REVIEWS

Autophagy in acute brain injury

Lorenzo Galluzzi1–5*, José Manuel Bravo-San Pedro1–5*, Klas Blomgren6 and
Guido Kroemer1–4,6–8
Abstract | Autophagy is an evolutionarily ancient mechanism that ensures the lysosomal
degradation of old, supernumerary or ectopic cytoplasmic entities. Most eukaryotic cells,
including neurons, rely on proficient autophagic responses for the maintenance of homeostasis in
response to stress. Accordingly, autophagy mediates neuroprotective effects following some
forms of acute brain damage, including methamphetamine intoxication, spinal cord injury and
subarachnoid haemorrhage. In some other circumstances, however, the autophagic machinery
precipitates a peculiar form of cell death (known as autosis) that contributes to the aetiology of
other types of acute brain damage, such as neonatal asphyxia. Here, we dissect the
context-specific impact of autophagy on non-infectious acute brain injury, emphasizing the
possible therapeutic application of pharmacological activators and inhibitors of this catabolic
process for neuroprotection.

The term autophagy (from ancient Greek autos, ‘self ’ degradation — to cope with perturbations in both the
1
Equipe 11 Labellisée Ligue
and phago, ‘to eat’ — literally meaning ‘self-eating’) refers intracellular and the extracellular microenvironments7,8.
Contre le Cancer, Centre de
Recherche des Cordeliers, to a group of evolutionarily old mechanisms whereby Importantly, such adaptive macroautophagic responses
75006 Paris, France. eukary­otic cells eliminate cytoplasmic material through can be driven by an excess of autophagic substrates or
2
INSERM, U1138, lysosomal degradation1. Three main types of autophagy by an accrued demand for autophagic products or func-
75006 Paris, France. have been described so far, and these differ from each tions, and this functional dichotomy determines cargo
3
Université Paris Descartes/
Paris V, Sorbonne Paris Cité,
other in the modalities through which autophagic sub- specificity 9. Thus, cells exposed to a chemical agent that
75006 Paris, France. strates are delivered to lysosomes. Specifically, cargo depolarizes mitochondria mount a macroautophagic
4
Université Pierre et Marie delivery during microautophagy involves the direct response specifically targeting damaged mitochondria
Curie/Paris VI, 75006 Paris, invagination of the lysosomal membrane2. By contrast, (which is known as mitophagy) and aimed at removing
France.
chaperone-mediated autophagy (CMA) depends on the an excess of potentially dangerous autophagic substrates.
5
Gustave Roussy
Comprehensive Cancer recognition of autophagic substrates bearing a KFERQ By contrast, cells subjected to nutrient deprivation activate
Institute, 94805 Villejuif, motif by cytosolic chaperones of the heat-shock protein a relatively nonspecific variant of macroautophagy to sat-
France. family, followed by the lysosomal-associated membrane isfy an increased need for autophagic products9. From here
6
Karolinska Institute, protein 2 (LAMP2)-dependent translocation of these sub- onwards, the term autophagy refers to macroautophagy
Department of Women’s and
Children’s Health, Karolinska
strates across the lysosomal membrane3. Finally, macro­ unless otherwise specified.
University Hospital Q2:07, autophagy relies on the progressive sequestration of Adaptive autophagy often mediates prominent
17176 Stockholm, Sweden. autophagic cargoes by double-membraned vesicles known cytoprotective and anti-inflammatory effects10 (BOX 1).
7
Metabolomics and Cell as autophagosomes, which, upon their closure, fuse with Accordingly, defects in autophagic responses contrib-
Biology Platforms, Gustave
lysosomes to form so‑called autolysosomes4 (FIG. 1). ute to various human pathologies that involve the loss
Roussy Comprehensive
Cancer Institute, Macroautophagy (the best characterized form of of cellular homeostasis and consequent demise of post­
94805 Villejuif, France. autophagy in mammalian cells) operates at baseline mitotic cells; such pathologies include cardiovascular and
8
Pôle de Biologie, Hopitâl levels to preserve the structure and activity of several renal diseases, as well as acute and chronic neurological
Européen George Pompidou, subcellular compartments, including the endoplasmic disorders11,12. Moreover, autophagic defects compromise
AP‑HP, 75015 Paris, France.
reticulum (ER) and mitochondria5. Accordingly, nor- inflammatory homeostasis and generally increase the
*These authors contributed
mal autophagic functions in the CNS are required for susceptibility of the host to infectious diseases13. In addi-
equally to this work.
neuronal survival in the absence of external perturba- tion, accumulating evidence indicates that functional
Correspondence to L.G.
and G.K.
tions6. Moreover, the molecular machinery for macro­ autophagic responses can, at least in some situations, pre-
deadoc@vodafone.it; autophagy responds to a wide panel of sensors that cipitate a peculiar form of regulated cell death (RCD) that
kroemer@orange.fr continuously monitor indicators of homeostasis7,8. Thus, has been called autosis14 (BOX 2). This evidence implies
doi:10.1038/nrn.2016.51 cells can adjust autophagic flux — that is, an ongoing that autophagy de facto mediates cytotoxic rather
Published online 3 Jun 2016 autophagic response functionally coupled to lysosomal than cytoprotective effects in specific settings15. The

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 1

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

a Chaperone-mediated autophagy (CMA) b Microautophagy

HSP70
KFERQ Bulk delivery
Acid hydrolase

Unfolded Cargo
protein
Chaperone

Selective
delivery
HSP90

LAMP2
Fragment of
unfolded protein

c Macroautophagy
Lysosomal degradation Autolysosome

Isolation
membrane

Mitochondrion

Phagophore

LC3

Autophagosome

Autophagic cargo

Figure 1 | General organization of autophagic responses. All types of autophagy culminate in theReviews
Nature delivery| Neuroscience
of
autophagic substrates to the lysosome and the degradation of these substrates by lysosomal hydrolases.
a | Chaperone-mediated autophagy involves the recognition of autophagic substrates bearing a KFERQ motif by
members of the heat shock protein (HSP) family. This step is followed by the lysosomal-associated membrane protein 2
(LAMP2)-dependent translocation of chaperoned autophagic cargoes across the lysosomal membrane. b | By contrast,
cargo delivery during microautophagy occurs upon the direct invagination of the lysosomal membrane. c | During
macroautophagy, cytoplasmic entities destined for disposal are progressively enwrapped by a double-membrane
organelle known as an autophagosome. Following closure, mature autophagosomes can fuse with lysosomes to form
so‑called autolysosomes. LC3 (official name: MAP1LC3B), microtubule-associated protein 1 light chain 3β.

Regulated cell death
(RCD). A form of cell death
that depends on genetically
encoded machinery. In
contrast to its accidental
counterpart, RCD can be
retarded or accelerated via
specific pharmacological or
genetic interventions.
precise mechanisms through which proficient autophagic
responses accelerate rather than retard RCD remain to
be elucidated.
In this Review, we discuss recent findings suggesting
that autophagy has a context-specific impact on differ-
ent types of non-infectious acute brain injury — includ-
ing excitotoxic challenges and epileptogenesis, exposure
to neurotoxins, neonatal asphyxia, adult stroke and
neuro­trauma — with particular emphasis on the possi-
bility of using pharmacological modulators (activators
or inhibitors) of autophagy for neuroprotection.
General regulation of autophagy
Autophagy mechanistically depends on the coordinated
activity of various members of the autophagy-­related
(ATG) protein family, which together underlie the
nucleation, elongation and closure of autophagosomes, as
well as the fusion of autophagosomes with lysosomes16.
ATG9 mediates an essential, yet poorly characterized,
function in early autophagosome formation. ATG3, ATG4
and ATG7 cooperate to catalyse the lipidation of micro-
tubule-associated protein 1 light chain 3β (MAP1LC3B;
also known as LC3), which subsequently accumulates
in autophagosomal membranes and binds to substrates
tagged for autophagic degradation. Moreover, ATG7
cooperates with ATG10 to promote the formation of an
ATG5–ATG12–ATG16L1 complex, which supports the
elongation of autophagosomes16.
Most of the signal transduction cascades that control
autophagy operate by influencing the activation status
of either of two multiprotein complexes: mechanistic
target of rapamycin (MTOR) complex 1 (MTORC1) or

2 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

a supramolecular complex containing beclin 1 (BECN1)
and phosphatidylinositol 3‑kinase catalytic subunit
type 3 (PIK3C3; also known as VPS34). MTORC1
contains MTOR and regulatory-associated protein of
MTOR (RAPTOR) and operates as a central suppres-
sor of autophagy. Conversely, the BECN1–VPS34 com-
plex promotes autophagy by catalysing the formation
of phosphatidylinositol 3‑phosphate16. For instance,
AMP-activated protein kinase (AMPK), which responds
to decreasing ATP levels upon phosphorylation by
serine/threonine kinase 11 (STK11; also known as LKB1)
as well as to phosphorylation by calcium/calmodulin-
dependent protein kinase kinase 2 (CAMKK2; also
known as CAMKKβ)17, triggers autophagy by inacti-
vating MTORC1 (REF. 18) and by activating VPS34 or
unc‑51‑like kinase 1 (ULK1), which is a key kinase for
the initiation of autophagic responses19–21. Conversely, the
stress sensor death-associated protein kinase 1 (DAPK1)
stimulates autophagy by releasing BECN1 from inhibi-
tory interactions with anti-apoptotic members of the B ell
lymphoma2 (BCL‑2) protein family 22.
Mitophagic responses to depolarized mitochondria
are generally associated with the PTEN-induced puta-
tive kinase 1 (PINK1)-dependent and parkin RBR E3
ubiquitin protein ligase (PARK2)-dependent ubiqui-
tylation of outer mitochondrial membrane proteins,
including mitofusin 1 (MFN1) and MFN2 (REF. 23).
Ubiquitylated MFN1 and MFN2 are degraded by the
proteasome, resulting in mitochondrial fragmentation23.
Moreover, ubiquitylated structures, including mito-
chondria, are bound by the autophagic adaptor seques-
tosome 1 (SQSTM1; also known as p62), which enables
their uptake by autophagosomes through lipidated
LC3 (REF. 24). Other signal transduction cascades reg-
ulate autophagy in many cell types, including neurons,
astrocytes and glial cells1,7. However, these pathways are

