Retinal Degenerative Diseases XIX

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Advances in Experimental Medicine and Biology 1415

John D. Ash · Eric Pierce · Robert E. Anderson ·

Catherine Bowes Rickman · Joe G. Hollyfield ·
Christian Grimm Editors

Retinal
Degenerative
Diseases XIX
Mechanisms and Experimental Therapy
Advances in Experimental Medicine
and Biology

Volume 1415

Series Editors
Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives
d’Aquitaine, CNRS and University of Bordeaux, Pessac Cedex, France
Haidong Dong, Departments of Urology and Immunology, Mayo Clinic
Rochester, MN, USA
Heinfried H. Radeke, Institute of Pharmacology and Toxicology, Clinic of
the Goethe University Frankfurt Main, Frankfurt am Main, Hessen,
Germany
Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical
Center, Tehran University of Medical Sciences, Tehran, Iran
Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital
Munich, Germany
Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of
Cardiovascular Sciences, School of Life Science, Shanghai University
Shanghai, China
Advances in Experimental Medicine and Biology provides a platform for
scientific contributions in the main disciplines of the biomedicine and the life
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2021 Impact Factor: 3.650 (no longer indexed in SCIE as of 2022)
John D. Ash • Eric Pierce
Robert E. Anderson
Catherine Bowes Rickman
Joe G. Hollyfield • Christian Grimm
Editors

Retinal Degenerative
Diseases XIX
Mechanisms and Experimental
Therapy
Editors
John D. Ash Eric Pierce
Department of Ophthalmology Ocular Genomics Institute
University of Pittsburgh School Department of Ophthalmology
of Medicine Massachusetts Eye and Ear Infirmary
Pittsburgh, PA, USA Harvard Medical School
Boston, MA, USA
Robert E. Anderson
Health Sciences Center Catherine Bowes Rickman
University of Oklahoma Health Department of Ophthalmology
Sciences Center Duke Medical Center
Oklahoma City, OK, USA Durham, NC, USA

Joe G. Hollyfield Christian Grimm

Department of Ophthalmology Laboratory for Retinal Cell Biology
Cleveland Clinic Lerner College Department of Ophthalmology
of Medicine University Hospital Zurich
Cleveland, OH, USA University of Zurich
Schlieren, Switzerland

ISSN 0065-2598     ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-031-27680-4    ISBN 978-3-031-27681-1 (eBook)
https://doi.org/10.1007/978-3-031-27681-1

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature
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whether the whole or part of the material is concerned, specifically the rights of translation,
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The editors are pleased dedicate this publication to the memory
of our long-time friend and colleague, Alan M. Laties. Except
for the most recent years, Alan attended each of these biennial
retinal degeneration meetings since they began in 1984. Early
on Alan recognized the importance of our attempt to provide a
continuing international platform for discussions and scientific
exchange to take place among investigators focused on retinal
degeneration research. Through his scientific leadership at the
Foundation Fighting Blindness (formerly the Retinitis
Pigmentosa Foundation), we received the first meeting grant to
partially cover some of the expenses of the RD meeting held in
San Francisco in 1988. The Foundation has provided
continuing support for each of the subsequent meetings in the
form of travel grant support for young investigators.
Born in Beverly, Massachusetts, the son of Russian immigrants,
he attended Harvard College (BA, 1954) and completed
medical school at Baylor College of Medicine (MD, 1959),
followed by a residency in ophthalmology in the Hospital of the
University of Pennsylvania (1961–63). A United States Public
Health Service Special Research Fellowship supported his
vi 

research training in the Institute of Neurological Sciences at

the University of Pennsylvania (1963–64). He joined the faculty
at the University of Pennsylvania in 1965 where he moved
through the academic ranks until retiring as Emeritus
Professor of Ophthalmology at the Perelman School of
Medicine in 2020. He held joint appointments in
Ophthalmology and Neurology where he was the Irene Heinz
Given and John LaPorte Given Research Professor and the
Harold G. Scheie Research Professor in Ophthalmology. He
served as neuro-ophthalmologist at the Hospital of the
University of Pennsylvania while pursuing basic research on
the autonomic innervation of the eye, eye growth, and
therapeutic approaches to eye diseases. He has published 140
original research papers, 30 review articles, and presented
numerous invited lectures at major university medical centers
around the world on a variety of topics critical to the treatment
of diseases of the eye. He was an inventor holding multiple
patents in the area of ophthalmology.
In the early 1970s, Alan was approached by the Retinitis
Pigmentosa Foundation to help them develop a scientific plan
to support targeted research that would lead to an
understanding of the causes of retinitis pigmentosa. At the time,
it was recognized that these diseases were inherited, but only in
a very limited way (autosomal dominant, recessive or
X-linked). At the time, no mutations causing RP had been
identified and the Human Genome Project would not be
initiated for another 20 years. Alan agreed and organized the
first Scientific Advisory Board for this Foundation and served
as Chairman. In this leadership role, Alan helped identify and
direct funding to the first laboratory focused on degenerative
retinal disease research, the Berman-Gund Laboratory at the
Massachusetts Eye and Ear Infirmary, Harvard University.
Research Centers focused on retinal degeneration would later
be expanded to many medical centers in North America,
England, and Europe. Alan recognized the importance and
need for animal models with these inherited retinal diseases
and directed funds from the Foundation to support the
development of the dog models with RP identified by Dr.
Gustavo Aguirre at the College of Veterinary Medicine. In the
early 1980s, Alan initiated a scientific plan for the Foundation
to identify the major genes responsible for RP. This led in 1989
to the discovery of a mutation in the rhodopsin gene
 vii

responsible for an autosomal dominant form of retinitis

pigmentosa. Discovery of mutations in other genes causing
retinitis pigmentosa quickly followed. With the discovery of
RP-65, a gene that causes a recessive form of RP, gene therapy
in a dog model with this recessive disorder could be quickly
initiated because of Dr. Laties’ early support from the
Foundation of these dog model lines. Dr. Laties’ early
leadership was hugely important to gene therapy clinical trials
and a number of other therapies related to these inherited
retinal diseases. To honor Dr. Laties, the Foundation Fighting
Blindness named their physicians’ and physician-scientists’
career development award the Alan Laties Career Development
Program and honored him with the inaugural Llura Liggett
Gund Lifetime Achievement Award.
Dr. Laties was a gifted scientist, outstanding leader, and
compassionate human who enriched the lives of his
contemporaries. He played a key role in nurturing and
expanding research in inherited retinal diseases. He is survived
by his wife Deena Gu, a distinguished artist, daughter Jane
Laties, sons Alex P. Laties and Nicholas P. Robinson, and a
brother, David.
Preface

The XIX International Symposium on Retinal Degeneration was held from

September 26 to October 2, 2021. The symposium was initially planned for
October of 2020 in Mendoza, Argentina. However, the global pandemic made
this meeting impossible. With the availability of vaccines, we decided in
March of 2021 that it would be possible to organize the meeting for late
September of 2021. From the beginning, we planned the meeting as an in-­
person meeting with the capability of switching to a hybrid or fully online
meeting depending on the state of the pandemic, and we moved the in-person
meeting to the United States to reduce travel complications for most attend-
ees. As the delta variant began to surge in the weeks leading up to the meet-
ing, we had to activate the hybrid meeting. The meeting platform we
established allowed both in-person and virtual platform talks as well as both
in-person and virtual attendance. The platform was organized so that all pre-
sentations were live and all participants were able to ask questions. All pre-
sentations, including posters, were recorded and made available 4 months
after the meeting. The in-person sessions were held in the Sonesta Nashville
Airport Hotel in Nashville, TN. Because of COVID concerns, the in-person
attendance was small (118 scientists) compared to previous meetings (~250
scientists), but the overall attendance increased to 344 attendees. The virtual
option was the main driver for the increase in attendance. The meeting pro-
gram included four outstanding keynote presentations from Michael Chiang,
Director of the National Eye Institute on Artificial intelligence for clinical
care and research; Douglas Wallace, National academy of Science member
and Professor at the University of Pennsylvania on Mitochondria and the eti-
ology of disease; David Gamm, Professor at the University of Wisconsin-­
Madison on Ultrathin micromolded 3D scaffolds for outer retina
reconstruction; Valeria Canto-Soler, Professor at the University of Colorado
on Human iPSC-derived 3D retinal tissue for stem cell-based therapies for
retinal degenerative diseases. Drs Chiang and Wallace presented via the vir-
tual platform, while Drs Gamm and Canto-Soler presented from the podium.
The program also included 41 platform talks, with 28 presented in person
from the podium and another 13 presented virtually. In addition, 143 posters
were presented as short talks on the virtual platform. Seventy-three of the
posters were also presented in person during two well-attended poster ses-
sions. New and important data were presented at the meeting, and we were
mentioned in a written article published on NPR, and several attendees were
interviewed by reporters from Science and other journals.

ix
x Preface

The RD2021 Travel award competition was highly successful at attracting

qualified applicants. We received a 35% increase in TA applications for a total
of 196. The applications were reviewed by a panel of 14 expert reviewers,
including 6 women, 8 men, and sceintists from a recognized underrepre-
sented minority (URM). Since funding from European sources is dedicated to
European early career scientists, we included three reviewers from Europe.
Many of the panel members have been prior travel awardees. Each applica-
tion was assigned four reviewers, and reviewers independently scored appli-
cations on a 1–9 scale. Based on scores, the applications are ranked and
slotted into funding sources based on funding agency criteria. We were able
to support full travel awards for 60 in-person early career scientists and
another 41 virtual early-career scientists. This is the largest pool of awardees
at an RD meeting. The awards were balanced between men and women. In
addition, we implemented a new diversity and inclusion policy and dedicated
a minimum of six awards to underrepresented minorities (URM). In the end,
we were able to fund 11 URMs to attend the RD meeting.
Although the pandemic made the RD2021 meeting more complex and
more challenging to organize, the RD2021 meeting was, by all accounts, a
terrific success.

Pittsburgh, PA, USA John D. Ash

Boston, MA, USA Eric Pierce
Oklahoma City, OK, USA Robert E. Anderson
Durham, NC, USA Catherine Bowes Rickman
Cleveland, OH, USA Joe G. Hollyfield
Schlieren, Switzerland Christian Grimm
Contents

Part I Age-related Macular Degeneration

High-Resolution Imaging Mass Spectrometry of Human

Donor Eye: Photoreceptors Cells and Basal Laminar
Deposit of Age-­Related Macular Degeneration������������������������������������   3
David M. G. Anderson, Ankita Kotnala, Jeffrey D. Messinger,
Nathan Heath Patterson, Jeffrey M. Spraggins, Christine A. Curcio,
Richard M. Caprioli, and Kevin L. Schey
The Noncanonical Role of Complement Factor H
in Retinal Pigment Epithelium (RPE) Cells and Implications
for Age-Related Macular Degeneration (AMD)������������������������������������   9
Angela Armento, David Adrian Merle, and Marius Ueffing
 acular Pigment Carotenoids and Bisretinoid A2E���������������������������� 15
M
Ranganathan Arunkumar and Paul S. Bernstein
Disturbed Matrix Metalloproteinases Activity in Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 21
Beatriz Martins and Rosa Fernandes
Current Views on Chr10q26 Contribution to Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 27
Navdeep Gogna, Lillian F. Hyde, Gayle B. Collin, Lisa Stone,
Jurgen K. Naggert, and Patsy M. Nishina
Untargeted Lipidomic Profiling of Aged Human Retina
With and Without Age-Related Macular Degeneration (AMD)���������� 37
Ankita Kotnala, David M. G. Anderson, Jeffrey D. Messinger,
Christine A. Curcio, and Kevin L. Schey
Decoding Race and Age-Related Macular Degeneration:
GPR 143 Activity Is the Key�������������������������������������������������������������������� 43
Dorothy Tung and Brian S. McKay
 eroxisome Proliferator-Activated Receptor Gamma
P
Coactivator-­1Alpha (PGC-1α): A Transcriptional Regulator at
the Interface of Aging and Age-Related Macular Degeneration? ������ 49
Freya M. Mowat

xi
xii Contents

Regulation of ABCA1 by miR-33 and miR-34a

in the Aging Eye �������������������������������������������������������������������������������������� 55
Florian Peters and Christian Grimm
 he Role of Gene Expression Regulation on Genetic Risk
T
of Age-­Related Macular Degeneration�������������������������������������������������� 61
Rinki Ratnapriya
Elastin Layer in Bruch’s Membrane as a Target
for Immunization or Tolerization to Modulate Pathology
in the Mouse Model of Smoke-Induced Ocular Injury������������������������ 67
Bärbel Rohrer, Nathaniel Parsons, Balasubramaniam Annamalai,
Crystal Nicholson, Elisabeth Obert, Bryan Jones,
and Andrew D. Dick
Repurposing Drugs for Treatment of Age-Related
Macular Degeneration ���������������������������������������������������������������������������� 73
Sarah G. Francisco and Sheldon Rowan

Part II Extracellular Vesicles

Extracellular Vesicle RNA Contents as Biomarkers

for Ocular Diseases���������������������������������������������������������������������������������� 81
Heran Getachew and Eric Pierce
Proteomics of Retinal Extracellular Vesicles: A Review
into an Unexplored Mechanism in Retinal Health
and AMD Pathogenesis���������������������������������������������������������������������������� 87
Adrian V. Cioanca, Riccardo Natoli, and Yvette Wooff

Part III Gene Editing

Prime Editing Strategy to Install the PRPH2

c.828+1G>A Mutation ���������������������������������������������������������������������������� 97
Salvatore Marco Caruso, Yi-Ting Tsai, Bruna Lopes da Costa,
Masha Kolesnikova, Laura A. Jenny, Stephen H. Tsang,
and Peter M. J. Quinn
Analysis of CRB1 Pathogenic Variants Correctable
with CRISPR Base and Prime Editing�������������������������������������������������� 103
Bruna Lopes da Costa, Laura A. Jenny, Irene H. Maumenee,
Stephen H. Tsang, and Peter M. J. Quinn
Generation of an Avian Myeloblastosis Virus (AMV)
Reverse Transcriptase Prime Editor������������������������������������������������������ 109
Yi-Ting Tsai, Bruna Lopes da Costa, Salvatore Marco Caruso,
Nicolas D. Nolan, Sarah R. Levi, Stephen H. Tsang,
and Peter M. J. Quinn
Contents xiii

Part IV Gene Therapy

Preexisting Neutralizing Antibodies against Different

Adeno-Associated Virus Serotypes in Humans
and Large Animal Models for Gene Therapy���������������������������������������� 117
Divya Ail and Deniz Dalkara
Optimization of Capillary-Based Western Blotting
for MYO7A ���������������������������������������������������������������������������������������������� 125
Kaitlyn R. Calabro, Sanford L. Boye, and Shannon E. Boye
AAV Serotypes and Their Suitability for Retinal
Gene Therapy ������������������������������������������������������������������������������������������ 131
Lynn J. A. Ebner and Christian Grimm
Gene Augmentation for Autosomal Dominant CRX-Associated
Retinopathies�������������������������������������������������������������������������������������������� 135
Chi Sun and Shiming Chen
 xnip Gene Therapy of Retinitis Pigmentosa Improves
T
Cone Health���������������������������������������������������������������������������������������������� 143
Yunlu Xue

