s
disease. Psychiatr Dev 7(4):297–315
Hoffman D, Wahlberg L, Aebischer P (1990) NGF
released from a polymer matrix prevents loss of
ChAT expression in basal forebrain neurons following
a fimbria-fornix lesion. Exp Neurol 110(1):39–44.
https://doi.org/10.1016/0014-4886(90)90049-x
Hohsfield LA, Ehrlich D, Humpel C (2014) Intravenous
infusion of nerve growth factor-secreting monocytes
supports the survival of cholinergic neurons in the
nucleus basalis of Meynert in hypercholesterolemia
Brown-Norway rats. J Neurosci Res 92(3):298–306.
https://doi.org/10.1002/jnr.23309
Hsu RS, Chen PY, Fang JH, Chen YY, Chang CW, Lu YJ,
Hu SH (2019) Adaptable microporous hydrogels of
propagating NGF-gradient by injectable building
blocks for accelerated axonal outgrowth. Adv Sci
(Weinh) 6(16):1900520. https://doi.org/10.1002/advs.
201900520
Hu X, Li R, Wu Y, Li Y, Zhong X, Zhang G, Kang Y,
Liu S, Xie L, Ye J, Xiao J (2020) Thermosensitive
heparin-poloxamer hydrogel encapsulated bFGF and
NGF to treat spinal cord injury. J Cell Mol Med 24
(14):8166–8178. https://doi.org/10.1111/jcmm.15478
Isaacson LG, Saffran BN, Crutcher KA (1990) Intracere-
bral NGF infusion induces hyperinnervation of cere-
bral blood vessels. Neurobiol Aging 11(1):51–55.
https://doi.org/10.1016/0197-4580(90)90062-5
Isaacson LG, Saffran BN, Crutcher KA (1992) Nerve
growth factor-induced sprouting of mature, uninjured
sympathetic axons. J Comp Neurol 326(3):327–336.
https://doi.org/10.1002/cne.903260302
Jonhagen ME (2000) Nerve growth factor treatment in
dementia. Alzheimer Dis Assoc Disord 14(Suppl 1):
S31–S38. https://doi.org/10.1097/00002093-
200000001-00006
Kamei N, Tanaka N, Oishi Y, Hamasaki T, Nakanishi K,
Sakai N, Ochi M (2007) BDNF, NT-3, and NGF
released from transplanted neural progenitor cells pro-
mote corticospinal axon growth in organotypic
cocultures. Spine (Phila Pa 1976) 32(12):1272–1278.
https://doi.org/10.1097/BRS.0b013e318059afab
Karami A, Eyjolfsdottir H, Vijayaraghavan S, Lind G,
Almqvist P, Kadir A, Linderoth B, Andreasen N,
Blennow K, Wall A, Westman E, Ferreira D,
Kristoffersen Wiberg M, Wahlund LO, Seiger A,
Nordberg A, Wahlberg L, Darreh-Shori T, Eriksdotter
M (2015) Changes in CSF cholinergic biomarkers in
response to cell therapy with NGF in patients with
Alzheimer’s disease. Alzheimers Dement 11
(11):1316–1328. https://doi.org/10.1016/j.jalz.2014.
11.008
Killick-Cole C, Woolley M, Johnson D, Lewis O,
Moore P, Fletcher J, Skinner P, Bienemann A, Gill S
(2019) SCIDOT-35. A novel model for the optimiza-
tion of drug-device combinations for the treatment of
brain tumors. Neuro-Oncology 21(Supplement_6):
185
vi279–vi279. https://doi.org/10.1093/neuonc/noz175.
1171
Kim HJ, Lee JH, Kim SH (2010) Therapeutic effects of
human mesenchymal stem cells on traumatic brain
injury in rats: secretion of neurotrophic factors and
inhibition of apoptosis. J Neurotrauma 27
(1):131–138. https://doi.org/10.1089/neu.2008-0818
Kim KY, Suh YH, Chang KA (2020) Therapeutic effects
of human amniotic epithelial stem cells in a transgenic
mouse model of Alzheimer’s disease. Int J Mol Sci 21
(7). https://doi.org/10.3390/ijms21072658
Koliatsos VE, Nauta HJ, Clatterbuck RE, Holtzman DM,
Mobley WC, Price DL (1990) Mouse nerve growth
factor prevents degeneration of axotomized basal fore-
brain cholinergic neurons in the monkey. J Neurosci 10
(12):3801–3813
Koliatsos VE, Applegate MD, Knusel B, Junard EO,
Burton LE, Mobley WC, Hefti FF, Price DL (1991a)
Recombinant human nerve growth factor prevents ret-
rograde degeneration of axotomized basal forebrain
cholinergic neurons in the rat. Exp Neurol 112
(2):161–173. https://doi.org/10.1016/0014-4886(91)
90066-l
Koliatsos VE, Clatterbuck RE, Nauta HJ, Knusel B,
Burton LE, Hefti FF, Mobley WC, Price DL (1991b)
Human nerve growth factor prevents degeneration of
basal forebrain cholinergic neurons in primates. Ann
Neurol 30(6):831–840. https://doi.org/10.1002/ana.
410300613
Kordower JH, Winn SR, Liu YT, Mufson EJ, Sladek JR Jr,
Hammang JP, Baetge EE, Emerich DF (1994) The
aged monkey basal forebrain: rescue and sprouting of
axotomized basal forebrain neurons after grafts of
encapsulated cells secreting human nerve growth fac-
tor. Proc Natl Acad Sci U S A 91(23):10898–10902.
https://doi.org/10.1073/pnas.91.23.10898
Kurakhmaeva KB, Djindjikhashvili IA, Petrov VE,
Balabanyan VU, Voronina TA, Trofimov SS,
Kreuter J, Gelperina S, Begley D, Alyautdin RN
(2009) Brain targeting of nerve growth factor using
poly(butyl cyanoacrylate) nanoparticles. J Drug Target
17(8):564–574. https://doi.org/10.1080/
10611860903112842
Kwak YD, Brannen CL, Qu T, Kim HM, Dong X, Soba P,
Majumdar A, Kaplan A, Beyreuther K, Sugaya K
(2006) Amyloid precursor protein regulates differenti-
ation of human neural stem cells. Stem Cells Dev 15
(3):381–389. https://doi.org/10.1089/scd.2006.15.381
Lambiase A, Rama P, Bonini S, Caprioglio G, Aloe L
(1998) Topical treatment with nerve growth factor for
corneal neurotrophic ulcers. N Engl J Med 338
(17):1174–1180. https://doi.org/10.1056/
NEJM199804233381702
Lambiase A, Tirassa P, Micera A, Aloe L, Bonini S (2005)
Pharmacokinetics of conjunctivally applied nerve
growth factor in the retina and optic nerve of adult
rats. Invest Ophthalmol Vis Sci 46(10):3800–3806.
https://doi.org/10.1167/iovs.05-0301
186
Lambiase A, Pagani L, Di Fausto V, Sposato V,
Coassin M, Bonini S, Aloe L (2007a) Nerve growth
factor eye drop administrated on the ocular surface of
rodents affects the nucleus basalis and septum: bio-
chemical and structural evidence. Brain Res 1127
(1):45–51. https://doi.org/10.1016/j.brainres.2006.09.
102
Lambiase A, Coassin M, Sposato V, Micera A,
Sacchetti M, Bonini S, Aloe L (2007b) NGF topical
application in patients with corneal ulcer does not
generate circulating NGF antibodies. Pharmacol Res
56(1):65–69. https://doi.org/10.1016/j.phrs.2007.03.
007
Lambiase A, Mantelli F, Sacchetti M, Rossi S, Aloe L,
Bonini S (2011) Clinical applications of NGF in ocular
diseases. Arch Ital Biol 149(2):283–292. https://doi.
org/10.4449/aib.v149i2.1363
Lammertink BH, Bos C, Deckers R, Storm G, Moonen
CT, Escoffre JM (2015) Sonochemotherapy: from
bench to bedside. Front Pharmacol 6:138. https://doi.
org/10.3389/fphar.2015.00138
Lee AC, Yu VM, Lowe JB 3rd, Brenner MJ, Hunter DA,
Mackinnon SE, Sakiyama-Elbert SE (2003) Controlled
release of nerve growth factor enhances sciatic nerve
regeneration. Exp Neurol 184(1):295–303. https://doi.
org/10.1016/s0014-4886(03)00258-9
Lee HJ, Lee JK, Lee H, Carter JE, Chang JW, Oh W, Yang
YS, Suh JG, Lee BH, Jin HK, Bae JS (2012a) Human
umbilical cord blood-derived mesenchymal stem cells
improve neuropathology and cognitive impairment in
an Alzheimer’s disease mouse model through modula-
tion of neuroinflammation. Neurobiol Aging 33
(3):588–602. https://doi.org/10.1016/j.neurobiolaging.
2010.03.024
Lee HJ, Lim IJ, Park SW, Kim YB, Ko Y, Kim SU
(2012b) Human neural stem cells genetically modified
to express human nerve growth factor (NGF) gene
restore cognition in the mouse with ibotenic acid-
induced cognitive dysfunction. Cell Transplant 21
(11):2487–2496. https://doi.org/10.3727/
096368912X638964
LeSauteur L, Wei L, Gibbs BF, Saragovi HU (1995) Small
peptide mimics of nerve growth factor bind TrkA
receptors and affect biological responses. J Biol
Chem 270(12):6564–6569. https://doi.org/10.1074/
jbc.270.12.6564
Li XB, Liao GS, Shu YY, Tang SX (2000) Brain delivery
of biotinylated NGF bounded to an avidin-transferrin
conjugate. J Nat Toxins 9(1):73–83
Li W, Huang A, Zhong Y, Huang L, Yang J, Zhou C,
Zhou L, Zhang Y, Fu G (2020) Laminin-modified
gellan gum hydrogels loaded with the nerve growth
factor to enhance the proliferation and differentiation
of neuronal stem cells. RSC Adv 10(29):17114–17122.
https://doi.org/10.1039/D0RA01723J
Liao GS, Li XB, Zhang CY, Shu YY, Tang SX (2001)
Pharmacological actions of nerve growth factor-
transferrin conjugate on the central nervous system. J
Nat Toxins 10(4):291–297
S. Mitra et al.
Lim F, Sun AM (1980) Microencapsulated islets as
bioartificial endocrine pancreas. Science 210
(4472):908–910. https://doi.org/10.1126/science.
6776628
Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B,
Masellis M, Herrmann N, Heyn C, Aubert I,
Boutet A, Smith GS, Hynynen K, Black SE (2018)
Blood-brain barrier opening in Alzheimer’s disease
using MR-guided focused ultrasound. Nat Commun 9
(1):2336. https://doi.org/10.1038/s41467-018-04529-6
Liu H, Wen W, Hu M, Bi W, Chen L, Liu S, Chen P, Tan
X (2013) Chitosan conduits combined with nerve
growth factor microspheres repair facial nerve defects.
Neural Regen Res 8(33):3139–3147. https://doi.org/
10.3969/j.issn.1673-5374.2013.33.008
Liu X, Li W, Fu X, Xu Y (2017) The immunogenicity and
immune tolerance of pluripotent stem cell derivatives.
Front Immunol 8:645. https://doi.org/10.3389/fimmu.
2017.00645
Liu PP, Xie Y, Meng XY, Kang JS (2019) History and
progress of hypotheses and clinical trials for
Alzheimer’s disease. Signal Transduct Target Ther
4:29. https://doi.org/10.1038/s41392-019-0063-8
Longo FM, Massa SM (2013) Small-molecule modulation
of neurotrophin receptors: a strategy for the treatment
of neurological disease. Nat Rev Drug Discov 12
(7):507–525. https://doi.org/10.1038/nrd4024
Longo FM, Manthorpe M, Xie YM, Varon S (1997) Syn-
thetic NGF peptide derivatives prevent neuronal death
via a p75 receptor-dependent mechanism. J Neurosci
Res 48(1):1–17. https://doi.org/10.1002/(sici)1097-
4547(19970401)48:1<1::aid-jnr1>3.0.co;2-k
Losurdo M, Pedrazzoli M, D’Agostino C, Elia CA,
Massenzio F, Lonati E, Mauri M, Rizzi L, Molteni L,
Bresciani E, Dander E, D’Amico G, Bulbarelli A,
Torsello A, Matteoli M, Buffelli M, Coco S (2020)
Intranasal delivery of mesenchymal stem cell-derived
extracellular vesicles exerts immunomodulatory and
neuroprotective effects in a 3xTg model of
Alzheimer’s disease. Stem Cells Transl Med 9
(9):1068–1084. https://doi.org/10.1002/sctm.19-0327
Machado A, Ferreira D, Grothe MJ, Eyjolfsdottir H,
Almqvist PM, Cavallin L, Lind G, Linderoth B,
Seiger A, Teipel S, Wahlberg LU, Wahlund LO,
Westman E, Eriksdotter M, Alzheimer’s Disease Neu-
roimaging Initiative (2020) The cholinergic system in
subtypes of Alzheimer’s disease: an in vivo longitudi-
nal MRI study. Alzheimers Res Ther 12(1):51. https://
doi.org/10.1186/s13195-020-00620-7
Mahoney MJ, Saltzman WM (1999) Millimeter-scale
positioning of a nerve-growth-factor source and
biological activity in the brain. Proc Natl Acad Sci U
S A 96(8):4536–4539. https://doi.org/10.1073/pnas.
96.8.4536
Mandal A, Pal D, Agrahari V, Trinh HM, Joseph M, Mitra
AK (2018) Ocular delivery of proteins and peptides:
challenges and novel formulation approaches. Adv
Drug Deliv Rev 126:67–95. https://doi.org/10.1016/j.
addr.2018.01.008
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Marcus M, Skaat H, Alon N, Margel S, Shefi O (2015)
NGF-conjugated iron oxide nanoparticles promote dif-
ferentiation and outgrowth of PC12 cells. Nanoscale 7
(3):1058–1066. https://doi.org/10.1039/c4nr05193a
Martinez-Serrano A, Bjorklund A (1998) Ex vivo nerve
growth factor gene transfer to the basal forebrain in
presymptomatic middle-aged rats prevents the devel-
opment of cholinergic neuron atrophy and cognitive
impairment during aging. Proc Natl Acad Sci U S A 95
(4):1858–1863. https://doi.org/10.1073/pnas.95.4.
1858
Martinez-Serrano A, Fischer W, Soderstrom S, Ebendal T,
Bjorklund A (1996) Long-term functional recovery
from age-induced spatial memory impairments by
nerve growth factor gene transfer to the rat basal fore-
brain. Proc Natl Acad Sci U S A 93(13):6355–6360.
https://doi.org/10.1073/pnas.93.13.6355
McGinley LM, Kashlan ON, Bruno ES, Chen KS, Hayes
JM, Kashlan SR, Raykin J, Johe K, Murphy GG,
Feldman EL (2018) Human neural stem cell transplan-
tation improves cognition in a murine model of
Alzheimer’s disease. Sci Rep 8(1):14776. https://doi.
org/10.1038/s41598-018-33017-6
Menei P, Pean JM, Nerriere-Daguin V, Jollivet C,
Brachet P, Benoit JP (2000) Intracerebral implantation
of NGF-releasing biodegradable microspheres protects
striatum against excitotoxic damage. Exp Neurol 161
(1):259–272. https://doi.org/10.1006/exnr.1999.7253
Minde J, Toolanen G, Andersson T, Nennesmo I, Remahl
IN, Svensson O, Solders G (2004) Familial insensitiv-
ity to pain (HSAN V) and a mutation in the NGFB
gene. A neurophysiological and pathological study.
Muscle Nerve 30(6):752–760. https://doi.org/10.
1002/mus.20172
Mitra S, Behbahani H, Eriksdotter M (2019) Innovative
therapy for Alzheimer’s disease-with focus on
biodelivery of NGF. Front Neurosci 13:38. https://
doi.org/10.3389/fnins.2019.00038
Montero CN, Hefti F (1988) Rescue of lesioned septal
cholinergic neurons by nerve growth factor: specificity
and requirement for chronic treatment. J Neurosci 8
(8):2986–2999
Montero CN, Hefti F (1989) Intraventricular nerve growth
factor administration prevents lesion-induced loss of
septal cholinergic neurons in aging rats. Neurobiol
Aging 10(6):739–743. https://doi.org/10.1016/0197-
4580(89)90011-0
Mufson EJ, Conner JM, Kordower JH (1995) Nerve
growth factor in Alzheimer’s disease: defective retro-
grade transport to nucleus basalis. Neuroreport 6
(7):1063–1066. https://doi.org/10.1097/00001756-
199505090-00028
Nagahara AH, Bernot T, Moseanko R, Brignolo L,
Blesch A, Conner JM, Ramirez A, Gasmi M,
Tuszynski MH (2009) Long-term reversal of choliner-
gic neuronal decline in aged non-human primates by
lentiviral NGF gene delivery. Exp Neurol 215
(1):153–159. https://doi.org/10.1016/j.expneurol.
2008.10.004
187
Nguyen TV, Shen L, Vander Griend L, Quach LN,
Belichenko NP, Saw N, Yang T, Shamloo M, Wyss-
Coray T, Massa SM, Longo FM (2014) Small mole-
cule p75NTR ligands reduce pathological phosphory-
lation and misfolding of tau, inflammatory changes,
cholinergic degeneration, and cognitive deficits in
AbetaPP(L/S) transgenic mice. J Alzheimers Dis 42
(2):459–483. https://doi.org/10.3233/JAD-140036
Oh B, Swaminathan V, Malkovskiy A, Santhanam S,
McConnell K, George PM (2020) Single-cell encapsu-
lation via click-chemistry alters production of para-
crine factors from neural progenitor cells. Adv Sci
(Weinh) 7(8):1902573. https://doi.org/10.1002/advs.
201902573
Olson L (1993) NGF and the treatment of Alzheimer’s
disease. Exp Neurol 124(1):5–15. https://doi.org/10.
1006/exnr.1993.1167
Olson L, Nordberg A, von Holst H, Backman L,
Ebendal T, Alafuzoff I, Amberla K, Hartvig P,
Herlitz A, Lilja A et al (1992) Nerve growth factor
affects 11C-nicotine binding, blood flow, EEG, and
verbal episodic memory in an Alzheimer patient (case
report). J Neural Transm Park Dis Dement Sect 4
(1):79–95. https://doi.org/10.1007/BF02257624
Orive G, Santos E, Poncelet D, Hernandez RM, Pedraz JL,
Wahlberg LU, De Vos P, Emerich D (2015) Cell
encapsulation: technical and clinical advances. Trends
Pharmacol Sci 36(8):537–546. https://doi.org/10.1016/
j.tips.2015.05.003
Orive G, Santos-Vizcaino E, Pedraz JL, Hernandez RM,
Vela Ramirez JE, Dolatshahi-Pirouz A,
Khademhosseini A, Peppas NA, Emerich DF (2019)
3D cell-laden polymers to release bioactive products in
the eye. Prog Retin Eye Res 68:67–82. https://doi.org/
10.1016/j.preteyeres.2018.10.002
Pakzaban P, Geller AI, Isacson O (1994) Effect of exoge-
nous nerve growth factor on neurotoxicity of and neu-
ronal gene delivery by a herpes simplex amplicon
vector in the rat brain. Hum Gene Ther 5(8):987–995.
https://doi.org/10.1089/hum.1994.5.8-987
Paradisi M, Alviano F, Pirondi S, Lanzoni G,
Fernandez M, Lizzo G, Giardino L, Giuliani A,
Costa R, Marchionni C, Bonsi L, Calza L (2014)
Human mesenchymal stem cells produce bioactive
neurotrophic factors: source, individual variability
and differentiation issues. Int J Immunopathol
Pharmacol 27(3):391–402. https://doi.org/10.1177/
039463201402700309
Pardridge WM (2019) Blood-brain barrier and delivery of
protein and gene therapeutics to brain. Front Aging
Neurosci 11:373. https://doi.org/10.3389/fnagi.2019.
00373
Park D, Yang YH, Bae DK, Lee SH, Yang G, Kyung J,
Kim D, Choi EK, Lee SW, Kim GH, Hong JT, Choi
KC, Lee HJ, Kim SU, Kim YB (2013) Improvement of
cognitive function and physical activity of aging mice
by human neural stem cells over-expressing choline
acetyltransferase. Neurobiol Aging 34
188
(11):2639–2646. https://doi.org/10.1016/j.
neurobiolaging.2013.04.026
Pentz R, Iulita MF, Ducatenzeiler A, Bennett DA, Cuello
AC (2020) The human brain NGF metabolic pathway
is impaired in the pre-clinical and clinical continuum of
Alzheimers disease. Mol Psychiatry. https://doi.org/10.
1038/s41380-020-0797-2
Piotrowicz A, Shoichet MS (2006) Nerve guidance
channels as drug delivery vehicles. Biomaterials 27
(9):2018–2027. https://doi.org/10.1016/j.biomaterials.
2005.09.042
Polak JM, Mantalaris S (2008) Stem cells bioprocessing:
an important milestone to move regenerative medicine
research into the clinical arena. Pediatr Res 63
(5):461–466. https://doi.org/10.1203/10.1203/PDR.
0b013e31816a8c1c
Powell EM, Sobarzo MR, Saltzman WM (1990) Con-
trolled release of nerve growth factor from a polymeric
implant. Brain Res 515(1–2):309–311. https://doi.org/
10.1016/0006-8993(90)90612-f
Pramanik S, Sulistio YA, Heese K (2017) Neurotrophin
signaling and stem cells-implications for neurodegen-
erative diseases and stem cell therapy. Mol Neurobiol
54(9):7401–7459. https://doi.org/10.1007/s12035-
016-0214-7
Qu M, Jiang X, Zhou X, Wang C, Wu Q, Ren L, Zhu J,
Zhu S, Tebon P, Sun W, Khademhosseini A (2020)
Stimuli-responsive delivery of growth factors for tissue
engineering. Adv Healthc Mater 9(7):e1901714.
https://doi.org/10.1002/adhm.201901714
Rafii MS, Baumann TL, Bakay RA, Ostrove JM, Siffert J,
Fleisher AS, Herzog CD, Barba D, Pay M, Salmon DP,
Chu Y, Kordower JH, Bishop K, Keator D, Potkin S,
Bartus RT (2014) A phase1 study of stereotactic gene
delivery of AAV2-NGF for Alzheimer’s disease.
Alzheimers Dement 10(5):571–581. https://doi.org/
10.1016/j.jalz.2013.09.004
Rafii MS, Tuszynski MH, Thomas RG, Barba D, Brewer
JB, Rissman RA, Siffert J, Aisen PS, Team ANS
(2018) Adeno-associated viral vector (serotype 2)-
nerve growth factor for patients with Alzheimer dis-
ease: a randomized clinical trial. JAMA Neurol 75
(7):834–841. https://doi.org/10.1001/jamaneurol.
2018.0233
Razavi S, Seyedebrahimi R, Jahromi M (2019)
Biodelivery of nerve growth factor and gold
nanoparticles encapsulated in chitosan nanoparticles
for schwann-like cells differentiation of human
adipose-derived stem cells. Biochem Biophys Res
Commun 513(3):681–687. https://doi.org/10.1016/j.
bbrc.2019.03.189
Rezai AR, Ranjan M, D’Haese PF, Haut MW, Carpenter J,
Najib U, Mehta RI, Chazen JL, Zibly Z, Yates JR,
Hodder SL, Kaplitt M (2020) Noninvasive hippocam-
pal blood-brain barrier opening in Alzheimer’s disease
with focused ultrasound. Proc Natl Acad Sci U S A 117
(17):9180–9182. https://doi.org/10.1073/pnas.
2002571117
S. Mitra et al.
Rocco ML, Soligo M, Manni L, Aloe L (2018) Nerve
growth factor: early studies and recent clinical trials.
Curr Neuropharmacol 16(10):1455–1465. https://doi.
org/10.2174/1570159X16666180412092859
Rodrigues BDS, Kanekiyo T, Singh J (2020) Nerve
growth factor gene delivery across the blood-brain
barrier to reduce beta amyloid accumulation in AD
mice. Mol Pharm 17(6):2054–2063. https://doi.org/
10.1021/acs.molpharmaceut.0c00218
Ruozi B, Belletti D, Bondioli L, De Vita A, Forni F,
Vandelli MA, Tosi G (2012) Neurotrophic factors
and neurodegenerative diseases: a delivery issue. Int
Rev Neurobiol 102:207–247. https://doi.org/10.1016/
B978-0-12-386986-9.00009-0
Saffran BN, Woo JE, Mobley WC, Crutcher KA (1989)
Intraventricular NGF infusion in the mature rat brain
enhances sympathetic innervation of cerebrovascular
targets but fails to elicit sympathetic ingrowth. Brain
Res 492(1-2):245–254. https://doi.org/10.1016/0006-
8993(89)90907-4
Sakiyama-Elbert SE, Hubbell JA (2000) Controlled
release of nerve growth factor from a heparin-
containing fibrin-based cell ingrowth matrix. J Control
Release 69(1):149–158. https://doi.org/10.1016/
s0168-3659(00)00296-0
Sakiyama-Elbert SE, Panitch A, Hubbell JA (2001) Devel-
opment of growth factor fusion proteins for cell-
triggered drug delivery. FASEB J 15(7):1300–1302.
https://doi.org/10.1096/fj.00-0564fje
Saltzman WM, Mak MW, Mahoney MJ, Duenas ET,
Cleland JL (1999) Intracranial delivery of recombinant
nerve growth factor: release kinetics and protein distri-
bution for three delivery systems. Pharm Res 16
(2):232–240. https://doi.org/10.1023/
a:1018824324275
Samal J, Hoban DB, Naughton C, Concannon R, Dowd E,
Pandit A (2015) Fibrin-based microsphere reservoirs
for delivery of neurotrophic factors to the brain.
Nanomedicine (Lond) 10(5):765–783. https://doi.org/
10.2217/nnm.14.221
Satizabal C, Beiser AS, Seshadri S (2016) Incidence of
dementia over three decades in the Framingham heart
study. N Engl J Med 375(1):93–94. https://doi.org/10.
1056/NEJMc1604823
Scheltens P, Blennow K, Breteler MM, de Strooper B,
Frisoni GB, Salloway S, Van der Flier WM (2016)
Alzheimer’s disease. Lancet 388(10043):505–517.
https://doi.org/10.1016/S0140-6736(15)01124-1
Schlachetzki JC, Pizzo DP, Morrissette DA, Winkler J
(2014) Intracerebroventricular administration of nerve
growth factor induces gliogenesis in sensory ganglia,
dorsal root, and within the dorsal root entry zone.
Biomed Res Int 2014:704259. https://doi.org/10.
1155/2014/704259
Schneider L (2020) A resurrection of aducanumab for
Alzheimer’s disease. Lancet Neurol 19(2):111–112.
https://doi.org/10.1016/S1474-4422(19)30480-6
Secnik J, Schwertner E, Alvarsson M, Hammar N,
Fastbom J, Winblad B, Garcia-Ptacek S, Religa D,
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Eriksdotter M (2020) Cholinesterase inhibitors in
patients with diabetes mellitus and dementia: an
open-cohort study of ~23 000 patients from the Swed-
ish Dementia registry. BMJ Open Diabetes Res Care 8
(1). https://doi.org/10.1136/bmjdrc-2019-000833
Seiger A, Nordberg A, von Holst H, Backman L,
Ebendal T, Alafuzoff I, Amberla K, Hartvig P,
Herlitz A, Lilja A et al (1993) Intracranial infusion of
purified nerve growth factor to an Alzheimer patient:
the first attempt of a possible future treatment strategy.
Behav Brain Res 57(2):255–261. https://doi.org/10.
1016/0166-4328(93)90141-c
Simone MD, De Santis S, Vigneti E, Papa G, Amadori S,
Aloe L (1999) Nerve growth factor: a survey of activity
on immune and hematopoietic cells. Hematol Oncol 17
(1):1–10. https://doi.org/10.1002/(sici)1099-1069
(199903)17:1<1::aid-hon635>3.0.co;2-l
Singh AP, Biswas A, Shukla A, Maiti P (2019) Targeted
therapy in chronic diseases using nanomaterial-based
drug delivery vehicles. Signal Transduct Target Ther
4:33. https://doi.org/10.1038/s41392-019-0068-3
Skeldal S, Sykes AM, Glerup S, Matusica D, Palstra N,
Autio H, Boskovic Z, Madsen P, Castren E, Nykjaer A,
Coulson EJ (2012) Mapping of the interaction site
between sortilin and the p75 neurotrophin receptor
reveals a regulatory role for the sortilin intracellular
domain in p75 neurotrophin receptor shedding and
apoptosis. J Biol Chem 287(52):43798–43809.
https://doi.org/10.1074/jbc.M112.374710
Sochocka M, Zwolinska K, Leszek J (2017) The infectious
etiology of Alzheimer’s disease. Curr Neuropharmacol
15(7):996–1009. https://doi.org/10.2174/
1570159X15666170313122937
Son AI, Opfermann JD, McCue C, Ziobro J, Abrahams JH
3rd, Jones K, Morton PD, Ishii S, Oluigbo C,
Krieger A, Liu JS, Hashimoto-Torii K, Torii M
(2017) An implantable micro-caged device for direct
local delivery of agents. Sci Rep 7(1):17624. https://
doi.org/10.1038/s41598-017-17912-y
Song B, Song J, Zhang S, Anderson MA, Ao Y, Yang CY,
Deming TJ, Sofroniew MV (2012) Sustained local
delivery of bioactive nerve growth factor in the central
nervous system via tunable diblock copolypeptide
hydrogel depots. Biomaterials 33(35):9105–9116.
https://doi.org/10.1016/j.biomaterials.2012.08.060
Song Z, Wang Z, Shen J, Xu S, Hu Z (2017) Nerve growth
factor delivery by ultrasound-mediated nanobubble
destruction as a treatment for acute spinal cord injury
in rats. Int J Nanomed 12:1717–1729. https://doi.org/
10.2147/IJN.S128848
Spiegel K, Agrafiotis D, Caprathe B, Davis RE, Dickerson
MR, Fergus JH, Hepburn TW, Marks JS, Van Dorf M,
Wieland DM et al (1995) PD 90780, a non peptide
inhibitor of nerve growth factor’s binding to the P75
NGF receptor. Biochem Biophys Res Commun 217
(2):488–494. https://doi.org/10.1006/bbrc.1995.2802
Springer JE, Collier TJ, Notter MF, Loy R, Sladek JR Jr
(1988a) Central nervous system grafts of nerve growth
factor-rich tissue as an alternative source of trophic
189
support for axotomized cholinergic neurons. Prog
Brain Res 78:401–407. https://doi.org/10.1016/s0079-
6123(08)60311-8
Springer JE, Collier TJ, Sladek JR Jr, Loy R (1988b)
Transplantation of male mouse submaxillary gland
increases survival of axotomized basal forebrain
neurons. J Neurosci Res 19(3):291–296. https://doi.
org/10.1002/jnr.490190303
Sugaya K (2008) Mechanism of glial differentiation of
neural progenitor cells by amyloid precursor protein.
Neurodegener Dis 5(3-4):170–172. https://doi.org/10.
1159/000113693
Sydow O, Hansson P, Young D, Meyerson B, Backlund
EO, Ebendal T, Farnebo LO, Freedman R,
Hamberger B, Hoffer B, Seiger A, Stromberq I,
Olson L (1995) Long-term beneficial effects of adrenal
medullary autografts supported by nerve growth factor
in Parkinson’s disease. Eur J Neurol 2(5):445–454.
https://doi.org/10.1111/j.1468-1331.1995.tb00154.x
Thomas CE, Ehrhardt A, Kay MA (2003) Progress and
problems with the use of viral vectors for gene therapy.
Nat Rev Genet 4(5):346–358. https://doi.org/10.1038/
nrg1066
Tirassa P, Triaca V, Amendola T, Fiore M, Aloe L (2003)
EGF and NGF injected into the brain of old mice
enhance BDNF and ChAT in proliferating
subventricular zone. J Neurosci Res 72(5):557–564.
https://doi.org/10.1002/jnr.10614
Tirassa P, Rosso P, Iannitelli A (2018) Ocular nerve
growth factor (NGF) and NGF eye drop application
as paradigms to investigate NGF neuroprotective and
reparative actions. Methods Mol Biol 1727:19–38.
https://doi.org/10.1007/978-1-4939-7571-6_2
Tresco PA, Winn SR, Aebischer P (1992) Polymer
encapsulated neurotransmitter secreting cells. Potential
treatment for Parkinson’s disease. ASAIO J 38
(1):17–23
Tuszynski MH (2000) Intraparenchymal NGF infusions
rescue degenerating cholinergic neurons. Cell Trans-
plant 9(5):629–636. https://doi.org/10.1177/
096368970000900508
Tuszynski MH, Gage FH (1995) Bridging grafts and tran-
sient nerve growth factor infusions promote long-term
central nervous system neuronal rescue and partial
functional recovery. Proc Natl Acad Sci U S A 92
(10):4621–4625. https://doi.org/10.1073/pnas.92.10.
4621
Tuszynski MH, U HS, Amaral DG, Gage FH (1990) Nerve
growth factor infusion in the primate brain reduces
lesion-induced cholinergic neuronal degeneration. J
Neurosci 10(11):3604–3614
Tuszynski MH, Roberts J, Senut MC, U HS, Gage FH
(1996) Gene therapy in the adult primate brain:
intraparenchymal grafts of cells genetically modified
to produce nerve growth factor prevent cholinergic
neuronal degeneration. Gene Ther 3(4):305–314
Tuszynski MH, Thal L, Pay M, Salmon DP, U HS,
Bakay R, Patel P, Blesch A, Vahlsing HL, Ho G,
Tong G, Potkin SG, Fallon J, Hansen L, Mufson EJ,
190
Kordower JH, Gall C, Conner J (2005) A phase 1 clini-
cal trial of nerve growth factor gene therapy for
Alzheimer disease. Nat Med 11(5):551–555. https://
doi.org/10.1038/nm1239
Tuszynski MH, Yang JH, Barba D, U HS, Bakay RA, Pay
MM, Masliah E, Conner JM, Kobalka P, Roy S,
Nagahara AH (2015) Nerve growth factor gene ther-
apy: activation of neuronal responses in Alzheimer
disease. JAMA Neurol 72(10):1139–1147. https://doi.
org/10.1001/jamaneurol.2015.1807
Varde NK, Pack DW (2004) Microspheres for controlled
release drug delivery. Expert Opin Biol Ther 4
(1):35–51. https://doi.org/10.1517/14712598.4.1.35
Venero JL, Hefti F, Knusel B (1996) Trophic effect of
exogenous nerve growth factor on rat striatal choliner-
gic neurons: comparison between intraparenchymal
and intraventricular administration. Mol Pharmacol
49(2):303–310
Wahlberg LU, Lind G, Almqvist PM, Kusk P, Tornoe J,
Juliusson B, Soderman M, Sellden E, Seiger A,
Eriksdotter-Jonhagen M, Linderoth B (2012) Targeted
delivery of nerve growth factor via encapsulated cell
biodelivery in Alzheimer disease: a technology plat-
form for restorative neurosurgery. J Neurosurg 117
(2):340–347. https://doi.org/10.3171/2012.2.
JNS11714
Wang Z, Wang Z, Lu WW, Zhen W, Yang D, Peng S
(2017) Novel biomaterial strategies for controlled
growth factor delivery for biomedical applications.
NPG Asia Mater 9(10):e435–e435. https://doi.org/10.
1038/am.2017.171
Wang SM, Lee CU, Lim HK (2019) Stem cell therapies for
Alzheimer’s disease: is it time? Curr Opin Psychiatry
32(2):105–116. https://doi.org/10.1097/YCO.
0000000000000478
Williams LR (1991) Hypophagia is induced by intracer-
ebroventricular administration of nerve growth factor.
Exp Neurol 113(1):31–37. https://doi.org/10.1016/
0014-4886(91)90143-z
Williams LR, Varon S, Peterson GM, Wictorin K,
Fischer W, Bjorklund A, Gage FH (1986) Continuous
infusion of nerve growth factor prevents basal fore-
brain neuronal death after fimbria fornix transection.
Proc Natl Acad Sci U S A 83(23):9231–9235. https://
doi.org/10.1073/pnas.83.23.9231
Williams LR, Vahlsing HL, Lindamood T, Varon S, Gage
FH, Manthorpe M (1987) A small-gauge cannula
device for continuous infusion of exogenous agents
into the brain. Exp Neurol 95(3):743–754. https://doi.
org/10.1016/0014-4886(87)90313-x
Winkler J, Ramirez GA, Kuhn HG, Peterson DA,
Day-Lollini PA, Stewart GR, Tuszynski MH, Gage
FH, Thal LJ (1997) Reversible Schwann cell hyperpla-
sia and sprouting of sensory and sympathetic neurites
after intraventricular administration of nerve growth
factor. Ann Neurol 41(1):82–93. https://doi.org/10.
1002/ana.410410114
Wu K, Meyer EM, Bennett JA, Meyers CA, Hughes JA,
King MA (2005) AAV2/5-mediated NGF gene
S. Mitra et al.
delivery protects septal cholinergic neurons following
axotomy. Brain Res 1061(2):107–113. https://doi.org/
10.1016/j.brainres.2005.08.056
Xhima K, Markham-Coultes K, Nedev H, Heinen S,
Saragovi HU, Hynynen K, Aubert I (2020) Focused
ultrasound delivery of a selective TrkA agonist rescues
cholinergic function in a mouse model of Alzheimer’s
disease. Sci Adv 6(4):eaax6646. https://doi.org/10.
1126/sciadv.aax6646
Xia B, Lv Y (2018) Dual-delivery of VEGF and NGF by
emulsion electrospun nanofibrous scaffold for periph-
eral nerve regeneration. Mater Sci Eng C Mater Biol
Appl 82:253–264. https://doi.org/10.1016/j.msec.
2017.08.030
Xie Y, Ye L, Zhang X, Cui W, Lou J, Nagai T, Hou X
(2005) Transport of nerve growth factor encapsulated
into liposomes across the blood-brain barrier: in vitro
and in vivo studies. J Control Release 105
(1–2):106–119. https://doi.org/10.1016/j.jconrel.2005.
03.005
Xie Y, Meeker RB, Massa SM, Longo FM (2019) Modu-
lation of the p75 neurotrophin receptor suppresses
age-related basal forebrain cholinergic neuron degen-
eration. Sci Rep 9(1):5273. https://doi.org/10.1038/
s41598-019-41654-8
Xu X, Yu H, Gao S, Ma HQ, Leong KW, Wang S (2002)
Polyphosphoester microspheres for sustained release
of biologically active nerve growth factor.
Biomaterials 23(17):3765–3772. https://doi.org/10.
1016/s0142-9612(02)00116-3
Xu D, Wu D, Qin M, Nih LR, Liu C, Cao Z, Ren J,
Chen X, He Z, Yu W, Guan J, Duan S, Liu F, Liu X,
Li J, Harley D, Xu B, Hou L, Chen ISY, Wen J,
Chen W, Pourtaheri S, Lu Y (2019) Efficient delivery
of nerve growth factors to the central nervous system
for neural regeneration. Adv Mater 31(33):e1900727.
https://doi.org/10.1002/adma.201900727
Yan Q, Matheson C, Sun J, Radeke MJ, Feinstein SC,
Miller JA (1994) Distribution of intracerebral
ventricularly administered neurotrophins in rat brain
and its correlation with trk receptor expression. Exp
Neurol 127(1):23–36. https://doi.org/10.1006/exnr.
1994.1076
Yang T, Liu H, Tran KC, Leng A, Massa SM, Longo FM
(2020a) Small-molecule modulation of the p75
neurotrophin receptor inhibits a wide range of tau
molecular pathologies and their sequelae in P301S
tauopathy mice. Acta Neuropathol Commun 8(1):156.
https://doi.org/10.1186/s40478-020-01034-0
Yang J, Wu S, Hou L, Zhu D, Yin S, Yang G, Wang Y
(2020b) Therapeutic effects of simultaneous delivery
of nerve growth factor mRNA and protein via
exosomes on cerebral ischemia. Mol Ther Nucleic
Acids 21:512–522. https://doi.org/10.1016/j.omtn.
2020.06.013
Yi X, Manickam DS, Brynskikh A, Kabanov AV (2014)
Agile delivery of protein therapeutics to CNS. J Con-
trol Release 190:637–663. https://doi.org/10.1016/j.
jconrel.2014.06.017
11 A Review of Techniques for Biodelivery of Nerve Growth Factor (NGF) to the. . .
Zassler B, Humpel C (2006) Transplantation of NGF
secreting primary monocytes counteracts NMDA-
induced cell death of rat cholinergic neurons in vivo.
Exp Neurol 198(2):391–400. https://doi.org/10.1016/j.
expneurol.2005.12.009
Zhang W, Wang PJ, Sha HY, Ni J, Li MH, Gu GJ (2014)
Neural stem cell transplants improve cognitive func-
tion without altering amyloid pathology in an APP/PS1
double transgenic model of Alzheimer’s disease. Mol
Neurobiol 50(2):423–437. https://doi.org/10.1007/
s12035-014-8640-x
Zhang W, Zhou G, Gao Y, Zhou Y, Liu J, Zhang L,
Long A, Zhang L, Tang P (2017) A sequential delivery
system employing the synergism of EPO and NGF
promotes sciatic nerve repair. Colloids Surf B
Biointerfaces 159:327–336. https://doi.org/10.1016/j.
colsurfb.2017.07.088
Zhao YZ, Jiang X, Xiao J, Lin Q, Yu WZ, Tian FR, Mao
KL, Yang W, Wong HL, Lu CT (2016) Using NGF
heparin-poloxamer thermosensitive hydrogels to
enhance the nerve regeneration for spinal cord injury.
Acta Biomater 29:71–80. https://doi.org/10.1016/j.
actbio.2015.10.014
191
Zhu CW, Livote EE, Scarmeas N, Albert M, Brandt J,
Blacker D, Sano M, Stern Y (2013) Long-term
associations between cholinesterase inhibitors and
memantine use and health outcomes among patients
with Alzheimer’s disease. Alzheimers Dement 9
(6):733–740. https://doi.org/10.1016/j.jalz.2012.09.
015
Zhu SP, Wang ZG, Zhao YZ, Wu J, Shi HX, Ye LB, Wu
FZ, Cheng Y, Zhang HY, He S, Wei X, Fu XB, Li XK,
Xu HZ, Xiao J (2016) Gelatin nanostructured lipid
carriers incorporating nerve growth factor inhibit endo-
plasmic reticulum stress-induced apoptosis and
improve recovery in spinal cord injury. Mol Neurobiol
53(7):4375–4386. https://doi.org/10.1007/s12035-
015-9372-2
Zilony-Hanin N, Rosenberg M, Richman M, Yehuda R,
Schori H, Motiei M, Rahimipour S, Groisman A,
Segal E, Shefi O (2019) Neuroprotective effect of
nerve growth factor loaded in porous silicon
nanostructures in an Alzheimer’s disease model and
potential delivery to the brain. Small 15(45):e1904203.
https://doi.org/10.1002/smll.201904203
 NGF Released from Blood Cells or
Collagen Hydrogels as a Therapeutic 12
Target in Alzheimer’s Disease?

Christian Humpel

Abstract Platelets are small anuclear cells and become

Alzheimer’s disease (AD) is a severe neurode-
generative disorder of the brain characterized
by extracellular beta-amyloid plaques,
intraneuronal tau inclusions, vascular
impairment, inflammation, neurodegeneration,
and memory loss. Acetylcholine is the most
important neurotransmitter for memory, and
cholinergic neurons selectively degenerate in
AD, and a loss of acetylcholine directly
correlates with cognitive decline. Nerve
growth factor (NGF) is the most potent growth
factor to support the survival of these cholin-
ergic neurons. Thus, researchers are interested
to deliver NGF directly into the brain to the
cholinergic neurons. As the brain is isolated by
the blood-brain barrier, the large protein NGF
cannot easily pass into the brain, and periph-
eral administration of NGF also causes severe
side effects. Blood cells may represent a potent
therapeutic strategy to deliver NGF into the
brain. Monocytes can be isolated and loaded
with NGF and may transmigrate into the brain.
As monocytes are precursors of microglia,
they may differentiate and release NGF but
also phagocyte and eliminate toxic plaques.
C. Humpel (*)
Laboratory for Psychiatry and Experimental Alzheimer’s
Research, Medical University Innsbruck, Innsbruck,
Austria
e-mail: christian.humpel@i-med.ac.at; http://www2.i-
med.ac.at/psychlab/
rapidly activated during vascular lesions, and
they may migrate to lesion sites and repair
blood vessels and also eliminate toxic beta-
amyloid depositions in vessels. In order to
guarantee a stable and slow release, the use
of biomaterials is of interest, especially colla-
gen hydrogels that may be useful to protect
these transmigrating blood cells. In this
review, I summarize advantages and
challenges of using transmigrating cells to
deliver NGF directly into the brain.
Keywords
NGF · Brain delivery · Cell migration ·
Biomaterial · Collagen · Therapeutic
12.1 Introduction
12.1.1 Pathology of Alzheimer’s
Disease
Alzheimer’s disease (AD) is a severe neurodegen-
erative disorder of the brain, characterized by
extracellular beta-amyloid (Aβ) depositions
(toxic beta-amyloid (42) plaques) and
intraneuronal hyperphosphorylated tau inclusions
(neurofibrillary tangles). These two pathologies
are accompanied by inflammatory processes
including reactive astroglia and microglia, break-
down of the blood-brain barrier (BBB), and

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 193
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_12
194
vascular dysfunctions and deposition of beta-
amyloid (40) in vessels (called cerebral amyloid
angiopathy, CAA). A major hallmark in AD is
also the cell death of neurons synthesizing and
releasing acetylcholine. The major neuronal
populations containing cholinergic neurons are
located in the septum and in the basal nucleus of
Meynert (nBM) and innervate the hippocampus
and whole cortex, respectively. In fact, a decline
of cholinergic neurons directly correlates with
cognitive decline and loss of memory. The
reasons for AD progression are not known yet.
Clinically, acetylcholinesterase inhibitors and the
NMDA antagonist memantine are so far the only
therapeutic choice.
12.1.2 NGF and the Cholinergic
Neurons in Alzheimer’s Disease
Growth factors have been shown to support the
survival of different subtypes of neurons in the
central and peripheral nervous system. The sur-
vival of cholinergic neurons is directly dependent
on the classical nerve growth factor (NGF) (Levi-
Montalcini et al. 1995). It is well known that in
AD, the NGF system is markedly impaired
playing a role in degeneration of neurons and
considered as a therapeutic molecule (Levi-
Montalcini et al. 1995). However, so far it is not
known if cell death of cholinergic neurons is a
primary event in AD or is caused by the dramatic
deposition of Aβ plaques in the cortex and hippo-
campus. In our lab, we have extensively studied
the cholinergic neurons of the nBM as
organotypic brain slices (Weis et al. 2001;
Humpel and Weis 2002). We and others showed
that NGF (>0.1 ng/ml) is required to support the
survival of the cholinergic neurons and such nBM
slices can be cultured for several weeks. We fur-
ther showed that the cholinergic neurons are
dependent on NGF during the whole culture
period and NGF can only be withdrawn for
2 weeks before starting again NGF delivery in a
continuous cycle (Weis et al. 2001; Humpel and
Weis 2002). Thus, it is well established that NGF
may be a useful neuroprotective therapeutic but
must be applied (a) early before the degeneration,
C. Humpel
(b) selectively to the target neurons, and
(c) during the whole lifespan.
12.1.3 Delivery of Therapeutics into
the Brain
In order to deliver protective proteins directly into
the tissue, several methods have been used: direct
infusion of growth factors; gene transfer of DNA
plasmids using, e.g., adeno- or retroviruses; or
transplantation of genetically modified cells
including the development and delivery of
biomaterials. The requirements for effective
delivery systems regarding NGF are (a) to effec-
tively stimulate cholinergic neurons, (b) to con-
tinuously deliver NGF over years, (c) to restrict
the distribution to sites of degeneration and to
avoid adverse effects, and (d) to simplify the
mode of delivery due to the growing disease
population.
Neurosurgical-Invasive Strategies Different
invasive strategies of NGF delivery to the brain
have been reported to show that indeed NGF
could be a promising therapeutic in AD, either
intracerebroventricular administration (Olson
1993, Eriksdotter-Jönhagen et al. 1998) or
ex vivo gene therapy using grafts of
NGF-secreting fibroblasts in AD forebrain
(Tuszynski et al. 2015) or intraparenchymal
viral gene delivery (Nagahara et al. 2009). How-
ever, it must be completely clear that such an
invasive strategy is limited only to a few selected
patients due to extreme costs and invasive sur-
gery. As we expect a very high number of AD
patients within the next 50 years (10 million in
Europe), it is fully clear that we must develop an
alternative to surgery and explore novel noninva-
sive delivery strategies.
Noninvasive Strategies Noninvasive strategies
must focus on novel delivery strategies, such as
intravenous, intranasal, or intraocular. In fact
many different strategies have been tested long
time ago, but did not succeed due to the complex-
ity of targeted delivery without any side effects:
peripheral delivery (Lapchak 1993), BBB
12 NGF Released from Blood Cells or Collagen Hydrogels as a Therapeutic. . .
transferrin receptor-mediated transcytosis with
OX-26 coupled to NGF (Granholm et al. 1994),
intranasal or intraocular application (Capsoni et al.
2009), NGF absorbed on poly(butyl cyanoacry-
late) nanoparticles (Kurakhmaeva et al. 2009),
NGF-loaded microspheres (Gu et al. 2009), or
cell-based strategies (Kramer et al. 1995).
12.2 Blood Cell-Derived
Therapeutics
The idea of cell-based strategies takes advantage
of the fact that blood cells transmigrate into the
brain during normal immune patrol and that, in
neurodegenerative diseases, the BBB is broken
and more cells can penetrate into the brain
(Kramer et al. 1995). Indeed, during an effective
immune response, the vascular permeability is
increased along with blood flow to the site of
inflammation or injury. This is accompanied by
the selective entry of immune cells from the
periphery. It has been well established that many
different cells can transmigrate into the brain (see
our review: Hohsfield and Humpel 2015), e.g.,
activated T lymphocytes, microglia, monocytes,
B cells, mast cells, stem cells, but also platelets
(Kniewallner et al. 2015). The general aim of
such a strategy is (a) to isolate blood cells from
patients; (b) to manipulate ex vivo, e.g., to load
NGF; and (c) to infuse the cells back into the
same patient (homotransplant), so that (d) the
cells can transmigrate into the brain and release
the protective/regenerative factor at the target of
interest.
12.2.1 Monocytes in the AD Brain
Monocytes play an important role during innate
and adaptive immune responses, and inflamma-
tion leads to an increased recruitment of
monocytes to peripheral tissues aiding in host
immune system defense and repair (Fig. 12.1a).
Monocytes are 5–10μm blood cells and are
identified by their expression of, e.g., CD14,
CD11b, CD115, F4/80, or Ly6C+. In fact, many
195
papers have been published on monocytes as
therapeutic cells in AD (see our review: Hohsfield
and Humpel 2015; Malm et al. 2010). Monocytes
can (a) be induced to differentiate into microglia
and macrophages, (b) be stimulated to migrate to
lesion sites or plaques, and (c) eliminate and
phagocytize plaques (Fig. 12.1b).
Differentiation of Monocytes It has been
suggested that monocytes are the precursors of
microglia and macrophages, and there is clear
evidence that different cytokines, e.g., tumor
necrosis factor alpha or granulocyte-macrophage
colony-stimulating factor, can convert the cells
into a microglia-like phenotype (Prinz et al.
2014; Hohsfield et al. 2013).
Migration of Monocytes Emerging evidence
from studies in stroke, brain trauma, and AD
indicate that these severe brain disorders can
lead to BBB breakdown and subsequent migra-
tion of blood-derived monocytes into the brain.
Monocytes are recruited to sites of Aβ plaque
deposition (triggered by Aβ40 and Aβ42) and can
eliminate Aβ deposits via phagocytosis (Fiala
et al. 2005; Giri et al. 2000; Humpel 2008). In
vivo it was reported that monocyte deposition is
only present in the brains of AD transgenic mice,
neighboring Aβ plaques, whereas little or no cell
deposition is present in the brains of control
animals (Lebson et al. 2010; Stalder et al. 2005).
Monocytes can be triggered to migrate to a lesion
site, and indeed monocyte chemoattractant
protein-1 (MCP-1) can induce migration of
monocytes (Fig. 12.1b). Thus, the release of
MCP-1 from activated microglial cells around
plaques may recruit monocytes to plaques.
Phagocytic Function of Monocyte-Derived
Microglia Extensive in vivo and in vitro evi-
dence indicates that blood-derived monocytes
can phagocytize Aβ (Hawkes and McLaurin
2009; Malm et al. 2010); however, the exact
mechanism of how monocytes clear Aβ remains
unclear (Fig. 12.1b). Some studies have
demonstrated that insoluble fibrillar Aβ can bind
to receptors on microglia and activate these cells
to produce a number of Aβ-degrading enzymes,
196 C. Humpel