Box 1 | Cytoprotection, hormesis and autophagy

One of the primary functions of autophagy in mammalian organisms is to support cellular viability in the course of
adaptive responses to stress10. In line with this notion, the pharmacological or genetic inhibition of autophagy usually
sensitizes mammalian cells (including neurons) to a wide panel of stressful conditions, including nutritional, physical and
chemical cues10.
The precise mechanisms through which autophagy exerts cytoprotective functions exhibit a high degree of context-
dependency. These mechanisms include, but are not limited to, the following: the provision of catabolic substrates for
bioenergetic reactions (for example, in response to nutrient deprivation); the supply of substrates for anabolic
reactions (such as DNA repair, upon exposure to DNA-damaging agents); the preservation of optimal energy
metabolism (associated with the co‑activation of mitophagy and mitochondrial biogenesis); the elimination of
potential sources of oxidative damage (such as permeabilized mitochondria and redox-active protein aggregates); and
the direct inhibition of lethal signal transducers or processes (such as mitochondrial outer membrane
permeabilization)1,192,193.
Notably, adaptive autophagic responses can lead to robust hormetic effects; that is, they do not simply contribute to the
restoration of baseline physiological conditions, but also establish a novel state of homeostasis characterized by an
accrued fitness and resistance to stress194 (see part a of the figure). The hormetic effects of autophagy are relatively broad,
meaning that cells mounting an adaptive autophagic response upon exposure to a specific insult normally exhibit
increased resistance to various stressful conditions194. However, although autophagy underlies the hormetic effects of
various sublethal insults, it rarely mediates the cytotoxicity of the same challenges imposed in a lethal manner. Thus, the
hormetic and lethal mechanisms triggered by specific insults often differ from each other, and the former (frequently
autophagy) generally inhibit the latter (frequently apoptosis or some forms of regulated necrosis)192.
Autophagy has also been associated with non-adaptive hormesis195 (see part b of the figure). Thus, cells that are
pharmacologically driven into an autophagic response in the absence of potentially dangerous insults can also develop an
accrued fitness and broad resistance to future stress195. The hormetic effects of autophagy explain (at least in part) why
several nutritional or pharmacological manipulations that induce autophagic responses — for example, caloric restriction
or the administration of the sirtuin 1 activator resveratrol or the mechanistic target of rapamycin complex 1 (MTORC1)
inhibitor rapamycin — extends lifespan in several model organisms, including mice196. Moreover, non-adaptive autophagy­-
dependent hormesis may provide the basis for using pharmacological activators of autophagy as neuroprotective agents in
individuals at increased risk of acute brain injury.

a b
Hormetic Hormetic Hormetic
Stress capacity stimulus capacity
Fitness

Fitness

Autophagic adaptor
Adaptive Adaptive
capacity capacity
A protein that physically
interacts with lipidated LC3 as
well as with autophagic
receptors, hence facilitating the
sequestration of autophagic Physiological Adaptive response Hormetic response Physiological Hormetic response
cargoes within forming conditions conditions
autophagosomes. Time Time

Nature Reviews | Neuroscience

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 3
©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

elicited by insults that have a limited role in the patho-
genesis of acute brain disorders, and hence will not be
discussed in further detail here.
Some of the biochemical reactions that accompany
autophagy are frequently exploited to experimentally
monitor autophagic responses in vitro and in vivo 25.
These reactions include the following: expression of
ATG5, BECN1 and LC3; phosphorylation of AMPK,
ULK1 or substrates of MTORC1; interaction of BECN1
with BCL‑2 family members; lipidation of LC3; lyso­
somal acidification; and the degradation of p62 or
autophagic substrates25. Importantly, several metabolic,
physical and chemical cues can promote morphological
and biochemical manifestations of autophagy, includ-
ing the cytoplasmic accumulation of autophagosomes
and autolysosomes (cytoplasmic vacuolization), as
well as LC3 lipidation10. However, cytoplasmic vac-
uolization and LC3 lipidation reflect the size of the
autophagosomal compartment, meaning that they also
increase when autophagosomes cannot fuse with lyso­
somes or when lysosomal functions are impaired25.
Monitoring autophagic flux is therefore crucial to
understand whether proficient autophagic responses
are ongoing or whether the autophagic machinery is
blocked25. This aspect of autophagic responses has pro-
found therapeutic implications. Indeed, when neuro-
toxicity is accompanied by a flux blockade, favouring
autophagosomal clearance or inhibiting the formation
of autophagosomes may mediate neuroprotection,
whereas pharmacologically boosting the formation of
autophagosomes or inhibiting lysosomal degradation
has detrimental effects. Unfortunately, autophagic flux
was not monitored in several of the reports presented
below, considerably complicating data interpretation.
Moreover, the pharmacological agents that are cur-
rently available for modulating autophagy are relatively

Box 2 | Autophagic cell death and autosis

The term autophagic cell death has long been used to refer to a type of
regulated cell death (RCD) that is characterized by extensive cytoplasmic
vacuolization197. However, the fact that cells die while manifesting
morphological (or biochemical) markers of autophagy does not necessarily
mean that autophagy causally promotes RCD, for at least two reasons15.
First, the accumulation of autophagosomes or LC3 lipidation may reflect
the blockade of lysosomal degradation rather than the upregulation of
autophagic flux25. Second, even the activation of proficient autophagic
responses (as monitored with flux assays) may represent an ultimate, but
unsuccessful, attempt of cells to restore homeostasis192. In line with this
notion, the pharmacological or genetic inhibition of autophagy most often
accelerates, rather than retards, stress-induced RCD, at least in
mammalian cells192.
Instances of bona fide autophagic cell death (a form of RCD that
mechanistically depends on the molecular machinery for autophagy)
were initially characterized in developing Drosophila melanogaster
larvae198. Accumulating evidence indicates that a specific variant of
autophagic cell death known as autosis also occurs in mammalian cells,
and may have important implications for acute brain injury14. Indeed,
various experimental conditions that promote autophagy, as well as
pathophysiologically relevant stimuli such as hypoxia–ischaemia,
induce autosis14. Cells undergoing autosis exhibit peculiar and
temporally consecutive sets of morphological features, which have
been categorized into three phases. In phase 1a, mild chromatin
condensation, nuclear membrane convolution and cytoplasmic
accumulation of autophagosomes (APs), autolysosomes (ALs) and empty
vacuoles (EVs) are observed (see the figure). Phase 1b involves the
separation of the inner and outer nuclear membrane, localized swelling
of the perinuclear space (PNS) and the occurrence of membrane-bound
densities in the PNS. Phase 2 is characterized by focal nuclear concavity
and focal ballooning of the PNS, accompanied by general signs of
necrosis, including swollen mitochondria, rare autophagosomes and
autolysosomes, and endoplasmic reticulum degradation.
In addition to depending on components of the autophagic molecular
machinery, as demonstrated with pharmacological inhibitors, such as
3-methyladenine (3-MA), and small interfering RNAs (siRNAs) targeting
components of the autophagy machinery (such as BECN1, ATG13 or
ATG14), autosis critically relies on the plasma membrane Na+/K+ ATPase,
and hence can be pharmacologically inhibited by various cardiac
glycosides, which target the α1 subunit of the Na+/K+ ATPase (ATP1A1).
It should be noted that the Na+/K+ ATPase expressed by some mouse
and rat tissues (including the cardiac muscle) is markedly more
resistant to inhibition by cardiac glycosides than its human
counterpart199 — partly because murine ATP1A1 binds to cardiac
glycosides with reduced affinity. However, the Na+/K+ ATPase expressed
in the mouse and rat brain is normally sensitive to the inhibitory activity
of several cardiac glycosides, including ouabain and neriifolin14,200.
Thus, cardiac glycosides may represent valuable pharmacological tools
for the neuroprotective inhibition of autosis in both preclinical and
clinical settings.

Autosis
Nutrient deprivation
Na +

↓ATP Na+/K+ ATPase Phase 1a Phase 1b Phase 2

K+ EV
AP
PNS
↓O2
Hypoxia–ischaemia

↑ BECN1
Mitochondrion AL

Autophagy-inducing ?
peptides ? • 3-MA ?
• BECN1-targeting siRNA
Cardiac glycosides
• ATG13-targeting siRNA
• ATG14-targeting siRNA

Nature Reviews | Neuroscience

4 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

nonspecific (BOX 3; FIG. 2; TABLE 1), and the use of such
non-selective compounds constitutes a caveat of several
studies discussed in this Review.
Autophagy in excitotoxicity
The term excitotoxicity refers to the neurotoxic effects
caused by the prolonged depolarization of postsynap-
tic terminals, which results in the sustained influx of
Ca2+ ions from the synaptic cleft26. Abrupt or prolonged
increases in cytoplasmic Ca2+ levels cannot be properly
buffered by the ER, and hence may trigger numerous
processes with potentially catastrophic consequences,
including the activation of various Ca2+-dependent
hydrolases, such as calpains, and mitochondrial permeabil-
ity transition (MPT)-driven RCD27. Excitotoxicity has
been implicated in the neurotoxic effects of seizures,
which are multifactorial (and generally symptomatic)
alterations of brain electrical activity that can occur
in short episodes or proceed uninterrupted for hours
(status epilepticus)28. Excitotoxic substances such as
glutamate 29, quinolinic acid 30, NMDA 31 and kainic
acid32–34 reportedly induce markers of autophagic and/or
mitophagic responses in rodents. However, whether
autophagy protects neurons from excitotoxic insults
remains unclear.
Evidence — particularly from in vitro studies —
suggests that autophagy may counteract or prevent the
detrimental effects of excitotoxicity. Pharmacological
activators of autophagy, including rapamycin and
trehalose, protected neonatal rat hippocampal neurons
from glutamate-induced excitotoxic death in vitro,
whereas chloroquine (which inhibits autophagy) had
no beneficial effects in this setting 35. Pyruvate (which
stimulates VPS34 activation and preserves bioenergetic
fitness) reduced the neurotoxicity of glutamate applied
to human neuroblastoma SH‑SY5Y cells — an effect that
could be blocked by the administration of the relatively
nonspecific VPS34 inhibitor 3‑methyl­adenine (3‑MA)36.
Lithium (which stimulates autophagy) limited the exci-
totoxic damage induced by kainic acid on spinal cord