Part V Human Retinal Degeneration

Factors Affecting Readthrough of Natural Versus

Premature Termination Codons ������������������������������������������������������������ 149
Avigail Beryozkin, Kerstin Nagel-Wolfum, Eyal Banin,
and Dror Sharon
Integrating Computational Approaches to Predict
the Effect of Genetic Variants on Protein Stability
in Retinal Degenerative Disease�������������������������������������������������������������� 157
Michelle Grunin, Ellen Palmer, Sarah de Jong, Bowen Jin,
David Rinker, Christopher Moth, John A. Capra,
Jonathan L. Haines, William S. Bush, and Anneke I. den Hollander
 etwork Biology and Medicine to Rescue: Applications
N
for Retinal Disease Mechanisms and Therapy�������������������������������������� 165
Anupam K. Mondal and Anand Swaroop
Non-syndromic Retinal Degeneration Caused by Pathogenic
Variants in Joubert Syndrome Genes���������������������������������������������������� 173
Riccardo Sangermano, Egle Galdikaité-Braziené,
and Kinga M. Bujakowska
Exonic Variants that Affect Splicing – An Opportunity
for “Hidden” Mutations Causing Inherited Retinal Diseases�������������� 183
Yogapriya Sundaresan, Eyal Banin, and Dror Sharon
 nhanced S-cone Syndrome, a Mini-review������������������������������������������ 189
E
Yiyi Wang, Jessica Wong, Jacque L. Duncan, Austin Roorda,
and William S. Tuten
xiv Contents

Part VI Inflammation

 he Role of Microglia in Inherited Retinal Diseases���������������������������� 197

T
Asha Kumari and Shyamanga Borooah
CD68: Potential Contributor to Inflammation and RPE
Cell Dystrophy������������������������������������������������������������������������������������������ 207
Mayur Choudhary and Goldis Malek
 ene Expression of Clusterin, Tissue Inhibitor
G
of Metalloproteinase-1, and Their Receptors in Retinal
Pigment Epithelial Cells and Müller Glial Cells Is
Modulated by Inflammatory Stresses���������������������������������������������������� 215
Mengmei Zheng, Eun-Jin Lee, Shinwu Jeong,
and Cheryl Mae Craft

Part VII Mechanisms of Degeneration

 xonal Transport Defects in Retinal Ganglion Cell Diseases�������������� 223

A
Iskalen Cansu Topcu Okan, Fatma Ozdemir, and Cavit Agca
 onnexins Biology in the Pathophysiology of Retinal Diseases���������� 229
C
Alejandro Ponce-Mora, Andrea Yuste, Giuliana Perini-Villanueva,
María Miranda, and Eloy Bejarano
 ole of Nuclear NAD+ in Retinal Homeostasis�������������������������������������� 235
R
Emily E. Brown, Michael J. Scandura, and Eric Pierce
Retinal Pigmented Epithelium-­Derived Ectopic Norrin Does
Not Promote Intraretinal Angiogenesis in Transgenic Mice���������������� 241
Andrea E. Dillinger and Ernst R. Tamm
Caveolin-1 in Müller Glia Exists as Heat-Resistant, High
Molecular Weight Complexes ���������������������������������������������������������������� 249
Eric N. Enyong, Jami Gurley, Virginie Sjoelung,
and Michael H. Elliott
 ole of VLC-PUFAs in Retinal and Macular Degeneration���������������� 257
R
Aruna Gorusupudi, Uzoamaka Nwagbo, and Paul S. Bernstein
Ocular Amyloid, Condensates, and Aggregates – Higher-Order
Protein Assemblies Participate in Both Retinal Degeneration
and Function�������������������������������������������������������������������������������������������� 263
Michael H. Hayes, DaNae R. Woodard, and John D. Hulleman
 hotoreceptor Ion Channels in Signaling and Disease������������������������ 269
P
Shivangi M. Inamdar, Colten K. Lankford, and Sheila A. Baker
The Role of Peripherin-2/ROM1 Complexes in Photoreceptor
Outer Segment Disc Morphogenesis������������������������������������������������������ 277
Tylor R. Lewis, Muayyad R. Al-Ubaidi, Muna I. Naash,
and Vadim Y. Arshavsky
Contents xv

Human Mutations in Arl3, a Small GTPase Involved

in Lipidated Cargo Delivery to the Cilia, Cause
Retinal Dystrophy������������������������������������������������������������������������������������ 283
Amanda M. Travis and Jillian N. Pearring
Genotype–Phenotype Association in ABCA4-Associated
Retinopathy���������������������������������������������������������������������������������������������� 289
Maximilian Pfau, Wadih M. Zein, Laryssa A. Huryn,
Catherine A. Cukras, Brett G. Jeffrey, Robert B. Hufnagel,
and Brian P. Brooks
Retinal Pathoconnectomics: A Window into
Neurodegeneration���������������������������������������������������������������������������������� 297
Rebecca L. Pfeiffer and Bryan W. Jones
The Role of Ceramide in Inherited Retinal
Disease Pathology������������������������������������������������������������������������������������ 303
Xinye Qian, Tanmay Srinivasan, Jessica He, and Rui Chen
 xtracellular Matrix: The Unexplored Aspects
E
of Retinal Pathologies and Regeneration���������������������������������������������� 309
Dmitri Serjanov and David R. Hyde
Role of TFEB in Diseases Associated with
Lysosomal Dysfunction���������������������������������������������������������������������������� 319
Hsuan-Yeh Pan and Mallika Valapala
Retinoic Acid Receptor-Related Orphan Receptors
(RORs) in Eye Development and Disease���������������������������������������������� 327
Felix Yemanyi, Kiran Bora, Alexandra K. Blomfield,
and Jing Chen

Part VIII Mechanisms of Degeneration – Animal Models

A Novel Mouse Model for Late-­Onset Retinal Degeneration

(L-ORD) Develops RPE Abnormalities Due
to the Loss of C1qtnf5/Ctrp5�������������������������������������������������������������������� 335
Shyamanga Borooah, Anil Chekuri, Shikha Pachauri,
Bhubananda Sahu, Marina Vorochikhina, John J. Suk,
Dirk-­Uwe Bartsch, Venkata R. M. Chavali, Monica M. Jablonski,
and Radha Ayyagari
 omparison of Mouse Models of Autosomal Dominant Retinitis
C
Pigmentosa Due to the P23H Mutation of Rhodopsin�������������������������� 341
Shannon R. Barwick and Sylvia B. Smith
Compensatory Cone-Mediated Mechanisms in Inherited
Retinal Degeneration Mouse Models: A Functional
and Gene Expression Analysis���������������������������������������������������������������� 347
Alicia A. Brunet, David M. Hunt, Carla Mellough,
Alan R. Harvey, and Livia S. Carvalho
xvi Contents

Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic

Reticulum (ER) Stress and Promotes ER Protein
Degradation in Cyclic Nucleotide-Gated
Channel Deficiency���������������������������������������������������������������������������������� 353
Fan Yang, Hongwei Ma, Rekha Garg, Alfred Lewin,
and Xi-Qin Ding
 ouse Choroid Proteome Revisited: Focus on Aging�������������������������� 359
M
Donita Garland, James Harnly, and Radha Ayyagari
Morphological and Functional Comparison
of Mice Models for Retinitis Pigmentosa ���������������������������������������������� 365
Prakadeeswari Gopalakrishnan, Avigail Beryozkin,
Eyal Banin, and Dror Sharon
 urrent Advancements in Mouse Models of Retinal Disease�������������� 371
C
T. J. Hollingsworth, Xiangdi Wang, Raven N. Simpson,
William A. White, Robert W. Williams,
and Monica M. Jablonski
Single-Cell Transcriptomic Profiling of Müller
Glia in the rd10 Retina���������������������������������������������������������������������������� 377
Duygu Sigurdsson and Christian Grimm

Methods for In Vivo Characterization of Proteostasis
in the Mouse Retina �������������������������������������������������������������������������������� 383
Yixiao Wang and Ekaterina S. Lobanova
Absence of PRCD Leads to Dysregulation in Lipid
Homeostasis Resulting in Disorganization
of Photoreceptor Outer Segment Structure������������������������������������������ 389
Sree I. Motipally and Saravanan Kolandaivelu

Expansion Microscopy of Mouse Photoreceptor Cilia ������������������������ 395
Abigail R. Moyel, Michael A. Robichaux, and Theodore Wensel
Rod Photoreceptor-Specific Ablation of Metformin Target,
AMPK, in a Preclinical Model of Autosomal
Recessive Retinitis Pigmentosa �������������������������������������������������������������� 403
Nicholas D. Nolan, Laura A. Jenny, Stephen H. Tsang,
and Xuan Cui
TLR2 Is Highly Overexpressed in Retinal Myeloid
Cells in the rd10 Mouse Model of Retinitis Pigmentosa ���������������������� 409
Alonso Sánchez-Cruz, Enrique J. de la Rosa,
and Catalina Hernández-Sánchez
Environmental Light Has an Essential Effect
on the Disease Expression in a Dominant RPE65 Mutation���������������� 415
Wenjing Wu, Yusuke Takahashi, Xiang Ma, Gennadiy Moiseyev,
and Jian-Xing Ma
Contents xvii

Microglia Preserve Visual Function in a Mouse Model

of Retinitis Pigmentosa with Rhodopsin-P23H Mutant ���������������������� 421
Chen Yu and Daniel R. Saban

Part IX Mechanisms of Degeneration – Metabolism

Measuring the Release of Lactate from Wild-Type

and rd1 Mouse Retina������������������������������������������������������������������������������ 429
Yiyi Chen, Laimdota Zizmare, Christoph Trautwein,
and François Paquet-Durand
Aerobic Glycolysis in Photoreceptors Supports Energy
Demand in the Absence of Mitochondrial Coupling���������������������������� 435
Daniel T. Hass, Celia M. Bisbach, Martin Sadilek,
Ian R. Sweet, and James B. Hurley

Redox Status in Retinitis Pigmentosa���������������������������������������������������� 443
L. Olivares-González, S. Velasco, I. Campillo, J. M. Millán,
and R. Rodrigo
Perspectives on Retinal Dolichol Metabolism,
and Visual Deficits in Dolichol Metabolism-Associated
Inherited Disorders���������������������������������������������������������������������������������� 449
Sriganesh Ramachandra Rao, Steven J. Pittler,
and Steven J. Fliesler
Retinal Metabolic Profile on IMPG2 Deficiency Mice
with Subretinal Lesions �������������������������������������������������������������������������� 457
Rong Xu, Yekai Wang, Jianhai Du, and Ezequiel M. Salido

Part X Neuroprotection

Glutathione Coating of Liposomes Enhances the Delivery

of Hydrophilic Cargo to the Inner Nuclear Layer
in Retinal Cultures ���������������������������������������������������������������������������������� 467
Gustav Christensen and François Paquet-Durand
Modification of Müller Glial Cell Fate and Proliferation
with the Use of Small Molecules ������������������������������������������������������������ 473
Marcus J. Hooper
A Potential Neuroprotective Role for Pyruvate Kinase
2 in Retinal Degeneration������������������������������������������������������������������������ 479
Jiaming Zhou, Michel Rasmussen, and Per Ekström

Part XI Photoreceptors

Critical Role of VEGF as a Direct Regulator

of Photoreceptor Function���������������������������������������������������������������������� 487
Jianyan Hu, Meili Zhu, Dai Li, Qiang Wu, and Yun-Zheng Le
xviii Contents


Lysine Ubiquitylation Drives Rhodopsin Protein Turnover���������������� 493
Allen P. F. Chen, Leon Chea, Eun-Jin Lee,
and Jonathan H. Lin
 Silico Prediction of MYO1C-­Rhodopsin Interactions
In
and Its Significance in Protein Localization
and Visual Function �������������������������������������������������������������������������������� 499
Glenn P. Lobo, Rakesh Radhakrishnan, Matthias Leung,
Andrew Gruesen, Hans-­Joachim Knölker, Frederik J. van Kuijk,
and Sandra R. Montezuma
A Ciliary Branched Actin Network Drives
Photoreceptor Disc Morphogenesis�������������������������������������������������������� 507
William J. Spencer and Vadim Y. Arshavsky

Part XII RPE


Revisiting the Daily Timing of POS Phagocytosis�������������������������������� 515
Antonio E. Paniagua, Harjas S. Sabharwal, Kausalya Kethu,
Andrew W. Chang, and David S. Williams
Inhibition of Bacterial Peptidoglycan Cytopathy
by Retina Pigment Epithelial PGRP2 Amidase������������������������������������ 521
Marlyn P. Langford, Laura A. Perilloux-Lyons,
and A. Scott Kavanaugh
Understanding Ischemic Retinopathies: The Role
of Succinate and Its Receptor in Retinal
Pigment Epithelium �������������������������������������������������������������������������������� 527
Bilge Esin Ozturk

The Amphipathic Helix in Visual Cycle Proteins: A Review���������������� 533
Sheetal Uppal, Eugenia Poliakov, Susan Gentleman,
and T. Michael Redmond

The Retinal Pigment Epithelium: Cells That Know the Beat!������������ 539
Elora M. Vanoni and Emeline F. Nandrot

Part XIII Stem Cell Models and Therapies

Retinal Organoids: A Human Model System

for Development, Diseases, and Therapies�������������������������������������������� 549
Sangeetha Kandoi and Deepak A. Lamba

Modeling Retinitis Pigmentosa with Patient-Derived iPSCs �������������� 555
Yeh Chwan Leong and Jane C. Sowden

Primary Retinal Cell Cultures as a Model to Study
Retina Biology������������������������������������������������������������������������������������������ 565
Germán A. Michelis, Luis E. Politi, and S. Patricia Becerra
Contents xix

Generation of CRB1 RP Patient-­Derived iPSCs

and a CRISPR/Cas9-­Mediated Homology-Directed
Repair Strategy for the CRB1 c.2480G>T Mutation���������������������������� 571
Bruna Lopes da Costa, Yao Li, Sarah R. Levi, Stephen H. Tsang,
and Peter M. J. Quinn
Inducing Neural Regeneration from Glia Using Proneural
bHLH Transcription Factors������������������������������������������������������������������ 577
Levi Todd

Index�������������������������������������������������������������������������������������������������������� 583
 Part I
Age-related Macular Degeneration
 High-Resolution Imaging Mass
Spectrometry of Human Donor Eye:
Photoreceptors Cells and Basal
Laminar Deposit of Age-­Related
Macular Degeneration

David M. G. Anderson, Ankita Kotnala,

Jeffrey D. Messinger, Nathan Heath Patterson,
Jeffrey M. Spraggins, Christine A. Curcio,
Richard M. Caprioli, and Kevin L. Schey

