REVIEWS

Compromised autophagy and

neurodegenerative diseases
Fiona M. Menzies1, Angeleen Fleming1,2 and David C. Rubinsztein1
Abstract | Most neurodegenerative diseases that afflict humans are associated with the
intracytoplasmic deposition of aggregate-prone proteins in neurons and with
mitochondrial dysfunction. Autophagy is a powerful process for removing such proteins and
for maintaining mitochondrial homeostasis. Over recent years, evidence has accumulated
to demonstrate that upregulation of autophagy may protect against neurodegeneration.
However, autophagy dysfunction has also been implicated in the pathogenesis of various
diseases. This Review summarizes the progress that has been made in our understanding of
how perturbations in autophagy are linked with neurodegenerative diseases and the
potential therapeutic strategies resulting from the modulation of this process.

Tauopathies
A group of neurodegenerative
diseases that are characterized
by the prominent accumulation
of tau protein in the CNS.
Ubiquitin–proteasome
system
A cellular protein degradation
system in which proteins
tagged with ubiquitin, a small
regulatory protein that is
covalently attached to a target
protein, are broken down by an
enzyme complex called the
proteasome.
1
Department of Medical
Genetics, University of
Cambridge, Cambridge
Institute for Medical
Research, Addenbrooke’s
Hospital, Hills Road,
Cambridge CB2 0XY, UK.
2
Department of Physiology,
Development and
Neuroscience, University of
Cambridge, Downing Street,
Cambridge CB2 3EG, UK.
Correspondence to D.C.R.
e‑mail: dcr1000@hermes.
cam.ac.uk
doi:10.1038/nrn3961
Intracytoplasmic protein misfolding and aggregation
are features of many late-onset neurodegenerative dis-
eases called proteinopathies. These proteinopathies
include Alzheimer disease (AD), Parkinson disease
(PD), tauopathies and the neurodegenerative diseases
caused by (CAG)n trinucleotide tract expansions that
encode abnormally long polyglutamine (polyQ) tracts,
such as Huntington disease (HD). In many of these
diseases, the proteins that accumulate are thought to
be toxic. This concept is supported by overexpression
mouse models of HD, the occurrence of autosomal
dominant tauopathies that are caused by mutations in the
gene encoding tau, and PD that is caused by triplication
of the α‑synuclein (SNCA) locus (reviewed in REFS 1,2).
Accordingly, reductions in the steady-state levels of these
proteins may be beneficial in targeting the root cause of
these diseases. Recent data suggest that the lifetime of
mutant huntingtin (mHTT), the protein that accumu-
lates in and causes HD, may be a superior predictor of
neuronal toxicity compared with its steady-state levels3,
supporting strategies that aim to enhance the clearance
of such proteins.
Although inducing clearance of intracytoplasmic
aggregate-prone proteins may represent a therapeutic
strategy, compromised clearance may exacerbate or con-
tribute to disease by increasing levels of key substrates
such as aggregate-prone proteins4,5 and dysfunctional
mitochondria6; enhancing susceptibility to cell death7,8;
and perturbing flux through the ubiquitin–proteasome
system 9. The genomics era has revealed many genes
associated with neurodegenerative diseases, either
as causative mutations or as risk factors for disease.
Investigations into the function of these genes have iden-
tified multiple intersections with the autophagy path-
way. In this Review, we examine the role of autophagy
— in particular, macroautophagy — in neurodegenera-
tion. We begin by briefly reviewing the core aspects of
autophagy biology, before focusing on studies from the
past 2 years that have revealed the diversity of autophagy
components that can be hijacked by disease-causing
lesions and how this process may be manipulated to
protect against neurodegeneration.
The autophagy machinery
Autophagy, literally meaning ‘self-eating’, is an intra-
cellular degradation pathway that is responsible for
the digestion and recycling of nutrients via autophago-
somes. These intracellular double-membraned struc-
tures engulf cytoplasmic proteins and organelles and
deliver them to the lysosome for degradation (FIG. 1).
The process is essential for cell survival during nutrient
starvation, as it provides cellular energy 10. Autophagy is
also a major pathway for the degradation of intracellu-
lar organelles and aggregate-prone proteins11. Through
both the recycling of nutrients and the degradation of
protein oligomers and damaged organelles, autophagy
serves a vital role in maintaining homeostasis within
the cell.
Initiation of autophagy. The initial step in the forma-
tion of autophagosomes is the fusion of vesicles that
have been proposed to arise from a variety of membrane
sources, including plasma membrane-derived endoso-
mal intermediates12, the endoplasmic reticulum (ER)13,14,

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H+ pH
ATP13A2 (PD) and PS1 (AD)
Trafficking
Degradation
ATG5–ATG12–ATG16L Cystatins (LSDs) and
LC3-II glucocerebrosidase
Receptor (LSDs and PD)
Recycling
Autophagic cargo Spastizin and Biogenesis
spatacsin (HSP) TFEB (multiple)

Initiation
Alsin (ALS), ATG7 (HD)
and spastizin (HSP) Precursor Pre-autophagosomal Autophagosome Lysosome Autolysosome
vesicle structures

VPS34
Beclin 1

Precursor formation Receptors

VPS35 and α-synuclein (PD), p62 and optineurin (ALS) Maturation of autophagosomes and lysosomes
PICALM (AD) and WIPI4 (BPAN) and HTT (HD) PICALM (AD), CHMP2B (CMT2) and SIGMAR1 (ALS)

Figure 1 | Overview of the autophagy pathway and the sites of action of disease-associated proteins.| Neuroscience
Nature Reviews This diagram
shows a simplified version of autophagy. Initiation of autophagy is signalled via the activity of the vacuolar protein
sorting 34 (VPS34) complex. Precursor vesicles fuse to form pre-autophagosomal structures that grow to eventually
become double-membraned autophagosomes. Substrates for degradation by autophagy are engulfed by these growing
membranes or may be sequestered into the forming structure by receptor proteins. The completed autophagosomes are
then trafficked to fuse with lysosomes. The acidic environment inside the lysosomes is maintained by ATPases. This low pH
is required for the correct function of the lysosomal degradative enzymes (depicted as scissors) and, therefore, the
breakdown of the autophagy substrates. Perturbations throughout the pathway, from initiation of autophagosome
formation to degradation in the autolysosomes, have been suggested to be involved in neurodegenerative diseases; some
disease-associated proteins function at multiple points in the process. The key points in the pathway and the selected
disease-associated proteins that are thought to act at these points are highlighted in boxes; the relevant disease is
indicated in parentheses. AD, Alzheimer disease; ALS, amyotrophic lateral sclerosis; ATG, autophagy protein; ATP13A2,
ATPase type 13A2; BPAN, β‑propeller protein-associated neurodegeneration; CHMP2B, charged multivesicular body
protein 2B; CMT2, Charcot–Marie–Tooth disease type 2; HD, Huntington disease; HSP, hereditary spastic paraplegia; HTT,
huntingtin; LC3, microtubule-associated protein 1 light chain 3; LSD, lysosomal storage disease; PD, Parkinson disease;
PICALM, phosphatidylinositol-binding clathrin assembly protein; PS1, presenilin 1; SIGMAR1, sigma non-opioid
intracellular receptor 1; TFEB, transcription factor EB; WIPI4, WD repeat domain phosphoinositide-interacting protein 4.