Box 3 | Pharmacological modulation of autophagy

Calpains
A family of Ca2+-dependent,
non-lysosomal cysteine
proteases involved in
processes as diverse as
long-term synaptic
potentiation and regulated cell
death.
Mitochondrial permeability
transition
(MPT). An abrupt increase in
the permeability of the inner
mitochondrial membrane to
small solutes, resulting in the
dissipation of the
mitochondrial transmembrane
potential and structural
breakdown of the organelle.
Nutrient deprivation is perhaps the most physiological stimulus that upregulates autophagic flux. It establishes a state of
bioenergetic and catabolic stress characterized by decreased intracellular levels of various metabolites, including ATP,
acetyl-CoA and several amino acids7. Declining ATP levels are paralleled by an increase in AMP, which promotes
autophagy by stimulating the ability of serine/threonine kinase 11 (STK11; also known as LKB1) to activate AMP-activated
protein kinase (AMPK). Low concentrations of acetyl-CoA stimulate autophagic responses as a consequence of reduced
E1A-binding protein p300 (EP300) activity. Finally, a shortage in amino acids triggers autophagy as it results in the
inactivation of mechanistic target of rapamycin complex 1 (MTORC1)7. As the systemic effects of nutrient deprivation are
pleiotropic and may introduce biases that are difficult to control, the effects of autophagy on acute brain injury have
been investigated with a panel of pharmacological agents with autophagy-inducing or -inhibiting properties25. However,
virtually all of these molecules also exhibit limited specificity, and some of them are sometimes administered
intraperitoneally or intravenously even though they might not readily cross the blood–brain barrier (TABLE 1); these two
issues considerably complicate data interpretation.
Commonly used inducers of autophagy include the following agents: rapamycin and curcumin, which potently inhibit
MTORC1; trehalose, which triggers MTORC1‑independent autophagy via hitherto unrecognized mechanisms; pyruvate,
which replenishes the acetyl-CoA pool and promotes the activation of death-associated protein kinase 1 (DAPK1); lithium
(alone or combined with valproate), which inhibits inositol(myo)-1(or 4)-monophosphatase 1 (IMPA1) and glycogen
synthase kinase 3β (GSK3β) and hence indirectly boosts the activity of phosphatidylinositol 3‑kinase catalytic subunit
type 3 (PIK3C3; also known as VPS34); the GSK3β inhibitor SB216763; and kaempferol, a phytoestrogen that reportedly
activates AMPK16. Additional molecules have been shown to promote signs of autophagy in the CNS (TABLE 1), but the
underlying molecular mechanisms remain to be elucidated. Moreover, autophagic flux has not been monitored in all these
settings, implying that some of these agents may actually inhibit (rather than stimulate) proficient autophagic responses.
Two main approaches are used for the experimental inhibition of autophagy: interfering with the formation of
autophagosomes and blocking lysosomal degradation. Molecules that inhibit VPS34 (hence preventing autophagy
initiation) include 3‑methyladenine (3‑MA), wortmannin, KU55933 and LY294002. Compared to 3‑MA and wortmannin
(which are considerably nonspecific), KU55933 and LY294002 exhibit an improved potency and selectivity for VPS34
(REF. 25). Taurine limits autophagy initiation by stimulating MTORC1 activity. β‑asarone inhibits mitogen-activated
protein kinase 8 (MAPK8; also known as JNK1), hence limiting the ability of JNK1 to release beclin 1 (BECN1) from
inhibitory interactions with B cell lymphoma (BCL‑2) family members16. Compound C (an AMPK inhibitor) also suppresses
autophagic responses at initiation, as do several proteins that stimulate AKT1 signalling (and hence MTORC1), such as
insulin-like growth factor 1 (IGF1), glial cell-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF)16.
Conversely, chloroquine, E64d, pepstatin, bafilomycin A1 and l‑asparagine all inhibit autophagy by blocking the fusion
of autophagosomes with lysosomes or by suppressing lysosomal functions16. As lysosomes participate in many biological
processes in addition to autophagy (for example, endocytosis), these molecules have multiple off-target effects25. Several
other molecules have been shown to decrease the abundance of autophagic markers in the CNS (TABLE 1). Although
some of these agents may modulate autophagic responses as a consequence of their antioxidant activity, the molecular
mechanisms that underlie these findings have not yet been clarified. Importantly, whereas blocking the formation of
autophagosomes is associated with a global decrease in autophagic markers, interfering with the fusion of
autophagosomes with lysosomes or inhibiting lysosomal functions generally causes an increase in the size of the
autophagosomal compartment, and hence manifests as a paradoxical increase in cytoplasmic vacuolization and LC3
lipidation. Thus, without robust flux studies, it is difficult to conclude whether a specific intervention actually promotes or
inhibits proficient autophagic responses.

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 5

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

RTK PI3K I Autophagy inducers* Autophagy inhibitors‡

LKB1 CAMKKβ MTORC1 AKT1 Initiation

↓ATP ↑Ca2+ • Kaempferol • Apelin-derived peptide
ULK1 • Rapamycin • GDNF
ATG13 ATG101 • Simvastatin • HGF
AMPK • IGF1
Initiation FIP200 • Pifitrin-α
• Salubrinal
ULK complex • Taurine
DAPK1 ATG9
AMBRA1
BECN1 BECN1 ATG14 Nucleation
VPS34 VPS15 Nucleation • Lithium • 3-methyladenine
JNK1 BCL-2 • Pyruvate • β-asarone
• SB216763 • KU55933
Pre-autophagosome • Valproate • LY294002
PI3K III complex • Wortmannin
ATG10
Elongation Forming
phagosome
ATG12 ATG12 Elongation
ATG5
ATG16L1 Retinoic acid
preLC3 ATG5 ATG12
ATG7 Fusion and degradation
ATG5–ATG12–ATG16L1 complex
ATG4 ATG3
• Bafilomycin A1
LC3 I LC3 II • Chloroquine
• E64d
• L-asparagine
Mitophagy PE PE LC3 II • Pepstatin
PE LC3 II

LC3 II Mitophagy
p62
PE LC3 II

PARK2 Ub Autophagosome Mdivi-1

Ub

Mitochondrion Changes in autophagic markers

Fragmentation LC3 II PE • Curcumin • Apocynin
Ub Ub • Deferoxamine • Caffeine
Fusion • Melatonin • Carnosine
Ub Ub Ub
PARK2 • N-acetyl-cysteine • Ceftriaxone
Ub MFN2 • Trehalose • GCEE
PINK1 Lysosome
MFN1 • Trichostatin A • Hydrogen sulfide
• Melatonin
Autolysosome • N-acetyl-serotonin
• NAD+
Ca 2+
• Necrostatin-1
PINK1 • Neriifolin
• Propofol
MPT Mitochondrion • Rosiglitazone
depolarization Degradation
• SN50

Figure 2 | Autophagy in the CNS. In response to decreasing ATP
concentrations or increases in cytosolic Ca2+ levels, neurons and other
cells of the CNS mount a canonical autophagic response driven by
AMP-dependent protein kinase (AMPK) and unc‑51-like kinase 1 (ULK1),
a process that is otherwise suppressed by mechanistic target of
rapamycin complex 1 (MTORC1). The functionality of this response
depends on the coordinated activation of a refined molecular
machinery that supports the nucleation, elongation and closure of
autophagosomes, as well as the fusion of autophagosomes with
lysosomes and lysosomal degradation. High cytosolic Ca2+ levels can
also promote mitochondrial permeability transition (MPT). In this
setting, several proteins of the outer mitochondrial membrane become
ubiquitylated, eventually resulting in mitochondrial fragmentation and
uptake by forming autophagosomes through lipidated LC3 (LC3 II). This
mitophagic response generally mediates robust neuroprotective effects.
Common pharmacological modulators of autophagy as well as
molecules that have been associated with an increase or decrease of
autophagic markers in the CNS are listed (see also TABLE 1). AMBRA1,
autophagy/beclin 1 regulator 1; ATG, autophagy-related; BCL-2, B cell
lymphoma 2; BECN1, beclin 1; CAMKKβ Nature(officialReviews | Neuroscience
name: CAMMK2),
calcium/calmodulin-dependent protein kinase kinase 2; DAPK1,
death-associated protein kinase 1; FIP200 (official name: RB1CC1), RB1
inducible coiled-coil 1; GCEE, γ‑glutamylcysteinyl ethyl ester; GDNF,
glial cell-derived neurotrophic factor; HGF, hepatocyte growth factor;
IGF1, insulin-like growth factor 1; JNK1 (official name: MAPK8),
mitogen-activated protein kinase 8; LC3 (official name: MAP1LC3B),
microtubule-associated protein 1 light chain 3β; LKB1 (official name:
STK11), serine/threonine kinase 11; MFN, mitofusin; p62 (official name:
SQSTM1), sequestosome 1; PARK2, parkin RBR E3 ubiquitin protein
ligase; PE, phosphatidylethanolamine; PI3K, phosphoinositide 3‑kinase;
PINK1, PTEN-induced putative kinase 1; RTK, receptor tyrosine kinase;
Ub, ubiquitin; VPS15, phosphoinositide 3‑kinase regulatory subunit 4;
VPS34 (official name: PIK3C3), phosphatidylinositol 3‑kinase catalytic
subunit type 3. *Or molecules associated with increased autophagic
markers in the CNS. ‡Or molecules associated with decreased
autophagic markers in the CNS.