Abstract nohistochemistry. Although both techniques

Pathologies of the retina are clinically visual-
ized in vivo with OCT and ex vivo with immu-
D. M. G. Anderson · N. H. Patterson · R. M. Caprioli
· K. L. Schey (*)
Department of Biochemistry, Vanderbilt University,
Nashville, TN, USA
Mass Spectrometry Research Center, Vanderbilt
University School of Medicine, Nashville, TN, USA
e-mail: k.schey@vanderbilt.edu
A. Kotnala
Department of Biochemistry, Vanderbilt University,
Nashville, TN, USA
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
J. D. Messinger · C. A. Curcio
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
J. M. Spraggins
Mass Spectrometry Research Center, Vanderbilt
University School of Medicine, Nashville, TN, USA
Department of Ophthalmology and Visual Sciences,
University of Alabama at Birmingham,
Birmingham, AL, USA
Department of Cell and Developmental Biology,
Vanderbilt University School of Medicine,
Nashville, TN, USA
provide valuable information on prognosis
and disease state, a comprehensive method for
fully elucidating molecular constituents pres-
ent in locations of interest is desirable. The
purpose of this work was to use multimodal
imaging technologies to localize the vast num-
ber of molecular species observed with
matrix-assisted laser desorption ionization
imaging mass spectrometry (MALDI IMS) in
aged and diseased retinal tissues. Herein,
MALDI IMS was utilized to observe molecu-
lar species that reside in photoreceptor cells
and also a basal laminar deposit from two
human donor eyes. The molecular species
observed to accumulate in these discrete
regions can be further identified and studied to
attempt to gain a greater understanding of bio-
logical processes occurring in debilitating eye
diseases such as age-related macular degener-
ation (AMD).
Keywords
Age-related macular degeneration · Macula ·
Retinal pigment epithelium · Photoreceptors ·
Basal lamina deposit · MALDI IMS

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 3

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_1
4 D. M. G. Anderson et al.
1 Introduction
Matrix-assisted laser desorption ionization
imaging mass spectrometry (MALDI IMS) can
localize and display the tissue distributions of
hundreds to thousands of molecules, at cellular
resolution, without the need for antibodies or
radioisotopes [1]. With effective co-registration
to multimodal optical imaging and optical
coherence topography (OCT) microscopy, these
distributions can be accurately correlated to
very small histological features of the neural
retina and retinal pigment epithelium (RPE).
MALDI IMS methods have been used to exam-
ine eye tissues including the retina [2–4], optic
nerve [4–6], lens [7, 8], and cornea [9]. Distinct
cell and synaptic layers of the retina have unique
layer-specific lipid and metabolite signatures
distinguished by IMS [4, 10, 11]. By applying
multimodal optical imaging technologies with
accurate registration and incorporating data-rich
IMS images [12], cellular and subcellular local-
ization of specific molecules informative to cell-
specific biochemistry can be observed. Human
retinal lipid composition studies have been per-
formed in the past. The results, while valuable,
provide limited information on cellular origin,
as experiments require dissections followed by
solvent extractions. MALDI IMS offers a
“molecular microscope” that localizes tissue
components in situ by molecular weights [11],
simultaneously providing hundreds to thou-
sands of spatially resolved signals. In this study,
we used a newly developed method of high-
accuracy registration [12] to co-­ register high
spatial resolution IMS images with OCT auto-
fluorescence and histological images of the
same tissue to examine subcellular localizations
and molecular features of photoreceptors and
AMD pathology.
2 Methods
This section has been summarized from Anderson
et al. [3]; for detailed explanations, please see
this reference.
2.1 Tissue Acquisition
and Characterization
Whole eyes were obtained from deceased human
donors via Advancing Sight Network (formerly
the Alabama Eye Bank) by the UAB authors.
2.2 Tissue Handling and Ex Vivo
Imaging
Methods were optimized for multimodal ex vivo
clinical imaging of the ocular fundus [13]. Globes
with lens and iris in place were immersed in buff-
ered 4% paraformaldehyde overnight. Iris and
lens were removed before imaging.
For imaging with OCT and scanning laser
ophthalmoscopy, globes were immersed in buffer
facing frontward within a custom-built chamber
with a 60-diopter lens [13]. Spectral domain
OCT images were captured with a Spectralis
(HRA&OCT, HRA2; Heidelberg Engineering).
Tissues were embedded in 2.5% carboxy-
methyl cellulose (Sigma C9481), and serial
10 μm cryosections were collected on Superfrost
glass slides and on large, 45 × 45 mm in-house,
polylysine-coated indium-tin-oxide (ITO) slides
(Delta Technologies Loveland, CO, USA).
2.3 MALDI IMS Analysis
The matrices 2,5-dihydroxyacetophenone (DHA)
and 1,5-diaminonaphthalene (DAN) (Sigma
Aldrich, St. Louis, MO, USA) were applied to
tissue sections by sublimation [14]. MALDI IMS
data were acquired with a laser spot size of
10–15 μm in full scan mode using a Bruker
SolariX 9.4T FTICR mass spectrometer (Bruker
Daltonics Billerica, MA, USA). Following data
acquisition, an advanced image registration
workflow [12] was performed. More detailed
information of the image registration process can
be found in publications by Patterson et al. [12]
and Anderson et al. [3]. Molecular identifications
were made using LC-MS of chloroform-­methanol
extracts from adjacent tissue sections.
High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…
3 Results
3.1 Signals Specific
to Photoreceptors and RPE
Figure 1a shows MALDI IMS and optical micros-
copy focusing on photoreceptors and their
­support cells. The RPE sends delicate processes
in the apical direction to contact photoreceptor
outer segments, near the RPE cell body for rods
and 10–15 μm above the cell body, to contact
cone outer segments, which are shorter. Figure 1a
is color-coded depiction of photoreceptor and
RPE compartments associated with IMS signals
in Fig. 1b (blue ONL, red inner segments, yellow
outer segments, green RPE).
Figure 1b shows MALDI IMS images over-
laid with H&E images from this donor. The pho-
toreceptors on the left side of the image are
attached to the RPE and detached from the RPE
on the right side, a common artifact which can
occur during sample preparation. In Fig. 1a, the
signal at m/z 818.575 was observed with high
abundance in the ONL and was identified as
PE(20:0_22:6) (blue). This region is comprised
of the photoreceptor cell bodies and processes of
Müller (radial) glia. A highly localized signal can
be observed with high abundance along a narrow
band aligned with photoreceptor inner segments
at m/z 1426.0 (red). This signal was identified as
Fig. 1 MALDI IMS signals consistent with localization
to photoreceptor and RPE compartments. (a) Schematic
diagram of outer retina, excerpted from Fig. 1a. Blue,
pink, yellow, and green bands indicate layers formed by
highly compartmentalized and vertically aligned photore-
ceptors and RPE cells in panels b, c. Layers: OPL outer
plexiform layer, ONL outer nuclear layer, ELM external
limiting membrane, RPE retinal pigment epithelium, BrM
Bruch’s membrane, R rod, C cone photoreceptors. (b–f)
5
a cardiolipin CL(70:5). Cardiolipins are highly
abundant in mitochondria, which are abundant in
the ellipsoid portion of photoreceptor inner seg-
ments. At the distal part of the photoreceptor
cells, outer segments are highly interleaved with
apical processes of RPE cells. A DHA-containing
PE(18:0_22:6) is observed at m/z 790.539 (yel-
low) in panel D which can be observed with high
abundance in the outer segments, while a signal
observed at m/z 728.596 (green) is localized
above and within the RPE.
3.2 Signals Specific to Basal
Laminar Deposit
Figure 2 shows multimodal imaging of a 93-year-­
old donor tissue with the imaging modalities
separated into panels. Figure 2a displays ex vivo
OCT hyperreflective foci (yellow arrowhead) and
an RPE elevation (green arrowhead) near the
fovea. Figure 2b shows that retinal layers are vis-
ible in H&E-stained sections after IMS data
acquisition. The inset magnifies BLamD (PASH
staining of an alternate section), clearly indicat-
ing thickened extracellular matrix between the
RPE plasma membrane and its native basal lam-
ina. Figure 2c shows autofluorescence of the ele-
vated RPE layer and anteriorly migrated RPE
cells, which account for high-risk-indicating
MALDI IMS images and H&E-stained tissue images
overlaid in perifoveal retina displaying signals from mul-
tiple lipid classes that localize to subcellular compart-
ments of the photoreceptor cells. (b) Overlay showing
four separate signals. (c) Localized to ONL. (d) Localized
to photoreceptor inner and outer segments. (e) Localized
to mitochondria-rich photoreceptor inner segments. (f)
Localized to RPE apical processes
6 D. M. G. Anderson et al.
Fig. 2 Imaging mass spectrometry (IMS) for molecularly
informed optical coherence tomography (OCT) and
tissue-­level target discovery. Asterisk, foveal pit; RPE,
retinal pigment epithelium. Color-coded arrowheads rep-
resent corresponding structures across modalities, in a
93-year-old human donor eye. (a) OCT B-scan shows
subretinal hyperreflective material (yellow) and an RPE
elevation (green). (b) H&E stained cryosection shows
hyperreflective foci of clinical OCT. Figure 2d
shows that a sphingomyelin-related lipid
(PE-Cer-NMe2(42:1)) at m/z 799.671 [15] is
highly abundant and localizes to BLamD and
RPE, building on previous histochemical and
chromatography findings of lipids in this deposit
[16, 17].
pigmented debris (yellow) and dysmorphic RPE overly-
ing BLamD. Insert, BLamD with basal mound (arrow).
Basal mounds contain soft druse material. Layers: GCL
ganglion cell, INL inner nuclear, HFL Henle fiber, ONL
outer nuclear. (c) Autofluorescent, pigmented debris (yel-
low) and dysmorphic RPE (green). (d) IMS reveals an m/z
signal at 799.673, restricted to BLamD and not detected in
the RPE
4 Conclusions
MALDI IMS combined with multimodal imag-
ing methods and ex vivo OCT provides a power-
ful tool to elucidate the molecular composition
and localization of molecular species in key
regions and pathology associated with ocular dis-
High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal…
ease. Understanding molecular processes occur-
ring in BLamD in early AMD is important as
they are early-identified histologic risk factors
for AMD progression [18] and are just now being
recognized clinically [19, 20].
Acknowledgments This project was supported by the
National Institutes of Health P41 GM103391 (R.M.C.)
and R01 EY027948 (C.A.C.). Support was also received
by a Research to Prevent Blindness Catalyst Award for
Innovative Research Approaches for Age-Related Macular
Degeneration to K.L.S.
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 The Noncanonical Role
of Complement Factor H in Retinal
Pigment Epithelium (RPE) Cells
and Implications for Age-Related
Macular Degeneration (AMD)

Angela Armento, David Adrian Merle,

and Marius Ueffing

Abstract pathway of the complement system, and the

Age-related macular degeneration (AMD) is a
complex degenerative disease of the retina.
Dysfunction of the retinal pigment epithelium
(RPE) occurs in early stages of AMD, and
progressive RPE atrophy leads to photorecep-
tor death and visual impairments that ulti-
mately manifest as geographic atrophy (GA),
one of the late-stage forms of AMD. AMD is
caused by a combination of risk factors includ-
ing aging, lifestyle choices, and genetic pre-
disposition. A gene variant in the complement
factor H gene (CFH) that leads to the Y402H
polymorphism in the factor H protein (FH)
confers the second highest risk for the devel-
opment and progression of AMD. FH is a
major negative regulator of the alternative
FH Y402H variant leads to increased comple-
ment activation, which is detectable in AMD
patients. For this reason, various therapeutic
approaches targeting the complement system
have been developed, however, so far with
very limited or no efficacy. Interestingly,
recent studies suggest roles for FH beyond
complement regulation. Here, we will discuss
the noncanonical functions of FH in RPE cells
and highlight the potential implications of
those functions for future therapeutic
approaches.
Keywords
Age-related macular degeneration (AMD) ·
Retinal pigment epithelium (RPE) cells ·
Intracellular complement factor H (CFH)

A. Armento (*) · M. Ueffing

Institute for Ophthalmic Research, Department for
Ophthalmology, Eberhard Karls University of
Tübingen, Tübingen, Germany
e-mail: angela.armento@uni-tuebingen.de;
marius.ueffing@uni-tuebingen.de
D. A. Merle
Institute for Ophthalmic Research, Department for
Ophthalmology, Eberhard Karls University of
Tübingen, Tübingen, Germany
Department of Ophthalmology, Medical University of
Graz, Graz, Austria
e-mail: david.merle@medunigraz.at
1 Introduction
Age-related macular degeneration (AMD) is a
complex and progressive disease of the macula,
which affects mainly the elderly population,
especially in the developed countries [1, 2]. Due
to demographic changes in western societies,
AMD incidence is constantly rising, and the con-
comitant visual impairment threatens not only