Risk factors
Factors (in this article,
alterations in DNA sequences)
that may increase the chance
of developing a disease.
Lysosome
An intracellular
membrane-bound organelle
containing hydrolytic enzymes
that are capable of breaking
down proteins and other
cellular components.
the Golgi15,16 and mitochondria17. These vesicles coalesce
to form a flattened membrane sac called a phagophore.
The fusion of additional vesicles results in the forma-
tion of a cup-shaped double membrane that surrounds
and eventually engulfs portions of cytoplasm as the two
edges of the structure come together and fuse (FIG. 1).
Various autophagy (ATG) proteins are essential
for the formation of autophagosomes. The vesicles
that give rise to the phagophore contain a complex
of ATG5–ATG12–ATG16L1, the formation of which
requires the catalytic activities of ATG7 and ATG10.
In addition, the phagophore membrane contains
ATG9, the only multipass transmembrane protein in
the ATG family. Recently, it was shown that ATG9- and
ATG16L1‑containing vesicles arise independently via
clathrin-mediated endocytosis but subsequently fuse in
the recycling endosome18.
A second enzyme cleavage and conjugation pathway
that is essential to autophagosome biogenesis involves
the processing of microtubule-associated protein 1 light
chain 3 (LC3). LC3 is a protein that has been proposed
to recruit membrane to the developing phagophore. LC3
is first cleaved by ATG4B to form LC3‑I, which is then
conjugated to phosphatidyl­ethanolamine by ATG7 and
ATG3 to form LC3‑II19. As the phagophore enlarges
and approaches closure, the ATG5–ATG12–ATG16L1
complex dissociates from the outer membrane,
whereas LC3‑II remains associated with the completed
autophagosome. Following closure, autophagosomes
are trafficked by dynein motors along microtubules20 to
the perinuclear region where they fuse with the lyso-
some and their contents are degraded. SNAREs (solu-
ble NSF attachment protein receptors) have a critical
role in autophagosome formation and degradation.
The primary function of these proteins is to facilitate
vesicle fusion, and different members of the SNARE
family are critical for various steps of the process.
For example, vesicle-associated membrane protein 3
(VAMP3)18 is important for ATG9–ATG16L1 vesicle
fusion; VAMP7, syntaxin 7 and syntaxin 8 (REF. 21) are
important for phagophore elongation; and VAMP7,
VAMP8 and VTI1B (vesicle transport through interac-
tion with t‑SNAREs homologue 1B)22,23 are involved in
autophagosome–lysosome fusion.

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Endocytic pathway
A general term to describe the
vesicle trafficking routes by
which cells internalize
molecules from the plasma
membrane.
NATURE REVIEWS | NEUROSCIENCE
Control over the initiation of autophagy is mediated
through a protein complex comprising ULK1 or ULK2,
ATG13, ATG101 and the focal adhesion kinase family
interacting protein of 200 kDa (FIP200)24. This ULK1/
ULK2–ATG13–FIP200 complex serves as both an initiator
of autophagy and a sensor for upstream signalling through
three major pathways: the mTOR complex 1 (mTORC1),
adenosine monophosphate (AMP)-activated protein
kinase (AMPK) and p53 pathways24,25. Phosphorylation of
ULK1 or ULK2 and ATG13 by mTORC1 inactivates this
complex, whereas AMPK activates autophagy through the
direct phosphorylation of ULK1 at sites that are distinct
from those targeted by mTORC1. p53 can also control
autophagy through transcriptional regulation. Activated
p53 transcriptionally upregulates pro-autophagic proteins
such as ULK1, ULK2, AMPK and tuberous sclerosis 2
protein (TSC2)25, whereas transcriptionally inactive, cyto-
plasmic p53 negatively regulates autophagy through direct
binding with FIP200 (REF. 26), which may block formation
of the ULK complex.
The ULK1/ULK2–ATG13–FIP200 complex controls
the initiation of autophagy through the PI3K complex,
which comprises the vacuolar protein sorting 34 (VPS34;
also known as PIK3C3) and its accessory partners VPS15
(also known as PIK3R4), beclin 1 and ATG14L (also
known as barkor). Activation of ULK1 results in the phos-
phorylation of beclin 1 and a subsequent increase in the
activity of the VPS34 complex 27. This complex is required
for the formation of phosphatidylinositol 3‑phosphate
(PtdIns3P) on nascent autophagosomes, which facilitates
the recruitment of PtdIns3P‑binding proteins such as
WD repeat domain phosphoinositide-interacting pro-
tein 1 (WIPI1) and WIPI2 (both of which are mammalian
orthologues of yeast Atg18) to autophagosome mem-
branes. The recruitment of ATG16L1 to these structures
appears to occur through binding to WIPI proteins28.
ULK1 also regulates the recruitment of the transmem-
brane protein ATG9 to the phagophore29,30. Recent data
suggest that PtdIns5P can substitute for the functions of
PtdIns3P in autophagosome biogenesis31.
Autophagy receptors. Until recently, autophagy was
thought to be a non-selective degradation pathway, but
an emerging field of research has revealed a series of
receptor proteins (which have sometimes been referred
to as adaptor proteins; for clarification on terminology,
see REF. 32) that confer substrate specificity on this pro-
cess. These receptor proteins bind to a diverse range of
cargoes including bacteria and viruses; however, more
pertinently, they have also been shown to bind to aggre-
gated proteins33–35. Receptor proteins — such as p62 (also
known as sequestosome 1), next to BRCA1 gene 1 pro-
tein (NBR1), nuclear domain 10 protein 52 (NDP52; also
known as CALCOCO2) and optineurin — are able to
bind to their cargo selectively. For example, such proteins
may recognize ubiquitylated proteins (the cargoes) via
ubiquitin-binding domains and bind to LC3 family mem-
bers on autophagosomes through their LC3‑interacting
region motifs36 (FIG. 1). Selective targeting of autophagic
cargoes can also be mediated by autophagy recep-
tors that form a bridge between the cargo–autophagy
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receptor complex and components of the autophagosome
membrane (for example, ATG5 and PtdIns3P36) (FIG. 1).
Autophagy-linked FYVE protein (ALFY) may function
in this way. ALFY is localized to the nuclear envelope
under basal conditions but redistributes to colocalize
with ubiquitin-positive cytoplasmic structures upon pro-
teasome inhibition, and with ATG5 or LC3‑positive cyto-
plasmic puncta under amino acid starvation conditions37.
The FYVE domain of ALFY is able to bind to PtdIns3P,
but it also contains additional domains, notably five
WD40 repeats, which are required for its interaction with
Atg5 (REF. 38), and a pleckstrin homology‑like domain
and a BEACH domain, which together interact with p62
(REF. 39). These interactions suggest that ALFY has a scaf-
folding role in the formation of p62‑labelled aggregates,
which can then be targeted to autophagosomes.
Additional autophagy proteins have been implicated
in the selective degradation of mitochondria (for example,
BNIP3L (BCL‑2/adenovirus E1B 1 19 kDa protein-inter-
acting protein 3‑like), Atg32 and p62) and peroxisomes
(for example, Atg30 and p62). Although Atg32 and Atg30
have only been characterized in yeast and it is unclear
whether mammalian homologues of these proteins exist,
BNIP3L, a mitochondrial membrane protein contain-
ing a LC3‑interacting region motif, has been shown in
mammalian cells to play a part in the targeted clearance of
damaged mitochondria40,41 (see BOX 1 and FIG. 2 for further
details on mitophagy and neurodegeneration).
Autophagosome trafficking and degradation. The mat-
uration of autophagosomes and the final steps of the
autophagic pathway have been the subject of a recent
expert review 42. Once formed, autophagosomes are
transported along microtubules to the lysosome-rich
peri­nuclear region. In neurons, autophagosomes formed
at the distal tip of axons are transported towards the
cell body by dynein-mediated retrograde transport 43.
Autophagosomes may fuse with vesicles from the endocytic
pathway (early or late endosomes) to form amphisomes,
which subsequently fuse with lysosomes, or they may fuse
directly with lysosomes. This final step of the autophagy
process is mediated by VAMP7, VAMP8 and VTI1B,
members of the SNARE family of proteins (reviewed in
REF. 44). Efficient degradation of autophagy substrates
can only occur if the acidic lysosomal pH is correctly
maintained and the degrading enzymes are functioning
correctly. Sufficient lysosomes must also be available for
fusion with autophagosomes because this process con-
sumes the lysosomes. They can be regenerated through
lysosomal reformation, in which tubules formed on auto­
lysosomes mature into new lysosomes45. Transcription
factor EB (TFEB) has been identified as a master regula-
tor for lysosomal biogenesis46; as such, this protein has an
important role in the control of autophagy 47.
Autophagy failure in neurodegeneration
The vast majority of neurodegenerative diseases, includ-
ing sporadic forms of such diseases, have a genetic
component, and the assessment of the cellular func-
tions of these genes has implicated autophagic dys-
function in their pathogenesis. These findings have
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Box 1 | Mitophagy in neurodegenerative disease