Fig. 12.1 Blood-derived cells may support survival of monocytes can be loaded with nerve growth factor
cholinergic neurons (a), phagocytize and eliminate (NGF) and may penetrate a damaged blood-brain barrier
plaques (b), or repair brain vessels (c). (a) Primary in Alzheimer patients. These NGF-loaded monocytes may
12 NGF Released from Blood Cells or Collagen Hydrogels as a Therapeutic. . .
including insulysin, neprilysin, and others
(Frenkel et al. 2013). We have shown that
monocyte-derived microglia can phagocytize
FITC-labeled Aβ (Hohsfield and Humpel
2015a). In a previous paper, we (Hohsfield and
Humpel 2015a) showed that monocytes derived
from young 2-week-old mice were effective to
eliminate plaques in a transgenic APP_SweDI
mouse model after tail vein infusion. In fact, the
number of small (5–20μm) and large (>40μm)
Aβ plaques was slightly reduced. This clearly
points to the migratory and phagocytic capacity
of especially young monocytes in the brain. Much
more work is necessary to prove that indeed
monocytes can counteract plaque pathologies in
humans.
Transmigration of NGF-Loaded Monocyte-
s Unfortunately, the transfection of primary
monocytes is difficult. In a previous work, we
(Hohsfield et al. 2013a) used electroporation, the
lipids effectene and FuGene, nucleofection,
BioPORTER delivery, as well as a lentiviral
transfection in order to load NGF into monocytes.
The most efficient method was lentiviral transfec-
tion giving 16 ng/ml NGF release within 24 hours
per one million monocytes. Using nucleofection
or BioPORTER, the efficiency was 20 less
(0.6–0.8 ng/ml NGF release), and nucleofection
also damaged monocytes, while in BioPORTER,
they were saved. Anyhow BioPORTER
ä
Fig. 12.1 (continued) migrate to cholinergic nerve
terminals, where NGF is released from monocytes and
acts as a therapeutic growth factor for cholinergic neurons
to support their survival. This is of particular interest as
every neuron is located close to a vessel capillary
(20–50μm distance). (b) Monocytes have the capacity to
differentiate into microglia or macrophages and migrate to
lesion sites when activated by, e.g., macrophage
chemoattractant protein-1 (MCP-1). In the Alzheimer
brain, such monocyte-derived microglia/macrophages
may phagocytize and eliminate beta-amyloid plaques. (c)
Platelets have the capacity to migrate to damaged vessels,
clotting the lesion site. Platelets loaded with the beta-
amyloid degrading-enzyme neprilysin may migrate to ves-
sel lesions and degrade plaques in vessels (the cerebral
amyloid angiopathy). Platelets co-loaded with NGF may
197
NGF-loaded monocytes could partly recover sur-
vival of cholinergic nBM neurons (Hohsfield
et al. 2014). In vivo, we (Hohsfield et al. 2014)
infused monocytes (5  106 cells, 100μl via the
dorsal penile vein i.v. 1/week) into mice and
found that a majority was captured in the lung,
liver, and spleen 24 h after a single injection.
However, after repeated infusion (2 months
weekly), monocytes were found in the cortex/
hippocampus and slightly increased the number
of cholinergic neurons in the nBM after infusion
of NGF-loaded monocytes (Fig. 12.1A).
12.2.2 Platelets in the AD Brain
Platelets (thrombocytes) are responsible for
maintaining the integrity of blood vessels and
promoting hemostasis, and they rapidly migrate
to a damaged vessel. As in AD the vessels are
damaged and the BBB is broken, platelets are an
important cell for vessel repair. They are very
small cells of 3–5μm size and do not have a
nucleus. Platelets are characterized by different
integrins, e.g., CD71, CD62P, or CD31
(Kniewallner et al. 2014). Platelets are of particu-
lar interest in AD since they contain high amounts
of amyloid-precursor protein (APP) and release
Aβ40 into the bloodstream (Evin et al. 2003),
which may play a role in blood clotting and vessel
repair. Furthermore, studies have shown that the
also migrate to cholinergic neurons and support their sur-
vival. (d) Blood cells may be loaded into colla-
gen hydrogels or other biomaterials, where they are
protected and released within time; alternatively, biomate-
rial may be chemically modified to be blood stable (e.g., as
microspheres); finally incorporation of targeting
molecules (e.g., antibodies, magnetic beads, integrins)
into biomaterials may allow selective transmigration
through the blood-brain barrier and targeting to specific
brain areas. Taken together, an approach where blood cells
are derived from humans, manipulated ex vivo, protected
with biomaterials, and then injected into the same patient
(as a homotransplant) may provide a novel therapeutic
approach to counteract Alzheimer pathologies (I thank
Lindsay Hohsfield for help with drawings)
198
larger 130 kDa APP isoform is significantly
reduced in platelets isolated from AD patients,
implicating their potential role in altered APP
metabolism (Padovani et al. 2001). Thus, there
is clear evidence that platelets could become an
interesting target in AD, either diagnostic with
APP as a biomarker or therapeutic as a tool for
vessel repair. In a previous study, we
(Kniewallner et al. 2015) infused platelets into
the brain (5  108, 100μl i.v. tail vein, 1/
week) and found that the majority was trapped
in the liver and lung 24 h after infusion. Only a
minority entered the brain, but those were seen in
the ventricle and partly close to a plaque. We
(Kniewallner et al. 2014) further showed that
platelets can be loaded with NGF using ultra-
sound giving 6.5 ng/mg NGF content. Indeed
those NGF-loaded platelets can rescue cell death
of cholinergic neurons in organotypic brain slices
(Kniewallner et al. 2014). Much more work is
necessary to evaluate if platelets can have a ther-
apeutic function in mouse models or even
humans. We favor the idea to load platelets with
the plaque-degrading enzyme neprilysin, which
could be useful to eliminate plaques in the vessels
and counteract symptoms of CAA (Fig. 12.1c).
12.3 Collagen Hydrogels as
Therapeutics
12.3.1 Collagen Hydrogels in Brain
Repair
The use of biomaterials provides new
opportunities for protection and regeneration of
the brain, and hydrogels and microspheres from
various natural or synthetic biomaterials can be
efficiently loaded with therapeutically active
agents, e.g., growth factors (see our review:
Ucar and Humpel 2018). Thereby, these
hydrogels and/or micro-/nanospheres provide
application of relevant therapeutics in a localized
manner, which minimizes unwanted side effects
that are observed with systemic administration
and enhances the effectiveness of the therapy.
Collagen has been extensively applied directly
upon different tissues, either as fluid collagen or
C. Humpel
as sponges or hydrogels, and is known for its
excellent biocompatibility due to its low toxicity
and poor immunogenic reactions. Collagen
hydrogels (CollH) are easily and rapidly
generated using different chemical cross-linkers
such as glutaraldehyde, formaldehyde, or poly-
ethylene glycol (PEG), as well as physical treat-
ment with dry heat and ultraviolet irradiation
(Friess 1998; Caliari and Burdick 2016). CollH
can be efficiently loaded with therapeutically
active substances and release them in a
degradation-based fashion, in which the sub-
stance is released along with the degradation of
the collagen (Foidl et al. 2018). CollH are
injectable, which provides direct application to
the target site of action rather than a systemic
administration route. As the bioavailability of
many therapeutics to the brain is greatly restricted
by the BBB, injectable CollH may be a minimally
invasive method of administration that provides
local, controlled, and prolonged release of the
growth factor with less systemic side effects. In
addition to difficulties in passage through the
BBB, many small molecules are unstable in the
body and degrade rapidly before they reach their
target. Application of therapeutics within CollH
will prevent rapid degradation of the therapeutics
before they reach the brain and may also be useful
for systemic applications.
12.3.2 NGF Released from Collagen
Hydrogels
CollH have been widely used to deliver growth
factors to different tissues. Coating of collagen
sponges with fibroblast growth factor-2 facilitated
early dermal and epidermal wound healing
(Marks et al. 1991). Platelet-derived growth
factors loaded into collagen sponges enhanced
capillary formation after 1 week (Royce et al.
1995), and bone morphogenetic proteins loaded
into collagen sponges promoted osseous regener-
ation (Kenley et al. 1993). Collagen hollow
spheres fabricated with a template method were
useful to load large amounts of growth factors,
e.g., NGF (Kraskiewicz et al. 2013) or vascular
endothelial growth factor (VEGF) (Nagai et al.
12 NGF Released from Blood Cells or Collagen Hydrogels as a Therapeutic. . .
2010), without causing any toxicity in vitro and
in vivo. It is well known that NGF can be released
from different types of collagen-associated bio-
material: Jin et al. (2013) reported that NGF
released from collagen and hyaluronan hydrogels
can stimulate neurite fiber growth for peripheral
nerve repair. Zeng et al. (2014) incorporated NGF
into collagen-chitosan hydrogels and observed
sustained release of NGF within 4 weeks in vitro.
Others reported controlled release of NGF but
with a focus on hollow fibrin microspheres
(Samal et al. 2015) or highly cross-linked colla-
gen spheres (Kraskiewicz et al. 2013). In our
group, we (Foidl et al. 2018) showed that NGF
can be loaded into CollH and release sufficient
biological active NGF in organotypic brain slices
to counteract cholinergic neurodegeneration.
Much more work needs to be done to show the
usefulness of CollH especially via the systemic
application route.
12.4 Outlook and Hope
The topic that “NGF can be used as a therapeutic
in AD” is now nearly 30–40 years old but did not
succeed to become a human therapy. The prob-
lem is the complexity of application and the
targeting to the brain area of interest. In addition,
most studies with NGF have been conducted too
late, at a time where most of the cholinergic
neurons are already gone. So what to protect if
there is nothing to protect? But still, the story is
ongoing, and indeed there is hope that NGF could
become a therapeutic factor in AD.
Safety and Biocompatibility First, it is important
to test the biocompatibility and stability of the
therapeutic strategy in ex vivo and in vivo
models. This will give us information if (a) the
therapeutic has neurotoxic effects, induces reac-
tive gliosis, or causes microglial immune
reactions and on (b) how long is the therapeutic
stable and (c) how long can cells survive after
application. This is extremely important espe-
cially when such a therapy will be transferred to
humans. The major concern of applying NGF is
the side effect at the spinal cord neurons resulting
in uncontrolled nerve fiber growth and pain.
199
Thus, the targeting directly into the brain area of
interest is the most important issue to be solved.
Delivery Methods As discussed earlier, noninva-
sive delivery of therapeutics is essential when
thinking about an AD strategy. As we expect
millions of AD patients, an invasive surgical
strategy will not be useful. In addition, we must
also develop strategies to protect the cholinergic
neurons and/or to eliminate plaques already at the
beginning of the disease. As AD starts already
30 years before plaque development, such a strat-
egy must be done in younger individuals without
any marked cognitive impairment (mild cognitive
impairment, MCI). Again, a simple and easy
application strategy is essential. Four modes of
application are suggested: intravenous, intrana-
sal, intraocular, and intracerebrospinal. Intra-
venous peripheral application provides a simple
and easy noninvasive strategy, but the most seri-
ous problems are stability in blood, trapping in
liver-spleen-lung or elsewhere, high metabolism,
side effects on peripheral neurons, low delivery
into the brain, low passage through BBB, or high
amounts of factor needed. Intranasal or intraocu-
lar application may take advantage of the fact that
the BBB is more open at those sites and factors
can easier pass. In fact, NGF could be applied as a
“NGF spray” via the nose and may reach the brain
via the olfactory bulb. Intraspinal application is
an alternative, as the cerebrospinal fluid (CSF) is
in direct contact with the brain. In fact, CSF is
routinely used for analysis of different brain-
derived biomarkers and allows to diagnose AD
with high specificity. Such an application may
target the therapeutic of interest better to the
brain. But, even if the therapeutic reaches the
brain, it must be targeted to the brain area of
interest, and this is the hippocampus or the cortex,
if NGF is considered to be taken up by nerve
terminals as a retrograde-acting growth factor.
Blood-Stable Collagen Hydrogels An impor-
tant step in the delivery process could be to stabi-
lize the growth factor or cell in the blood or nasal
fluid or CSF. Especially long-term repeated appli-
cation may make this necessary. Thus,
researchers develop methods to encapsulate cells
200
or drugs in biomaterials, e.g., microspheres or
nanospheres. Such small vehicles may easily
transport a drug of interest into the brain and are
also stabilized. On the other hand, such a bioma-
terial is not degradable and can be concentrated in
cells and cause neurotoxic effects or immune
responses. Thus, the use of biodegradable mate-
rial, e.g., collagen, could overcome such potential
problems, because it is degraded over time and
eliminated by the cells.
Targeting to the Brain As mentioned previ-
ously, targeting of the therapeutic to the brain
and more specifically to the brain area of interest
is the most important challenge. This issue must
involve a transdisciplinary approach including
neurobiologists, chemists, and biotechnologists.
The delivery vehicle (be it a blood cell, a micro-
sphere, or a collagen) must be chemically
modified to target the BBB. One may think of
magnetically labeled vehicles which could be
targeted to the brain with strong magnets. In
fact, magnetic stimulation is a well-established
approach in clinics, e.g., by using MRI or TMS
(transmagnetic stimulation). It is suggested to
load vehicles with a therapeutic and magnetic
beads and to magnetically stimulate the brain
area of interest. Strong magnets could be applied
outside of the brain and force the magnetically
labeled vehicles to migrate into the brain area of
interest.
12.5 Conclusion
An approach where blood cells are derived from
humans, manipulated ex vivo, protected with
biomaterials, and then injected into the same
patient (as a homotransplant) may provide a
novel therapeutic strategy to counteract
Alzheimer pathologies. Blood cells or growth
factors loaded into biomaterials (e.g., collagen
or microspheres) may protect the cells in blood
and may enhance specific transmigration through
the BBB and selective physical targeting to spe-
cific brain areas.
C. Humpel
References
Caliari SR, Burdick JA (2016) A practical guide to
hydrogels for cell culture. Nat Methods 13(5):405–414
Capsoni S, Covaceuszach S, Ugolini G, Spirito F,
Vignone D, Stefanini B, Amato G, Cattaneo A (2009)
Delivery of NGF to the brain: intranasal versus ocular
administration in anti-NGF transgenic mice. J
Alzheimers Dis 16:371–388
Eriksdotter-Jönhagen M, Nordberg A, Amberla K,
Bäckman L, Ebendal T, Meyerson B, Olson L, Seiger
SM, Theodorsson E, Viitanen M, Winblad B, Wahlund
LO (1998) Intracerebroventricular infusion of nerve
growth factor in three patients with Alzheimer’s dis-
ease. Dement Geriatr Cogn Disord 9:246–257
Evin G, Zhu AHR, Masters CL, Li QX (2003) Proteolytic
processing of the Alzheimer's disease amyloid precur-
sor protein in brain and platelets. J Neurosci Res
74:386–392
Fiala M, Lin J, Ringman J, Kermani-Arab V, Tsao G,
Patel A, Lossinsky AS, Graves MC, Gustavson A,
Sayre J, Sofroni E, Suarez T, Chiappelli F, Bernard G
(2005) Ineffective phagocytosis of amyloid-beta by
macrophages of Alzheimer's disease patients. J
Alzheimers Dis 7:221–232
Foidl BM, Ucar B, Schwarz A, Rebel AL, Pandit A,
Humpel C (2018) Nerve growth factor released from
collagen scaffolds protects axotomized cholinergic
neurons of the basal nucleus of Meynert in organotypic
brain slices. J Neurosci Methods 295:77–86
Frenkel D, Wilkinson K, Zhao L, Hickman SE, Means TK,
Puckett L, Farfara D, Kingery ND, Weiner HL, El
Khoury J (2013) Scara1 deficiency impairs clearance
of soluble amyloid-β by mononuclear phagocytes and
accelerates Alzheimer’s-like disease progression. Nat
Commun 4:2030
Friess W (1998) Collagen – Biomaterial for drug delivery.
Eur J Pharm Biopharm 45(2):113–136. https://doi.org/
10.1016/S0939-6411(98)00017-4
Giri R, Shen Y, Stins M, Du Yan S, Schmidt AM, Stern D,
Kim KS, Zlokovic B, Kalra VK (2000) Beta-amyloid-
induced migration of monocytes across human brain
endothelial cells involves RAGE and PECAM-1. Am J
Physiol Cell Physiol 279:C1772–C1781
Granholm AC, Bäckman C, Bloom F, Ebendal T, Gerhardt
GA, Hoffer B, Mackerlova L, Olson L, Söderström S,
Walus LR, Friden P (1994) NGF and anti-transferrin
receptor antibody conjugate: short and long-term
effects on survival of cholinergic neurons in intraocular
septal transplants. J Pharmacol Exp Ther 268:448–459
Gu H, Long D, Song C, Li X (2009) Recombinant human
NGF-loaded microspheres promote survival of basal
forebrain cholinergic neurons and improve memory
impairments of spatial learning in the rat model of
Alzheimer's disease with fimbria-fornix lesion.
Neurosci Lett 453:204–209
Hawkes CA, McLaurin J (2009) Selective targeting of
perivascular macrophages for clearance of beta-
12 NGF Released from Blood Cells or Collagen Hydrogels as a Therapeutic. . .
amyloid in cerebral amyloid angiopathy. Proc Natl
Acad Sci U S A 106:1261–1266
Hohsfield L, Humpel C (2015) Migration of blood cells to
beta-amyloid plaques in Alzheimer's disease. Review.
Exp Gerontol 65:8–15
Hohsfield L, Humpel C (2015a) Intravenous infusion of
monocytes isolated from 2-week-old mice enhances
clearance of beta-amyloid plaques in an Alzheimer
mouse model. PlosONE 10(4):e0121930. https://doi.
org/10.1371/journal.pone.0121930. eCollection 2015
Hohsfield LA, Ammann C, Humpel C (2013) Inflamma-
tory status of transmigrating primary rat monocytes in
a novel perfusion model simulating blood flow. J
Neuroimmunol 258(1–2):17–26
Hohsfield LA, Geley S, Reindl M, Humpel C (2013a) The
generation of NGF-secreting primary rat monocytes: a
comparison of different transfer methods. J Immunol
Methods 391(1–2):112–124
Hohsfield LA, Ehrlich D, Humpel C (2014) Intravenous
infusion of nerve growth factor-secreting monocytes
supports the survival of cholinergic neurons in the
nucleus basalis of Meynert in hypercholesterolemia
Brown-Norway rats. J Neurosci Res 92:298–306
Humpel C (2008) Basolateral aggregated rat amyloidbeta
(1-42) potentiates transmigration of primary rat
monocytes through a rat blood-brain barrier. Curr
Neurovasc Res 5:185–192
Humpel C, Weis C (2002) Nerve growth factor and cho-
linergic CNS neurons studied in organotypic brain
slices: implications in Alzheimer’s disease? Journal
Neural Transmission Supplement 62:253–263
Jin J, Limburg S, Joshi SK, Landman R, Park M, Zhang Q,
Kim HT, Kuo AC (2013) Peripheral nerve repair in rats
using composite hydrogel-filled aligned nanofiber
conduits with incorporated nerve growth factor. Tissue
Eng Part A 19(19–20):2138–2146
Kenley RA, Yim K, Abrams J, Ron E, Turek T, Marden
LJ, Hollinger JO (1993) Biotechnology and bone graft
substitutes. Pharm Res 10:1393–1401
Kniewallner K, Grimm N, Humpel C (2014) Platelet-
derived nerve growth factor supports the survival of
cholinergic neurons in organotypic rat brain slices.
Neurosci Lett 574:64–69
Kniewallner KM, Ehrlich D, Kiefer A, Marksteiner J,
Humpel C (2015) Platelets in the Alzheimers disease
brain: do they play a role in cerebral amyloid
angiopathy? Curr Neurovasc Res 12:4–14
Kramer R, Zhang Y, Gehrmann J, Gold R, Thoenen H,
Wekerle H (1995) Gene transfer through the blood-
nerve barrier: NGF-engineered neuritogenic T
lymphocytes attenuate experimental autoimmune neu-
ritis. Nature Med 1:1162–1166
Kraskiewicz H, Breen B, Sargeant T, McMahon S, Pandit
A (2013) Assembly of protein-based hollow spheres
encapsulating a therapeutic factor. ACS Chem
Neurosci 4:1297–1304
Kurakhmaeva KB, Djindjikhashvili IA, Petrov VE,
Balabanyan VU, Voronina TA, Trofimov SS,
Kreuter J, Gelperina S, Begley D, Alyautdin (2009)
201
Brain targeting of nerve growth factor using poly(butyl
cyanoacrylate) nanoparticles. J Drug Target 17
(8):564–574
Lapchak PA (1993) Nerve growth factor pharmacology:
application to the treatment of cholinergic
neurodegeneration in Alzheimer’s disease. Exp Neurol
124(1):16–20
Lebson L, Nash K, Kamath S, Herber D, Carty N, Lee DC,
Li Q, Szekeres K, Jinwal U, Koren J, Dickey CA,
Gottschall PE, Morgan D, Gordon MN (2010) Traf-
ficking CD11b-positive blood cells deliver therapeutic
genes to the brain of amyloid-depositing transgenic
mice. J Neurosci 30:9651–9658
Levi-Montalcini R, Dal Toso R, della Valle F, Skaper SD,
Leon A (1995) Update of the NGF saga. J Neurol Sci
130(2):119–127
Malm T, Koistinaho M, Muona A, Magga J, Koistinaho J
(2010) The role and therapeutic potential of monocytic
cells in Alzheimer's disease. Glia 58:889–900
Marks MG, Doillon C, Silvert FH (1991) Effects of
fibroblasts and basic fibroblast growth factor on facili-
tation of dermal wound healing by type I collagen
matrices. J Biomed Mater Res 25:683–696
Nagahara AH, Bernot T, Moseanko R, Brignolo L,
Blesch A, Conner JM, Ramirez A, Gasmi M,
Tuszynski MH (2009) Long-term reversal of choliner-
gic neuronal decline in aged non-human primates by
lentiviral NGF gene delivery. Exp Neurol 215
(1):153–159
Nagai N, Kumasaka N, Kawashima T, Kaji H,
Nishizawa M, Abe T (2010) Preparation and charac-
terization of collagen microspheres for sustained
release of VEGF. J Mater Sci Mater Med
21:1891–1898
Olson L (1993) NGF and the treatment of Alzheimer’s
disease. Exp Neurol 124:5–15
Padovani A, Pastorino L, Borroni B, Colciaghi F,
Rozzini L, Monastero R, Perez J, Pettenati C,
Mussi M, Parrinello G, Cottini E, Lenzi L,
Trabucchi M, Cattabeni F, Di Luca M (2001) Amyloid
precursor protein in platelets: a peripheral marker for
the diagnosis of sporadic AD. Neurology
57:2243–2248
Prinz M, Tay TL, Wolf Y, Jung S (2014) Microglia:
unique and common features with other tissue
macrophages. Acta Neuropathol 128(3):319–331
Royce PM, Kato T, Ohsaki KI, Miura A (1995) The
enhancement of cellular infiltration and vascularisation
of a collagenous dermal implant in the rat by platelet-
derived growth factor BB. J Dermatol Sci 10:42–52
Samal J, Deirdre B, Naughton C, Concannon R, Dowd E,
Pandit A (2015) Fibrin-based microsphere reservoirs
for delivery of neurotrophic factors to the brain.
Nanomedicine (Lond) 10(5):765–783
Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach
GJ, Deller T, Coomaraswamy J, Staufenbiel M,
Landmann R, Jucker M (2005) Invasion of
hematopoietic cells into the brain of amyloid precursor
protein transgenic mice. J Neurosci 25:11125–11132
202
Tuszynski MH, Yang JH, Barba D, Hoi-Sang U, Bakay
RA, Pay MM, Masliah E, Conner JM, Kobalka P,
Roy S, Nagahara AH (2015) Nerve growth factor
gene therapy activates neuronal responses in
Alzheimer’s disease. JAMA Neurol 72(10):1139–1147
Ucar B, Humpel C (2018) Collagen for brain repair: thera-
peutic perspectives – a mini-review. Neural Regen Res
13(4):595–598
C. Humpel
Weis C, Marksteiner J, Humpel C (2001) Nerve growth
factor and glial cell line-derived neurotrophic factor
restore the cholinergic neuronal phenotype in
organotypic brain slices of the basal nucleus of
Meynert. Neuroscience 102:129–138
Zeng W, Rong M, Hu X, Xiao W, Qi F, Huang J, Luo Z
(2014) Incorporation of chitosan microspheres into
collagen-chitosan scaffolds for the controlled release
of nerve growth factor. PLoS One 9:e101300
 Part IV
Complex Behaviors
 When Nerve Growth Factor Met
Behavior
Daniela Santucci, Arianna Racca, and Enrico Alleva
Abstract
Since its first characterization in the early
1950s, the role of the polypeptidic nerve
growth factor (NGF) in controlling behavior
remained elusive. Since the mid-1980s, we
undertook a series of experiments aimed at
elucidating the biological role(s) played by
neurotrophins, particularly NGF, in adult
rodents. At the beginning, we concentrated
on the submandibular salivary gland of the
male mouse, which was known to store mas-
sive amount of NGF. We found that under
specific stress conditions, the salivary NGF is
released in the bloodstream: intermale fighting
between isolated males was the first reported
context in which salivary NGF was released,
thus providing a physiological significance for
its presence in the adult, territorial males. We
also found that dominant males release less
NGF than subordinates and provided a loop-
type model which includes intermale social
confrontation, adrenal gland size, and func-
tional status, corticosterone release, a model
resulting in likelihood to be stabilized in a
“dominant” or a “subordinate” social status.
A variety of social anxiety contexts of
mammals, humans included, has been
D. Santucci (*) · A. Racca · E. Alleva
Reference Center for Behavioural Sciences and Mental
Health, Istituto Superiore di Sanità, Rome, Italy
e-mail: Daniela.santucci@iss.it; arianna.racca@guest.iss.
it; enrico.alleva@iss.it
# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_13
13
described since then, and further studies car-
ried out on humans showed that NGF is
released in the bloodstream of parachutists at
their first skydiving experience and in the case
of ranking high on the Passionate Love Scale
(amour fou). Ethological data from lab rodents
helped in understanding NGF function in sub-
tly controlling social “status” of male mice: the
considerations about the interplay among neu-
robiological, physiological, and behavioral
factors in structuring the dominant vs subordi-
nate phenotypes may well apply to other ver-
tebrate species, specifically addressing the
underlying role of neurotrophins in relating
behavior and brain neuroplasticity.
13.1 Introduction
It was July 1983 when, in central Rome, in the
elegant attic of Viale di Villa Massimo 3–5, fol-
lowing an almost 10-year-long discussion with
Rita Levi-Montalcini (RLM) about the mysteri-
ous, yet likely physiological, role of NGF stored
in the submaxillary “salivary” glands of adult
male mice, in collaboration wither her closest
fellow Luigi Aloe, we decided to attempt what
is now regarded as a crucial experiment
(Fig. 13.1).
The origin of NGF release into the circulation
was in the early Eighty highly debated, and we
205
206
tested whether, under an ethological evolutionary
perspective, intermale aggressive behavior could
represent the “elusive” triggering factor causing
NGF release. We found the mysterious trigger.
This was indeed the case: NGF was quite
rapidly released after about 25–30 separate inter-
male “territorial-type” fighting episodes occur-
ring in previously isolated adult male mice
during a single 20-min session (subjects with a
previous parental experience showed peak levels;
isolation per se is an enhancer). More interest-
ingly, by exploiting a dyadic 10-repeated
encounters paradigm, with intermale encounters
occurring in a “neutral cage,” devoid of any
marking scent odor, subordinated males released
about twice as much NGF as “dominants.” Rather
surprisingly, a series of non-social stressors, such
as a very mild level of escapable or inescapable
foot shock, cold water swimming, or forced biting
of a wooden stick, all failed to elicit NGF release.
Only changes in social bonding, including
pre-copula arousal and maternal nest-defending
(this against a potentially infanticidal male
attacker), triggered a high level of NGF release
in the bloodstream (Branchi et al. 2004).
NGF was discovered, biochemically
characterized, and searched for its physiological
role(s) by Levi-Montalcini already at the begin-
ning of the 1950s (Levi-Montalcini 1987a, b).
Since NGF discovery, which opened a vast ave-
nue in the early and late exploration and exploita-
tion of neurotrophic/trophic proteins in a wide
variety of biomedical endpoints, no sound expla-
nation was available during almost three decades
to disentangle NGF physiological role in adult
mammals. Most neurobiologists explained its
presence in adult subjects as a kind of long-term
remains of a molecule having exerted a pivotal
physiological role during early or middle
ontogeny.
This also provided for a while an “unstable,”
fallacious early explanation for the presence of
NGF receptors even in the adult brain.
NGF was originally described by RLM and
colleagues as a protein selectively acting on sym-
pathetic peripheral neurons (initially in bird
embryos), and only decades later, it was found
to be also exerting some physiological role on
D. Santucci et al.
neurons located in the central nervous system
and, in an explosive, unusual advancement of
new knowledge, a jump which illuminated a vari-
ety of neuroethological phenomena, on a variety
of immune cell types (Alleva and Calamandrei
1990); for the evolution of growth factor proteins,
we refer to the enlightening paper by
Black (1986).
Aloe, in close collaboration with our ISS
group, was the first to demonstrate that NGF is
released in humans following stress. We
exploited Holger Ursin’s model (CAT; Ursin
and Eriksen 2004) of intensive training and the
very first analysis, with a really original perspec-
tive, of skydiving experience of military
parachutists as a potent, rather unique, stressor.
To jump from an airplane in the void is really a
neuromotor, emotional, and even cognitive per-
formance violating one of the most basic survival
rules.
Even neonates, immediately after parturition,
show a kind of fixed-action pattern (Lorenz 1935)
which triggers a compulsive response to avoid
falling down (Fox 1965).
Therefore, even if “cultural” (inner motivation,
military hard training, sport necessity, war con-
text, intermale attempt to emerge, etc.) produces
such an anti-Darwinian performance act, the NGF
release remains however an adjustment response
to cope with a particularly severe stress situation.
In the subsequent decades, various laboratories
found that even much lower-level stressors were
enough to trigger NGF release in the bloodstream.
Even presently, the most likely possibility to store
and release NGF in the human species remains
some cellular elements belonging to the immune
system (Torcia et al. 1996).
At the end of prolonged and standardized
training, however with some individual
variability in its duration (a skillful officer even-
tually decided that the single parachutist is ready
to jump), one evening, a young parachutist of a
selected (also by temperamental attitudes) Air-
borne Brigade of the Italian army (Esercito
Italiano) was eventually given the possibility by
expert military trainers to eventually launch from
an airplane the following morning.
13 When Nerve Growth Factor Met Behavior
Fig. 13.1 Laurea honoris
causa in Biomedical
Sciences to Luigi Aloe,
“Alma Mater” Bologna
University (Italy) 22/11/
1989
Immediately after being informed that his
training was ended and the jumping event was
scheduled, blood NGF levels start to raise, in
anticipation of the highly emotional skydiving
experience that will follow the subsequent morn-
ing. Social competition among young men, living
in sexually segregated groups, may well exert a
major triggering role in such a sudden NGF
release (Branchi et al. 2004).
After so many years, it is still surprising to
observe in how many cases NGF is released
mainly following social and stressful events.
Racing horses—likely because of equine social
competition—release NGF in the bloodstream
upon running in a furious game.
In humans, Aloe’s group showed that during
labor and lactation, NGF was fivefold enhanced.
The fatiguing burden of prolongedly assisting a
cognitively impaired close relative raises NGF
blood levels up to 200% (Hadjiconstantinou
et al. 2001). Also during the recognized stressful
phase of smoking habit cessation, NGF levels
grow rapidly, almost doubling in only 3 days
(Lang et al. 2002).
Behavioral and neurobiology scientists of this
Third Millennium are revisiting several phenom-
ena, such as social competition, proudness, and
even love, often considered as unique to humans
and thus dealing with emotions and passions.
207
Those phenomena were only considered in the
realm of social sciences for many decades, a
trend that progressively evolved when “cogni-
tive” sciences, mainly cognitive ethology, started
to emerge. Some major bias still affects these
developing science areas, since it is well known
that jumping from a different (regularly lower)
level of analysis, especially in a typical reduction-
istic, even paleo-positivistic, perspective, is still
very often a classical mistake, obscuring the deli-
cate relationship between the expression of indi-
vidual behavioral phenotypes and their
physiological, lower-level determinants (molecu-
lar, nuclear, cellular, tissue, organ, system, full
individual), just to mention some major descrip-
tive hierarchies and their various levels of analy-
sis. We do recommend the enlightening reflection
by Steven Rose (1981).
These were rarely approached in the past by
means of standardized methods of behavioral
neurobiology, cognitive ethology, experimental
psychology, endocrinology, or functional neuro-
anatomy, just to mention some sub-fields where
the mistaken explanations violating level-of-anal-
ysis considerations are rather common. Human
geneticist Luigi “Luca” Cavalli-Sforza in his orig-
inal reconstruction of human evolution (Cavalli-
Sforza 1996; Diamond 1998) well testified of this
dualistic, cross-pollinating approach,
208
encompassing the gap between the Two Cultures,
Hard Sciences vs Humanities. A third culture,
melting social-type phenomena with more
“hard-science” methodologies, may be eventually
the most useful response for contemporary analy-
sis of behavioral (e.g., coping response to a vari-
ety of stressors administered under different
contexts) phenomena.
Psychoneuroendocrinology already devoted
years ago an entire issue to the “neurobiology of
love” in 1998 (Vol. 23, Issue 8). This editorial
effort included a paper by Emanuele et al.,
exploiting a rather reductionist, yet relevant, sim-
ilar approach, concomitantly assessing the levels
of the four most characterized neurotrophins
(NGF, BDNF, NT-3, and NT-4) in the human
circulation to disentangle the neurobiological
bases of what is described as “romantic love.”
(Incidentally, in some psychiatric manuals, such
a “state” or a syndrome (!) is considered a
pre-alerting condition, possibly followed, even
very rarely and with a marked sex bias, by a
suicidal act).
This is in line with several other analyses
aimed at attempting to understanding the physio-
logical, sometimes “key,” regulators underlying
such a complex behavioral phenomenon, possibly
not unique to the human species, that mixes
feelings, passions, emotions, mental states, inner
desires, and personalized motivations. The most
intriguing finding in the paper by Emanuele et al.
is that exclusively NGF levels were affected by
such a “falling in love,” further demonstrating the
unique role of this proteic neurotrophin (histori-
cally, the first prototype of this “family” of tro-
phic molecules) in regulating social bonding in
mammals, including human beings (Emanuele
2011). Again, we refer to Black (1986), for the
evolutionary meaning of neurotrophic bonding
and their likely taxonomic progression.
Rita Levi-Montalcini fell in love with NGF
several decades ago, when she astonishingly
observed the magnificent “halo effect” produced
in vitro when even minimal amounts of this “bio-
logically powerful” protein reached an explanted
sensory ganglion of the chick embryo, rather rap-
idly stimulating massive dendritic growth. This
happened in Rio de Janeiro during the yearly
D. Santucci et al.
Carnival, in the early 1950s. Her enduring love
for the biological properties of this protein,
almost two decades after her Nobel Prize, lasted
until the end of her life.
13.2 Early Phases of Development:
The Mother-Pups Interaction
The importance of the role of neurotrophins in
directing brain growth and neuronal functionality
has been increasingly recognized.
In the last three decades, in the scientific
Rome-based group, coordinated and inspired by
Rita, the studies carried out by William T. “Bill”
Mobley, then at Stanford Medical School on the
NGF role on developing postnatal cholinergic
central nervous system neurons, represented a
veritable step: a neurotrophin reportedly
regulating (selectively) early adrenergic develop-
ment of sympathetic sensory neurons in birds was
in fact demonstrated to speed up CNS
cholinergics in rats.
When Mobley visited Rome at a special inti-
mate dinner in Rita’s apartment, such former
selective properties lose their specificity. Her pro-
found and durable scientific and personal rela-
tionship with Eric Shooter, then Mobley’s
supervisor at Stanford, also played a relevant
role since he magnified Mobley’s results as well
as his scientific attitudes.
Much later, it was observed that variants of
maternal care in mammals can promote hippo-
campal synaptogenesis and spatial learning and
memory through systems known to mediate
experience-dependent neural ontogeny. Offspring
of highly competent rat mothers showing high
levels of pup licking and grooming and arched-
back nursing (the triadic ethogrammatic score to
evaluate maternal behavior in rodents) had
increased expression of mRNA BDNF and
strengthened hippocampal cholinergic innerva-
tion, spatial learning, and memory capability.
A variety of studies also revealed a role for
neurotrophic proteins as “mediators” of CNS and
peripheral nervous system (PNS) structural
changes, not limited to the early developmental
phases. In fact, the continuous adult plasticity of
13 When Nerve Growth Factor Met Behavior
neuronal nets from synaptogenesis to trophic and
differentiative phenomena is also molded by
neurotrophic actions.
13.3 Social Milieu and NGF Release
In general, when summarizing a vast variety of
data, collected not exclusively on vertebrates,
including the human species, a “lack of controlla-
bility” could act to trigger NGF release or to
enhance its magnitude. Further evidence of the
association between increased NGF levels and a
“subordinate-like” profile emerged from a study
performed long time ago in our laboratory. Lac-
tating female mice are known to display intraspe-
cific aggression patterns toward male intruders to
actively defend their litter. In fact, in the mouse
species as in bears and in many feline species,
male attack pups of lactating females to kill them.
When the physical and bio-behavioral acts
involved in lactation (nipple stimulation, peptide
release, maternal behaviors, etc.) are interrupted,
the mother often undergoes estral phase; there-
fore, the infanticidal adult male may eventually
fecundate her to spread his gene while eliminating
the genetic contribution of the former father, the
one that made the mother pregnant.
NGF levels in lactating female mice were pos-
itively correlated with the degree to which sex-
and species-specific defensive behavioral patterns
were displayed by the mother: when confronting
a potentially infanticide male intruder, lactating
females show the greater amount of defensive
behavioral acts also displaying higher levels of
circulating plasma NGF (Alleva et al. 1996a).
Female aggression, usually of a very high inten-
sity and targeted at vulnerable (e.g., ventral, head)
parts of the body, is considered a seemingly
“uncontrollable” protective reaction of the mother
toward potentially dangerous predators of her
pups (Alleva 1993). These include adult male
mice showing highly cannibalistic attacking
behavior toward her litter.
Moreover, systemic administration of physio-
logical levels of exogenous NGF causes a “sub-
ordinate-like” profile in adult males. NGF-treated
subjects spend proportionally less time fighting
209
than controls, had lowered levels of attack-type
ethogrammatic elements, and showed a longer
latency to attack counterpart (Bigi et al. 1992).
NGF administration inhibits a reliable indicator of
“ambiguous,” transitional element of the inter-
male aggressive pattern, i.e., aggressive
grooming, a behavioral item belonging to the
mouse aggressive repertoire consisting of
grooming the back of the opponent and indicative
of a marked motivation to attack. In fact, follow-
ing repeated and elongated episode of aggressive
grooming, some veritable biting episodes tended
to occur.
13.4 Adrenal Pathway
One probable way by which NGF could exert a
physiological role seemed to be, since our first
studies, via the adrenal gland, known since a long
time to be among the main targets of the NGF
released following intermale aggressive behavior.
Moreover, repeated (once for ten consecutive
days) exogenous NGF administration results in
marked adrenal gland hypertrophy (Aloe et al.
1986; Bigi et al. 1992). Repeated administrations
(6–10 consecutive days) cause a dose-dependent
increase in both adrenal weight and volume, med-
ullary and cortical layers undergoing a marked
volume and area increase.
Level changes in the production of adrenal
hormones (in particular, raised corticosterone
levels) resulting from increased levels of blood
NGF could, in turn, affect the expression of
aggressive behavior. Rodent data show that both
ACTH and glucocorticoid hormones affect ago-
nistic behavior, yet the negative feedback action
between these hormones reveals that their effects
are complex; also depending on the profile of
their plasma release, glucocorticoids directly pro-
mote submissiveness, hierarchical state well sig-
naled in rodents because of very evident postures
and/or vocal emission; at the same time, they
increase aggression indirectly through a negative
feedback suppression of ACTH synthesis.
Leshner and colleagues (Leshner et al. 1980)
reported that ACTH facilitates submissiveness in
mice. A direct action of NGF on the peripheral
210
neural substrates regulating aggressive behavior
can then be also hypothesized.
NGF release may in fact exert some inhibitory
feedback effect on aggressive behavior. The
higher NGF release scored in subordinate
subjects (Maestripieri et al. 1990) and their hyper-
trophic adrenals led to hypothesize a “regulative
loop” involving NGF-promoted adrenal hormone
trigger to stimulate and facilitate a submissive
profile (Alleva et al. 1996a; Leshner et al. 1980).
Such a proposed functional loop, maintained and
regulated by both by a finely tune interplay
between behavioral and neurobiological
components, was detailed in Alleva et al. 1996b.
Other mouse studies lend further support to the
idea that NGF expression may be regulated by
activation modulated by social behavior
(Spillantini et al. 1989). It was in fact reported
that intermale mouse fighting leads to a marked
increase in mRNA NGF production in hypotha-
lamic neurons. Elevated levels of mRNA NGF,
which followed a single, separated fighting epi-
sode, were already evident at 30 min, peaked
about 3 h later, and declined to control levels
between 6 and 12 h. Such a drastic increase in
mRNA NGF in the hypothalamic area was
paralleled by an increase in biologically active
NGF; notably, NGF antibodies markedly
inhibited neurite outgrowth elicited from sensory
and sympathetic ganglia from hypothalamic
tissues extracted from isolated fighting mice.
Labeled cells were observed in the magnocellular
preoptic and ventrolateral hypothalamic nuclei.
Importantly, no specific labeling of glial cells
was observed in this or other brain regions
supporting the view that only neuronal cells
were involved. The increase in hypothalamic
NGF following intermale aggressive behavior is
not abolished by sialoadenectomy, surgical abla-
tion of the salivary glands, suggesting as expected
that the NGF scored in the hypothalamic area is
not of salivary origin (Aloe et al. 1986).
Several lines of converging evidence, from
both intact and injured CNS structures, have
shown that NGF is expressed in the hypothalamic
zones, although its definite roles in regulating
these structures are still elusive (Spillantini et al.
1989).
D. Santucci et al.
It has however been demonstrated that admin-
istration of exogenous NGF promotes structural
recovery following specific hypothalamic dam-
age; also, genes encoding NGF and its receptor
(s) are expressed in the hypothalamus. Various
lines of evidence support the hypothesis that
hypothalamic NGF might be involved in neuro-
endocrine function, thereby acting to regulate
behavioral outcomes. Paracrine and/or autocrine
loops have been repeatedly suggested.
The relevance of the observed changes in NGF
levels at the hypothalamic level upon intraspecific
male “territorial” fighting and the mechanisms
involved is however still a matter of speculation.
Alleva and Aloe (1989) hypothesized that the
rapid increase in expression levels of brain
NGF, following a psychosocial stressful event,
could allow some subtle phenomena of adult
brain plasticity to rapidly being activated (see
Figs. 13.2 and 13.3).
NGF could contribute to subtle structural
changes, such as emergence of dendritic spines
or collateral sprouting, eventually sculpturing the
structure of neural connections in the mature
brain. In other words, NGF, as most likely other
neurotrophins, represent a major, or at least not
minor, key regulator, translating behavioral
events or states in structural changes of neural
architecture. It is however rather obvious that
NGF expression in the hypothalamus might influ-
ence levels of other peptides and hormones pres-
ent in this structure, all cooperating in structural
changes in neural networking. A series of interac-
tive effects between NGF and thyroid hormones,
ACTH, and peptides have, in fact, been reported
since a long time.
13.5 The Most Famous Sibling: The
Brain-Derived Neurotrophic
(BDNF) Factor
BDNF appeared on the scientific scene much later
than NGF.
It is rarely mentioned that any the
“primigenial” NGF sources were tumor tissue,
bull semen, and most importantly Naja naja
snake “venom.” Reportedly (Rita
13 When Nerve Growth Factor Met Behavior 211

NON-PSYCHOSOCIAL
Unescapable footshock
Escapable footshock NO
Cold water swimming NO*
Restraint stress NO
Forced biting NO

PSYCHOSOCIAL
Male fighting YES (high, 100-300 ng/ml serum)
Female fighting YES (low, 3-13 ng/ml serum)
Pre-copula arousal YES (low, 2-9 ng/ml serum)
Running competition YES (prominent neurite outgrowth)

* In rats published observations indicated a decrease of NGF level and NGF receptor expression in the striatum following cold-water swimming stress while
an increase was shown in the hippocampus.
Aloe L, Tirassa P, Alleva E (1994) Cold water swimming stress alters NGF and low-affinity NGF receptor distribution in developing rat brain. Brain Res
Bull:173-178.
** Matsuda H, Koyama H, Oikawa M et al (1991) Nerve Growth Factor-like Activity detected in Equine Peripheral Blood after Running Exercise. J
Vet Med 38:557-559.

Fig. 13.2 Non-psychosocial and psychosocial contexts in which NGF is released in the bloodstream

Levi-Montalcini, in verbis), it occurred that the
biochemist Stanley Cohen, who shared in 1986
the Nobel Prize with RLM, had the enlightening
idea, by exploiting a purely “comparative”
approach to look at mouse submandibular “sali-
vary” glands, as the analogous structure of snake
venom glands, in search of an easily accessible
and available in laboratories source of NGF. It
was indeed the case, and such a rich,
concentrated, and anatomically “friendly” source
of the protein made it quite comfortable to extract,
to purify, and therefore to biochemically charac-
terize the protein; this last was Cohen’s job when
Rita hired him at Washington University. Many

Mouse/ Rat (nearly absent) submandibolar salivary gland

Snake Naja naja venom*

Bull, Rabbit, Camel, Llama, Alpa seminal plasma **

Horse unknown

*a neurotropic effect to enhance neuromotor impairment; Cohen S, Levi-Montalcini R (1956) A Nerve Growth-stimulating
factor isolated from snake venom. Proc Natl Acad Sci USA 42:572-574.
** Castellini C, Mattioli S, Dal Bosco A, et al. (2020) A, Role of NGF on sperm traits: A review, Theriogenology 150:210-214.

Fig. 13.3 NGF sources in various vertebrate species

212
years later, in Hans Thoenen laboratory, and by
concentrating a rather large quantity of brain tis-
sue from pigs, the BDNF molecule was isolated
and relatively rapidly characterized from a bio-
chemical point of view.
Years later, NT3 and NT4 were added to the
“taxonomical” unit named neurotrophins.
A pivotal study showed that the protein BDNF
elicits dramatic sprouting of basal dendrites
associated with a concomitant regression of den-
dritic spines. When compared to green fluorescent
protein (GFP)-transfected controls, such newly
originated dendrites and spines were highly
unstable. Experiments utilizing Trk receptor
antibodies, K252a, along with overexpression of
NGF molecules demonstrated that such an ele-
vated level of instability was due to secreted
BDNF interacting with extracellular TrkB
receptors. In other words, BDNF promotes struc-
tural instability in dendrites and spines. When
those connection bonds are restricted to particular
portions of a dendritic arbor, they may well coop-
erate in translating activity patterns into specific,
yet minute, brain structural changes.
In addition to the governing neuronal survival
and differentiation, neurotrophins reportedly play
a role in synapse development and plasticity.
More in general, their trophic effects, addressing
neural growth toward target in PNS and CNS
areas, have been demonstrated, mainly for NGF.
Exposure to BDNF triggers long-term potenti-
ation (LTP) in CA1 synapses of the neonatal
hippocampal zones, which otherwise display
only short-term potentiation. A putative, yet
quite robust, explanation points to an attenuation
of synaptic fatigue induced by repeated high-
frequency stimulation. Such a selective potentia-
tion of high-frequency transmission by BDNF
appears to play a direct role correcting BDNF
effects on LTP, rather than indirectly inducing
release of additional diffusible factors. The pref-
erential BDNF potentiation of highly active
synapses may well be implicated in the
Hebbian-type mechanisms involved in synaptic
plasticity.
Following the report by Gottschalk et al.
(1999), who initially described the biologically
transducing events mediating BDNF modulation
D. Santucci et al.
of high-frequency synaptic transmission, the
neurotrophin BDNF is nowadays well known to
stand out among members of the neurotrophin
family, for its faculty to regulate synaptic plastic-
ity and, in particular, for generating sustained
structural and functional changes at hippocampal
synapses underlying learning and memory.
Recent advances show how BDNF function is
controlled and diversified by molecular and cellu-
lar mechanisms including trafficking and subcel-
lular compartmentalization of different BDNF
mRNA species, pre- vs postsynaptic release of
BDNF, control of BDNF signaling by tropomyo-
sin receptor kinase B (TrkB) receptor interactors,
and conversion of pro-BDNF to mature BDNF
and BDNF propeptide (De Vincenti et al. 2019;
Yang et al. 2020). Altogether, those complex
mechanisms and processes provide general
framework of how neurotrophic activities regu-
late environmental inputs, coping responses, sen-
sory motor integration, neuronal plasticity, and
neural tissue continuous reorganization.
13.6 Conclusion
Despite reported, long-term claims in the biomed-
ical literature, few evidences are given for future
therapeutical uses of neurotrophins; NGF, in par-
ticular, was claimed a remedy for curing
Alzheimer’s disease, Huntington’s disease,
neuroimmune disturbances, etc. However, the
hyperalgesic side effect of systemically
administered NGF, firstly recorded by us in
mice (Levi-Montalcini et al. 1990) and by others
in rats, is consistently emerging as a potential
avenue for treating diabetic neuropathic pain or,
more in general, for chronic pain conditions.
Coming to behavioral disturbances, there are
series of clinical, preclinical, and animal studies
assuming a role for neurotrophic growth factors
(GFs) in the etiology and drug treatment of clini-
cal conditions related to human mood disorders.
Convergent evidence accumulated in the last
decades indicates that neuroplastic mechanisms
involving GFs (and BDNF in particular) are in
some way altered in depressive disorders and in
animal models of stress.
13 When Nerve Growth Factor Met Behavior
More in general, all data overall substantiate
the view that GF plays an important role in the
pathophysiology of several psychiatric disorders
and in the reinterpretable mechanism of action of
psychotropic drugs and that, conversely, drugs
that selectively stimulate the production of
neurotrophins might represent a new generation
of, e.g., antidepressants.
The present contribution, which essentially
summarizes some decades of experimental work
mainly carried out by exploiting animal models
and, among them, the mouse for its species-
specific social structure (Bronson 1979), was
prominent when compared with the various rat
strains, this latter species being in fact
characterized by a colonial-type social organiza-
tion (Alleva et al. 1995).
However, after several years of animal studies,
a progressive amount of evidence was
accumulated for the human species, where NGF
and the other neurotrophins BDNF, NT3, and
NT4 play physiological and pathophysiological
roles rather similar to that observed in
non-human mammals.
We just mention that Branchi and co-workers
(Alboni et al. 2017) have much more recently
accumulated sound evidences that the renewed
brain plasticity and the continuous neural network
reorganization, and even minute processes such
as synaptogenesis, are involved in common
human psychopathological disorders (mainly,
depression), providing a framework for an origi-
nal reinterpretation for these psychiatric
conditions.
Perhaps more importantly, these lines of evi-
dence provided new insights on the recognized
variable therapeutical efficacy of the most com-
mon antidepressant drugs, presently prescribed
very commonly. The very high proportion of
non-responder patients can hopefully benefit of
these new interpretative steps elucidating opaque
aspects of neurotrophin-served mechanisms and
processes linking specific social and non-social
behavioral patterns, synthesis, storage, and
release of proteic agents and structural brain
(and sometimes PNS) plasticity.
213
Acknowledgments The authors would like to acknowl-
edge Stella Falsini and Francesco Ciabattoni for format
and critical reading of the manuscript. We would also
express our deep gratitude to Dr. Luigi Aloe for sharing
his passion for science with us.
References
Alboni S, van Dijk RM, Poggini S et al (2017) Fluoxetine
effects on molecular, cellular and behavioral
endophenotypes of depression are driven by the living
environment. Mol Psychiatry 22(4):552–561. https://
doi.org/10.1038/mp.2015.142
Alleva E (1993) Assessment of aggressive behaviour in
rodents. In: Conn PM (ed) Paradigms for the study of
behavior, vol 14. Academic Press, New York, pp
111–137
Alleva E, Aloe L (1989) Physiological roles of nerve
growth factor in adult rodents: a biobehavioral perspec-
tive. Int J Comp Psychol 2:147–162
Alleva E, Calamandrei G (1990) On the functional role of
polypeptide growth factors in rodent neurobehavioral
development. Acta Neurobiol Exp (Wars) 50
(4–5):341–352
Alleva E, Fasolo A, Lipp HP, Nadel L et al (1995)
Behavioural brain research in naturalistic and semi-
naturalistic settings: possibilities and perspectives.
Kluwer Academic Publishers, Dordrecht, p 466
Alleva E, Aloe L, Cirulli F et al (1996a) Serum NGF levels
increase during lactation and following maternal
aggression in mice. Physiol Behav 59(3):461–466.
https://doi.org/10.1016/0031-9384(95)02083-7
Alleva E, Petruzzi S, Cirulli F et al (1996b) NGF regu-
latory role in stress and coping of rodents and humans.
Pharmacol Biochem Behav 54(1):65–72. https://doi.
org/10.1016/0091-3057(95)02111-6
Aloe L, Alleva E, Böhm A et al (1986) Aggressive behav-
ior induces release of nerve growth factor from mouse
salivary gland into the bloodstream. Proc Natl Acad
Sci U S A 83(16):6184–6187. https://doi.org/10.1073/
pnas.83.16.6184
Bigi S, Maestripieri D, Aloe L et al (1992) NGF decreases
isolation-induced aggressive behavior, while increas-
ing adrenal volume, in adult male mice. Physiol Behav
51(2):337–343. https://doi.org/10.1016/0031-9384
(92)90150-z
Black IB (1986) Trophic molecules and evolution of the
nervous system. Proc Natl Acad Sci U S A 83
(21):8249–8252. https://doi.org/10.1073/pnas.83.21.
8249
Branchi I, Francia N, Alleva E (2004) Epigenetic control
of neurobehavioural plasticity: the role of
neurotrophins. Behav Pharmacol 15(5–6):353–362.
https://doi.org/10.1097/00008877-200409000-00006
Bronson FH (1979) The reproductive ecology of the house
mouse. Q Rev Biol 54(3):265–299. https://doi.org/10.
1086/411295
214
Cavalli-Sforza LG (1996) Geni, popoli e lingue. Adelphi,
Milano, p 354
De Vincenti AP, Ríos AS, Paratcha G et al (2019)
Mechanisms that modulate and diversify BDNF
functions: implications for hippocampal synaptic plas-
ticity. Front Cell Neurosci 13:135. https://doi.org/10.
3389/fncel.2019.00135
Diamond J (1998) Armi Acciaio e Malattie, Breve storia
del mondo negli ultimi tredicimila anni. Einaudi,
Torino, p 366
Emanuele E (2011) NGF and romantic love. Arch Ital Biol
149(2):265–268. https://doi.org/10.4449/aib.v149i2.
1367
Fox WM (1965) Reflex-ontogeny and behavioural devel-
opment of the mouse. Anim Behav 13(2):234–241.
https://doi.org/10.1016/0003-3472(65)90041-2
Gottschalk WA, Jiang H, Tartaglia N et al (1999) Signal-
ing mechanisms mediating BDNF modulation of syn-
aptic plasticity in the hippocampus. Learn Mem 6
(3):243–256
Hadjiconstantinou M, McGuire L, Duchemin AM et al
(2001) Changes in plasma nerve growth factor levels
in older adults associated with chronic stress. J
Neuroimmunol 116(1):102–106. https://doi.org/10.
1016/s0165-5728(01)00278-8
Lang UE, Gallinat J, Kuhn S et al (2002) Nerve growth
factor and smoking cessation. Am J Psychiatry 159
(4):674–675. https://doi.org/10.1176/appi.ajp.159.4.
674-a
Leshner AI, Korn SJ, Mixon JF et al (1980) Effects of
corticosterone on submissiveness in mice: some tem-
poral and theoretical considerations. Physiol Behav 24
(2):283–288. https://doi.org/10.1016/0031-9384(80)
90087-6
Levi-Montalcini R (1987a) The nerve growth factor:
35 years later (Nobel lecture). Angew Chem Int Ed
D. Santucci et al.
Engl 26(8):707–716. https://doi.org/10.1002/anie.
198707073
Levi-Montalcini R (1987b) The nerve growth factor:
thirty-five years later. EMBO J 6(5):1145–1154
Levi-Montalcini R, Aloe L, Alleva E (1990) A role for
nerve growth factor in nervous, endocrine and immune
systems. Prog Neuro-Endocrinol Immunol 3:1–10
Lorenz K (1935) Der Kumpan in der Umwelt des Vogels,
J. Orn. In: Schiller C (ed) Instinctive behavior, vol 83.
International University Press, New York, pp 137–289
Maestripieri D, De Simone R, Aloe L et al (1990) Social
status and nerve growth factor serum levels after ago-
nistic encounters in mice. Physiol Behav 47
(1):161–164. https://doi.org/10.1016/0031-9384(90)
90056-a
Rose SPR (1981) From causations to translations: what
biochemists can contribute to the study of behavior. In:
Bateson PPG, Klopfer PH (eds) Perspectives in ethol-
ogy. Springer, Boston, MA. https://doi.org/10.1007/
978-1-4615-7575-7_8
Spillantini MG, Aloe L, Alleva E et al (1989) Nerve
growth factor mRNA and protein increase in hypothal-
amus in a mouse model of aggression. Proc Natl Acad
Sci U S A 86(21):8555–8559. https://doi.org/10.1073/
pnas.86.21.8555
Torcia M, Bracci-Laudiero L, Lucibello M et al (1996)
Nerve growth factor is an autocrine survival factor for
memory B lymphocytes. Cell 85(3):345–356. https://
doi.org/10.1016/s0092-8674(00)81113-7
Ursin H, Eriksen HR (2004) The cognitive activation
theory of stress. Psychoneuroendocrinology 29
(5):567–592. https://doi.org/10.1016/S0306-4530(03)
00091-X
Yang T, Nie Z, Shu H et al (2020) The role of BDNF on
neural plasticity in depression. Front Cell Neurosci
14:82. https://doi.org/10.3389/fncel.2020.00082
 Cross Talk of BDNF and GDNF in Spinal
Substantia Gelatinosa (Lamina II): Focus 14
on Circuitry