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 9

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_2
10
the individual’s quality of life but also poses a
significant burden on healthcare systems world-
wide [3]. The early stages of the disease are char-
acterized by the presence of enlarging drusen in
the retina. With disease progression, early AMD
advance to late stages: wet or dry AMD. Wet
AMD is defined by the presence of neovascular-
ization in the retina, while dry AMD is limited to
progressive RPE and retinal atrophy, called geo-
graphic atrophy (GA), without newly formed
vessels. Around 10% of the cases constitute wet
AMD, and intravitreal anti-VEGF is effective in
treating most cases. However, only recently,
one approved treatment has become available for
dry AMD in the US [2, 8].
The main pathogenetic driver for AMD is
advanced age. Specific genetic factors can pre-
dispose for AMD or protect from it. Unhealthy
lifestyle habits, like smoking or malnutrition,
can further increase the risk for AMD. The com-
bination of those risk factors leads to pathologi-
cal changes in the retina, involving a plethora of
cellular mechanisms promoting disease pro-
gression [4].
2 Complement System in AMD
Among the processes involved in AMD, the com-
plement system is associated to AMD at different
levels. First of all, many single nucleotide poly-
morphisms (SNPs) associated with AMD cluster
into genes of the alternative pathway of the com-
plement system with the Complement Factor H
(CFH) gene carrying the second highest risk for
AMD [5]. The most common risk haplotype
causes an amino acid substitution from tyrosine
to histidine at position 402 (called Y402H) in
proteins coded by CFH, which are full length FH
and a truncated version, FHL-1 [6, 7]. The com-
plement system is a part of the innate immune
system designed to recognize and mediate the
removal of pathogens or waste material [8]. The
eye is an immune-privileged organ, meaning that
inflammation needs to be strictly limited, and
therefore uncontrolled complement activation is
deleterious [8]. FH is a major negative regulator
of the complement system, and the AMD variant
A. Armento et al.
402H of FH causes uncontrolled complement
activation in vitro and in vivo [9]. Moreover,
complement factors accumulate in drusen [10],
and increased complement system activation has
been detected in AMD patients, independent of
their genotype [11].
Due to the strong implications of complement
system activation in AMD pathology, various
complement inhibitors have been tested in clini-
cal trials. However, most agents that entered
phase II were discontinued due to a reported lack
of efficacy. Interestingly, in some clinical trials, a
group of subjects showed improvements, while
others did not [12]. Apparently, one of the sus-
pected reasons for the observed lack of efficacy is
a lack of patient’s stratification and identification
of those patients that are likely to benefit from
complement inhibition. Moreover, several stud-
ies point to a lack of understanding of the role of
FH in AMD pathology, especially in RPE cells,
which are degenerating in AMD.
3 Noncanonical Role
of Complement Factor H
in RPE Cells
RPE cells exert several functions vital for retinal
homeostasis, which are impaired in AMD [13].
First of all, RPE cells are responsible for phago-
cytosis of shed photoreceptor outer segments
(POS) and recycling of visual pigments, needed
for a proper visual cycle. This process is energy
intensive, requiring constant mitochondrial res-
piration and glycolysis within RPE cells. Due to
a lack of the inner retina vasculature in the mac-
ula, RPE cells are vital to ascertain nutrient sup-
ply from the choriocapillaris. Any pathological
disruption or perturbance of metabolic activity
of RPE cells would affect the amount of nutri-
ents that can reach the neuroretina. Indeed, the
so-­called bioenergetics crisis of RPE cells has
been described for AMD pathology [14].
Moreover, RPE cells quench short UV light as
well as the continuous influx of peroxidized lip-
ids from phagocytosed photoreceptor outer seg-
ments and thus need a high degree of antioxidant
capacity as well as perfect functioning of their
The Noncanonical Role of Complement Factor H in Retinal Pigment Epithelium (RPE) Cells and…
mitochondria to protect the retina from exces-
sive oxidative stress.
Our recent work demonstrates that FH is
endogenously produced by RPE cells. Within
RPE cells, FH is acting locally supporting retinal
homeostasis [15, 16]. This function of FH is con-
text dependent, and we suggest that locally pro-
duced FH exerts specific functions at the
RPE/retina interface. Most of these newly dis-
covered functions (summarized in Fig. 1) affect a
balanced regulation of immune competence and
surveillance as well as regulating oxidative stress
response and energy metabolism, mechanisms
which are part of AMD pathology [8].
FH plays a protective role against oxidative
stress in RPE cells. Exogenously applied recom-
binant FH protects RPE cells from damage
induced by the exposure to lipid oxidation prod-
ucts [17, 18]. In parallel, loss of endogenous FH
in RPE cells, via CFH silencing, also led to
increased vulnerability to oxidative stress,
increased levels of oxidized lipids, and reduced
viability [15]. Interestingly, addition of recombi-
nant FH in CFH-silenced RPE cells did not cause
any rescuing effects [15]. This finding indicates
that, although exogenous FH is protective, a
proper intracellular FH function must be present
to ascertain physiological balance and resilience
to stress. This phenomenon may be explained by
impaired mitochondrial function seen in iPSC-­
RPE cells carrying the CFH Y402H polymor-
phism [19] and CFH-silenced hTERT-RPE1 cells
[15]. In both models, major processes involved in
energy production, glycolysis, and mitochondrial
respiration are impaired. In addition, a misbal-
ance in the degree of mitochondria turnover
seems to be involved, with an exaggerated
increase in mitophagy [15, 19]. Moreover, it has
been shown in iPSC-RPE carrying CFH 402H
variant that mitochondria are enlarged and accu-
mulating [20]. Mitochondria are essential for a
correct response to oxidative stress; therefore,
damage or dysfunction of these organelles is del-
eterious in such an oxidative milieu like the ret-
ina. Accumulation of oxidized lipids has been
reported in RPE cells under FH dysregulation
and induced oxidative stress, when FH function
was impaired as a consequence of CFH silencing
11
[15] or in the presence of the CFH 402H variant
[20]. Moreover, accumulation of lipids was
shown also in cfh Y402H mouse models of AMD,
especially when subjected to high cholesterol
diet [21].
The mechanism of action of intracellular FH
is not fully elucidated. Based on the data gener-
ated in iPSC-RPE cells carrying the CFH 402H
variant, it has been speculated that more than one
signaling pathway mediates the effects of FH
[19]. Specifically, it has been postulated that the
NF-kB pathway and mTOR pathway are likely
involved, in concomitance with autophagy and
proteasome activity dysregulation, which ulti-
mately lead to mitochondrial damage [19].
In our model of CFH-silenced hTERT-RPE1
cells, we could verify these hypotheses. Indeed,
we found that in absence of endogenous FH, both
the NF-kB and mTOR pathway are upregulated,
with NF-kB triggering pro-inflammatory cyto-
kine expression [22] and the mTOR pathway
modulating mitochondrial respiration, but not
glycolysis [23]. The mTOR pathway is a major
regulator of autophagy [24]. Dysfunction in the
autophagy-lysosomal axis in iPSC-RPE cells
with CFH high risk was reported but is not regu-
lated by this pathway. Its dysfunction rather
depends on the accumulation of complement
activation products [25]. Based on our analyses
of the intracellular FH protein interactions that
form a functional interactome in RPE cells, we
suggest a regulatory role of FH in interaction
with the ubiquitin proteasome system (UPS) and
RB1/E2F signaling, adding a new level of com-
plexity in the understanding of FH mechanism of
action [23]. Interestingly, and based on studies in
other cell types than RPE, it has already been
suggested that FH has a noncanonical intracellu-
lar function, independent from complement acti-
vation. In clear renal cell carcinoma cells and
kidney endothelial cells, FH knockdown impairs
cellular homeostasis, at least partially via NF-kB
activation [26, 27].
The synopsis of these findings indicates that
understanding the function of FH in a specific
cell and tissue context is key. In the context of
AMD, it is important to advance our understand-
ing on how RPE cells interact with the neuroret-
12
Fig. 1 Schematic representation of the noncanonical functions of FH in RPE cells
ina once intracellular FH dysregulation or a CFH
402H high risk variant perturbs intracellular
homeostasis within these cells. In this regard, we
have recently established a novel coculture model
of RPE and neuroretina. Using this system, we
show that CFH-silenced RPE cells promote reti-
nal degeneration, which could not be rescued by
the addition of exogenous FH. Moreover, we
showed that the damage occurred at the level of
mitochondrial activity and lipid oxidation in the
photoreceptors, rather than a canonical inflam-
mation or complement activation [16].
4 Future Perspective
Recent works of several groups, including ours,
emphasize a noncanonical and intracellular role
of FH in RPE cells. Besides their reduced capac-
ity to dampen alternative complement activity,
CFH risk polymorphisms such as Y402H impair
RPE homeostasis and are likely contributing to
promote AMD. In consequence, a simple comple-
A. Armento et al.
ment pathway inhibitor strategy to treat AMD
progression may not suffice. Rational re-­balancing
strategies in a form of combinatorial therapies
may be needed to target several drivers of AMD
molecular pathology in a systems-­oriented fash-
ion simultaneously. More specifically, the increase
of alternative complement pathway activity at the
Bruch’s membrane/RPE interface may have to be
considered concomitantly with pro-inflammatory,
oxidative stress-­ related perturbation and meta-
bolic disbalance of RPE and neuroretina to
achieve effective future therapeutic regimes for
dry AMD. Last but not least, a very thorough
stratification of patient groups may be needed to
move to an individualized and rational therapy for
dry AMD of the future.
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 Macular Pigment Carotenoids
and Bisretinoid A2E

Ranganathan Arunkumar and Paul S. Bernstein

Abstract Keywords

Lutein (L), zeaxanthin (Z), and meso-­ Abca4 · A2E · Antioxidant · Bisretinoids ·
zeaxanthin (MZ) are the three macular pig- Carotenoids · Lipofuscin · Macular pigments
ments (MP) carotenoids that uniquely · Photo-oxidation · Retina · Retinal pigment
accumulate in the macula lutea region of the epithelium
human retina. L and Z are obtained by humans
through dietary intake. The third MP, MZ, is
rarely present in diet, and its abundance in the 1 Introduction
human fovea is due to the metabolic conver-
sion of dietary L by the retinal pigment epithe- Carotenoids are natural pigments synthesized by
lium’s RPE65 enzyme. The major functions of photosynthetic bacteria, algae, yeast, and plants.
MP in ocular health are to filter high-intensity, L, Z, and MZ are oxygenated carotenoids (xan-
phototoxic blue light and to act as effective thophylls) obtained by humans through dietary
antioxidants for scavenging free radicals. The intake of leafy greens, fruits, and vegetables.
pyridinium bisretinoid, N-retinylidene-N-­ Among the 15 major dietary carotenoids detected
retinylethanolamine (A2E), contributes to in human serum, these three xanthophyll carot-
drusen formation in dry age-related macular enoids selectively accumulate with a unique dis-
degeneration (AMD) and to the autofluores- tribution in the macular region of human retina
cent flecks in autosomal recessive Stargardt [1]. The MP’s total concentration is about 1 mM
disease (STGD1). Retinal carotenoids attenu- at the fovea, and its concentration declines to less
ate A2E formation and can directly and indi- than 10 μM in the peripheral retina [2]. The ratio
rectly alleviate A2E-mediated oxidative of L:Z:MZ in the peripheral retina is about 3:1:0,
damage. In this chapter, we review these more and the concentration of total carotenoids rises
recently recognized interconnections between 100-fold in the macula lutea with a change in the
MP carotenoids and A2E bisretinoids. ratio of L:Z:MZ of about 1:1:1, as measured by
high-performance liquid chromatography
(HPLC) [3]. More recently, high-resolution con-
R. Arunkumar · P. S. Bernstein (*) focal resonance Raman microscopy imaging of
Department of Ophthalmology and Visual Sciences, the human retina from our laboratory has showed
John A. Moran Eye Center, University of Utah that the ratio of Z + MZ:L can be greater than 9:1
School of Medicine, Salt Lake City, UT, USA at the foveal center, while L is more diffusely dis-
e-mail: paul.bernstein@hsc.utah.edu

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 15

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_3
16 R. Arunkumar and P. S. Bernstein

tributed across the macula at a relatively lower aided by conjugated double-bond structure with
concentration [4]. Retinal carotenoids mainly the hydrophilic group on the ionone ring [2]. The
localize to the outer plexiform (Henle fiber) layer absorption maximum of L is around 445 nm, and
and the inner plexiform layer, with axial exten- Z is around 450 nm, corresponding with the haz-
sion from the inner limiting membrane to the ardous blue light range of 430–500 nm.
outer limiting membrane. The specificity in the Depending on the retinal carotenoid concentra-
distribution of retinal carotenoids in the human tion, they are estimated to absorb 40–90% of
retina may be due to the selective uptake of reti- incident short-wavelength, high-energy photo-
nal carotenoids by carotenoid binding proteins toxic blue light [8].
StARD3 (L binding protein) and GSTP1 (Z and Retinal carotenoids with conjugated double
MZ binding protein). The other major factors that bond (C=C) structures are associated with greater
influence the MP carotenoid distributions and singlet oxygen quenching. Z has 11 conjugated
concentrations between individuals are the double bonds and is 50% better at quenching sin-
amount of oral intake of carotenoids and their glet oxygen relative to L with its 10-conjugated
bioavailability [5]. L is present in higher concen- double bonds [9]. MZ, a stereoisomer of Z, with
trations in the diet, but it is converted to MZ by an identical 11-conjugated double-bond structure
the RPE65 enzyme, and the preferential accumu- would be expected to possess the same antioxi-
lation of Z and MZ at the foveal center may be dant properties as Z, but it exhibited better singlet
due to their higher antioxidant potential relative oxygen quenching properties relative to L and Z
to L. The fovea is prone to photo-damage caused [9]. Furthermore, the antioxidant activity of all
by light-induced oxidative stress from reactive three retinal carotenoids as a mixture showed bet-
oxygen species, and singlet oxygen can be gener- ter singlet oxygen quenching ability than any of
ated by photo-oxidation of A2E and other bisreti- the individual MP carotenoids. Retinal carot-
noid components of lipofuscin [6]. A2E acts as a enoids not only quench singlet oxygen, but they
photosensitizer in the presence of short-­ also quench superoxide anion radicals and
wavelength blue light and oxygen that generate hydroxyl radicals, the major causative agents of
reactive oxygen caused by photo-oxidation and lipid peroxidation and light-induced damage [10,
photo-degradation, resulting in retinal degenera- 11]. Generally, retinal carotenoids are better
tion and apoptosis of photoreceptor and RPE hydroxyl radical scavengers than superoxide
cells [7]. Supplementation with retinal carot- anion scavengers, and Z exhibits better hydroxyl
enoids potentially attenuates harmful blue light radical scavenging activity than lutein [10]. MP
and effectively scavenges singlet oxygen and carotenoids protect the retina, which is vulnera-
reactive free radicals in ocular tissues and in bio- ble to oxidative damage caused by exposure to
chemical assays. light, high concentration of oxygen, abundant
photosensitizers, and high concentrations of
polyunsaturated fatty acids (PUFAs).
2 Structure and Function
of the Retinal Carotenoids
3 A2E Bisretinoids
Retinal carotenoids are characterized by the pres-
ence of hydroxyl (O-H) functional groups In the visual cycle, the activation of rhodopsin by
attached at the 3 and 3′ positions of terminal ion- a photon of light during phototransduction results
one rings connected by an isoprenoid backbone in the isomerization of 11-cis-retinal to all-trans-­
structure (Fig. 1). The presence of hydroxyl retinal in photoreceptor outer segments and the
groups and the conjugated double bonds struc- release of all-trans-retinal, which is subsequently
ture determine the light-absorbing properties and reduced to all-trans-retinol and transported to the
antioxidant activities of retinal carotenoids. The RPE cells. In the RPE, the all-trans-retinol is
light filtering function of retinal carotenoids is either stored as a fatty acid ester or is converted
Macular Pigment Carotenoids and Bisretinoid A2E
HO
Lutein
HO
Zeaxanthin
HO
Meso-Zeaxanthin
Fig. 1 Structures of the macular carotenoids: lutein (L), zeaxanthin (Z), meso-zeaxanthin (MZ), and N-retinylidene-N-­
retinylethanolamine (A2E)
back to an 11-cis-retinoid that is transported back
to the photoreceptor outer segment, which can
bind to opsin to produce a regenerated visual pig-
ment. Some all-trans-retinal in the photoreceptor
outer segments reacts with phosphatidylethanol-
amine (PE) to form N-retinylidene-PE (NRPE),
which is transported or “flipped” by the ATP-­
binding cassette, subfamily A, member 4
(ABCA4) protein present in photoreceptor outer
segments. ABCA4 translocates NRPE across the
lipid bilayer from the luminal side to the cyto-
plasmic side of the disc membrane. NRPE
released on the cytoplasmic side is hydrolyzed
into all-trans-retinal and reduced to all-trans-­
retinol by retinol dehydrogenases (RDHs) [12]. If
ABCA4 does not effectively translocate NRPE, it
reacts with one more molecule of all-trans-­retinal
to form a toxic bisretinoid, dihydropyridinium-­
A2PE. Dihydropyridinium-A2PE is further
oxidized either to A2-dihydropyridine-PE
­ loss of membrane integrity and perturbs mem-
(A2-DHP-PE) by eliminating one hydrogen or to
phosphatidylethanolamine-pyridinium bisreti-
noid (A2PE) by eliminating two hydrogens in the
acidic and oxidizing environment (Fig. 2). During
phagocytosis of photoreceptor outer segments by
17
OH
OH
OH
N
OH
A2E
RPE, A2PE is further hydrolyzed to A2E by
phospholipase D inside RPE phagosomes.
Mutation or dysfunction in the ABCA4 gene
leads to excessive accumulation of A2E and other
bisretinoids in RPE lipofuscin, resulting in reti-
nal cell death and vision loss. A2E is reported to
accumulate with aging and dry AMD and in
STGD1 [13].
A2E (C42H58ON; molecular weight, 592) is a
well-characterized component of lipofuscin. It
exhibits absorbance in both the UV and visible
regions of the spectrum [A2E: absorbance max-
ima (λmax) are 440 and 340 nm, and iso-A2E’s
λmax are 430 and 340 nm]. A2E’s hydrophobic
side arms and positively charged hydrophilic
head group confer an amphiphilic structure
(Fig. 1). A2E has a detergent-like structure that
may influence membrane properties and inhibit
the lysosomal degradation of lipids. A2E induces
brane stability, and it inhibits cytochrome C oxy-
genase and the ATP-driven proton pump. A2E
photo-oxidation products can activate the com-
plement system and cause inflammation and
DNA damage [12, 13].
18
Fig. 2 The visual cycle and bisretinoid formation in pho-
toreceptor outer segments and RPE. When a photon of
light is captured by the visual chromophore, its 11-cis-­
retinal (11-cis-RAL) Schiff base chromophore photoi-
somerizes to all-trans-retinal (all-trans-RAL). The
released all-trans-RAL from opsin is reduced to all-trans-­
retinol (all-trans-ROL) by retinol dehydrogenases
(RDHs). Alternatively, some all-trans-RAL reacts with
phosphatidylethanolamine (PE) in the photoreceptor outer
segments to form N-retinylidene-PE (NRPE), which is
transported by ABCA4. The bisretinoid synthesis path-
way (red) is initiated when NRPE, rather than hydrolyzing
to all-trans-ROL and PE, reacts with a second molecule of
retinaldehyde. A multistep pathway leads to the formation
of the intermediate dihydropyridinium-A2PE. Auto-­
oxidation of dihydropyridinium-A2PE with loss of two
4 Retinal Carotenoids
Attenuate Bisretinoids
in Various Systems
4.1 In Vitro
A2E and its precursor A2PE can be irradiated
with 430 nm blue light and analyzed by fast atom
bombardment mass spectroscopy (FAB-MS).
The FAB-MS spectra after irradiation not only
showed a molecular ion peak at m/z 592 and 1223
attributable to A2E and A2PE, but they also
R. Arunkumar and P. S. Bernstein
hydrogens generates A2PE, and loss of one hydrogen gen-
erates A2-DHP-PE; phosphate hydrolysis of the latter
produces A2-DHP-E. A2E, lysoA2PE, and A2-GPE are
produced from A2PE. Reaction of the all-trans-RAL
dimer with PE with the formation of a Schiff base linkage
generates all-trans-retinal dimer-PE (atRALdi-PE), and
phosphate hydrolysis of the latter yields all-trans-retinal
dimer-ethanolamine (atRALdi-E). All-trans-ROL is
transported into RPE cells where all-trans-ROL is esteri-
fied to fatty acids to make all-trans retinyl esters (all-­
trans-­RE) by the enzyme lecithin retinol acyl transferase
(LRAT). All-trans-RE is converted to 11-cis retinol
(11-cis-ROL) by the RPE65 isomerohydrolase. 11-cis
retinol dehydrogenase (11cRDH) oxidizes 11-cis-ROL to
11-cis-RAL which enters the photoreceptor outer seg-
ments for the regeneration of opsin
showed additional peaks at m/z 608, 624, 640,
656, 672, and 688, as well as 1239, 1255, 1271,
and 1286. The additional higher m/z peaks are
photo-oxidation products of A2E and A2PE
formed by the insertion of oxygen atoms at the
C=C bonds along the side arms of A2E and
A2PE. When A2E and A2PE are irradiated at
430 nm in the presence of L and Z, the additional
photo-oxidized peaks are absent [14]. Singlet
oxygen quenching abilities of retinal carotenoids
were studied in the presence of endoperoxide
1,4-dimethylnapthalene (1 mM), which releases
Macular Pigment Carotenoids and Bisretinoid A2E 19