Mitochondria have long been studied as key organelles in the
pathogenesis of many neurodegenerative diseases, particularly Parkinson
disease (PD). Recently, much of this research has focused on the potential
contribution of altered clearance of dysfunctional mitochondria by
autophagy (termed mitophagy). This research has centred on two genes
that are the major causes of autosomal recessive PD, namely parkin
(PARK2) and phosphatase and tensin homologue-induced putative
kinase 1 (PINK1; also known as PARK6), which have been shown in
Drosophila melanogaster to act via the same pathway146–148.
In functional mitochondria, which are able to maintain their
mitochondrial membrane potential, PINK1 is imported via the translocase
of the outer membrane (TOM) complex and the translocase of the inner
membrane (TIM23) complex (FIG. 2). It is then cleaved by mitochondrial
processing peptide (MPP) and presenilins-associated rhomboid-like
protein (PARL) before being translocated back to the cytoplasm for
degradation by the proteasome149–151. However, in the absence of
a mitochondrial membrane potential, PINK1 accumulates on the
mitochondrial outer membrane where it recruits parkin152–155, resulting in
the targeting of mitochondria for degradation by autophagy. The exact
mechanism by which mitophagy is promoted downstream of parkin
recruitment has yet to be established (reviewed in REF. 156). A major
hypothesis for the mechanism is that parkin ubiquitylates a number of
mitochondrial outer membrane proteins, such as voltage-dependent
anion-selective channel protein 1 (VDAC1), TOM20 and mitochondrial
RHO GTPase 1 (MIRO1)152,157, which then recruit p62 to the mitochondrial
outer membrane where autophagsomes subsequently form, although this
role for VDAC1 has been disputed158. PINK1 and parkin are also reported
to have roles in mitochondrial fission and fusion, as well as in
mitochondrial trafficking (reviewed in REF. 159). These roles could
represent either mechanisms by which PINK1 and parkin act to ultimately
control mitophagy or independent processes that affect mitochondrial
health and function.
Investigations into the role for the PINK1–parkin pathway in
mitophagy have mostly been carried out in non-neuronal cultured
cells overexpressing parkin and treated with carbonyl cyanide
m‑chlorophenylhydrazone (CCCP) to induce mitochondrial
depolarization, and it is not clear how relevant this process is to
disease progression. However, recent work has shown that
endogenous parkin enables mitochondrial clearance157. This is further
supported by work using mt‑KR, a photoactivatable protein that
produces local increases in reactive oxygen species. Using this
construct to damage mitochondria in the distal axons of cultured
neurons, it was observed that the PINK1–parkin pathway induced
autophagy of these damaged mitochondria in the axons160.
An alternative mechanism controlling mitophagy is the externalization
of cardiolipin, a phospholipid that is normally only present on the
mitochondrial inner membrane. Cardiolipin is able to bind to microtubule-
associated protein 1 light chain 3 (LC3)161, thereby promoting autophagy.
However, this effect has only been observed in cells treated with
mitochondrial toxins such as rotenone and 6‑hydroxydopamine, which
did not cause the same increase in PINK1 levels that are seen after CCCP
treatment. This suggests that different causes of mitochondrial damage
may result in mitophagy via different mechanisms.
When considering the role of mitophagy in PD, it must be asked how
mutations in PINK1 or PARK2, which account for only a small proportion
of PD cases, relate to other forms of the disease. A recent study has
addressed this, suggesting that sterol regulatory element-binding
transcription factor 1 (SREBF1), a gene identified by a genome-wide
association study as a risk factor for PD162, has a role in parkin recruitment
to damaged mitochondria and mitophagy163 and could thus represent a
link between mitophagy and sporadic PD.
Mitophagy might have further disease relevance because xeroderma
pigmentosum group A, a DNA repair disorder associated with
neurodegeneration, has also been linked to defective mitophagy that is
associated with increased cleavage of PINK1 (REF. 164). Importantly,
impaired autophagy flux will also affect mitophagy and perturb the
clearance of dysfunctional mitochondria, which may increase the cellular
levels of reactive oxygen species and the propensity to caspase activation.
Although mitophagy is not the focus of this Review (and has been the
subject of a recent excellent review156), these consequences are important
to consider as pathogenic mechanisms in the diseases in which autophagy
is compromised.