Adalberto Merighi

Abstract functional interplay in the regulation of

BDNF and GDNF display the notable qualities
of undergoing a regulated secretion in neurons
and being anterogradely transported to nerve
terminals, where they can modulate fast syn-
aptic transmission. That BDNF positively
modulates nociception and/or pain is today
widely accepted, as the growth factor can
start and maintain physiological and patholog-
ical pain. The contribution of GDNF to
nociception is by far most elusive, but evi-
dence is accumulating that the molecule
displays analgesic activity, at least in rodents.
Here I resume the current knowledge on the
spinal cord circuits in which these two factors
may act as modulators of pain-related synaptic
transmission, focusing on their structural and
nociception and pain.
14.1 Introduction
Neurotrophic factors are an assorted group of
molecules supporting the development, mainte-
nance, and differentiation of immature and
mature neurons. Some neurotrophic factors also
affect synaptic activity and plasticity and thus
fulfil the criteria to be included in the group of
neuromodulators (Gibon and Barker 2017). Sev-
eral neurotrophic factors have received attention
as modulators of nociception and players in the
maladaptive changes leading to chronic pain

A. Merighi (*)
Department of Veterinary Sciences, University of Turin,
Grugliasco, Italy
e-mail: adalberto.merighi@unito.it

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 215
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_14
216
under pathological conditions (Khan and Smith
2015; Ossipov 2011). The nerve growth factor
(NGF) was the first to be studied as a spinal
nociceptive modulator (Merighi et al. 2004), but
afterward, a much higher interest was raised by
another neurotrophin, the brain-derived
neurotrophic factor (BDNF), on the basis of sev-
eral histological, functional, and pharmacological
observations (Merighi et al. 2004; Merighi et al.
2008b; Bardoni and Merighi 2009). More
recently, evidence has been accumulating also
for an intervention of the glial cell line-derived
neurotrophic factor (GDNF) in the modulation of
spinal nociception (Merighi 2016). Here I recon-
sider the spinal cord circuitry in relation to the
intervention of BDNF and GDNF in the modula-
tion of the physiological processing of nocicep-
tive stimuli and in the maladaptive changes that
occur in pathological conditions.
14.1.1 BDNF and Its Receptors
BDNF, like the other members of the
neurotrophin family, i.e., NGF, neurotrophin-3
(NT-3), and neurotrophin-4/neurotrophin-5
(NT-4/NT-5), interacts with two different types
of membrane receptors: a group of tyrosine kinase
receptors, referred to as tropomyosin receptor
kinases (Trks), and a common receptor, p75NTR
(Chao and Hempstead 1995).
There are three different Trk isoforms, named
TrkA, TrkB, and TrkC, with different affinities
for the various neurotrophins: NGF preferentially
binds to TrkA, BDNF and NT-4/NT-5 bind to
TrkB, and NT-3 is associated with TrkC but
also binds to TrkA and TrkB, even though with
a low affinity (Chao and Hempstead 1995). Trk
receptors have an extracellular domain that binds
the neurotrophin(s) and an intracellular domain
that possesses tyrosine kinase activity (Reichardt
2006). BDNF binding prompts receptor dimeriza-
tion, phosphorylation of the cytoplasmic tyrosine
residues, and activation of downstream effectors
(Koerber 2009). However, Trk receptors may be
also active in the absence of the neurotrophins,
following trans-activation via several G protein-
coupled receptors (Reichardt 2006).
A. Merighi
Splicing variants exist for TrkB and TrkC.
These truncated receptors are devoid of the tyro-
sine kinase domain but still can evoke Ca2+
transients and trigger intracellular signals (Blum
and Konnerth 2005). Interestingly, several studies
indicate that truncated Trk isoforms typically
counteract the function of the respective full-
length receptors, e.g., by inhibiting dimerization
or disrupting intracellular responses (Gold and
Gebhart 2010).
The low-affinity p75NTR also is made of an
extracellular and an intracellular domain. The
latter includes a death domain acting on several
cytoplasmic proteins. Expression of p75NTR
decreases in parallel with development but
increases after injury (Mogil 2019). p75NTR does
not directly activate the neurotrophins but
interacts with Trks giving rise to several different
downstream effects (Mogil 2019).
14.1.2 GDNF and Its Receptors
GDNF is a member of a family of related ligands,
referred to as GDNF family of ligands (GFLs)
that also consists of neurturin, artemin, and
persephin. All members of the family have struc-
tural links with the components of the superfam-
ily of the transforming growth factor β
(Airaksinen and Saarma 2002). GFLs signal
through a multicomponent receptor complex
consisting of the tyrosine kinase transmembrane
receptor Ret and a high-affinity ligand-binding
glycosylphosphatidylinositol-anchored
co-receptor referred to as GDNF family receptor
α (GFRα). Each molecule of the GFL family ties
with a specific GFRα isoform, GFRα1 in the case
of GDNF, characterized by a distinct structure of
the cysteine-rich repeats in the ligand-binding
domains (Cervero and Laird 1999). GFRα-Ret
activation mainly signals through Ras/MAPK,
phosphatidylinositol 3-kinase (PI3K)/Akt, and
PLCγ, which play a key role in neurite outgrowth
and cell survival in the nervous system
(McMahon et al. 1995; Gebhart 2000; Sengupta
2009).
Consistent with a broader expression of GFRα
than Ret in neurons, GFLs can also transduce
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry
through GFRα independently from Ret (Sengupta
2009).
14.2 General Features
of the Substantia Gelatinosa
The substantia gelatinosa of Rolando covers the
apex of the posterior (dorsal) horn of the spinal
gray matter and owes its name to its gelatinous,
semitransparent aspect in fresh preparations of the
spinal cord (see, for review, Merighi 2018). It
consists of small neurons and glia and a rich
neuropil but very few myelinated fibers. In
Rexed’s laminar subdivision of the spinal gray
matter, the substantia gelatinosa corresponds to
lamina II. Although dissimilar in shape and exten-
sion, lamina II can be detected at all spinal levels
and constitutes most of the dorsal part of the head
of the dorsal horn. The gelatinosa receives a direct
input from the primary afferent fibers (PAFs)
originating from the primary sensory neurons
(PSNs) of the dorsal root ganglia (DRGs) and
reaching the spinal cord through the dorsal roots
of the spinal nerves. A substantial fraction of
these fibers derives from nociceptors and
thermoreceptors, but some others originate from
low-threshold mechanoreceptors (LTMs).
Neurons form a fine network inside lamina
II. Their short axons make synapses with each
other or with neurons in other laminae of the
gray matter, including the contralateral lamina
II. Notably, rather than directly contributing pro-
jection fibers to the ascending anterolateral sys-
tem or the spinothalamic tracts (STTs) that
convey nociceptive information to higher centers,
the substantia gelatinosa controls nociception at
the interface between first-order DRG neurons
and second-order projection neurons in laminae
I and III–V of the spinal cord. The publication of
the Gate Theory of Pain (Melzack and Wall 1965)
was seminal to sustain the pivotal role of lamina II
in the control of pain, but more recent studies
have implicated the substantia gelatinosa also in
the integration of pruritic stimuli.
In humans (Schoenen 1982), monkey (Light
and Perl 1979), cat (Light and Perl 1979; Rexed
1952, 1954), rat (Light and Perl 1979; Lorenzo
217
et al. 2008; Pan and Pan 2004), and mouse
(Woodbury et al. 2000), lamina II can be further
subdivided into two zones (sublaminae), an outer
and inner lamina II (lamina IIo and IIi). In rodents,
lamina IIi further consists of a dorsal (IIid) and a
ventral (IIiv) part that display differences in the
type(s) of interneurons herein contained, their
neurochemistry, and connections with the
endings of PAFs (Ribeiro-Da-Silva and De
Koninck 2008). The input to the substantia
gelatinosa not only derives from the PAFs
originating from the DRG neurons but also from
the descending fibers deriving from several areas
of the brain and other spinal cord neurons, among
which are the antenna neurons (Schoenen 1982).
These latter are interlaminar neurons with cell
bodies in laminae III–IV, dendrites that travel
dorsally into lamina II and lamina I, and, at least
in some cases, axons that go up to the brain along
the STT (Surmeier et al. 1988) or the dorsal
columns (Brown and Fyffe 1981). The output of
lamina II chiefly reaches neurons in laminae I, III,
and IV (Bennett et al. 1980; Gobel 1975, 1978a;
Light and Kavookjian 1988), but, at least in rat
(Giesler et al. 1978) and monkey (Willis et al.
1978), there are projections also to the brainstem
or the thalamus.
14.2.1 Neurons of the Substantia
Gelatinosa
In lamina II reside several populations of
interneurons (Merighi 2018). These neurons are
intralaminar, i.e., they have processes that do not
span outside lamina II, or interlaminar, i.e., they
connect different laminae of the spinal cord
dorsal horn.
The islet cells form the best described distinct
population of inhibitory neurons, based on their
morphology, neurochemistry, and function. They
are GABA-, glutamic acid decarboxylase (GAD)-
, and vesicular GABA transporter (VGAT)-
immunoreactive and form GABAergic synapses
(Lu and Perl 2003; Zheng et al. 2010). The islet
cells are solely inhibitory, as demonstrated by the
lack of expression of the vesicular glutamate
transporter 2 (VGLUT2) gene (Punnakkal et al.
218
2014). They chiefly display a tonic firing pattern
and are presynaptic to transient-firing central cells
(Lu and Perl 2003) and postsynaptic to C and Aδ
PAFs (Grudt and Perl 2002; Hantman et al. 2004;
Lu and Perl 2003, 2005; Yasaka et al. 2007) and
to the central cells (Zheng et al. 2010). C PAFs
excite the islet cells directly, whereas Aδ PAFs
inhibit them indirectly, via a polysynaptic path
(Yasaka et al. 2007). Remarkably, islet cells
establish dendro-axonic synapses onto the PAF
C boutons at V1 profiles in glomeruli (Ribeiro-
da-Silva 2015). This is, unfortunately, one of the
few information that we have about the origin of
glomerular dendrites, as we will discuss in a
following section of this chapter. Local inputs to
the islet cells are both excitatory and inhibitory
and derive almost exclusively from other
substantia gelatinosa neurons (Kato et al. 2007).
Central cells look like islet cells but are
smaller. They may be glutamatergic (Lu and
Perl 2003, 2005) or GABAergic (Hantman et al.
2004; Hantman and Perl 2005; Maxwell et al.
2007; Todd and McKenzie 1989; Zheng et al.
2010) and show various firing patterns (tonic,
transient IA, and transient non-IA).
Glutamatergic tonic and transient non-IA central
cells synapse onto the vertical cells (Lu and Perl
2003, 2005), whereas GABAergic tonic central
cells make synapses onto vertical and islet cells
(Hantman et al. 2004; Zheng et al. 2010). Central
cells connect indirectly to PAFs. They receive an
excitatory or inhibitory synaptic input from C
PAFs, but a merely inhibitory input from Aδ
PAFs (Grudt and Perl 2002; Hantman et al.
2004; Lu and Perl 2003; Yasaka et al. 2007).
The stalked cells are a rodent subpopulation of
large vertical cells. Many of them are
glutamatergic excitatory interneurons with
delayed firing (Lu and Perl 2005) that are immu-
noreactive for VGLUT2, but not GABA. At least
some large vertical cells contain one or more
neuropeptides (Bennett et al. 1982; Cruz and
Basbaum 1985; Ribeiro-Da-Silva et al. 1991;
Todd et al. 2003). They are interlaminar neurons,
and those cells having their cell body in lamina IIo
are chiefly presynaptic to lamina I spinothalamic
nociceptive neurons (Gobel 1978b) expressing
the NK1 receptor (Polgár et al. 2010).
A. Merighi
Functionally, stalked/large vertical cells may be
either nociceptive-specific or wide dynamic range
(WDR) neurons (Bennett et al. 1980). They also
receive excitatory and inhibitory inputs from both
C (directly) and Aδ (indirectly) PAFs (Grudt and
Perl 2002; Hantman et al. 2004; Lu and Perl
2003; Yasaka et al. 2007). Based on earlier ultra-
structural observations, it was inferred that PAFs
contact the dendrites of large vertical cells specif-
ically at type Ia glomeruli (Maxwell et al. 2007),
but this has, at present, only remained speculative
and does not exclude that contacts occur also at
other types of glomeruli. The dendritic spines of
the large vertical cells form several synapses with
VGLUT1-immunoreactive boutons. At least a
few of these synapses possibly originate from A
LTMs (Yasaka et al. 2014).
The small vertical cells are GABAergic,
GAD-immunoreactive inhibitory neurons mainly
residing in lamina IIo (Grudt and Perl 2002;
Maxwell et al. 2007; Yasaka et al. 2007). They
have a smaller dendritic tree than the larger
glutamatergic vertical cells. Another difference
is that the small vertical cells display a tonic firing
pattern instead of delayed firing. At least some
small vertical cells expressing dynorphin have
been linked to mechanical nociception (Cruz
and Basbaum 1985; Duan et al. 2014).
Radial cells are excitatory glutamatergic
neurons that have a characteristic shape with
compact discoidal dendritic trees and IA firing
(Grudt and Perl 2002; Yasaka et al. 2007; Yasaka
et al. 2010). Some of them are instead inhibitory,
have a more dispersed dendritic tree, and are
immunoreactive for GABA (Maxwell et al.
2007; Todd and McKenzie 1989). C PAFs
(directly) and Aδ PAFs (indirectly) provide excit-
atory and inhibitory inputs to the radial cells
(Grudt and Perl 2002; Hantman et al. 2004; Lu
and Perl 2003; Yasaka et al. 2007).
14.3 Nociceptive Circuits
in Substantia Gelatinosa
Separate routes in mammals process nociceptive
stimuli conveyed from somatic or visceral organs
to cortical centers. In particular, somatic sensory
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry
fibers from the skin and the locomotor apparatus
follow the trigeminal and spinal nerves, whereas
sensory nerves in viscera originate from the sym-
pathetic (via the white communicating branches
of the thoracic and lumbar nerves) and parasym-
pathetic (via certain cranial nerves and the
splanchnic pelvic nerves) autonomic nervous sys-
tem. Visceral afferents, for the most, do not reach
the substantia gelatinosa, but terminate in deeper
laminae of the spinal cord dorsal horn (Cervero
and Connell 1984). However, lamina II contains a
few terminals of visceral origin, which are sparser
and span over a larger number of spinal segments
than the cutaneous afferents (Sugiura et al. 1989).
Given the primary importance of lamina II in the
processing of somatic stimuli, I will focus the
following discussion on this matter.
14.3.1 Spinal Somatic Nociceptive
Circuits
The PSNs of the DRGs collect nociceptive and
other types of sensory stimuli from all parts of the
body except the head and the proximal regions of
the neck, which are innervated by the PSNs of the
trigeminal nerve. The main somatic pain pathway
originating from the DRG neurons is the
spinothalamic pathway that is crucial for the sen-
sory discriminative aspects of pain perception, in
other words for the animal to be aware of which
part of the body is suffering pain. It consists of a
polysynaptic chain of three key neurons, often
referred to as the first-, second-, and third-order
sensory neurons. First-order neurons are the PSNs
of the DRGs. Second-order neurons lie in laminae
I and V of the dorsal horn of the spinal cord. They
give rise to axons that cross the midline and
ascend all along the spinal cord and brainstem to
reach the thalamus. Finally, the ventral postero-
lateral nucleus of the thalamus contains the third-
order sensory neurons that eventually send their
axons to the cerebral cortex. Modulation of noci-
ceptive signals can occur all along this
polyneuronal chain but takes place mainly at the
level of the substantia gelatinosa (Merighi 2018).
219
14.3.2 Localization of BDNF and Its
Preferred Receptor TrkB1
in DRGs and Dorsal Horn
of the Spinal Cord
Initial localization studies demonstrated, in sev-
eral species, the presence of the BDNF mRNA
and protein in a subpopulation of PSNs of the
DRGs (Ernfors et al. 1992; Josephson et al.
2001; Widenfalk et al. 2001). In laboratory
rodents, BDNF+ DRG neurons are for the most
of small to medium size (Michael et al. 1997;
Salio et al. 2005; Salio and Ferrini 2016) and
peptidergic, i.e., they also express the calcitonin
gene-related peptide (CGRP), which typically
characterizes the nociceptors encoding thermal
pain (Groenewegen 1988; Bushnell and Duncan
1989), alone or in combination with other
peptides. On the other hand, the non-peptidergic
PSNs, a set of nociceptors chiefly involved in the
transduction of mechanical pain, which can be
labelled using the isolectin B4 (IB4), do not
express BDNF (Groenewegen 1988; Bushnell
and Duncan 1989). Accordingly, BDNF-positive
PAFs in the superficial dorsal horn display the
same localization of the CGRP-expressing PAFs
in lamina I and lamina IIo (Salio and Ferrini 2016;
Conner et al. 1997). BDNF has also been detected
in PSNs other than those labelled after CGRP or
IB4 immunostaining (Salio and Ferrini 2016). In
keeping with this observation, by using a BDNF-
LAcZ reporter transgenic mouse, Basbaum’s
group has confirmed a wider expression of
BDNF than formerly reported (Borsook and
Becerra 2011). Specifically, these authors
described the occurrence of the neurotrophin in
large myelinated PAFs, suggesting a still poorly
understood role of BDNF also in the processing
of non-nociceptive stimuli (Salio and Ferrini
2016).
The TrkB protein and mRNA are detectable in
DRG neurons of variable size (Josephson et al.
2001; Widenfalk et al. 2001; Salio et al. 2005;
Thompson and Bushnell 2012). Among these
trkB+ DRG neurons, we have shown a subset of
1
Unless otherwise stated, I will refer to full-length func-
tional trkB receptors in this and in the following sections.
220
CGRP+/BDNF+ nociceptors, suggesting the
occurrence of an autocrine activation loop at
their terminals in the substantia gelatinosa (Salio
et al. 2005). By using a transgenic TrkBtauEGFP
mouse, the trkB receptor was also demonstrated
in Aδ LTMs (Li et al. 2011), which, however,
mainly terminate in lamina III of the dorsal horn.
Sparse propriospinal neurons express BDNF
and/or TrkB. The BDNF mRNA was localized
only in the deep dorsal horn of the adult rat
(Widenfalk et al. 2001; Conner et al. 1997). Con-
versely, most spinal cord neurons express the
trkB protein (Widenfalk et al. 2001; Bradbury
et al. 1998).
14.3.3 Localization of GDNF
and GFRa1/Ret in DRGs
and the Dorsal Horn
of the Spinal Cord
Like BDNF, GDNF is chiefly expressed by small-
to medium-sized DRG neurons (Salio and Ferrini
2016; Bennett et al. 1998), although the GDNF-
expressing neurons form a distinct and smaller
subpopulation of peptidergic neurons than that
expressing BDNF (Salio and Ferrini 2016). In
mouse, GDNF+ DRG neurons also express
CGRP and somatostatin (Salio and Ferrini
2016). The central projections of these neurons
terminate in laminae I–IIo (Salio and Ferrini
2016; Holstege et al. 1998; Ohta et al. 2001;
Salio et al. 2014).
The RET mRNA is detected in more than 50%
of the lumbar DRG neurons (Bennett et al. 1998,
2000; Kashiba et al. 2003). More than two thirds
of the RET-expressing lumbar (Kashiba et al.
2003; Molliver et al. 1997) or thoracic (Josephson
et al. 2001) neurons also display the GDNF-
specific co-receptor GFRα1 at their membrane.
Remarkably, while the DRG peptidergic neurons
mainly express GDNF, it is the non-peptidergic
IB4+ neurons that express the GFRα1/Ret recep-
tor complex (Molliver and Snider 1997).
Consistent with the expression of GFRα1/Ret
in non-peptidergic PSNs, GDNF receptors have
been detected in PAFs of lamina IIi (Salio et al.
2014; Jongen et al. 2007). Ultrastructurally,
A. Merighi
GFRα1 receptors are expressed by IB4+ PAFs
that, in lamina II, lay ventrally to the GDNF+
peptidergic terminals (Salio and Ferrini 2016;
Salio et al. 2014).
There is not convincing proof for a
propriospinal source of GDNF. On the other
hand, GFRα1/Ret are expressed by some dorsal
horn neurons, comprising a population of sup-
posed projection neurons in lamina I that also
express the substance P-preferred receptor NK1
(Jongen et al. 2007).
14.3.4 Role of BDNF and GDNF
in Nociception
Although localization studies in PSNs and
substantia gelatinosa powerfully advocate an
involvement of both BDNF and GDNF in
nociception, the functional relevance of the two
trophic factors in pain physiology remains elu-
sive. The main reasons for this have up to now
been the scarcity of knowledge of the spinal cord
circuits made by the two trophic factors and their
receptors, the nonexistence of specific drugs
targeting the two molecules, as well as the limited
information that could be obtained from standard
knockout animals. More recently, on the other
hand, the progress and readiness of conditional
mutant mice have opened new paths to question
the influence of BDNF and GDNF in pain
transmission.
Studies on the conditional blockade of a
mutant TrkB receptor in mice concluded that
BDNF did not intervene in the processing of
acute heat, mechanical, or chemical pain sensitiv-
ity. The role of BDNF in nociception was also
studied in a conditional transgenic mouse strain in
which a tamoxifen injection could selectively
destroy virtually all PSNs expressing BDNF
(Borsook and Becerra 2011). In these mice, only
minor effects could be observed, such as an unim-
portant increase in the heat thresholds in males
but not females, and no effects on mechanical or
cold sensitivity, irrespective of gender (Borsook
and Becerra 2011).
A different situation follows the ablation of
Ret by conditional deletion in nociceptors
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry 221

expressing the Nav1.8 channel (Golden et al. 14.4.1 BDNF and the Excitatory Path
2010): in this case, mice display an augmented
sensitivity to cold and an augmented response to The occurrence or lack of TrkB receptors allows
formalin-induced pain, thus indicating a constitu- for further subdividing BDNF containing PAFs
tive inhibitory role of Ret in spinal nociception. into two subcategories. The first, where TrkB
As I will discuss in the following paragraph, the receptors are absent, is crucial to generate a
findings ex vivo obtained in my laboratory in long-lasting excitation in lamina IIo following
wild-type animals (Salio et al. 2014) are fully two different paths that ultimately converge onto
consistent with these observations in the gelatinosa large vertical/stalked cells. The first
transgenic mice. path (Fig. 14.1—path 1) comprises a series of
three excitatory synapses. The first synapse is
made by a peptidergic BDNF+ C fiber that also
expresses substance P and the transient receptor
14.4 BDNF and GDNF Circuits
potential vanilloid receptor 1 (TRPV1). It initiates
in the Substantia Gelatinosa
the circuit by providing input to an excitatory
central cell in lamina II. The central cell, in turn,
Neuronal circuitries in lamina II and other
is presynaptic to a large vertical cell, which sends
laminae of the superficial dorsal horn remain,
its axon outside lamina II to make the third excit-
for the most, vague. Not only one of the main
atory synapse of the path by contacting a STT
reasons for this substantial lack of information is
projection neuron in lamina I. The other path
our present difficulty to identify functionally dis-
(Fig. 14.1—path 2) also starts with a peptidergic
tinct populations of interneurons, but also the
BDNF+ C fiber but is more complex as it consists
unfeasibility to provide a complete reconstruction
of a chain of four neurons that includes two
of the exact wiring of the substantia gelatinosa
inhibitory islet cells in series that are inserted
neurons has an unquestionable influence. Here I
between the C PAF and the large vertical cell.
try to put together and discuss the data collected
Thus, as the first islet cell drives an inhibition of
in my laboratory with those obtained by other
the second islet cell, the ultimate result is an
colleagues on the circuits made by the PAFs
excitation of the large vertical/stalked cell onto
containing BDNF or GDNF in lamina II and
which the path also converges. As PAFs involved
propose a hypothetical model of parallel
in both circuits contain a blend of messengers
processing of somatosensory information based
with different molecular weight, release can
on the interplay between the two molecules.
occur selectively in dependency of the frequency
According to this model, there are two pathways
and firing pattern of activation (Merighi 2017). In
with opposite functions to process sensory infor-
nociceptive fibers, the firing pattern, rather than
mation in the gelatinosa. One of these is excit-
just the firing rate, is important to encode the
atory and is triggered by a subgroup of PAFs that,
intensity of noxious stimuli. Thus, it was
besides the fast excitatory transmitter glutamate,
demonstrated that, in dependence with the
may release BDNF together with other
strength of nociceptive stimulation of the C fibers,
pronociceptive modulators, chiefly substance
the pronociceptive modulator substance P may be
P. The other is, instead, inhibitory and involves
released together with glutamate above a certain
another subgroup of PAFs that release the fast
threshold of activation (Adelson et al. 2009).
transmitter glutamate but also GDNF together
Notably, substance P mediates the central sensiti-
with the anti-nociceptive modulator somatostatin
zation of nociceptors (Seybold 2009). Yet, NK1
(Merighi 2018).
receptors mainly lay outside the substantia
gelatinosa, and, thus, the peptide’s modulatory
222
Fig. 14.1 Schematic representation of the circuits
modulated by BDNF and GDNF in the substantia
gelatinosa of the spinal cord. C PAFs containing BDNF
(yellow) may be subdivided into two groups on the pres-
ence or absence of TrkB receptors (autoreceptors). They
also contain the neuropeptides CGRP and substance
P. The C PAFs containing GDNF (light blue) also contain
CGRP and somatostatin. Both BDNF and GDNF are
synthesized by subsets of DRG neurons and anterogradely
A. Merighi
transported to terminals. Under basal conditions or in
nociceptive (physiological) pain, the peptidergic C fibers
only release glutamate, but when a certain threshold of
stimulation is reached, they can also release BDNF or
GDNF with their co-stored neuropeptides according to
the pattern and rate of C fiber activation (bottom).
Interactions chiefly occur at the level of lamina II synaptic
glomeruli (colored circles). See text for further
information
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry
effects in lamina II are supposed to be inexistent
or primarily extrasynaptic, i.e., taking place
through volume transmission (Liu and
Sandkühler 1995). It is also worth of note that
substance P is co-stored with BDNF (and CGRP)
in large dense core vesicles (LGVs) within
peptidergic PAF terminals (Salio et al. 2007).
Therefore, one can reasonably suppose that the
two modulators are co-released from these
terminals upon LGV exocytosis (Merighi 2017).
Different from NK1 receptors, TrkB receptors
are highly expressed in lamina II, both pre- and
postsynaptically (Salio et al. 2005). We have pre-
viously demonstrated that the neurotrophin can
modulate sensory information in the substantia
gelatinosa with a combination of excitatory
(Merighi et al. 2008a) and inhibitory (Bardoni
et al. 2007) effects. PAFs can trigger the activa-
tion of the STT projection neurons indirectly,
along a minimally bi-neuronal chain of two
gelatinosa excitatory neurons, a central and a
vertical cell in sequence (Lu and Perl 2005;
Yasaka et al. 2007). It is remarkable that the
first- to second-order sensory neuron synapses in
lamina II do not simultaneously express TrkB
receptors pre- and postsynaptically (Salio et al.
2005). This indicates that the activation of den-
dritic postsynaptic TrkB receptors takes place at
simple axo-dendritic synapses or, more likely, at
the type Ib glomeruli (Fig. 14.1) made by the
peptidergic PAFs (Salio et al. 2005). Alterna-
tively, or in addition, BDNF can stimulate pre-
synaptic TrkB autoreceptors (Merighi et al.
2008a) at a diverse subset of type Ib glomeruli
(Fig. 14.1), as I will discuss later. As certain C
PAFs containing BDNF also express TRPV1
(above), they can release BDNF following capsa-
icin stimulation, i.e., a condition that mimics
inflammation (Merighi et al. 2008a). Remarkably
and in accord with the electrophysiological data
obtained after recordings from pairs of gelatinosa
neurons or stimulating inhibitory neurons with
the neurotrophin, the tetra-synaptic pathway
involving the two islet cells (inhibition of inhibi-
tion) (Fig. 14.1—path 2) may be the anatomical
substrate for the long-lasting excitation of several
223
lamina II neurons that occurs after capsaicin-
stimulated release of BDNF (Merighi et al.
2008a). The same pathway is also compatible
with the observed increase in the frequency of
GABAergic/glycinergic mIPSCs and reduction
of eIPSCs in lamina II neurons after BDNF chal-
lenge, under pharmacological block of excitatory
neurotransmission (Bardoni et al. 2007). This cir-
cuit thus may be the structural and functional
substrate for a disinhibition (i.e., excitation) of
the large vertical/stalked cells that is mediated
synergistically by BDNF and substance P acting
as co-transmitters after co-release by a subset of
peptidergic PAFs. Additionally, it may be central
in the long-lasting central sensitization of the
gelatinosa synapses under certain inflammatory
conditions and/or neuropathic pain (Merighi
et al. 2008a, b).
14.4.2 GDNF and the Inhibitory Path
The peptidergic PAFs containing GDNF
(together with somatostatin) arise from a subpop-
ulation of LA4-immunoreactive type C DRG
neurons that, before the discovery of GDNF-
expressing DRG neurons and the association of
GDNF with nociception, were assumed to have
atypical neurochemical and functional features
(Averill et al. 1995). These neurons are roughly
equivalent in number to those expressing BDNF
and substance P (Merighi 2018). Their central
projections, i.e., the C PAFs formed by these
cells, contain glutamate as the chief excitatory
neurotransmitter together with GDNF and
somatostatin that negatively modulate
nociception. In keeping with the well-known
simultaneous presence of transmitters with
contrasting function in these fibers, a study has
demonstrated that somatostatin-expressing PAFs
are pruritoceptive, rather than nociceptive, and
that the peptide, instead, counteracts nociception
(Huang et al. 2018). Therefore, as the C fibers
directly excite the islet, central, radial, and verti-
cal cells (Yasaka et al. 2007), it seems possible
that, under basal conditions, the GDNF/
224
somatostatin peptidergic C PAFs exert a tonic
inhibition onto the projection neurons in lamina
I. The underlying circuitry (Fig. 14.1—path 3)
comprises a type Ib glomerulus made by the C
bouton of the GDNF/somatostatin C PAF and an
inhibitory central cell that forms an axo-dendritic
synapse onto the vertical cell and, thus, reduces
the outflow of excitatory information from lamina
II. Like it is the case for BDNF/substance P C
PAFs, tonic inhibition through this circuit may be
modulated by the differential release of the fast
transmitter glutamate and the slow-acting
modulators GDNF and somatostatin according
to the intensity of the C fiber activation. GDNF
could also hyperpolarize the C boutons of IB4+
non-peptidergic PAFs expressing GFRα1 at type
Ia glomeruli. The ensuing experimental proofs
support such a possibility: (1) somatostatin
promotes central analgesia (Pintér et al. 2006)
via tonic inhibition of C and Aδ PAFs (Guo
et al. 2008; Wang et al. 2009) and decrease of
the glutamate-dependent stimulation of these
fibers (Luo et al. 2010); (2) GDNF, binding to
GFRα1, exerts a sustained inhibition of the
glutamatergic excitatory drive of the gelatinosa
neurons (Salio et al. 2014); and (3) the large
vertical cells are postsynaptic to non-peptidergic
PAFs at type Ia glomeruli (Maxwell et al. 2007),
thus being the principal targets of IB4+
non-peptidergic PAFs. It is also possible that
synaptic interactions are much more composite,
as the vertical cells, in turn, give rise to V1
profiles at the same type of glomeruli (Merighi
2018). Additionally, peptidergic PAFs containing
GDNF and somatostatin may be inhibitory at type
Ib glomeruli of lamina IIid that have GFRα1-
immunoreactive dendrites (Salio et al. 2014).
These dendrites are likely to stem from lamina
I–II neurons, perhaps the transient or tonic central
cells, which express GFRα1 (Ortega-de San Luis
and Pascual 2016). In keeping with this prospect,
intrathecal GDNF inhibited the thermal hypersen-
sitivity induced by substance P (Malcangio et al.
2002), and GDNF presynaptically reduced the
release of substance P and CGRP from
capsaicin-stimulated PAFs (Salio et al. 2014).
A. Merighi
14.4.3 Cross Talk Between BDNF
and GDNF in Nociceptive
Pathways
As discussed in the previous sections, BDNF and
GDNF are expressed by two distinct populations
of peptidergic nociceptors (Salio and Ferrini
2016), and, while absolutely pronociceptive or
anti-nociceptive effects cannot be specifically
linked to one or the other neurotrophic factor,
there is a broad agreement that in neuropathic
pain, BDNF is pronociceptive, while GDNF is
anti-nociceptive (Merighi et al. 2008b; Merighi
2018; Merighi 2016). Many studies in the previ-
ous years have discovered how pathological
situations disturb the expression of BDNF and
GDNF in the nervous system, thus proving the
intervention of both molecules in the shift from
physiology to pathology. In several
circumstances, the levels of the two factors
change along divergent directions, an observation
that underpins the notion that BDNF and GDNF
play alternative/complementary roles in the noci-
ceptive system. Nonetheless, only few studies
have focused onto the direct interactions between
BDNF and GDNF in the modulation of central
neurotransmission. Based on the structural and
functional observations resumed previously, I
here propose that the interaction between the
two molecules in lamina II of the spinal cord
primarily takes place at synaptic glomeruli that
are particularly abundant in the substantia
gelatinosa (Ribeiro-Da-Silva and Coimbra
1982). Glomeruli are crucial to the process of
presynaptic modulation of sensory information
collected from periphery and conveyed to the
superficial dorsal horn (Ribeiro-da-Silva 2015;
Willis and Coggeshall 2004). A central axonal
bouton (C bouton) made by a PAF, surrounded
by several dendrites and a few axon terminals,
forms the glomeruli, which are separated by an
incomplete glial envelope from the surrounding
neuropil. There are three types of glomeruli in
primates, whereas four types have been described
in rodents (Merighi et al. 2008b). Rat (Coimbra
et al. 1974; Ribeiro-Da-Silva et al. 1985; Ribeiro-
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry 225

Da-Silva and Coimbra 1982) and mouse (Hiura glomeruli with a C bouton expressing GFRα1
et al. 1991; Marker et al. 2006; Salio et al. 2005; (Salio et al. 2014).
Salio et al. 2014) glomeruli only display slight
The peptidergic C PAFs that contain a mix of
ultrastructural differences. In their seminal work,
fast-acting (glutamate) and slow-acting
Ribeiro-da-Silva and Coimbra (1982) divided rat
transmitters are thus crucial in regulating the out-
glomeruli in two types (I and II) with different
flow of nociceptive information from the
ultrastructure (Ribeiro-Da-Silva and Coimbra
substantia gelatinosa in relation to the possibility
1982). Subsequent studies added substantial
that the two types of transmitters are differentially
information onto the differences in the neuro-
released according to the pattern of firing, even-
chemistry of the two categories. Thus, type I
tually leading to the release of glutamate without
glomeruli made by the unmyelinated C PAFs
or with coexisting growth factors or peptides
may be non-peptidergic (type Ia) or peptidergic
(Merighi 2017). It seems possible that under
(type Ib) on the absence or presence of LGVs
basal conditions and in nociceptive pain, only
containing neuropeptides and other high MW
glutamate is released from these fibers, albeit
modulators such as BDNF or GDNF at their C
one can hold that, as discussed previously, the
boutons. Type II glomeruli can also be further
peptidergic C PAFs containing GDNF exert a
subdivided into type IIa and type IIb based on
tonic inhibition onto the peptidergic C fibers
fine ultrastructural features. The C bouton in type
expressing BDNF at the type Ib glomeruli
IIa originates from a poorly myelinated Aδ PAF,
possessing GFRα1 peripheral dendrites
whereas that in type IIb derives from a myelinated
(Fig. 14.1) and thus that GDNF might be released
Aαβ PAF (Coimbra et al. 1984). Remarkably, the
also under basal conditions. When long-lasting
C boutons in type II glomeruli do not contain high
pain ensues, high MW transmitters are released
MW modulators and are, consequently, devoid
together with glutamate. In this case, the
of LGVs.
peptidergic C PAFs containing BDNF release
In relation to their BDNF or GDNF content
the growth factor that exerts its pronociceptive
and expression of the related receptors, type I
effects by enhancing the excitatory drive of lam-
glomeruli form four distinct subpopulations
ina II output large vertical cells, with the interpo-
(Fig. 14.1):
sition of a central cell (Fig. 14.1—path 1) and/or
1. The type Ia glomeruli made by with the intervention of two islet cells in series
non-peptidergic C fibers that are labelled by (Fig. 14.1—path 2). The pronociceptive effect to
IB4 and express GFRα1 (Salio et al. 2014). BDNF can be further increased by stimulation of
2. The type Ib glomeruli made by peptidergic C the TrkB autoreceptors that are present at certain
fibers that are endowed with LGVs containing type Ib glomeruli, as the activation of these pre-
BDNF herein stored with CGRP and sub- synaptic TrkB receptors leads to an additional
stance P (Salio et al. 2007). This second release of glutamate and substance P from C
group of glomeruli also expresses TrkB at PAFs (Merighi et al. 2008a). The effect of
their C boutons (Salio et al. 2005) and BDNF onto nociceptive transmission can be
GFRα1 (Salio et al. 2014) at dendrites. counteracted by GDNF in several different ways
3. The type Ib glomeruli made by peptidergic C (Merighi 2018):
fibers that are provided with LGVs containing
1. The GDNF released at the C boutons of
BDNF, CGRP, and substance P, but devoid of
peptidergic type Ib glomeruli acts onto the
TrkB, which is, instead, expressed at some
GFRα1 glomerular dendrites. These dendrites
glomerular dendrites (Salio et al. 2005).
may belong to a galanin-expressing inhibitory
4. The type Ib glomeruli made by peptidergic C
lamina II interneuron (Merighi 2018). This
fibers that are provided with LGVs containing
results in depressing the excitation of the
GDNF, CGRP, and somatostatin. The overall
large vertical cells (Fig. 14.1—path 3).
picture is completed by a number of type IIa
226
2. The GDNF released at the C boutons of
peptidergic type Ib glomeruli acts onto the
IB4+ non-peptidergic C PAFs expressing
GFRα1 and engaged in type Ia glomeruli.
3. The GDNF released at the C boutons of
peptidergic type Ib glomeruli acts onto the
IB4+ non-peptidergic Aδ PAFs expressing
GFRα1 and engaged in type IIa glomeruli.
4. The GDNF released at the C boutons of
peptidergic type Ib glomeruli acts onto the
Aβ PAFs expressing GFRα1 in lamina III.
14.5 Concluding Remarks
I have here tried to provide a comprehensive
portrait of the neuronal circuitry in lamina II that
represents the structural substrate for the
interactions of BDNF and GDNF in the mature
spinal cord. This portrait is somewhat speculative
as our knowledge of the synaptic connections
between the PAFs and the lamina II neurons is
incomplete, and even less information is available
on the connections between neurons inside the
gelatinosa. This paucity of knowledge depends
on several reasons: (1) the difficulty in achieving
a sound morphological and functional classifica-
tion of the lamina II neurons, (2) the complexity
in recording simultaneously from two neurons
and to combine electrophysiological results with
structural information, (3) the still challenging
immunocytochemical approach at the ultrastruc-
tural level, and (4) the even more challenging
feasibility to perform reliable quantitative studies.
Yet, we are beginning to understand the impor-
tance of high MW neurotransmission in the spinal
somatosensory system and, in particular, the
importance of the cross talk between different
subsets of peptidergic PAFs in the modulation
of physiological pain and in the transition to
long-lasting pathological pain.
References
Adelson D, Lao L, Zhang G, Kim W, Marvizòn JC (2009)
Substance P release and neurokinin 1 receptor
A. Merighi
activation in the rat spinal cord increases with the firing
frequency of C-fibers. Neuroscience 161:538–553
Airaksinen MS, Saarma M (2002) The GDNF family:
signalling, biological functions and therapeutic value.
Nat Rev Neurosci 3:383–394
Averill S, McMahon SB, Clary DO, Reichardt LF,
Priestley JV (1995) Immunocytochemical localization
of trkA receptors in chemically identified subgroups of
adult rat sensory neurons. Eur J Neurosci 7
(7):1484–1494
Bardoni R, Merighi A (2009) BDNF and trkB mediated
mechanisms in the spinal cord. In: Malcangio M
(ed) Synaptic plasticity in pain. Springer, New York
Bardoni R, Ghirri A, Salio C, Prandini M, Merighi A
(2007) BDNF-mediated modulation of GABA and
glycine release in dorsal horn lamina II from postnatal
rats. Dev Neurobiol 67:960–975
Bennett GJ, Abdelmoumene M, Hayashi H, Dubner R
(1980) Physiology and morphology of substantia
gelatinosa neurons intracellularly stained with horse-
radish peroxidase. J Comp Neurol 194:809–827
Bennett GJ, Ruda MA, Gobel S, Dubner R (1982) Enkeph-
alin immunoreactive stalked cells and islet cells in
lamina IIb of the cat substantia gelatinosa. Brain Res
240:162–166
Bennett DL, Michael GJ, Ramachandran N, Munson JB,
Averill S, Yan Q, McMahon SB, Priestley JV (1998) A
distinct subgroup of small DRG cells express GDNF
receptor components and GDNF is protective for these
neurons after nerve injury. J Neurosci 18:3059–3072
Bennett DL, Boucher TJ, Armanini MP, Poulsen KT,
Michael GJ, Priestley JV, Phillips HS, McMahon SB,
Shelton DL (2000) The glial cell line-derived
neurotrophic factor family receptor components are
differentially regulated within sensory neurons after
nerve injury. J Neurosci 20:427–437
Blum R, Konnerth A (2005) Neurotrophin-mediated rapid
signaling in the central nervous system: mechanisms
and functions. Physiology 20:70–78
Borsook D, Becerra L (2011) CNS animal fMRI in pain
and analgesia. Neurosci Biobehav Rev 35:1125–1143
Bradbury EJ, Burnstock G, McMahon SB (1998) The
expression of P2X3 purinoreceptors in sensory
neurons: effects of axotomy and glial-derived
neurotrophic factor. Mol Cell Neurosci 12:256–268
Brown AG, Fyffe RE (1981) Form and function of dorsal
horn neurones with axons ascending the dorsal
columns in cat. J Physiol 321:31–47
Bushnell MC, Duncan GH (1989) Sensory and affective
aspects of pain perception: is medial thalamus
restricted to emotional issues? Exp Brain Res 78
(2):415–418
Cervero F, Connell LA (1984) Distribution of somatic and
visceral primary afferent fibres within the thoracic spi-
nal cord of the cat. J Comp Neurol 230:88–98
Cervero F, Laird JMA (1999) Visceral pain. Lancet
353:2145–2148
Chao MV, Hempstead BL (1995) p75 and Trk: a
two-receptor system. Trends Neurosci 18:321–326
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry
Coimbra A, Sodré-Borges BP, Magalhaes MM (1974) The
substantia gelatinosa Rolandi of the rat. Fine structure,
cytochemistry (acid phosphatase) and changes after
dorsal root section. J Neurocytol 3:199–217
Coimbra A, Ribeiro-Da-Silva A, Pignatelli D (1984)
Effects of dorsal rhizotomy on the several types of
primary afferent terminals in laminae I-III of the rat
spinal cord. An electron microscope study. Anat
Embryol (Berl) 170:279–287
Conner JM, Lauterborn JC, Yan Q, Gall CM, Varon S
(1997) Distribution of brain-derived neurotrophic fac-
tor (BDNF) protein and mRNA in the normal adult rat
CNS: evidence for anterograde axonal transport. J
Neurosci 17:2295–2313
Cruz L, Basbaum AI (1985) Multiple opioid peptides and
the modulation of pain: immunohistochemical analysis
of dynorphin and enkephalin in the trigeminal nucleus
caudalis and spinal cord of the cat. J Comp Neurol
240:331–348
Duan B, Cheng L, Bourane S, Britz O, Padilla C, Garcia-
Campmany L, Krashes M, Knowlton W, Velasquez T,
Ren X, S-á R, B-á L, Wang Y, Goulding M, Ma Q
(2014) Identification of spinal circuits transmitting and
gating mechanical pain. Cell 159:1417–1432
Ernfors P, Merlio JP, Persson H (1992) Cells expressing
mRNA for Neurotrophins and their receptors during
embryonic rat development. Eur J Neurosci
4:1140–1158
Gebhart GF (2000) J.J. Bonica lecture--2000: physiology,
pathophysiology, and pharmacology of visceral pain.
Reg Anesth Pain Med 25:632–638
Gibon J, Barker PA (2017) Neurotrophins and proneuro-
trophins: focus on synaptic activity and plasticity in the
brain. Neuroscientist 23:587–604
Giesler GJ Jr, Cannon JT, Urca G, Liebeskind JC (1978)
Long ascending projections from substantia gelatinosa
Rolandi and the subjacent dorsal horn in the rat. Sci-
ence 202:984–986
Gobel S (1975) Golgi studies of the substantia gelatinosa
neurons in the spinal trigeminal nucleus. J Comp
Neurol 162:397–416
Gobel S (1978a) Golgi studies of the neurons in layer I of
the dorsal horn of the medulla (trigeminal nucleus
caudalis). J Comp Neurol 180:375–394
Gobel S (1978b) Golgi studies of the neurons in layer II of
the dorsal horn of the medulla (trigeminal nucleus
caudalis). J Comp Neurol 180:395–413
Gold MS, Gebhart GF (2010) Nociceptor sensitization in
pain pathogenesis. Nat Med 16(11):1248–1257
Golden JP, Hoshi M, Nassar MA, Enomoto H, Wood JN,
Milbrandt J, Gereau RW, Johnson EM Jr, Jain S (2010)
RET signaling is required for survival and normal
function of nonpeptidergic nociceptors. J Neurosci
30:3983–3994
Groenewegen HJ (1988) Organization of the afferent
connections of the mediodorsal thalamic nucleus in
the rat, related to the mediodorsal-prefrontal topogra-
phy. Neuroscience 24:379–431
227
Grudt TJ, Perl ER (2002) Correlations between neuronal
morphology and electrophysiological features in the
rodent superficial dorsal horn. J Physiol 540:189–207
Guo Y, Yao FR, Cao DY, Pickar JG, Zhang Q, Wang HS,
Zhao Y (2008) Somatostatin inhibits activation of
dorsal cutaneous primary afferents induced by anti-
dromic stimulation of primary afferents from an adja-
cent thoracic segment in the rat. Brain Res 1229:61–71
Hantman AW, Perl ER (2005) Molecular and genetic
features of a labeled class of spinal substantia
gelatinosa neurons in a transgenic mouse. J Comp
Neurol 492:90–100
Hantman AW, van den Pol AN, Perl ER (2004) Morpho-
logical and physiological features of a set of spinal
substantia gelatinosa neurons defined by green fluores-
cent protein expression. J Neurosci 24:836–842
Hiura A, Ishizuka H, Villalobos EL (1991) GABAergic
neurons in the mouse superficial dorsal horn with spe-
cial emphasis on their relation to primary afferent cen-
tral terminals. Arch Histol Cytol 54:195–206
Holstege JC, Jongen JL, Kennis JH, Rooyen-Boot AA,
Vecht CJ (1998) Immunocytochemical localization of
GDNF in primary afferents of the lumbar dorsal horn.
Neuroreport 9:2893–2897
Huang J, Polgár E, HJr S, Mishra SK, Tseng PY,
Iwagaki N, Boyle KA, Dickie AC, Kriegbaum MC,
Wildner H, Zeilhofer HU, Watanabe M, Riddell JS,
Todd AJ, Hoon MA (2018) Circuit dissection of the
role of somatostatin in itch and pain. Nat Neurosci
21:707–716
Jongen JL, Jaarsma D, Hossaini M, Natarajan D, Haasdijk
ED, Holstege JC (2007) Distribution of RET immuno-
reactivity in the rodent spinal cord and changes after
nerve injury. J Comp Neurol 500:1136–1153
Josephson A, Widenfalk J, Trifunovski A, Widmer HR,
Olson L, Spenger C (2001) GDNF and NGF family
members and receptors in human fetal and adult spinal
cord and dorsal root ganglia. J Comp Neurol
440:204–217
Kashiba H, Uchida Y, Senba E (2003) Distribution and
colocalization of NGF and GDNF family ligand recep-
tor mRNAs in dorsal root and nodose ganglion neurons
of adult rats. Brain Res Mol Brain Res 110:52
Kato G, Kawasaki Y, Ji RR, Strassman AM (2007) Differ-
ential wiring of local excitatory and inhibitory synaptic
inputs to islet cells in rat spinal lamina II demonstrated
by laser scanning photostimulation. J Physiol
580:815–833
Khan N, Smith MT (2015) Neurotrophins and neuropathic
pain: role in pathobiology. Molecules 20:10657–10688
Koerber HR (2009) Nociceptors: adequate stimulus. In:
Binder MD, Hirokawa N, Windhorst U (eds) Encyclo-
pedia of neuroscience. Springer, Berlin, Heidelberg, pp
2873–2876
Li L, Rutlin M, Abraira VE, Cassidy C, Kus L, Gong S,
M-á J, Luo W, Heintz N, Koerber H, Woodbury C,
Ginty DD (2011) The functional organization of cuta-
neous low-threshold mechanosensory neurons. Cell
147:1615–1627
228
Light AR, Kavookjian AM (1988) Morphology and ultra-
structure of physiologically identified substantia
gelatinosa (lamina II) neurons with axons that termi-
nate in deeper dorsal horn laminae (III-V). J Comp
Neurol 267:172–189
Light AR, Perl ER (1979) Reexamination of the dorsal
root projection to the spinal dorsal horn including
observations on the differential termination of coarse
and fine fibers. J Comp Neurol 186:117–131
Liu XG, Sandkühler J (1995) The effects of extrasynaptic
substance P on nociceptive neurons in laminae I and II
in rat lumbar spinal dorsal horn. Neuroscience
68:1207–1218
Lorenzo LE, Ramien M, St.Louis M, De Koninck Y,
Ribeiro-da-Silva A (2008) Postnatal changes in the
Rexed lamination and markers of nociceptive afferents
in the superficial dorsal horn of the rat. J Comp Neurol
508:592–604
Lu Y, Perl ER (2003) A specific inhibitory pathway
between substantia gelatinosa neurons receiving direct
C-fiber input. J Neurosci 23:8752–8758
Lu Y, Perl ER (2005) Modular organization of excitatory
circuits between neurons of the spinal superficial dorsal
horn (laminae I and II). J Neurosci 25:3900–3907
Luo R, Guo Y, Cao DY, Pickar JG, Li L, Wang J, Zhao Y
(2010) Local effects of octreotide on glutamate-evoked
activation of Aδ and C afferent fibers in rat hairy skin.
Brain Res 1322:50–58
Malcangio M, Getting SJ, Grist J, Cunningham JR,
Bradbury EJ, Charbel IP, Lever IJ, Pezet S, Perretti
M (2002) A novel control mechanism based on GDNF
modulation of somatostatin release from sensory
neurones. FASEB J 16:730–732
Marker CL, Lujan R, Colon J, Wickman K (2006) Distinct
populations of spinal cord lamina II interneurons
expressing G-protein-gated potassium channels. J
Neurosci 26:12251–12259
Maxwell DJ, Belle MD, Cheunsuang O, Stewart A, Morris
R (2007) Morphology of inhibitory and excitatory
interneurons in superficial laminae of the rat dorsal
horn. J Physiol 584:521–533
McMahon SB, Dmitrieva N, Koltzenburg M (1995) Vis-
ceral pain. Br J Anaesth 75:132–144
Melzack R, Wall PD (1965) Pain mechanisms: a new
theory. Science 150:971–980
Merighi A (2016) Targeting the glial-derived neurotrophic
factor and related molecules for controlling normal and
pathologic pain. Exp Opin Ther Targets 20:193–208
Merighi A (2017) Neuropeptides and Coexistence. In:
Reference module in neuroscience and biobehavioral
psychology. Elsevier. isbn:9780128093245,
Amsterdam. https://doi.org/10.1016/B978-0-12-
809324-5.02310-5
Merighi A (2018) The histology, physiology, neurochem-
istry and circuitry of the substantia gelatinosa Rolandi
(lamina II) in mammalian spinal cord. Prog Neurobiol
169:91–134
Merighi A, Carmignoto G, Gobbo S, Lossi L, Salio C,
Vergnano AM, Zonta M (2004) Neurotrophins in
A. Merighi
spinal cord nociceptive pathways. Prog Brain Res
146:291–321
Merighi A, Bardoni R, Salio C, Lossi L, Ferrini F,
Prandini M, Zonta M, Gustincich S, Carmignoto G
(2008a) Presynaptic functional trkB receptors mediate
the release of excitatory neurotransmitters from pri-
mary afferent terminals in lamina II (substantia
gelatinosa) of postnatal rat spinal cord. Dev Neurobiol
68:457–475
Merighi A, Salio C, Ghirri A, Lossi L, Ferrini F, Betelli C,
Bardoni R (2008b) BDNF as a pain modulator. Prog
Neurobiol 85:297–317
Michael GJ, Averill S, Nitkunan A, Rattray M, Bennett
DLH, Yan Q, Priestley JV (1997) Nerve growth factor
treatment increases brain-derived neurotrophic factor
selectively in trk-a expressing dorsal root ganglion
cells and in their central terminations within the spinal
cord. J Neurosci 17:8476–8490
Mogil JS (2019) The translatability of pain across species.
Phil Trans R Soc London B374:20190286
Molliver DC, Snider WD (1997) Nerve growth factor
receptor TrkA is down-regulated during postnatal
development by a subset of dorsal root ganglion
neurons. J Comp Neurol 381:428–438
Molliver DC, Wright DE, Leitner ML, Parsadanian AS,
Doster K, Wen D, Yan Q, Snider WD (1997)
IB4-binding DRG neurons switch from NGF to
GDNF dependence in early postnatal life. Neuron
19:849–861
Ohta K, Inokuchi T, Gen E, Chang J (2001) Ultrastructural
study of anterograde transport of glial cell line-derived
neurotrophic factor from dorsal root ganglion neurons
of rats towards the nerve terminal. Cells Tiss Organs
169:410–421
Ortega-de San Luis C, Pascual A (2016) Simultaneous
detection of both GDNF and GFRα1 expression
patterns in the mouse central nervous system. Front
Neuroanat 10:73
Ossipov MH (2011) Growth factors and neuropathic pain.
Curr Pain Headache Rep 15:185–192
Pan YZ, Pan HL (2004) Primary afferent stimulation dif-
ferentially potentiates excitatory and inhibitory inputs
to spinal lamina II outer and inner neurons. J
Neurophysiol 91:2413–2421
Pintér E, Helyes Z, Szolcsanyi J (2006) Inhibitory effect of
somatostatin on inflammation and nociception.
Pharmacol Ther 112:440–456
Polgár E, Al Ghamdi KS, Todd AJ (2010) Two
populations of neurokinin 1 receptor-expressing pro-
jection neurons in lamina I of the rat spinal cord that
differ in AMPA receptor subunit composition and den-
sity of excitatory synaptic input. Neuroscience
167:1192–1204
Punnakkal P, von Schoultz C, Haenraets K, Wildner H,
Zeilhöfer HU (2014) Morphological, biophysical and
synaptic properties of glutamatergic neurons of the
mouse spinal dorsal horn. J Physiol 592:759–776
Reichardt LF (2006) Neurotrophin-regulated signalling
pathways. Philos Trans R Soc Lond B 361:1545–1564
14 Cross Talk of BDNF and GDNF in Spinal Substantia Gelatinosa (Lamina II): Focus on Circuitry
Rexed B (1952) The cytoarchitectonic organisation of the
spinal cord of the cat. J Comp Neurol 96:415–496
Rexed B (1954) A cytoarchitectonic atlas of the spinal
cord in the cat. J Comp Neurol 100:297–379
Ribeiro-da-Silva A (2015) Chapter 7 – Substantia
gelatinosa of the spinal cord – Paxinos, George. In:
The rat nervous system, 4th edn. Academic Press, San
Diego, pp 97–114
Ribeiro-Da-Silva A, Coimbra A (1982) Two types of
synaptic glomeruli and their distribution in laminae
I-III of the rat spinal cord. J Comp Neurol
209:176–186
Ribeiro-Da-Silva A, De Koninck Y (2008) Morphological
and neurochemical organization of the spinal dorsal
horn. In: Albright TD, Albright TD, Masland RH et
al (eds) The senses: a comprehensive reference. Aca-
demic Press, New York, pp 279–310
Ribeiro-Da-Silva A, Pignatelli D, Coimbra A (1985) Syn-
aptic architecture of glomeruli in superficial dorsal
horn of rat spinal cord, as shown in serial
reconstructions. J Neurocytol 14:203–220
Ribeiro-Da-Silva A, Pioro EP, Cuello AC (1991) Sub-
stance P- and enkephalin-like immunoreactivities are
colocalized in certain neurons of the substantia
gelatinosa of the rat spinal cord: an ultrastructural
double-labeling study. J Neurosci 11:1068–1080
Salio C, Ferrini F (2016) BDNF and GDNF expression in
discrete populations of nociceptors. Ann Anat
207:55–61
Salio C, Lossi L, Ferrini F, Merighi A (2005) Ultrastruc-
tural evidence for a pre- and post-synaptic localization
of full length trkB receptors in substantia gelatinosa
(lamina II) of rat and mouse spinal cord. Eur J Neurosci
22:1951–1966
Salio C, Averill S, Priestley JV, Merighi A (2007)
Costorage of BDNF and neuropeptides within individ-
ual dense-core vesicles in central and peripheral
neurons. Dev Neurobiol 67:326–338
Salio C, Ferrini F, Muthuraju S, Merighi A (2014) Presyn-
aptic modulation of spinal nociceptive transmission by
glial cell line-derived neurotrophic factor (GDNF). J
Neurosci 34:13819–13833
Schoenen J (1982) Dendritic organization of the human
spinal cord: the dorsal horn. Neuroscience
7:2057–2087
Sengupta JN (2009) Visceral pain: the neurophysiological
mechanism. Handbook Exp Pharmacol 194:31–74
Seybold VS (2009) The role of peptides in central sensiti-
zation. In: Canning BJ, Spina D (eds) Sensory nerves.
Springer, Berlin, Heidelberg, pp 451–491
Sugiura Y, Terui N, Hosoya Y (1989) Difference in distri-
bution of central terminals between visceral and
somatic unmyelinated (C) primary afferent fibers. J
Neurophysiol 62:834–840
Surmeier DJ, Honda CN, Willis WD Jr (1988) Natural
groupings of primate spinothalamic neurons based on
229
cutaneous stimulation. Physiological and anatomical
features. J Neurophysiol 59:833–860
Thompson SJ, Bushnell MC (2012) Rodent functional and
anatomical imaging of pain. Neurosci Lett
520:131–139
Todd AJ, McKenzie J (1989) GABA-immunoreactive
neurons in the dorsal horn of the rat spinal cord. Neu-
roscience 31:799–806
Todd AJ, Hughes DI, Polgàr E, Nagy GG, Mackie M,
Ottersen OP, Maxwell DJ (2003) The expression of
vesicular glutamate transporters VGLUT1 and
VGLUT2 in neurochemically defined axonal
populations in the rat spinal cord with emphasis on
the dorsal horn. Eur J Neurosci 17:13–27
Wang J, Guo Y, Cao DY, Luo R, Ma SJ, Wang HS, Pickar
JG, Zhao Y (2009) Tonic inhibition of somatostatin on
C and Aδ afferent fibers in rat dorsal skin in vivo. Brain
Res 1288:50–59
Widenfalk J, Lundstromer K, Jubran M, Brene S, Olson L
(2001) Neurotrophic factors and receptors in the imma-
ture and adult spinal cord after mechanical injury or
kainic acid. J Neurosci 21:3457–3475
Willis WD Jr, Coggeshall RE (2004) Structure of the
dorsal horn. In: Sensory mechanisms of the spinal
cord – primary afferent neurons and the spinal dorsal
horn, 3rd edn. Kluver Academic/Plenum Publisher,
New York, pp 155–186
Willis WD, Leonard RB, Kenshalo DRJ (1978)
Spinothalamic tract neurons in the substantia
gelatinosa. Science 202:986–988
Woodbury CJ, Ritter AM, Koerber HR (2000) On the
problem of lamination in the superficial dorsal horn
of mammals: a reappraisal of the substantia gelatinosa
in postnatal life. J Comp Neurol 417:88–102
Yasaka T, Kato G, Furue H, Rashid MH, Sonohata M,
Tamae A, Murata Y, Masuko S, Yoshimura M (2007)
Cell-type-specific excitatory and inhibitory circuits
involving primary afferents in the substantia gelatinosa
of the rat spinal dorsal horn in vitro. J Physiol
581:603–618
Yasaka T, Tiong SY, Hughes DI, Riddell JS, Todd AJ
(2010) Populations of inhibitory and excitatory
interneurons in lamina II of the adult rat spinal dorsal
horn revealed by a combined electrophysiological and
anatomical approach. Pain 151:475–488
Yasaka T, Tiong SY, Polgár E, Watanabe M, Kumamoto
E, Riddell JS, Todd AJ (2014) A putative relay circuit
providing low-threshold mechanoreceptive input to
lamina I projection neurons via vertical cells in lamina
II of the rat dorsal horn. Mol Pain 10:1744–8069
Zheng J, Lu Y, Perl ER (2010) Inhibitory neurones of the
spinal substantia gelatinosa mediate interaction of
signals from primary afferents. J Physiol
588:2065–2075
 Part V
Clinical Medicine
 BDNF Gene as a Precision Skill of Obesity
Management 15
Helena Marcos-Pasero, Elena Aguilar-Aguilar,
Maria P. Ikonomopoulou, and Viviana Loria-Kohen