6 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Ischaemic preconditioning
A hormetic process in which
sublethal hypoxic challenges
establish resistance to
subsequent exposure to lethal
hypoxia, and which is known to
robustly activate autophagy.
motor neurons from chick embryos37 or from mice38.
Furthermore, the depletion of BECN1 or high-mobility
group box 1 (HMGB1), a nuclear protein that stimulates
autophagy upon release in the cytoplasm, increased the
sensitivity of cultured rat neurons to NMDA39,40. Small
interfering RNA (siRNA)-mediated downregulation of
BECN1 also abolished the protective effects of ischaemic
preconditioning on mouse cortical neurons that were
subsequently exposed to a neurotoxic glutamate chal-
lenge41,42. Similar results were obtained with 3‑MA and
the VPS34 inhibitor KU55933 in mice42. Indirect evi-
dence for a neuroprotective role of autophagy in mod-
els of excitotoxicity has been collected in vivo, as mice
lacking both copies of NHL repeat-containing 1 (Nhlrc1)
exhibited autophagic defects and are more sensitive to
kainic acid‑triggered seizures than wild-type mice43.
Similarly, autophagy-stimulating manganese com-
plexes of curcumin and diacetylcurcumin limited kainic
acid‑driven seizures in rats44. In these studies, however,
cell death was not directly monitored, implying that
autophagy may affect epileptogenesis independently of
its ability to limit the demise of stressed neurons.
Additional data indicate that autophagic defects have
an aetiological role in epileptogenesis. Thus, activators
of autophagy may mediate therapeutic effects in this set-
ting (Supplementary information S1 (table)). In humans,
autophagy-suppressing mutations in genes encoding
endogenous MTORC1 inhibitors, including tuberous
sclerosis 1 (TSC1), TSC2 and phosphatase and tensin
homologue (PTEN), are associated with an increased
predisposition to epilepsy 28. Accordingly, post-mortem
or surgical samples from individuals with tuberous scle-
rosis exhibited profound autophagic defects45, and mice
lacking copies of Tsc1, Tsc2 or Pten in cortical neurons,
hippocampal neurons or glial cells exhibited frequent
episodes of spontaneous seizures that were associated
with premature mortality 45–48. Tsc1−/−, Tsc2−/− or Pten−/−
mouse neurons or glial cells also exhibited profound
autophagic defects, and the systemic administration of
rapamycin considerably alleviated the severity of sei-
zures in, and improved the survival of, these diseased
mice45–49. Finally, the conditional deletion of Atg7 from
cortical and hippocampal mouse neurons also resulted
in spontaneous recurrent seizures45.
Nevertheless, apparently at odds with the notion
that autophagy may help to protect neurons from exci-
totoxicity, 3‑MA and the lysosomal inhibitor E64d25
inhibited manifestations of autophagy, including LC3
lipidation and p62 degradation, and protected striatal
neurons (ex vivo and in vivo) from the toxic effects of
quinolinic acid and kainic acid30,33,50. Moreover, 3‑MA,
LY294002 (another VPS34 inhibitor), carnosine (an
endogenous pleiotropic dipeptide) and insulin-like
growth factor 1 (IGF1; which promotes MTORC1
signalling) limited various autophagic markers and
inhibited the demise of cultured neurons from the rat
striatum, cerebellum and cortex exposed to NMDA or
kainic acid51–54. Comparable results were obtained when
rat cerebellar granule neurons were administered with
an Atg7‑targeting siRNA51, and when rat cortical neu-
rons were transfected with a lentiviral vector coding for
Becn1- or Atg7‑targeting short hairpin RNAs (shRNAs)53.
Consistent with this result, rat cortical neurons engi-
neered to overexpress BECN1 or ATG7 were sensitized
to the excitotoxic effects of kainic acid53. Furthermore,
mice receiving an Atg7‑specific siRNA directly in the
hippocampus were less sensitive to the administration
of kainic acid than were animals subjected to control,
non-targeting RNA interference 34 (Supplementary
information S2 (table)).
Interestingly, the neurotoxic effects of glutamate
have been partially attributed to the transcriptional
downregulation and calpain-dependent degradation
of MFN2 and consequent mitochondrial dysfunc-
tion55,56. Accordingly, primary Mfn2−/− mouse motor
neurons displayed mitochondrial defects at baseline
and were more sensitive to the neurotoxic effects of
glutamate than were control Mfn2 fl/fl neurons. By
contrast, MFN2‑overexpressing neurons (which are
expected to exhibit a reduced propensity to mitophagy)
were more resistant to glutamate-mediated excito-
toxicity than their wild-type counterparts56. Whole-
body overexpression of MFN2 also protected mice
from the loss of spinal cord motor neurons induced
by the intraventricular administration of glutamate56.
However, neither 3‑MA nor the lysosomal inhibitor
bafilomycin A1 blocked the degradation of MFN2 and
consequent mitochondrial dysfunction triggered by
glutamate in this setting 56, casting additional uncer-
tainty on the involvement of autophagy in this pro-
cess. Notably, rat cortical neurons exposed to a brief
glutamate pulse exhibited PARK2 accumulation in the
ER and mitochondria, but did not show signs of mito-
phagy unless an antioxidant (N‑acetyl-cysteine) was
co-administered57.
These results suggest that, at least in some circum-
stances, glutamate and other excitotoxic agents might
block autophagic responses by causing mitochondrial
dysfunction and hence establishing profound oxidative
stress. Supporting this notion, a cytoplasmic pool of
ATM serine/threonine kinase (ATM), which activates
autophagy in response to oxidative (but not genotoxic)
stress58, has been implicated in the cytotoxic response
of cortical, hippocampal and cerebellar neurons to
intraperitoneal kainic acid administration in mice59.
Unfortunately, however, it has not yet been investigated
whether the activation of cytoplasmic ATM by the oxi-
dative stress caused by kainic acid indeed triggers an
autophagic response.
Taken together, these observations indicate that the
actual impact of autophagy on excitotoxicity remains to
be clarified (Supplementary information S1,S2 (tables)).
Although differences in the type, intensity and dura-
tion of the excitotoxic challenge may explain (at least
partially) the heterogeneity of the findings discussed
above29, we fear that methodological issues have a prom-
inent confounding role in this setting. Thus, additional
investigations involving specific strategies of autophagy
inhibition and accurate flux assessments are urgently
awaited to elucidate whether proficient autophagic
responses limit or support the detrimental effects of
excitotoxicity.

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 7

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Table 1 | Pharmacological modulation of autophagy in models of acute brain injury

Agent Target Phase Blood–brain barrier Comments
permeability
Inducers (and molecules associated with increased levels of autophagic markers)
Curcumin Unknown Unknown Permeant Reported to inhibit autophagy in some settings
Cystatin C Unknown Unknown Impermeant Endogenous inhibitor of cysteine proteases
Deferoxamine Fe ions*
2+
Elongation* Permeant Expected to inhibit autophagy as a result of limited ROS
availability
Kaempferol AMPK Initiation Unknown AMPK activation secondary to bioenergetic impairment
Lithium IMPA1 and Nucleation Permeant Relatively nonspecific agent, inhibits several other enzymes
GSK3β
Melatonin ROS* Elongation* Permeant Expected to suppress autophagy upon ATG4 inhibition
N‑acetyl-cysteine ROS* Elongation* Permeant Expected to suppress autophagy upon ATG4 inhibition
Pyruvate DAPK1 Nucleation Permeant Promotes BECN1 release from inhibitory interactions with BCL‑2
Rapamycin MTORC1 Initiation Permeant Potent MTORC1 inhibitor with clinical applications
Retinoic acid RARα* Transcription* Impermeant Reported to specifically inhibit CMA in some settings
and elongation
SB216763 GSK3β Nucleation Permeant Mixed inhibitor of GSK3α and GSK3β
Simvastatin HMGCR Initiation Permeant Inhibits AKT1–MTORC1 signalling
Trehalose Unknown Unknown Permeant Disaccharide that stimulates MTORC1‑independent autophagy
Trichostatin A Unknown Transcription* Impermeant Mixed inhibitor of class I and class II (but not class III) HDACs
Valproate GSK3β and Transcription* Permeant Relatively nonspecific agent, inhibits several other enzymes
HDACs and nucleation
Inhibitors (and molecules associated with decreased levels of autophagic markers)
3‑methyladenine VPS34 Nucleation Permeant Considerably nonspecific class III PI3K inhibitor
β‑asarone JNK1 Nucleation Permeant Limits BECN1 release from inhibitory interactions with BCL‑2
Apelin-derived APLNR Initiation Permeant Stimulates AKT1–MTORC1 signalling
peptide
Apocynin NADPH Elongation* Permeant Expected to limit ROS availability, hence blocking ATG4
oxidase* activation
Bafilomycin A1 Vacuolar H+ Lysosomal Permeant Nonspecific inhibitor of lysosomal functions
ATPase acidification
Caffeine Unknown Unknown Permeant Mostly reported to promote MTORC1‑dependent autophagy
Carnosine ROS* Elongation* Permeant Expected to limit ROS availability, hence blocking ATG4
activation
Ceftriaxone Unknown Unknown Permeant β-lactam antibiotic approved for use in humans
Chloroquine H+ ions Lysosomal Permeant Nonspecific inhibitor of lysosomal functions
acidification
E64d CAPN1 and Lysosomal Unknown Nonspecific inhibitor of lysosomal functions
CTSB proteolysis
GCEE ROS* Elongation* Unknown Expected to limit ROS availability, hence blocking ATG4
activation
GDNF GFRA1 Initiation Impermeant Stimulates AKT1–MTORC1 signalling
HGF MET Initiation Permeant Stimulates AKT1–MTORC1 signalling
Hydrogen sulfide Unknown Unknown Permeant Also reported to promote MTORC1‑dependent autophagy
IGF1 IGF1R Initiation Permeant Stimulates AKT1–MTORC1 signalling
KU55933 VPS34 Nucleation Unknown Exhibits improved selectivity for VPS34
l‑asparagine Unknown Fusion Impermeant May block autophagy more selectively than lysosomal inhibitors
LY294002 VPS34 Nucleation Permeant Exhibits improved selectivity for VPS34
Mdivi‑1 DNML1 Mitophagy Permeant Specific inhibitor of mitochondrial fragmentation
Melatonin ROS* Elongation* Permeant Expected to limit ROS availability, hence blocking ATG4
activation

8 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Table 1 (cont.) | Pharmacological modulation of autophagy in models of acute brain injury

Agent Target Phase Blood–brain barrier Comments
permeability
N‑acetyl-serotonin ROS* Elongation* Permeant Precursor of melatonin, expected to reduce ROS availability
NAD+ SIRT1* Unknown Permeant Expected to promote autophagy through SIRT1
Necrostatin‑1 RIPK1* Unknown Permeant Potent inhibitor of some forms of necroptosis
Neriifolin Na+/K+ ATPase Elongation* Impermeant Cardiac glycoside that potently inhibits autosis
Pepstatin Aspartyl Lysosomal Unknown Generally used in combination with E64d
proteases proteolysis
Pifithrin‑α p53 Transcription Permeant May also activate autophagy in specific settings
Propofol ROS* Elongation* Permeant Expected to limit ROS availability, hence blocking ATG4
activation
Rosiglitazone PPARγ* Unknown Impermeant Antidiabetic drug expected to promote (not inhibit) autophagy
Salubrinal GADD34 Initiation Permeant Blocks autophagy initiation by ER stress
SN50 NF‑κB* General* Unknown Cell-permeant peptide that inhibits NF‑κB nuclear translocation
Taurine MTORC1 Initiation Permeant Endogenous sulfonic acid with multiple biological functions
Wortmannin VPS34 Nucleation Impermeant Considerably nonspecific class III PI3K inhibitor
AMPK, AMP-activated protein kinase; APLNR, apelin receptor; ATG4, autophagy-related 4 cysteine peptidase; BCL-2, B cell lymphoma 2; BECN1, beclin 1; CAPN1,
calpain 1; CMA, chaperone-mediated autophagy; CTSB, cathepsin B; DAPK1, death-associated protein kinase 1; DNM1L, dynamin 1‑like; ER, endoplasmic reticulum;
GADD34 (official name: PPP1R15A), protein phosphatase 1 regulatory subunit 15A; GCEE, γ‑glutamylcysteinyl ethyl ester; GDNF, glial cell-derived neurotrophic
factor; GFRA1, GDNF family receptor α1; GSK3α, glycogen synthase kinase 3α; GSK3β, glycogen synthase kinase 3β; HDAC, histone deacetylase; HGF, hepatocyte
growth factor; HMGCR, 3‑hydroxy‑3‑methylglutaryl-CoA reductase; IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; IMPA1, inositol(myo)-1(or
4)-monophosphatase 1; JNK1 (official name: MAPK8), mitogen-activated protein kinase 8; MET, MET proto-oncogene, receptor tyrosine kinase (HGF receptor);
MTORC1, mechanistic target of rapamycin complex 1; NF‑κB, nuclear factor-κB; p53 (official name: TP53), tumour protein p53; PI3K, phosphoinositide 3‑kinase;
PPARγ, peroxisome proliferator-activated receptor-γ; RARα, retinoic acid receptor-α; RIPK1, receptor-interacting protein kinase 1; ROS, reactive oxygen species;
SIRT1, sirtuin 1; VPS34 (official name: PIK3C3), phosphatidylinositol 3‑kinase catalytic subunit type 3. *To be confirmed.