Polymorphisms
The existence of multiple
variants of a DNA sequence
that occur within different
individuals in the population,
with no sequence being
regarded as the standard
sequence.
been complemented by studies showing that autophagy
markers are perturbed in post-mortem tissue in many
neurodegenerative diseases. However, in many cases, the
molecular mechanisms have yet to be determined, and
it remains to be ascertained whether this truly repre-
sents dysfunctional autophagy or whether compromised
autophagy contributes to disease progression.
Here, we do not discuss the evidence for altered
autophagy disease-by‑disease as we have done previously48
but, rather, consider how recent research has revealed a
number of key points in the autophagic pathway that can
be affected by disease-causing mutations. To date, there
has only been one disease — β‑propeller protein-associ-
ated neurodegeneration (BPAN; also known as neuro­
degeneration with brain iron accumulation 5 (see below))
— in which the causal mutation occurs in a gene that is
proposed to function solely in autophagy (although other
functions of this gene may emerge with further research).
Indeed, most of the proteins discussed in this Review have
functions beyond a role in autophagy. Many of these func-
tions may be pertinent to the pathogenesis of neurodegen-
erative disease but are, for the most part, not discussed
here. We do not suggest that autophagy dysfunction is
the single mechanism underlying the pathogenesis of
neurodegenerative diseases. Instead, we aim to highlight it
as a common aspect of these complex diseases and discuss
the new information coming to light about the intersec-
tion of autophagy and neurodegeneration.
Mutations in core autophagy genes. Loss-of-function
mutations have been identified in the gene encoding the
β‑propeller scaffold protein WIPI4 (WDR45) in BPAN49.
WDR45 is a core autophagy gene, encoding one of four
homologues of yeast Atg18, involved in autophago-
some formation. Parents or siblings of patients with
WDR45 mutations do not share the mutation, suggest-
ing that such mutations occur de novo50. Lymphoblastoid
cell lines derived from these patients have reduced
autophagic activity 50, which may result from disrupted
ATG9A vesicle translocation at the autophagosome for-
mation site owing to altered WIPI4 activity 51.
Given the importance of autophagy in cellular func-
tion, mutations in core autophagy genes are likely to have
severe consequences. Thus, it is perhaps unlikely that
late-onset neurodegenerative diseases will be caused by
recessive mutations in autophagy genes, and it is more
likely that polymorphisms in these genes contribute to the
age of onset and disease progression of such disorders.