Abstract various GWASs. Finally, we assess gene-diet

The scarcity of the results obtained for the
treatment of obesity leads us to consider new
strategies, contemplating all the factors
involved in the development of the disease.
One of the key molecules for controlling
body weight and energy homeostasis is the
brain-derived neurotrophic factor (BDNF).
This work summarizes the mechanisms in
which BDNF gene regulates this multifactorial
disease. In addition, we discuss the role of
other BDNF polymorphisms as genetic
determinants of obesity. In this context, a
total of 14 SNPs near or inside BDNF/
BDNF-AS related to BMI were identified in
interaction as a novel tool to prevent obesity
and formulate solid and personalized
nutritional management. Our research group
has performed the first study on the association
of BDNF-AS rs925946 polymorphism and cal-
cium intake as potential modulators of the
nutritional status. Although these results
should be confirmed in future studies, they
open the path for new prevention
opportunities.
15.1 Introduction

Obesity is a complex, multifactorial, and

H. Marcos-Pasero · E. Aguilar-Aguilar
Nutrition and Clinical Trials Unit, GENYAL Platform,
IMDEA-Food Institute, CEI UAM + CSIC, Madrid, Spain
e-mail: helena.marcos@imdea.org; elena.aguilar@imdea.
org
M. P. Ikonomopoulou
Translational Venomics Group, IMDEA-Food, CEI UAM
+CSIC, Madrid, Spain
Institute for Molecular Bioscience, The University of
Queensland, St Lucia, Australia
e-mail: maria.ikonomopoulou@imdea.org
V. Loria-Kohen (*)
Nutrition and Clinical Trials Unit, GENYAL Platform,
IMDEA-Food Institute, CEI UAM + CSIC, Madrid, Spain
Department of Nutrition and Food Science, Faculty of
Pharmacy, Complutense University of Madrid, Madrid,
Spain
e-mail: viviana.loria@imdea.org; vloria@ucm.es
preventable disease whose high prevalence
makes it an important public health problem.
According to the World Health Organization
(WHO), more than 1.9 billion individuals over
18 years had overweight, and more than 650 mil-
lion were obese in 2016. In addition, the preva-
lence of obesity has been tripled since 1975
(Obesity and Overweight 2019). Obesity
increases the risk of a large number of cardiovas-
cular, metabolic, and pulmonary diseases and
deaths (Hruby and Hu 2015).
At early ages these values are also alarming. It
is estimated that more than 340 million children
between 5 and 19 years old are overweight or
obese and that prevalence has been increased

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 233
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_15
234
from 4% in 1975 to 18% in 2016 (Obesity and
Overweight 2019).
Obesity and its many medical complications
emerge from the dysregulation of energy homeo-
stasis. Excessive weight gain might also arise
from alterations in reward systems of the brain
that drive consumption of calorie-dense and pal-
atable foods in the absence of an energy require-
ment (Rios 2014).
Energy balance leads us to hypothesize that
obesity is caused by a mere increase of energy
intake. However, the evidence does not seem to
support this argument. On the contrary, there are
indicators that the origin of this disease is
underlined by numerous interrelated factors
(genetic, social, economic, etc.) that make obesity
a highly complex condition (Hruby and Hu
2015).
The scarcity of the results obtained from obe-
sity treatment leads us to consider new strategies,
contemplating all the factors involved in the
development of this disease in order to find
novel therapeutic avenues for its treatment and
prevention as well as its associated medical
afflictions.
15.2 Implication of the BDNF
in Obesity and Eating
Disorders
Complex interactions between the brain and
peripheral tissues mediate the effective control
of energy balance and body weight. One of the
key molecules for body weight control and
energy homeostasis is the brain-derived
neurotrophic factor (BDNF). This neurotrophin
(NT) has been extensively studied to elucidate its
function as a potential target against obesity
(Lebrun et al. 2006; Rosas-Vargas et al. 2011).
The first study which suggested a role for
BDNF in the regulation of food intake was carried
out in 1992 by Lapchak and Hefti, who described
that the central infusion of BDNF reduces weight
gain in rats (Lapchak and Hefti 1992). Later on, it
was shown that infusion of BDNF in the lateral
ventricle of adult rats caused dose-dependent,
severe appetite suppression and weight loss
(Pelleymounter et al. 1995). In general, different
H. Marcos-Pasero et al.
studies have suggested that BDNF may regulate
obesity via different mechanisms.
15.2.1 Role of BDNF in Energy
Homeostasis and Food Intake
Different studies using mouse models argue in
favor of an endocrine role of BDNF in energy
homeostasis to modulate food intake and
glycemia. In db/db mice (nonfunctional leptin
receptor mice provide a model for obesity and
non-insulin-dependent diabetes mellitus), contin-
uous treatment with BDNF over several days has
been shown to decrease food intake and lower
blood glucose to normoglycemic levels. BDNF
treatment sustained body temperature and oxygen
consumption during food restriction, probably
due to an increase in energy expenditure
(Nakagawa et al. 2000). In addition, BDNF-
treated db/db mice and vehicle-treated pair-fed
controls display a similar pattern of weight loss.
Similar observation on energy expenditure has
been made on the acute effects of BDNF in db/
db mice, where a single injection largely
prevented fasting-induced hypothermia and cold
exposure-induced hypothermia whereas the body
weight and blood glucose were equally reduced in
treated and vehicle-treated groups. BDNF treat-
ment has also been shown to increase whole-body
glucose turnover, and to improve serum lipid
parameters in db/db mice. All these results high-
light that BDNF treatment improves the erratic
energy balance, which characterizes db/db mice
(Lebrun et al. 2006).
All together show that BDNF mediates the
central control of food intake, which acts as an
anorexigenic factor. Most studies on the role of
BDNF in food intake regulation are focused on
the hypothalamus as the probable food intake and
energy balance target. Until recently, two sites in
the adult brain have been highlighted for the
anorectic role of BDNF: (1) the ventromedial
nucleus in the hypothalamus, where its mRNA
levels are modulated by the nutritional state and
melanocortin signaling, and (2) the dorsal vagal
complex in the hindbrain, where exogenous pro-
tein infusion induces anorexia and endogenous
protein levels are modulated by nutritional status,
15 BDNF Gene as a Precision Skill of Obesity Management 235

peripheral leptin, and cholecystokinin treatments 15.2.3 Genetic Alterations

(Lebrun et al. 2006). in the Genes Encoding BDNF or
Its High-Affinity Receptor TrkB
(Tropomyosin-Related Kinase
15.2.2 Participation of BDNF B)
on Metabolism
Mutations in the BDNF gene and its receptor
TrkB lead to severe obesity in humans. The
Contradicting studies concerning the action of
WAGR (Wilms’ tumor, aniridia, genitourinary
BDNF on metabolism exist in the literature. In
anomalies, and mental retardation) disorder is
this respect, clinical data indicate that decreased
characterized by contiguous gene syndrome and
BDNF plasma levels, which are associated with
displays obesity when deletions extend into the
birth weight and body mass index, may influence
BDNF locus (Han 2016). Deletions of chromo-
the development and pathophysiology of child-
some 11p ranged from 1.0 to 26.5 Mb, and 58%
hood obesity. Similarly, diminished BDNF serum
of the patients had heterozygous BDNF deletions.
levels impair glucose metabolism and correlate
These patients had significantly higher BMI
with obesity and diabetic complications in adults.
z-scores throughout childhood than did patients
Contrary to these findings, fasting serum BDNF
with intact BDNF length (Han et al. 2008).
concentrations have been found to be high in
Different variants of the BDNF-encoding gene
obese women. Furthermore, recent results do not
have been described in human patients with AN
link BDNF and insulin resistance or cardiovascu-
or BN. The most frequently encountered variant
lar risk factors in obese children but demonstrate
(196G/A) leads to a single amino acid substitu-
a positive correlation between the gene alterations
tion (Val66Met) in the pro-region of BDNF. It has
and changes of leptin, probably due to fat mass
been shown that this mutation affects the sorting
(Briana and Malamitsi-Puchner 2018). Neverthe-
of BDNF into the nerve terminals and markedly
less, a recent meta-analysis developed by
reduces its activity-dependent secretion (Chen
Sandrini and colleagues concluded that obese
et al. 2004). Mutations in the gene encoding
patients have normal levels of BDNF and
TrkB have also been linked to eating disorders.
emphasized that data evaluation may be
A de novo mutation leading to a single amino acid
influenced by the procedure of sampling,
substitution (Y722C) on the gene encoding TrkB
handling, and storage and these could lead to a
has been described in a young boy exhibiting
difficulty in the results’ interpretation. Hence,
severe obesity. This mutation has been shown to
standardization of the procedure is urgently
considerably alter the signaling capabilities of the
needed for strong, affordable, and reliable
receptor TrkB (Lebrun et al. 2006). In addition, a
conclusions (Sandrini et al. 2018).
genome-wide association study revealed a link
Correlation studies between BDNF serum
between polymorphisms of BDNF and obesity
levels and eating disorders in humans have also
(Thorleifsson et al. 2009). Likewise, a recent
been undertaken. It has been demonstrated that
meta-analysis developed by Akbarian and
BDNF serum levels are significantly reduced in
colleagues suggests that some polymorphisms in
female patients with anorexia nervosa (AN) or
BDNF including rs925946, rs10501087, rs6265,
bulimia nervosa (BN) (Nakazato et al. 2003;
and rs988712 can be considered as genetic
Monteleone et al. 2005). It has been further
determinants of obesity (Akbarian et al. 2018).
suggested that since BDNF exerts a satiating
The presence of polymorphisms in the BDNF
effect, this may be an adaptation to the decreased
associated with obesity will be discussed deeply
caloric ingestion in these patients (Lebrun et al.
in the following section.
2006).
236
15.2.4 Epigenetic Programming
of BDNF
The interactions between environmental and
genetic factors are reflected by epigenetic
changes, including DNA methylation and histone
modifications. These variations are particularly
important at early life stages due to their potential
impact on individuals’ physiological develop-
ment (Rosas-Vargas et al. 2011). Recent evidence
suggests that BDNF may have essential functions
during pregnancy by modulating fetal growth and
participating in the development and maturation
of central and peripheral fetal organs, including
the placenta. This gene probably acts as a nutrient
functional sensor for the placenta and may consti-
tute a link between maternal nutrition and fetal
nutrients’ demand. Furthermore, the diminished
BDNF signaling is linked with energy balance
dysregulation. Indeed, a close association has
been described between BDNF and adult
nutritional status in the central nervous system.
Both the functional loss of one copy of BDNF and
a de novo mutation of the full-length TrkB recep-
tor have been associated with severe obesity in
humans. BDNF is downregulated in preterm
infants, with tendency for obesity and develop-
ment of metabolic syndrome later in life (Briana
and Malamitsi-Puchner 2018).
15.2.5 BDNF and Hedonic Eating
In addition to its role in the homeostatic regula-
tion of energy balance, BDNF may suppress
hedonic feeding of palatable food through the
reward system. This system begins in the ventral
tegmental area (VTA) and joins to the nucleus
accumbens. Mice with deficient BDNF-to-TrkB
signaling exhibit hyperphagia while on high-fat
diets because of the larger meal size. Mice lacking
BDNF synthesis in CaMKIIα-expressing neurons
show an extreme hypersensitivity of the dopa-
mine receptor D1 as it is shown that peripheral
injection of D1 receptor agonist normalizes the
hyperphagia observed in high-fat diets. We
hypothesize that the effect of BDNF deficiency
H. Marcos-Pasero et al.
on the dopamine receptor D1 could prevent dopa-
mine release in the nucleus accumbens and the
dorsal striatum as a compensatory effect. As the
VTA has been identified as an important source of
BDNF contributing to hedonic eating regulation,
it would be of interest to evaluate whether BDNF
acts on TrkB-expressing neurons in the VTA or
the nucleus accumbens to regulate the consump-
tion of palatable food. Thus, it is necessary to
understand the changes in the reward circuit’s
structure and function when BDNF signaling is
defective and promotes hyperphagia on high-fat
diets and leads to obesity (Xu and Xie 2016).
15.2.6 BDNF as a Mediator
in the Effects of Exercise
and Food Intake
on Neuroplasticity
and the Vulnerability
of the Brain and Peripheral
Organs to Metabolic Morbidity
and Obesity
Recent evidence suggests that running and other
types of aerobic exercise can enhance cognitive
performance and increase serum BDNF levels in
human subjects. Running also lessens anxiety and
depression in animal models by BDNF-mediated
mechanisms. This is consistent with the role of
BDNF in alleviating anxiety and depression in
humans. Dietary energy restriction can exert anxi-
olytic effects via the involvement of BDNF and
ketone bodies. All these aspects might have a
crucial effect on the obesity control (Marosi and
Mattson 2014).
15.3 SNPs of the BDNF Associated
with the Obesity
During the last few decades, significant changes
have been taken place in high-throughput geno-
mics and thus advanced on the whole-genome
sequencing data through new molecular
technologies and new analytical methods. The
facilitation of recruiting unrelated individuals
has contributed to the novel genomics research
15 BDNF Gene as a Precision Skill of Obesity Management
through the performance of a large population-
based biobanks-genetic database for public
availability.
These achievements have been possible thanks
to genome-wide association studies (GWASs).
The primary goal of these studies is to better
understand the biology of the disease, under the
study of associations between common traits and
common variants. In the context of polygenic or
common diseases, many loci affecting the trait
segregate in the population should be examined.
GWAS, up until now, has identified more than
10,000 robust associations among SNPs and
diseases or traits, proving highly replicable, both
within and between populations (Hebbring 2019;
Visscher et al. 2017).
At least, 500 human adiposity-related loci
were identified by GWAS (Loos 2018). In the
majority of these genes, mutations result in mono-
genic obesity and common obesity, including
MC4R, BDNF, PCSK1, POMC, SH2B1, LEPR,
and NTRK2 (Goodarzi 2018). The latest meta-
analysis of GWAS for BMI in European
populations ( p < 1
108) revealed
941 BMI-related SNPs. In that study, there were
over 250,000 European participants and have
been identified more than 100 new BMI-related
SNPs, a fact that highlights the necessity for a
large sample size to increase prediction accuracy
and to provide insights into complex diseases
(Yengo et al. 2018).
The GWAS results have been reported in vari-
ous databases. This paper has at length discussed
the researches published in Ensembl (www.
ensembl.org/index.html), GWAS Catalog (www.
ebi.ac.uk/gwas/), and GWAS Central (www.
gwascentral.org). We identified 14 SNPs near or
inside BDNF/BDNF-AS related to BMI:
rs10501087, rs10767654, rs10767664,
rs11030100, rs11030102, rs11030104,
rs16917237, rs2030323, rs2049045, rs4923460,
rs6265, rs7103411, rs7481311, and rs925946
(Fig. 15.1).
These genetic variants and their GWAS-effect
size are shown in Table 15.1.
Conversely, there are only four BMI-related
SNPs in BDNF/BDNF-AS as a result of the latest
meta-analysis and review on the topic: rs925946,
237
rs10501087, rs6265, and rs988712. This study
analyzed the results from 35 cross-sectional
observational and case-control studies where all
kinds of BDNF/BDNF-AS SNPs were studied in
relation to BMI. The overall results by means of β
and OR showed a statistically significant associa-
tion between the SNPs and the BMI (Akbarian
et al. 2018).
However, inconsistencies in the researches’
results impose the need for further study. Thus,
a summary of the published studies available in
the literature from all the BMI-related SNPs of
BDNF/BDNF-AS is presented below:
Note that we found no entries for rs10767654,
rs11030100, rs11030102, rs2049045, rs492346,
rs7103411, or rs7481311.
15.3.1 rs10501087
This genetic variant is significantly linked to BMI
in the latest meta-analysis and review of
BMI-related SNPs of BDNF/BDNF-AS
(Akbarian et al. 2018). Results are coincident
with other studies involving both adulthood and
childhood populations of European descents (Jiao
et al. 2011; Zhao et al. 2009), and with extreme
obesity specifically among French and German
children (Scherag et al. 2010).
Interestingly, the Black Women’s Health
Study in African-American women has found no
correlation between rs10501087 and BMI (Ruiz-
Narváez et al. 2016), maybe due to differences
caused by race, ethnicity, or country of origin.
This genetic variant has been highlighted in a
genetic risk score (GRS) for the study of Spanish
obesity in two prospective representatives to the
central population cohort studies, Pizarra in the
South of Spain and Zona Urbana de Valladolid
(Martínez-García et al. 2013).
15.3.2 rs10767664
The rs10767664 variation has been associated
with BMI in the GOYA study composed of two
large Danish cohorts (Paternoster et al. 2011) as
well as in another European population-based
238 H. Marcos-Pasero et al.

Fig. 15.1 Plotted

ideogram of all BMI-related
SNPs in or near BDNF/
BDNF-AS
(11-chromosome).
Footnote: Lines were
plotted on the chromosome
corresponding to the base-
pair location of each
BDNF/BDNF-AS SNP,
and the line connects to
colored circles representing
the same phenotype
associated with that SNP
(BMI) [PhenoGram (Wolfe
et al. 2013)]

work (Vimaleswaran et al. 2012) that included a
gene-centric meta-analysis study (Guo et al.
2013) and a population of Saudi Arabians
(Alharbi et al. 2014). However, in other studies
such an association has not been found (Wang
et al. 2011; McCaffery et al. 2013).
In fact, this association has reported
conflicting data in children and in elderly
populations. For example, a longitudinal study
shows a distinct effect on BMI during childhood
in the community of Bogalusa in Louisiana (Mei
et al. 2012). These results are in line with the data
reported in other pediatric cohorts composed of
South Brazilian children (Zandoná et al. 2017).
Despite these data, this association proved not to
be significant for Cardiovascular Risk in the
Young Finns Study of a European cohort
(Juonala et al. 2011), or in Chinese children
(Wang et al. 2016). In the case of elderly, this
polymorphism has been strongly associated with
BMI in the AGES-Reykjavik comprising 4,846
individuals of European ancestry (Murphy et al.
2013).
This variant has also been studied in the con-
text of other obesity-related traits, such as the age
of onset of menarche (Fernández-Rhodes et al.
2013) and type 2 diabetes mellitus in Caucasian
women (De et al. 2017a). Common homozygous
AA showed a daily increase of 100 kcal in their
energy intake in the Look AHEAD (Action for
Health in Diabetes) clinical trial (McCaffery et al.
2012), and a better improvement in glucose,
 15

Table 15.1 Summary of results from the SNPs of the GWAS of BMI inside or near BDNF/BDNF-AS
Risk
Variant allele Mapped gene Location Consequence MAF Beta CI p Study accession
rs10501087 T BDNF-AS 11:27648561 Intron variant 0.230 0.072 unit increase [0.043–0.101] 7
107 Ng et al. (2017)
rs10767654 T LINC00678, BDNF- 11:27618676 Intron variant 0.327 0.054 unit decrease [0.040–0.070] 2
1012 Tachmazidou et al.
AS (2017)
rs10767664 A BDNF, AC103796.1 11:27704439 Intron variant 0.239 0.190 kg/m2 [0.130–0.250] 5
1026 Speliotes et al. (2010)
increase
rs11030100 T BDNF-AS, BDNF 11:27656039 3 prime UTR variant 0.228 0.038 kg/m2 [0.032–0.044] 1
1028 Akiyama et al. (2017)
decrease
rs11030102 C BDNF-AS, BDNF 11:27660049 Noncoding transcript exon 0.114 0.036 unit decrease [0.022–0.051] 1
106 Tachmazidou et al.
variant (2017)
rs11030104 A BDNF, BDNF-AS 11:27662970 Intron variant 0.222 0.047 unit increase [0.038–0.058] 2
1020 Wen et al. (2014)
0.042 kg/m2 [0.034–0.049] 7
1030 Locke et al. (2015)
increase
rs16917237 T BDNF-AS, BDNF 11:27680836 Intron variant 0.221 0.040 unit decrease UN 1
1034 Winkler et al. (2015)
rs2030323 C AC103796.1, BDNF 11:27706992 Intron variant 0.239 0.044 kg/m2 [0.034–0.055] 9
1016 Graff et al. (2017)
increase
BDNF Gene as a Precision Skill of Obesity Management

rs2049045 G BDNF-AS, BDNF 11:27672694 Intron variant 0.062 0.042 kg/m2 [0.029–0.057] 9
1010 Graff et al. (2017)
increase
rs4923460 G BDNF-AS 11:27635242 Intron variant 0.254 0.041 kg/m2 [0.029–0.053] 5
1011 Graff et al. (2017)
increase
rs6265 C BDNF-AS, BDNF 11:27658369 Missense variant 0.201 0.040 unit increase [0.036–0.045] 3
1068 Turcot et al. (2018)
G 4.58% SD increase [3.070–6.090] 5
1010 Thorleifsson et al.
(2009)
T 0.04 kg/m2 [0.034–0.046] 2
1051 Akiyama et al. (2017)
decrease
rs7103411 T BDNF, BDNF-AS 11:27678578 Intron variant 0.247 0.041 kg/m2 [0.033–0.05] 2
1022 Graff et al. (2017)
increase
rs7481311 T BDNF-AS 11:27561582 Intron variant 0.247 3.15% SD increase [1.780–4.520] 8
106 Thorleifsson et al.
(2009)
rs925946 T BDNF-AS 11:27645655 Intron variant 0.252 3.85% SD increase [2.620–5.080] 9
1010 Thorleifsson et al.
(2009)
BDNF-AS, BDNF antisense RNA; LINC00678, long intergenic nonprotein coding RNA 678; AC103796.1, novel transcript, sense overlapping BDNF
MAF Minor allele frequency in 1000 genomes combined population, UN Undefined
239
240
insulin, and HOMA-IR levels after
biliopancreatic surgery (De et al. 2016) in com-
parison with minority allele carriers. The associa-
tion between insulin levels and rs10767664 has
also been found in a clinical trial using European
obese patients after weight loss caused by a
hypocaloric diet enriched with monounsaturated
fatty acids (De et al. 2017b) and with standard
hypocaloric diet (De et al. 2018).
This genetic variant has also been included as
a genetic risk score for the study of BMI, greater
weight gain, and satiety (Llewellyn et al. 2014;
Badsi et al. 2014; Rukh et al. 2016).
15.3.3 rs11030104
The association between rs11030104 and BMI
has been reported in the Pakistanis PROMIS
cohort (Ahmad et al. 2015) and in Korean people
(Lee et al. 2017). Other obesity-related traits were
investigated in relation with this polymorphism,
including the levels of high-density lipoproteins
in adults (Ramos-Lopez et al. 2018), and the
satiety response in European preschoolers
(Monnereau et al. 2017) showing significant asso-
ciation for the former but nominal for the later.
15.3.4 rs16917237
This genetic variant has been documented in rela-
tion with eating disorders. Specifically, patients
with bulimia nervosa TT homozygous exhibited
higher BMI and minimum registered weight than
GT and GG genotypes (Gamero-Villarroel et al.
2014).
15.3.5 rs2030323
Without a direct reference to BMI association,
this variant has been studied in the context of
another obesity-related trait. An association has
been found between energy intake and rs2030323
in the Diabetes Prevention Program, where an
increase of 100–150 kcal per risk allele is noted
(McCaffery et al. 2017). In Chinese children and
H. Marcos-Pasero et al.
adolescents, a borderline statistical significance
with leptin levels has been reported (Fu et al.
2017a).
This genetic variant has also been investigated
as a genetic risk score for the study of BMI and
sugar-sweetened beverage consumption in two
Swedish cohorts (Brunkwall et al. 2016).
15.3.6 rs6265 (Val66Met)
The rs6265 (Val66Met) variant is significantly
associated with BMI in the only meta-analysis
on genetic variants from BDNF/BDNF-AS
(Akbarian et al. 2018) and in Korean adults
(Hong et al. 2012). Intriguingly, there are reports
on differences in sex stratifications. For example,
Met66 allele is associated with obesity only in
Belgian and British women (Beckers et al. 2008;
Shugart et al. 2009), while in the Boston Puerto
Rican Health Study, men with GG genotype had
higher BMI, waist and hip circumferences, and
weight than other studied genotypes. An interac-
tion has been observed between rs6265 and BMI
due to the consumption of polyunsaturated fatty
acids and with waist circumference (n-3:n-6
ratio). On the contrary, in the case of Puerto
Rican women, GG genotypes are associated
with lower BMI, hip circumference, and weight
(Ma et al. 2012). Similar observations have been
documented in European (Zhao et al. 2009), Chi-
nese (Wu et al. 2010), American (Mei et al.
2012), Mexican (Martínez-Ezquerro et al. 2017),
and Brazilian (Zandoná et al. 2017) pediatric
cohorts.
This genetic variant has also been studied in
the context of other obesity-related traits. Adults
that carry this variant allele are associated with
both an increased risk of gaining weight (border-
line statistical significance p ¼ 0.051)
(McCaffery et al. 2013) and an increased intrinsic
motivation during exercise (Caldwell et al. 2014).
On the other hand, this polymorphism is
associated with abdominal obesity in Chinese
children and adolescences (Xi et al. 2013; Fu
et al. 2017b). Met allele has also been associated
with AN, restrictive AN, binge eating/purging
AN as well as BN (Ribasés et al. 2004; Dardennes
15 BDNF Gene as a Precision Skill of Obesity Management
et al. 2007; Ribasés et al. 2003), and rumination
(Beevers et al. 2009). However, these results are
not significant in adolescents and preadolescents
(Arija et al. 2010). In reference to the diet, there
are evidence to support that GG genotypes are
associated with higher caloric intake added to a
diet containing higher ratio of meat, eggs, nuts,
beans, and dairy products (McCaffery et al.
2012), although no documented evidence is
recorded in relation to the intake of any macronu-
trient and rs6265 (Park et al. 2013).
This genetic variant has also been included in
two genetic risk scores for the study of childhood
BMI (Klimentidis et al. 2011; Mitchell et al.
2013).
15.3.7 rs925946
This genetic variant has been associated with
obesity, weight, and BMI in European,
European-American, and African-American
cohorts (Thorleifsson et al. 2009; Sandholt et al.
2010) as well as with the only BMI-related meta-
analysis on genetic variants from BDNF/BDNF-
AS (Akbarian et al. 2018) and with a greater
impact on adults than children (den Hoed et al.
2010), which could explain the contradictory
results in pediatric populations. No significant
association has been found with BMI in a
European-American descent pediatric cohort
(Zhao et al. 2009), nor in the European Inter99
cohort (Andersson et al. 2010). However, in
French and German populations, there is an asso-
ciation with the early onset of extreme obesity
(Scherag et al. 2010), and weight as well as with
height and BMI in the European ALSPAC cohort
(Elks et al. 2010). An association has also been
found for babies being born small for gestational
age (Morgan et al. 2010).
In adolescents, rs925946 is associated with the
metabolic syndrome in Europeans (Dušátková
et al. 2013), and with BMI in Australians (border-
line significance p ¼ 0,053) (Liu et al. 2010).
Also, adolescents’ carriers of the minor allele
appeared to absorb less dietetic calcium
(Dušátková et al. 2015). Similar results have
recently been reported in children of the Spanish
241
GENYAL cohort by our research group. We also
observed an interaction between rs925946 and
BMI as well as calcium and dairy product intake
(Marcos-Pasero et al. 2019).
Table 15.2 shows SNPs with other than BMI
associations according to GWAS described
above.
15.4 Precision Nutrition
for Personalizing Nutritional
Recommendations
The Precision Nutrition (PN) is a modern and
multidimensional concept, which contains indi-
vidual characteristics that define the phenotype
(e.g., sex, age, current diseases, biochemical
parameters, etc.), behavioral lifestyle factors
(e.g., dietary habits and patterns, food behavior,
physical activity, drugs, stress, etc.), preferences
(e.g., food, culinary techniques, flavors, etc.), and
genetics. Other conditions, such as resources,
environmental, and sustainability, are also con-
sidered in PN (Corella and Ordovás 2018). Thus,
the PN emerges due to the integration of many
fields of investigation and with a broad definition.
The origin of PN emerged as a part of Preci-
sion Medicine focused on discoursing the hetero-
geneity in nutrient metabolism between
subgroups and the inter-individual variability in
responses to diet interventions (Ramos-Lopez
et al. 2017).
The pillars of PN have been categorized by the
International Society of Nutrigenetics/
Nutrigenomics (ISNN) in stratified,
individualized, and genotype-directed nutrition
in order to show the role of genetic variations
and individual dietary responses, as well as the
role of nutrients in gene expression (de Toro-
Martín et al. 2017).
The main goal of the PN is to prevent diseases
and formulate solid and personalized nutritional
prescriptions, in other words to establish tailored
dietary instructions regarding multiple variables
to satisfactorily anticipate individual responses to
diet. Nevertheless, clinicians show uncertainty in
the use of PN recommendations, so it is necessary
further interventional trials to be conducted in
242
Table 15.2 Other GWAS-related associations between BDNF/BDNF-AS SNPs
rs10501087
rs10767654
rs11030102
rs11030104
rs16917237
rs2030323
rs2049045
rs6265
rs7103411
rs7481311
rs925946
order to increase the level of evidence, strength,
and reproducibility of the findings (Laddu and
Hauser 2019). The Food4Me project is focused
on this area. It tries to establish the relevant
modus operandi of PN in preventing and manag-
ing chronic diseases such as obesity (de Toro-
Martín et al. 2017).
On the basis of genomic background, studying
gene-diet interactions in obesity requires more
exhaustive analyses including different kinds of
individual phenotypes, genotypes, and dietary
factors (Heianza and Qi 2017).
In this context, our research group performed
the first study on the association between the
BDNF-AS rs925946 polymorphism and calcium
intake as potential modulators of the nutritional
status. The objective of this study was to evaluate
the association between calcium/dairy intake, the
BDNF-AS rs925946 polymorphism, and
nutritional status in a group of schoolchildren as
H. Marcos-Pasero et al.
Morning person (Kichaev et al. 2019)
Waist circumference (Tachmazidou et al. 2017)
Weight (Tachmazidou et al. 2017)
Educational attainment (Lee et al. 2018)
BMI in smokers (Justice et al. 2017)
BMI in nonsmokers (Justice et al. 2017)
Grip strength (Tikkanen et al. 2018)
Coronary artery disease (van der Harst and Verweij 2018)
Age of onset of menopause (Horikoshi et al. 2018)
Age of onset of menarche (Kichaev et al. 2019)
Obesity (Berndt et al. 2013)
Snoring (Jansen et al. 2019)
Tobacco cessation (Liu et al. 2019)
Risk tolerance (Karlsson Linnér et al. 2019)
Hip circumference (Shungin et al. 2015)
Smoker status (Liu et al. 2019)
Smoking behavior (Tobacco and Genetics Consortium 2010)
Age of tobacco use initiation (Liu et al. 2019)
Weight (Thorleifsson et al. 2009)
Risky behaviors (Karlsson Linnér et al. 2019)
Cigarettes smoked per day (Liu et al. 2019)
Visceral adipose tissue (Karlsson et al. 2019)
Age of onset of menarche (Perry et al. 2014)
Weight (Thorleifsson et al. 2009)
Diastolic blood pressure (Surendran et al. 2016)
Weight (Thorleifsson et al. 2009)
part of the GENYAL study to childhood obesity
prevention (www.clinicaltrials.gov
NCT03419520). A total of 221 schoolchildren
from 6 different public schools among the Com-
munity of Madrid, Spain, were included. 52.50%
were girls (n ¼ 116) and 47.50% boys (n ¼ 105)
between 6 and 9 years old. Anthropometric and
dietary data were collected, and children were
genotyped according to the rs925946 polymor-
phism obtained from saliva samples. An inverse
significant association was observed between the
schoolchildren’s BMI and the mg of calcium
intake/day (β ¼ 0.002 (0.003, 0.0003)
p ¼ 0.019) (data adjusted for sex and age). The
distribution caused by genotypes according to the
genetic variant rs925946 showed that 55.91% of
the participants were GG common homozygous
(n ¼ 123), 36.82% heterozygous (n ¼ 81), and
7.27% TT homozygous (n ¼ 16). No significant
differences were observed regarding the BMI
15 BDNF Gene as a Precision Skill of Obesity Management
between both genotypes (BMI
(GG) ¼ 16.97  0.47 kg/m2 and
(GT + TT) ¼ 17.00  0.540 kg/m2).
A significant interaction was observed
between the genetic variant and the BMI. Indeed,
GG common homozygous reduced on average
0.34 kg/m2 the BMI for each 100 mg of calcium
(β ¼ 0.003 (0.006, 0.001), p ¼ 0.004).
However, this effect was not observed in T allele
carriers, where the consumption of 100 mg of
calcium was not associated with the BMI
(β ¼ 1.3e-4 (0.0022, 0.0024), p ¼ 0.93).
To our knowledge, this is the first time that the
association between BDNF-AS rs925946 and cal-
cium intake as potential modulators of the
nutritional status has been described, which
opens up the avenue for future investigations in
this area. We hypothesize that the presence of the
SNP may cause failures in the control of the
energy balance and conditioning the effect of
calcium on satiety or on other mechanisms
involved in body weight control which may pro-
mote obesity development. The determination of
differential effects of calcium/dairy consumption
on the nutritional status in accordance with genet-
ics may contribute to the personalization of future
nutritional advice. Nevertheless, these results
should be confirmed in future prospective studies
with a larger sample size (Marcos-Pasero et al.
2019).
15.5 Conclusion and Future
Perspectives
The identification of diverse individual nutritional
needs and responses to diet is changing standard
of nutritional care. The integration of all the
factors involved (phenotype, lifestyle factors,
preferences, and, indisputably, genetic) will cre-
ate new treatment options for obesity and other
complex diseases.
Acknowledgments MPI is supported by the TALENTO
Program from the Regional Madrid Government (2018-
T1/BIO-11262).
243
References
Ahmad S, Zhao W, Renström F, Rasheed A, Samuel M,
Zaidi M et al (2015) Physical activity, smoking, and
genetic predisposition to obesity in people from
Pakistan: the PROMIS study. BMC Med Genet [Inter-
net] 16:114–114. Available from: https://europepmc.
org/articles/PMC4683724/ [cited 2019 Oct 31]
Akbarian S-A, Salehi-Abargouei A, Pourmasoumi M,
Kelishadi R, Nikpour P, Heidari-Beni M (2018) Asso-
ciation of Brain-derived neurotrophic factor gene
polymorphisms with body mass index: A systematic
review and meta-analysis. Adv Med Sci [Internet] 63
(1):43–56. Available from: http://www.sciencedirect.
com/science/article/pii/S1896112617300536 [cited
2019 Oct 30]
Akiyama M, Okada Y, Kanai M, Takahashi A,
Momozawa Y, Ikeda M et al (2017) Genome-wide
association study identifies 112 new loci for body
mass index in the Japanese population. Nat Genet
[Internet] 49:1458. Available from: https://doi.org/10.
1038/ng.3951
Alharbi KK, Richardson TG, Khan IA, Syed R,
Mohammed AK, Boustred CR et al (2014) Influence
of adiposity-related genetic markers in a population of
Saudi Arabians where other variables influencing obe-
sity may be reduced. Dis Markers [Internet]
2014:758232. Available from: https://europepmc.org/
articles/PMC4251424/ [cited 2019 Oct 31]
Andersson EA, Pilgaard K, Pisinger C, Harder MN,
Grarup N, Færch K et al (2010) Do gene variants
influencing adult adiposity affect birth weight? A
population-based study of 24 loci in 4,744 Danish
individuals. PloS One [Internet] 5(12):e14190–
e14190. Available from: https://europepmc.org/
articles/PMC2995733/ [cited 2019 Nov 5]
Arija V, Ferrer-Barcala M, Aranda N, Canals J (2010)
BDNF Val66Met polymorphism, energy intake and
BMI: a follow-up study in schoolchildren at risk of
eating disorders. BMC Public Health [Internet]
10:363–363. Available from: http://europepmc.org/
abstract/MED/20573217 [cited 2019 Nov 4]
Badsi MN, Mediene-Benchekor S, Ouhaibi-Djellouli H,
Lardjam-Hetraf SA, Boulenouar H, Meroufel DN et al
(2014) Combined effect of established BMI loci on
obesity-related traits in an Algerian population sample.
BMC Genet [Internet] 15:128–128. Available from:
https://europepmc.org/articles/PMC4247883/ [cited
2019 Oct 31]
Beckers S, Peeters A, Zegers D, Mertens I, Gaal LV, Van
Hul W (2008) Association of the BDNF Val66Met
variation with obesity in women. Mol Genet Metab
[Internet] 95(1):110–112. Available from: http://
www.sciencedirect.com/science/article/pii/
S1096719208001741 [cited 2019 Nov 4]
Beevers CG, Wells TT, JE MG (2009) The BDNF
Val66Met polymorphism is associated with rumination
in healthy adults. Emot Wash DC [Internet] 9
244
(4):579–584. Available from: http://europepmc.org/
abstract/MED/19653783 [cited 2019 Nov 4]
Berndt SI, Gustafsson S, Mägi R, Ganna A, Wheeler E,
Feitosa MF et al (2013 May) Genome-wide meta-anal-
ysis identifies 11 new loci for anthropometric traits and
provides insights into genetic architecture. Nat Genet
45(5):501–512
Briana DD, Malamitsi-Puchner A (2018) Developmental
origins of adult health and disease: the metabolic role
of BDNF from early life to adulthood. Metabolism
81:45–51
Brunkwall L, Chen Y, Hindy G, Rukh G, Ericson U,
Barroso I et al (2016) Sugar-sweetened beverage con-
sumption and genetic predisposition to obesity in
2 Swedish cohorts. Am J Clin Nutr [Internet] 104
(3):809–815. Available from: https://europepmc.org/
articles/PMC4997292/ [cited 2019 Nov 4]
Caldwell AH, Bryan AD, Hagger MS (2014) What keeps a
body moving? The brain-derived neurotrophic factor
val66met polymorphism and intrinsic motivation to
exercise in humans. J Behav Med [Internet] 37
(6):1180–1192. Available from: http://europepmc.org/
abstract/MED/24805993 [cited 2019 Nov 4]
Chen Z-Y, Patel PD, Sant G, Meng C-X, Teng KK,
Hempstead BL et al (2004) Variant brain-derived
neurotrophic factor (BDNF) (Met66) alters the intra-
cellular trafficking and activity-dependent secretion of
wild-type BDNF in neurosecretory cells and cortical
neurons. J Neurosci 24(18):4401–4411
Corella D, Ordovás JM (2018) The role of omics in preci-
sion nutrition: strengths and weaknesses. Nutr Hosp.
35(Spec No 4):10–18
Dardennes RM, Zizzari P, Tolle V, Foulon C, Kipman A,
Romo L et al (2007) Family trios analysis of common
polymorphisms in the obestatin/ghrelin, BDNF and
AGRP genes in patients with Anorexia nervosa: asso-
ciation with subtype, body-mass index, severity and
age of onset. Psychoneuroendocrinology [Internet] 32
(2):106–113. Available from: http://europepmc.org/
abstract/MED/17197106 [cited 2019 Nov 4]
de Toro-Martín J, Arsenault BJ, Després J-P, Vohl M-C
(2017 Aug) Precision nutrition: a review of
personalized nutritional approaches for the prevention
and Management of Metabolic Syndrome. Nutrients
22:9(8)
De DL, Izaola O, Primo D, Pacheco D (2016) Effect of the
rs10767664 variant of the brain-derived neurotrophic
factor gene on weight change and cardiovascular risk
factors in morbidly obese patients after biliopancreatic
diversion surgery. J Nutr Nutr [Internet] 9
(2–4):116–122. Available from: http://europepmc.org/
abstract/MED/27544754 [cited 2019 Oct 31]
De DL, Romero E, Izaola O, Primo D, Aller R (2017a)
Cardiovascular risk factors and insulin resistance after
two hypocaloric diets with different fat distribution in
obese subjects: effect of the rs10767664 gene variant in
brain-derived neurotrophic factor. J Nutr Nutr [Inter-
net] 10(5–6):163–171. Available from: http://
H. Marcos-Pasero et al.
europepmc.org/abstract/MED/29339649 [cited 2019
Oct 31]
De DL, Aller R, Izaola O, Primo D, Romero E (2017b)
rs10767664 gene variant in brain-derived neurotrophic
factor is associated with diabetes mellitus type 2 in
Caucasian females with obesity. Ann Nutr Metab
[Internet] 70(4):286–292. Available from: http://
europepmc.org/abstract/MED/28595187 [cited 2019
Oct 31]
De DL, Fernández HO, Izaola O, Primo D, Aller R (2018)
RS 10767664 gene variant in Brain Derived
Neurotrophic Factor (BDNF) affect metabolic changes
and insulin resistance after a standard hypocaloric diet.
J Diabetes Complications [Internet] 32(2):216–220.
Available from: http://europepmc.org/abstract/MED/
29174117 [cited 2019 Oct 31]
den Hoed M, Ekelund U, Brage S, Grontved A, Zhao JH,
Sharp SJ et al (2010 Nov) Genetic susceptibility to
obesity and related traits in childhood and adolescence:
influence of loci identified by genome-wide association
studies. Diabetes 59(11):2980–2988
Dušátková L, Zamrazilová H, Sedláčková B, Včelák J,
Hlavatý P, Aldhoon Hainerová I et al (2013) Associa-
tion of obesity susceptibility gene variants with meta-
bolic syndrome and related traits in 1,443 Czech
adolescents. Folia Biol (Praha) 59(3):123–133
Dušátková L, Zamrazilová H, Aldhoon-Hainerová I,
Sedláčková B, Včelák J, Hlavatý P et al (2015) A
common variant near BDNF is associated with dietary
calcium intake in adolescents. Nutr Res N Y N 35
(9):766–773
Elks CE, Loos RJF, Sharp SJ, Langenberg C, Ring SM,
Timpson NJ et al (2010) Genetic markers of adult
obesity risk are associated with greater early infancy
weight gain and growth. PLoS Med 7(5):e1000284
Fernández-Rhodes L, Demerath EW, Cousminer DL,
Tao R, Dreyfus JG, Esko T et al (2013) Association
of adiposity genetic variants with menarche timing in
92,105 women of European descent. Am J Epidemiol
[Internet] 178(3):451–460. Available from: https://
europepmc.org/articles/PMC3816344/ [cited 2019 Oct
31]
Fu J, Li G, Li L, Yin J, Cheng H, Han L et al (2017a) The
role of established East Asian obesity-related loci on
pediatric leptin levels highlights a neuronal influence
on body weight regulation in Chinese children and
adolescents: the BCAMS study. Oncotarget [Internet]
8(55):93593–93607. Available from: https://
europepmc.org/articles/PMC5706821/ [cited 2019
Nov 4]
Fu LW, Zhang MX, Wu LJ, Gao LW, Mi J (2017b) Gene-
gene interaction on central obesity in school-aged chil-
dren in China. Zhonghua Liu Xing Bing Xue Za Zhi
Zhonghua Liuxingbingxue Zazhi [Internet] 38
(7):883–888. Available from: http://europepmc.org/
abstract/MED/28738459 [cited 2019 Nov 4]
Gamero-Villarroel C, Gordillo I, Carrillo JA, García-
Herráiz A, Flores I, Jiménez M et al (2014) BDNF
genetic variability modulates psychopathological
15 BDNF Gene as a Precision Skill of Obesity Management
symptoms in patients with eating disorders. Eur Child
Adolesc Psychiatry [Internet] 23(8):669–679. Avail-
able from: https://doi.org/10.1007/s00787-013-0495-6
[cited 2019 Oct 31]
Goodarzi MO (2018) Genetics of obesity: what genetic
association studies have taught us about the biology of
obesity and its complications. Lancet Diabetes
Endocrinol 6(3):223–236
Graff M, Scott RA, Justice AE, Young KL, Feitosa MF,
Barata L et al (2017) Genome-wide physical activity
interactions in adiposity ― A meta-analysis of 200,452
adults. PLOS Genet [Internet] 13(4):e1006528. Avail-
able from: https://doi.org/10.1371/journal.pgen.
1006528
Guo Y, Lanktree MB, Taylor KC, Hakonarson H, Lange
LA, Keating BJ (2013) Gene-centric meta-analyses of
108 912 individuals confirm known body mass index
loci and reveal three novel signals. Hum Mol Genet
[Internet] 22(1):184–201. Available from: https://
europepmc.org/articles/PMC3522401/ [cited 2019
Oct 31]
Han JC (2016) Rare syndromes and common variants of
the brain-derived Neurotrophic factor gene in human
obesity. Prog Mol Biol Transl Sci 140:75–95
Han JC, Liu Q-R, Jones M, Levinn RL, Menzie CM,
Jefferson-George KS et al (2008) Brain-derived
neurotrophic factor and obesity in the WAGR syn-
drome. N Engl J Med 359(9):918–927
Hebbring S (2019) Genomic and Phenomic research in the
21st century. Trends Genet [Internet] 35(1):29–41.
Available from: http://www.sciencedirect.com/sci
ence/article/pii/S0168952518301689 [cited 2019 Oct
28]
Heianza Y, Qi L (2017) Gene-diet interaction and preci-
sion nutrition in obesity. Int J Mol Sci 18(4):787
Hong K-W, Lim J-E, Go MJ, Shin YS, Ahn Y, Han B-G
et al (2012) Recapitulation of the association of the
Val66Met polymorphism of BDNF gene with BMI in
Koreans. Obesity [Internet] 20(9):1871–1875. Avail-
able from: https://www.onlinelibrary.wiley.com/doi/
abs/10.1038/oby.2011.352 [cited 2019 Nov 4]
Horikoshi M, Day FR, Akiyama M, Hirata M,
Kamatani Y, Matsuda K et al (2018) Elucidating the
genetic architecture of reproductive ageing in the Japa-
nese population. Nat Commun 9(1):1977
Hruby A, Hu FB (2015) The epidemiology of obesity: a
big picture. PharmacoEconomics 33(7):673–689
Jansen PR, Watanabe K, Stringer S, Skene N, Bryois J,
Hammerschlag AR et al (2019) Genome-wide analysis
of insomnia in 1,331,010 individuals identifies new
risk loci and functional pathways. Nat Genet 51
(3):394–403
Jiao H, Arner P, Hoffstedt J, Brodin D, Dubern B,
Czernichow S et al (2011) Genome wide association
study identifies KCNMA1 contributing to human obe-
sity. BMC Med Genomics [Internet] 4:51. Available
from: https://europepmc.org/articles/PMC3148553/
[cited 2019 Oct 30]
245
Juonala M, Juhola J, Magnussen CG, Würtz P, Viikari JS,
Thomson R et al (2011) Childhood environmental and
genetic predictors of adulthood obesity: the cardiovas-
cular risk in young Finns study. J Clin Endocrinol
Metab [Internet] 96(9):E1542–E1549. Available
from: https://europepmc.org/articles/PMC3167668/
[cited 2019 Oct 30]
Justice AE, Winkler TW, Feitosa MF, Graff M, Fisher VA,
Young K et al (2017) Genome-wide meta-analysis of
241,258 adults accounting for smoking behaviour
identifies novel loci for obesity traits. Nat Commun
8:14977
Karlsson Linnér R, Biroli P, Kong E, Meddens SFW,
Wedow R, Fontana MA et al (2019) Genome-wide
association analyses of risk tolerance and risky
behaviors in over 1 million individuals identify
hundreds of loci and shared genetic influences. Nat
Genet 51(2):245–257
Karlsson T, Rask-Andersen M, Pan G, Höglund J,
Wadelius C, Ek WE et al (2019) Contribution of genet-
ics to visceral adiposity and its relation to cardiovascu-
lar and metabolic disease. Nat Med 25(9):1390–1395
Kichaev G, Bhatia G, Loh P-R, Gazal S, Burch K, Freund
MK et al (2019) Leveraging polygenic functional
enrichment to improve GWAS power. Am J Hum
Genet 104(1):65–75
Klimentidis YC, Chen GB, López-Alarcón M, Harris JJ,
Duarte CW, Fernández JR (2011) Associations of obe-
sity genes with obesity-related outcomes in multiethnic
children. Arch Med Res [Internet] 42(6):509–514.
Available from: https://europepmc.org/articles/
PMC3541020/ [cited 2019 Nov 4]
Laddu D, Hauser M (2019) Addressing the nutritional
phenotype through personalized nutrition for chronic
disease prevention and management. Prog Cardiovasc
Dis 62(1):9–14
Lapchak PA, Hefti F (1992) BDNF and NGF treatment in
lesioned rats: effects on cholinergic function and
weight gain. Neuroreport 3(5):405–408
Lebrun B, Bariohay B, Moyse E, Jean A (2006) Brain-
derived neurotrophic factor (BDNF) and food intake
regulation: a minireview. Auton Neurosci Basic Clin
126–127:30–38
Lee JS, Cheong HS, Shin HD (2017) BMI prediction
within a Korean population. PeerJ [Internet] 5:e3510–
e3510. Available from: https://europepmc.org/articles/
PMC5493974/ [cited 2019 Oct 31]
Lee JJ, Wedow R, Okbay A, Kong E, Maghzian O, Zacher
M et al (2018) Gene discovery and polygenic predic-
tion from a genome-wide association study of educa-
tional attainment in 1.1 million individuals. Nat Genet
50(8):1112–1121
Liu JZ, Medland SE, Wright MJ, Henders AK, Heath AC,
Madden PA et al (2010) Genome-wide association
study of height and body mass index in Australian
twin families. Twin Res Hum Genet Off J Int Soc
Twin Stud [Internet] 13(2):179–193. Available from:
https://europepmc.org/articles/PMC3232006/ [cited
2019 Nov 5]
246
Liu M, Jiang Y, Wedow R, Li Y, Brazel DM, Chen F et al
(2019) Association studies of up to 1.2 million
individuals yield new insights into the genetic etiology
of tobacco and alcohol use. Nat Genet 51(2):237–244
Llewellyn CH, Trzaskowski M, Van CJ, Plomin R, Wardle
J (2014) Satiety mechanisms in genetic risk of obesity.
JAMA Pediatr [Internet] 168(4):338–344. Available
from: https://europepmc.org/articles/PMC3981891/
[cited 2019 Oct 31]
Locke AE, Kahali B, Berndt SI, Justice AE, Pers TH, Day
FR et al (2015) Genetic studies of body mass index
yield new insights for obesity biology. Nature 518
(7538):197–206
Loos RJ (2018) The genetics of adiposity. Mol Genet
Basis Metab Dis [Internet] 50:86–95. Available from:
http://www.sciencedirect.com/science/article/pii/
S0959437X17301259
Ma XY, Qiu WQ, Smith CE, Parnell LD, Jiang ZY,
Ordovas JM et al (2012) Association between BDNF
rs6265 and obesity in the Boston Puerto Rican health
study. J Obes [Internet] 2012:102942. Available from:
http://europepmc.org/abstract/MED/23326649 [cited
2019 Nov 4]
Marcos-Pasero H, Aguilar-Aguilar E, de la Iglesia R,
Espinosa-Salinas I, Gómez-Patiño M, Colmenarejo G
et al (2019) Association of calcium and dairy product
consumption with childhood obesity and the presence
of a brain derived neurotropic factor-antisense (BDNF-
AS) polymorphism. Clin Nutr 38(6):2616–2622
Marosi K, Mattson MP (2014) BDNF mediates adaptive
brain and body responses to energetic challenges.
Trends Endocrinol Metab TEM 25(2):89–98
Martínez-Ezquerro JD, Rendón-Macías ME, Zamora-
Mendoza G, Serrano-Meneses J, Rosales-
Rodríguez B, Escalante-Bautista D et al (2017) Asso-
ciation between the brain-derived neurotrophic factor
Val66Met polymorphism and overweight/obesity in
pediatric population. Arch Med Res [Internet] 48
(7):599–608. Available from: http://europepmc.org/
abstract/MED/29506764 [cited 2019 Nov 4]
Martínez-García F, Mansego ML, Rojo-Martínez G, De
GM-S, Morcillo S, Soriguer F et al (2013) Impact of
obesity-related genes in Spanish population. BMC
Genet [Internet] 14:111. Available from: https://
europepmc.org/articles/PMC4222487/ [cited 2019
Oct 30]
McCaffery JM, Papandonatos GD, Peter I, Huggins GS,
Raynor HA, Delahanty LM et al (2012) Obesity sus-
ceptibility loci and dietary intake in the look ahead
trial. Am J Clin Nutr [Internet] 95(6):1477–1486.
Available from: https://europepmc.org/articles/
PMC3349457/ [cited 2019 Oct 30]
McCaffery JM, Papandonatos GD, Huggins GS, Peter I,
Kahn SE, Knowler WC et al (2013) FTO predicts
weight regain in the look AHEAD clinical trial. Int J
Obes 2005 [Internet] 37(12):1545–1552. Available
from: https://europepmc.org/articles/PMC3750057/
[cited 2019 Oct 31]
H. Marcos-Pasero et al.
McCaffery JM, Jablonski KA, Franks PW, Delahanty LM,
Aroda V, Marrero D et al (2017) Replication of the
association of BDNF and MC4R variants with dietary
intake in the diabetes prevention program. Psychosom
Med [Internet] 79(2):224–233. Available from: https://
europepmc.org/articles/PMC5285480/ [cited 2019
Nov 4]
Mei H, Chen W, Jiang F, He J, Srinivasan S, Smith EN
et al (2012) Longitudinal replication studies of GWAS
risk SNPs influencing body mass index over the course
of childhood and adulthood. PLoS One 7(2):e31470
Mitchell JA, Hakonarson H, Rebbeck TR, Grant SFA
(2013 Jun) Obesity-susceptibility loci and the tails of
the pediatric BMI distribution. Obes Silver Spring Md
21(6):1256–1260
Monnereau C, Jansen PW, Tiemeier H, Jaddoe VW, Felix
JF (2017) Influence of genetic variants associated with
body mass index on eating behavior in childhood.
Obes Silver Spring Md [Internet] 25(4):765–772.
Available from: https://europepmc.org/articles/
PMC5496668/ [cited 2019 Oct 31]
Monteleone P, Fabrazzo M, Martiadis V, Serritella C,
Pannuto M, Maj M (2005 Jun) Circulating brain-
derived neurotrophic factor is decreased in women
with anorexia and bulimia nervosa but not in women
with binge-eating disorder: relationships to co-morbid
depression, psychopathology and hormonal variables.
Psychol Med 35(6):897–905
Morgan AR, Thompson JM, Murphy R, Black PN, Lam
WJ, Ferguson LR et al (2010) Obesity and diabetes
genes are associated with being born small for gesta-
tional age: results from the Auckland Birthweight Col-
laborative study. BMC Med Genet [Internet] 11:125.
Available from: https://europepmc.org/articles/
PMC2928774/ [cited 2019 Nov 5]
Murphy RA, Nalls MA, Keller M, Garcia M, Kritchevsky
SB, Tylavsky FA et al (2013) Candidate gene associa-
tion study of BMI-related loci, weight, and adiposity in
old age. J Gerontol A Biol Sci Med Sci [Internet] 68
(6):661–666. Available from: https://europepmc.org/
articles/PMC3660116/ [cited 2019 Oct 30]
Nakagawa T, Tsuchida A, Itakura Y, Nonomura T,
Ono M, Hirota F et al (2000) Brain-derived
neurotrophic factor regulates glucose metabolism by
modulating energy balance in diabetic mice. Diabetes
49(3):436–444
Nakazato M, Hashimoto K, Shimizu E, Kumakiri C,
Koizumi H, Okamura N et al (2003) Decreased levels
of serum brain-derived neurotrophic factor in female
patients with eating disorders. Biol Psychiatry 54
(4):485–490
Ng MCY, Graff M, Lu Y, Justice AE, Mudgal P, Liu C-T
et al (2017) Discovery and fine-mapping of adiposity
loci using high density imputation of genome-wide
association studies in individuals of African ancestry:
African Ancestry Anthropometry Genetics Consor-
tium. PLOS Genet [Internet] 13(4):e1006719. Avail-
able from: https://doi.org/10.1371/journal.pgen.
1006719
15 BDNF Gene as a Precision Skill of Obesity Management
Park SL, Cheng I, Pendergrass SA, Kucharska-Newton
AM, Lim U, Ambite JL et al (2013) Association of
the FTO obesity risk variant rs8050136 with percent-
age of energy intake from fat in multiple racial/ethnic
populations: the PAGE study. Am J Epidemiol [Inter-
net] 178(5):780–790. Available from: https://
europepmc.org/articles/PMC3755639/ [cited 2019
Nov 4]
Paternoster L, Evans DM, Nohr EA, Holst C,
Gaborieau V, Brennan P et al (2011) Genome-wide
population-based association study of extremely over-
weight young adults--the GOYA study. PloS One
[Internet] 6(9):e24303. Available from: https://
europepmc.org/articles/PMC3174168/ [cited 2019
Oct 30]
Pelleymounter MA, Cullen MJ, Wellman CL (1995)
Characteristics of BDNF-induced weight loss. Exp
Neurol 131(2):229–238
Perry JR, Day F, Elks CE, Sulem P, Thompson DJ,
Ferreira T et al (2014) Parent-of-origin-specific allelic
associations among 106 genomic loci for age at men-
arche. Nature 514(7520):92–97
Ramos-Lopez O, Milagro FI, Allayee H, Chmurzynska A,
Choi MS, Curi R et al (2017) Guide for current
Nutrigenetic, Nutrigenomic, and Nutriepigenetic
approaches for precision nutrition involving the pre-
vention and Management of Chronic Diseases
Associated with obesity. J Nutr Nutr 10(1–2):43–62
Ramos-Lopez O, Riezu-Boj JI, Milagro FI, Cuervo M,
Goni L, Martinez JA (2018) Prediction of blood lipid
phenotypes using obesity-related genetic
polymorphisms and lifestyle data in subjects with
excessive body weight. Int J Genomics [Internet]
2018:4283078. Available from: https://europepmc.
org/articles/PMC6276413/ [cited 2019 Oct 31]
Ribasés M, Gratacòs M, Armengol L, De RC, Badía A,
Jiménez L et al (2003) Met66 in the brain-derived
neurotrophic factor (BDNF) precursor is associated
with anorexia nervosa restrictive type. Mol Psychiatry
[Internet] 8(8):745–751. Available from: http://
europepmc.org/abstract/MED/12888803 [cited 2019
Nov 4]
Ribasés M, Gratacòs M, Fernández-Aranda F, Bellodi L,
Boni C, Anderluh M et al (2004) Association of BDNF
with anorexia, bulimia and age of onset of weight loss
in six European populations. Hum Mol Genet [Inter-
net] 13(12):1205–1212. Available from: http://
europepmc.org/abstract/MED/15115760 [cited 2019
Nov 4]
Rios M (2014) Neurotrophins and the regulation of energy
balance and body weight. Handb Exp Pharmacol
220:283–307
Rosas-Vargas H, Martínez-Ezquerro JD, Bienvenu T
(2011) Brain-derived neurotrophic factor, food intake
regulation, and obesity. Arch Med Res 42(6):482–494
Ruiz-Narváez EA, Haddad SA, Rosenberg L, Palmer JR
(2016) Birth weight modifies the association between
central nervous system gene variation and adult body
mass index. J Hum Genet [Internet] 61(3):193–198.
247
Available from: https://europepmc.org/articles/
PMC4808432/ [cited 2019 Oct 30]
Rukh G, Ahmad S, Ericson U, Hindy G, Stocks T,
Renström F et al (2016) Inverse relationship between
a genetic risk score of 31 BMI loci and weight change
before and after reaching middle age. Int J Obes 2005
[Internet] 40(2):252–259. Available from: https://
europepmc.org/articles/PMC4753358/ [cited 2019
Oct 31]
Sandholt CH, Sparsø T, Grarup N, Albrechtsen A,
Almind K, Hansen L et al (2010) Combined analyses
of 20 common obesity susceptibility variants. Diabetes
[Internet] 59(7):1667–1673. Available from: https://
europepmc.org/articles/PMC2889766/ [cited 2019
Nov 5]
Sandrini L, Di Minno A, Amadio P, Ieraci A, Tremoli E,
Barbieri SS (2018) Association between obesity and
circulating brain-derived Neurotrophic factor (BDNF)
levels: systematic review of literature and meta-
analysis. Int J Mol Sci 19(8):2281
Scherag A, Dina C, Hinney A, Vatin V, Scherag S, Vogel
CI et al (2010) Two new Loci for body-weight regula-
tion identified in a joint analysis of genome-wide asso-
ciation studies for early-onset extreme obesity in
French and German study groups. PLoS Genet [Inter-
net] 6(4):e1000916. Available from: https://
europepmc.org/articles/PMC2858696/ [cited 2019
Oct 30]
Shugart YY, Chen L, Day IN, Lewis SJ, Timpson NJ,
Yuan W et al (2009) Two British women studies
replicated the association between the Val66Met poly-
morphism in the brain-derived neurotrophic factor
(BDNF) and BMI. Eur J Hum Genet EJHG [Internet]
17(8):1050–1055. Available from: http://europepmc.
org/abstract/MED/19209189 [cited 2019 Nov 4]
Shungin D, Winkler TW, Croteau-Chonka DC, Ferreira T,
Locke AE, Mägi R et al (2015) New genetic loci link
adipose and insulin biology to body fat distribution.
Nature 518(7538):187–196
Speliotes EK, Willer CJ, Berndt SI, Monda KL,
Thorleifsson G, Jackson AU et al (2010) Association
analyses of 249,796 individuals reveal 18 new loci
associated with body mass index. Nat Genet 42
(11):937–948
Surendran P, Drenos F, Young R, Warren H, Cook JP,
Manning AK et al (2016) Trans-ancestry meta-
analyses identify rare and common variants associated
with blood pressure and hypertension. Nat Genet 48
(10):1151–1161
Tachmazidou I, Süveges D, Min JL, Ritchie GRS,
Steinberg J, Walter K et al (2017) Whole-genome
sequencing coupled to imputation discovers genetic
signals for anthropometric traits. Am J Hum Genet
[Internet] 100(6):865–884. Available from: http://
www.sciencedirect.com/science/article/pii/
S0002929717301593
Thorleifsson G, Walters GB, Gudbjartsson DF,
Steinthorsdottir V, Sulem P, Helgadottir A et al
(2009) Genome-wide association yields new sequence
248
variants at seven loci that associate with measures of
obesity. Nat Genet 41(1):18–24
Tikkanen E, Gustafsson S, Amar D, Shcherbina A,
Waggott D, Ashley EA et al (2018) Biological insights
into muscular strength: genetic findings in the UK
Biobank. Sci Rep 8(1):6451
Tobacco and Genetics Consortium (2010) Genome-wide
meta-analyses identify multiple loci associated with
smoking behavior. Nat Genet 42(5):441–447
Turcot V, Lu Y, Highland HM, Schurmann C, Justice AE,
Fine RS et al (2018) Protein-altering variants
associated with body mass index implicate pathways
that control energy intake and expenditure in obesity.
Nat Genet [Internet] 50(1):26–41. Available from:
https://doi.org/10.1038/s41588-017-0011-x
van der Harst P, Verweij N (2018 Feb 02) Identification of
64 novel genetic loci provides an expanded view on the
genetic architecture of coronary artery disease. Circ
Res 122(3):433–443
Vimaleswaran KS, Tachmazidou I, Zhao JH, Hirschhorn
JN, Dudbridge F, Loos RJ (2012) Candidate genes for
obesity-susceptibility show enriched association
within a large genome-wide association study for
BMI. Hum Mol Genet [Internet] 21(20):4537–4542.
Available from: https://europepmc.org/articles/
PMC3607467/ [cited 2019 Oct 30]
Visscher PM, Wray NR, Zhang Q, Sklar P, McCarthy MI,
Brown MA et al (2017) 10 years of GWAS discovery:
biology, function, and translation. Am J Hum Genet
[Internet] 101(1):5–22. Available from: http://www.
sciencedirect.com/science/article/pii/
S0002929717302409 [cited 2019 Oct 28]
Wang K, Li WD, Zhang CK, Wang Z, Glessner JT, Grant
SF et al (2011) A genome-wide association study on
obesity and obesity-related traits. PloS One [Internet] 6
(4):e18939. Available from: https://europepmc.org/
articles/PMC3084240/ [cited 2019 Oct 30]
Wang HJ, Hinney A, Song JY, Scherag A, Meng XR,
Grallert H et al (2016) Association of common variants
identified by recent genome-wide association studies
with obesity in Chinese children: a case-control study.
BMC Med Genet [Internet] 17:7. Available from:
https://europepmc.org/articles/PMC4724138/ [cited
2019 Oct 31]
Wen W, Zheng W, Okada Y, Takeuchi F, Tabara Y,
Hwang J-Y et al (2014) Meta-analysis of genome-
wide association studies in East Asian-ancestry
populations identifies four new loci for body mass
H. Marcos-Pasero et al.
index. Hum Mol Genet [Internet] 23(20):5492–5504.
Available from: https://doi.org/10.1093/hmg/ddu248
[cited 2019 Oct 29]
Winkler TW, Justice AE, Graff M, Barata L, Feitosa MF,
Chu S et al (2015) The influence of age and sex on
genetic associations with adult body size and shape: a
large-scale genome-wide interaction study. PLOS
Genet [Internet] 11(10):e1005378. Available from:
https://doi.org/10.1371/journal.pgen.1005378
World Health Organization (WHO). Obesity and Over-
weight. [Internet]. [cited 2019 Nov 6]. Available
from: https://www.who.int/news-room/factsheets/
detail/obesity-and-overweight
Wolfe D, Dudek S, Ritchie MD, Pendergrass SA (2013)
Visualizing genomic information across chromosomes
with PhenoGram. BioData Min [Internet] 6:18. Avail-
able from: https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC4015356/ [cited 2019 Oct 30]
Wu L, Xi B, Zhang M, Shen Y, Zhao X, Cheng H et al
(2010) Associations of six single nucleotide
polymorphisms in obesity-related genes with BMI
and risk of obesity in Chinese children. Diabetes [Inter-
net] 59(12):3085–3089. Available from: http://
europepmc.org/abstract/MED/20843981 [cited 2019
Nov 4]
Xi B, Cheng H, Shen Y, Chandak GR, Zhao X, Hou D et al
(2013) Study of 11 BMI-associated loci identified in
GWAS for associations with central obesity in the
Chinese children. PLoS One 8(2):e56472
Xu B, Xie X (2016) Neurotrophic factor control of satiety
and body weight. Nat Rev Neurosci 17(5):282–292
Yengo L, Sidorenko J, Kemper KE, Zheng Z, Wood AR,
Weedon MN et al (2018) Meta-analysis of genome-
wide association studies for height and body mass
index in 700000 individuals of European ancestry.
Hum Mol Genet 27(20):3641–3649
Zandoná MR, Sangalli CN, Campagnolo PDB, Vitolo
MR, Almeida S, Mattevi VS (2017) Validation of
obesity susceptibility loci identified by genome-wide
association studies in early childhood in South
Brazilian children. Pediatr Obes [Internet] 12
(1):85–92. Available from: https://onlinelibrary.wiley.
com/doi/abs/10.1111/ijpo.12113 [cited 2019 Oct 31]
Zhao J, Bradfield JP, Li M, Wang K, Zhang H, Kim CE
et al (2009 Dec) The role of obesity-associated loci
identified in genome-wide association studies in the
determination of pediatric BMI. Obes Silver Spring
Md 17(12):2254–2257
 The Science of Love: State of the Art
16
Donatella Marazziti, Stefania Palermo, and Federico Mucci