singlet oxygen with a half-life time of 5 h. @ 3.1-fold in the zeaxanthin-supplemented

25 °C. The endoperoxide was incubated with
A2E (500 μM) with and without retinal carot-
enoids (500 μM to 4 mM). A2E oxidation was
measured using HPLC, and it was found that reti-
nal carotenoids effectively quench singlet oxygen
products in a concentration-dependent manner.
Furthermore, Z was observed to be a better sin-
glet oxygen quencher than L [14].
group. A2E levels increased sixfold in the
unsupplemented control group, whereas there
was minimal increase in A2E in the MP carot-
enoid supplemented group, and their A2E lev-
els were significantly lower than the control
group [16].
4.5 Rodent Model

4.2 Cell Culture Until recently, the protective effects of macular

carotenoids against bisretinoids had not been
The protective effects of retinal carotenoids on studied in small laboratory mammals because
photo-oxidation of A2E were studied in ARPE-­ non-primates do not accumulate substantial
19 cells grown in Dulbecco’s Modified Eagle amounts of carotenoids in ocular tissues due to
Medium (DMEM) medium with 10 μM A2E for the presence of very active carotenoid cleavage
10 days. The cells accumulated A2E and were enzymes (Bco1 and Bco2). This led us to cross
incubated with L or Z (10 μM), and they were our Bco2−/− “macular pigment mice” that accu-
then exposed to blue light (430 nm). Cells incu- mulate dietary MP carotenoids in their retinas
bated with retinal carotenoids completely attenu- with a mouse model of STGD that accumulates
ated A2E photo-oxidation and inhibited A2E with increasing age. Abca4−/−/Bco2−/− mice
proteasome inactivation [15]. fed with L or Z have lower levels of A2E and iso-­
A2E compared to the control group with no
carotenoid supplementation, and there was a sta-
4.3 Human Donor Eyes tistically significant inverse correlation between
retinal carotenoids and A2E and iso-A2E levels
A2E concentrations in human cadaver eyes in RPE/choroid (Fig. 3). Furthermore, L and Z
increase with age in both the macula and in the supplementation improved visual performance in
peripheral retina, and A2E levels are threefold Abca4−/−/Bco2−/− mice compared to the control
lower in the macula region compared to the group [13].
peripheral retina despite excess light exposure
and high metabolic activity. Moreover, A2E con-
centrations are inversely related to retinal carot- 5 Summary
enoid concentrations [16]. Lipofuscin and A2E
levels are significantly lower in the fovea region The macular pigment carotenoids are distinct
where retinal carotenoids are present in abun- from other nutrients due to their selective accu-
dance [17]. mulation and unique spatial distribution in the
fovea, the region most vulnerable to excess light
and high metabolic oxygen. While it is widely
4.4 Avian Model appreciated that the beneficial properties of MP
carotenoids are in part mediated by their blue
The association between retinal carotenoids light-absorbing and antioxidant properties, it is
and A2E was studied in Japanese quail, whose now becoming increasingly understood that
retinal carotenoids are present as fatty acid attenuation of A2E formation and oxidation by L,
esters in distinct photoreceptor oil drops. After Z, and MZ are important protective effects as
16 weeks of feeding, total carotenoids rose well, further emphasizing their valuable role as
1.6-fold in the lutein-supplemented group and safe and cost-effective nutritional interventions
20
Fig. 3 Distribution of RPE/choroid A2E + iso-A2E levels
in relation to retinal carotenoids in Abca4−/−/Bco2−/− mice.
There was a statistically significant inverse correlation
between retinal carotenoids and A2E and iso-A2E levels
in RPE/choroid (p values: *p < 0.05). Red line refers to L
with R2 = 0.815, blue line refers to Z with R2 = 0.825, and
the orange dotted line refers to combined regression plot
of L, Z, and placebo group with R2 = 0.904. [13]
against visual loss from age-related and inherited
macular diseases.
Conflict of Interest No authors have any conflicts of
interest.
Grant Support EY-11600 and EY-14800; Research to
Prevent Blindness.
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 Disturbed Matrix
Metalloproteinases Activity
in Age-Related Macular
Degeneration

Beatriz Martins and Rosa Fernandes

Abstract ulation and TIMPs’ significant regulatory

Matrix metalloproteinases (MMPs) are a
tightly regulated family of proteolytic
enzymes that break down extracellular matrix
(ECM) and basement membrane components.
Because it is associated with development,
morphogenesis, tissue remodeling, and repair,
ECM remodeling is an important mechanism.
MMPs are thought to act as a double-edged
sword, as they contribute to maintaining pho-
toreceptors/retinal pigment epithelium (RPE)/
Bruch’s membrane (BM)/choroid complex
homeostasis and also contribute to the onset
and progression of age-related macular degen-
eration (AMD). Polymorphisms and/or altered
expression in MMPs and their tissue inhibitors
(TIMPs) are associated with age-related mac-
ular degeneration (AMD). Here, we review
the evidence for MMPs’ role in the onset and
progression of AMD via addressing their reg-
functions.
Keywords
Age-related macular degeneration · Matrix
metalloproteinases · Extracellular matrix ·
Bruch’s membrane · Geographic atrophy ·
Choroidal neovascularization
1 Introduction
Matrix metalloproteinases (MMPs), also known
as matrixins, belong to an important class of
zinc-dependent endopeptidases, the metzincins
superfamily [1]. This superfamily includes 21
human disintegrins and metalloproteinase
domain (ADAMs) and 19 secreted disintegrin
and metalloproteinase thrombospondin domain
(ADAMTSs), in addition to 23 MMPs expressed

B. Martins R. Fernandes (*)

Faculty of Medicine, Coimbra Institute for Clinical
and Biomedical Research (iCBR), University of
Coimbra, Coimbra, Portugal
Institute of Pharmacology and Experimental
Therapeutics, Faculty of Medicine, University of
Coimbra, Coimbra, Portugal
Faculty of Medicine, Coimbra Institute for Clinical
and Biomedical Research (iCBR), University of
Coimbra, Coimbra, Portugal
Institute of Pharmacology and Experimental
Therapeutics, Faculty of Medicine, University of
Coimbra, Coimbra, Portugal
Association for Innovation and Biomedical Research
on Light and Image (AIBILI), Coimbra, Portugal
e-mail: rcfernandes@fmed.uc.pt