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PINK1
Proteasome
TOM Mitochondrion
TIM23
MPP PARL
H+ Cardiolipin
MIRO1 Phagopore
Ubiquitin
VDAC1
Parkin LC3-II
ATG5–ATG12–ATG16L
Figure 2 | Initiation signals for mitophagy. In mitochondria that are able to maintain
Nature
their membrane potential (as shown in the upper half of the Reviews
diagram), | Neuroscience
phosphatase and
tensin homologue-induced putative kinase 1 (PINK1) is imported across the
mitochondrial membrane by the translocase of the outer membrane (TOM) complex
and the translocase of the inner membrane (TIM23) complex before being cleaved by
mitochondrial processing peptidase (MPP) and presenilins-associated rhomboid-like
protein (PARL) and then targeted for degradation by the proteasome. In the absence of
a membrane potential (lower half of the diagram), PINK1 cannot be transported across
the membrane and instead recruits parkin to the membrane, resulting in the
ubiquitylation of other mitochondrial outer membrane proteins (such as
voltage-dependent anion-selective channel protein 1 (VDAC1) and mitochondrial RHO
GTPase 1 (MIRO1)), phagophore formation and subsequent degradation. Alternatively,
externalization of cardiolipin onto the outer membrane following mitochondrial
depolarization has also been reported to result in phagophore formation. ATG,
autophagy protein; LC3, microtubule-associated protein 1 light chain 3.
Support for this assertion can be found in HD. The age
of onset of HD is closely correlated with the polyQ repeat
length, which accounts for 70% of the variance; however,
other genetic factors have been proposed to contribute
to age of onset. One such gene is ATG7: the V471A poly-
morphism has been associated with an earlier onset of
disease by 4–6 years in certain populations52,53. However,
it has yet to be demonstrated whether this polymorphism
has functional importance in terms of autophagic activity.
Beclin 1 interactions. As noted above, beclin 1 is req­
uired for the early steps of autophagosome formation.
Neurodegeneration-associated proteins are now coming
to light that may affect the interactions of beclin 1 with
its protein partners and influence its ability to medi-
ate autophagy. Overexpression of the E46K variant of
α‑synuclein, which causes a rare autosomal dominant
form of PD, leads to decreased levels of phosphorylated
BCL‑2, which then enhances the interaction between
beclin 1 and BCL‑2 and therefore results in autophagy
inhibition54. Regulation of the beclin 1–BCL‑2 inter-
action has also been suggested to be compromised in
HD. HTT has been demonstrated to interact with Ras
homologue enriched in striatum (RHES), which is selec-
tively expressed in the striatum, where HD pathology
NATURE REVIEWS | NEUROSCIENCE
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is observed55. In turn, RHES has been demonstrated
to enhance autophagy via its interaction with beclin 1,
blocking the inhibitory interaction of beclin 1 with
BCL‑2. Importantly, mHTT prevents this autophagy
activation56.
Beclin 1 is also involved in the maturation of
autophago­somes through its role in the ultraviolet radia-
tion resistance-associated gene protein (UVRAG)–
rubicon–beclin 1 complex. Beclin 1 also interacts with
spastizin57 (encoded by ZFYVE26, which is mutated
in hereditary spastic paraplegia (HSP) type 15), and
disease-associated mutations disrupt this interaction58.
Autophagosomes accumulate in cells lacking spastizin or
expressing mutant forms of this protein58. This accumula-
tion has been proposed to result from loss of function of
the UVRAG–rubicon–beclin 1 complex 58 but may also be
due to defects in lysosomal function (see below).
Mutations in genes involved in trafficking. Membrane
trafficking is a vital element of autophagy, and the
identification of proteins involved in such trafficking
is currently an area of intensive research. Many neuro­
degenerative disease-causing mutations have been iden-
tified in genes that can be classed as trafficking genes,
and an attractive hypothesis for the pathogenic mecha-
nism of late-onset neurodegenerative diseases is that the
mutations are likely to impair autophagy efficiency.
Phosphatidylinositol-binding clathrin assembly
protein (PICALM) was identified through genome-
wide association studies to be associated with AD59,60.
Moreover, PICALM has been reported to be abnormally
cleaved in the brains of patients with AD, resulting in
a decreased level of the full-length protein61. PICALM
is required for the endocytosis of VAMP2 and VAMP3,
which are necessary for autophagosome formation,
and VAMP8, which is important for autophagosome–
lysosome fusion. In the absence of PICALM, decreased
fusion of autophagosome precursor vesicles is observed
(VAMP2- and VAMP3‑dependent processes), leading
to impaired autophagosome biogenesis, along with a
decrease in the fusion of autophagosomes with lyso­
somes (a VAMP8‑dependent process), resulting in an
overall reduction in autophagic flux 62. These defects
contribute to the accumulation of tau, an autophagy
substrate and a probable contributor to AD pathology.
The small GTPase RAB protein family comprises
well-known regulators of membrane trafficking and
fusion events. Mutations in RAB7, which encodes a
late endosomal RAB protein known to be involved in
autophagy (reviewed in REF. 63), have been reported
to cause Charcot–Marie–Tooth type 2B disease 64.
Similarly, the gene encoding alsin (ALS2) is mutated
in some cases of recessive amyotrophic lateral sclerosis
(ALS)65. Alsin is an activator of RAB5 (REF. 66), which is
required for the clearance of aggregate-prone proteins
by autophagy 67. Missense mutations in ALS2 result in
mislocalization of the protein and a decreased fusion
of autophagosomes with endosomes68. Hexanucleotide-
repeat expansions in a non-coding region of chromo-
some 9 open reading frame 72 (C9ORF72) have been
reported as a major genetic factor in patients with ALS
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Multivesicular bodies and in patients with frontotemporal dementia (FTD).
A type of late endosome Recently, C9ORF72 has been suggested to have a
containing internal vesicles. role in endosomal trafficking, and it contains DENN
(differentially expressed in normal and neoplastic
Lewy bodies
Protein aggregates found
cells) domains, which are normally associated with
within neurons in patients with RABGEFs, activators of endocytic RAB proteins69. In
certain neurodegenerative neuronal cell lines, C9ORF72 colocalizes with several
diseases, including Parkinson RAB proteins, and small interfering RNA (siRNA)-
disease and Lewy body
mediated knockdown of C9ORF72 inhibited shiga toxin
dementia.
trafficking from the plasma membrane to the Golgi and
endocytosis of tropomyosin-related kinase B (TRKB),
as well as increased the levels of LC3‑II70. However, the
importance of these mechanisms in the pathogenesis of
ALS is unclear. This is because mutations in C9ORF72
are found in non-coding regions, and it is considered
that repeat associated non-ATG-mediated translation
(RAN) of arginine-rich dipeptides are the toxic entities
in the case of this mutation71.
Mutations in charged multivesicular body pro-
tein 2B (CHMP2B) can cause FTD72. CHMP2B is part of
the ESCRT-III (endosomal secretory complex required
for transport III) complex, and CHMP2B mutations
result in disruption of this complex, causing defects in
the endosome–lysosome pathway. Overexpression of
mutant CHMP2B has been demonstrated to result in
the accumulation of autophagosomes, suggesting that
their maturation is blocked73. This finding is also con-
sistent with the role of ESCRT-III in the formation of
multi­vesicular bodies, which can fuse with autophago-
somes before the fusion of autophagosomes with
lysosomes.
VPS35 is a component of retromer, which mediates
endosome-to‑Golgi trafficking (reviewed in REF. 74).
Retromer is also required for the endosomal recruit-
ment of the actin nucleation-promoting WAS protein
family homologue (WASH) complex that is required for
the formation of actin patches and efficient protein sort-
ing 75. Recently, the D620N substitution in VPS35 has
been identified as causing autosomal dominant PD76,
and this mutation impairs binding of the WASH com-
plex to endosomes. As a consequence, ATG9 trafficking
Box 2 | Chaperone-mediated autophagy
Chaperone-mediated autophagy (CMA) is a form of lysosomal degradation in which
substrates are delivered directly to the lysosome rather than being trafficked via
autophagosomes as occurs in macroautophagy. CMA substrates are recognized by a
pentapeptide motif based on the charge of the amino acids, allowing a motif to also be
generated by post-translational modifications. Proteins to be degraded are bound by
the heat shock protein HSC70 and are targeted to the lysosome, where they interact
with a multiprotein complex, which includes lysosomal-associated membrane
glycoprotein 2 (LAMP2). They are unfolded and translocated directly across the
lysosomal membrane. This process is not the focus of this Review and has been
reviewed elsewhere165–167; however, it is important to note that CMA dysfunction has
also been implicated in the pathogenesis of neurodegenerative diseases. CMA is able to
degrade proteins associated with neurodegeneration, such as α-synuclein168,
leucine-rich repeat serine/threonine-protein kinase 2 (LRRK2)169 and tau170, and
disease-associated mutant versions of these proteins are less efficiently degraded by
CMA and in fact block the degradation of other substrates by CMA168,169. It is also
important to note that the defects in lysosomal function outlined in this Review are
equally likely to affect CMA and macroautophagy and may thus contribute to
neurodegeneration by perturbation in CMA.
350 | JUNE 2015 | VOLUME 16
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is disrupted, resulting in the inhibition of the early
stages of autophagosome formation and decreased
α‑synuclein clearance75. The ATG9 mislocalization that
results from VPS35-D620N is reminiscent of that seen
to result from increased levels of α-synuclein77, the pro-
tein that accumulates in Lewy bodies in PD. Increased
levels of α‑synuclein are sufficient to cause PD78, and
this protein has been shown to inhibit RAB1A79, lead-
ing to an alteration of ATG9 localization and decreased
autophagosome formation in cell culture and in vivo77.
The identification of the autophagy defect caused by
VPS35-D620N could be a mechanistic feature that
is shared by other neuro­degenerative diseases. For
example, mutations in the gene encoding strumpellin
(KIAA0196; a WASH complex subunit) have been iden-
tified in some cases of HSP80, and mutations in the gene
encoding SWIP (also known as KIAA1033; another
WASH complex subunit) have been associated with
autosomal recessive intellectual disability 81. Moreover,
another gene mutated in PD, DNAJC13 (also known
as RME8)82, encodes a protein that associates with the
WASH complex through binding to the same subunit as
does VPS35 (REF. 80).
Mutations in the 3ʹ untranslated region and the cod-
ing region of sigma non-opioid intracellular receptor 1
(SIGMAR1) can cause forms of ALS and FTD83,84, and
levels of SIGMAR1 are decreased in the spinal cord of
patients with sporadic ALS85. Loss of Sigmar1 in mice
results in motor deficits86, and treatment with a SIGMAR1
agonist improves motor function and survival in a mutant
superoxide dismutase 1 (SOD1) ALS mouse model87.
SIGMAR1 is a chaperone that is located at the ER–mito-
chondria interface and has many functions, including
a role in trafficking. Knockdown of SIGMAR1 impairs
vesicle trafficking from the ER to the Golgi and results in a
decrease in the fusion of autophagosomes with lysosomes
and therefore a reduction in the degradation of autophagy
substrates88.
Lysosomal defects in neurodegenerative diseases.
Lysosomal storage disorders (LSDs) are the most com-
mon neurodegenerative diseases of childhood and com-
prise more than 50 diseases. The clinical phenotypes
of these diseases vary; however, they frequently show
progressive CNS defects. Because the final step in the
autophagic process is the fusion of autophagic vesicles
with lysosomes and the degradation of the contents of
these autolysosomes, lysosomal defects are highly likely
to affect the autophagic capacity of the cell (reviewed in
REF. 89), both in terms of macro­autophagy, as we discuss
here, and in terms of chaperone-mediated autophagy
(BOX 2). Although the defects causing LSDs can vary
widely from one type of LSD to another (LSDs may
arise as a consequence of defects in lyso­somal enzymes,
lysosomal membrane-associated proteins or even non-
lysosomal-associated enzymes), all cases studied to
date show impaired autophagy, suggesting that this
is the common pathogenic mechanism in these dis-
eases. A LSD that has been widely studied in terms of
its autophagic characteristics is Niemann–Pick disease
type C1 (BOX 3).
www.nature.com/reviews/neuro