Abstract positive effects of love can be extremely bene-

In these last decades, emotions and feelings, ficial for both mental and physical health.
neglected for centuries by experimental However, available data are still limited,
sciences, have become the topic of extensive and the proposed models, although supported
neuroscientific research. Currently, love, the by converging data, should be considered
most typically human feeling, can be viewed speculative and oversimplified. The hope is
as the result of different phases (steps), each that neuroscience will permit to shed light on
regulated by evolutionary well-conserved and love, one of the most intriguing, and still
integrated neural substrates. We have pro- largely unknown mysteries of human nature.
posed that the early stage, generally called
romantic love, is the result of the activation
of the brain limbic structures regulating fear/
anxiety reactions leading to changes of major
Abbreviations
neurotransmitters, such as increased mono-
amine levels and decreased serotonin
BD bipolar disorder
concentrations. The second stage of love is
BDNF brain-derived neurotrophic factor
mainly underlain by the structures regulating
dACC dorsal anterior cingulate cortex
the attachment system and involving oxytocin
ECR Experiences in Close Relationships
and vasopressin neuropeptides and
fMRI functional magnetic resonance imaging
neurotrophins. This would explain why the
NGF nerve growth factor
NTs neurotrophins
OCD obsessive-compulsive disorder
D. Marazziti (*)
Department of Clinical and Experimental Medicine OT oxytocin
Section of Psychiatry, University of Pisa, Pisa, Italy ReHo regional homogeneity
UniCamillus - Saint Camillus International University of rsfMRI resting state functional magnetic
Health and Medical Sciences, Rome, Italy resonance imaging
e-mail: dmarazzi@psico.med.unipi.it VP vasopressin
S. Palermo 5-HT 5-hydroxytryptamine (serotonin)
Department of Clinical and Experimental Medicine
Section of Psychiatry, University of Pisa, Pisa, Italy
F. Mucci
Department of Biotechnology, Chemistry and Pharmacy,
University of Siena, Siena, Italy

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 249
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_16
250
16.1 Introduction
Love is an exquisite human attribute that is
embedded in human nature. Love springs and
develops because human beings are born with
the innate drive toward the reward intrinsic to
loving and to be loved. Love is a process, specifi-
cally an integrated neurobiobehavioral process
which promotes proximity between two unrelated
individuals, reproduction, and kids’ rearing.
Moreover, it reduces feelings of stress and anxi-
ety while triggering a deep sense of safety, joy,
and reward. Therefore, love is a blend of brain
mechanisms and related physical phenomena, as
well as cultural and social factors that may shape
some features and regulate its expression. If some
components of this complex process can be
also found in mammals, what makes love typical
of humans is the evidence that the formation of
pair bonding in our species is related not only to
reproduction, but also to the creation of group
structures, social organizations, and interactions.
Nature should have provided us mechanisms of
increasing complexity for assuring that the focus
is limited to one (or a few) partner(s) only, and the
sense of safety is formed and maintained, while
rewarding the individuals with that feeling of
pleasure and completeness that we call love. The
sum of emotions + behaviors + subjective aware-
ness of the whole processes constitutes, perhaps,
the essence of love (Marazziti 2009, 2015; De
Boer et al. 2012).
The consistency of love across different
individuals suggests that it is regulated by shared
biological mechanisms that only recently have
been investigated. Modern brain imaging and
neurobiological techniques have revealed that
people in romantic relationships, the first step of
love, bear a striking similarity to those with psy-
chiatric disorders, which is not surprising, just
considering that love involves the same systems
that regulate anxiety and fear responses, and
related neurotransmitters (Marazziti et al. 1999;
Bartels and Zeki 2000; Fisher 1992; Fisher et al.
2016). The next step, that is, attachment, involves
the nonapeptide oxytocin and dopamine areas
regulating the reward process.
D. Marazziti et al.
Nature follows its rules in all processes, that is
to say, accomplishment of its scopes. Therefore,
love is moral from the point of view of the nature,
but while being regulated by the same and strong
neurobiological systems involved in species sur-
vival, its intents may collide with human rules. If
love may represent the most extraordinary and
joyful experience that nature invented for
humans, on the other side, it may provoke not
only extreme individual suffering, but also family
destructions, or disasters like wars, suicides, and
murders.
The aim of the present paper is to provide a
careful and updated literature review on the neu-
robiology of love together with authors’ personal
contributions in this fascinating domain.
16.1.1 The First Step of Love:
Attraction
Attraction is the biological term to denote what is
commonly called romantic love or falling in love.
The evidence that is experienced by every
individual all over the world strongly indicates
that it is possibly underlaid by neurobiological
underpinnings (Jankowiak and Fischer 1992).
Available data suggest that generally it lasts
between 6 months and 2 years that is considered
the necessary time required to mate, to reach a
pregnancy, and to take care of the newborns
before they become mature to survive alone.
Attraction is independent from bonding, having
sex, or being reciprocated.
Our hypothesis is that attraction is a basic
emotion that shares some neurobiological
substrates with fear and anxiety (LeDoux 2000;
Marazziti and Cassano 2003; Marazziti 2015). As
such, nobody can decide by his/her will to be
attracted by somebody; rather, it is driven by
casual and strong sensorial stimuli occurring
more or less on the same time in two unrelated
individuals. Indeed, following internal (such as
hormonal variations) and external (such as life
events) changes, our brain, in particular amyg-
dala, a group of subcortical nuclei regulating
fight/flight responses fears and emotions,
becomes particularly predisposed to “normal”
16 The Science of Love: State of the Art
Fig. 16.1 The circuitry of
romantic love. (a) Shorter
pathway, (b) longer
pathway
stimuli coming from another person. These
stimuli are first processed by the thalamus, and
then sent to amygdala through two pathways: a
direct and a longer one involving an intermediate
step represented by the cortex (Fig. 16.1). The
amygdala modulates the incoming stimuli while
detecting its emotional quality (good or bad?) and
organizes all outputs, such as neurovegetative
reactions and hormonal responses, in order to
prepare the individual to fight or flight. After
6 milliseconds, the activated cortex, through the
longer pathway, provides the subjective aware-
ness of the bodily changes that everybody can
relate to being in love, in spite of the evidence
that, in terms of brain circuits, they are the same
of fear or anxiety. Love can be considered, there-
fore, “a fear without fear” (Marazziti 2015).
Altered mental state and intrusive thoughts
about the partner are the specific features of
attraction. Altered mental state resembles
alternating phases of the bipolar disorder (BD):
people can experience a raised energy, changes in
both the appetite and in the need of sleep, lower-
ing of rational judgment, or mood swings
according to the reciprocation or not by the part-
ner. Such characteristics would suggest increased
functioning of the norepinephrine and dopamine
systems, as well as increased opioid peptides and
a decrease in serotonin (5-HT). The lower func-
tioning of the 5-HT system may explain intrusive
thoughts regarding the partner and repetitive
251
behaviors to gain his/her attention, while
suggesting a common alteration with obsessive-
compulsive disorder (OCD) patients (Marazziti
et al. 1999).
The temporary state of “madness” experienced
by people in the phase of romantic love may have
an evolutionary purpose, as it pushes the individ-
ual to become more impulsive, to overcome fear
and neophobia, and to willingly accept to be close
to a stranger that becomes the most extraordinary
one all over the world. That is to say, it allows the
individual to get out of their comfort zone to
experience an emotional storm.
Romantic love is a stressful event, as shown by
the hormonal changes it entails (Marazziti and
Canale 2004), and, as such it has the function of
outgoing individual boundaries, creating new
bonds, and evolving. However, there is a cost to
pay due to the significant changes of
neurotransmitters and related brain structures: a
greater risk of developing a full-blown psychiatric
disorder.
In any case, human beings cannot escape from
falling in love: it is embedded in our nature that if
we love and will be loved, we will be rewarded by
a deep joy and by increasing chance of survival.
Not surprisingly, some authors reported higher
levels of two neurotrophins (NTs), specifically
nerve growth factor (NGF) and brain-derived
neurotrophic factor (BDNF) in plasma of roman-
tic lovers (Emanuele et al. 2006).
252
Unfortunately, all described hormonal and
neurobiological do not last forever, and the
“altered state of mind” returns to a more stable
and calm condition. In parallel, if the partner does
not reciprocate or the emotions do not last, the
relation comes to an end; otherwise, the couple is
ready to move toward experiencing the next
step of love that is romantic attachment.
16.1.2 Romantic Attachment
Romantic attachment is the social process
keeping together two individuals for a long
period, sometimes forever. It is believed generally
that it is triggered and maintained by the neuro-
peptide oxytocin (OT) that seems to be involved
in the initiation and maintenance of pair bonding,
infant attachment, and maternal behavior, while
vasopressin (VP) would show opposite activities
(Carter 1998; Insel and Young 2001). Both OT
and VP are produced in the hypothalamus and
released by the posterior pituitary (Acher 1996).
Oxytocin would be produced in the hypothalamus
when the attraction phase starts, and it is sup-
posed to “transform” the “anxiety/fear” reactions
in a sense of well-being, reward, and joy (Kosfeld
et al. 2005). This is possible through the reduction
of the stress systems, and the activation of the
reward processing regulated by the neurotrans-
mitter dopamine (Heinrichs et al. 2003). At the
same time, OT is released in the bloodstream and
conveys information to peripheral organs, where
it is also produced in some of them, and returns to
the brain, closing a loop linking brain
mechanisms to periphery. According to the
polyvagal theory, the activity of OT on the
nucleus vagus, especially on the ventral complex,
the so-called smart vagus would permit to provide
an explanation of the different emotions and
bodily changes occurring during a social bonding
such as love (Porges and Furman 2011). One of
our studies confirmed the relationship between
romantic attachment and OT in 45 healthy
subjects (Marazziti et al. 2006). In addition, the
first double-blind studies conducted with func-
tional magnetic resonance imaging (fMRI) con-
firmed the role of OT in romantic attachment
D. Marazziti et al.
while showing activation of brain area rich in
OT receptors when individuals look at pictures
of the partners (Bartels and Zeki 2000; Fisher
et al. 2002). Subsequent authors confirmed these
findings while highlighting how RR is a strong
motivational state that follows the neuronal cir-
cuitry of reward and pleasure (Aron et al. 2005),
and how long-term love activates the attachment
system (Acevedo et al. 2012).
Besides OT and VP, also NTs seem to play a
role in this phase, as shown in a study
investigating the possible relationship between
BDNF plasma levels and romantic attachment in
24 healthy adults (12 men, 12 women), as
assessed by the “Experiences in Close
Relationships” (ECR) questionnaire (Brennan
et al. 1998). The results highlighted the presence
of a strong negative correlation between BDNF
levels and the ECR avoidance scale, while
suggesting a role of BDNF in promoting social
bonding through the decrease of fear and avoid-
ance behaviors. On the contrary, this correlation
was not present among men. The authors
hypothesized that perhaps the role of BDNF is
gender-related and influenced by the interactions
between hormones and genes (Marazziti et al.
2009). Interestingly, estrogens are supposed to
induce BDNF synthesis in different brain regions
(Sasahara et al. 2007). Therefore, BDNF may be
more active in women, which are basically more
anxious than men (Kessler et al. 2001; Afifi
2007), so that NTs would protect neurons from
stress-induced damage (Bergström et al. 2008).
According to some authors (Insel 2003; Fisher
et al. 2016), love resembles an addiction, on the
basis of a shared activation of dopamine-rich
regions involved in the reward system and drug
or behavioral addictions, and of overlapping
symptoms, such as a sense of euphoria, craving,
and physical and emotional dependence. Love
can be a positive addiction or a negative one
when not reciprocated or when it becomes toxic,
for example, leading to depression, stalking, sui-
cide, homicide, and other crimes (Fisher et al.
2016).
Song et al., using resting state functional mag-
netic resonance imaging (rsfMRI), aimed at
investigating how romantic love may affect the
16 The Science of Love: State of the Art
Fig. 16.2 The
dopaminergic circuitry of
the social brain (VTA
ventral tegmental area,
NAcc nucleus accumbens)
brain and its functional architecture during rest.
Participants consisted of 34 people (the “in-love
group”) intensely in love for 4–18 months,
34 people who ended their romantic relationship
recently (2–17 months earlier), and 32 people
who had never fallen in love. The “in-love
group,” in comparison with the control group,
showed an increased ReHo (regional homogene-
ity) on the left dorsal anterior cingulate cortex
(dACC), and an increased functional connectivity
within both the reward network (caudate, nucleus
accumbens, etc.) and the social cognition network
(temporoparietal junction, posterior cingulate cor-
tex, etc.) (Fig. 16.2). All the mentioned increases
were positively correlated with the duration of the
“being in love” state (Song et al. 2015).
16.2 Conclusions
The present paper is a review on some of the
major findings on the neurobiology of love. This
is an emerging and intriguing field of research
that only recently has become the topic of inten-
sive scientific investigations and has received
benefits from the application of the most
advanced methods provided by neuroscience.
The scattered data that have been gathered and
are accumulating at an increasing amount, albeit
preliminary and fragmentary, permit to set up an
initial framework and hypotheses that need to be
tested.
253
The scope of the science is not to destroy the
poetry of love, as somebody claimed; on the
contrary, our opinion is that a deeper understand-
ing of the neurobiology of love would permit to
get access to our unique humanity, to love in a
more rewarding and joyful way, and perhaps to
prevent the suffering and dramatic consequences
of unrequited love or separations.
References
Acevedo BP, Aron A, Fisher HE, Brown LL (2012) Neu-
ral correlates of long-term intense romantic love. Soc
Cogn Affect Neurosci 7(2):145–159. https://doi.org/
10.1093/scan/nsq092
Acher R (1996) Molecular evolution of fish neurohypo-
physial hormones: neutral and selective evolutionary
mechanisms. Gen Comp Endocrinol 102(2):157–172.
https://doi.org/10.1006/gcen.1996.0057
Afifi M (2007) Gender differences in mental health.
Singap Med J 48(5):385–391
Aron A, Fisher HE, Mashek DJ, Strong G, Li H, Brown
LL (2005) Reward, motivation, and emotion systems
associated with early-stage intense romantic love. J
Neurophysiol 94:327–337. https://doi.org/10.1152/jn.
00838.2004
Bartels A, Zeki S (2000) The neural basis of romantic love.
Neuroreport 11(17):3829–3834. https://doi.org/10.
1097/00001756-200011270-00046
Bergström A, Jayatissa MN, Mørk A, Wiborg O (2008)
Stress sensitivity and resilience in the chronic mild
stress rat model of depression; an in situ hybridization
study. Brain Res 1196:41–52. https://doi.org/10.1016/
j.brainres.2007.12.025
Brennan KA, Clark CL, Shaver PR (1998) Self-report
measurement of adult attachment: an integrative over-
view. In: Simpson JA, Rholes WS (eds) Attachment
254
theory and close relationships. New York, Guilford
Press, pp 46–76
De Boer A, van Buel EM, Ter Horst GJ (2012) Love is
more than just a kiss: a neurobiological perspective on
love and affection. Neuroscience 201:114–124. https://
doi.org/10.1016/j.neuroscience.2011.11.017
Carter CS (1998) Neuroendocrine perspectives on social
attachment and love. Psychoneuroendocrinology 23
(8):779–818. https://doi.org/10.1016/s0306-4530(98)
00055-9
Emanuele E, Politi P, Bianchi M, Minoretti P, Bertona M,
Geroldi D (2006) Raised plasma nerve growth factor
levels associated with early-stage romantic love.
Psychoneuroendocrinology 31(3):288–294. https://
doi.org/10.1016/j.psyneuen.2005.09.002
Fisher E (1992) Anatomy of love. Fawcett Columbine,
New York
Fisher HE, Aron A, Mashek D, Li H, Brown LL (2002)
Defining the brain systems of lust, romantic attraction,
and attachment. Arch Sex Behav 31(5):413–419.
https://doi.org/10.1023/a:1019888024255
Fisher HE, Xu X, Aron A, Brown LL (2016) Intense,
passionate, romantic love: a natural addiction? How
the fields that investigate romance and substance abuse
can inform each other. Front Psychol 7:687. https://doi.
org/10.3389/2Ffpsyg.2016.00687
Heinrichs M, Baumgartner T, Kirschbaum C, Ehlert U
(2003) Social support and oxytocin interact to suppress
cortisol and subjective responses to psychosocial
stress. Biol Psychiatry 54(12):1389–1398. https://doi.
org/10.1016/s0006-3223(03)00465-7
Insel TR (2003) Is social attachment an addictive disorder?
Physiol Behav 79:351–357. https://doi.org/10.1016/
s0031-9384(03)00148-3
Insel TR, Young LJ (2001) The neurobiology of attach-
ment. Nat Rev Neurosci 2:129–136. https://doi.org/10.
1038/35053579
Jankowiak WR, Fischer EF (1992) A cross-cultural per-
spective on romantic love. Ethnology 31(2):149–155.
https://doi.org/10.2307/3773618
Kessler RC, Keller MB, Wittchen HU (2001) The epide-
miology of generalized anxiety disorder. Psychiatr Clin
North Am 24:19–39. https://doi.org/10.1016/s0193-
953x(05)70204-5
Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E
(2005) Oxytocin increases trust in humans. Nature
435:673–676. https://doi.org/10.1038/nature03701
LeDoux JE (2000) Emotion circuits in the brain. Annu Rev
Neurosci 23:155–184. https://doi.org/10.1146/
annurev.neuro.23.1.155
D. Marazziti et al.
Marazziti D (2009) Neurobiology and hormonal aspects of
romantic attachment. In: De Haan M, Gunnar MN
(eds) Handbook of developmental and social neurosci-
ence. New York, Guilford Press, pp 265–280
Marazziti D (2015) “Beyond emotion: love as an encoun-
ter of myth and drive” by Lubomir Lamy. Emotion Rev
8(2):110–112. https://doi.org/10.1177/
2F1754073915594437
Marazziti D, Canale D (2004) Hormonal changes when
falling in love. Psychoneuroendocrinology 29
(7):931–936. https://doi.org/10.1016/j.psyneuen.2003.
08.006
Marazziti D, Cassano GB (2003) The neurobiology of
attraction. J Endocrinol Investig 26:58–61
Marazziti D, Akiskal HS, Rossi A, Cassano GB (1999)
Alteration of the platelet serotonin transporter in
romantic love. Psychol Med 29(3):741–745. https://
doi.org/10.1017/s0033291798007946
Marazziti D, Dell’Osso B, Baroni S, Mungai F, Catena M,
Rucci P, Albanese F, Giannaccini G, Betti L,
Fabbrini L, Italiani P, Del Debbio A, Lucacchini A,
Dell’Osso L (2006) A relationship between oxytocin
and anxiety of romantic attachment. Clin Pract
Epidemiol Ment Health: CP & EMH 2:28. https://doi.
org/10.1186/1745-0179-2-28
Marazziti D, Roncaglia I, Del Debbio A, Bianchi C,
Massimetti G, Origlia N, Domenici L, Piccinni A,
Dell’Osso L (2009) Brain-derived neurotrophic factor
in romantic attachment. Psychol Med 39
(11):1927–1930. https://doi.org/10.1017/
S0033291709990742
Porges SW, Furman SA (2011) The early development of
the autonomic nervous system provides a neural plat-
form for social behavior: a polyvagal perspective.
Infant Child Dev 20(1):106–118. https://doi.org/10.
1002/icd.688
Sasahara K, Shikimi H, Haraguchi S, Sakamoto H,
Honda S, Harada N, Tsutsui K (2007) Mode of action
and functional significance of estrogen-inducing den-
dritic growth, spinogenesis, and synaptogenesis in the
developing Purkinje cell. J Neurosci 27:7408–7417.
https://doi.org/10.1523/JNEUROSCI.0710-07.2007
Song H, Zou Z, Kou J, Liu Y, Yang L, Zilverstand A,
d’Oleire Uquillas F, Zhang X (2015) Love-related
changes in the brain: a resting-state functional mag-
netic resonance imaging study. Front Hum Neurosci
9:71. https://doi.org/10.3389/fnhum.2015.00071
 NGF and Retinitis Pigmentosa: Structural
and Molecular Studies 17
Maria Luisa Rocco, Laura Calzà, and Luigi Aloe

Abstract reduces/stops the progression of photoreceptor

Nerve growth factor (NGF) is a neuroprotective
molecule performing not only on central and
peripheral neurons but also on cells of the
visual system. Human retinitis pigmentosa
(RP) is a major cause of blindness worldwide,
and a resolute therapy is still lacking. Recent
studies have shown that ocular NGF adminis-
tration exerts a protective action on damaged
retinal cells of mammalians, including human
beings, although whether NGF also protects
photoreceptors is not clear.
We used the Royal College of Surgeons
(RCS) strain in this study. The RCS is a rodent
affected by inherited retinitis pigmentosa
(RP) during postnatal life. For this study, we
investigated whether ocular NGF treatment
M. L. Rocco (*)
IRET Foundation, Ozzano Emilia, Bologna, Italy
Institute of Translational Pharmacology, CNR, Rome,
Italy
L. Calzà
Health Science and Technologies Interdepartmental
Center for Industrial Research (HST-ICIR), University of
Bologna, Ozzano Emilia, Bologna, Italy
Department of Pharmacy and Biotechnology, University
of Bologna, Bologna, Italy
Montecatone Rehabilitation Institute, Montecatone,
Bologna, Italy
e-mail: laura.calza@unibo.it
L. Aloe
IRET Foundation, Ozzano Emilia, Bologna, Italy
degeneration of rats with RP.
This study was carried out in vitro on
isolated photoreceptors to further investigate
the action on these cells and whether the action
is direct or mediated.
The results indicate that ocular NGF admin-
istration can protect photoreceptors from
degeneration into a model developing
inherited RP and that the NGF action is direct.
In this regard, we observed that binding of
NGF to its receptor modulates expression of
rhodopsin, a specific biological marker for
photoreceptor survival and functionality.
Part of the data reported in this chapter has
been published in a previous study.
Abbreviations
NGF Nerve Growth Factor
RCS Royal College of Surgeons
RP Retinitis Pigmentosa
RPE Retinal Pigment Epithelium
TrkA Tyrosine Kinase Receptor
17.1 Introduction
We used the Royal College of Surgeons (RCS)
strain in this study. The RCS is a rodent affected
by inherited retinitis pigmentosa (RP) during
postnatal life (D’Cruz et al. 2000). The primary

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 255
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_17
256
pathological event in many forms of retinal
degeneration is the degeneration of photoreceptor
cells, while one of the most common retinal
degenerations is retinitis pigmentosa (RP). RP is
a group of inherited human diseases characterized
by progressive degeneration of photoreceptor
cells and loss of photoreceptor function (Dowling
and Sidman 1962; Faktorovich et al. 1990). A
number of recent studies report that the differen-
tiation, survival, and function of retinal cells are
regulated by survival growth factors, including
neurotrophins (Amendola and Aloe 2002;
Amendola et al. 2003; Pagani et al. 2006). NGF
is a potent stimulant of the tropism and wound
healing process in the eye, and basically, all
structures of the eye can produce NGF and even
upregulate NGF synthesis in the event of a lesion.
In recent years, Aloe et al. have demonstrated that
NGF topical administration plays an important
role in the survival of photoreceptor cells, as
well as that NGF exercises a significant effect
on expression of its receptors, blood vessel distri-
bution, and the degeneration of the outer nuclear
layer (ONL) (Aloe 2004; Aloe et al. 2008, 2012).
In this study, we investigated whether ocular
NGF treatment reduces/stops the progression of
photoreceptor degeneration of rats with RP
(Rocco et al. 2015). During the early stages of
photoreceptor degeneration, rats were treated
with NGF eye drops (200 ug/ml) three times a
day for 10 consecutive days. The controls were
treated with doses of vehicle solution for the same
length of time (Rocco et al. 2015). At the end of
the treatment, the eyes were surgically removed
and used for histological, immunohistochemical,
biochemical, and molecular studies (Rocco et al.
2015). Compared to controls, it emerged that
NGF-treated retinas showed altered expression
of apoptotic markers, enhanced presence of
NGF receptors, and reduced loss of
photoreceptors (Rocco et al. 2015). Our study
was carried out in vitro on isolated photoreceptors
to further investigate the action on these cells and
whether the latter is direct or mediated (Rocco
et al. 2015).
M. L. Rocco et al.
17.2 Methods
NGF Isolation and Purification For these stud-
ies, the molecule 2.5S NGF was purified in our
laboratory from adult CD1 mouse submaxillary
gland (75gr). The biological activity of NGF is
tested on PC12 cells, plated on 24-well plates at a
density of 10,000 cells/wells in Dulbecco’s
Modified Eagle Medium (DMEM), containing
10% heat-inactivated horse serum and 5% bovine
serum. Cells plated on glass dishes, which had
previously coated with polylysine, were cultured
in the presence of and with different
concentrations of NGF for 2–4 days.
Figure 17.1a–b illustrates representative
photomicrographs showing PC12 cells cultured
in vitro for four consecutive days in the absence
(Fig. 17.1a) and in the presence of 10 ng/ml of
NGF (Fig. 17.1b). The presence of NGF
stimulates neuritis outgrowth. For further details
see Bocchini and Angeletti (1969).
NGF and ANA Biological Activity
on PC12 Before using the NGF on isolating
photoreceptors, we first tested the biological
activity on PC12 cell line routinely used for test-
ing the biological activity of NGF. In the absence
of NGF, the cells are unable to produce neuritis
outgrowth, while in the presence of 50 ng/ml
NGF, they respond with neuritis outgrowth, and
in the presence or 50 ng/ml NGF, preincubated
with 500 ng/ml anti-NGF antibody (ANA) that
inhibit the NGF biological activity, these cells are
unable to produce neuritis. Together these
observations indicate that both NGF and ANA
are biologically and functionally active (for the
results see Rocco et al. (2015)).
Western Blotting Analysis For Western blot-
ting analysis, tissues were homogenized with
ultrasonication in RIPA buffer (50 mM Tris,
pH 7.5; 150 mM NaCl; 5 mM EDTA; 1% Triton
X-100; 0.1% SDP; 0.5% DOC (sodium
deoxycholate); 1 mM PMSF; 1 μg/ml leupeptin),
centrifuged at 4
C for 20 min at 13000 rpm; then
the supernatant was stored at 20
C. Samples
(30 μg of total protein) were dissolved in loading
17 NGF and Retinitis Pigmentosa: Structural and Molecular Studies
Fig. 17.1 PC12 cells in the absence (untreated) or pres-
ence of NGF. Untreated cells are unable to produce neuri-
tis outgrowth, (a) while in the presence of 50 ng/ml NGF,
they respond with neuritis outgrowth (b red arrow). Scale
bar: 15 μm. Phase-contrast images, 40/objective. Confo-
cal microscopy images of DAPI (c green channel),
buffer (0.1 M Tris–HCl buffer, pH 6.8, containing
0.2 M dithiothreitol (DTT), 4% Sodium dodecyl
sulfate (SDS), 20% glycerol, and 0.1%
bromophenol blue), separated by 8% or 12%
SDP-PAGE, and electrophoretically transferred
to PVDF membrane overnight. The membranes
were incubated for 1 h at room temperature with
blocking buffer constituted by 5% nonfat dry
milk in TBS-T (10 mM Tris, pH 7.5, 100 mM
NaCl, and 0.1% Tween-20). Membranes were
washed three times for 10 min each at room
temperature in TBS-T followed by incubation at
4
C with primary antibodies overnight with the
antibodies shown in the table. Membranes were
washed three times for 10 min each at room
temperature in TBS-T and incubated for 1 h
with horseradish peroxidase-conjugated
anti-rabbit IgG 1:4000 or horseradish
peroxidase-conjugated anti-mouse IgG as the sec-
ondary antibody (Cell Signaling Technology,
MA, USA) at room temperature. The blots were
developed with an ECL chemiluminescent horse-
radish peroxidase (HRP) substrate as the chromo-
phore (Millipore, MA, USA). The public ImageJ
software was used to evaluate band density,
which was expressed as arbitrary units of gray
level. The ImageJ program determines the optical
density of the bands using a grayscale sharehold-
ing operation. The optical density of polyclonal
rabbit anti-GAPDH 1:4000 (Santa Cruz
257
rhodopsin (c red channel), NGF-treated RCS retinas. Rho-
dopsin and DAPI immunoreactivity appeared in photore-
ceptor cells was clearly increased and the outgrowth of
neurites after NGF treatment (c yellow arrow). Scale bar:
15 μm. 60/objective; zoom 5; 1024  1024 pixels; 10 Hz
acquisition speed
Biotechnology, CA, USA) bands was used as a
normalizing factor. For each gel blot, the
normalized values were then expressed as per-
centage of relative normalized controls and used
for statistical evaluation. Statistical evaluations
were performed using the GraphPad Prism pack-
age for Windows and data expressed as means 
SEM of eight different corneas (for the results see
Rocco et al. (2015)).
Relative Quantitative Real-Time PCR For
RT-PCR analysis of TrkA and p75NTR, total
RNA from the cornea was extracted using SV
Total Isolation System (Promega Italia, Milan,
Italy). RNA was quantified by spectrophotometer
at 260 nm and stored at 80
C before use. RNA
was converted into cDNA in a 20 μl reverse
transcription reaction containing 2γ of total
RNA; 1X reverse transcriptase buffer (50 mM
Tris–HCl, pH 8.3; 75 mM KCl; 3 mM MgCl2;
10 mM DTT); 0.5 mM dNTPs; 200 ng oligo
(dT) 15; 0.5 U RNasin ribonuclease inhibitor;
and 200 U of M-MLV RT (Promega Italia,
Milan, Italy). Reactions were incubated at 42
C
for 60 min, heated at 95
C for 5 min, and then
cooled at 4
C for a minimum of 5 min and a
maximum of 30 min. The polymerase chain reac-
tion (PCR) was analyzed using the 7900HT Fast
Real-Time PCR System (PE Applied Biosystems)
and FAM-labeled probe specific for the NTrk1
258
(Rn00572130_m1) and Ngfr (Rn00561634_m1)
(PE Applied Biosystems). The housekeeping
gene, glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) (4352338E) (PE Applied
Biosystems), was co-amplified with TrkA and
p75NTR and used to normalize sample
variability. The cDNA was amplified under the
following conditions: 1 cycle at 50
C for 2 min
and at 95
C for 10 min, followed by 40 cycles at
95
C for 15 s and at 60
C for 1 min. The amount
of mRNA of each gene was calculated using the
standard curve method and adjusted for the
expression of GAPDH. Relative quantification
was performed using the ΔΔCt method (for the
results see Rocco et al. (2015)).
Immunohistochemistry For immunohisto-
chemistry eye globes of each experimental
group were removed and fixed in 4% paraformal-
dehyde dissolved in 0.1 M phosphate buffer,
pH 7.4, for 24 h and then left overnight in the
same buffer containing 20% sucrose. Coded
sections of each eye were then sectioned with a
cryostat, at 20 μm thickness. Sections were first
exposed to 0.03% of hydrogen peroxide (H2O2)
and 10% of methanol for 20 min, followed by
exposure to 0.1 M PBS containing 10% of horse
or goat serum for 1 h, and then incubated over-
night at 4
C with polyclonal rabbit anti-TrkA
1:100 (Santa Cruz, CA, USA) and monoclonal
mouse anti-p75 1:100 (Santa Cruz, CA, USA).
Sections, after having been washed, were then
exposed to biotinylated anti-mouse or anti-rabbit
IgG 1:300 (anti-IgG and avidin-conjugated horse-
radish peroxidase complex) which were pur-
chased by Vector Laboratories (Burlingame,
CA, USA) with 2% of goat or horse serum,
depending on the animal in which the secondary
antibody was produced, for 2 h at room tempera-
ture, and immunoperoxidase staining was
performed using an ABC (1:100) (Avidin-Biotin
Complex Solution, Vectastain Elite Kit (Vector
Laboratories, CA, USA) for 2 h at room tempera-
ture. Signals were visualized by DAB (3,3-
0
-diaminobenzidine) as a chromate. Stained
sections were visualized using a Zeiss Axiophot
M. L. Rocco et al.
microscope equipped with a 40X objective
(Rocco et al. 2015).
Immunofluorescence For immunofluorescence
eye globes of each experimental group were
removed and fixed in 4% paraformaldehyde
dissolved in 0.1 M phosphate buffer, pH 7.4, for
24 h and then left overnight in the same buffer
containing 20% of sucrose. Coded sections of
each eye were then sectioned with a cryostat, at
20 μm thickness. Sections were first washed with
1% of PBS two times for 5 min and then
incubated overnight at 4
C with polyclonal rabbit
anti-TrkA 1:100 (Santa Cruz, CA, USA) and
monoclonal mouse anti-rhodopsin 1:100
(Neomarkers, CA, USA). After washing with
PBST, the sections were incubated for 1 h at
room temperature with Alexa Fluor 488 donkey
anti-rabbit IgG (1: 200) and Alexa Fluor 555 don-
key anti-mouse IgG (1: 200). After two rinses in
PBS and 10-min incubation with the DAPI solu-
tion (Molecular Probes, Invitrogen, Italy) for
nuclei visualization, sections were coverslipped
and examined under a confocal laser scanning
microscope (Leica SP5, Leica Microsystems,
Germany) under a sequential mode to avoid
cross talk between channels. The confocal image
acquisition was conducted so that all samples
were imaged using consistent settings for laser
power and detector gain (for the results see
Rocco et al. (2015)).
Cell Culture We first tested the biological activ-
ity on PC12 cells as reported under Material and
Method Section. As shown in Fig. 17.1a–b, it was
found that the presence of 10 ng of NGF/ml of
culture medium was sufficient to stimulate neurite
outgrowth from these cells, whereas sister cells
cultured in the absence of NGF were unable to do
it. Based on these observations, we tested the
effect of the purified NGF on photoreceptor
response and survival. For cell culture eye globes
of each experimental group were removed, and
the retinal was isolated. The cells were washed
with 6 ml of 10 μg/ml in 0.1 M phosphate buffer
and, after that, were centrifuged for 1 min at
1000 rpm. After digestion in a 0.25% trypsin for
17 NGF and Retinitis Pigmentosa: Structural and Molecular Studies
30 min at 37
C, the trypsin action was stopped
with 10% of serum retinal, after was washed with
10 μg/ml in 0.1 M phosphate buffer, pelleted and
resuspended for 15 min in culture medium
(DMEM/F12, 1:1 Invitrogen), and supplemented
with 5% FBS and 1% penicillin/streptomycin.
The supernatant was released and the pellet was
resuspended with 1 ml of culture medium.
Finally, the two supernatants were centrifuged
together for 10 min at 1000 rpm, and 6 ml of
culture medium was added with 6 ml of culture
medium. The cells (2.5  106 for plate) remained
undisturbed at 37
C in a 5% CO2 atmosphere for
48h before the treatments (for the results see
Rocco et al. (2015)).
Photoreceptor Sorting To verify the effect of
NGF on the photoreceptor, the cells were selected
and separated through from whole retinal cultures
by EasySep Magnet separation with the use of
tetrameric antibody complexes recognizing
rhodopsin-bearing photoreceptors, in the absence
of columns (EasySep Magnet separation;
Stemcell Technologies, Voden Medical
Instruments SpA, Milan, Italy). Enriched
photoreceptors (~80–90%) were cultured, treated,
and analyzed as previously reported. Purity was
assessed by a digital-based flow cytometry plat-
form (MACSQuant Analyzer, Miltenyi, GmbH,
Germany). Both untreated and NGF-treated cells
(6 days) were postfixed in buffered 4% PFA and
analyzed for trkANGFR and p75NTR expression
(for the results see Rocco et al. (2015)).
Cell Death Studies The cells (2.5  106 for
plate) remained undisturbed at 37
C in a 5%
CO2 atmosphere for 48h before the treatments.
After the treatment the cells were homogenized
with ultrasonication in RIPA buffer (50 mM Tris,
pH 7.5; 150 mM NaCl; 5 mM EDTA; 1% Triton
X-100; 0.1% SDP; 0.5% DOC (sodium
deoxycholate); 1 mM PMSF; 1 μg/ml leupeptin),
centrifuged at 4
C for 20 min at 13000 rpm;
then the supernatant was stored at 20
C.
Samples (20 μg of total protein) were dissolved
in loading buffer (0.1 M Tris–HCl buffer, pH 6.8,
containing 0.2 M dithiothreitol (DTT), 4%
259
Sodium dodecyl sulfate (SDS), 20% glycerol,
and 0.1% bromophenol blue), separated by 8%
or 12% SDP-PAGE, and electrophoretically
transferred to PVDF membrane overnight. The
membranes were incubated for 1 hr at room tem-
perature with blocking buffer constituted by 5%
nonfat dry milk in TBS-T (10 mM Tris, pH 7.5,
100 mM NaCl, and 0.1% Tween-20). Membranes
were washed three times for 10 min each at room
temperature in TBS-T followed by incubation at
4
C with Bax (B8429, Sigma, Missouri, USA)
and Bcl-2 (sc-7382, Santa Cruz Biotechnology,
Inc., Europe) antibodies overnight with the
antibodies shown in the table. Membranes were
washed three times for 10 min each at room
temperature in TBS-T and incubated for 1 h
with horseradish peroxidase-conjugated
anti-rabbit IgG 1:4000 or horseradish
peroxidase-conjugated anti-mouse IgG as the sec-
ondary antibody (Cell Signaling Technology,
MA, USA) at room temperature. The blots were
developed with an ECL chemiluminescent horse-
radish peroxidase (HRP) substrate as the chromo-
phore (Millipore, MA, USA). The public ImageJ
software was used to evaluate band density,
which was expressed as arbitrary units of gray
level. The ImageJ program determines the optical
density of the bands using a grayscale sharehold-
ing operation. The optical density of polyclonal
rabbit anti-GAPDH 1:4000 (Santa Cruz Biotech-
nology, CA, USA) bands was used as a
normalizing factor. For each gel blot, the
normalized values were then expressed as per-
centage of relative normalized controls and used
for statistical evaluation. Statistical evaluations
were performed using the GraphPad Prism pack-
age for Windows and data expressed as means 
SEM of eight different corneas (for the results see
Rocco et al. (2015)).
17.3 Results
This chapter presents only two previously unpub-
lished figures, which summarize the essence of a
previously published work (Rocco et al. 2015).
260
Animals were housed in animal facilities and
treated according to the experimental procedure
approved by the Ethical Commission on Animal
Experimentation of the National Research Coun-
cil of our Institution (CNR, Rome). All efforts
were made to reduce the number of animals
used for experimentation, and to minimize animal
suffering. For the in vivo study, rats were grouped
as untreated (n ¼ 30; RCS) and NGF treated
(n ¼ 30; NGF-RCS). RCS rats were treated with
NGF eye drops (200 μg/mL; 5 μL) for 3 times a
day over 21 consecutive days, starting at day
20 of postnatal age. The NGF administration
enhanced both NGF mRNA and protein in the
retina. Similarly, both trkANGFR and p75NTR
levels significantly increased, as quantified by
Western blot analysis and gene expression. The
phosphorylated trkANGFR form (p-trkANGFR)
was overexpressed compared to total trkANGFR
indicating the activation of a specific receptor
(Rocco et al. 2015). To assess whether NGF acts
on the photoreceptor function, retinal sections of
NGF-treated and untreated eyes were
immunostained with anti-rhodopsin, a specific
marker of these cells and of both NGF receptors.
The confocal microscopy revealed increased
trkANGFR immunoreactivity in the retina of
NGF-treated RCS rats against untreated ones
(Fig. 17.2a, b, c and d). As shown in Fig. 17.2a,
b, c, and d, the ONL thickness of rats treated with
ocular NGF (c–d) is higher and markedly stained
with rhodopsin (b–d), compared to the lower
thickness and poor immunostaining in the ONL
of control rats (a–b). A significant enhancement
of both receptors was localized in the ON layer of
NGF-treated RCS retinas, alongside
photoreceptors and in proximity of rhodopsin
immunoreactivity (for the results see Rocco
et al. (2015)). Overall, these observations
revealed a critical protective link between the
NGF effect and photoreceptor survival. To ana-
lyze the effect of the NGF administration on cell
survival, expression of the apoptotic markers Bax
and Bcl-2 – pro- and anti-apoptotic markers,
respectively – was measured (for the results see
Rocco et al. (2015)). All data produced in vivo
showed a positive effect of NGF treatment on the
retinal tissue and, above all, on the ON layer,
M. L. Rocco et al.
through activation of NGF signaling on cell
survival. This is further supported by increased
neuritis outgrowth and rhodopsin expression after
treatment (Fig. 17.1c) (Rocco et al. 2015).
17.3.1 The Effect of NGF Exposure
on Survival of p10 Retinal Cells
We thereafter tested the effect of NGF on primary
cultures obtained from P10 retinas dissected out,
dissociated, and plated for 12 h or 6d depending
on the experiment. As a result, the cells quickly
adhered to the plate (A, time 0 or 12 h), and some
of them resulted in rhodopsin positive (Fig. 17.1c
red staining). These rhodopsin-positive cells
showed a few elongations. Culturing was
performed over 6d in medium alone, with the
result that a reduced number of rhodopsin-
positive cells were detected compared to the ini-
tial plating. Upon 6d NGF exposure (Fig. 17.1c),
the rhodopsin-positive cell number was compara-
ble to that of plating, and interestingly,
elongations occurred in most of the rhodopsin-
positive cells. The effect is clearly visible when
comparing NGF treated with untreated fields.
These observations indicated that NGF stimulates
the survival of photoreceptors (for the results see
Rocco et al. (2015)).
17.3.2 The Effect of NGF
on Rhodopsin-Positive Neuritis
Outgrowth and trkANGFR
Expression
To validate the specificity of the NGF effect on
these retinal cells, primary cultures were seeded/
cultured over 6d in medium alone or
supplemented with NGF, neutralizing anti-NGF
antibodies (ANA) or NGF preincubated with
ANA (NGF/ANA), highlighting the specificity
of the addition of exogenous NGF on survival
and neuritis outgrowth of dissociated P10 retinal
cells as compared to controls. This NGF effect
was detectable neither in the presence of
neutralizing anti-NGF antibodies nor when the
cells were exposed to NGF preincubated with
17 NGF and Retinitis Pigmentosa: Structural and Molecular Studies 261