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 21

J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_4
22
in human tissue [2]. MMPs typically have an
80-amino acid propeptide, a 170-amino acid cat-
alytic metalloproteinase domain, a variable-­
length linker peptide (hinge region), and a
200-amino acid hemopexin (Hpx) domain. A
detailed description of MMPs structure and an
overview of their substrate preferences and their
association with extracellular matrix (ECM) com-
ponents and inhibitors can be found in other
excellent reports [2–4].
The MMPs are usually secreted as zymogens
by a variety of cells into the extracellular space
[3] or stay tethered to their plasma membrane [5].
In humans, there are six membrane-type (MT)-
MMPs [5]. In the generation of active MMPs,
enzymatic cleavages of the zymogens are needed.
Active MMP-2, for example, is made from pro-­
MMP-­2 by MMP-14 cleavage, while pro-MMP-9
is cleaved by MMP-3 [5, 6].
MMPs are mainly classified based on their
substrate preference and domain organization,
being grouped into gelatinases (MMP-2, MMP-­
9), collagenases (MMP-1, MMP-8, MMP-13),
stromelysins (MMP-3, MMP-10), MT-MMPs
(MMP-14, MMP-15, MMP-16, MMP-17), and
others [7]. Under normal conditions, both MMPs
and the tissue inhibitors of metalloproteinases
(TIMPs) have an important role in the regulation
of the turnover of ECM [2, 8]. A dysregulation of
these components can both contribute to patho-
logical ECM remodeling and serve as a nexus for
a variety of pathways involved in age-related
macular degeneration (AMD) pathogenesis.
2 MMPs Are Implicated in AMD
AMD is a retinal degenerative disease that is a
leading cause of central vision loss in the elderly.
This disease, in its early stages, is characterized
by the accumulation of drusen that causes pro-
gressive degeneration of the photoreceptors and
retinal pigment epithelium (RPE). The disease
can progress to geographic atrophy (GA), an
advanced form of dry AMD, and/or choroidal
neovascularization (CNV), also known as wet
AMD [9, 10].
B. Martins and R. Fernandes
The specific interplay of pathophysiological
events that converge to AMD and related degen-
eration mechanisms remain to be fully elucidated
[11]. Nevertheless, several studies have been
highlighting the alterations in the regulation of
ECM, by dysregulation of the activity of MMPs
and TIMPs, as one of the main mechanisms
involved in the development of AMD [12]. This
modulation of ECM turnover is associated with
the other molecular mechanisms involved in the
pathophysiology of the disease since some stud-
ies have shown that both oxidative stress and acti-
vation of the complement system can modulate
the activity of ECM components [13, 14].
Additionally, the formation of drusen, the hall-
mark of AMD, is also believed to be associated
with a dysregulation of the ECM components,
especially in the RPE and Bruch’s membrane
(BM) [15]. These alterations in ECM modulation
caused by dysregulation of MMP/TIMP complex
can also be influenced not only by environmental
but also by genetic factors. For example, poly-
morphisms in MMP-2 (rs243865) and in MMP-9
(rs3918424 and rs3918241) are associated with
increased risk of AMD, mainly in younger
patients (<65 years) [16–18]. Other MMP-2
polymorphisms (rs243865, rs243866, and
rs2287074) are associated with a lower likeli-
hood of AMD development [19, 20]. There are
also some TIMPs polymorphisms associated
with AMD. Despite some polymorphisms in
TIMP-2 (rs817909) and in TIMP-3 (rs5754227)
appear to have a protective effect, other polymor-
phisms in TIMP-3 (rs713685 and rs743751) are
more frequent in AMD patients [18, 21, 22]. As a
result, these findings highlight evidence that
changes in ECM turnover are essential mecha-
nisms implicated in the development of AMD
and may help explain the susceptibility to the dis-
ease’s late stages.
2.1 MMPs in Dry AMD
BM is a five-layered ECM located at the inter-
face of the retina and choriocapillaris. BM plays
an important role in cell-cell communication and
Disturbed Matrix Metalloproteinases Activity in Age-Related Macular Degeneration
regulates the exchange of oxygen and nutrients
between the choroid and the outer retina [23].
However, BM undergoes a number of age-related
alterations that may have a role in AMD patho-
genesis. Especially noteworthy, the accumulation
of cellular debris and ECM proteins leads BM to
thicken and to the formation of drusen [24].
Several studies have been demonstrated that
aging causes structural and functional changes in
the BM due to a decrease in the activity of the
gelatinase components of the MMP system. In
this case, increased levels of high molecular
weight components of the MMPs pathway
(HMW1 and HMW2) reduce the pool of the acti-
vated gelatinases MMP-2 and MMP-9 and con-
tribute to reduced matrix degradation and
thickness of BM [25–27]. Another study revealed
that higher TIMP-3 concentrations in the drusen
block MMPs activity, lowering proteolytic activ-
ity within the drusen [28]. Oxidative stress
reduces MMP-2 activity and leads to the forma-
tion of sub-RPE deposits that can be involved in
the formation of drusen during AMD develop-
ment [29].
Nevertheless, several other studies also
detected increased levels of other MMPs in
patients with GA. MMP-7 levels were found to
be lower in these patients, but MMP-9 and
TIMP-1 levels were higher [30, 31]. In vitro and
in vivo models of dry AMD led to increased
expression, secretion, and activation of MMP-1,
MMP-3, and MMP-9 and are associated with
decreased RPE cell viability [32–34]. Moreover,
MMP-9’s role in the breakdown of the RPE bar-
rier has been highlighted, as silencing of this
MMP can improve the barrier integrity [35, 36].
Regarding genetic alterations, only a few stud-
ies have related polymorphisms specifically with
dry AMD. Liutkeviciene et al. detected that a
polymorphism in the MMP-2 gene (rd243865) is
associated with the development of hard drusen
in AMD patients [37].
2.2 MMPs in Wet AMD
The choroid is a network of blood vessels, located
below the BM that is critical for nutrients and
23
oxygen supply to the outer retina. The oxidative
and inflammatory processes that occur in late
stages of AMD might promote a pro-angiogenic
environment, resulting in CNV, which is the hall-
mark of wet AMD. Neovessels from choroid,
also referred as CNV, develop beneath the RPE
or penetrate into the subretinal space in patients
with wet AMD [38, 39].
Similar to GA and BM thickness, in this case
also dysregulation of ECM turnover appears to
have a close relation with CNV. Increased activ-
ity of MMPs can degrade some components of
BM, essential to the growth of new vessels [40,
41]. Several studies have shown that patients with
wet AMD present increased levels of MMP-9 in
aqueous humor, vitreous, and plasma [42–44].
This increase in the levels of MMP-9 contributes
to CNV since this MMP can increase VEGF lev-
els (pro-angiogenic factor) by decreasing PEDF
levels (anti-angiogenic factor), both secreted by
the RPE [45]. MMP-2, MMP-7, and MMP-13
have likewise shown a rise in their expression and
levels in patients with CNV lesions [18, 46, 47].
Moreover, cellular and animal models of CNV
lesions reported increased expression of MMP-2
and MMP-9, which were involved in the forma-
tion of experimental CNV. Additionally, the inhi-
bition of these two gelatinases appears to be a
good strategy to treat CNV, since their elimina-
tion is associated with decreased CNV lesion size
[48–50]. Some studies have also been underlin-
ing the alterations in TIMPs activity as an impor-
tant mechanism underlying CNV. On both wet
AMD patients and animal models, decreased lev-
els of TIMPs (TIMP-2 and TIMP-3) contribute to
CNV susceptibility [18, 51].
Regarding genetic alterations, several studies
identified various polymorphisms on both MMPs
and TIMPs that are related to CNV and wet
AMD. Microsatellites in the promoter of MMP-­
9, along with other polymorphisms on the
MMP-9 gene (rs142450006, rs3918424), have
been associated with increased risk of CNV pro-
gression and wet AMD development [17, 52, 53].
Also polymorphisms on MMP-1 (rs1799750),
MMP-2 (rs243865), MMP-7 (rs11568818), and
MMP-20 (rs10895322) are associated with
increased CNV lesion size and development of
24
wet AMD [54–56]. Finally, a polymorphism in
the TIMP-3 gene (rs9621532) has a protective
effect and is associated with decreased risk of
wet AMD [57].
3 Concluding Remarks
Due to their ability to break down ECM constitu-
ents, MMPs are relevant components in many
pathological processes of AMD. Specifically,
they play an important role in the accumulation
of drusen and degeneration of photoreceptors/RPE
in dry AMD and pathological vessel growth in
wet AMD. TIMPs control changes in their
expression and activity. To better understand the
role of each MMP in AMD pathogenesis, further
research is needed.
Acknowledgments Funding provided by the Global
Ophthalmology Awards Program (GOAP), a Bayer spon-
sored initiative committed to supporting ophthalmic
research across the world. Thanks are also due to FCT
(Portugal) and Strategic Projects UIDB/04539/2020 and
UIDP/04539/2020 (CIBB), COMPETE-FEDER (POCI-
01-0145-FEDER-007440), and Centro 2020 Regional
Operational Program: BRAINHEALTH 2020
(CENTRO-01-0145- FEDER-­000008). The authors also
acknowledge FCT for the doctoral research grant
2020.04811.BD (to B.M.).
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 Current Views on Chr10q26
Contribution to Age-Related
Macular Degeneration
Navdeep Gogna, Lillian F. Hyde, Gayle B. Collin,
Lisa Stone, Jurgen K. Naggert,
and Patsy M. Nishina
Abstract
Age-related macular degeneration (AMD) is
the leading cause of blindness in the global
aging population. Familial aggregation and
genome-wide association (GWA) studies have
identified gene variants associated with AMD,
implying a strong genetic contribution to
AMD development. Two loci, on human Chr
1q31 and 10q26, respectively, represent the
most influential of all genetic factors. While
the role of CFH at Chr 1q31 is well estab-
lished, uncertainty remains about the genes
ARMS2 and HTRA1, at the Chr 10q26 locus.
Since both genes are in strong linkage disequi-
librium, assigning individual gene effects is
difficult. In this chapter, we review current lit-
erature about ARMS2 and HTRA1 and their
relevance to AMD risk. Future studies will be
necessary to unravel the mechanisms by which
they contribute to AMD.
Keywords
AMD · ARMS2 · HTRA1 · Linkage disequi-
librium · Genetics
N. Gogna (*) · L. F. Hyde · G. B. Collin · L. Stone ·
J. K. Naggert · P. M. Nishina
The Jackson Laboratory, Bar Harbor, ME, USA
e-mail: navdeep.gogna@jax.org
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
J. D. Ash et al. (eds.), Retinal Degenerative Diseases XIX, Advances in Experimental Medicine
and Biology 1415, https://doi.org/10.1007/978-3-031-27681-1_5
1 Introduction
Age-related macular degeneration (AMD) is the
leading cause of irreversible central vision loss in
aging populations [1, 2]. By 2050, an estimated
22 million people in the USA [3], and, by 2040,
288 million people worldwide [4], will be
affected with AMD. Early stages of AMD are
characterized by the presence of few (<20)
medium-sized drusen and/or retinal pigment epi-
thelium (RPE) abnormalities [5–7]. Drusen,
lipid-rich, protein-containing deposits that accu-
mulate between the RPE and Bruch’s membrane,
are considered a “hallmark” of AMD [8].
Intermediate AMD exhibits at least one large
druse, multiple medium-size drusen, or geo-
graphic atrophy (GA) that does not extend to the
center of the macula [5]. Advanced or late AMD
is either non-neovascular (dry, atrophic, or non-
exudative) or neovascular (wet or exudative) [9].
Dry AMD, characterized by drusen and GA,
extends to macula’s center and affects the chorio-
capillaris, RPE cells, and photoreceptors (PRs)
without leakage of blood or serum into the retina
and macula [10]. In contrast, wet AMD is associ-
ated with RPE detachment and choroidal neovas-
cularization, leading to leakage and fibrovascular
scarring [11].
AMD is a complex, multifactorial disease
[12–24]. Risk factors identified from case-­
control, cross-sectional, and prospective cohort
studies [10, 25–34] include age [35], family his-
27
28
tory [28, 36], dietary choices [30, 37–40], race
and ethnicity [41–43], gender [21], and smoking
[25, 44–46]. In addition, genetic factors explain
46–71% of the variation in overall disease sever-
ity [47]. To date, a total of 52 independently asso-
ciated common and rare gene variants
(p < 5 × 10−8), distributed across 34 loci, have
been associated with AMD risk [48–51].
Genome-wide association (GWA) and family-­
based linkage studies identified genetic variants
in the complement factor H (CFH) gene on
Chr1q32 and two tightly linked genes—ARMS2
and HTRA1—on Chr10q26 as major contributors
to AMD risk [52–65]. Together, these increase
AMD predisposition by more than 40-fold [66].
While significant progress has been made toward
understanding the role of CFH in AMD risk [67–
70], uncertainty remains regarding the gene(s)
responsible at the Chr10q26 locus. Strong link-
age disequilibrium between ARMS2 and HTRA1
genes has made it difficult to distinguish individ-
ual gene effects by statistical methods [71].
Moreover, PLEKHA1 is also found in linkage
disequilibrium with ARMS2 [55]. Several studies
suggest that ARMS2/HTRA1 genetic variations
are more strongly associated with AMD risk than
PLEKHA1 [56, 72, 73]. In this chapter, we review
existing information about ARMS2 and HTRA1
and their association with AMD.
2 
ARMS2
The ARMS2 gene, age-related maculopathy sus-
ceptibility 2, is only present in higher primates
[74]. ARMS2 encodes an 11-kDa protein [75];
however the function, localization, and native
structure of the protein have not been firmly
established. An in silico approach to predict the
structure of ARMS2 concluded that it has 15
putative phosphorylation sites, four putative gly-
cation sites of ε-amino groups of lysine, two
putative O-linked glycation sites, and three
potential binding sites [76]. The alanine at posi-
tion 69 is predicted to be a functional residue for
protein binding.
N. Gogna et al.
2.1 Reported Tissue, Cellular
and Subcellular Expression
of ARMS2/ARMS2
The expression of ARMS2 was detected in pla-
cental tissue [55], at low levels in retinal samples
[56, 73] and later, ubiquitously, in human tissues
[77]. The Bgee gene expression database (v14.2)
currently lists 62 distinct human tissues with
varying ARMS2 mRNA levels [78].
Kanda et al. expressed human retinal ARMS2
mRNA in COS-1, ARPE-19, and JEG cells.
Subcellular fractionation, followed by co-­staining
with MitoTracker and cytochrome C oxidase
subunit IV, located ARMS2 to the mitochondrial
(Mt) outer membrane [73]. Subsequently, another
group co-stained ARMS2 with retinoschisin (an
extracellular protein that stains PR inner seg-
ments) and Mt cytochrome c oxidase subunit II in
human retinal sections. ARMS2 localized in the
Mt-enriched ellipsoid region of the inner seg-
ments of both rod and cone PR cells [79].
However, conflicting results were presented by
Wang et al. [80]. Using immunofluorescence
(IF), verified by immunoblotting, endogenous
ARMS2 in ARPE-19 cells and overexpressed
GFP-tagged and HIS-tagged exogenous ARMS2
in COS7 cells were found to localize in the cyto-
sol and not in Mt or any other organelle [80]. The
group further performed in silico analyses to
identify the protein features required for Mt tar-
geting and concluded that ARMS2 lacked canon-
ical mitochondrial targeting sequences [80].
Kortvely et al. transfected HEK293 cells with
plasmid constructs coding for normal or risk vari-
ant ARMS2. Immunostaining findings suggested
that ARMS2 is a secreted protein and a compo-
nent of the extracellular matrix (ECM), found
mainly in choroid pillars of human eye [81].
Using a yeast two-hybrid system, followed by
coprecipitation, ARMS2 was found to bind sev-
eral ECM proteins [81]. Further, ARMS2 colo-
calized with the endoplasmic reticulum (ER) in
transfected ARPE-19 cells expressing ARMS2.
Adding a small N-terminal tag to ARMS2 did not
have any effect, whereas the same tag at the
Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration
C-terminus abolished ER colocalization, indicat-
ing that the C-terminus is integral to proper
­targeting [81]. A follow-up study concluded that
ARMS2 is secreted via an unconventional, Golgi-­
independent pathway. Blocking the canonical ER
to Golgi transport did not affect ARMS2 secre-
tion. Instead, ARMS2 was shown to colocalize
with calnexin-positive and protein disulfide
isomerase-negative vesicle-like structures and,
with GRASP65, a marker for unconventional
protein secretion. Exon 2 of ARMS2 codes for
eight amino acids (-SIIHTAAR) at the
C-terminus. By generating a set of mutants, the
two isoleucines were found to be indispensable
for correct targeting. The intracellular distribu-
tion of the A69S risk allele was similar to the
non-risk allele, with both protein variants secreted
into the culture medium [82].
Micklisch et al. found endogenous ARMS2
gene expression in human blood-derived mono-
cytes and in human-induced pluripotent stem
cell-derived microglia (iPSdM) by PCR and laser
scanning microscopy. Contrary to the study by
Kortvely et al. which showed translation of the
ARMS2 A69S variant, this study reported that
ARMS2 protein was absent in patients homozy-
gous for the ARMS2 AMD risk variant A69S or
the non-risk variant R38X [75].
2.2 
ARMS2 Risk Variants and Their
Putative Role in AMD
The missense variant, rs10490924 (G>T:A69S)
[73, 80, 82], encoding an alanine-to-serine sub-
stitution at position 69 (A69S) is the most fre-
quently observed ARMS2 risk allele in the human
population. Little is known about the effect of
this mutation on ARMS2 protein structure and
function. One study reports that the A69S varia-
tion in ARMS2 creates a new putative phosphory-
lation site that breaks a predicted α-helix [73]. It
has been suggested that the A69S change affects
ARMS2’s function in either Mt [73, 79] or its
role in the cytoskeleton in COS7cells [80]. A
recent study implicated ARMS2 as a surface com-
plement regulator [75]. Recombinant ARMS2,
expressed in Pichia pastoris, was shown to bind
29
to human apoptotic and necrotic cells and initiate
properdin-mediated complement activation and
C3b surface opsonization for phagocytosis [75].
Two additional variants of ARMS2 have been
reported: an insertion-deletion (indel) polymor-
phism (NM_001099667.1:c.*372_815del443
ins54) in the 3′untranslated region (3′-UTR) and
a coding nonsense polymorphism (R38X) [79].
The ARMS2 indel lies within the HTRA1 cis-­
regulatory region, upstream of its transcription