Box 3 | Niemann–Pick disease type C1 — excessive or defective autophagy?
The study of the role of autophagy in Niemann–Pick disease type C1 (NPC1) highlights
not only the difficulties associated with interpreting the contribution of autophagy
function and dysfunction to a disease but also the challenges of understanding the
potential of autophagy modulation as a therapeutic approach. NPC1 is caused by
mutations in NPC1, which result in the loss of correct trafficking of cholesterol within
the cell and the accumulation of unesterified cholesterol and glycosphingolipids in
lysosomes and late endosomes (which would normally be trafficked on to the Golgi and
endoplasmic reticulum for further processing). This mislocalization disrupts both
lysosomes and endosomes, as well as other membrane compartments, which do not
receive the cholesterol they require for their correct membrane composition171.
A role for autophagy in NPC1 was first suggested after autophagosomes were found
to accumulate in degenerating Purkinje cells in mice with mutant NPC1 (REF. 172), and
autophagy was initially thought to be upregulated in this disease (reviewed in REF. 171).
However, with improved knowledge of the autophagic pathway and how to measure
autophagy activity, such as the use of tandem red fluorescent protein–GFP-tagged
microtubule-associated protein 1 light chain 3 (LC3)173, it has become clear that
autophagy is defective in this disease, leading to the proposal that autophagy was
both induced and defective174,175. These studies suggested that treatment with either
cholesterol-depleting drugs (such as cyclodextrin) or autophagy inhibitors (such as
3‑methyladenine) may be beneficial. Cyclodextrin was suggested to decrease the
accumulation of autophagosomes as it restored autophagic function175. However, more
recent studies have argued against the evidence for any upregulation of autophagy in
NPC1 (REFS 176–178), demonstrating that the increase in autophagic markers is solely a
result of decreased maturation of autophagosomes. Furthermore, it was suggested that
cyclodextrin treatment in fact inhibits autophagy. A proposed therapeutic strategy for
this disease involved treatment with low-dose cyclodextrin to alleviate the cholesterol
build‑up, along with autophagy inducers such as rapamycin177 or carbamazepine176.
The LSD Gaucher disease is caused by homozygous
mutations in glucocerebrosidase (GBA). Heterozygosity
for GBA mutations (Gaucher disease carrier status) is
the most common known genetic risk factor associated
with PD90. Loss of GBA results in lysosomal dysfunction
owing to the accumulation of its substrate, glucocere-
broside. Mice lacking Gba have defective autophagy
and accumulation of potential autophagy substrates
in the brain, such as p62, monomeric and oligomeric
forms of α‑synuclein, and ubiquitylated proteins, along
with decreased mitochondrial function and decreased
mitophagy 91. Patients with sporadic PD have decreased
levels of GBA in affected regions of the brain, along with
increased levels of α-synuclein92,93.
Lysosomal membrane permeabilization (LMP) may
also cause lysosomal dysfunction in LSDs (reviewed in
REF. 94). LMP leads to a release of cathepsins into the
cytoplasm, which has been associated with calpain acti-
vation and apoptotic cell death, and may also lead to a
block in autophagy. For example, in Niemann–Pick dis-
ease type A, which is caused by mutations in the gene
encoding acid sphingomyelinase, increases in sphingo-
myelin levels lead to LMP and a concomitant decrease
in autophagosome degradation95. Although there is clear
evidence for a defect in autophagy in LSDs, the specific
contribution of autophagy defects versus lysosomal dys-
function has not been parsed out. In addition to clas-
sical LSDs, it is now becoming evident that lysosomal
dysfunction may play a part in other neurodegenerative
diseases.
Kufor–Rakeb syndrome and some early-onset forms
of PD are associated with mutations in the gene encoding
lysosomal ATPase type 13A2 (ATP13A2)96. Fibroblasts
NATURE REVIEWS | NEUROSCIENCE
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
derived from patients with ATP13A2 mutations show
increased lysosomal pH and reduced processing of lyso-
somal proteases from their precursor to mature forms.
This results in decreased protease activity 97 and an
increase in the number of autophagic vesicles owing to a
block in fusion with lysosomes. Mutations in presenilin 1
(PS1) in AD can also result in an increase in lysosomal
pH owing to the role of the presenilin holoprotein (rather
than the cleaved form that is active in the γ‑secretase
complex) in the glycosylation of the lyso­somal v‑ATPase
subunit V0a1, which results in decreased autophagic
turnover 98,99. Although the exact mechanism that leads
to this decrease in turn­over is unclear 100,101, there does
seem to be a consensus regarding the idea that PS1 has a
role in autophagic clearance98,99,102,103.
TFEB enhances both lysosomal biogenesis and
autophagosome formation46,47. Mutant forms of the andro-
gen receptor, which cause the polyQ disorder spinobul-
bar muscular atrophy (SBMA; also known as Kennedy
disease), interact with and inhibit TFEB, with conse-
quent effects on the autophagy–lysosome pathway 104.
A role for altered TFEB activity has also been suggested
in PD because α‑synuclein has been reported to inter-
act with TFEB; in dopaminergic neurons from patients
with PD, TFEB seems to be sequestered in the cytoplasm
(away from its target of action in the nucleus)105. In AD,
enhanced levels of acid sphingomyelinase, one of a group
of sphingomyelin-metabolizing enzymes, have been
shown in fibroblasts, brain and plasma of patients106.
This increase in acid sphingomyelinase causes a decrease
in TFEB levels. The genetic reduction of acid sphingo­
myelinase in heterozygous knockout mice, or pharma-
cological inhibition of this enzyme with amitriptyline,
protected against disease development in the APP/PS1
mouse model of AD, reducing the accumulation of LC3‑II
and p62 that is observed in this model106.
Finally, two genes in which mutations have been iden-
tified in autosomal recessive HSP are implicated in the
regeneration of lysosomes. The genes encode spastizin
(ZFYVE26) and spatacsin (SPG11), which act together in
a complex that is required for the initiation of lysosomal
reformation. Loss or mutation of either gene results in a
decrease in lysosomal number and an accumulation of
autolyososomes107.
Autophagy receptors for aggregate-prone proteins. p62
and other autophagy receptors are components of neu-
ronal protein aggregates in various neurodegenerative
diseases33,108–110. p62 and LC3 were shown to colocalize
within a shell formed around mHTT aggregates in vitro,
and knockdown of p62 enhanced mHTT toxicity, provid-
ing the first evidence for its role in the selective clearance
of mHTT aggregates33. Indeed, in Drosophila melanogaster
models of neurodegeneration, loss of p62 exacerbates
the degeneration of the eye caused by overexpression
of mHTT, a truncated form of ataxin 3 containing an
expanded polyQ repeat (MJDtr‑Q78; associated with
another polyQ disease, spinocerebellar ataxia type 3) or
TAR DNA-binding protein 43 (TDP43; associated with
ALS)111. However, these observations have not trans-
lated simply into vertebrate models. In a mouse model
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REVIEWS
of SBMA in which a polyQ-expanded androgen recep-
tor was overexpressed, knockout of p62 enhanced the
disease phenotype, whereas p62 over­expression was
protective112. However, p62 overexpression, rather than
enhancing clearance of the mutant protein, converted it
from a soluble to an insoluble form, thereby increasing
the number of nuclear inclusions observed in the brain112.
Furthermore, depletion of p62 in mouse models of HD
has been shown to ameliorate rather than enhance disease
signs113. Although the number of nuclear mHTT aggre-
gates was decreased in p62-knockout mice expressing
mHTT, there was an increase in the levels of cytoplasmic
aggregates and no apparent change in total HTT levels in
the brain113.
Another autophagy receptor implicated in the clear-
ance of mHTT aggregates is ALFY, which co‑immuno­
precipitates with p62, ATG5 and LC3 within mHTT
aggregates in mammalian cells expressing exon 1 of HTT
or in fibroblasts from patients with HD38. Furthermore,
in mammalian cells expressing mHTT, knockdown
and overexpression of ALFY led to an accumulation of
aggregates and a reduction in the number of aggregates,
respectively, and similar effects were observed in vivo in a
D. melanogaster model of polyQ toxicity 38. Together, these
findings provide good functional evidence that ALFY
plays a part in facilitating autophagic clearance of polyQ
protein aggregates.
Recently, a new class of receptors, the Cue5–TOLLIP
proteins (CUET proteins; also known as CUE-domain
targeting adaptors), were identified in yeast from a
screen for novel ubiquitin–Atg8 (LC3) interactors114.
Of these, Cue5 has a putative mammalian homologue,
namely Toll-interacting protein (TOLLIP). TOLLIP
was observed to colocalize with LC3 in mammalian
cells, and its over­expression enhanced the clearance of
mHTT, suggesting that this protein and maybe other
yet‑to‑be‑identified CUET homologues act as mam-
malian autophagy receptors. Although both p62 and
CUET proteins contain ubiquitin-binding domains,
ubiquitylation is not the only signal for selective recog-
nition. p62 can also bind to non-ubiquitylated protein
aggregates and target these for autophagic degrada-
tion115. In addition, NDP52, already known as a selec-
tive autophagy receptor from work in bacteria116, has
recently been shown to co‑immuno­precipitate with
human tau through its SKICH domain. Although it
contains a Lim‑L domain that is capable of binding
ubiquitylated targets, this domain was not required for
its interaction with phosphorylated tau. NDP52 expres-
sion is upregulated in response to nuclear factor eryth-
roid 2‑related factor 2 (NRF2) activation, which leads to
enhanced clearance of phosphorylated tau both in vitro
and in vivo, providing good evidence that NDP52 can
mediate selective autophagic clearance of phosphorylated
tau in response to NRF2 activation117.
Another protein that has been shown to have a role
as an autophagy receptor is HTT itself. In the presence
of mHTT, autophagosomes are able to form but seem
to lack content, suggesting a failure in the sequestration
of substrates118. Further investigation of this phenom-
enon led to the discovery that wild-type HTT is able to
352 | JUNE 2015 | VOLUME 16
© 2015 Macmillan Publishers Limited. All rights reserved
act as a scaffold for selective autophagy by interacting
with p62 and facilitating its association with LC3 and
ubiquitylated substrates119. Wild-type HTT was also
shown to be involved in autophagy initiation: domains
of the protein distinct from those shown to interact with
p62 are able to interact with ULK1, which reduces the
inhibitory interaction of ULK1 with mTOR and leads to
autophagy activation119.
Mutations in the genes encoding p62 and another
receptor protein, optineurin, have been associated with
ALS110,120, along with other diseases such as Paget dis-
ease of bone and open-angle glaucoma. The proposed
functions for p62 and optineurin in the clearance of
aggregate-prone proteins suggest a clear pathogenic
mechanism for forms of ALS; however, mutations in
the genes encoding p62 and optineurin are located
throughout the genes, affecting multiple domains121.
Although mutations affecting the ubiquitin-binding
or LC3‑interacting domain of optineurin have been
demonstrated to inhibit its function as a receptor 122, it
has yet to be established whether mutations affecting
other regions of the proteins have the same functional
consequences.
Autophagy as a therapeutic target
In recent years, numerous studies have demonstrated
that the aggregate-prone proteins at the heart of neuro-
degenerative disease toxicity are autophagy substrates
and that pharmacological upregulators of autophagy
can be beneficial in both cell and animal models of
these diseases, in which they are able to reduce both
intracytoplasmic aggregates and associated cell death
(reviewed in REF. 123). However, understanding the
exact mechanisms by which autophagy may be com-
promised in neurodegeneration is vital in the quest
for therapeutics that act on the pathway. In the pres-
ence of a defect, pharmacological upregulators acting
upstream would not enhance aggregate-prone protein
clearance, and where autophagosome clearance is dis-
rupted, enhancing formation may exacerbate rather
than ameliorate pathology. Increasing our knowledge
of the function and dysfunction of autophagy in neu-
rodegenerative disease will facilitate the design of
therapeutic interventions that can bypass a block in the
autophagic pathway, should it exist. Achieving this goal
is feasible given the diverse mechanisms of action of
the autophagy-modulating agents that have so far been
identified and the existence of others for which the
mechanisms are not yet completely understood.
The mTOR inhibitor rapamycin was the first drug
to be identified as an autophagy inducer and, as such,
has been at the forefront of studies establishing the role
of autophagy upregulation as a therapeutic strategy
for neurodegeneration, along with more soluble ana-
logues (rapalogues) such as temsirolimus (previously
known as CCI‑779)124–128. However, mTOR has diverse
autophagy-independent functions, and its inhibition
has adverse effects in patients, including immuno­
suppression and impaired wound healing. Efforts have
therefore been made to identify mTOR-independent
upregulators of autophagy.
www.nature.com/reviews/neuro