Autophagy in acute neurotoxic responses
Neurotoxic responses can be subdivided into two groups
according to their kinetics of manifestation. Acute neuro-
toxicity manifests symptomatically over periods of hours
to days after a single exposure to neurotoxins, whereas
the symptoms of chronic neurotoxicity become clinically
evident years to decades later, and generally stem from
continued exposure11,27. In particular, chronic neuro-
toxic responses are associated with the progressive loss
of specific neuronal populations, eventually resulting in
degenerative disorders such as Parkinson disease11. The
beneficial effects of autophagy in neurodegeneration have
been convincingly established11, and will not be discussed
in further detail here. However, some of the neurotoxins
used in pharmacological models of neurodegeneration,
including rotenone, 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahy-
dropyridine (MPTP), 6‑hydroxydopamine (6‑OHDA),
paraquat and lactacystin, also trigger acute neuro­toxicity
in association with autophagic responses, particularly
when used in high doses.
Rotenone establishes a Parkinson disease‑like state
in rats and mice, and is also frequently used in experi-
mental settings to inhibit complex I of the mitochondrial
respiratory chain8. Acute exposure of neuronal cells lines
— including mouse hybrid Mes 23.5 cells60, rat PC12
cells61 and human SH‑SY5Y cells62–64 — to rotenone
resulted in rapid RCD accompanied by signs of auto-
phagy. Supporting a cytoprotective role of autophagy,
pharmacological activators of autophagy, including rapa-
mycin63, deferoxamine (a chelator of Fe2+ ions that triggers
autophagy through poorly characterized mechanisms)64
and kaempferol (a phytoestrogen that activates AMPK)62,
limited the acute neurotoxic effects of rotenone on
SH‑SY5Y cells. In Mes 23.5 cells exposed to rotenone, the
depletion of sestrin 2 (SESN2), an upstream inhibitor of
MTORC1, compromised autophagy induction and aggra-
vated neurotoxicity, whereas SESN2 overexpression acti-
vated a robust, AMPK-dependent autophagic response
while limiting RCD60. Similar results have been obtained
with rat H9C2 cardiomyocytes treated with an siRNA
specific for glyceraldehyde‑3‑phosphate dehydrogenase
(Gapdh)65, an enzyme that also exerts autophagy-regu-
lating functions66. Conversely, the depletion of tyrosine
3‑monooxygenase/tryptophan 5‑monooxygenase acti-
vation protein-ε (YWHAE; also known as 14‑3‑3ε), a
protein that inhibits autophagy by sequestering BECN1
into inactive complexes67, exacerbated the acute neuro-
toxic effects of rotenone on PC12 cells61 (Supplementary
information S1,S2 (tables)).
MPTP is frequently used to establish a Parkinson
disease‑like disorder in rodents owing to its severe and
rather specific toxicity for dopaminergic neurons of the
substantia nigra11. There is some evidence suggesting
that autophagy may mitigate the neurotoxic effects of
acute MPTP exposure. Specifically, the siRNA-medi-
ated depletion of ATG7 nearly abolished the demise
of rat glioma C6 cells following acute MPTP exposure,
as did the administration of melatonin, which also
suppressed several manifestations of autophagy in rat
cells and interfered with the ability of chronic MPTP
exposure to establish a Parkinson disease‑like state in
mice68. According to one study, acute MPTP exposure
displaced BECN1 from inhibitory interactions with
BCL‑2 and induced cell death in human neuroblastoma

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 9

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Trimethyltin
A tin-containing organic
compound with prominent
neurotoxic activity towards the
hippocampus.
Locomotor sensitization
Long-lasting exacerbation of
a psychostimulant-induced
locomotor response, elicited by
the repeated intermittent
administration of the same
psychoactive agent.
SK‑N‑SH cells69. However, the authors of this study
did not test whether autophagy itself was modulated
in these conditions. Independent data demonstrated
that the activation of autophagy by systemic adminis-
tration of rapamycin or valproate plus lithium limited
the ability of MPTP to cause long-term symptoms of
Parkinson disease in mice70,71; however, acute neuro-
toxicity was not investigated. Another study provided
correlative evidence in support of a cytoprotective role
of autophagy in acute MPTP neurotoxicity in vivo 72.
In this experimental scheme, investigators relied on
the administration of mesenchymal stem cells (which
were suggested to activate autophagy), but they did not
attempt to modulate autophagy by pharmacological or
genetic means, casting doubts on the actual relevance
of their findings.
Similarly, it is difficult to conclude whether autophagy
limits or exacerbates the acute toxicity of 6‑OHDA, para-
quat or lactacystin. The rat striatum was protected from
6‑OHDA in the presence of 3‑MA73; by contrast, both
3‑MA and the depletion of parkinson protein 7 (PARK7;
a component of the molecular machinery for mitophagy
also known as DJ‑1) exacerbated the death of SH‑SY5Y
cells acutely exposed to paraquat74,75, and rapamycin lim-
ited the acute cytotoxic potential of lactacystin on PC12
cells76 (Supplementary information S1,S2 (tables)). Taken
together, these observations indicate that the relevance
of autophagy in the acute neurotoxic effects of agents
commonly used to generate animal models of Parkinson
disease remains to be determined.
Accumulating evidence suggests that autophagy has
an important role in the response of the CNS to psycho-
active molecules, including cocaine, methamphetamine,
ethanol and the atypical antipsychotic olanzapine, as well
as to experimental toxins such as trimethyltin. Cocaine
has recently been reported to trigger conventional auto-
phagic responses in human SVGA astrocytes, mouse
immortalized BV‑2 microglial cells, primary rat micro-
glial cells and primary mouse cortical neurons, as well
as in the mouse brain in vivo77–79, but to robustly activate
MTORC1 (and hence to inhibit autophagy) in neurons
from the nucleus accumbens80. Rapamycin markedly
reduced locomotor sensitization (one of the neurobehav-
ioural symptoms of cocaine administration) in rats81, as
did the deletion of either Mtor or Raptor from specific
neuronal populations in mice80. Apparently at odds with
these findings, however, inhibition of autophagy with
wortmannin also decreased the ability of microglial
cells to secrete neuroinflammatory factors in response
to cocaine77, whereas 3‑MA and bafilomycin A1 limited
cocaine-induced RCD in human SVGA astrocytes78 and
in primary mouse cortical neurons79. Moreover, siRNAs
targeting Atg5, Becn1 or Gapdh markedly limited the
demise of primary mouse cortical neurons acutely
exposed to cocaine79. Taken together, these data indicate
that some aspects of the neurotoxicity of cocaine, such as
locomotor neuron sensitization, are attenuated by auto-
phagy (Supplementary information S1 (table)), whereas
other aspects, including neuroinflammation and RCD,
depend on the activation of autophagic responses
(Supplementary information S2 (table)).
The neurotoxic effects of methamphetamine were
associated with morphological signs of autophagy as
early as 1994 (REF. 82). It was only more than a decade
later, however, that autophagy was suggested to mediate
the neuroprotective effects in this setting, when 3‑MA
and the expression of a dominant-negative form of
VPS34 were found to exacerbate the demise of rat PC12
and human SH‑SY5Y cells exposed to low concentra-
tions of methamphetamine83,84. Corroborating these
findings, the downregulation of PINK1 aggravated the
demise of methamphetamine-stimulated PC12 cells,
whereas PINK1 overexpression exerted robust neuro-
protective effects that correlated with the activation of
mitophagy 85. Similar results were obtained in rat N27
dopaminergic cells subjected to the siRNA-mediated
downregulation of LC3 or treated with 3‑MA86, as well
as in human non-neuronal cells in which autophagy
was inhibited with BECN1‑targeting siRNAs, 3‑MA
or chloroquine87. It has also been proposed that caf-
feine may boost the neurotoxicity of methampheta-
mine88 by inhibiting autophagy 89. However, caffeine
triggers robust autophagic responses in neurons and
many other cell types90, implying that the link between
caffeine and methamphetamine should be interpreted
with caution. Further substantiating the neuroprotec-
tive activity of autophagy in this setting, rapamycin
limited neurotoxicity in rats that received metham-
phetamine in combination with antiretroviral agents
and purified HIV‑1 envelope glycoproteins (which
were used together as a model of drug addiction in
patients with HIV‑1 treated with antiretroviral drugs)91
(Supplementary information S1 (table)).
Apparently at odds with these observations, data
obtained in rat N27 cells92, rat PC12 cells93, human
SK‑N‑SH cells94 and rat primary cortical neurons95 sug-
gest that autophagy contributes to (rather than coun-
teracts) the acute neurotoxicity of methamphetamine.
These findings, however, are mostly correlative (that
is, no experimental modulation of the process was
undertaken)92–94 or present other problematic aspects.
For example, both bafilomycin A1 and rapamycin were
counter-intuitively found to aggravate the detrimen-
tal effects of methamphetamine on neurite length, but
autophagic flux was not investigated in this study 95
(Supplementary information S2 (table)). Therefore,
it seems that autophagy mainly counteracts the acute
neurotoxicity of methamphetamine.
Similar considerations can be made regarding the
impact of autophagy on the acute neurotoxic effects
of ethanol, olanzapine and trimethyltin. Indeed, both
wortmannin and a BECN1‑targeting shRNA aggra-
vated the acute toxicity of ethanol in SH‑SY5Y cells96,
and ischaemic preconditioning led to robust neuropro-
tective effects in the same model97. Interestingly, chronic
ethanol administration caused the downregulation of
various components of the autophagic machinery in the
cerebral cortex of wild-type mice, but not in mice lacking
the gene encoding Toll-like receptor 4 (Tlr4)98. Taken
together, these data suggest that TLR4 signalling may
promote the chronic neurotoxicity of ethanol by inhib-
iting autophagy. As the damage-associated molecular

10 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.