Fig. 17.2 Confocal

microscopy images of
DAPI (upper panel a–c) and
TrkANGFR (lower panel
b–d), in untreated (a–b) or
NGF-treated RCS retinas
(c–d). TrkANGFR (b–d)
and DAPI (a–c)
immunoreactivity appeared
mainly in the outer nuclear
layer (ONL) and in
surrounding cells was
clearly increased and the
thickness of the layer after
NGF treatment (c–d). Scale
bar: 15 μm. 60/objective;
1024  1024 pixels; 10 Hz
acquisition speed

ANA. Survival and neuritis outgrowth were

17.4 Discussion
quantified in three optic fields from 20 untreated
and NGF-treated wells (for the results see Rocco
The aim of this study was to provide further
et al. (2015)).
supporting data regarding the role of NGF on
The NGF-exposed rhodopsin-positive cells
the cells of the anterior and posterior segments
(photoreceptor) survived culturing over 6d,
of the eye. The first convincing data indicating the
implying that unless daily supply of NGF is
critical protective role of NGF on retinal cells was
provided, they will degenerate. This hypothesis
initially demonstrated in fish by Turner and
might be supported by the observation that these
Delaney (1979), and in mammalians’ visual
cells express trkANGFR. The NGF-exposed cells
cells a few years later. It emerged that NGF
co-expressed both trkANGFR and rhodopsin. A
administration induces modification of presynap-
widespread neuritis outgrowth and a higher num-
tic elements in adult visual system (Colafrancesco
ber of trkANGFR-positive cells were observed
et al. 2011; Conner and Varon 1994; Cuello et al.
upon NGF stimulation, as compared to untreated
2010; Cuello et al. 1992; Curtis et al. 1995);
or ANA- and/or ANA/NGF-treated cells. Indeed,
prevents the shift in ocular dominance distribu-
high trkANGFR and rhodopsin were
tion of visual cortical neurons and promotes func-
co-expressed in NGF-treated cultures and
tional recovery of RGCs after ischemia
co-localized in ANA-treated cultures. Moreover,
(Siliprandi et al. 1993); delays retinal cell degen-
the confocal analysis indicates that NGF
eration in rodents with inherited retinopathy
stimulates neuritis outgrowth from photoreceptor
(Amendola and Aloe 2002; Liang et al. 2012);
(for the results see Rocco et al. (2015)).
and reduces retinal damages in rabbits affected by
Part of the data reported in this chapter has
ocular hypertension, while NGF antibodies can
been published in a previous study (Rocco et al.
exacerbate the damaging action of RGCs
2015).
(Lambiase et al. 2007). A few years later, it was
reported that in the case of purified NGF topical
application on purified NGF patients with
262
advanced glaucoma running the imminent risk of
loss of visual functions (Coassin et al. 2008;
Lambiase et al. 2009), NGF promoted progres-
sive improvement of the functionality of the inner
retinal layer and of the parameters of the post-
retinal neural conduction becoming manifest dur-
ing the treatment period, and maintained it for
3 months after discontinuation of treatment;
visual acuity significantly improved in patients
in whom it had remained unchanged after several
months’ follow-up.
To understand the role of NGF more deeply on
the protective mechanisms of corneal and retinal
cells, we used intravitreal administration of
purified αNGF and examined the effect induced
in corneal and retinal cells. The results showed
that the αNGF administration has no significant
effect on the endogenous presence of NGF in the
retina and considerably reduces the level of NGF
in the cornea. One possible interpretation of these
different effects might be either that corneal cells
are more vulnerable than retinal cells to the pres-
ence of αNGF or alternatively that retinal cells
express large concentration of NGF. Western blot
analysis and immunostaining analyses revealed
that αNGF administration induces no significant
alteration of the high-affinity NGF receptor and
TrkANGFR in the retina and enhances expression
of TrkANGFR protein in the cornea, thus
suggesting different vulnerability of retinal and
corneal cells to αNGF exposure. However, the
possibility that αNGF administration induces
changes in other retinal and corneal biological
mediators cannot be excluded. Indeed, it was
found that the expression of rhodopsin and
inflammatory markers in the retina is significantly
downregulated as a result of αNGF administra-
tion. The observed significant decrease in some
cytokine expression has no explanation. Interest-
ingly, the overexpression to IL-21 may possibly
be related to the activation of Muller cells, which
needs to be investigated to better understand
αNGF action on visual cells. These findings indi-
cate that αNGF administration reaches both cor-
neal and retinal cells and assert that the latter can
cause downregulation of endogenous NGF
expression and retinal cell deficit, thus providing
a useful experimental approach of noninvasive
M. L. Rocco et al.
administration for delivering αNGF to further
investigate the structural, biochemical, and
molecular role of retinal and corneal cells. Over-
all, the information resulting from this study is
that under physiological conditions, NGF
regulates not only normal survival of corneal
and retinal cells but also NGF-responsive cell
degeneration under NGF-deprivation conditions.
To conclude with, our data demonstrated that
cross talk between photoreceptor activity and
NGF signaling actually exists, and reinforced
the concept that NGF is crucial for the regular
activity of the visual system and, in particular, of
photoreceptors.
Acknowledgments L.A. by Fondazione IRET, Ozzano
Emilia, Bologna, Italy.
References
Aloe L (2004) NGF and molecules in health and disease.
Prog Brain Res 2004, 146
Aloe L, Tirassa P, Lambiase A (2008) The topical applica-
tion of nerve growth factor as a pharmacological tool
for human corneal and skin ulcers. Pharmacol Res
57:253–258
Aloe L, Rocco ML, Bianchi P, Manni L (2012) Nerve
growth factor: from the early discoveries to the poten-
tial clinical use. J Transl Med 10:239
Amendola T, Aloe L (2002) Developmental expression of
nerve growth factor in the eye of rats affected by
inherited retinopathy: correlative aspects with retinal
structural degeneration. Arch Ital Biol 140:81–90
Amendola T, Fiore M, Aloe L (2003) Postnatal changes in
nerve growth factor and brain derived neurotrophic
factor levels in the retina, visual cortex, and geniculate
nucleus in rats with retinitis pigmentosa. Neurosci Lett
345:37–40
Bocchini V, Angeletti PU (1969) The nerve growth factor:
purification as a 30,000-molecular-weight protein.
Proc Natl Acad Sci U S A 64:787–794
Coassin M, Lambiase A, Sposato V, Micera A, Bonini S,
Aloe L (2008) Retinal p75 and bax overexpression is
associated with retinal ganglion cells apoptosis in a rat
model of glaucoma. Graefes Arch Clin Exp
Ophthalmol 246:1743–1749
Colafrancesco V, Coassin M, Rossi S, Aloe L (2011)
Effect of eye NGF administration on two animal
models of retinal ganglion cells degeneration. Ann Ist
Super Sanita 47:284–289
Conner JM, Varon S (1994) Nerve growth factor
influences the distribution of sympathetic sprouting
17 NGF and Retinitis Pigmentosa: Structural and Molecular Studies
into the hippocampal formation by implanted superior
cervical ganglia. Exp Neurol 130:15–23
Cuello AC, Maysinger D, Garofalo L (1992) Trophic
factor effects on cholinergic innervation in the cerebral
cortex of the adult rat brain. Mol Neurobiol 6:451–461
Cuello AC, Bruno MA, Allard S, Leon W, Iulita MF
(2010) Cholinergic involvement in Alzheimer's dis-
ease. A link with NGF maturation and degradation. J
Mol Neurosci 40:230–235
Curtis R, Adryan KM, Stark JL, Park JS, Compton DL,
Weskamp G, Huber LJ, Chao MV, Jaenisch R, Lee KF
et al (1995) Differential role of the low affinity
neurotrophin receptor (p75) in retrograde axonal trans-
port of the neurotrophins. Neuron 14:1201–1211
D'Cruz PM, Yasumura D, Weir J, Matthes MT,
Abderrahim H, LaVail MM, Vollrath D (2000) Muta-
tion of the receptor tyrosine kinase gene Mertk in the
retinal dystrophic RCS rat. Hum Mol Genet 9:645–651
Dowling JE, Sidman RL (1962) Inherited retinal dystro-
phy in the rat. J Cell Biol 14:73–109
Faktorovich EG, Steinberg RH, Yasumura D, Matthes
MT, LaVail MM (1990) Photoreceptor degeneration
in inherited retinal dystrophy delayed by basic fibro-
blast growth factor. Nature 347:83–86
Lambiase A, Coassin M, Sposato V, Micera A,
Sacchetti M, Bonini S, Aloe L (2007) NGF topical
application in patients with corneal ulcer does not
generate circulating NGF antibodies. Pharmacol Res
56:65–69
263
Lambiase A, Aloe L, Centofanti M, Parisi V, Bao SN,
Mantelli F, Colafrancesco V, Manni GL, Bucci MG,
Bonini S et al (2009) Experimental and clinical evi-
dence of neuroprotection by nerve growth factor eye
drops: implications for glaucoma. Proc Natl Acad Sci
U S A 106:13469–13474
Liang XL, Li J, Chen F, Ding XY, Yang XX, Long LX
(2012) A comparing study of quantitative staining
techniques for retinal neovascularization in a mouse
model of oxygen-induced retinopathy. Int J
Ophthalmol 5:1–6
Pagani L, Manni L, Aloe L (2006) Effects of electroacu-
puncture on retinal nerve growth factor and brain-
derived neurotrophic factor expression in a rat model
of retinitis pigmentosa. Brain Res 1092:198–206
Rocco ML, Balzamino BO, Petrocchi Passeri P, Micera A,
Aloe L (2015) Effect of purified murine NGF on
isolated photoreceptors of a rodent developing retinitis
pigmentosa. PlosOne 21:10
Siliprandi R, Canella R, Carmignoto G (1993) Nerve
growth factor promotes functional recovery of retinal
ganglion cells after ischemia. Invest Ophthalmol Vis
Sci 34:3232–3245
Turner JE, Delaney RK (1979) Retinal ganglion cell
response to axotomy and nerve growth factor antise-
rum in the regenerating visual system of the newt
(Notophthalmus viridescens): an ultrastructural mor-
phometric analysis. Brain Res 177:35–47
 NGF in Inflammatory
and Neurodegenerative Diseases 18
of the Eye: New Findings Supporting
Neuroprotection and Proper Tissue
Remodeling in Vitreoretinal Disorders

Graziana Esposito, Bijorn Omar Balzamino, Luca Bruno,

Andrea Cacciamani, and Alessandra Micera

Abstract disease-associated biomarkers and allow the

Nerve growth factor (NGF) plays a crucial role
in retinal disorders, as suggested by in vitro/
in vivo models. The major effect embraces the
neuroprotective activity on retinal ganglion
cells (RGCs) undergoing degeneration, as
observed in experimental diabetic retinopathy,
age-related and diabetic macular degeneration,
and some vitreoretinal diseases. Focused
experiments suggested that locally applied
NGF (intravitreal delivery) not only allowed
the counteraction of RGC degeneration but
also provided data for a whole retina restora-
tion. The currently available retinal microsur-
gery allows the collection of human aqueous
and more interesting vitreous (vitreal reflux)
humors. The recent biomolecular analysis
highlights the possibility to identify
G. Esposito · B. O. Balzamino · L. Bruno · A. Cacciamani
Research and Development Laboratory for Biochemical,
Molecular and Cellular Applications in Ophthalmological
Sciences IRCCS – Fondazione Bietti, Rome, Italy
e-mail: graziana.esposito@fondazionebietti.it; bijorn.
balzamino@fondazionebietti.it; luca.
bruno@fondazionebietti.it; andrea.
cacciamani@fondazionebietti.it
A. Micera (*)
Research and Development Laboratory for Biochemical,
Molecular and Cellular Applications in Ophthalmological
Sciences IRCCS – Fondazione Bietti, Rome, Italy
Head of Research Laboratories in Ophthalmology,
IRCCS – Fondazione Bietti, Rome, Italy
e-mail: alessandra.micera@fondazionebietti.it
monitoring of retinal impairments with sustain
to the retinal imaging. Coupled to other solu-
ble mediators, NGF has been quantified in
aqueous (slightly expressed) from diabetic
retinopathy-suffering patients (cataract sur-
gery) and vitreal reflux (significantly impaired)
of diabetic macular degeneration-suffering
patients (intravitreal surgery). Although the
reasons of these NGF impairments are not
fully comprehended, some retinal cells (glial
cells, bipolar neurons, and RGCs) have been
recognized partially responsible for these local
changes.
Taken together, the recent progress in the
ocular microsurgeries might be associated with
sampling of small amount of ocular humors,
allowing the collection of biochemical informa-
tion about diseased retina and the monitoring of
treatment. The chance to detect NGF and like-
wise other neuroprotective or pro-/anti-inflam-
matory factors in these fluids would open to the
possibility to identify biomarkers of early diag-
nosis or monitoring of retinal disease evolution/
therapy (precision medicine).
18.1 Introduction
Since the discovery occurred in 1950 by Rita
Levi-Montalcini and Stanley Cohen, nerve

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 265
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_18
266
growth factor (NGF) has represented the proto-
type of the neurotrophic family (neurotrophins)
(Aloe and Chaldakov 2013). The signal pathway,
a peculiar aspect of this NT family (NGF, BDNF,
NT-3, NT-4, and NT-5), is driven by two distinct
receptors (the tyrosine kinase receptors trkANGFR,
trkBBDNFR, trkCNTR and the pan-neurotrophin
p75NTR receptor), working in concert (high-
affinity binding; heterodimers) or alone (single
binding; homodimers) to downstream the indica-
tion inside the cell. Both survival and death
signals can be produced to regulate tissue homeo-
stasis and functional parainflammation, mainly
for NGF (Micera et al. 2007).
18.1.1 NGF and Eye: The Prototype
of a Prodigious Family
Originally, NGF was believed to stimulate the
survival, growth, and differentiation of cells
populating the central nervous system and periph-
eral nervous system (CNS and PNS) throughout
development and adulthood. Later, NGF was
demarcated as a pleiotropic factor exerting
biological effects to non-neuronal cells, opening
to new considerations for a modulatory role of
NGF alongside the neuro-immuno-endocrine
systems (Aloe and Chaldakov 2013). Over the
last decades, several aspects in NGF biological
effects were highlighted, and those related to the
visual system, from the cornea to the conjunctiva,
with respect to epithelial-stromal cross talk
(keratocytes, fibroblast/myofibroblast), were
highlighted as opening the way to the application
in those human corneal diseases orphan of tangi-
ble treatment (Micera et al. 2004).
18.1.2 NGF and NGF Receptors:
Distribution and Function
in Posterior Eye Segment
NGF and receptors (trkANGFR-p75NTR) were
observed in several ocular fluids and tissues,
including aqueous/vitreous, posterior lens, retina,
and optic nerve (Micera et al. 2007). Retinal
ganglion cells (RGCs), bipolar neurons,
G. Esposito et al.
amacrine, photoreceptors, and glial cells synthe-
size and release into the microenvironment the
mature NGF form for autocrine/paracrine cell
function, homeostatic, as well as survival/protec-
tive effects under physiopathological states (Aloe
and Chaldakov 2013). The release of NGF into
ocular fluids occurs locally by structural, acces-
sory, and local or infiltrating immune cells (Aloe
and Chaldakov 2013). A pre-proNGF protein
(140 kDa) is quickly cleaved to a proNGF form
(32–25 kDa), the result of specific protease action
(intracellularly by furins and extracellularly by
several proteases including metalloproteinases
(MMP)-7, which in turn can be cleaved to pro-
duce a mature NGF (13.5 kDa) (Masoudi et al.
2009). Intracellularly, trkANGFR-NGF binding
activates mainly pro-survival pathways, while
p75NTR-proNGF triggers inflammatory and apo-
ptotic pathways. NGF prefers the high-affinity
binding which includes trkANGFR- and p75NTR-
coupled receptor, although a ratio between hetero
and homo binding can also occur producing mod-
ulation in the final cell activity. Finally, the
mature NGF form binds specifically the tyrosine
kinase receptor (trkANGFR), with or without
cooperation of both receptors, while the proNGF
form binds and activates selectively the
pan-receptor p75NTR (Micera et al. 2007).
NGF expression inside retinal layers has been
reported in subsequent years and strongly
associated with protective tasks, especially for
RGC. A general concept was produced after the
in vitro/in vivo/ex vivo findings of NGF
applications on the visual system, regarding the
possibility of NGF to reduce or counteract
neurodegeneration of photoreceptors, neuron
loss, and Müller cell gliosis. Different strategies
aimed at increasing endogenous NGF availability
by exogenous NGF administration (Sivilia et al.
2009). The possibility for exogenous NGF to
reach both the retina and optic nerve was tested
in animal models (Lambiase et al. 2005). Modern
techniques allowed to specifically trace the topi-
cal administration of NGF, confirming the possi-
bility of a retinal targeting by a noninvasive
approach. The benefits of local instillation of
NGF eye drops could be twice: a dual effect in
improving the quality of a “healthy” ocular
18 NGF in Inflammatory and Neurodegenerative Diseases of the Eye: New. . .
surface epithelium and reaching the retina. Infor-
mation from animal models opened to a wider
usage of topical NGF applications inside the eye
upon pathological states. Other recent experimen-
tal studies opened to the use of intravitreal NGF
administration to counteract retina degeneration
(Rocco et al. 2017). More recent studies
highlighted the NGF ability to promote survival
of retinal and even brain cells when NGF was
applied intraocularly and/or topically (Aloe and
Chaldakov 2013).
18.2 NGF and Vitreoretinal
Disorders
Vitreoretinal disorders are increasing in the last
decade. The vitreous and retina can be affected by
a variety of insults, and a number of vitreoretinal
surgery techniques have been developed in the
last 10 years, to counteract vision loss. Not all
vitreoretinal impairments are age-related
diseases: a direct consequence of certain diseases
(diabetes, myopia) or even trauma (crash, colli-
sion) or lifestyling (amateur activities/sport) can
exert a major influence, resulting in sight-
threatening and severe impact on person’s daily
activities and quality of life. At the biomolecular
level, diabetic retinopathy (DR) and age-related
macular degeneration (AMD) are directly
associated with alterations of the blood-retinal
barrier (BRB), merely the inner BRB for DR
and the outer BRB for AMD (Vieira-Potter et al.
2016). Dysfunction of BRB occurs by increased
microvascular permeability and culminates in
diabetic macular edema (DME), also leading to
decreased visual acuity (Vieira-Potter et al. 2016).
At the extracellular level, neovascularization and
increase of several pro-/antiangiogenic factors
have been detected, including vascular endothe-
lial growth factor (VEGF) (Wang et al. 2016;
Vujosevic et al. 2016).
Current treatments for DR and DME are
repeated anti-VEGF injections into vitreal cham-
ber to reduce the high VEGF levels (Vieira-Potter
et al. 2016). Those injections have been
associated with decrease of VEGF and increase
of other mediators, while no significant changes
267
were observed for NGF levels, as discussed in
detail below (Cacciamani et al. 2019).
From experimental studies, NGF changes
were observed in the vitreous from animal
models, including reeler (defected retinogenesis)
mice and RCS (diabetic retina) rats (Balzamino
et al. 2014; Rocco et al. 2017). Vitreous from
DR-suffering patients showed changes in the nor-
mal levels of NGF, BDNF, NT-3, and NT-4, as
observed from the analysis of vitreous/vitreal
reflux samples collected during the intravitreal
injections or vitrectomy (therapeutic vitreoretinal
surgery) (Cacciamani et al. 2016). A correlation
between vitreal NGF and inflammatory mediators
was reported in these patients and further
associated with inflammatory/oxidative stress
conditions (Cacciamani et al. 2019).
Retinal cell layers are structurally organized to
accomplish the vision function in a proper and
smart way. As the principal glia of the retina,
Müller cells keep in touch with all neuronal
subsets, providing a unique support in neuronal
networking and allowing a link with vessels
through their processes, forming/maintaining the
BRB, regulating retinal glutamate levels, and
stabilizing blood flow. Müller cells represent an
important source of VEGF, NGF, basic fibroblast
growth factor-2 (bFGF2), tumor necrosis factor,
and other soluble mediators with neuroprotective
effects (Wang et al. 2016). From experimental
in vitro/in vivo studies, both NGF receptors are
differentially expressed by resting and activated
Müller cells, sustaining the involvement of NGF
signaling in Müller cells’ functions (Balzamino
et al. 2014).
When infiltration of inflammatory/immune
cells occurs inside the retinal layers, the choroid
represents the “main” entrance implying that the
“systemic inflammatory status” can severely
affect the retinal one. The plethora of soluble
mediators includes growth and angiogenic-
angiostatic factors, neurogenic components, as
well as neurotrophins, in addition to the common
inflammatory path (cytokines and chemokines)
(Wang et al. 2016; Vujosevic et al. 2015;
Cacciamani et al. 2019). This wide-range influx
can severely perturbates the conservative asset of
retinal layers, and the local parainflammation can
268
quickly shift to inflammation with angiogenesis
and neovascularization, opening to harmful
consequence.
18.2.1 NGF as Bystander or Partaker
in the Cross Talk Between
Resident and Infiltrating Cells
Inflamed retina is a suitable microenvironment to
study the interplay between inflammation,
neuroprotection, and matrix remodeling. All
these steps can discern a simple and reversible
damage from a neurodegenerative one. Diabetes
is a neurodegenerative disease characterized by
chronic hyperglycemia that can cause damage of
body microvasculature and thereafter silent
insults to the retinal microvasculature with vision
deficits (Vieira-Potter et al. 2016). The long-
lasting insults occurring at the retinal layers, due
to non-stabilized glycemia, can offend continu-
ously the retinal layers, with a reduced retinal
thickness, neurofilament abnormality, and
declined ERG, all steps culminating in retinal
cell death and blindness (Vieira-Potter et al.
2016).
The eye comprises two fluid systems, whose
fluid density is regulated by circulation and drain-
age, guaranteeing homeostasis and perfect func-
tion (Pizzirani and Gong 2015). One is in front of
aqueous and the other behind of vitreous lens,
which compositions and clearness are the result
of gender, age, systemic and local factors, and
life-styling features. Ciliary body system
contributes massively to aqueous composition,
keeping functioned both sclerocorneal and retinal
structures. So, glucose and other systemic
components (complement, albumin, insulin, etc.)
are equilibrated and drained (Vieira-Potter et al.
2016).
The early expression/production of NTs might
represent an attempt to trigger neuronal survival
signaling to quickly compensate the neuronal
insult and counteract as early as the initial neuro-
degenerative process. The association between
DR and altered NGF, BDNF, NT-3, and NT-4
levels and/or expression is consistent with the
hypothesis (Mohamed and El-Remessy 2015).
G. Esposito et al.
Upon a chronic insult, NGF levels occur highly
expressed at beginning while lowing at late states
of disease. This NGF expression is often
associated with inflammation, erroneously
pointed as inducing inflammation. By itself,
NGF does not trigger inflammation, although
being a proliferating factor for different inflam-
matory cells (Aloe and Chaldakov 2013). Even if
a protein signature is quite visible for DR, DME,
AMD, and vitreoretinal diseases in general, actu-
ally no biomarker or a few biomarker signatures
detectable in ocular fluids can predict the ongoing
of an inflamed retina (Wang et al. 2016). There-
fore, indications in changes of NGF and VEGF
levels in vitreal fluids might represent an addi-
tional information for monitoring the current ther-
apy (Cacciamani et al. 2019).
In recent years, increasing data support the
contribution of matrix enzymes in the remodeling
of inflamed retina. The well-known process of
conversion from pro- to mature NGF is driven
by furins and MMP-7, intracellularly localized.
An impaired MMP-7 activity might cause an
imbalanced expression between these two forms
in both the retina and circulating, as observed for
diabetic retinopathy (Ali et al. 2011). As the early
retinal neurodegeneration is linked to a decrease
of NGF expression, the exogenous supply might
avoid retinal damage as reported for animal
models of diabetes (Ali et al. 2011). The NGF
content in aqueous and vitreous from diabetic
subjects is shown in Fig. 18.1.
18.2.2 Neuroprotection: NGF-VEGF
Race for RGC, Photoreceptor,
Bipolar, and Müller Cell
Protection
Supported by several clinical trials and a current
routine approach, anti-VEGF remains a mainstay
to control posterior segment conditions like
neovascular (or wet) age-related macular degen-
eration (nAMD) (Cacciamani et al. 2019). While
various molecules suitable for retinal protection
have shown efficacy in maintaining vision and
preventing disease progression, there is still
much debate on the current treatment and ideal
18 NGF in Inflammatory and Neurodegenerative Diseases of the Eye: New. . .
Fig. 18.1 NGF levels in vitreoretinal disorders. The anti-
VEGF intravitreal injection represents a routine therapeu-
tic approach in several retinal disorders, to delay but not
reverse/cure the damage of the retina. (a) Representative
SD-OCT (spectral domain optical coherence tomography)
scan image from a DME retina. Briefly, OCT is a nonin-
vasive, noncontact imaging modality used to visualize and
monitor changes of retinal layers. (b) The higher expres-
sion in vitreal reflux is consistent with the inflammatory
regimen/most efficacious intervals of
administration.
New indications suggest that Müller cells are
active partakers in DR and vitreoretinal diseases
(Zhang et al. 2019). Essential during
retinogenesis and adult homeostasis, Müller
cells are present in the entire depth of neural
retina (Zhang et al. 2019). As observed in vitro,
Müller cell-derived NGF can increase VEGF
expression, providing an active boost for physio-
logical retinal neovascularization and cell prolif-
eration via activating ERK1/2 and AKT signaling
(Wang et al. 2016). The retinal layers, mainly
crossed by Müller cells, are continuously per-
fused by a variety of neurotropic factors (Zhang
et al. 2019). A new insight does not exclude
Müller-derived NGF as an ancillary
neuroprotective agent. Merely, NGF release into
retinal microenvironment can sustain neurons and
other retinal subsets (Rocco et al. 2015). In an
autocrine/paracrine manner, NGF can sustain
Müller cell activation and consequently different
signaling pathways involved also in Müller cells
spreading through the specific trkANGFR signal
pathway (PI3K, phosphatidylinositol 3-kinase;
Akt, protein kinase B; and ERK1/2, extracellular
269
condition (DR). Briefly, vitreal reflux was collected during
intravitreal surgery, while vitreous was sampled at the time
of pars plana vitrectomy, a commonly employed technique
in vitreoretinal surgery. (c) NGF concentration in the
vitreal samples from pucker and macular holes. Vitreal
samples were collected during pars plana vitrectomy.
NGF levels were quantified by ELISA, with antibodies
that do not recognize the other neurotrophins (b–c)
signal-regulated kinase) and finally the VEGF
gene activation (Wang et al. 2016).
The neuronal counterpart of the retinal tissue
interacts with vasculature through a complex net-
work responsible for guaranteeing tissue homeo-
stasis and preserving retinal network functionality
(Segatto et al. 2019). Among those factors,
VEGF, by means of VEGFR-1 and VEGFR-2-
receptors, and NGF, through specific and
pan-neurotrophin receptors, might be additional
main players. Some few studies on treated
patients indicate that anti-VEGF treatment nega-
tively influences the survival of retinal neuronal
and the chronic neutralization of local VEGF
might prime neuron loss (Fogli et al. 2018). This
would suggest that the neuroprotective effect of
VEGF is actually a debate: it is quite clear that a
complete neutralization of VEGF is followed by
other tissue alterations as well as angiogenesis
and neovascularization can offend seriously reti-
nal network. VEGF acts as a potent trophic factor
for retinal neurons, facilitating both proliferation
and differentiation of retinal progenitors into
specialized neurons, while its overt production
promotes new vessel formations with deleterious
effects on whole retinal network (Fogli et al.
2018). Recently, NGF has been proposed as
270
important angiogenic growth factors, as direct
enhancers of VEGF expression (Nakamura et al.
2011). While NGF stimulates VEGF production,
there is no evidence for VEGF induction of NGF,
highlighting the importance of NGF in clinical
applications. Anti-VEGF therapy is widely used
as the primary treatment for counteracting retinal
neovascularization (DR, AMD, CNV, and retinal
vein occlusion) (Cacciamani et al. 2016). By the
way, experimental data showed a risen apoptosis
of retinal cells after anti-VEGF administration,
clearly visible with a reduced thickness and
NGF production (Fogli et al. 2018). Thus, the
purpose of our studies was to evaluate the
human vitreous reflux, obtained during anti-
VEGF administration, in the modulation of dif-
ferent biomarkers (Cacciamani et al. 2016).
18.2.3 Tissue Remodeling: NGF
and Extracellular-Matrix
Interplay
Chronic (Th1- and Th2-mediated) inflammatory
conditions might influence the local innervation
as well as trigger the development of complex
neurogenic mechanisms including the release of
neuromediators (VIP, NPY, and CGRP),
representing a novel mechanism in the pathogen-
esis of ocular surface (Micera et al. 2004). The
same mechanisms might be activated in autoim-
mune and neurodegenerative states, including
type I and type II diabetes. In experimental
models of DR, NGF and its pro-forms and own
receptors were found overexpressed in
GFAP- and Iba1-bearing cells, in association
with several intracellular pathways (Rocco et al.
2015). Other tissue remodeling proteins have
been associated with retinal remodeling upon
inflammation. A specific function for OPN in
homeostatic processes is to facilitate the survival
of stressed endothelial cells, possibly by
occupying un-ligated integrins and suppressing
integrin-mediated death (Dinice et al. 2020). In
other studies, on Müller cells, exposure to high
glucose increased Cyr61 expression, inhibited
cell viability/migration, and induced apoptosis,
suggesting the potential role of Cyr61 in Müller
G. Esposito et al.
cell degeneration (Zhou et al. 2014). Both degen-
eration and Cyr61 overexpression in Müller cells
take critical parts in the pathogenesis of
DR. Elevated Cyr61 levels were quantified in
the vitreous from PDR patients (Zhou et al.
2014). Recently, our group observed a correlation
between NGF, VEGF, OPN, and cyr61 at least in
ERMs (Dinice et al. 2020). This association
should be verified in Müller cells exposed to
vitreal samples from DR, including the
proliferative forms, AMD and CNV.
18.3 Conclusion and Future
Perspectives
Several lines of evidence highlight a protective
effect of NGF against retinal cell loss, with spe-
cific emphasis on reduction of retinal ganglion
cell degeneration, photoreceptor death, and
Müller cell gliosis. A better understanding of the
mechanisms underlying the effects of NGF and
proNGF as well as NGF/BDNF/NT4/p75NTR in
the modulation of retinal neurodegeneration and
gliosis will help to develop efficient therapeutic
strategies for various retinal diseases. Different
strategies, including increasing the availability
of endogenous NGF, administration of exoge-
nous NGF, activation of trkANGFR, and inhibition
of p75NTR, have been reviewed highlighting the
potential aspects in NGF neuroprotection. Our
studies showing the increased NGF levels inside
the vitreal chamber would suggest an initial
attempt of NGF to protect neuronal cells from
degeneration. A potential therapeutic intervention
with NGF for the treatment of retinal neurodegen-
erative diseases can be therefore suggested as an
implementation of endogenous NGF. The major
limitation of NT treatment for CNS diseases is
represented by the difficulty of crossing the
blood-brain barrier and the impossibility to
deliver NTs systemically, particularly NGF
(Giannaccini et al. 2018). Alternatives for exoge-
nous delivery of NTs has been proposed for the
treatment of retinal disorders: intravitreal injec-
tion and the nanoscale MNP-protein hybrid rep-
resent excellent tools for bypassing the limit of
BRB and implementing ocular drug delivery
18 NGF in Inflammatory and Neurodegenerative Diseases of the Eye: New. . .
Fig. 18.2 Different routes of drug administration in the
eye: a glimpse to NGF. The draw summarizes all accessi-
ble intraocular sites as indicated: topical absorption (eye
drops and emulsions/gels) in the anterior segment and
related to the posterior segment, the intravitreal injections
(ophthalmic solutions/suspensions, implants, nanotech-
nology), periocular administration (implants or injections),
and finally the systemic delivery (oral and intravenous).
strategies for neuroprotection and therapy
(Giannaccini et al. 2018). As above introduced,
the eye has two fluid systems and own barriers,
providing a limited and immune-privileged sys-
tem (Pizzirani and Gong 2015). The anterior seg-
ment includes tear film, conjunctiva, cornea,
aqueous, and iris, and the posterior segment
contains the vitreous humor and the underneath
retina with retinal pigment epithelium (RPE) and
choroid. The lens represents the physical edge
between those two compartments that are in
271
The periocular route includes subconjunctival, subtenon,
peribulbar, and retrobulbar sites of injection. Except for
the systemic route, all the others have been tested for NGF
and anti-NGF at both experimental and clinical levels.
Highlighted in red are future directions on experimental
animals, comprising the anti-VEGF/NGF-combined
intravitreal injection or the use of NGF nanoparticles lay-
ered in vitreal chamber
touch by fluid circulation. The poor permeability
of the cornea/sclera and BRB makes the systemic
administration of NTs ineffective in reaching the
retina as well as the short half-life of NTs ideally
bypassed by multiple injections (Gaudana et al.
2010). For this reason, several attempts were car-
ried out to find out alternatives to reach the retina,
such as topical application as well as intravitreal,
periocular, subretinal, or systemic administration
(Fig. 18.2). To combine anti-VEGF/NGF therapy
or use NGF nanoparticle delivery represents the
272
starting points for our future investigations in
experimental models.
Acknowledgments Many thanks to Tiziana Pacchetti for
precious assistance in collecting fluids and tissues at sur-
gery and Angelica Napoli for drawing Fig. 17.2. This work
was partially supported by MaBIOS Project (Regione
Lazio; LR13/2015; FILAS-RU-2014-112), Italian Minis-
try of Health (ricerca corrente), and 5xMille (2015 and
2016) to IRCCS Fondazione Bietti.
References
Ali TK, Al-Gayyar MMH, Matragoon S et al (2011) Dia-
betes induced peroxynitrite impairs the balance of
pro-nerve growth factor and nerve growth factor, and
causes neurovascular injury. Diabetologia 54(3):
657–668
Aloe L, Chaldakov GN (2013) The multiple life of nerve
growth factor: tribute to rita levi-montalcini
(1909–2012). Balkan Med J 30(1):4–7
Balzamino BO, Biamonte F, Esposito G et al (2014)
Characterization of NGF, trkA (NGFR), and p75
(NTR) in retina of mice lacking Reelin glycoprotein.
Int J Cell Biol 2014:725928. https://doi.org/10.1155/
2014/725928
Cacciamani A, Parravano M, Scarinci F et al (2016) A
simple spontaneous Vitreal reflux collecting procedure
during intravitreal injection: set-up and validation stud-
ies. Curr Eye Res 41(7):971–976. https://doi.org/10.
3109/02713683.2015.1080282
Cacciamani A, Esposito G, Scarinci F, Parravano M,
Dinice L, Di Nicola M, Micera A (2019) Inflammatory
mediators in the vitreal reflux of patients with diabetic
macular edema. Graefes Arch Clin Exp Ophthalmol
257(1):187–197. https://doi.org/10.1007/s00417-018-
4169-4
Dinice L, Cacciamani A, Esposito G et al (2020)
Osteopontin in vitreous and idiopathic epiretinal
membranes. Graefes Arch Clin Exp Ophthalmol 258
(7):1503–1513. https://doi.org/10.1007/s00417-020-
04685-w
Fogli S, Del Re M, Rofi E, Posarelli C, Figus M, Danesi R
(2018) Clinical pharmacology of intravitreal anti-
VEGF drugs. Eye (Lond) 32(6):1010–1020. https://
doi.org/10.1038/s41433-018-0021-7
Gaudana R, Ananthula HK, Parenky A, Mitra AK (2010)
Ocular drug delivery. AAPS J 12(3):348–360. https://
doi.org/10.1208/s12248-010-9183-3
Giannaccini M, Usai A, Chiellini F, Guadagni V,
Andreazzoli M, Ori M, Pasqualetti M, Dente L, Raffa
V (2018) Neurotrophin-conjugated nanoparticles pre-
vent retina damage induced by oxidative stress. Cell
Mol Life Sci 75(7):1255–1267. https://doi.org/10.
1007/s00018-017-2691-x
Lambiase A, Tirassa P, Micera A, Aloe L, Bonini S (2005)
Pharmacokinetics of conjunctivally applied nerve
G. Esposito et al.
growth factor in the retina and optic nerve of adult
rats. Invest Ophthalmol Vis Sci 46(10):3800–3806.
https://doi.org/10.1167/iovs.05-0301
Masoudi R, Ioannou MS, Coughlin MD et al (2009)
Biological activity of nerve growth factor precursor is
dependent upon relative levels of its receptors. J Biol
Chem 284(27):18424–18433. https://doi.org/10.1074/
jbc.M109.007104
Micera A, Lambiase A, Aloe L, Bonini S, Levi-Schaffer F,
Bonini S (2004) Nerve growth factor involvement in
the visual system: implications in allergic and neuro-
degenerative diseases. Cytokine Growth Factor Rev 15
(6):411–417. https://doi.org/10.1016/j.cytogfr.2004.
09.003
Micera A, Lambiase A, Stampachiacchiere B et al (2007)
Nerve growth factor and tissue repair remodeling: trkA
(NGFR) and p75(NTR), two receptors one fate. Cyto-
kine Growth Factor Rev 18(3–4):245–256. https://doi.
org/10.1016/j.cytogfr.2007.04.004
Mohamed R, El-Remessy AB (2015) Imbalance of the
nerve growth factor and its precursor: implication in
diabetic retinopathy. J Clin Exp Ophthalmol 6(5):483.
https://doi.org/10.4172/2155-9570.1000483
Nakamura K, Tan F, Li Z, Thiele CJ (2011) NGF activa-
tion of TrkA induces vascular endothelial growth fac-
tor expression via induction of hypoxia-inducible
factor-1α. Mol Cell Neurosci 46(2):498–506. https://
doi.org/10.1016/j.mcn.2010.12.002
Pizzirani S, Gong H (2015) Functional anatomy of the
outflow facilities. Vet Clin North Am Small Anim
Pract 45(6):1101–1126. https://doi.org/10.1016/j.
cvsm.2015.06.005
Rocco ML, Balzamino BO, Petrocchi Passeri P, Micera A,
Aloe L (2015) Effect of purified murine NGF on
isolated photoreceptors of a rodent developing retinitis
pigmentosa. PLoS One 10(4):e0124810. https://doi.
org/10.1371/journal.pone.0124810
Rocco ML, Balzamino BO, Esposito G et al (2017) NGF/
anti-VEGF combined exposure protects RCS retinal
cells and photoreceptors that underwent a local wors-
ening of inflammation. Graefes Arch Clin Exp
Ophthalmol 255(3):567–574. https://doi.org/10.1007/
s00417-016-3567-8
Segatto M, Fico E, Gharbiya M et al (2019) VEGF inhibi-
tion alters neurotrophin signalling pathways and
induces caspase-3 activation and autophagy in rabbit
retina. J Cell Physiol 234(10):18297–18307. https://
doi.org/10.1002/jcp.28462
Sivilia S, Giuliani A, Fernández M, Turba ME, Forni M,
Massella A, De Sordi N, Giardino L, Calzà L
(2009 May 27) Intravitreal NGF administration
counteracts retina degeneration after permanent carotid
artery occlusion in rat. BMC Neurosci 10:52. https://
doi.org/10.1186/1471-2202-10-52
Vieira-Potter VJ, Karamichos D, Lee DJ (2016) Ocular
complications of diabetes and therapeutic approaches.
Biomed Res Int 2016:3801570. https://doi.org/10.
1155/2016/3801570
18 NGF in Inflammatory and Neurodegenerative Diseases of the Eye: New. . .
Vujosevic S, Micera A, Bini S, Berton M, Esposito G,
Midena E (2015) Aqueous humor biomarkers of
Müller cell activation in diabetic eyes. Invest
Ophthalmol Vis Sci 56(6):3913–3918. https://doi.org/
10.1167/iovs.15-16554
Vujosevic S, Micera A, Bini S, Berton M, Esposito G,
Midena E (2016) Proteome analysis of retinal glia
cells-related inflammatory cytokines in the aqueous
humour of diabetic patients. Acta Ophthalmol 94
(1):56–64. https://doi.org/10.1111/aos.12812
Wang J, He C, Zhou T, Huang Z, Zhou L, Liu X (2016)
NGF increases VEGF expression and promotes cell
273
proliferation via ERK1/2 and AKT signaling in Müller
cells. Mol Vis 22:254–263. eCollection 2016
Zhang L, Li X, Lin X, Wu M (2019) Nerve growth factor
promotes the proliferation of Müller cells
co-cultured with internal limiting membrane by
regulating cell cycle via Trk-a/PI3K/Akt pathway.
BMC Ophthalmol 19(1):130. https://doi.org/10.
1186/s12886-019-1142-x
Zhou F, Zhang Y, Chen D et al (2014) Potential role of
Cyr61 induced degeneration of human Müller cells in
diabetic retinopathy. PLoS One 9(10):e109418. https://
doi.org/10.1371/journal.pone.0109418
 Part VI
Veterinary Medicine
 Nerve Growth Factor (NGF) and Animal
Reproduction 19
Margherita Maranesi, Cristiano Boiti, and Massimo Zerani

Abstract luteotrophic action, while, in induced

Stimuli that lead to the release of
gonadotropin-releasing hormone (GnRH) and
pituitary gonadotropins and, consequently,
ovulation in mammals fall into two broad
categories. In the first, high plasma oestrogen
concentrations induce the events that trigger
ovulation, a characteristic of spontaneous
ovulators. In the second, nerve stimuli occur-
ring during mating reach the hypothalamus
and trigger the release of GnRH and ovulation
with a neuroendocrine reflex that characterizes
induced ovulators.
In this review, we will give an overview of
the distribution of NGF and its expression in
the different tissues of the male accessory sex
glands, the main sites of NGF production.
Next, we will highlight the role of NGF in
sperm function and its potential
cryopreserving role in artificial insemination
techniques. Finally, we will evaluate the
functions of NGF in ovulation, particularly in
induced ovulators. Overall, the information
obtained so far indicates that NGF is widely
distributed in organs that regulate the repro-
ductive activity, in both males and females. In
spontaneous ovulators, NGF exerts mainly a
M. Maranesi (*) · C. Boiti · M. Zerani
Dipartimento di Medicina Veterinaria, Università di
Perugia, Perugia, PG, Italy
e-mail: margherita.maranesi@unipg.it; massimo.
zerani@unipg.it
ovulators it is the main ovulation-inducing
factor. A better understanding of the role of
NGF in reproduction would be of great inter-
est, since it could help finding innovative ther-
apeutic aids to improve mammalian fertility.
19.1 Introduction
Ovulation in domestic animals can be induced
(reflex) or spontaneous. The hypothalamic-
pituitary-gonadal (HPG) axis is involved in both
cases. In spontaneous ovulation species, the
oestrogens cyclically produced by ovarian
follicles exert a positive feedback on the hypotha-
lamic nuclei that are responsible for the produc-
tion of GnRH, determining the preovulatory peak
that leads to ovulation. In induced ovulation spe-
cies, the sexual act triggers, by means of afferent
neuronal pathways, the activation of the HPG axis
leading to ovulation (Seibel 1986; Milligan
1982). In the induced ovulation species such as
rabbits, cats, camelids, squirrels, and ferrets, there
is not a real oestrous cycle. Different signals,
which may be tactile, olfactory, and visual, assist
the ovulatory response in these species. However,
the stimulation of the genital tract during mating
turns out to be decisive for triggering the LH peak
and ovulation (Bakker and Baum 2000).

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 277
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_19
278 M. Maranesi et al.