Several compounds have been identified that regu-
late autophagy via the cAMP–EPAC (exchange fac-
tor directly activated by cAMP 1)–phospholipase Cε
(PLCε)–inositol‑1,4,5‑trisphosphate (Ins(1,4,5)P3) path-
way and the Ca2+–calpain–Gsα pathway129. One of these
compounds, rilmenidine, which acts via Gi‑coupled imi-
dazoline receptors, was subsequently shown to decrease
aggregate load and disease signs in a transgenic mouse
model of HD130 and is now in safety trials in patients with
this disease (European Clinical Trials Database number:
2009‑018119‑14). Several other drugs that act on these
pathways have since been shown to be effective in mam-
malian models of neurodegeneration. Lithium acts to
induce autophagy by reducing Ins(1,4,5)P3 levels through
the inhibition of inositol monophosphatase131 and is
protective in mouse models of tauopathy 132, whereas
carbamazepine, which inhibits inositol synthesis, has
been shown to be protective in mouse models of AD133.
Additionally, inhibitors of calpain activation, such as
calpastatin, are able to promote the clearance of aggre-
gate-prone proteins by autophagy and are protective in
mouse and D. melanogaster models of HD and tauopa-
thy 134. Another mTOR-independent autophagy regula-
tor, the disaccharide trehalose, acts through unknown
mechanisms to promote autophagy 135. Trehalose is
neuro­protective in mouse models of tauopathy 136,137 and
prolongs lifespan in mouse models of ALS138,139.
Box 4 | New concepts and areas for investigation
Glial autophagy
Although much of the focus of the field so far has been on neuronal autophagy, recent
studies suggest that autophagy in glia may be similarly important in maintaining
neuronal health and in the pathogenesis of neurodegenerative diseases. Many
neurological diseases are associated with inflammatory responses, and autophagy is
required for the maintenance of mitochondrial architecture during inflammation in
astrocytes179. Studies in the Long–Evans shaker rat, a model of demyelinating disease180,
show that autophagy can protect vulnerable oligodendrocytes. In humans, duplications
of or point mutations in the gene encoding peripheral myelin protein 22 (PMP22) in
Schwann cells cause the progressive demyelinating disease Charcot–Marie–Tooth
disease type 1A (CMT1A). The levels of the aberrant PMP22 and Schwann cell health
appear to be regulated by autophagy in mouse models of CMT1A, and autophagy
upregulation may be a suitable strategy to consider for this class of diseases181.
In lysosomal storage disorders, astrocytic autophagy may be important for neuronal
health. Astrocyte-specific loss of the enzyme impaired in multiple sulfatase deficiency
causes a defect in autophagosome maturation. As the ability of these astrocytes to
support normal neurons is compromised, it is interesting to speculate whether this non-
cell-autonomous mechanism can be partially attributed to the autophagy defect182.
Autophagy and secretion of proteins
Recent studies have highlighted the ways in which autophagy may regulate various
secretory processes183. This concept may be of relevance to neurodegenerative diseases,
particularly those in which cell‑to‑cell spreading of toxic proteins such as tau and
α‑synuclein have been implicated184. However, the current status of this field is complex.
In Parkinson disease, it seems that lysosomal impairment or defective autophagosome–
lysosome fusion causes increased exosomal α‑synuclein release, which can be
reconciled with the resulting increased pool of autophagosomes185–187. However, in the
context of Alzhiemer disease, the situation appears to be different. In mouse studies, the
secretion of amyloid‑β and the extracellular plaque load were reduced when autophagy
was blocked by deletion of Atg7 in relevant neurons. However, the authors suggest that
this was associated with increased intracellular amyloid‑β levels and enhanced
pathology188. This study reiterates the possibility that intracellular amyloid‑β may be a
major toxic entity. However, it is important to note that in all these cases the secretory–
excretory route has not been precisely defined189.
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REVIEWS
The combined evidence from different compounds
and different models of neurodegeneration strongly sup-
port the idea that upregulation of autophagy is a valid
therapeutic strategy for neurodegenerative disease.
However, upregulating autophagosome biogenesis is
unlikely to be suitable or safe for all neurodegenerative
diseases. For example, in diseases in which autophago-
some degradation is slowed owing to lysosomal lesions,
increasing autophagosome formation will result in largely
unproductive formation of autophagosomes that are not
properly cleared and therefore a marked increase in
autophagosomes, which may be deleterious. It has been
suggested that one approach to ameliorate lyso­somal
function in some of these diseases is to enhance the
activity of TFEB. Viral transduction of TFEB has dem-
onstrated promising results in mouse models of LSDs140,
PD105 and tauopathies141. Another strategy for the treat-
ment of LSDs may be to reduce the activity of cystatins,
which are endogenous inhibitors of lysosomal cathepsin
proteases. This strategy has also yielded promising results
in mouse models of AD142.
Future challenges
As our understanding of the autophagic process
increases, we are beginning to identify new areas that
will be important to investigate in the quest to under-
stand the role of autophagy in neurodegenerative dis-
ease (BOX 4). One of the major difficulties in studying
autophagic processes in the brain in vivo is to define
flux on the basis of ‘snapshot’ data. For example, if
a study finds that there are increased numbers of
autophagosomes in the brain in a mouse model or an
individual with a particular disease, this does not mean
that autophagy is increased. An increase in autophago-
some numbers could be due to increased formation
(which can lead to increased delivery to lysosomes of
substrates) or could be due to impaired lysosomal deg-
radation or trafficking of completed autophagosomes
(as occurs in LSDs). In mice, at least, good progress has
been made in adapting methods to differentiate between
these scenarios. Hetz and colleagues143 performed intra-
ventricular injections of adenoviruses encoding mono-
meric tandem mCherry–GFP–LC3, which were then
expressed in central and peripheral neurons. Before
lysosome fusion, the LC3 vesicles were autophago-
somes and exhibited both red and green fluorescence,
but after fusion, the vesicles were autolysosomes and
exhibited red fluorescence only (as GFP was more rap-
idly quenched in the acidic environment). Assessing the
numbers of the two types (colours) of vesicles enabled a
more reliable assessment of autophagic flux.
Another important issue relates to the interpretation
of whether autophagy is causing cell death in certain
diseases in which increased numbers of autophago-
somes are observed. First, one needs to know whether
there is increased formation of autophagosomes or
decreased degradation. If there is increased formation,
then one needs to use a number of genetic manipu-
lations of autophagy genes to perturb autophagy in
order to test whether the increased autophagy is caus-
ally involved in the toxic process. This is preferable to
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REVIEWS