Caspase 3
A cysteine protease that
precipitates apoptotic variants
of regulated cell death,
irrespective of the subcellular
compartment in which they are
initiated.
Cardiac glycoside
A natural compound, such as
digoxin or digitoxin, that exerts
positive inotropic effects and
suppresses autosis as it inhibits
the plasma membrane Na+/K+
ATPase.
NATURE REVIEWS | NEUROSCIENCE
REVIEWS
pattern (DAMP) HMGB1 (REF. 99) is one of the main Taken together, these data provide limited experi-
endogenous ligands for TLR4, it is tempting to specu- mental support for the hypothesis that autophagy medi-
late that neurons succumbing to acute exposure to eth- ates bona fide neuroprotective effects in the course of
anol may be the source of TLR4‑activating HMGB1, neonatal hypoxia–ischaemia. Moreover, work from
which in turn limits the ability of remaining neurons several independent groups overtly argues against this
and glial cells to mount proficient autophagic responses interpretation. Indeed, 3‑MA limited the demise of pri-
and therefore survive chronic ethanol neurotoxicity; mary rat cortical neurons, rat hippocampal slices and
however, this remains to be formally demonstrated. SH‑SY5Y cells exposed to oxygen and glucose deprivation
Irrespectively, human SH‑SY5Y cells subjected to the (OGD) in vitro114,115. Similarly, newborn rats receiving an
depletion of BECN1 and LC3 exhibited increased sensi- intracerebro­ventricular injection of 3‑MA after ischaemia
tivity to olanzapine100, as did mice exposed to olanzapine (more than 4 hours after the ischaemic episode) exhibited
in the presence of chloroquine100. Similar results were considerably smaller lesions compared with control ani-
obtained in neuronal and astrocytic cultures exposed to mals116. Comparable results were obtained with newborn
trimethyltin alone or in combination with pharmaco- rats treated with the cardiac glycoside neriifolin, a bona fide
logical modulators of autophagy, including 3‑MA, bafi- inhibitor of autosis (BOX 2), immediately after carotid
lomycin A1, chloroquine, rapamycin and lithium101,102. artery occlusion14. Moreover, 7‑day-old rats receiving a
Overall, these observations favour the interpretation lentiviral vector encoding a Becn1‑specific shRNA in the
that autophagic responses limit the acute neurotoxic striatum exhibited twice the amount of intact striatal tis-
effects of various psychoactive agents (Supplementary sue 24 hours after a hypoxic–ischaemic challenge than did
information S1 (table)). rats receiving a control shRNA53. An even higher degree of
long-term protection was afforded to pyramidal neurons
Autophagy in neonatal asphyxia in the hippocampal CA regions of mice in which Atg7 was
Newborn babies who have died as a consequence of specifically deleted from neurons106. Extending these find-
hypoxic–ischaemic encephalopathy exhibit ultrastruc- ings, the neuron-specific deletion of Atg7 inhibited RCD
tural and biochemical manifestations of autophagy in in the hippocampus, thalamus, cortex and striatum of
various cerebral areas, encompassing the basal gan- newborn mice subjected to neonatal hypoxia–ischaemia,
glia, thalamus, cortex and hippocampus103,104. Such globally reducing tissue loss by approximately 40%104.
autophagic markers include cytoplasmic vacuoliza- Notably, the possibility that such neuroprotection would
tion, BECN1 upregulation, LC3 lipidation and p62 originate from a preconditioning effect, reflecting the
degradation103,104. Similar findings were observed in hormetic adaptation of neurons to the absence of ATG7,
the brains of mice and rats experimentally subjected was experimentally discarded104. Newborn mice lacking
to hypoxia–ischaemia in the perinatal period53,103–107. both copies of the gene encoding the mitophagy recep-
Generally, rodent neurons manifesting signs of auto- tor BCL‑2/adenovirus E1B 19 kDa interacting protein 3
phagy upon neonatal asphyxia also exhibit morpho- (Bnip3) exhibited much smaller infarcts than did their
logical and biochemical markers of RCD and ongoing wild-type counterparts at 7 and 28 days (but not at 1 and
cellular degeneration. These signs include axonal and/or 3 days) after hypoxia–ischaemia117. Indeed, infarct size
somatodendritic swelling, mitochondrial fragmenta- 1 day after the hypoxic–ischaemic challenge was much
tion, chromatin fragmentation and condensation, and larger in Bnip3−/− mice than in wild-type mice, perhaps
caspase 3 activation103,105,108. Based on these correlative indicating that the rapid mitophagic removal of dam-
observations alone, however, one cannot conclude aged mitochondria is a cytoprotective mechanism in the
whether autophagic responses in the neonatal brain first phases of neonatal hypoxia–ischaemia. Irrespective
mediate neuroprotective or neurotoxic effects. of this possibility, the observations discussed here lend
The first study to investigate the effects of pharma- solid support to the hypothesis that autophagy aetiolog-
cological modulators of autophagy in newborn rats ically contributes to the pathological effects of perinatal
subjected to hypoxia–ischaemia observed that post-­ asphyxia (Supplementary information S2 (table)).
ischaemic administration of rapamycin efficiently limited Interestingly, it has been shown that the midbrain
damage in this model109. Such a neuroprotective effect of releases endogenous cardiac glycosides (namely, ouabain
rapamycin could be blocked by the subsequent admin- or endobain) in response to hypoxia–ischaemia118. It is
istration of 3‑MA110, suggesting that this effect origi- therefore tempting to speculate that the brain has evolved
nated from an autophagic response. However, although a safeguard mechanism that inhibits autophagic responses
the post-ischaemic administration of 3‑MA and wort- when they may have neurotoxic consequences (such as
mannin changed the molecular mechanism by which during hypoxic–ischaemic challenges), but not when they
neurons died (switching it from caspase 3‑dependent are in place to preserve neuronal homeostasis (such as
apoptosis to necrosis)109,111, neither of these inhibitors under baseline conditions or in response to neurotoxins).
of autophagy truly limited damage in newborn rats sub- This hypothesis awaits urgent experimental verification.
jected to a hypoxic–ischaemic episode109 (Supplementary
information S1 (table)). Further complicating the issue, Autophagy in adult stroke
lithium (which normally triggers autophagy) was neuro­ Brains of adult mice119–121 and rats54,122–141 subjected to
protective in a rat model of neonatal hypoxia–ischaemia, an ischaemic challenge exhibit various ultrastructural
but reduced LC3 lipidation, which is consistent with and biochemical markers of autophagy, including
autophagy inhibition, rather than activation112,113. cytoplasmic vacuolization, MTORC1 inhibition, LC3
ADVANCE ONLINE PUBLICATION | 11
©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Metformin
An oral medication currently
licensed for the treatment of
specific forms of diabetes.
Metformin activates
AMP-activated protein kinase
and inhibits respiratory
complex I.
Thapsigargin
A natural compound that
promotes endoplasmic
reticulum stress by operating
as a non-competitive inhibitor
of the sarco/endoplasmic
reticulum Ca2+ ATPase.
Tunicamycin
A mixture of homologous
nucleoside antibiotics that
induce endoplasmic reticulum
stress by blocking N‑linked
glycosylation.
Ischaemic penumbra
Ischaemic tissue potentially
destined to infarction but not
irreversibly injured in the
course of stroke. The ischaemic
penumbra is the main target of
acute therapeutic interventions
in stroke patients.
lipidation, p62 degradation and BECN1 and LAMP2
upregulation. At least two lines of evidence support
the tenet that autophagy mediates considerable neuro­
protective effects in adult rodents exposed to acute
ischaemic episodes such as those caused by transient
middle carotid artery occlusion (tMCAO; usual dura-
tion: 30 minutes to 3 hours) or permanent MCAO
(pMCAO) (Supplementary information S1 (table)).
First, genetic interventions that limit autophagic
proficiency — including the deletion of mitochondrial
superoxide dismutase 2 (Sod2)121 or S‑antigen, retina and
pineal gland (arrestin) (Sag)142, or the administration of a
viral vector encoding a Tsc1‑targeting shRNA143 — aggra-
vated neuronal loss in rodents subjected to tMCAO or
pMCAO. Similarly, the stereotaxic delivery of a viral vec-
tor overexpressing nicotinamide phosphoribosyltrans-
ferase (NAMPT)134,144, a manipulation that efficiently
upregulates autophagy, had remarkable neuroprotective
effects in adult rats subsequently subjected to tMCAO.
Moreover, inhibition of autophagy — caused by either the
homozygous deletion of protein kinase AMP-activated
α2 catalytic subunit (Prkaa2) or sirtuin 1 (Sirt1), or by
the downregulation of ATG7 or TSC1 — exacerbated
the neurotoxic effects of OGD in primary mouse144
or rat 143,145 cortical neurons. Interestingly, the siRNA-
mediated downregulation of LAMP2 (which is an essen-
tial mediator of CMA), but not depletion of ATG5 (which
is specifically involved in macroautophagy), increased
the susceptibility of mouse Neuro2a cells to stan-
dalone hypoxia in vitro, whereas the activation of CMA
with mycophenolic acid led to robust neuroprotective
effects131. These findings perhaps indicate that distinct
forms of autophagy differentially contribute to situations
as multifactorial as an ischaemic insult in vivo.
Second, the robust neuroprotective effects of pre-
conditioning, elicited by hypoxia or other interventions
(including hyperbaric oxygen, the anaesthetic isoflu-
rane, metformin, thapsigargin and tunicamycin), appear to
rely on the establishment of sublethal ER stress138 cou-
pled with the activation of hormetic autophagy (BOX 1).
Accordingly, mice lacking sphingosine kinase 2 (SPHK2;
a protein that activates BECN1) were insensitive to iso-
flurane-induced preconditioning 41,42, and Park2+/− mice,
which exhibit a selective defect in mitophagy, could
not establish normal preconditioning in response to
thapsigargin and tunicamycin146. The ability of isoflu-
rane, hypoxia, thapsigargin and tunicamycin to protect
mouse cortical neurons against OGD was also abolished
when neurons were previously transfected with a Becn1-
or an Atg7‑targeting siRNA42,146. Moreover, autophagy
activation (with rapamycin) or suppression (with 3‑MA,
bafilomycin A1, mdivi‑1, KU55933 or AMPK inhibitors)
enhanced or blunted, respectively, neuroprotection in
various experimental models of preconditioning. These
models include the following: mice pretreated with
thapsigargin or tunicamycin and subsequently subjected
to tMCAO146; rats subjected to ischaemic precondition-
ing followed by pMCAO132,135,139,147; rats administered
with metformin and then exposed to pMCAO133; and
rats placed in hyperbaric oxygen and then subjected to
tMCAO137 (Supplementary information S1 (table)).
Interestingly, specific cerebral areas may benefit in
a preferential manner from the activation of autophagy
in the course of, or after, an ischaemic insult. The post-
ischaemic administration of a chemical inhibitor of gly-
cogen synthase kinase 3β (GSK3β) activated autophagy
in the microglia of rats subjected to pMCAO148. This
effect was associated with neuroprotection coupled with
an autophagy-dependent anti-inflammatory effect 148.
Methylene blue and pinocembrin (two antioxidants)
administered after tMCAO also conferred remarkable
neuroprotection in rats, and this protective effect was
linked to the specific activation of autophagy in the
ischaemic penumbra140,141. These latter observations suggest
that the beneficial effects of autophagy activators admin-
istered after ischaemia may reflect an improved salvage of
the ischaemic penumbra (Supplementary information S1
(table)). However, additional studies measuring auto-
phagy in specific cerebral areas are required to validate
this hypothesis.
Activation of autophagy with rapamycin limited infarct
size in mouse models of tMCAO and pMCAO; however,
similar data were obtained with the autophagy inhibitor
chloroquine (although neuroprotection was slightly less
remarkable)149. Moreover, both 3‑MA and mdivi‑1 (an
inhibitor of mitophagy) exacerbated the loss of neurons
associated with tMCAO in mice, yet exerted a neuro-
protective or null effect, respectively, in mice subjected
to pMCAO145. These latter observations cast doubts on
the hypothesis that autophagy always exerts neuroprotec-
tive activity in the course of adult stroke (Supplementary
information S1,S2 (tables)).
Indeed, additional data support the tenet that auto-
phagy contributes to neurotoxicity in rodent models of
adult stroke. In particular, the VPS34 inhibitor 3‑MA
and the lysosomal inhibitor bafilomycin A1 limited
the infarct area in mice subjected to tMCAO150, in rats
subjected to pMCAO123,151 or tMCAO129, and in rats sub-
jected to global ischaemia in the four-vessel occlusion
model124. Similarly, the expression of a Becn1‑targeting
shRNA152 and the downregulation of eva‑1 homologue A
(EVA1A), a lysosomal protein involved in autophagic
responses122, lowered the sensitivity of rats to tMCAO.
Bafilomycin A1 and 3‑MA and also limited the demise
of rat astrocytes caused by pMCAO in vivo or OGD
in vitro128. Furthermore, several molecules that protect
neurons in rodents subjected to tMCAO, pMCAO or
global ischaemia also inhibited autophagy. These mol-
ecules include melatonin and its precursor N‑acetyl-
serotonin127,130, propofol (an intravenous anaesthetic)129,
carnosine54, β‑asarone (an ether found in some plants)125,
edavarone (a free radical scavenger)120, 2‑methoxye-
stradiol (an endogenous metabolite of oestrogen that
inhibits hypoxia-inducible factor 1)124, NAD+ (REF. 150),
glial cell-derived neurotrophic factor (GDNF) and
hepatocyte growth factor (HGF)126. Finally, the brains of
mice lacking nuclear factor of κ-light poly­peptide gene
enhancer in B cells 1 (Nfkb1) manifested exacerbated
autophagic responses and were more sensitive to tMCAO
than the brains of wild-type mice119 (Supplementary
information S2 (table)). However, no causal relation-
ship between the neuroprotective activity of all these