start site [83, 84], and is suggested to regulate
HTRA1 expression. In heterozygous retinal and
placental samples from human donors, Fritsche
et al. showed that this indel mutation destabilizes
the ARMS2 transcript by removing the polyade-
nylation signal and inserting a 54-bp element
known to mediate rapid mRNA turnover [79].
The group validated their findings by developing
recombinant ARMS2 isoforms, quantifying heter-
ologous ARMS2 expression in vitro and conclud-
ing that the indel polymorphism in ARMS2 leads
to reduced mRNA levels [83].
The ARMS2 variant identified by Fritsche
et al., rs2736911(C>T:R38X), is predicted to
result in a premature stop codon (R38X) and a
truncated protein [79]. Yang et al. observed a
50% decrease in mRNA in patients heterozygous
for R38X [85], presumably due to nonsense-­
mediated mRNA decay. They reasoned that the
loss of ARMS2 is insufficient to explain AMD
susceptibility as both the indel and R38X variants
result in a decrease in ARMS2, but only the for-
mer confers risk to AMD, while the latter is
mildly protective [85, 86]. This led to a hypothe-
sis of dual causality, in which concomitant down-
regulation of ARMS2 and upregulation of HTRA1
explains the disease [85]. Similar results were
obtained in another study that showed reduced
ARMS2 expression in human postmortem retinal
and RPE samples, heterozygous for R38X [83].
However, a subsequent investigation failed to
detect a rs2736911-dependent alteration in
ARMS2 expression [87].
Since ARMS2 is not found in mice, to gain a
better understanding of ARMS2 function, two
transgenic mouse models expressing human
ARMS2 and ARMS2 A69S were developed. The
constructs used a ubiquitous cytomegalovirus
Another random document with
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The car passed the ornamental ponds, in which the colliers threw
their newspapers, and took the private drive to the house. It stood
above, aside, a very pleasant stucco building from the middle of the
eighteenth century. It had a beautiful alley of yew trees, that had
approached an older house, and the hall stood serenely spread out,
winking its Georgian panes as if cheerfully. Behind, there were really
beautiful gardens.
Connie liked the interior much better than Wragby. It was much
lighter, more alive, shapen and elegant. The rooms were panelled
with creamy-painted panelling, the ceilings were touched with gilt,
and everything was kept in exquisite order, all the appointments
were perfect, regardless of expense. Even the corridors managed to
be ample and lovely, softly curved and full of life.
But Leslie Winter was alone. He had adored his house. But his park
was bordered by three of his own collieries. He had been a generous
man in his ideas. He had almost welcomed the colliers in his park.
Had the miners not made him rich! So, when he saw the gangs of
unshapely men lunging by his ornamental waters—not on the private
part of the park, no, he drew the line there—he would say: "the
miners are perhaps not so ornamental as deer, but they are far more
profitable."
But that was in the golden—monetarily—latter half of Queen
Victoria's reign. Miners were then "good working men."
Winter had made this speech, half apologetic, to his guest, the then
Prince of Wales. And the Prince had replied, in his rather guttural
English:
"You are quite right. If there were coal under Sandringham, I would
open a mine on the lawns, and think it first-rate landscape
gardening. Oh, I am quite willing to exchange roe-deer for colliers, at
the price. Your men are good men too, I hear."
But then, the Prince had perhaps an exaggerated idea of the beauty
of money, and the blessings of industrialism.
However, the Prince had been a King, and the King had died, and
now there was another King, whose chief function seemed to be, to
open soup-kitchens.
And the good working men were somehow hemming Shipley in. New
mining villages crowded on the park, and the squire felt somehow
that the population was alien. He used to feel, in a good-natured but
quite grand way, lord of his own domain and of his own colliers. Now,
by a subtle pervasion of the new spirit, he had somehow been
pushed out. It was he who did not belong any more. There was no
mistaking it. The mines, the industry had a will of its own, and this
will was against the gentleman-owner. All the colliers took part in the
will, and it was hard to live up against it. It either shoved you out of
the place, or out of life altogether.
Squire Winter, a soldier, had stood it out. But he no longer cared to
walk in the park after dinner. He almost hid, indoors. Once he had
walked, bare-headed, and in his patent-leather shoes and purple silk
socks, with Connie down to the gate, talking to her in his well-bred
rather haw-haw fashion. But when it came to passing the little gangs
of colliers who stood and stared without either salute or anything
else, Connie felt how the lean, well-bred old man winced, winced as
an elegant antelope stag in a cage winces from the vulgar stare. The
colliers were not personally hostile: not at all. But their spirit was
cold, and shoving him out. And deep down, there was a profound
grudge. They "worked for him." And in their ugliness, they resented
his elegant, well-groomed, well-bred existence. "Who's he!" It was
the difference they resented.
And somewhere, in his secret English heart, being a good deal of a
soldier, he believed they were right to resent the difference. He felt
himself a little in the wrong, for having all the advantages.
Nevertheless he represented a system, and he would not be shoved
out.
Except by death. Which came on him soon after Connie's call,
suddenly. And he remembered Clifford handsomely in his will.
The heirs at once gave out the order for the demolishing of Shipley.
It cost too much to keep up. No one would live there. So it was
broken up. The avenue of yews was cut down. The park was
denuded of its timber, and divided into lots. It was near enough to
Uthwaite. In the strange, bald desert of this still-one-more no-man's-
land, new little streets of semi-detacheds were run up, very
desirable! The Shipley Hall Estate!
Within a year of Connie's last call, it had happened. There stood
Shipley Hall Estate, an array of red-brick semi-detached "villas" in
new streets. No one would have dreamed that the stucco hall had
stood there twelve months before.
But this is a later stage of King Edward's landscape gardening, the
sort that has an ornamental coal mine on the lawn.
One England blots out another. The England of the Squire Winters
and the Wragby Halls was gone, dead. The blotting out was only not
yet complete.
What would come after? Connie could not imagine. She could only
see the new brick streets spreading into the fields, the new erections
rising at the collieries, the new girls in their silk stockings, the new
collier lads lounging into the Pally or the Welfare. The younger
generation were utterly unconscious of the old England. There was a
gap in the continuity of consciousness, almost American: but
industrial really. What next?
Connie always felt there was no next. She wanted to hide her head
in the sand: or at least, in the bosom of a living man.
The world was so complicated and weird and gruesome! The
common people were so many, and really, so terrible. So she
thought as she was going home, and saw the colliers trailing from
the pits, grey-black, distorted, one shoulder higher than the other,
slurring their heavy ironshod boots. Underground grey faces, whites
of eyes rolling, necks cringing from the pit roof, shoulders out of
shape. Men! Men! Alas, in some ways patient and good men. In
other ways, non-existent. Something that men should have was bred
and killed out of them. Yet they were men. They begot children. One
might bear a child to them. Terrible, terrible thought! They were good
and kindly. But they were only half, only the grey half of a human
being. As yet, they were "good." But even that was the goodness of
their halfness. Supposing the dead in them ever rose up! But no, it
was too terrible to think of. Connie was absolutely afraid of the
industrial masses. They seemed so weird to her. A life with utterly no
beauty in it, no intuition, always "in the pit."
Children from such men! Oh God, oh God!
Yet Mellors had come from such a father. Not quite. Forty years had
made a difference, an appalling difference in manhood. The iron and
the coal had eaten deep into the bodies and souls of the men.
Incarnate ugliness, and yet alive! What would become of them all?
Perhaps with the passing of the coal they would disappear again, off
the face of the earth. They had appeared out of nowhere in their
thousands, when the coal had called for them. Perhaps they were
only weird fauna of the coalseams. Creatures of another reality, they
were elementals, serving the elements of coal, as the metal workers
were elementals, serving the element of iron. Men not men, but
animas of coal and iron and clay. Fauna of the elements, carbon,
iron, silicon: elementals. They had perhaps some of the weird
inhuman beauty of minerals, the lustre of coal, the weight and
blueness and resistance of iron, the transparency of glass.
Elemental creatures, weird and distorted, of the mineral world! They
belonged to the coal, the iron, the clay, as fish belong to the sea and
worms to dead wood. The anima of mineral disintegration!
Connie was glad to be home, to bury her head in the sand. She was
glad even to babble to Clifford. For her fear of the mining and iron
Midlands affected her with a queer feeling that went all over her, like
influenza.
"Of course I had to have tea in Miss Bentley's shop," she said.
"Really! Winter would have given you tea."
"Oh yes, but I daren't disappoint Miss Bentley."
Miss Bentley was a sallow old maid with a rather large nose and
romantic disposition, who served tea with a careful intensity worthy
of a sacrament.
"Did she ask after me?" said Clifford.
"Of course!—May I ask your Ladyship how Sir Clifford is!—I believe
she ranks you even higher than Nurse Cavell!"
"And I suppose you said I was blooming."
"Yes! And she looked as rapt as if I had said the heavens had
opened to you. I said if she ever came to Tevershall she was to
come and see you."
"Me! Whatever for! See me!"
"Why yes, Clifford. You can't be so adored without making some
slight return. Saint George of Cappadocia was nothing to you, in her
eyes."
"And do you think she'll come?"
"Oh, she blushed! and looked quite beautiful for a moment, poor
thing! Why don't men marry the women who would really adore
them?"
"The women start adoring too late. But did she say she'd come?"
"Oh!" Connie imitated the breathless Miss Bentley, "your Ladyship, if
ever I should dare to presume!"
"Dare to presume! how absurd! But I hope to God she won't turn up.
And how was her tea?"
"Oh, Lipton's and very strong! But Clifford, do you realise you are the
Roman de la rose of Miss Bentley and lots like her?"
"I'm not flattered, even then."
"They treasure up every one of your pictures in the illustrated
papers, and probably pray for you every night. It's rather wonderful."
She went upstairs to change.
That evening he said to her:
"You do think, don't you, that there is something eternal in
marriage?"
She looked at him.
"But Clifford, you make eternity sound like a lid or a long, long chain
that trailed after one, no matter how far one went."
He looked at her, annoyed.
"What I mean," he said, "is that if you go to Venice, you won't go in
the hopes of some love affair that you can take au grand sérieux, will
you?"
"A love affair in Venice au grand sérieux? No, I assure you! No, I'd
never take a love affair in Venice more than au très petit sérieux."
She spoke with a queer kind of contempt. He knitted his brows,
looking at her.
Coming downstairs in the morning, she found the keeper's dog
Flossie sitting in the corridor outside Clifford's room, and whimpering
very faintly.
"Why Flossie!" she said softly, "What are you doing here?"
And she quietly opened Clifford's door. Clifford was sitting up in bed,
with the bed-table and typewriter pushed aside, and the keeper was
standing attention at the foot of the bed. Flossie ran in. With a faint
gesture of head and eyes, Mellors ordered her to the door again, and
she slunk out.
"Oh, good morning Clifford!" Connie said. "I didn't know you were
busy." Then she looked at the keeper, saying good morning to him.
He murmured his reply, looking at her as if vaguely. But she felt a
whiff of passion touch her, from his mere presence.
"Did I interrupt you, Clifford? I'm sorry."
"No, it's nothing of any importance."
She slipped out of the room again, and up to the blue boudoir on the
first floor. She sat in the window, and saw him go down the drive,
with his curious, silent motion, effaced. He had a natural sort of quiet
distinction, an aloof pride, and also a certain look of frailty. A hireling!
One of Clifford's hirelings! "The fault, dear Brutus, is not in our stars,
but in ourselves, that we are underlings."
Was he an underling? Was he? What did he think of her?
It was a sunny day, and Connie was working in the garden, and Mrs.
Bolton was helping her. For some reason, the two women had drawn
together, in one of the unaccountable flows and ebbs of sympathy
that exist between people. They were pegging down carnations, and
putting in small plants for the summer. It was work they both liked.
Connie especially felt a delight in putting the soft roots of young
plants into a soft black puddle, and cradling them down. On this
spring morning she felt a quiver in her womb too, as if the sunshine
had touched it and made it happy.
"It is many years since you lost your husband?" she said to Mrs.
Bolton, as she took up another little plant and laid it in its hole.
"Twenty-three!" said Mrs. Bolton, as she carefully separated the
young columbines into single plants. "Twenty-three years since they
brought him home."
Connie's heart gave a lurch, at the terrible finality of it. "Brought him
home!"
"Why did he get killed, do you think?" she asked. "He was happy
with you?"
It was a woman's question to a woman. Mrs. Bolton put aside a
strand of hair from her face, with the back of her hand.
"I don't know, my Lady! He sort of wouldn't give in to things: he
wouldn't really go with the rest. And then he hated ducking his head
for anything on earth. A sort of obstinacy, that gets itself killed. You
see he didn't really care. I lay it down to the pit. He ought never to
have been down pit. But his dad made him go down, as a lad; and
then, when you're over twenty, it's not very easy to come out."
"Did he say he hated it?"
"Oh no! Never! He never said he hated anything. He just made a
funny face. He was one of those who wouldn't take care: like some
of the first lads as went off so blithe to the war and got killed right
away. He wasn't really wezzle-brained. But he wouldn't care. I used
to say to him: 'You care for nought nor nobody!' But he did! The way
he sat when my first baby was born, motionless, and the sort of fatal
eyes he looked at me with, when it was over! I had a bad time, but I
had to comfort him. 'It's all right, lad, it's all right!' I said to him. And
he gave me a look, and that funny sort of smile. He never said
anything. But I don't believe he had any right pleasure with me at
nights after; he'd never really let himself go. I used to say to him:
'Oh, let thysen go, lad!'—I'd talk broad to him sometimes. And he
said nothing. But he wouldn't let himself go, or he couldn't. He didn't
want me to have any more children. I always blamed his mother, for
letting him in th' room. He'd no right t'ave been there. Men makes so
much more of things than they should, once they start brooding."
"Did he mind so much?" said Connie in wonder.
"Yes, he sort of couldn't take it for natural, all that pain. And it spoilt
his pleasure in his bit of married love. I said to him: If I don't care,
why should you? It's my look-out!—But all he'd ever say was: It's not
right!"
"Perhaps he was too sensitive," said Connie.
"That's it! When you come to know men, that's how they are: too
sensitive in the wrong place. And I believe, unbeknown to himself, he
hated the pit, just hated it. He looked so quiet when he was dead, as
if he'd got free. He was such a nice looking lad. It just broke my heart
to see him, so still and pure looking, as if he'd wanted to die. Oh, it
broke my heart, that did. But it was the pit."
She wept a few bitter tears, and Connie wept more. It was a warm
spring day, with a perfume of earth and of yellow flowers, many
things rising to bud, and the garden still with the very sap of
sunshine.
"It must have been terrible for you!" said Connie.
"Oh, my Lady! I never realised at first. I could only say: Oh my lad,
what did you want to leave me for!—That was all my cry. But
somehow I felt he'd come back."
"But he didn't want to leave you," said Connie.
"Oh, no, my Lady! That was only my silly cry. And I kept expecting
him back. Especially at nights. I kept waking up thinking: Why he's
not in bed with me!—It was as if my feelings wouldn't believe he'd
gone. I just felt he'd have to come back and lie against me, so I
could feel him with me. That was all I wanted, to feel him there with
me, warm. And it took me a thousand shocks before I knew he
wouldn't come back, it took me years."
"The touch of him," said Connie.
"That's it, my Lady! the touch of him! I've never got over it to this day,
and never shall. And if there's a heaven above, he'll be there, and
will lie up against me so I can sleep."
Connie glanced at the handsome, brooding face in fear. Another
passionate one out of Tevershall! The touch of him! For the bonds of
love are ill to loose!
"It's terrible, once you've got a man into your blood!" she said.
"Oh, my Lady! And that's what makes you feel so bitter. You feel
folks wanted him killed. You feel the pit fair wanted to kill him. Oh, I
felt, if it hadn't been for the pit, an' them as runs the pit, there'd have
been no leaving me. But they all want to separate a woman and a
man, if they're together."
"If they're physically together," said Connie.
"That's right my Lady! There's a lot of hard-hearted folks in the world.
And every morning when he got up and went to th' pit, I felt it was
wrong, wrong. But what else could he do? What can a man do?"
A queer hate flared in the woman.
"But can a touch last so long?" Connie asked suddenly. "That you
could feel him so long?"
"Oh my Lady, what else is there to last? Children grows away from
you. But the man, well—! But even that they'd like to kill in you, the
very thought of the touch of him. Even your own children! Ah well!
We might have drifted apart, who knows. But the feeling's something