chemical strategies because all the drugs currently used
to inhibit (or induce) autophagy have off-target effects.
One also needs to consider that manipulations of some
autophagy genes may have consequences unrelated to
autophagy (for example, beclin 1 indirectly regulates p53
levels144). Recent data suggest additional non-autophagy
roles of beclin 1 in retromer trafficking and phago­cytosis
of amyloid‑β in microglia, and this appears to be disease-
relevant because reduced beclin 1 and retromer levels
have been observed in AD brains145.
These challenges are difficult enough to negotiate in
cell culture and mouse experiments. However, ultimately,
we need to understand human diseases, many of which
are complex and cannot be faithfully modelled in animals.
Thus, a major challenge for the future will be to develop
markers that enable inferences regarding autophagic flux
in vivo and in post-mortem samples. Such new methods
will not only be helpful for understanding disease patho-
genesis but also serve as a powerful tool for monitoring
target engagement in therapeutic trials.
Conclusions
There are now substantial data showing how autophagy
may be compromised in various neurodegenerative
diseases and extensive preclinical support for the use of
autophagy inducers in certain neurodegenerative dis-
ease models. Further understanding of the basic biology
of autophagy will provide additional insights into how
disease-causing protein variants may affect this critical
degradation pathway. Similarly, further drug discovery
efforts coupled with the development of relevant bio-
markers will facilitate the testing of whether autophagy
induction can affect the genesis or progression of some
neurodegenerative diseases.

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Acknowledgements
The authors are grateful for funding from a Wellcome Trust
Principal Research Fellowship (D.C.R.), a Wellcome Trust and
Medical Research Council (MRC) Strategic Grant on neuro­
degeneration, a Wellcome Trust Strategic Award to the
Cambridge Institute for Medical Research, the Alzheimer’s
disease National Institute for Health Research (NIHR)
Biomedical Research Unit at Addenbrooke’s Hospital, the Tau
Consortium and Alzheimer’s Research UK.
Competing interests statement
The authors declare competing interests: see Web version for
details.
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