12 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

17‑Allylamino-
demethoxygeldanamycin
(17‑AAG). A derivative of
geldanamycin that targets heat
shock protein 90 kDa-α family
class A member 1 (HSP90AA1;
also known as HSP90).
Necroptosis
A specific type of regulated cell
death that depends on the
activity of receptor-interacting
serine/threonine kinase 3
(RIPK3) and mixed lineage
kinase domain-like (MLKL).
experimental interventions and autophagy inhibition has
been established, implying that the latter may represent an
epiphenomenon of the former.
In summary, the current literature does not allow us
to conclude whether autophagy mediates bona fide cyto-
protective or cytotoxic effects in adult rodents subjected
to ischaemic episodes. As autophagy appears to mediate
neurotoxic effects in the course of neonatal hypoxia–
ischaemia, developmental stage may partially account
for the apparently contradictory findings reported here.
However, we fear that in many cases, drawing solid con-
clusions is complicated by the use of pharmacological
agents with limited specificity and the lack of accurate
flux determinations. It will be important to tackle the
impact of autophagy on adult stroke with robust meth-
odological approaches that take these two points into
adequate consideration.
Autophagy in neurotrauma
Several observational studies have associated distinct
forms of neurotrauma — including spinal cord injury
(SCI), subarachnoid haemorrhage (SAH) and traumatic
brain injury (TBI) — with morphological and biochem-
ical markers of autophagy (see above), both in experi-
mental samples from rodents153–160 and in human biopsy
samples153. Although only two of these studies attempted
to elucidate whether these biomarkers reflected the acti-
vation of a proficient autophagic response or the blockade
of autophagic degradation, both favoured the latter inter-
pretation over the former 154,159. Indeed, motor neurons
of rats subjected to contusive SCI and cortical neurons of
mice subjected to TBI exhibited an accumulation
of LC3‑containing autophagosomes and defective p62
degradation starting at day 1 post-trauma154,159.
Results from various interventional studies in ani-
mal models of neurotrauma support the tenet that
autophagy exerts robust cytoprotective effects (but is
usually impaired) in the course of neurotrauma, and
hence favours functional recovery. Rapamycin pre-
served neuronal survival, promoted axonal regeneration
towards the lesion and improved the restoration of hind-
limb function in rodents subjected to SCI161,162. Similar
effects were observed with other autophagy-activating
interventions, including the post-SCI administration of
a splicing isoform of human vascular endothelial growth
factor A (VEGFA165)163, retinoic acid (which also limited
the disruption of the blood–spinal cord barrier)164 and
simvastatin (an anti-atherosclerotic drug)165. Notably,
the neuroprotection afforded by VEGFA165 and retinoic
acid was abolished by the concomitant administration
of 3‑MA163 and chloroquine164, respectively, reinforcing
the mechanistic link with autophagy in this setting.
Rapamycin limited spinal cord damage and improved the
recovery of locomotor activity post-SCI, even in diabetic
rats, which are generally more sensitive to neurological
damage than wild-type rats166. Moreover, both 3‑MA
administration and the depletion of BECN1 exacerbated
the percentage of rat primary spinal cord neurons suc-
cumbing to mechanical injury in vitro, whereas the over-
expression of BECN1 or the addition of rapamycin to
the culture medium exerted neuroprotective effects167.
Pharmacological activation of autophagy with rapamy-
cin, melatonin, simvastatin, trichostatin A or recombi-
nant cystatin C (CST3; an endogenous cysteine protease
inhibitor) also limited brain damage in rats subjected
to SAH168–172. In this setting, inhibiting autophagy with
3‑MA or wortmannin either aggravated SAH-dependent
acute neurodegeneration168,170 or abolished neuroprotec-
tion conferred by autophagy-activating interventions172.
Taken together, these observations suggest that autophagy
efficiently counteracts acute neurodegeneration caused by
SCI and SAH (Supplementary information S1 (table)).
Accumulating evidence indicates that proficient auto-
phagic responses also protect the CNS from the delete-
rious effects of TBI. Becn1+/− mice exhibited decreased
neuronal survival and limited functional recovery
upon hemicerebellectomy compared with wild-type
mice, and such effects were insensitive to rapamycin
administration173. Conversely, rapamycin improved
the hemicerebellectomy-induced neurological deficits
in wild-type mice173, highlighting a mechanistic link
between the neuroprotective effects of rapamycin and
autophagy. Rapamycin administration post-TBI also
limited neurological damage in mice subjected to closed-
head injury174 and weight-drop damage175. Similar results
were obtained in mice and rats receiving other phar-
macological activators of autophagy, including mela-
tonin176, methylene blue177, luteolin (a plant flavonoid)178
and 17‑allylamino-demethoxygeldanamycin (17‑AAG)179.
Importantly, the neuroprotective effects of melatonin and
17‑AAG were abolished in the presence of 3‑MA176,179, and
those of 17‑AAG were exacerbated by the concomitant
administration of rapamycin179. Similarly, 3‑MA blunted
the well-characterized beneficial effects of post-injury
hypothermia in a fluid percussion rat model of TBI180,
suggesting that at least part of the neuroprotective activity
of hypothermia may be associated with the activation of
autophagy (Supplementary information S1 (table)).
Nevertheless, various pharmacological inhibitors of
autophagy also have neuroprotective activity in rodent
models of TBI. In particular, post-injury chloroquine
reduced brain oedema and improved neurological recov-
ery in rats subjected to controlled cortical impact 181.
Moreover, pretreatment with 3‑MA or bafilomycin A1
attenuated neuronal damage and improved behavioural
outcomes in a mouse weight-drop model of TBI182.
Similar observations were made in mice receiving (pre-
TBI) an autophagy-blocking peptide derived from the
adipocytokine apelin183. In addition, the ability of various
molecules to limit histological damage and behavioural
deficits in mice or rats subjected to TBI has been correl-
atively linked to the inhibition of autophagy. Such agents
include hydrogen sulfide184, the chemical inhibitor of
necroptosis necrostatin-1 (REF. 185), the β-lactam anti­
biotic ceftriaxone186, the antioxidant γ‑glutamylcysteinyl
ethyl ester 187 and the antidiabetic drug rosiglitazone188
(Supplementary information S2 (table)). However, no
mechanistic connections have been established between
the neuroprotective activity of these molecules and
autophagy inhibition, and some of these compounds
are actually known to activate autophagy, at least in spe-
cific settings. Thus, the precise role of autophagy in the

NATURE REVIEWS | NEUROSCIENCE ADVANCE ONLINE PUBLICATION | 13

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Acute neurotoxicity Traumatic damage Adult stroke Excitotoxic challenge Neonatal asphyxia

mAMP SAH tMCAO Glutamate Hypoxic–ischaemic

Ethanol SCI pMCAO NMDA challenge
Cocaine TBI 4VO Kainic acid

Autophagy-dependent Ambiguous impact Autophagy-mediated

neuroprotection on acute brain damage neurotoxicity

Figure 3 | Impact of autophagy on acute brain injury. Neurons die in response to acuteNature injury while activating
Reviews | Neuroscience
a robust autophagic response. In some pathophysiologically relevant settings, including intoxication by various
psychoactive agents, spinal cord injury (SCI) and subarachnoid haemorrhage (SAH), autophagy mediates a clear
neuroprotective activity (green). In other scenarios, such as neonatal hypoxic–ischaemic encephalopathy, it is evident
that autophagic cell death by autosis actively contributes to neuronal demise (red). However, it remains to be
determined whether autophagy has a cytoprotective or a cytotoxic role in neurons subjected to various other forms of
acute injury, including cocaine intoxication, adult stroke and traumatic brain injury (TBI) (blue). This dichotomy reflects
the existence of a highly heterogeneous (and sometimes contradictory) literature based on experimental models and
biomarkers of autophagy that, at least in some cases, do not allow clear conclusions to be drawn. 4VO, four-vessel
occlusion; mAMP, methamphetamine; pMCAO, permanent middle cerebral artery occlusion; tMCAO, temporary
middle cerebral artery occlusion.