Despite our understanding of the factors to be widely distributed in central neurons, NGF
inducing ovulation in these species that has was subsequently found to be also expressed in
improved in recent years, much of the intricate several other cell types and organs such as the
mechanistic details still remains elusive and immune (Ehrhard et al. 1993), vascular endothe-
represents one of the most fascinating themes of lial, and muscular smooth cells (Yamamoto et al.
reproductive physiology. 1996), the lung (Ricci et al. 2007), the adrenal,
The nerve growth factor (NGF) was the first and the thyroid (Saruta et al. 2010; Ceccanti et al.
member of the neurotrophin family to be discov- 2013). Remarkably, among the numerous body
ered (Levi-Montalcini 1987), followed by brain- sources of NGF, the pivotal paper of Ratto et al. in
derived growth factor (BDNF) and other 2012 demonstrated the NGF is highly conserved
neurotrophins (NT3, NT4). Together, these among species in the seminal fluid identified as
neurotrophins support survival, development, dif- the OIF.
ferentiation, and synaptic plasticity of both cen-
tral and peripheral neurons and, therefore, play an
essential biological role (Thoenen and Barde
19.2 Male
1980; Snider 1994). Recently, however, several
studies have shown that NGF also influences the
19.2.1 Expression of NGF in Male
reproductive function of both males and females
Accessory Glands
(Bogle et al. 2018; Ratto et al. 2019).
In the mid-1980s, an ovulation-inducing factor
In 1980, Harper and Thoenen firstly described the
(OIF) was identified in the seminal plasma of
distribution of NGF in mammalian male sex
camelids, which are induced ovulators (Chen
organs and its relevance for male reproductive
et al. 1985). Later, OIF was found in the seminal
functions. From there on, several works on this
plasma of other induced ovulators such as koalas
topic were realized (see, e.g. Adams et al. 2016;
(Johnston et al. 2004), rabbits (Silva et al. 2011),
Bogle et al. 2018).
alpacas, llamas, and further camelids (Adams
NGF and its receptors have been detected in
et al. 2005; Silva et al. 2014; Ulloa-Leal et al.
different organs of the male reproductive tract
2014; Berland et al. 2016; El Allali et al. 2017).
(Shikata et al. 1984; MacGrogan et al. 1991; Li
OIF was even detected in the seminal plasma of
et al. 2005; Wang et al. 2011). In a recent work,
species with spontaneous ovulation, such as cat-
Adams et al. (2016) evidenced the immunoreac-
tle, pigs, and horses (Adams et al. 2016).
tivity of NGF in the secretory epithelium of sev-
Subsequent biochemical studies identified NGF
eral male accessory glands: the ampulla, prostate,
as the major OIF component of seminal plasma
bulbourethral gland, vesicular gland, and
that induced ovulation in llamas (Silva et al.
coagulating gland. Then, Bogle et al. (2018)
2014) and alpacas (Kershaw-Young et al. 2012).
extended this research to other organs and tissues
Two classes of receptors mediate the
including the testis, epididymis, ductus deferens,
biological effects of NGF. The first is a high-
ampulla, and penis of llamas, cattle, bison, elks,
affinity-specific 140-kDa transmembrane
white-tailed deer, and rats. They reported that
neurotrophic tyrosine kinase receptor
differences in the NGF expression among species
1 (NTRK1; also known as tropomyosin receptor
were likely related to differences in the anatomy
kinase A—TrKA) and the second a low-affinity
and function of accessory sex glands (Adams
75-kDa receptor, p75, or nerve growth factor
et al. 2005; Ratto et al. 2012).
receptor (NGFR) belonging to the tumour necro-
The perinuclear distribution of NGF in several
sis factor family receptor (Johnson et al. 1986;
cell types of the male genital apparatus,
Kaplan et al. 1991; Dechant and Barde 1997;
demonstrates protein translation of NGF and stor-
Schecterson and Bothwell 2010). Although
age into secretory vesicles (Bogle et al. 2018;
initially investigated extensively for its nerve
Wang et al. 2011).
growth-promoting effects and then demonstrated
19 Nerve Growth Factor (NGF) and Animal Reproduction
In llamas, NGF is particularly expressed in the
prostate gland rather than in other accessory
glands (Bogle et al. 2018). In testicles of llamas,
NGF immunoreactivity was localized only in the
vascular endothelium and connective tissue of the
stroma and rete testes, but not in the interstitial
cells (Bogle et al. 2018).
In rats, NGF was mainly expressed in
coagulating glands (especially in the connective
tissue), but a strong staining was observed also in
interstitial cells of the testes. In the same species,
Squillacioti et al. (2009) found that the prostate
and seminal vesicles were the major sites of NGF
expression.
In bulls, NGF mRNA abundance was highest
in the vesicular gland, intermediate in the
ampulla, and lowest in the bulbourethral and
prostate glands. By immunohistochemistry, the
most intense NGF staining was found in the
ampulla and vesicular glands (Hofmann and
Unsicker 1982; Adams et al. 2016; Stewart et al.
2018) with a strong immune reaction in the apical
region of the glandular epithelium (Bogle et al.
2018). On the other hand, there was a lesser NGF
staining intensity in the prostate and
bulbourethral glands (Harper and Thoenen 1980;
Harper et al. 1982; Bogle et al. 2018).
In rabbits, the highest levels of NGF and
NTRK1 transcripts were found in the prostate,
while intermediate expressions were found in
the testis. In these animals, the main source of
NGF is the prostate as suggested by the higher
NGF biological activity (Harper and Thoenen
1980), the widespread distribution of NGF in all
prostate glandular cells, and its relatively high
mRNA abundance (Maranesi et al. 2015;
Sanchez-Rodriguez et al. 2019). Based on immu-
nohistochemical and molecular studies, the pros-
tate gland is thought to be the main source of
seminal NGF also in llamas, alpacas, and guinea
pigs, while the ampulla and prostate are richer in
elks and white-tailed deer, respectively, and the
ampulla and vesicular glands in cattle and bison
(Adams et al. 2016; Bogle et al. 2018).
In the epididymis, the caudal region seems to
have greater NGF expression than the other
segments (Jin et al. 2010), especially in the con-
nective tissue. Similarly, the penis of different
279
species displays a positivity in the connective
tissue and smooth muscle (Bogle et al. 2018). In
rabbits, no positivity for NGF was found in the
epididymis at different ages of sexual maturity
(22 and 37 weeks); conversely, TrKA immunore-
activity was evidenced only in younger rabbits at
22 weeks, within stereocilia and epithelial cells
(Sanchez-Rodriguez et al. 2019). In the testis of
rabbits, intense immunostaining for NGF and its
receptors was observed in the perinuclear region
and cytoplasm of Leydig, Sertoli, and germinal
cells at different stages of development (Maranesi
et al. 2015).
One of the most intriguing findings was the
NGF positivity of vascular endothelium of the
male genital apparatus (Bogle et al. 2018). NGF
could play an important role in the vascularization
of genital tissues, as shown for female llamas,
where NGF extracted from seminal plasma
increased luteal vasculature (Silva et al. 2017),
or female rats, where NGF contributed to ovarian
vascularization via the vascular endothelial
growth factor (VEGF—Julio-Pieper et al. 2006).
19.2.2 NGF and Sperm Function
Several authors have assessed the role of NGF
and its receptors in spermatogenesis and sperm
function of several animal species and humans
(Li and Zhou 2013; Adams et al. 2016; Bogle
et al. 2018; Jin et al. 2006; Levine et al. 2000).
Strong immune reactions for NGF and NGFR
were found in germinal cells and spermatozoa,
suggesting an important intervention of these
molecules in all steps of spermatogenesis
(Maranesi et al. 2015). Transcript corresponding
to both NGF and TrkA has been detected in
human foetal testis, and the NGF protein was
mainly localized to Sertoli and interstitial cells
(Robinson et al. 2003). Functional assays
performed with a mouse tumour Leydig cell line
have revealed that NGF increased cellular steroid
production, indicating that the neurotrophin could
regulate testis morphogenesis (Müller et al.
2006). This NGF role in promoting proliferation
and differentiation of Leydig cells as well as
testosterone production was demonstrated also
280
in rats by Zhang et al. (2013). In addition, Perrard
et al. (2007) proposed that NGF has a role also in
regulating the meiosis of rat spermatocytes.
Remarkably, the seminal plasma of oligoasthe-
nozoospermic men had lower NGF content than
that of fertile men (Li et al. 2010a, b), suggesting
a relevant role of NGF in sperm function.
Increased plasma concentrations of NGF report-
edly affect the pattern and intensity of TrkA and
p75 in rat testicles, suggesting that the
NGF/TrkA/p75 system has a role in male repro-
ductive physiology (Levanti et al. 2006).
In all species insofar studied, NGF stimulates
sperm motility (Lin et al. 2015; Shi et al. 2012;
Saeednia et al. 2015; Castellini et al. 2019) and
viability (Li et al. 2010a) and also facilitates
sperm cell acrosome reactions (Li and Zhou
2013). These NGF functions are mediated in a
time- and dose-dependent manner (Jin et al.
2010).
The great differences in the abundance of
OIF/NGF among species suggest a greater physi-
ological importance in camelids. In fact, NGF
represented approximately 27% of total protein
in llama/alpaca ejaculates (1.2  0.21 mg/dl;
Bogle et al. 2018), 1.5% of total protein in rabbits
(ranging from 1894  277 pg/ml to
151.9  9.25μg/ml; Maranesi et al. 2015, 2018),
and less than 1% of total protein in bovine
ejaculates (0.10  0.03 mg/dl; Bogle et al. 2018).
In a recent study, Bezerra et al. (2019)
described the proteome of seminal plasma in
rabbits. Interestingly, in this work sperm motility,
viability, and morphology were associated with
specific seminal proteins. The in silico analysis
pointed out to NGF interactions with some
proteins involved in acrosome reaction and
sperm capacitation (Bezerra et al. 2019). The
NGF content in rabbit seminal plasma during
winter decreases four times compared with the
other seasons (Casares-Crespo et al. 2018).
Zhang et al. (2015) reported similar results in
wild ground squirrels where the production of
NGF by testes decreased during the nonbreeding
season and increased in the breeding season.
Interestingly, also in squirrel females, the ovarian
NGF transcript was higher in the breeding than in
nonbreeding season (Maranesi et al. 2020). Such
M. Maranesi et al.
seasonal changes found in male rabbits agree with
the findings of Schneidgenova et al. (2011) that
showed less sperm motility and concentration
during the winter season and may reflect the nat-
ural fluctuation of breeding capability during
the year.
Using an immunofluorescent technique, NGF
immunoreactivity was localized to the sperm
head and tail, whereas that of TrkA was detected
in the acrosomal cap, nucleus, and tail regions. In
addition, in vitro exogenous NGF administration
significantly affected the secretion of leptin, cell
viability, and sperm apoptosis (Li et al. 2010a).
Squillacioti et al. (2009) observed that testicular
steroids likely regulated the expressions of NGF
and NTRK1 receptor in the accessory sex glands
because castration enhanced their expression.
NGF increased Leydig cell steroid production in
an in vitro system (Müller et al. 2006). By con-
trast, NGF expression and concentration in semi-
nal plasma and blood plasma remain constant
throughout the maturation process, independently
of testosterone level (Sanchez-Rodriguez et al.
2019).
19.2.3 NGF and Preservation
of Spermatozoa
Spermatozoa are highly sensitive to several types
of chemical/physical stress due to reactive oxy-
gen species, osmotic changes, and different
temperatures applied during cryopreservation or
freeze-drying procedures (Koshimoto and Mazur
2002). Semen conservation methods can affect
DNA methylation, thus influencing gene expres-
sion and animal fertility (Aurich et al. 2016;
Verma et al. 2014). For this reason, the addition
of chelating or antioxidant agents is mandatory to
improve and maintain sperm quality during the
preservation procedures and to avoid the deleteri-
ous effects of ROS (Gadea et al. 2011; Kalthur
et al. 2011; Mercati et al. 2020).
Previous studies demonstrated that addition of
exogenous NGF to the incubation medium had
significant effects on rabbit (Castellini et al.
2019), bovine (Li et al. 2010a) and human
(Saeednia et al. 2016) sperm cell viability, and
19 Nerve Growth Factor (NGF) and Animal Reproduction
also exhibits trophic activity in the rescue of
Sertoli cell viability mediated by the TrkA recep-
tor protein (Chen et al. 1997).
In seminal plasma, there was an abundance of
NGF mRNA positively associated with the main-
tenance of post-thaw functional membrane integ-
rity in bull spermatozoa, indicating that it could
be used to assess cryotolerance (Shilpa et al.
2017). In another study, there were greater
abundances of spermatozoa NGF mRNA in
bulls with greater quality semen (<25%
discarded ejaculates) compared with those with
lesser quality semen (>40% discarded
ejaculates); relative abundances of NGF mRNA
were also positively associated with pre-freeze
mitochondrial membrane potential and post-
thaw sperm velocity variables in bulls (Parthipan
et al. 2017). In both normozoospermic and
asthenozoospermic men, supplementation of
freezing extender with NGF at 0.5 ng/mL
improved sperm viability and motility and
decreased DNA fragmentation (Saeednia et al.
2015, 2016). Consistently, treatment of frozen-
thawed bull sperm cells with 40–80 ng/mL NGF
increased both leptin secretion and sperm mem-
brane integrity (Li et al. 2010a). Based on all the
previous findings, NGF may be an optimal candi-
date protein to enhance sperm cryopreservation
techniques in domestic animals and men, a prop-
osition that should be better analysed in the
future.
19.3 Female
19.3.1 NGF and Ovulation in Females
Recently, NGF and its cognate receptors, NTRK1
and NGFR, as well as BDNF, were detected in the
uterus of several spontaneous ovulatory species,
including rodents, chiropters, and farm animals
such as rat, guinea pig, mouse, bat, pig, and horse
(Varol et al. 2000; Brauer et al. 2000; Wessels
et al. 2014). The uterine expression of
the neurotrophin system varied greatly across
the oestrous cycle and during pregnancy under
the influence of oestrogen and progesterone
(P4) in golden hamsters, ewes, rat, and mouse
281
(Shi et al. 2006; Mirshokraei et al. 2013; Lobos
et al. 2005; Bjorling et al. 2002).
The rabbit uterus expresses NGF and its cog-
nate receptors, NTRK1 as well as NGFR
(Maranesi et al. 2016, 2018). In addition, NGF
induces the synthesis and secretion of both
PGF2alpha and PGE2 by the uterus in vitro, via
selective modulation of nitric oxide synthase and
PGE2–9-ketoreductase activities (Maranesi et al.
2016).
Evidence documented in cattle suggests that
NGF may have a role as a luteotrophic factor in
spontaneous ovulators (Carrasco et al. 2016;
Tribulo et al. 2015) and intramuscular injection
at the time of artificial insemination of purified
ß-NGF from bovine seminal plasma increased
pregnancy rate up to 16% (Stewart et al. 2018).
In induced ovulators in which OIF/NGF was
first described (llamas and alpacas), an endocrine
mechanism was postulated to explain the ovula-
tory response induced by deposition of fresh
homologous seminal plasma into the genital
tract (Ratto et al. 2012). According to this mecha-
nism, seminal plasma NGF is absorbed through
the genital tract and reaches the pituitary and/or
the hypothalamus via the bloodstream to elicit the
release of LH that, in turn, induces ovulation
(Adams et al. 2005). Indeed, it has been recently
reported that, in llamas, significant increases
occur in circulating NGF levels 15 min after the
intrauterine deposition of seminal plasma,
followed by a LH surge 1 hour later (Berland
et al. 2016). More intriguingly, Berland et al.
(2016) using urethrostomized males, for the first
time, unequivocally demonstrated that copulation
alone did not induce ovulation in the absence of
seminal plasma, thus challenging the concept of
reflex ovulation in llamas.
The observation that seminal plasma OIF
elicited LH release from the pituitary in vitro
and induced ovulation in various mammalian spe-
cies (Adams et al. 2016) further supported the
concept that the endocrine mechanisms involved
in spontaneous ovulation also included stimuli
derived from the male reproductive tract. More-
over, a study using recombinant human NGF in
alpacas confirmed a dose-related effect on ovula-
tion rate (Stuart et al. 2015), similar to that
282
obtained by exogenous GnRH administration
(Silva et al. 2012).
In a recent work, Ratto et al. (2019) described
the presence NGF immunoreactivity in tanycytes
of the median eminence in llamas, where GnRH
nerve terminals are concentrated. In the same
work, a greater relative abundance of kisspeptin
fibres was detected in association with GnRH
fibres in the median eminence region. Moreover,
NGF may act through the hypothalamic
kisspeptin neurons to elicit the preovulatory LH
surge and ovulation and may be similarly impor-
tant in the spontaneous and induced ovulators
(Inoue et al. 2011; El Allali et al. 2017).
Studies conducted in rabbits, however, have
yielded contradictory results.
Recently, several studies (Maranesi et al.
2015; García-García et al. 2017; Casares-Crespo
et al. 2018; Maranesi et al. 2018; Castellini et al.
2019) have confirmed the presence of NGF in
rabbit semen. This molecule was found at rela-
tively high concentrations in rabbit seminal
plasma, ranging from 1894  277 pg/ml to
151.9  9.25μg/ml (Maranesi et al. 2015, 2018),
closer to those in bovine seminal plasma
(100  30μg/mL), but much lower than those
reported in lama/alpaca 1.2  0.21 mg/ml
(Bogle et al. 2018).
Despite these concentrations, Silva et al.
(2011) did not detect ovulation in rabbits after
intramuscular injections of rabbit seminal plasma
at different doses, although the same procedure
induced ovulation in llamas. Conversely,
Cervantes et al. (2015) reported that the intramus-
cular injection of rabbit seminal plasma induced
ovulation in group-housed, but not in individually
housed rabbits. An experiment conducted by
Rebollar et al. (2012) confirmed ovulation in
75% of rabbits after intravaginal deposition of
raw semen without treatment with a GnRH ana-
logue. No increase in blood plasma LH concen-
tration was observed. Quite surprisingly,
however, there was no ovulation in those
inseminated with raw semen after lumbar intra-
epidural anaesthesia.
A recent study (García-García et al. 2017)
reported that only one of the six female rabbits
treated intramuscularly with 24μg of murine NGF
M. Maranesi et al.
ovulated, and again no preovulatory elevation in
blood plasma LH was detected. In the same study,
mechanical stimulation of the vagina increased
the ovulation rate by 50% in NGF-treated females
(García-García et al. 2017), suggesting that
besides OIF/NGF, these females need physical
stimulation to ovulate.
Similarly, Maranesi et al. (2018) reported ovu-
lation rates of 67% (4/6) and 17% (1/6) in female
rabbits inseminated with raw semen in
non-anaesthetized and lumbar-anaesthetized
females, respectively; in all these females, ovula-
tion was preceded by an increase in plasma LH
concentration during the 2 h after artificial insem-
ination. Lumbar anaesthesia or administration of
COX inhibitors before artificial insemination
blocked the increase in plasma LH concentration,
attenuated the systemic blood rise of NGF, and
reduced the ovulation rate. These authors
postulated that other seminal plasma cytokines,
besides NGF, might contribute to local stimula-
tion of the female reproductive tract and ovulation
in the rabbit (Maranesi et al. 2018).
Based on these findings, the same group pro-
posed a novel paracrine mechanism driven by raw
semen OIF, likely NGF, in the uterus/cervix,
which reinforces the neuroendocrine reflex pro-
voked by vaginal stimuli during natural mating
(Fig. 19.1). According to such a mechanism,
(a) semen-derived NGF stimulates de novo syn-
thesis of NGF in the uterine wall; (b) both seminal
NGF and uterine NGF are absorbed into the
bloodstream and act directly on the ovary rather
than on the pituitary and/or hypothalamus; and
(c) semen-derived NGF and locally synthesized
NGF stimulate uterine/cervix sensory neurons,
which trigger GnRH neurons in the hypothala-
mus. In sensory neuron stimulation (nervous
pathway), prostaglandins and nitric oxide are
synthesized by the uterus after binding of NGF
to NTRK1 and/or NGFR receptors. NGF-induced
prostaglandins would then stimulate directly or
indirectly (via local chemical mediators) uterine/
cervix neurons that reach, via spinal cord afferent
pathways, the hypothalamic centres responsible
for the LH surge that induces ovulation. Addition-
ally, NGF could directly stimulate uterine/cervix
terminals of visceral afferent fibres through
19 Nerve Growth Factor (NGF) and Animal Reproduction
Fig. 19.1 Schematic representation of the suggested
endocrine (left yellow area) and nervous (right green
area) NGF-/OIF-mediated pathways involved in inducing
ovulation in rabbits. The inserts magnify NGF/OIF mech-
anism at the uterine level: line thickness represents the
different NGF amounts of either exogenous (seminal
plasma) or endogenous (uterus/cervix) origin that enter
283
the circulating blood and/or act on sensory nervous
terminals. The question marks indicate putative target or
way. GnRH gonadotropin-releasing hormone, LH
luteinizing hormone, NGF nerve growth factor, NTRK1
neurotrophic tyrosine kinase receptor 1, OIF ovulation-
inducing factor. Figure from Maranesi et al. (2018)
284
binding to NTRK1 receptors and/or indirectly via
downstream activation of prostaglandin and/or
nitric oxide synthesis (Maranesi et al. 2016).
This experimental evidence suggests that the
neuro reflex triggered by coitus and a seminal
plasma male factor stimulating the endocrine
mechanism are both involved in inducing ovula-
tion during natural mating or artificial insemina-
tion in rabbits.
Finally, it is worth recollecting here that NGF
and its cognate receptors, NTRK1 and NGFR,
were immunolocalized in several rabbit ovarian
structures, including corpus luteum (Zerani et al.
2021), granulosa and theca cells (Maranesi et al.
2018), similar to results previously reported for
the ovary of Japanese Shiba goats (Ren et al.
2005) and other species (review: Linher-Melville
and Li 2013). The wide distribution of NGF
receptors in ovarian structures provides the
grounds for a local paracrine and/or endocrine
action of NGF on the ovary. Indeed, Silva et al.
(2011) reported an increase in the total number of
antral and haemorrhagic anovulatory follicles, but
no ovulation, in rabbits treated with an intramus-
cular dose of rabbit seminal plasma compared to
control those treated with PBS. Similar
haemorrhagic follicles were obtained with murine
NGF (García-García et al. 2017) or after super-
ovulation of those with eCG (García-Ximénez
and Vicente 1990).
19.4 Conclusions
NGF and its cognate receptors are expressed,
although differently, in all male accessory sex
glands, in the genital tract of several females in
the hypothalamus, pituitary, and ovary of all spe-
cies insofar studied. These findings, together with
results from in vitro studies, indicate that the NGF
system can regulate male and female reproduction
with different mechanistic processes. In addition,
the relatively high concentrations of NGF in the
seminal plasma in all species examined so far
suggest that this neurotrophin can exert direct
and/or indirect effects at several levels of the
female reproductive axis acting on ovarian
structures and/or on the release of gonadotropins.
M. Maranesi et al.
There are increasing evidences that several
components of the seminal plasma are involved
in both male and female reproductive processes
(Maranesi et al., unpublished results). Besides
NGF, a well-known ovulation-inducing factor,
other seminal plasma cytokines may stimulate
the female reproductive tract and induce ovula-
tion, thus enhancing the reproductive success. A
better knowledge of these complex biological
mechanisms could be useful to improve reproduc-
tive performance of farm animals as well as
humans.
References
Adams GP, Ratto MH, Huanca W, Singh J (2005)
Ovulation-inducing factor in the seminal plasma of
alpacas and llamas. Biol Reprod 73(3):452–457
Adams GP, Ratto MH, Silva ME, Carrasco RA (2016)
Ovulation-inducing factor (OIF/NGF) in seminal
plasma: a review and update. Reprod Dom Anim
51:4–17
Aurich C, Schreiner B, Ille N, Alvarenga M, Scarlet D
(2016) Cytosine methylation of sperm DNA in horse
semen after cryopreservation. Theriogenology 86
(5):1347–1352
Bakker J, Baum MJ (2000) Neuroendocrine regulation of
GnRH release in induce ovulators. Front
Neuroendocrinol 21:220–262
Berland MA, Ulloa-Leal C, Barría M, Wright H, Dissen
GA, Silva ME et al (2016) Seminal plasma induces
ovulation in llamas in the absence of a copulatory
stimulus: role of nerve growth factor as an ovulation-
inducing factor 1. Endocrinology 57(8):3224–3232
Bezerra MJB, Arruda-Alencar JM, Martins JAM, Viana
AGA, Viana Neto AM, Rêgo JPA et al (2019) Major
seminal plasma proteome of rabbits and associations
with sperm quality. Theriogenology 2019
(128):156–166
Bjorling DE, Beckman M, Clayton MK, Wang ZY (2002)
Modulation of nerve growth factor in peripheral organs
by estrogen and progesterone. Neuroscience
110:155–167
Bogle OA, Carrasco RA, Ratto MH, Singh J, Adams GP
(2018) Source and localization of ovulation-inducing
factor/nerve growth factor in male reproductive tissues
among mammalian species. Biol Reprod 99
(6):1194–1204
Brauer MM, Shockley KP, Chávez R, Richeri A,
Cowen T, Crutcher KA (2000) The role of NGF in
pregnancy-induced degeneration and regeneration of
sympathetic nerves in the Guinea pig uterus. J Auton
Nerv Syst 79:19–27
Carrasco R, Singh J, Adams GP (2016) The dynamics of
trkA expression in the bovine ovary are associated with
19 Nerve Growth Factor (NGF) and Animal Reproduction
a luteotrophic effect of ovulation inducing factor/nerve
growth factor (OIF/NGF). Reprod Biol Endocrinol
14:47–57
Casares-Crespo L, Fernandez-Serrano P, Vicente JS,
Marco-Marco-Jimenez F, Viudes-Castro MP (2018)
Rabbit seminal plasma proteome: the importance of
the genetic origin. Anim Reprod Sci 189:30e42
Castellini C, Mattioli S, Dal Bosco A, Collodel G,
Pistilli A, Stabile AM et al (2019) In vitro effect of
nerve growth factor on the main traits of rabbit sperm.
Reprod Biol Endocrinol 17(1):93
Ceccanti M, De Nicolo S, Mancinelli R, Chaldakov G,
Carito V, Ceccanti M et al (2013) NGF and BDNF
long-term variations in the thyroid, testis and adrenal
glands of a mouse model of fetal alcohol spectrum
disorders. Ann Ist Super Sanita 49:383–390
Cervantes MP, Palomino JM, Adams GP (2015) In vivo
imaging in the rabbit as a model for the study of
ovulation-inducing factors. Lab Anim 49:1–9
Chen BX, Yuen ZX, Pan G (1985) Semen-induced ovula-
tion in the Bactrian camel (Camelus bactrianus).
Reproduction 73(2):335–339
Chen Y, Dicou E, Djakiew D (1997) Characterization of
nerve growth factor precursor protein expression in rat
round spermatids and the trophic effects of nerve
growth factor in the maintenance of Sertoli cell viabil-
ity. Mol Cell Endocrinol 127(2):129–136
Dechant G, Barde YA (1997) Signalling through the
neurotrophin receptor p75NTR. Curr Opin Neurobiol
7:413–418
Ehrhard PB, Erb P, Graumann U, Otten U (1993) Expres-
sion of nerve growth factor and nerve growth factor
receptor tyrosine kinase Trk in activated CD4-positive
T cell clones. Proc Natl Acad Sci U S A
90:10984–10988
El Allali K, El Bousmaki N, Ainani H, Simonneaux V
(2017) Effect of the camelid’s seminal plasma
ovulation-inducing factor/ß-NGF: a kisspeptin target
hypothesis. Front Vet Sci 4:99
Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez
FA, Gardon JC (2011) Reduced glutathione content in
human sperm is decreased after cryopreservation:
effect of the addition of reduced glutathione to the
freezing and thawing extenders. Cryobiology 62
(1):40–46
García-García RM, Masdeu M, Sánchez-Rodriguez A,
Millán P, Arias Alvarez M, Sakr OG et al (2017)
ß-nerve growth factor identification in male rabbit gen-
ital tract and seminal plasma and its role in ovulation
induction in rabbit does. Ital J Anim Sci 17:442–453
García-Ximénez F, Vicente JS (1990) Effect of PMSG
treatment to mating interval in the superovulatory
response of primiparous rabbits. J Appl Rabbit Res
13:71–73
Harper GP, Thoenen H (1980) The distribution of nerve
growth factor in the male sex organs of mammals. J
Neurochem 34:893e903
Harper GP, Glanville RW, Thoenen H (1982) The purifi-
cation of nerve growth factor from bovine seminal
plasma. J Biol Chem 275:8541e8
285
Hofmann H-D, Unsicker K (1982) The seminal vesicle of
the bull: a new and very rich source of nerve growth
factor. Eur J Biochem 128:421–426
Inoue N, Sasagawa K, Ikai K, Sasaki Y, Tomikawa J,
Oishi S et al (2011) Kisspeptin neurons mediate reflex
ovulation in the musk shrew (Suncus murinus). Proc
Natl Acad Sci U S A 108(42):17527–17532
Jin W, Arai KY, Shimizu K, Kojima C, Itoh M, Watanabe
G et al (2006) Cellular localization of NGF and its
receptors trkA and p75LNGFR in male reproductive
organs of the Japanese monkey, Macaca fuscata
fuscata. Endocrine 29(1):155–160
Jin W, Tanaka A, Watanabe G, Matsuda H, Taya K (2010)
Effect of NGF on the motility and acrosome reaction of
golden hamster spermatozoa in vitro. J Reprod Dev
56:437–443
Johnson D, Lanahan A, Buck CR, Sehgal A, Morgan C,
Mercer E et al (1986) Expression and structure of the
human NGF receptor. Cell 47:545–554
Johnston SD, O’Callaghan P, Nilsson K, Tzipori G,
Curlewis JD (2004) Semen-induced luteal phase and
identification of a LH surge in the koala (Phascolarctos
cinereus). Reproduction 128:629–634
Julio-Pieper M, Lara HE, Bravo JA, Romero C (2006)
Effects of nerve growth factor (NGF) on blood vessels
area and expression of the angiogenic factors VEGF
and TGFbeta1 in the rat ovary. Reprod Biol Endocrinol
4:57
Kalthur G, Raj S, Thiyagarajan A, Kumar S, Kumar P,
Adiga SK (2011) Vitamin E supplementation in
semen-freezing medium improves the motility and
protects sperm from freeze-thaw-induced DNA dam-
age. Fertil Steril 95(3):1149–1151
Kaplan DR, Martin-Zanca D, Parada LF (1991) Tyrosine
phosphorylation and tyrosine kinase activity of the trk
protooncogene product induced by NGF. Nature
350:158–160
Kershaw-Young CM, Druart X, Vaughan J, Maxwell WM
(2012) ß-nerve growth factor is a major component of
alpaca seminal plasma and induces ovulation in female
alpacas. Reprod Fertil Dev 24(8):1093–1097
Koshimoto C, Mazur P (2002) Effects of warming rate,
temperature, and antifreeze proteins on the survival of
mouse spermatozoa frozen at an optimal rate. Cryobi-
ology 45(1):49–59
Levanti MB, Germanà A, de Carlos F, Ciriaco E, Vega JA,
Germanà G (2006) Effects of increased nerve growth
factor plasma levels on the expression of TrkA and p75
in rat testicles. J Anat 208:373–379
Levi-Montalcini R (1987) The nerve growth factor
35 years later. Science 237:1154–1162
Levine E, Cupp AS, Skinner MK (2000) Role of
neurotropins in rat embryonic testis morphogenesis
(cord formation). Biol Reprod 62(1):132–142
Li C, Zhou X (2013) The potential roles of neurotrophins
in male reproduction. Reproduction 145(4):R89–R95
Li C, Watanabe G, Weng Q, Jin W, Furuta C, Suzuki AK
et al (2005) Expression of nerve growth factor (NGF),
and its receptors TrkA and p75 in the reproductive
organs of the adult male rats. Zool Sci 22(8):933–937
286
Li C, Sun Y, Yi K, Ma Y, Sun Y, Zhang W et al (2010a)
Detection of nerve growth factor (NGF) and its specific
receptor (TrkA) in ejaculated bovine sperm, and the
effects of NGF on sperm function. Theriogenology 74
(9):1615–1622
Li C, Zheng L, Wang C, Zhou X (2010b) Absence of nerve
growth factor and comparison of tyrosine kinase recep-
tor a levels in mature spermatozoa from oligoastheno-
zoospermic, asthenozoospermic and fertile men. Clin
Chim Acta 411(19–20):1482–1486. https://doi.org/10.
1016/j.cca.2010.06.002. Epub 2010 Jun 11. PMID:
20542022
Lin K, Ding XF, Shi CG, Zeng D, QuZong S, Liu SH et al
(2015) Nerve growth factor promotes human sperm
motility in vitro by increasing the movement distance
and the number of a grade spermatozoa. Andrologia 47
(9):1041–1046
Linher-Melville K, Li J (2013) The roles of glial cell line-
derived neurotrophic factor, brain-derived
neurotrophic factor and nerve growth factor during
the final stage of folliculogenesis: a focus on oocyte
maturation. Reproduction 145(2):R43–R54
Lobos E, Gebhardt C, Kluge A, Spanel-Borowski K
(2005) Expression of nerve growth factor (NGF)
isoforms in the rat uterus during pregnancy: accumula-
tion of precursor proNGF. Endocrinology
146:1922–1929
MacGrogan D, Desprès G, Romand R, Dicou E (1991)
Expression of the beta-nerve growth factor gene in
male sex organs of the mouse, rat, and Guinea pig. J
Neurosci Res 28(4):567–573
Maranesi M, Zerani M, Leonardi L, Pistilli A, Arruda-
Alencar J, Stabile AM et al (2015) Gene expression
and localization of NGF and its cognate receptors
NTRK1 and NGFR in the sex organs of male rabbits.
Reprod Domest Anim 50(6):918–925
Maranesi M, Parillo F, Leonardi L, Rebollar PG,
Alonso B, Petrucci L et al (2016) Expression of nerve
growth factor and its receptors in the uterus of rabbits:
functional involvement in prostaglandin synthesis.
Domest Anim Endocrinol 56:20–28
Maranesi M, Petrucci L, Leonardi L, Piro F, Rebollar PG,
Millán P et al (2018) New insights on a NGF-mediated
pathway to induce ovulation in rabbits (Oryctolagus
cuniculus). Biol Reprod 98(5):634–643
Maranesi M, Palermo FA, Bufalari A, Mercati F,
Paoloni D, Cocci P et al (2020) Seasonal expression
of NGF and its cognate receptors in the ovaries of Grey
squirrels (Sciurus carolinensis). Animals (Basel)
10:1558
Mercati F, Domingo P, Pasquariello R, Dall'Aglio C, Di
Michele A, Forti K et al (2020) Effect of chelating and
antioxidant agents on morphology and DNA methyla-
tion in freeze-drying rabbit (Oryctolagus cuniculus)
spermatozoa. Reprod Domest Anim 55(1):29–37
Milligan SR (1982) Induced ovulation in mammals. In:
Finn CA (ed) Oxford review in reproductive biology.
Clarendon press, London, pp 1–46
Mirshokraei P, Hassanpour H, Rahnama A, Foster WG
(2013) Gene expression of BDNF and its receptors,
M. Maranesi et al.
TrkB and p75 in the uterus and oviduct of pregnant and
non-pregnant ewes. Res Vet Sci 95:164–168
Müller D, Davidoff MS, Bargheer O, Paust HJ, Pusch W,
Koeva Y et al (2006) The expression of neurotrophins
and their receptors in the prenatal and adult human
testis: evidence for functions in Leydig cells.
Histochem Cell Biol 126:199–211
Parthipan S, Selvaraju S, Somashekar L, Arangasamy A,
Sivaram M, Ravindra JP (2017) Spermatozoal
transcripts expression levels are predictive of semen
quality and conception rate in bulls (Bos taurus).
Theriogenology 98:41–49
Perrard MH, Vigier M, Damestoy A, Chapat C,
Silandre D, Rudkin BB et al (2007) Beta-nerve growth
factor participates in an auto/paracrine pathway of
regulation of the meiotic differentiation of rat
spermatocytes. J Cell Physiol 210:51e62
Ratto MH, Leduc YA, Valderrama XP, van Straaten KE,
Delbaere LT, Pierson RA et al (2012) The nerve of
ovulation-inducing factor in semen. Proc Natl Acad Sci
U S A 109:15042–15047
Ratto MH, Berland MA, Silva ME, Adams G (2019) New
insights of the role of β-NGF in the ovulation mecha-
nism of induced ovulating species. Reproduction 157
(5):R199–R207
Rebollar PG, Dal Bosco A, Millan P, Cardinali R,
Brecchia G, Sylla L et al (2012) Ovulating induction
methods in rabbit does: the pituitary and ovarian
responses. Theriogenology 77:292–298
Ren L, Medan MS, Weng Q, Jin W, Li C, Watanabe G et al
(2005) Immunolocalization of nerve growth factor
(NGF) and its receptors (TrkA and p75LNGFR) in
the reproductive organs of shiba goats. J Reprod Dev
51:399–404
Ricci A, Graziano P, Bronzetti E, Saltini C,
Sciacchitano S, Cherubini E et al (2007) Increased
pulmonary neurotrophin protein expression in idio-
pathic interstitial pneumonias. Sarcoidosis Vasc Dif-
fuse Lung Dis 24(1):13–23
Robinson LL, Townsend J, Anderson RA (2003) The
human fetal testis is a site of expression of
neurotrophins and their receptors: regulation of the
germ cell and peritubular cell population. J Clin
Endocrinol Metab 88:3943–3951
Saeednia S, Bahadoran H, Amidi F, Asadi MH, Naji M,
Fallahi P et al (2015) Nerve growth factor in human
semen: effect of nerve growth factor on the
normozoospermic men during cryopreservation pro-
cess. Iran J Basic Med Sci 18(3):292–299
Saeednia S, Shabani Nashtaei M, Bahadoran H,
Aleyasin A, Amidi F et al (2016) Effect of nerve
growth factor on sperm quality in asthenozoosprmic
men during cryopreservation. Reprod Biol Endocrinol
14(1):29
Sanchez-Rodriguez A, Arias-Alvarez M, Timón P,
Bautista JM, Rebollar PG, Lorenzo PL et al (2019)
Characterization of β-nerve growth factor-TrkA system
in male reproductive tract of rabbit and the relationship
between β-NGF and testosterone levels with seminal
quality during sexual maturation. Theriogenology
126:206–213
19 Nerve Growth Factor (NGF) and Animal Reproduction
Saruta J, Sato S, Tsukinoki K (2010) The role of
neurotrophins related to stress in saliva and salivary
glands. Histol Histopathol 25:1317–1330
Schecterson LC, Bothwell M (2010) Neurotrophin
receptors: old friends with new partners. Dev
Neurobiol 70:332–338
Schneidgenova M, Vašíček J, Cupka P, Chrenek P (2011)
Is it necessary to control seasonal quality of the rabbit
ejaculate? Slovak J Anim Sci 44:48–51
Seibel MM (1986) Luteinizing hormone and ovulation
timing. J Reprod Med 31(8):754–759
Shi Z, Arai KY, Jin W, Weng Q, Watanabe G, Suzuki AK
et al (2006) Expression of nerve growth factor and its
receptors NTRK1 and TNFRSF1B is regulated by
estrogen and progesterone in the uteri of golden
hamsters. Biol Reprod 74:850–856
Shi C-G, Lin K, Xu X-B, Zhang S-C, Wang N, Fan M
(2012) Evidence for the involvement of NGF in human
sperm motility. J Biomed Sci Eng 5(9):534–541
Shikata H, Utsumi N, Hiramatsu M, Minami N,
Nemoto N, Shikata T (1984) Immunohistochemical
localization of nerve growth factor and epidermal
growth factor in Guinea pig prostate gland. Histochem-
istry 80(4):411–413
Shilpa M, Selvaraju S, GirishKumar V, Parthipan S,
Binsila KB, Arangasamy A et al (2017) Novel insights
into the role of cell-free seminal mRNAs on semen
quality and cryotolerance of spermatozoa in bulls
(Bos taurus). Reprod Fertil Dev 29(12):2446–2456
Silva M, Niño A, Guerra M, Letelier C, Valderrama XP,
Adams GP et al (2011) Is an ovulation-inducing factor
(OIF) present in the seminal plasma of rabbits? Anim
Reprod Sci 127(3–4):213–221
Silva ME, Recabarren MP, Recabarren SE, Adams GP,
Ratto MH (2012) Ovarian estradiol modulates the
stimulatory effect of ovulation inducing factor (OIF)
on pituitary LH secretion in llamas. Theriogenology
77:1873–1882
Silva M, Ulloa-Leal C, Norambuena C, Fernandez A,
Adams GP, Ratto MH (2014) Ovulation-inducing fac-
tor (OIF/NGF) from seminal plasma origin enhances
Corpus luteum function in llamas regardless the pre-
ovulatory follicle diameter. Anim Reprod Sci 148
(3–4):221–227
Silva M, Ulloa-Leal C, Valderrama XP, Bogle OA, Adams
GP, Ratto MH (2017) Nerve growth factor from semi-
nal plasma origin (spβ-NGF) increases CL vasculari-
zation and level of mRNA expression of steroidogenic
enzymes during the early stage of Corpus luteum
development in llamas. Theriogenology 103:69–75
Snider WD (1994) Functions of the neurotrophins during
nervous system development: what the knockouts are
teaching us. Cell 77:627–638
Squillacioti C, De Luca A, Paino S, Langella E, Mirabella
N (2009) Effect of castration on the expression of the
NGF and NTRK1 in the vas deferens and accessory
male genital glands of the rat. Eur J of Histochem
53:239–248
287
Stewart JL, Mercadante VRG, Diaz NW, Canisso IF,
Yau P, Imai B et al (2018) Nerve growth factor-beta,
purified from bull seminal plasma, enhances corpus
luteum formation and conceptus development in Bos
taurus cows. Theriogenology 106:30–38
Stuart CC, Vaughan JL, Kershaw-Young CM,
Wilkinson J, Bathgate R, de Graaf SP (2015) Effects
of varying doses of β-nerve growth factor on the timing
of ovulation, plasma progesterone concentration and
corpus luteum size in female alpacas (Vicugna pacos).
Reprod Fertil Dev 27(8):1181–1186
Thoenen H, Barde YA (1980) Physiology of nerve growth
factor. Physiol Rev 60:1284–1335
Tribulo P, Bogle O, Mapletoft RJ, Adams GP (2015)
Bioactivity of ovulation inducing factor (or nerve
growth factor) in bovine seminal plasma and its effects
on ovarian function in cattle. Theriogenology 83
(9):1394–1401
Ulloa-Leal C, Bogle OA, Adams GP, Ratto MH (2014)
Luteotrophic effect of ovulation-inducing factor/nerve
growth factor present in the seminal plasma of llamas.
Theriogenology 81(8):1101–1107.e1
Varol FG, Duchemin AM, Neff NH, Hadjiconstantinou M
(2000) Nerve growth factor (NGF) and NGF mRNA
change in rat uterus during pregnancy. Neurosci Lett
294:58–62
Verma A, Rajput S, De S, Kumar R, Chakravarty AK,
Datta TK (2014) Genome-wide profiling of sperm
DNA methylation in relation to buffalo (Bubalus
bubalis) bull fertility. Theriogenology 82(5):750-9.e1
Wang H, Dong Y, Chen W, Hei J, Dong C (2011) Expres-
sion and localization of nerve growth factor (NGF) in
the testis of alpaca (llama pacos). Folia Histochem
Cytobiol 49:55–61
Wessels JM, Wu L, Leyland NA, Wang H, Foster WG
(2014) The brain uterus connection: brain derived
neurotrophic factor (BDNF) and its receptor (Ntrk2)
are conserved in the mammalian uterus. PLoS One 9:
e94036
Yamamoto M, Sobue G, Yamamoto K, Terao S, Mitsuma
T (1996) Expression of mRNAs neurotrophic factors
(NGF, BDNF, NT-3 and GDNF) and their receptors
(p75NGFR, NTRK1, TrkB, and Trkc) in the adult
human peripheral nervous system and non-neural
tissues. Neurochem Res 21:929–938
Zhang L, Wang H, Yang Y, Liu H, Zhang Q, Xiang Q et al
(2013) NGF induces adult stem Leydig cells to prolif-
erate and differentiate during Leydig cell regeneration.
Biochem Biophys Res Commun 436(2):300–305
Zhang H, Wang Y, Zhang J, Wang L, Li Q, Sheng X et al
(2015) Testicular expression of NGF, TrkA and p75
during seasonal spermatogenesis of the wild ground
squirrel (Citellus dauricus Brandt). Eur J Histochem 59
(3):2522
Zerani M, Polisca A, Boiti C, Maranesi M (2021) Current
knowledge on the multifactorial regulation of corpora
lutea lifespan: the rabbit model. Animals (Basel) 11
(2):296
 Neurotrophins in the Brain of Teleost
Fish: The State of the Art 20
Paolo de Girolamo and Livia D’Angelo

Abstract multifaceted roles of neurotrophins in the

Neurotrophins are evolutionary well-
conserved molecules, and fish constitute valu-
able vertebrate models to explore their pleio-
tropic role in the brain. In addition to an
introduction on the evolutionary importance
of using fish in biomedicine and their neuro-
anatomy in comparison with mammals, here
we review the available literature on the
molecular evolution of neurotrophins and
their receptors in teleost fish as well as their
role in the fish brain, from the early stages of
development until adulthood and aging.
Among neurotrophins, BDNF is the most
well studied in the brain of teleost fish, and
we report data on the functional involvement
of the BDNF/TrkB system in the development
of the visual system and in the mechanisms of
adult brain regeneration. With the exception of
neuroanatomical expression, much less is
known about the role of the other members
of neurotrophin family in fish brain. We hope
that this chapter opens new avenues leading to
a better understanding of the complex and
P. de Girolamo · L. D’Angelo (*)
Department of Veterinary Medicine and Animal
Production, University of Naples Federico II, Naples, Italy
e-mail: paolo.degirolamo@unina.it; livia.dangelo@unina.
it
brain of fish and other vertebrates.
20.1 Introduction
The discrete degree of molecular and functional
conservation of neurotrophins in teleostean fish
(Heinrich and Lum 2000) has attracted numerous
researchers to address studies on the related
conserved functional roles during the develop-
ment of the fish central nervous system (CNS)
as well as its maintenance in adult and aged brain.
Molecular evolution studies of neurotrophins and
their receptors represent a clear example of stud-
ies for analyzing coevolution processes of these
gene families and their correlation with the
increased complexity of the vertebrate nervous
system. Fish share enough commonalities to jus-
tify conducting research relevant to humans,
although fish diverged from humans more than
400 million years ago. A vast amount of literature
has convincingly demonstrated the usefulness of
fish for improving our understanding of the
molecular and cellular mechanisms leading to
pathological conditions, and for the development
of new diagnostic and therapeutic tools (Schartl
2014); Aleström et al. 2020).

# The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 289
L. Calzà et al. (eds.), Recent Advances in NGF and Related Molecules, Cellular Neuroscience, Neural
Circuits and Systems Neuroscience 1331, https://doi.org/10.1007/978-3-030-74046-7_20
290
20.1.1 Evolutionary Aspects of Fish
as Model Organisms
in Biomedicine
The most powerful and today used laboratory fish
species is zebrafish (Danio rerio), thanks also to
the advanced and refined development of genetic
techniques for modeling human-acquired disease.
These freshwater fish provide a number of excep-
tional advantages as laboratory organisms. First,
they are small, can be bred and maintained in
large numbers, and are at low cost (Aleström
et al. 2020). Second, they offer the opportunity
to combine the analytical clarity of developmental
biology with the power of genetics, and trans-
genic lines can be easily and quickly obtained
(Mann and Bhatia 2019). Furthermore, they
offer the great advantage of high-throughput
approaches such as whole-genome mutagenesis
or chemical library drug screens (Cornet et al.
2018). Despite these undisputable advantages,
the zebrafish as “genetically domesticated
models” has also some limitations, particularly
in studies of pleiotropy, multigenic inheritance,
variable penetrance, and so on. (Schartl 2014).
Furthermore, the selective breeding of laboratory
animals makes very difficult or impossible to
observe and investigate the effect of the natural
environment on human model phenotypes
(Albertson et al. 2009). Indeed, natural selection
is a potent generator of variation, and in this
perspective, other fish models are emerging as
“evolutionary mutant models” (Albertson et al.
2019). This concept was postulated on the
evidences that in species or populations of many
animal groups, including fish, the adaptation to a
specific environment may result in an adaptive
phenotype, which resemble maladaptive human
diseases (Albertson et al. 2009). This phenome-
non constitutes a complementary approach to the
genetic manipulation of a “domesticated” labora-
tory organism, to discover genes and mechanisms
involved in human diseases. In addition, in this
sense, teleost fish, being the largest group of
vertebrates, offer great opportunities residing in
the biodiversity of the different environment and
adaptive morphophysiological features.
P. de Girolamo and L. D’Angelo
Evolutionary mutant models can be further
categorized into two main classes:
(a) evolutionary mutants with adaptive disease
phenotypes and (b) fish that are afflicted with
the same disease as humans, and thus develop a
true illness (Albertson et al. 2009; Schartl 2014).
Examples of models in the first category are
represented by Antarctic icefish, mimicking
“spontaneous” anemia-like disease and
osteopenia. The Antarctic icefish family is unique
among vertebrates which do not express the
developmental program for erythrocyte forma-
tion. They do not make hemoglobin, nor they
produce erythrocytes, and therefore they model
harmful human blood diseases, including
anemias, hemoglobinopathies, and thalassemias.
Comparative genomic analysis with closely
related species, i.e., zebrafish, which produce
hemoglobin and have erythrocytes, has led to
the identification of candidate genes (Detrich
and Yergeau 2004) also for human anemia. Fur-
ther examples, among others, are represented
by the blind cavefish and the short-lived annual
killifish. The former evolved degenerated lens
and retinas, similar to humans with retinal degen-
eration diseases (Jeffery 2009), the latter will be
discussed more in detailed in this chapter.
Examples of medical condition models include
those fish species that naturally develop the same
disease in humans. Examples are the
Xiphophorus which develops true malignant
skin tumors from the naturally occurring large
pigment spots (Fernandez and Bowser 2010)
and eels which spontaneously in the wild develop
Wilms’ tumor, a malignant kidney tumor that
affects children at early age (Hu et al. 2011).
Beyond the opportunity of choosing between
laboratory fish and evolutionary mutant model
according to the scientific need, when dealing
with fish it should be taken into account their
evolutionary history, a key and relevant aspect.
Indeed, during evolution, teleost fish have
undergone a whole-genome duplication so that
fish have two copies of many genes of which
tetrapods have only one (Meyer and Schartl
1999; Postlethwait et al. 1998). Those duplicated
genes may be (1) orthologs, pairs of genes (one in
each of two different species) that evolved from a
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
single gene in the last common ancestor of those
two species, and/or (2) paralogs, genes that are
related to each other by gene duplication events
occurring within a lineage.
The duplication has resulted in altered gene
expression patterns or protein functions, such
that the complement of the expression domains
or protein functions of both fish paralogs is equiv-
alent to the single ortholog in other vertebrates.
For instance, if a mouse gene is expressed in the
liver and kidney, one copy of the equivalent sep-
arate fish gene would be expressed in the liver,
and the second copy would be expressed in the
kidney. This evolutionary process, which has
been termed subfunction partitioning or
subfunctionalization (Force et al. 1999), has
far-reaching implications for functional studies
of many genes. For example, when a mutation
in mouse inactivates a gene that has a role in early
development in addition to an adult organ-
specific function, it becomes impossible to study
the function of the adult organ in such mutants. In
fish, both functions can be partitioned between
duplicates, making it possible to separate both
phenotypes. Thus, the teleost whole-genome
duplication provides unique opportunities for
studies on genes involved in disease development
(Schartl 2014).
20.1.2 The Organization of the Brain
of Teleostean Fish
The brain of fish exhibits the typical arrangements
of subdivisions typical of most vertebrates: the
spinal cord extends rostrally with the
(1) brainstem, including the tegmentum of mes-
encephalon and diencephalon; (2) cerebellum and
(3) pair of optic lobes which cap the mesenceph-
alon; and (4) telencephalon consisting of paired
hemispheres with olfactory bulbs in ventro-rostral
position. The motor system mainly resides in the
spinal cord, except for few a pathways
descending from the brain and some prominent
brainstem reflexes, i.e., the Mauthner neuron sys-
tem for escape. The somatosensory information
291
reaches the brain primarily via specialized cranial
nerves (trigeminus, facialis, vagus, and the three
lateral line nerves), rather than through ascending
fibers of the spinal cord (Kotrschal et al. 1998).
The brainstem houses primarily the represen-
tation centers for all receiving somatosensory
stimuli, except for olfaction and vision. Somato-
sensory target areas are generally displaced dor-
sally and organized in columns, whereas motor
fibers originated from ventrally located centers. In
certain fish species, i.e., Cyprinids, characterized
by a well-developed taste system made up of
internal and skin taste buds, the areas of the dorsal
column form prominent bulges: the facial lobe
emerges from the fourth ventricle as a singular
lobe, and the vagal lobe is a pair of dorso-caudal
ridges along the fourth ventricle receiving tactile/
chemosensory nerves. In addition to several
ascending and descending fiber systems, the
brainstem houses the reticular formation, a ven-
trally located network of neurons that is essential
for the vital functions.
The mesencephalic tegmentum and dienceph-
alon house integrative systems for the brain
structures arising from the cerebellum, optic tec-
tum, and forebrain. In a rostrocaudal direction,
the tegmental third ventricle changes from a slit-
like gap to a narrow channel before opening into
the fourth ventricle. Several structures develop as
extensions to this ventricle. The inferior lobes of
the hypothalamus are paired, constitute the
ventro-caudal part of diencephalon, and serve as
multimodal integration centers. As in all
vertebrates, the hypothalamus converts sensory
inputs into hormonal and behavioral responses.
The cerebellum is a discretely well-developed
structure, organized in a corpus and a valvula, the
latter appearing as a rostral extension beneath the
optic tectum. Cerebellar structures are intimately
connected among each other and intervene in
spatial orientation, proprioception, motor coordi-
nation, and eye movement. The ventral cerebellar
surface area, the granular area, represents the
central acoustic area (Popper and Fay 1993). On
both sides of the corpus of cerebellum, there is a
parvocellular area called eminentia granularis,
292
which receives inputs from the inner ear and from
lateral line, whereas caudal and in continuation
with the molecular layer of the corpus, there is the
crista cerebelli, predominantly involved in
processing lateral line input (Davis and Northcutt
1983).
The optic tectum consists of paired, dorsal
hemispheres, separated from the tegmentum by
the ventricular space, receives projections from
contralateral retinal ganglion cells, and
participates in the bidirectional communication
with the brainstem. Below the optic lobes, in the
paramedian position, the torus longitudinalis
extends into the subtectal ventricle as a pair of
longitudinal cylinders. Its presumed functions
include postural control, detection of luminance
levels, and monitoring of saccadic movements
(Northmore et al. 1983). It has also a role as a
premotor center between the telencephalon and
the brainstem (Wullimann 1994).
The telencephalon arises from the rostral por-
tion of the embryonic neural tube forming two
hemispheres, which present a T-shaped ventricle
separating the two halves up to the dorsolateral
surface. Centrally, the two hemispheres are
closely attached to each other and may even be
fused. In addition to secondary olfactory
tract which terminates throughout the entire struc-
ture, virtually all sensory modalities project to its
dorsal portion through lemniscal pathways (Fin-
ger 1983), and hypothalamic as well as primary
olfactory input is received at the ventral forebrain.
The latter also contains the anterior commissure
with a peduncle of decussating tracts for a
two-way flow of information between the telen-
cephalon and the diencephalon as well as within
the telencephalon itself.
The telencephalon has attracted the attention
of numerous neuroscientists about its develop-
mental process, which has consequences on the
neuromorphological organization of homologous
telencephalic brain areas of tetrapods. More
recently, developmental and gene expression
studies have confirmed the theory of the “everted
telencephalon” (Nieuwenhuys 2009; Folgueira
et al. 2015), and identified the dorsal part of
P. de Girolamo and L. D’Angelo
telencephalon as the pallium, and the ventral
part as the subpallium (Nieuwenhuys and Meek
1990). According to this theory, the rostral neural
tube is thought to bend outward resulting in two
telencephalic hemispheres separated by an
unpaired ventricle and covered by a thin roof
plate, thus referred to as “everted” (Folgueira
et al. 2015). This eversion process results in an
unpaired ventricle and a different arrangement of
the homologous of pallium. The pallium in
teleosts has generally been subdivided into a
medial (Dm), dorsal (Dd), central (Dc), lateral
(Dl), and posterior (Dp) part (Nieuwenhuys
2009). However, the precise and exact
homologies with the pallium of tetrapods are
quite debated, because several models of homol-
ogy have been proposed based on connectional,
neurochemical, gene expression, and functional
data (Ganz et al. 2012; Mueller 2011; Wullimann
and Mueller 2004). More recently, a new pallial
subdivision and related homology to pallial nuclei
in tetrapods have been proposed, based on the
expression of conserved marker genes in
zebrafish (Ganz et al. 2015). According to the
authors, Dm is homologous to the pallial amyg-
dala in tetrapods, and the dorsal subdivision of Dl
is homologous to part of the hippocampal forma-
tion in mouse (Ganz et al. 2015). The combina-
tion of ablation and behavioral experiments
documented that animals fed, grew, and behaved
normally in most respects, but exhibited signifi-
cantly diminished rates of learning and were
unable to perform more complex social tasks
(Broglio et al. 2005; Rodriguez et al. 2002). An
analogous approach, based on molecular marker
analysis, has been used to also identify homolo-
gous areas of the subpallium, the ventral part of
telencephalic hemispheres. The nuclei homolo-
gous of striatum and pallidum are stretched
along the rostrocaudal axis of the telencephalon,
also caudal to the anterior commissure (Ganz
et al. 2012). Subpallial homologies have been
identified as follows: a striatal domain in ventro-
dorsal (Vd) and the dorsal part of ventro-ventral
(Vv) and a striatal-septal domain (dorsal and ven-
tral part of Vv) in the rostral telencephalon and
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
striatum (Vd, dorsal part of Vv), pallidum, and a
pallidal-septal domain (ventral part of Vv) in the
mid-telencephalon, caudal to anterior commis-
sure (Ganz et al. 2012).
20.1.3 Comparative Histological
Aspects Between the Brain
of Teleostean Fish
and Mammals
The adult teleost brain resembles more to the
embryonic than to the adult mammalian brain.
Studies on neurogenic and regenerative capacity
in the adult throughout the rostrocaudal axis of
the brain largely and accurately have
demonstrated this aspect. It has been suggested
that the addition and/or replacement/turnover of
neuronal subtypes is necessary for expanding the
existing sensory systems concomitantly with the
allometric growth and related increase in primary
sensory input (Kaslin et al. 2008). The great neu-
rogenic ability of fish brain resides mainly in the
persistence of neuronal progenitors through the
entire life.
20.1.3.1 Radial Glia
The neuronal progenitors have a radial glia mor-
phology, and express radial glia markers, such as
the glial fibrillary acidic protein (GFAP) (Lyons
et al. 2003). Cells expressing radial glia morphol-
ogy and markers act as progenitors over the entire
life. However, many radial progenitor cells,
which remain proliferative, do not express
GFAP as radial glia marker (März et al. 2010),
but rather those of neuroepithelial cells, i.e.,
nestin (Mahler and Driever 2007).
Neural stem and progenitor cells are located
within the brain in distinct neurogenic niches
differing in their location, cellular composition,
and proliferative behavior. Proliferating radial
glia, quiescent radial glia, and
neuroepithelial-like cells (Lindsey et al. 2018)
mainly characterize heterogeneity in the neural
stem and progenitor cell populations. This hetero-
geneity is hypothesized to reflect varying
capacities for neurogenesis, plasticity, and repair
between different neurogenic zones.
293
A remarkable difference between fish and
mammals is the absence of stellate astrocytes in
the former, and it has been hypothesized that
radial glia in fish may serve some specialized
roles of astrocytes in mammals. For example,
zebrafish radial glia (1) express aquaporin-4,
which is localized within the end feet of mamma-
lian astrocytes at the blood-brain barrier, although
in fish the localization is not polarized (Grupp
et al. 2010), and (2) express the glutamate trans-
porter Eaat2b, suggesting that radial glia in
zebrafish may serve functions related to
neurotransmitters, as in mammals (McKeown
et al. 2012). Alternatively, some features in the
“reactive astrocyte” response of mammalian brain
upon injury are conserved in fish glia (Neve et al.
2012) under similar experimental conditions. In
the course of injury, also zebrafish glia
overexpresses rat lipocalin-2, an autocrine medi-
ator of reactive astrocytosis in rodents, which
induces changes in the GFAP-expressing radial
glia (Lee et al. 2009).
20.1.3.2 Oligodendrocytes
The myelinating cells of the CNS of fish present
evolutionary conserved features common to all
vertebrates (Baraban et al. 2016). The mature
morphology of oligodendrocytes is plastic and
determined by the axonal environment. Indeed,
neuronal activity can affect distinct stages of oli-
godendrocyte development and the process of
myelination. Increasing evidences show that
new myelin sheaths can be made on previously
unmyelinated axons into adult life and there is a
certain degree of myelin sheet turnover through
life (Lyons and Talbot 2014). Molecular signature
during development and differentiation of
oligodendrocytes, as well as in the myelination
process, has been investigated, and
commonalities with mammals have been con-
firmed (Baraban et al. 2016). Altogether, these
studies are pivotal to define therapeutic strategies
for the treatment of demyelinating diseases.
20.1.3.3 Schwann Cells
Developmental mechanisms regulating the cor-
rect differentiation and myelination process have
been studied in zebrafish (Lyons and Talbot
294
2014). Precursors of Schwann cells, the
myelinating glia of peripheral nerves, derive
form the neuronal crest, and migrate with grow-
ing axons during nerve development. After
migration, the process of radial sorting begins,
and Schwann cells associate with individual
axons in the bundle of the developing nerve
(Jessen and Mirsky 2005; Raphael and Talbot
2011). As in mammals, in zebrafish Swann cells
form myelin by wrapping their cell membranes
around axons to generate a multilayered membra-
nous sheath that insulates and supports the axons
(Lyons and Talbot 2014). Myelinating Schwann
cells are postmitotic, but, in response to injury,
they lose contact with axons and undergo a pro-
cess of dedifferentiation (Glenn and Talbot 2013).
20.1.3.4 Microglia
Microglia represent the tissue-resident macro-
phage population of the fish brain, alike in
mammals. In zebrafish, a subpopulation of primi-
tive macrophages from the yolk sac colonize the
brain early during development from 48 h post-
fertilization onward. Once in the brain, these
macrophages then rapidly differentiate into early
microglia over the next 24 h post-fertilization
(hpf) (Herbomel et al. 2001). At 3 and 4 days
post-fertilization (dpf), microglia are fully func-
tional (Sieger et al. 2012), and share a significant
number of expressed genes with their adult
counterparts in zebrafish as well as with mouse
and human microglia (Mazzolini et al. 2019).
Mature microglia are present throughout the
parenchyma of the brain and spinal cord of fish
(Sieger and Peri 2013), with cell bodies largely
stationary. They actively extend and retract their
processes in the presence of perturbation in the
environment, becoming activated (Svahn et al.
2013).
20.1.4 Neuromediator Systems
of Teleostean Fish
Vertebrate neurochemistry is generally highly
conserved. Numerous and very detailed studies,
mainly addressed on zebrafish, have
demonstrated fish possess all primary
P. de Girolamo and L. D’Angelo
neuromediator systems, including transmitters,
receptors, transporters, and the enzymes required
for synthesis and metabolism of these mediators
(Stewart et al. 2014). Remarkably, the spatial and
temporal distribution of these systems has been
well characterized in both embryo/larva and adult
zebrafish for glutamate, γ-aminobutyric acid
(GABA), acetylcholine, and the aminergic
neurotransmitters dopamine (DA), noradrenaline
(NA), serotonin (5-hydroxytryptamine (5-HT)),
and histamine (Stewart et al. 2015). Similar
anatomical distribution of dopaminergic, norad-
renergic, and cholinergic neurons has been
reported in N. furzeri (Matsui et al. 2019; de
Girolamo et al. 2020). Neuroanatomical studies
have documented that these major neurotransmit-
ter systems are overall localized in homologous
brain areas of mammals, and drug treatment has
confirmed the conserved functions in the adult
CNS (Stewart et al. 2014). The pattern of distri-
bution of dopamine is slightly different when
compared to homologous brain areas of zebrafish,
because the gene encoding th is duplicated, and
th1 and th2 are differentially expressed
(Yamamoto et al. 2010). Additionally, dopami-
nergic neurons have not been identified in the
mesencephalon (Holzschuh et al. 2001) and ven-
tral midbrain (Tay et al. 2011). Finally, it is
important to consider that the fish-specific
genome duplication resulted in expressing more
neurotransmitter receptors than in other classes of
vertebrates. For example, zebrafish have an
estimated 122 G-protein-coupled receptors for
biogenic amines (such as 5-HT, NA, DA, HA,
adrenaline, and trace amines), compared with
57 in mouse and 44 in humans (Fredriksson and
Schioth 2005).
20.2 The Two Mainstream Fish
Models in Neurotrophin
Research: Zebrafish
and the African Turquoise
Killifish
The existence of functional NGF-related
neurotrophins in invertebrate lineages is
supported by studies in Drosophila and
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
C. elegans (Jaaro et al. 2001) as well as in
Lymnaea (Getz et al. 2020). Among nonmamma-
lian vertebrates, two mainstream model species
have been used to explore the role of
neurotrophins in the CNS: zebrafish and the
annual turquoise killifish, N. furzeri, both tropical
freshwater organisms belonging to the class of
Actinopterygii. While zebrafish is cyprinid,
originated from ponds and standing water bodies,
often connected to rice cultivation in India
(Parichy 2015), N. furzeri is a Cyprinodon-
tiformes living in the ephemeral pond, which
can be found during the monsoon season in
South-Eastern Africa (Mozambique and
Zimbabwe) (Reichard and Polačik 2019). The
origin of their different habitat results in a strong
evolutive adaptation, with different morphophy-
siological features (D’Angelo et al. 2016a).
Zebrafish is the canonical fish model for study-
ing human diseases. It offers the advantage of
using either the larval or the adult forms. At the
larval stage, it has the unique benefit of the optical
clarity, allowing visualization of individual genes
(fluorescently labeled or dyed) throughout the
developmental process using noninvasive imag-
ing techniques. The embryonic transparency also
helps in case of genetic manipulations. The
sequencing of genome has been completed
(Howe et al. 2013), and numerous protocols for
transgenesis, mutagenesis, and genome editing
are available (Albadri et al. 2017). Owing to the
small size of the larvae, high-throughput screen-
ing of neuroactive compounds can be easily
performed (Saleem and Kannan 2018). Adult
zebrafish have an integrated nervous system,
which is proposed to contain homologous brain
structures to those found in humans, as well as
equivalent cellular and synaptic structure and
functions (Imperatore et al. 2018). Zebrafish dis-
play (1) evolutionarily conserved nature of many
behaviors to resemble those of other species,
(2) simple and relatively complex forms of
learning, and good performance on cognitive
tasks dependent on short-term and long-term
memory (Gerlai 2016). Furthermore, zebrafish,
as all other teleosts, has attracted numerous
295
scientists to investigate the regulatory
mechanisms underlying the remarkable adult neu-
rogenic and regenerative capacity throughout the
rostrocaudal axis of the brain (Ganz and Brand
2016).
N. furzeri, a clear example of evolutionary
mutant model, is characterized by a remarkable
short lifespan (condensed in a few months), dur-
ing which it recapitulates typical hallmarks of
vertebrate aging (Harel et al. 2016). Interestingly,
the short lifespan can be modulated by pharma-
cological, dietary, and lifestyle interventions
(Cellerino et al. 2016). In the last few years, a
number of tools to work with this model have
been developed. First, the genome has been
sequenced and assembled (Valenzano et al.
2015; Reichwald et al. 2015). Second,
transcriptomes for a number of tissues (Petzold
et al. 2013; Baumgart et al. 2014) are available.
Third, several inbred lines are housed under lab
condition (Hu and Brunet 2018). Fourth, genome-
editing techniques are widely adopted to rapidly
produce stable transgenic lines (Harel et al. 2016).
Therefore, this species, which emerged as excel-
lent model for aging studies, is now broadly used
in disparate research fields, such as evolutionary
genomics (Reichwald et al. 2015), regenerative
medicine (Wendler et al. 2015; Wang et al.
2020a, b), developmental biology (Hu and Brunet
2018), and ecotoxicology (Philippe et al. 2018).
However, N. furzeri remains as dominant model
organism for aging studies, because of the unique
so far described feature of a rapid as well as
spontaneous aging process. The brain of
N. furzeri has been studied with reference to its
morphology (D’Angelo 2013). The major cellular
phenotypes observed during aging are (1) a dra-
matic reduction of stem cell activity and
age-dependent reduction of adult neurogenesis
(Tozzini et al. 2012); (2) glial hypertrophy
(gliosis), visualized as overexpression of GFAP
(Tozzini et al. 2012); and (3) neuronal degenera-
tion, as measured by Fluoro-Jade B staining and
accumulation of lipofuscin (Terzibasi Tozzini
et al. 2012). At transcriptomic level, comparison
with human brain datasets revealed conserved
296 P. de Girolamo and L. D’Angelo