different. It's 'appen better never to care. But there, when I look at
women who's never really been warmed through by a man, well,
they seem to me poor dool-owls after all, no matter how they may
dress up and gad. No, I'll abide by my own. I've not much respect for
people."
 CHAPTER XII
Connie went to the wood directly after lunch. It was really a lovely
day, the first dandelions making suns, the first daisies so white. The
hazel thicket was a lacework of half-open leaves, and the last dusty
perpendicular of the catkins. Yellow celandines now were in crowds,
flat open, pressed back in urgency, and the yellow glitter of
themselves. It was the yellow, the powerful yellow of early summer.
And primroses were broad, and full of pale abandon, thick-clustered
primroses no longer shy. The lush, dark green of hyacinths was a
sea, with buds rising like pale corn, while in the riding the forget-me-
nots were fluffing up, and columbines were unfolding their ink-purple
riches, and there were bits of bluebird's eggshell under a bush.
Everywhere the bud-knots and the leap of life!
The keeper was not at the hut. Everything was serene, brown
chickens running lustily. Connie walked on towards the cottage,
because she wanted to find him.
The cottage stood in the sun, off the wood's edge. In the little garden
the double daffodils rose in tufts, near the wide-open door, and red
double daisies made a border to the path. There was the bark of a
dog, and Flossie came running.
The wide-open door! so he was at home. And the sunlight falling on
the red-brick floor! As she went up the path, she saw him through the
window, sitting at the table in his shirtsleeves, eating. The dog
wuffed softly, slowly wagging her tail.
He rose, and came to the door, wiping his mouth with a red
handkerchief, still chewing.
"May I come in?" she said.
"Come in!"
The sun shone into the bare room, which still smelled of a mutton
chop, done in a dutch oven before the fire, because the dutch oven
still stood on the fender, with the black potato-saucepan on a piece
of paper beside it on the white hearth. The fire was red, rather low,
the bar dropped, the kettle singing.
On the table was his plate, with potatoes and the remains of the
chop; also bread in a basket, salt, and a blue mug with beer. The
tablecloth was white oil-cloth. He stood in the shade.
"You are very late," she said. "Do go on eating!"
She sat down on a wooden chair, in the sunlight by the door.
"I had to go to Uthwaite," he said, sitting down at table but not eating.
"Do eat," she said.
But he did not touch the food.
"Shall y'ave something?" he asked her. "Shall y'ave a cup of tea? t'
kettle's on t' boil." He half rose again from his chair.
"If you'll let me make it myself," she said rising. He seemed sad, and
she felt she was bothering him.
"Well, teapot's in there,"—he pointed to a little, drab corner
cupboard; "an' cups. An' tea's on t' mantel ower yer 'ead."
She got the black teapot, and the tin of tea from the mantelshelf. She
rinsed the teapot with hot water, and stood a moment wondering
where to empty it.
"Thrown it out," he said, aware of her. "It's clean."
She went to the door and threw the drop of water down the path.
How lovely it was here, so still, so really woodland. The oaks were
putting out ochre yellow leaves; in the garden the red daisies were
like red plush buttons. She glanced at the big, hollow sandstone slab
of the threshold, now crossed by so few feet.
"But it's lovely here," she said. "Such a beautiful stillness, everything
alive and still."
He was eating again, rather slowly and unwillingly, and she could
feel he was discouraged. She made the tea in silence, and set the
teapot on the hob, as she knew the people did. He pushed his plate
aside and went to the back place; she heard a latch click, then he
came back with cheese on a plate, and butter.
She set the two cups on the table, there were only two.
"Will you have a cup of tea?" she said.
"If you like. Sugar's in th' cupboard, an' there's a little cream-jug.
Milk's in a jug in th' pantry."
"Shall I take your plate away?" she asked him. He looked up at her
with a faint ironical smile.
"Why ... if you like," he said, slowly eating bread and cheese. She
went to the back, into the penthouse scullery, where the pump was.
On the left was a door, no doubt the pantry door. She unlatched it,
and almost smiled at the place he called a pantry; a long narrow
whitewashed slip of a cupboard. But it managed to contain a little
barrel of beer, as well as a few dishes and bits of food. She took a
little milk from the yellow jug.
"How do you get your milk?" she asked him, when she came back to
the table.
"Flints! They leave me a bottle at the warren end. You know, where I
met you!"
But he was discouraged.
She poured out the tea, poising the cream-jug.
"No milk," he said; then he seemed to hear a noise, and looked
keenly through the doorway.
"'Appen we'd better shut," he said.
"It seems a pity," she replied. "Nobody will come, will they?"
"No unless it's one in a thousand, but you never know."
"And even then it's no matter," she said. "It's only a cup of tea.
Where are the spoons?"
He reached over, and pulled open the table drawer. Connie sat at
table in the sunshine of the doorway.
"Flossie!" he said to the dog, who was lying on a little mat at the stair
foot. "Go an' hark, hark!"
He lifted his finger, and his "hark!" was very vivid. The dog trotted out
to reconnoitre.
"Are you sad today?" she asked him.
He turned his blue eyes quickly, and gazed direct on her.
"Sad! no, bored! I had to go getting summonses for two poachers I
caught, and oh well, I don't like people."
He spoke cold, good English, and there was anger in his voice.
"Do you hate being a gamekeeper?" she asked.
"Being a gamekeeper, no! So long as I'm left alone. But when I have
to go messing around at the police station, and various other places,
and waiting for a lot of fools to attend to me ... oh well, I get mad ..."
and he smiled, with a certain faint humour.
"Couldn't you be really independent?" she asked.
"Me? I suppose I could, if you mean manage to exist on my pension.
I could! But I've got to work, or I should die. That is, I've got to have
something that keeps me occupied. And I'm not in a good enough
temper to work for myself. It's got to be a sort of job for somebody
else, or I should throw it up in a month, out of bad temper. So
altogether I'm very well off here, especially lately...."
He laughed at her again, with mocking humour.
"But why are you in a bad temper?" she asked. "Do you mean you
are always in a bad temper?"
"Pretty well," he said, laughing. "I don't quite digest my bile."
"But what bile?" she said.
"Bile!" he said. "Don't you know what that is?" She was silent, and
disappointed. He was taking no notice of her.
"I'm going away for a while next month," she said.
"You are! Where to?"
"Venice."
"Venice! With Sir Clifford? For how long?"
"For a month or so," she replied. "Clifford won't go."
"He'll stay here?" he asked.
"Yes! He hates to travel as he is."
"Ay, poor devil!" he said, with sympathy.
There was a pause.
"You won't forget me when I'm gone, will you?" she asked. Again he
lifted his eyes and looked full at her.
"Forget?" he said. "You know nobody forgets. It's not a question of
memory."
She wanted to say: "What then?" but she didn't. Instead, she said in
a mute kind of voice: "I told Clifford I might have a child."
Now he really looked at her, intense and searching.
"You did?" he said at last. "And what did he say?"
"Oh, he wouldn't mind. He'd be glad, really, so long as it seemed to
be his." She dared not look up at him.
He was silent a long time, then he gazed again on her face.
"No mention of me, of course?" he said.
"No. No mention of you," she said.
"No, he'd hardly swallow me as a substitute breeder.—Then where
are you supposed to be getting the child?"
"I might have a love affair in Venice," she said.
"You might," he replied slowly. "So that's why you're going?"
"Not to have the love affair," she said, looking up at him, pleading.
"Just the appearance of one," he said.
There was silence. He sat staring out of the window, with a faint grin,
half mockery, half bitterness, on his face. She hated his grin.
"You've not taken any precautions against having a child then?" he
asked her suddenly. "Because I haven't."
"No," she said faintly. "I should hate that."
He looked at her, then again with the peculiar subtle grin out of the
window. There was a tense silence.
At last he turned to her and said satirically:
"That was why you wanted me then, to get a child?"
She hung her head.
"No. Not really," she said.
"What then, really?" he asked rather bitingly.
She looked up at him reproachfully, saying: "I don't know." He broke
into a laugh.
"Then I'm damned if I do," he said.
There was a long pause of silence, a cold silence.
"Well," he said at last. "It's as your Ladyship likes. If you get the
baby, Sir Clifford's welcome to it. I shan't have lost anything. On the
contrary, I've had a very nice experience, very nice indeed!" and he
stretched in a half suppressed sort of yawn. "If you've made use of
me," he said, "it's not the first time I've been made use of; and I don't
suppose it's ever been as pleasant as this time; though of course
one can't feel tremendously dignified about it." He stretched again,
curiously, his muscles quivering, and his jaw oddly set.
"But I didn't make use of you," she said, pleading.
"At your Ladyship's service," he replied.
"No," she said. "I liked your body."
"Did you?" he replied, and he laughed. "Well then, we're quits,
because I liked yours."
He looked at her with queer darkened eyes.
"Would you like to go upstairs now?" he asked her, in a strangled
sort of voice.
"No, not here. Not now!" she said heavily, though if he had used any
power over her, she would have gone, for she had no strength
against him.
He turned his face away again, and seemed to forget her.
"I want to touch you like you touch me," she said. "I've never really
touched your body."
He looked at her, and smiled again. "Now?" he said.
"No! No! Not here! At the hut. Would you mind?"
"How do I touch you?" he asked.
"When you feel me."
He looked at her, and met her heavy, anxious eyes.
"And do you like it when I feel you?" he asked, laughing at her still.
"Yes, do you?" she said.
"Oh, me!" Then he changed his tone. "Yes," he said. "You know
without asking." Which was true.
She rose and picked up her hat. "I must go," she said.
"Will you go?" he replied politely.
She wanted him to touch her, to say something to her, but he said
nothing, only waited politely.
"Thank you for the tea," she said.
"I haven't thanked your Ladyship for doing me the honours of my
teapot," he said.
She went down the path, and he stood in the doorway, faintly
grinning. Flossie came running with her tail lifted. And Connie had to
plod dumbly across into the wood, knowing he was standing there
watching her, with that incomprehensible grin on his face.
She walked home very much downcast and annoyed. She didn't at
all like his saying he had been made use of; because in a sense it
was true. But he oughtn't to have said it. Therefore, again, she was
divided between two feelings; resentment against him, and a desire
to make it up with him.
She passed a very uneasy and irritated teatime, and at once went up
to her room. But when she was there it was no good; she could
neither sit nor stand. She would have to do something about it. She
would have to go back to the hut; if he was not there, well and good.
She slipped out of the side door, and took her way direct and a little
sullen. When she came to the clearing she was terribly uneasy. But
there he was again, in his shirtsleeves, stooping, letting the hens out
of the coops, among the chicks that were now growing a little gawky,
but were much more trim than hen-chickens.
She went straight across to him.
"You see I've come!" she said.
"Ay, I see it!" he said, straightening his back, and looking at her with
a faint amusement.
"Do you let the hens out now?" she asked.
"Yes, they've sat themselves to skin and bone," he said. "An' now
they're not all that anxious to come out an' feed. There's no self in a
sitting hen; she's all in the eggs or the chicks."
The poor mother hens; such blind devotion! even to eggs not their
own! Connie looked at them in compassion. A helpless silence fell
between the man and the woman.
"Shall us go i' th' 'ut?" he asked.
"Do you want me?" she asked, in a sort of mistrust.
"Ay, if you want to come."
She was silent.
"Come then!" he said.
And she went with him to the hut. It was quite dark when he had shut
the door, so he made a small light in the lantern, as before.
"Have you left your underthings off?" he asked her.
"Yes!"
"Ay, well, then I'll take my things off too."
He spread the blankets, putting one at the side for a coverlet. She
took off her hat, and shook her hair. He sat down, taking off his
shoes and gaiters, and undoing his cord breeches.
"Lie down then!" he said, when he stood in his shirt. She obeyed in
silence, and he lay beside her, and pulled the blanket over them
both.
"There!" he said.
And he lifted her dress right back, till he came even to her breasts.
He kissed them softly, taking the nipples in his lips in tiny caresses.
"Eh, but tha'rt nice, tha'rt nice!" he said, suddenly rubbing his face
with a snuggling movement against her warm belly.
And she put her arms round him under his shirt, but she was afraid,
afraid of his thin, smooth, naked body, that seemed so powerful,
afraid of the violent muscles. She shrank, afraid.
And when he said, with a sort of little sigh: "Eh, tha'rt nice!"
something in her quivered, and something in her spirit stiffened in
resistance: stiffened from the terribly physical intimacy, and from the
peculiar haste of his possession. And this time the sharp ecstacy of
her own passion did not overcome her; she lay with her hands inert
on his striving body, and do what she might, her spirit seemed to
look on from the top of her head, and the butting of his haunches
seemed ridiculous to her, and the sort of anxiety of his penis to come
to its little evacuating crisis seemed farcical. Yes, this was love, this
ridiculous bouncing of the buttocks, and the wilting of the poor
insignificant, moist little penis. This was the divine love! After all, the
moderns were right when they felt contempt for the performance; for
it was a performance. It was quite true, as some poets said, that the
God who created man must have had a sinister sense of humour,
creating him a reasonable being, yet forcing him to take this
ridiculous posture, and driving him with blind craving for this
ridiculous performance. Even a Maupassant found it a humiliating
anticlimax. Men despised the intercourse act, and yet did it.
Cold and derisive her queer female mind stood apart, and though
she lay perfectly still, her impulse was to heave her loins, and throw
the man out, escape his ugly grip, and the butting overriding of his
absurd haunches. His body was a foolish, impudent, imperfect thing,
a little disgusting in its unfinished clumsiness. For surely a complete
evolution would eliminate this performance, this "function."
And yet when he had finished, soon over, and lay very very still,
receding into silence, and a strange, motionless distance, far, farther
than the horizon of her awareness, her heart began to weep. She
could feel him ebbing away, ebbing away, leaving her there like a
stone on a shore. He was withdrawing, his spirit was leaving her. He
knew.
And in real grief, tormented by her own double consciousness and
reaction, she began to weep. He took no notice, or did not even
know. The storm of weeping swelled and shook her, and shook him.
"Ay!" he said, "It was no good that time. You wasn't there." So he
knew! Her sobs became violent.
"But what's amiss?" he said. "It's once in a while that way."
"I ... I can't love you," she sobbed, suddenly feeling her heart
breaking.
"Canna ter? Well, dunna fret! There's no law says as tha's got to.
Ta'e it for what it is."
He still lay with his hand on her breast. But she had drawn both her
hands from him.
His words were small comfort. She sobbed aloud.
"Nay, nay," he said. "Ta'e the thick wi' th' thin. This wor' a bit o' thin
for once."
She wept bitterly, sobbing: "But I want to love you, and I can't. It only
seems horrid."
He laughed a little, half bitter, half amused.
"It isna horrid," he said, "even if tha thinks it is. An' tha canna ma'e it
horrid. Dunna fret thysen about lovin' me. Tha'lt niver force thysen to
't. There's sure to be a bad nut in a basketful. Tha mun ta'e th' rough
wi' th' smooth."
He took his hand away from her breast, not touching her. And now
she was untouched she took an almost perverse satisfaction in it.
She hated the dialect: the thee and the tha and the thysen. He could
get up if he liked, and stand there above her buttoning down those
absurd corduroy breeches, straight in front of her. After all, Michaelis
had had the decency to turn away. This man was so assured in
himself, he didn't know what a clown other people found him, a half-
bred fellow.
Yet, as he was drawing away, to rise silently and leave her, she clung
to him in terror.
"Don't! Don't go! Don't leave me! Don't be cross with me! Hold me!
Hold me fast!" she whispered in blind frenzy, not even knowing what
she said, and clinging to him with uncanny force. It was from herself
she wanted to be saved, from her own inward anger and resistance.
Yet how powerful was that inward resistance that possessed her!
He took her in his arms again and drew her to him, and suddenly she
became small in his arms, small and nestling. It was gone, the
resistance was gone, and she began to melt in a marvellous peace.
And as she melted small and wonderful in his arms, she became
infinitely desirable to him, all his blood-vessels seemed to scald with
intense yet tender desire, for her, for her softness, for the penetrating
beauty of her in his arms, passing into his blood. And softly, with that
marvellous swoon-like caress of his hand in pure soft desire, softly
he stroked the silky slope of her loins, down, down between her soft
warm buttocks, coming nearer and nearer to the very quick of her.
And she felt him like a flame of desire, yet tender, and she felt
herself melting in the flame. She let herself go. She felt his penis
risen against her with silent amazing force and assertion, and she let
herself go to him. She yielded with a quiver that was like death, she
went all open to him. And oh, if he were not tender to her now, how
cruel, for she was all open to him and helpless!
She quivered again at the potent inexorable entry inside her, so
strange and terrible. It might come with the thrust of a sword in her