upregulation of ribosome and lysosome, and expression suggests that subfunctionalization

complement activation and conserved and, to some degree, neo-functionalization have
downregulation of synapse, mitochondrion, occurred after the duplication (Dethleffsen et al.
proteasome, and spliceosome (Baumgart et al. 2003).
2014). Based on tyrosine kinase domain comparisons
of the zebrafish and mammalian receptors, one of
the fish genes was most related to TrkA, two to
TrkB, and the remaining two to TrkC. Consis-
20.2.1 Evolutionary Aspects
tently, the correspondence between the zebrafish
of Neurotrophins in Teleost Fish
and the mammalian receptors was maintained
when analyzing their extracellular domains,
Neurotrophins and their high-affinity Trk
except for the TrkA, whose extracellular domain
receptors have been found in each class of
is yet unknown (Martin et al. 1995). Similarly, in
vertebrates (Heinrich and Lum 2000). Accord-
fully sequenced genome of the pufferfish
ingly, neurotrophins evolved early in vertebrate
Tetraodon (Chiu et al. 2004) and N. furzeri
history during two rounds of genome duplication
(Reichwald et al. 2015), there are also five Trk
that characterized the origin and evolution of the
receptors, one of the A class, and two of each B
subphylum (Lundin et al. 2003; Hallböök et al.
and C classes. This situation correlates well with
2006). The phylogeny of Trk receptors is consis-
the doubling of the genome that occurred early
tent with this general pattern as well (Hallböök
during the evolution of teleost fishes (Benito-
1999). More in details, orthologs of NGF, BDNF,
Gutiérrez et al. 2006) and implies that one of the
NT-3, and NT-4/5 as well as of TrkA, TrkB, and
duplicated TrkA genes was lost early in the fish
TrkC are known in teleost fish (Götz et al. 1994;
lineage. Two paralogs of TrkB and of TrkC
Martin et al. 1995; Hallböök 1999; Dethleffsen
receptor have been identified in zebrafish (Martin
et al. 2003). A complete set of the “core” four
et al. 1995), pufferfish (Crollius 2006), and the
neurotrophins were originally retrieved in two
African turquoise killifish (N. furzeri) (Reichwald
pufferfish (Takifugu rubripes and Tetraodon
et al. 2015; Leggieri et al. 2019).
nigroviridis) and the zebrafish (Danio rerio)
Differently from Trk receptor, the nerve
genome assemblies. Recently, they were
growth factor receptor (Ngfr), alias p75 NT recep-
identified also in N. furzeri.
tor, represents a real “case study” in molecular
First phylogenetic analyses revealed the exis-
evolution. The teleost ngfr- and ngfr-related
tence of bony fishes’ neurotrophins not known in
genes have been discretely characterized mainly
other vertebrates: neurotrophin-6 (NT-6) in
during development (Brosamle and Halpern
platyfish (Xiphophorus maculatus and
2009; Han et al. 2014; Pinzón-Olejua et al.
Xiphophorus helleri) (Götz et al. 1994) and
2014), and to a less extent in adult animals
neurotrophin-7 (NT-7) in zebrafish (Nilsson
recapitulating some humanlike disease states,
et al. 1998) and carp (Cyprinus carpio) (Lai
such as anxiety behavior associated with type
et al. 1998). Identification of just one NT-6/NT-
2 diabetes (Wang et al. 2020a, b). From an
7 ortholog in the two pufferfish as well as in
evolutive perspective, the most recent theory,
zebrafish genome assemblies shows that NT-6
based on elegant synteny and phylogenetic stud-
and NT-7 are orthologs, and the gene should be
ies, supports that after the divergence of the rayfin
denoted NT-6/7 in analogy with the NT-4/5
and lobefin lineage, the whole-genome duplica-
nomenclature (Hallböök et al. 2006; Dethleffsen
tion resulted in duplication of both ngfr1 and
et al. 2003). The NT-6/7 is similar to NGF and is
ngfr2. This then gave rise to ngfr1a and ngfr1b,
phylogenetically distant from the human pseudo
which both persist in zebrafish, and to ngfr2a and
NT-6 genes, closely related to NT-4/5. NT-6/7
ngfr2b, the latter of which was lost in the teleost
and fish NGF bind and activate TrkA, and their
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
lineage (Catchen et al. 2011). However, in the
case of the African turquoise killifish, ngfr has
been annotated in single copy (de Girolamo et al.
2020).
20.2.2 Neurotrophins in the Brain
of Teleost Fish
The transcription activity and phenotypic charac-
terization of neurotrophins have been investigated
in developing as well as mature neural
phenotypes. The most well-investigated member
of the neurotrophin family is BDNF and its
related receptor, TrkB. However, data on tran-
scriptional activity and phenotypic characteriza-
tion of all other related members are available
either in the developing or in the adult brain of
fish. A summary of the brain localization of the
different neurotrophins in various fish species is
reported in Table 20.1, and an overview of locali-
zation in the adult brain of N. furzeri is depicted in
Fig. 20.1.
20.2.3 bdnf/trkB
The gene encoding for bdnf in zebrafish is about
90% identical to its mammalian counterpart
(Hashimoto and Heinrich 1997), and it is more
compact and complex in its exon organization
and primary transcript processing. Five
promoters, and possibly as many as six, have
been identified in zebrafish (Heinrich and
Pagtakhan 2004). Of the seven 50 noncoding
exons associated with promoters, two have been
found only in the embryo before transcription
starts (they must be parts of maternally transmit-
ted transcripts); the other two, promoters 1b and
1d, could not be detected at any stage or in any
adult organ. The four active promoters appeared
differentially expressed in the adult organs
(Heinrich and Pagtakhan 2004). At amino acid
levels, the degree of conservation between teleos-
tean fish and mammals is very high (Lanave et al.
2007; Tettamanti et al. 2010).
In developing zebrafish, BDNF transcripts
were first measurable 13 hours post fertilization
297
(hpf). Their abundance relative to total transcripts
increased sixfolds between 0 day and 2 dpf and
again onefold by 6 dpf (Hashimoto and Heinrich
1997). The expression of bdnf in the developing
brain seemed to be structure-specific (De Felice
et al. 2014): genetic markers (shh, pax2a, emx1,
krox20, lhx2b, and lhx9) were instrumental to
identify major topological domains of bdnf-posi-
tive cells in the pallium, hypothalamus, posterior
tuberculum, and optic tectum (De Felice et al.
2014; Sahu et al. 2019). However, the expression
of bdnf in the 5 days post fertilization (dpf)
embryos likely depended upon the strain, as it
was higher in the larvae of AB strain compared
to larvae of the strain Tubingen long fin (van den
Bos et al. 2017). De Felice et al. (2014) also
identified the relative timing of BDNF transcrip-
tion in the eye and optic tectum, hypothesizing an
involvement of BDNF in a mechanism for coor-
dinated development of the retinotectal system. A
few years later, Hall and Tropepe (2018)
demonstrated the role of BDNF in a zebrafish
model of in vivo visual experience-dependent
neuronal survival. The authors found that
restricting the visual experience, by exposing
embryo at dim light or treating larvae with a
glutamate antagonist, reduced BDNF expression
in the periventricular gray zone. However,
supplementing larvae with exogenous BDNF
from the early dim light treatment prevented
subsequent neuronal loss in the following days,
suggesting that adaptive neurogenic growth in the
zebrafish optic tectum exhibits a similar mecha-
nism of sensory input-mediated neuronal sur-
vival, via neural activity-dependent BDNF
production (Hall and Tropepe 2018).
In the larval (7 dpf) and adult stages, bdnf is
widely expressed throughout the whole brain of
zebrafish. It is mainly localized in the forebrain
(dorsal telencephalon, preoptic area, dorsal thala-
mus, posterior tuberculum, hypothalamus,
synencephalon), optic tectum, cerebellum, and
medulla oblongata (Gatta et al. 2016; Cacialli
et al. 2016; Sahu et al. 2019), in regions that are
homolog to bdnf-expressing areas in the mamma-
lian brain. Most remarkably, when investigating
the cell phenotype expressing bdnf, transcripts
were observed in mature neurons (MAP2 and
298 P. de Girolamo and L. D’Angelo

Table 20.1 Summary of the brain localization of the different neurotrophins and Trk receptors in Danio rerio,
N. furzeri, and Xiphophorus reported in embryonic and adult stages
Nothobranchius
Danio rerio furzeri Xiphophorus
Embryo Adult Adult Embryo Adult
bdnf mRNA Telencephalon/ Telencephalon/ Telencephalon/ N/A N/A
diencephalon diencephalon diencephalon
Optic tectum Optic tectum Optic tectum
Cerebellum and Cerebellum and Cerebellum and
medulla oblongata medulla oblongata medulla oblongata
Protein N/A Cerebellum Telencephalon/ N/A N/A
diencephalon
Mesencephalon/
optic tectum
Cerebellum and
medulla oblongata
trkB mRNA Telencephalon/ N/A N/A N/A
diencephalon
Optic tectum
Cerebellum and
medulla oblongata
Protein N/A Cerebellum Telencephalon/ N/A N/A
diencephalon
Cerebellum
ngf mRNA Optic tectum Telencephalon/ Telencephalon/ N/A N/A
diencephalon diencephalon
Optic tectum Mesencephalon/
optic tectum
Cerebellum and Cerebellum and
medulla oblongata medulla oblongata
Protein N/A Cerebellum Telencephalon/ N/A N/A
diencephalon
Mesencephalon/
optic tectum
Cerebellum and
medulla oblongata
nt-6 mRNA N/A N/A Telencephalon/ Cerebellum Telencephalon/
diencephalon diencephalon
Mesencephalon/ Mesencephalon/
optic tectum optic tectum
Cerebellum and Cerebellum and
medulla oblongata medulla oblongata
Protein N/A N/A N/A N/A N/A
trkA mRNA Hindbrain N/A Telencephalon/ N/A N/A
diencephalon
Cerebellum and
medulla oblongata
Protein N/A Cerebellum Telencephalon/ N/A N/A
diencephalon
nt-3 mRNA Telencephalon/ N/A N/A N/A N/A
diencephalon
Optic tectum
Cerebellum and
medulla oblongata
Protein N/A N/A N/A N/A
(continued)
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art 299

Table 20.1 (continued)

Nothobranchius
Danio rerio furzeri Xiphophorus
Embryo Adult Adult Embryo Adult
trkC mRNA Diencephalon N/A N/A N/A N/A
Hindbrain
Protein N/A Cerebellum Telencephalon/ N/A N/A
diencephalon
nt-4 mRNA N/A N/A Telencephalon/ N/A N/A
diencephalon
Cerebellum and
medulla oblongata
Protein N/A N/A N/A N/A N/A
N/A: not available in literature

acetylated-tubulin markers) and never expressed
by radial glial cells (aromatase B marker) or
proliferating cells (PCNA marker) (Cacialli et al.
2016). Similar to zebrafish, the identification and
characterization of the cDNA fragment encoding
the BDNF protein and the localization of BDNF
mRNA and protein have been investigated in the
whole brain of adult brain of N. furzeri (D’Angelo
et al. 2014b). For protein distribution, a sensitive
immunohistochemical technique, along with
highly specific affinity-purified antibodies to
BDNF, was employed. Both BDNF mRNA and
protein were detected in neurons and identifiable
glial cells. Interestingly, both in zebrafish and
N. furzeri, BDNF was also localized in the brain
areas involved in adult neurogenesis, suggesting a
specific role for this neurotrophic factor in
controlling cell proliferation. Studies that are
more recent have contributed to reinforce the
hypothesis that bdnf/trkb system is highly
involved in the neurogenic as well as reparative
abilities of adult fish brain (Lucini et al. 2018). An
experimental model of injured telencephalon in
the adult zebrafish has documented that bdnf
levels increased significantly the first day post-
lesion in mature neurons around the lesion area,
and then sharply decreased from the fourth day
post-lesion onward. The opposite regulation was
reported in the contralateral uninjured telence-
phalic hemisphere: few mature neurons expressed
bdnf 1 day post-lesion, and then they progres-
sively and significantly increased until 15 days

Fig. 20.1 Schematic

localization of the different
neurotrophins and Trk
receptors in the adult brain
of N. furzeri
300
post-lesion (Cacialli et al. 2018). The expression
of bdnf, however, was not seen in proliferating
cells (PCNA), and notably the timing of expres-
sion peak of pcna occurred later than bdnf peak
(Cacialli et al. 2018).
The role of bdnf in brain regenerative ability of
zebrafish has been also indirectly demonstrated
by using ANA-12, an antagonist of Trkb, the
BDNF-specific tyrosine kinase receptor (Anand
and Mondal 2018). Intraperitoneally
pre-treatment of ANA-12 before telencephalic
stab injury reduced significantly the expression
levels of PCNA over different telencephalic
areas (Anand and Mondal 2018). Sato et al.
(2017) have proposed another evidence of
involvement of bdnf/trkB system in brain regen-
eration by using a different experimental model,
the optic nerve crush. In this experimental para-
digm, the proliferative neuroepithelial-like neural
stem cells in the optic tectum decreased or
increased proportionally to the projections of the
optic nerves from adult zebrafish retinas.
After crush, while bdnf expression decreased
in the optic tectum in the operation side, trkb was
expressed in both sides after nerve crush (Sato
et al. 2017) without significant differences. Treat-
ment with 7,8-DHF, a Trkb agonist, increased cell
proliferation in the mitotic region of the optic
tectum. This confirmed that the lesion-induced
decrease of neuroepithelial-like neural stem cell
proliferation in the optic tectum was rescued by
TrkB signal activation (Sato et al. 2017).
Concerning trkB expression, the two paralogs
(trkB1 and trkB2) were expressed very early in
the somitogenesis and brain development (Martin
et al. 1995; Nittoli et al. 2018). trkB1 occurred in
several neurons in the posterior telencephalon,
hypothalamus, ventral thalamus, ventral tegmen-
tum, ventral hindbrain, cranial ganglia, and spinal
cord at 24 hpf (Nittoli et al. 2018). trkB2 was
expressed in the posterior telencephalon, ventro-
rostral thalamus, ventral tegmentum, and ventral
hindbrain at 24 hpf. At 96 hpf the expression
increased remarkably over the entire brain (Nittoli
et al. 2018). According to Nittoli and colleagues,
the pattern of expression of trkB1 was quite
overlapping to bdnf, and both were co-expressed
in cranial nerves. Sahu et al. (2019) detected both
P. de Girolamo and L. D’Angelo
trkB1 and trkB2 expression in the peripheral sym-
pathetic ganglia at 5 dpf and, only at 6 dpf,
observed staining in the brain, with the isoform
trkB2 more abundantly distributed unlike trkB1.
Thus, trkB2 expression started early and
corresponded to bdnf expression pattern,
suggesting that trkB2 is the key receptor for
BDNF in the zebrafish brain (Sahu et al. 2019).
The central role of bdnf/trkb2 during develop-
ment was also investigated by a double approach,
including a mutant and morphant zebrafish model
for trkB2, to unravel the function of this receptor
in the maintenance of the two major aminergic
systems (serotonin and dopamine) (Sahu et al.
2019). In both experimental models, specific
subsets of the dopaminergic and serotonergic
neuronal populations reduced drastically with
consequences at behavioral level. Overexpression
of the full-length trkB2 mRNA reversed the
effect. Overall, these results confirmed that, also
in zebrafish, the appropriate development and
maintenance of dopamine and serotonin neuronal
populations rely on the bdnf/trkB system (Sahu
et al. 2019).
20.2.4 ngf and nt-6/trkA
The genomic organization and transcript structure
of ngf and nt-6 in the teleost zebrafish share a high
similarity with the mouse ngf and suggest that
teleost NT-6 has evolved from a common ances-
tor after a single fish-specific duplication of NGF
(Dethleffsen et al. 2003). Furthermore, NGF
proteins in zebrafish and turquoise killifish were
reported in mature and two precursor forms
(D’Angelo et al. 2014a; Gatta et al. 2016; Cacialli
et al. 2019).
In the developing brain of zebrafish, first posi-
tive ngf neurons appeared at 24 hpf in the optic
tectum, and later the expression reduced and was
manly observed in the pharyngeal arches (Nittoli
et al. 2018). In the adult brain, both NGF mRNA
and protein were detected in the perikaryon of
mature neurons in all brain areas, including
Purkinje cells of the valvula of cerebellum
(Gatta et al. 2016), whereas only pro-NGF protein
was distributed in the neuronal projections
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
(Cacialli et al. 2019). The majority of
NGF-positive cells were indeed mature neurons
(MAP2 positive). NGF was not distributed in
radial glial cells (aromatase B cells) nor in
proliferating cells (PCNA marker), although
PCNA and aromatase B cells were closely
intermingled with NGF cells. Therefore, it
seemed that mature neurons in the zebrafish
brain were expressing NGF mRNA and storing
pro-NGF (Cacialli et al. 2019).
Consistently with the observations in
zebrafish, in the teleost fish, N. furzeri NGF was
morphologically detected in neurons widespread
throughout all brain regions. On the other hand,
only some glial cells lining the mesencephalic
and rhombencephalic ventricles expressed NGF
(D’Angelo et al. 2014a).
The expression of fish-specific neurotrophin
nt-6 appeared in the developing brain of zebrafish
at 16 hpf in two clusters of cells adjacent to the
anterior and posterior of the inner ear primordium
and persisted until 48 hpf in some otic vesicle
patches. After, expression seemed to be lost
(Nittoli et al. 2018). In the brain of N. furzeri,
nt-6 expression persisted during adult as well as
old stages. Remarkably, the expression levels
were comparable between young and old animals,
and nt-6 was discretely expressed in mature
neurons of all brain regions (Leggieri et al.
2019). Similarly to ngf, the cellular phenotype
of nt-6 expression was represented by mature
neurons (MAP2 marker) but not by developing
neurons or glial cells (Leggieri et al. 2019). NT-6
mRNA was documented in the valvula of cere-
bellum during the developing stages of
Xiphophorus, a phylogenetically close fish spe-
cies, and the expression was confirmed in the
fully adult brain (Götz et al. 1994).
In spite of the teleost-specific duplication, the
genomes of fish contain only one trkA gene. Dur-
ing development of zebrafish, the expression of
trkA appeared at 24 hpf in two domains of cranial
nerve ganglia flanking the hindbrain, and in the
spinal cord (Nittoli et al. 2018). At 6 dpf, ntrkA
was expressed in the trigeminal ganglion and in
the rostral hindbrain (Martin et al. 1995). In the
adult brain, the TrkA protein has been reported in
the cerebellum of zebrafish (Gatta et al. 2016) and
301
in the forebrain of N. furzeri (D’Angelo et al.
2012). In addition, in the African turquoise killi-
fish, trkA appeared more expressed than ngfr in
young and old animals, without a significant
age-dependent change. trkA and ngfr receptors
were not expressed in cholinergic neurons, as
widely demonstrated in mammals (de Girolamo
et al. 2020).
20.2.5 nt-3/trkC
Phylogenetic analysis based on nucleotides and
mature amino acid sequences of NT-3 confirmed
that this neurotrophin evolved in teleost fish from
the common ancestor of NGF and NT-6. In
zebrafish, the NT-3 protein was expressed as
mature (Auer et al. 2015) and pro-NT-3 (Gatta
et al. 2016).
nt-3 expression appeared at 12 hpf in
zebrafish, and was distributed in different devel-
oping organs, including the sense organs. How-
ever, at 72 hpf a discrete bilateral group of
positive neuronal cell types was observed in the
brain and cranial nerve region (Nittoli et al.
2018). It has been elegantly demonstrated that in
larvae mutated for Kif5A, characterized by a dis-
ruption of presynaptic activity, there is an
upregulation of nt-3 in postsynaptic tectal cells.
This, in turn, promoted exuberant branching of
retinal axons by signaling through the TrkC
receptor. The increased production of nt-3 was
also observed when neuronal activity was
blocked, for example, by toxins. The lack of
neuronal activity in retinal axons therefore
seemed to increase the production of nt-3, which
then stimulated axonal branching (Auer et al.
2015). In the adult brain of zebrafish, NT-3 was
distributed in the majority of Purkinje cells of
valvular and body of cerebellum, less in the
molecular and granular layers (Gatta et al. 2016).
The two paralogs of trkC, trkC1, and trkC2
displayed a different expression. trkC1 expres-
sion started at 1 dpf and was localized in cells of
telencephalon including also diencephalon, pitui-
tary gland, hindbrain, cranial ganglia, otic vesicle,
and spinal cord (Martin et al. 1995; Auer et al.
2015; Nittoli et al. 2018). The localization in the
302
trigeminal ganglia is still debated (Pan et al. 2012;
Nittoli et al. 2018). Between 48 and 96 hpf, trkC1
was expressed diffusely in the brain (Nittoli et al.
2018). trkC2 had a more restricted expression at
24 hpf, specifically in a small group of neurons in
the telencephalon and midbrain, whereas in the
hindbrain it appeared at 48 hpf (Nittoli et al.
2018). In the adult brain of zebrafish, a faint
immunoreactivity to TrkC protein has been
reported in the molecular layer of cerebellum
(Gatta et al. 2016), whereas in the adult brain of
N. furzeri, TrkC positivity has been observed in
the most aboral region of the telencephalon,
magnocellular preoptic nucleus, and few neurons
of hypothalamus (D’Angelo et al. 2012).
20.2.6 nt-4/5
nt-4/5 is the less studied neurotrophin in fish. It
was expressed during zebrafish development
(Auer et al. 2015; Nittoli et al. 2018), and the
first expression appeared during somitogenesis,
at 12 hpf. With the exception of a signal in the
cranial nerves at 24 hpf, this neurotrophin seemed
more localized in non-neuronal tissues during
development (Nittoli et al. 2018). In the adult
brain, nt-4/5 was studied only in N. furzeri,
where it was observed in neurons and glial cells
of the forebrain and hindbrain, and very scarcely
in the midbrain (D’Angelo et al. 2016b).
20.3 Conclusion and Future
Perspectives
The last two decades have seen an increasing
interest in the use of teleost fish, mainly zebrafish
and the African turquoise killifish, as valuable
model organisms to explore the pleiotropic action
of neurotrophins in the CNS during development,
adulthood, and aging. This is corroborated by
several observations:
1. The CNS of fish and mammals displays
several homologies, despite some specific
morphological features and a less complex
P. de Girolamo and L. D’Angelo
organization in fish. This is fundamental to
translate novel insights to mammals and helpful
to elucidate complex and unknown mechanisms
of the neurobiology of disease and aging.
2. Neurotrophins are evolutionary well-
conserved molecules, in terms of nucleotides
and amino acid sequences. This is particularly
true for BDNF.
3. All neurotrophins and their specific receptors
are expressed at the early stages of brain devel-
opment and in adult brain. There are
differences in the spatiotemporal distribution
of all neurotrophins in zebrafish and N. furzeri.
However, it seems that in both species, the cell
phenotype expressing neurotrophins is
represented by mature neurons.
4. In zebrafish, the BDNF/TrkB system regulates
the appropriate development of visual system
and participates to the regenerative ability of
the brain after traumatic injury.
More studies are needed to better explore the
functional role also of other members of
neurotrophin family. Data available on NGF,
NT-3, NT-4/5, and NT-6 in the brain are mainly
limited to the neuroanatomical expression,
whereas very little is known about their functional
role. Future studies are thus mandatory to better
characterize their involvement during develop-
ment and neuronal maintenance in adult brain.
The great variety of morphophysiological
features of fish species combined to the growing
availability of genetic and imaging assays can
help to identify neural pathways and circuits
implicated in neurotrophin-related physiology
and pathological states in vertebrates. Remark-
ably, fish offer the opportunity to dissect and
better comprehend the regulatory mechanisms of
neurotrophins in the brain during developmental
and adult stages, as well as to conduct longitudi-
nal studies over aging in short time (Baumgart
et al. 2016). In addition, pharmacological and
environmental interventions can help to comple-
ment analyses and may have implications for
translational neuroscience research and modeling
CNS disorders (Stewart et al. 2014).
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art 303

References telencephalon of adult zebrafish. J Comp Neurol 526

(4):569–582
Cacialli P, Gatta C, D’Angelo L, Leggieri A, Palladino A,
Albadri S, Del Bene F, Revenu C (2017) Genome editing
de Girolamo P, Pellegrini E, Lucini C (2019) Nerve
using CRISPR/Cas9-based knock-in approaches in
growth factor is expressed and stored in central
zebrafish. Methods 121–122:77–85
neurons of adult zebrafish. J Anat 235(1):167–179
Albertson RC, Cresko W, Detrich HW 3rd, Postlethwait
Catchen JM, Braasch I, Postlethwait JH (2011) Conserved
JH (2009) Evolutionary mutant models for human
synteny and the zebrafish genome. Methods Cell Biol
disease. Trends Genet 25(2):74–81
104:259–285
Aleström P, D’Angelo L, Midtlyng PJ, Schorderet DF,
Cellerino A, Valenzano DR, Reichard M (2016) From the
Schulte-Merker S, Sohm F, Warner S (2020)
bush to the bench: the annual Nothobranchius fishes as
Zebrafish: Housing and husbandry recommendations.
a new model system in biology. Biol Rev 91:511–533
Lab Anim 54(3):213–224. https://doi.org/10.1177/
Chiu CH, Dewar K, Wagner GP, Takahashi K, Ruddle F,
0023677219869037
Ledje C, Bartsch P, Scemama JL, Stellwag E, Fried C,
Anand SK, Mondal AC (2018) TrkB receptor antagonism
Prohaska SJ, Stadler PF, Amemiya CT (2004) Bichir
inhibits stab injury induced proliferative response in
HoxA cluster sequence reveals surprising trends in
adult zebrafish (Danio rerio) brain. Neurosci Lett
ray-finned fish genomic evolution. Genome Res 14
672:28–33
(1):11–17
Auer TO, Xiao T, Bercier V, Gebhardt C, Duroure K,
Cornet C, Di Donato V, Terriente J (2018) Combining
Concordet JP, Wyart C, Suster M, Kawakami K,
zebrafish and CRISPR/Cas9: toward a more efficient
Wittbrodt J, Baier H, Del Bene F (2015) Deletion of
drug discovery pipeline. Front Pharmacol 9:703
a kinesin I motor unmasks a mechanism of homeostatic
Crollius HR (2006) The tetraodon genome. Genome Dyn
branching control by neurotrophin-3. elife 15:4. https://
2:154–164
doi.org/10.7554/eLife.0506
D’Angelo L (2013) Brain atlas of an emerging teleostean
Baraban M, Mensch S, Lyons DA (2016) Adaptive
model: Nothobranchius furzeri. Anat Rec (Hoboken)
myelination from fish to man. Brain Res 1641
296(4):681–691
(Pt A):149–161
D’Angelo L, de Girolamo P, Cellerino A, Tozzini ET,
Baumgart M, Groth M, Priebe S, Savino A, Testa G,
Castaldo L, Lucini C (2012) Neurotrophin Trk
Dix A, Ripa R, Spallotta F, Gaetano C, Ori M,
receptors in the brain of a teleost fish, Nothobranchius
Terzibasi Tozzini E, Guthke R, Platzer M, Cellerino
furzeri. Microsc Res Tech 75(1):81–88
A (2014) RNA-seq of the aging brain in the short-lived
D’Angelo L, Castaldo L, Cellerino A et al (2014a) Nerve
fish N. furzeri - conserved pathways and novel genes
growth factor in the adult brain of a teleostean model
associated with neurogenesis. Aging Cell 13:965–974
for aging research: Nothobranchius furzeri. Ann Anat
Baumgart M, Priebe S, Groth M, Hartmann N, Menzel U,
196:183–191
Pandolfini L, Koch P, Felder M, Ristow M, Englert C,
D’Angelo L, De Girolamo P, Lucini C, Terzibasi ET,
Guthke R, Platzer M, Cellerino A (2016) Longitudinal
Baumgart M, Castaldo L, Cellerino A (2014b) Brain-
RNA-Seq analysis of vertebrate aging identifies mito-
derived neurotrophic factor: mRNA expression and
chondrial complex I as a small-molecule-sensitive
protein distribution in the brain of the teleost
modifier of lifespan. Cell Syst 2(2):122–132
Nothobranchius furzeri. J Comp Neurol 522
Benito-Gutiérrez E, Garcia-Fernàndez J, Comella JX
(5):1004–1030
(2006) Origin and evolution of the Trk family of
D’Angelo L, Lossi L, Merighi A, de Girolamo P (2016a)
neurotrophic receptors. Mol Cell Neurosci 31
Anatomical features for the adequate choice of experi-
(2):179–192
mental animal models in biomedicine: I. Fishes Ann
Broglio C, Gomez A, Duran E et al (2005) Hallmarks of a
Anat 205:75–84
common forebrain vertebrate plan: specialized pallial
D’Angelo L, Avallone L, Cellerino A, de Girolamo P,
areas for spatial, temporal and emotional memory in
Paolucci M, Varricchio E, Lucini C (2016b)
actinopterygian fish. Brain Res Bull 66(4–6):277–281
Neurotrophin-4 in the brain of adult Nothobranchius
Brosamle C, Halpern ME (2009) Nogo-Nogo receptor
furzeri. Ann Anat 207:47–54
signalling in PNS axon outgrowth and pathfinding.
Davis RE, Northcutt RG (eds) (1983) Fish neurobiology,
Mol Cell Neurosci 40:401–409
vol II. The University of Michigan Press, Ann Arbor
Cacialli P, Gueguen MM, Coumailleau P, D’Angelo L,
De Felice E, Porreca I, Alleva E, De Girolamo P,
Kah O, Lucini C, Pellegrini E (2016) BDNF expres-
Ambrosino C, Ciriaco E, Germanà A, Sordino P
sion in larval and adult zebrafish brain: distribution and
(2014) Localization of BDNF expression in the devel-
cell identification. PLoS One 11(6):e0158057
oping brain of zebrafish. J Anat 224(5):564–574
Cacialli P, D’Angelo L, Kah O, Coumailleau P, Gueguen
de Girolamo P, Leggieri A, Palladino A, Lucini C,
MM, Pellegrini E, Lucini C (2018) Neuronal expres-
Attanasio C, D’Angelo L (2020) Cholinergic system
sion of brain derived neurotrophic factor in the injured
and NGF receptors: insights from the brain of the
304
short-lived fish Nothobranchius furzeri. Brain Sci 10
(6):394
Dethleffsen K, Heinrich G, Lauth M, Knapik EW, Meyer
M (2003) Insert-containing neurotrophins in teleost
fish and their relationship to nerve growth factor. Mol
Cell Neurosci 24(2):380–394
Detrich HW, Yergeau DA (2004) Comparative genomics
in erythropoietic gene discovery: synergisms between
the Antarctic icefishes and the zebrafish. Methods Cell
Biol 77:475–503
Fernandez AA, Bowser PR (2010) Selection for a domi-
nant oncogene and large male size as a risk factor for
melanoma in the Xiphophorus animal model. Mol Ecol
19(15):3114–3123
Finger TE (1983) Organization of the teleost cerebellum.
In: Davis RE, Northcutt RG (eds) Fish neurobiology,
vol I. The University of Michigan Press, Ann Arbor, pp
261–284
Folgueira M, Bayley P, Navratilova P, Becker TS, Wilson
SW, Clarke JD (2015) Morphogenesis underlying the
development of the everted teleost telencephalon. Neu-
ral Dev 10:22
Force A, Lynch M, Pickett FB, Amores A, Yan YL,
Postlethwait J (1999) Preservation of duplicate genes
by complementary, degenerative mutations. Genetics
151:1531–1545
Fredriksson R, Schioth HB (2005) The repertoire of G-
protein-coupled receptors in fully sequenced genomes.
Mol Pharmacol 67:1414–1425
Ganz J, Brand M. 2016. Adult neurogenesis in fish. Cold
Spring Harb Perspect Biol. 8(7). Pii: a019018
Ganz J, Kaslin J, Freudenreich D, Machate A, Geffarth M,
Brand M (2012) Subdivisions of the adult zebrafish
subpallium by molecular marker analysis. J Comp
Neurol 520(3):633–655
Ganz J, Kroehne V, Freudenreich D, Machate A,
Geffarth M, Braasch I, Kaslin J, Brand M (2015)
Subdivisions of the adult zebrafish pallium based on
molecular marker analysis. F1000Res 3:308
Gatta C, Altamura G, Avallone L, Castaldo L,
Corteggio A, D’Angelo L, de Girolamo P, Lucini C
(2016) Neurotrophins and their Trk-receptors in the
cerebellum of zebrafish. J Morphol 277(6):725–736
Gerlai R (2016) Learning and memory in zebrafish (Danio
rerio). Methods Cell Biol 134:551–586
Getz AM, Janes TA, Visser F, Zaidi W, Syed NI (2020)
Neurotrophic factors and target-specific retrograde sig-
naling interactions define the specificity of classical
and neuropeptide cotransmitter release at identified
Lymnaea synapses. Sci Rep 10(1):13526
Glenn TD, Talbot WS (2013) Signals regulating
myelination in peripheral nerves and the Schwann
cell response to injury. Curr Opin Neurobiol
23:1041–1048
Götz R, Köster R, Winkler C, Raulf F, Lottspeich F,
Schartl M, Thoenen H (1994) Neurotrophin-6 is a
new member of the nerve growth factor family. Nature
372(6503):266–269
P. de Girolamo and L. D’Angelo
Grupp L, Wolburg H, Mack AF (2010) Astroglial
structures in the zebrafish brain. J Comp Neurol
518:4277–4287
Hall ZJ, Tropepe V (2018) Visual experience facilitates
BDNF-dependent adaptive recruitment of new neurons
in the postembryonic optic tectum. J Neurosci 38
(8):2000–2014
Hallböök F (1999) Evolution of the vertebrate
neurotrophin and Trk receptor gene families. Curr
Opin Neurobiol 9:616–621
Hallböök F, Wilson K, Thorndyke M, Olinski RP (2006)
Formation and evolution of the chordate neurotrophin
and trk receptor genes. Brain Behav Evol 68:133–144
Han HW, Chou CM, Chu CY, Cheng CH, Yang CH, Hung
CC, Hwang PP, Lee SJ, Liao YF, Huang CJ (2014)
The Nogo-C2/Nogo receptor complex regulates the
morphogenesis of zebrafish lateral line primordium
through modulating the expression of dkk1b, a Wnt
signal inhibitor. PLoS One 9(1):e86345
Harel I, Valenzano DR, Brunet A (2016) Efficient genome
engineering approaches for the short-lived African tur-
quoise killifish. Nat Protoc 11:2010–2028
Hashimoto M, Heinrich G (1997) Brain-derived
neurotrophic factor gene expression in the developing
zebrafish. Int J Dev Neurosci 15(8):983–997
Heinrich G, Lum T (2000) Fish neurotrophins and Trk
receptors. Int J Dev Neurosci 18(1):1–27
Heinrich G, Pagtakhan CJ (2004) Both 50 and 30 flanks
regulate zebrafish brain-derived neurotrophic factor
gene expression. BMC Neurosci 5:19
Herbomel P, Thisse B, Thisse C (2001) Zebrafish early
macrophages colonize cephalic mesenchyme and
developing brain, retina, and epidermis through a
M-CSF receptor-dependent invasive process. Dev
Biol 238:274–288
Holzschuh J, Ryu S, Aberger F, Driever W (2001) Dopa-
mine transporter expression distinguishes dopaminer-
gic neurons from other catecholaminergic neurons in
the developing zebrafish embryo. Mech Dev
101:237–243
Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C,
Muffato M, Collins JE, Humphray S, McLaren K,
Matthews L, McLaren S, Sealy I, Caccamo M,
Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ,
White S, Chow W, Kilian B, Quintais LT, Guerra-
Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH,
Eyre T, Redmond S, Banerjee R, Chi J, Fu B,
Langley E, Maguire SF, Laird GK, Lloyd D,
Kenyon E, Donaldson S, Sehra H, Almeida-King J,
Loveland J, Trevanion S, Jones M, Quail M, Willey D,
Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J,
Clee C, Oliver K, Clark R, Riddle C, Elliot D,
Threadgold G, Harden G, Ware D, Begum S,
Mortimore B, Kerry G, Heath P, Phillimore B,
Tracey A, Corby N, Dunn M, Johnson C, Wood J,
Clark S, Pelan S, Griffiths G, Smith M, Glithero R,
Howden P, Barker N, Lloyd C, Stevens C, Harley J,
Holt K, Panagiotidis G, Lovell J, Beasley H,
Henderson C, Gordon D, Auger K, Wright D,
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
Collins J, Raisen C, Dyer L, Leung K, Robertson L,
Ambridge K, Leongamornlert D, McGuire S,
Gilderthorp R, Griffiths C, Manthravadi D, Nichol S,
Barker G, Whitehead S, Kay M, Brown J, Murnane C,
Gray E, Humphries M, Sycamore N, Barker D,
Saunders D, Wallis J, Babbage A, Hammond S,
Mashreghi-Mohammadi M, Barr L, Martin S,
Wray P, Ellington A, Matthews N, Ellwood M,
Woodmansey R, Clark G, Cooper J, Tromans A,
Grafham D, Skuce C, Pandian R, Andrews R,
Harrison E, Kimberley A, Garnett J, Fosker N,
Hall R, Garner P, Kelly D, Bird C, Palmer S,
Gehring I, Berger A, Dooley CM, Ersan-Ürün Z,
Eser C, Geiger H, Geisler M, Karotki L, Kirn A,
Konantz J, Konantz M, Oberländer M, Rudolph-
Geiger S, Teucke M, Lanz C, Raddatz G,
Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C,
Yang F, Schuster SC, Carter NP, Harrow J, Ning Z,
Herrero J, Searle SM, Enright A, Geisler R, Plasterk
RH, Lee C, Westerfield M, de Jong PJ, Zon LI,
Postlethwait JH, Nüsslein-Volhard C, Hubbard TJ,
Roest Crollius H, Rogers J, Stemple DL (2013) The
zebrafish reference genome sequence and its relation-
ship to the human genome. Nature 496(7446):498–503
Hu CK, Brunet A (2018) The African turquoise killifish: a
research organism to study vertebrate aging and dia-
pause. Aging Cell 17:e12757
Hu Q, Gao F, Tian W, Ruteshouser EC, Wang Y, Lazar A,
Stewart J, Strong LC, Behringer RR, Huff V (2011)
Wt1 ablation and Igf2 upregulation in mice result in
Wilms tumors with elevated ERK1/2 phosphorylation.
J Clin Invest 121:174–183
Imperatore R, D’Angelo L, Safari O, Motlagh HA,
Piscitelli F, de Girolamo P, Cristino L, Varricchio E,
di Marzo V, Paolucci M (2018) Overlapping distribu-
tion of orexin and endocannabinoid receptors and their
functional interaction in the brain of adult zebrafish.
Front Neuroanat 12:62
Jaaro H, Beck G, Conticello SG, Fainzilber M (2001)
Evolving better brains: a need for neurotrophins?
Trends Neurosci 24:79–85
Jeffery WR (2009) Chapter 8. Evolution and development
in the cavefish Astyanax. Curr Top Dev Biol
86:191–221
Jessen KR, Mirsky R (2005) The origin and development
of glial cells in peripheral nerves. Nat Rev Neurosci
6:671–682
Kaslin J, Ganz J, Brand M (2008) Proliferation,
neurogenesis and regeneration in the non-mammalian
vertebrate brain. Philos Trans R Soc Lond Ser B Biol
Sci 363:101–122
Kotrschal K, Van Staaden MJ, Huber R (1998) Fish
brains: evolution and environmental relationships.
Fish Biol Fish 8:373–408
Lai KO, Fu WY, Ip FC, Ip NY (1998) Cloning and
expression of a novel neurotrophin, NT-7, from carp.
Mol Cell Neurosci 11(1–2):64–76
305
Lanave C, Colangelo AM, Saccone C, Alberghina L
(2007) Molecular evolution of the neurotrophin family
members and their Trk receptors. Gene 394:1–12
Lee S, Park JY, Lee WH, Kim H, Park HC, Mori K, Suk K
(2009) Lipocalin-2 is an autocrine mediator of reactive
astrocytosis. J Neurosci 29:234–249
Leggieri A, Attanasio C, Palladino A, Cellerino A,
Lucini C, Paolucci M, Terzibasi Tozzini E, de
Girolamo P, D’Angelo L (2019) Identification and
expression of Neurotrophin-6 in the brain of
Nothobranchius furzeri: one more piece in
Neurotrophin research. J Clin Med 8(5):Pii: E595
Lindsey BW, Hall ZJ, Heuzé A, Joly JS, Tropepe V,
Kaslin J (2018) The role of neuro-epithelial-like and
radial-glial stem and progenitor cells in development,
plasticity, and repair. Prog Neurobiol 170:99–114
Lucini C, D’Angelo L, Cacialli P, Palladino A, de
Girolamo P (2018) BDNF, brain, and regeneration:
insights from zebrafish. Int J Mol Sci 19(10):Pii:
E3155
Lundin LG, Larhammar D, Hallböök F (2003) Numerous
groups of chromosomal regional paralogies strongly
indicate two genome doublings at the root of the
vertebrates. J Struct Funct Genom 3:53–63
Lyons DA, Talbot WS (2014) Glial cell development and
function in zebrafish. Cold Spring Harb Perspect Biol 7
(2):a020586
Lyons DA, Guy AT, Clarke JD (2003) Monitoring neural
pro-genitor fate through multiple rounds of division in
an intact vertebrate brain. Development
130:3427–3436
Mahler J, Driever W (2007) Expression of the zebrafish
intermediate neurofilament nestin in the developing
nervous system and in neural proliferation zones at
post-embryonic stages. BMC Dev Biol 7:89
Mann A, Bhatia S (2019 Sep 16) Zebrafish: A powerful
model for understanding the functional relevance of
noncoding region mutations in human genetic diseases.
Biomedicines 7(3):Pii: E71
Martin SC, Marazzi G, Sandell JH, Heinrich G (1995) Five
Trk receptors in the zebrafish. Dev Biol 169
(2):745–758
Matsui H, Kenmochi N, Namikawa K (2019) Age- and
α-Synuclein-dependent degeneration of dopamine and
noradrenaline neurons in the annual killifish
Nothobranchius furzeri. Cell Rep 26(7):1727–1733.e6
Mazzolini J, Le Clerc S, Morisse G, Coulonges C, Kuil
LE, van Ham TJ, Zagury JF, Sieger D (2019) Gene
expression profiling reveals a conserved microglia sig-
nature in larval zebrafish. Glia 68(2):298–315
McKeown KA, Moreno R, Hall VL, Ribera AB, Downes
GB (2012) Disruption of Eaat2b, a glutamate trans-
porter, results in abnormal motor behaviors in devel-
oping zebrafish. Dev Biol 362:162–171
Meyer A, Schartl M (1999) Gene and genome duplications
in vertebrates: the one-to-four (to-eight in fish) rule
and the evolution of novel gene functions. Curr Opin
Cell Biol 11:699–704
306
Mueller T (2011) The conserved bauplan of the teleostean
telencephalon. Brain Behav Evol 78(4):259–260
März M, Chapouton P, Diotel N, Vaillant C, Hesl B,
Takamiya M, Lam CS, Kah O, Bally-Cuif L, Strähle
U (2010) Heterogeneity in progenitor cell subtypes in
the ventricular zone of the zebrafish adult telencepha-
lon. Glia 58:870–888
Neve LD, Savage AA, Koke JR, García DM (2012)
Activating transcription factor 3 and reactive astrocytes
following optic nerve injury in zebrafish. Comp
Biochem Physiol C Toxicol Pharmacol 155:213–218
Nieuwenhuys R (2009) The structural organization of the
forebrain: a commentary on the papers presented at the
20th annual Karger workshop ‘Forebrain evolution in
fishes’. Brain Behav Evol 74(1):77–85
Nieuwenhuys R, Meek J (1990) The telencephalon of
Actinopterygian fishes. In: Jones EG, Peters A (eds)
Comparative structure and evolution of the cerebral
cortex. Plenum Press, New York, pp 31–73
Nilsson AS, Fainzilber M, Falck P, Ibáñez CF (1998)
Neurotrophin-7: a novel member of the neurotrophin
family from the zebrafish. FEBS Lett 424(3):285–290
Nittoli V, Sepe RM, Coppola U, D’Agostino Y, De
Felice E, Palladino A, Vassalli QA, Locascio A,
Ristoratore F, Spagnuolo A, D’Aniello S, Sordino P
(2018) A comprehensive analysis of neurotrophins and
neurotrophin tyrosine kinase receptors expression dur-
ing development of zebrafish. J Comp Neurol 526
(6):1057–1072
Northmore DPM, Williams B, Vanegas H (1983) The
teleostean torus longitudinalis: responses related to
eye movements, visuotopic mapping and functional
relations with the optic tectum. J Comp Physiol A
150:39–50
Pan YA, Choy M, Prober DA, Schier AF (2012) Robo2
determines subtype-specific axonal projections of tri-
geminal sensory neurons. Development 139
(3):591–600
Parichy DM (2015) Advancing biology through a deeper
understanding of zebrafish ecology and evolution. elife
25:4
Petzold A, Reichwald K, Groth M, Taudien S,
Hartmann N, Priebe S, Shagin D, Englert C, Platzer
M (2013) The transcript catalogue of the short-lived
fish Nothobranchius furzeri provides insights into age-
dependent changes of mRNA levels. BMC Genomics
14:185
Philippe C, Hautekiet P, Grégoir AF, Thoré ESJ,
Pinceel T, Stoks R, Brendonck L, Boeck G (2018)
Combined effects of cadmium exposure and tempera-
ture on the annual killifish (Nothobranchius furzeri).
Environ Toxicol Chem 37:2361–2371
Pinzón-Olejua A, Welte C, Abdesselem H, Málaga-
Trillo E, Stuermer CA (2014) Essential roles of
zebrafish rtn4/Nogo paralogues in embryonic develop-
ment. Neural Dev 9:8
Popper AN, Fay RR (1993) Sound detection and
processing by fish: critical review and major research
questions. Brain, Behav. Evolution 41:14–38
P. de Girolamo and L. D’Angelo
Postlethwait JH, Yan Y-L, Gates M, Horne S, Amores A,
Brownlie A, Donovan A, Egan E, Force A, Gong Z,
Goutel C, Fritz A, Kelsh R, Knapik E, Liao E, Paw B,
Ransom D, Singer A, Thomson M, Abduljabbar TS,
Yelick P, Beier D, Joly J-S, Larhammar D, Talbot WS
et al (1998) Vertebrate genome evolution and the
zebrafish gene map. Nat Genet 18:345–349
Raphael AR, Talbot WS (2011) New insights into signal-
ing during myelination in zebrafish. Curr Top Dev Biol
97:1–19
Reichard M, Polačik M (2019). Nothobranchius furzeri, an
‘instant’ fish from an ephemeral habitat. Elife 8:Pii:
e41548
Reichwald K, Petzold A, Koch P, Downie BR,
Hartmann N, Pietsch S, Baumgart M, Chalopin D,
Felder M, Bens M, Sahm A, Szafranski K,
Taudien S, Groth M, Arisi I, Weise A, Bhatt SS,
Sharma V, Kraus JM, Schmid F, Priebe S, Liehr T,
Görlach M, Than ME, Hiller M, Kestler HA, Volff JN,
Schartl M, Cellerino A, Englert C, Platzer M (2015)
Insights into sex chromosome evolution and aging
from the genome of a short-lived fish. Cell 163
(6):1527–1538
Rodriguez F, Lopez JC, Vargas JP et al (2002) Conserva-
tion of spatial memory function in the pallial forebrain
of reptiles and ray-finned fishes. J Neurosci 22
(7):2894–2903
Sahu MP, Pazos-Boubeta Y, Pajanoja C, Rozov S,
Panula P, Castrén E (2019) Neurotrophin receptor
Ntrk2b function in the maintenance of dopamine and
serotonin neurons in zebrafish. Sci Rep 9(1):2036
Saleem S, Kannan RR (2018) Zebrafish: an emerging real-
time model system to study Alzheimer’s disease and
neurospecific drug discovery. Cell Death Discov 4:45
Sato Y, Yano H, Shimizu Y, Tanaka H, Ohshima T (2017)
Optic nerve input-dependent regulation of neural stem
cell proliferation in the optic tectum of adult zebrafish.
Dev Neurobiol 77(4):474–482
Schartl M (2014) Beyond the zebrafish: diverse fish spe-
cies for modeling human disease. Dis Model Mech 7
(2):181–192
Sieger D, Peri F (2013) Animal models for studying
microglia: the first, the popular, and the new. Glia
61:3–9
Sieger D, Moritz C, Ziegenhals T, Prykhozhij S, Peri F
(2012) Long range Ca2+ waves transmit brain-damage
signals to microglia. Dev Cell 22:1138–1148
Stewart AM, Braubach O, Spitsbergen J, Gerlai R, Kalueff
AV (2014) Zebrafish models for translational neurosci-
ence research: from tank to bedside. Trends Neurosci
37:264–278
Stewart AM, Ullmann JF, Norton WH, Parker MO,
Brennan CH, Gerlai R, Kalueff AV (2015) Molecular
psychiatry of zebrafish. Mol Psychiatry 20(1):2–17
Svahn AJ, Graeber MB, Ellett F, Lieschke GJ, Rinkwitz S,
Bennett MR, Becker TS (2013) Development of
ramified microglia from early macrophages in the
zebrafish optic tectum. Dev Neurobiol 73:60–71
20 Neurotrophins in the Brain of Teleost Fish: The State of the Art
Tay TL, Ronneberger O, Ryu S, Nitschke R, Driever W
(2011) Comprehensive catecholaminergic projectome
analysis reveals single-neuron integration of zebrafish
ascending and descending dopaminergic systems. Nat
ComTmun 2:171
Tettamanti G, Cattaneo AG, Gornati R, de Eguileor M,
Bernardini G, Binelli G (2010) Phylogenesis of
brain-derived neurotrophic factor (BDNF) in
vertebrates. Gene 450(1–2):85–93
Tozzini ET, Baumgart M, Battistoni G, Cellerino A (2012)
Adult neurogenesis in the short-lived teleost
Nothobranchius furzeri: localization of neurogenic
niches, molecular characterization and effects of
aging. Aging Cell 11(2):241–251
Valenzano DR, Benayoun BA, Singh PP, Zhang E, Etter
PD, Hu CK, Clément-Ziza M, Willemsen D, Cui R,
Harel I, Machado BE, Yee MC, Sharp SC, Bustamante
CD, Beyer A, Johnson EA, Brunet A (2015) The
African turquoise killifish genome provides insights
into evolution and genetic architecture of lifespan.
Cell 163:1539–1554
van den Bos R, Mes W, Galligani P, Heil A, Zethof J,
Flik G, Gorissen M (2017) Further characterisation of
differences between TL and AB zebrafish (Danio
rerio): gene expression, physiology and behaviour at
day 5 of the larval stage. PLoS One 12(4):e0175420
307
Wang W, Hu CK, Zeng A, Alegre D, Hu D, Gotting K,
Ortega Granillo A, Wang Y, Robb S, Schnittker R,
Zhang S, Alegre D, Li H, Ross E, Zhang N,
Brunet A, Sánchez AA (2020a) Changes in
regeneration-responsive enhancers shape regenerative
capacities in vertebrates. Science 369(6508):eaaz3090
Wang J, Li Y, Lai K, Zhong Q, Demin KA, Kalueff AV,
Song C (2020b) High-glucose/high-cholesterol diet in
zebrafish evokes diabetic and affective pathogenesis:
the role of peripheral and central inflammation,
microglia and apoptosis. Prog Neuro-
Psychopharmacol Biol Psychiatry 96:109752
Wendler S, Hartmann N, Hoppe B, Englert C (2015)
Age-dependent decline in fin regenerative capacity in
the short-lived fish Nothobranchius furzeri. Aging Cell
14:857–866
Wullimann MF (1994) The teleostean torus longitudinalis:
a short review on its structure, histochemistry, connec-
tivity, possible function and phylogeny. Europ J
Morphol 32:235–242
Wullimann MF, Mueller T (2004) Teleostean and mam-
malian forebrains contrasted: evidence from genes to
behavior. J Comp Neurol 475:143–162
Yamamoto K, Ruuskanen JO, Wullimann MF, Vernier P
(2010) Two tyrosine hydroxylase genes in vertebrates.
New dopaminergic territories revealed in the zebrafish
brain. Mol Cell Neurosci 43:394–402