Systemic lupus
erythematosus
An autoimmune disease that
has a complex aetiology,
involves multiple organs,
progresses in an unpredictable
manner and is usually treated
with systemic
immunosuppressants.
response of the CNS to TBI remains to be elucidated
(Supplementary information S1,S2 (tables)). Part of the
apparent discrepancy between the results discussed here
may be accounted for by context-dependent parameters,
including whether autophagy was modulated according
to a prophylactic or therapeutic paradigm. However, we
fear that method­ological issues concerning the specific-
ity of autophagy-modulating experimental interventions
and the absence of accurate flux assessments may be the
main cause of such heterogeneity.
Conclusions and perspectives
As discussed above, the impact of autophagy on certain
forms of acute brain damage, including intoxication
with various psychoactive drugs, SCI, SAH and neona-
tal hypoxia–ischaemia has been clarified. However, how
autophagic responses modulate the neurotoxic effects of
various other forms of acute brain damage — includ-
ing excitotoxicity, cocaine intoxication, adult stroke and
TBI — remains to be precisely elucidated (FIG. 3). Indeed,
the data published in the past two decades on the topic
are relatively heterogeneous and sometimes overtly
contradictory. Although part of this apparent discrep-
ancy may stem from the heterogeneity of experimental
variables (including the type, intensity and duration of
neuro­toxic challenges, and the sex and developmental
stage of animals, among others), at least four important
points should also be taken into consideration.
First, the intensity and duration of the stimulus may
determine whether autophagy contributes to the demise
of neurons exposed to acute stress (when neurotoxic
conditions are intense or prolonged) or instead exerts
prominent cytoprotective and hormetic effects (when
neurotoxic conditions are weak or restricted in time)29
(BOX 1). Second, most of the studies presented above
lacked accurate flux determinations25. Third, in many of
the studies discussed above, autophagy was modulated
with pharmacological agents that exhibit a low degree
of specificity (such as 3‑MA and lysosomal inhibitors)25,
or with genetic approaches targeting a single component
of the autophagic machinery, even though each of these
proteins is likely to participate in processes unrelated to
autophagy 189. Moreover, standard genetic approaches
involve the lifelong inhibition of autophagy in a specific
tissue, which may be associated with a general rewir-
ing of cellular metabolism7. Fourth, at least part of the
long-term functional impairment caused by acute brain
injury results from local inflammatory responses that
are initiated by microglia in response to danger signals
emitted by dying neurons190. It therefore remains possi-
ble that the effects attributed to activators or inhibitors
of autophagy (at least partially) arise from: the impact of
autophagy on the release of danger signals99; the extran-
euronal control of inflammation by autophagy 13; and/or
the ability of pharmacological modulators of autophagy to
influence inflammatory processes as an off-target effect.
One relevant example of a pharmacological modulator of
autophagy that has an anti-inflammatory effect is chloro-
quine, as its anti-inflammatory activity is harnessed for
the treatment of systemic lupus erythematosus191. It will be
important to determine to what extent the cytoprotective
and anti-inflammatory functions of autophagy influence
the context-dependent impact of this process on acute
brain injury.
Collectively, these caveats suggest that the current
literature on this topic should be interpreted with cau-
tion. Moreover, these considerations highlight the urgent
need for a novel wave of investigations that attempt to
dissect the actual impact of autophagy on acute brain
injury with rigorous tools. Such tools include flux assays
that measure actual autophagic proficiency (as opposed
to the accumulation of autophagic markers), pharma-
cological agents with improved specificity and genetic
strategies that target at least two distinct components of
the autophagic machinery in an inducible manner. We
surmise that such experimental approaches will even-
tually clarify the actual effects of autophagy in various
forms of acute brain injury.

14 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.

1. Kaur, J. & Debnath, J. Autophagy at the crossroads of
catabolism and anabolism. Nat. Rev. Mol. Cell Biol.
16, 461–472 (2015).
2. Li, W. W., Li, J. & Bao, J. K. Microautophagy: lesser-
known self-eating. Cell. Mol. Life Sci. 69, 1125–1136
(2012).
3. Cuervo, A. M. & Wong, E. Chaperone-mediated
autophagy: roles in disease and aging. Cell Res. 24,
92–104 (2014).
4. Lamb, C. A., Yoshimori, T. & Tooze, S. A. The
autophagosome: origins unknown, biogenesis
complex. Nat. Rev. Mol. Cell Biol. 14, 759–774
(2013).
5. Galluzzi, L. et al. Autophagy in malignant
transformation and cancer progression. EMBO J. 34,
856–880 (2015).
6. Komatsu, M. et al. Loss of autophagy in the central
nervous system causes neurodegeneration in mice.
Nature 441, 880–884 (2006).
This study elegantly demonstrates that the
neuron-specific loss of a component of the
autophagic machinery (ATG7) in mice causes an
accelerated neurodegenerative process associated
with behavioural defects and early mortality.
7. Galluzzi, L., Pietrocola, F., Levine, B. & Kroemer, G.
Metabolic control of autophagy. Cell 159,
1263–1276 (2014).
8. Galluzzi, L., Bravo-San Pedro, J. M. & Kroemer, G.
Organelle-specific initiation of cell death. Nat. Cell
Biol. 16, 728–736 (2014).
9. Sica, V. et al. Organelle-specific initiation of autophagy.
Mol. Cell 59, 522–539 (2015).
10. Kroemer, G., Marino, G. & Levine, B. Autophagy and
the integrated stress response. Mol. Cell 40,
280–293 (2010).
11. Menzies, F. M., Fleming, A. & Rubinsztein, D. C.
Compromised autophagy and neurodegenerative
diseases. Nat. Rev. Neurosci. 16, 345–357 (2015).
12. Choi, A. M., Ryter, S. W. & Levine, B. Autophagy in
human health and disease. N. Engl. J. Med. 368,
651–662 (2013).
13. Deretic, V., Saitoh, T. & Akira, S. Autophagy in
infection, inflammation and immunity. Nat. Rev.
Immunol. 13, 722–737 (2013).
14. Liu, Y. et al. Autosis is a Na+,K+-ATPase-regulated
form of cell death triggered by autophagy-inducing
peptides, starvation, and hypoxia–ischemia. Proc.
Natl Acad. Sci. USA 110, 20364–20371 (2013).
This study is the first morphological and
biochemical characterization of autosis, and the
first demonstration of its pathophysiological
relevance in the course of neonatal
hypoxia–ischaemia.
15. Galluzzi, L. et al. Essential versus accessory aspects of
cell death: recommendations of the NCCD 2015. Cell
Death Differ. 22, 58–73 (2015).
16. Noda, N. N. & Inagaki, F. Mechanisms of autophagy.
Annu. Rev. Biophys. 44, 101–122 (2015).
17. Hoyer-Hansen, M. et al. Control of macroautophagy
by calcium, calmodulin-dependent kinase kinase-β,
and Bcl‑2. Mol. Cell 25, 193–205 (2007).
18. Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K. L. TSC2 is
phosphorylated and inhibited by Akt and
suppresses mTOR signalling. Nat. Cell Biol. 4,
648–657 (2002).
19. Kim, J. et al. Differential regulation of distinct Vps34
complexes by AMPK in nutrient stress and autophagy.
Cell 152, 290–303 (2013).
20. Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by
AMP-activated protein kinase connects energy sensing
to mitophagy. Science 331, 456–461 (2011).
These authors demonstrate a direct,
MTORC1‑independent connection between the
regulator of autophagy, AMPK and one of the
upstream components of the molecular machinery
of autophagy, ULK1.
21. Russell, R. C. et al. ULK1 induces autophagy by
phosphorylating Beclin‑1 and activating VPS34 lipid
kinase. Nat. Cell Biol. 15, 741–750 (2013).
22. Zalckvar, E. et al. DAP-kinase-mediated phosphorylation
on the BH3 domain of beclin 1 promotes dissociation of
beclin 1 from Bcl‑XL and induction of autophagy. EMBO
Rep. 10, 285–292 (2009).
23. Tanaka, A. et al. Proteasome and p97 mediate
mitophagy and degradation of mitofusins induced by
Parkin. J. Cell Biol. 191, 1367–1380 (2010).
24. Narendra, D., Kane, L. A., Hauser, D. N., Fearnley, I. M.
& Youle, R. J. p62/SQSTM1 is required for Parkin-
induced mitochondrial clustering but not mitophagy;
VDAC1 is dispensable for both. Autophagy 6,
1090–1106 (2010).
NATURE REVIEWS | NEUROSCIENCE
25. Klionsky, D. J. et al. Guidelines for the use and
interpretation of assays for monitoring autophagy
(3rd edition). Autophagy 12, 1–222 (2016).
This report provides a comprehensive and detailed
set of guidelines on how to select the most
appropriate assays to monitor autophagic responses
in vitro and in vivo, and on how to correctly interpret
the results.
26. Mehta, A., Prabhakar, M., Kumar, P., Deshmukh, R. &
Sharma, P. L. Excitotoxicity: bridge to various triggers in
neurodegenerative disorders. Eur. J. Pharmacol. 698,
6–18 (2013).
27. Galluzzi, L., Blomgren, K. & Kroemer, G. Mitochondrial
membrane permeabilization in neuronal injury. Nat.
Rev. Neurosci. 10, 481–494 (2009).
28. Giorgi, F. S., Biagioni, F., Lenzi, P., Frati, A. & Fornai, F.
The role of autophagy in epileptogenesis and in epilepsy-
induced neuronal alterations. J. Neural Transm. (Vienna)
122, 849–862 (2015).
29. Stamoula, E. et al. Low dose administration of
glutamate triggers a non-apoptotic, autophagic
response in PC12 cells. Cell Physiol. Biochem. 37,
1750–1758 (2015).
30. Wang, Y. et al. p53 induction contributes to excitotoxic
neuronal death in rat striatum through apoptotic and
autophagic mechanisms. Eur. J. Neurosci. 30,
2258–2270 (2009).
31. Borsello, T., Croquelois, K., Hornung, J. P. & Clarke, P. G.
N‑methyl-d‑aspartate-triggered neuronal death in
organotypic hippocampal cultures is endocytic,
autophagic and mediated by the c‑Jun N‑terminal
kinase pathway. Eur. J. Neurosci. 18, 473–485 (2003).
32. Shacka, J. J. et al. Kainic acid induces early and
transient autophagic stress in mouse hippocampus.
Neurosci. Lett. 414, 57–60 (2007).
33. Wang, Y. et al. An autophagic mechanism is involved in
apoptotic death of rat striatal neurons induced by the
non‑N‑methyl-d‑aspartate receptor agonist kainic acid.
Autophagy 4, 214–226 (2008).
34. Chang, C. F. et al. Melatonin attenuates kainic acid-
induced neurotoxicity in mouse hippocampus via
inhibition of autophagy and α-synuclein aggregation.
J. Pineal Res. 52, 312–321 (2012).
35. Kulbe, J. R., Mulcahy Levy, J. M., Coultrap, S. J.,
Thorburn, A. & Bayer, K. U. Excitotoxic glutamate
insults block autophagic flux in hippocampal neurons.
Brain Res. 1542, 12–19 (2014).
36. Tian, J. et al. Protection of pyruvate against glutamate
excitotoxicity is mediated by regulating DAPK1 protein
complex. PLoS ONE 9, e95777 (2014).
37. Caldero, J. et al. Lithium prevents excitotoxic cell death
of motoneurons in organotypic slice cultures of spinal
cord. Neuroscience 165, 1353–1369 (2010).
38. Fulceri, F. et al. Autophagy activation in glutamate-
induced motor neuron loss. Arch. Ital. Biol. 149,
101–111 (2011).
39. Perez-Carrion, M. D. et al. Dendrimer-mediated siRNA
delivery knocks down Beclin 1 and potentiates NMDA-
mediated toxicity in rat cortical neurons. J. Neurochem.
120, 259–268 (2012).
40. Perez-Carrion, M. D. & Cena, V. Knocking down
HMGB1 using dendrimer-delivered siRNA unveils its
key role in NMDA-induced autophagy in rat cortical
neurons. Pharm. Res. 30, 2584–2595 (2013).
41. Yung, L. M. et al. Sphingosine kinase 2 mediates
cerebral preconditioning and protects the mouse
brain against ischemic injury. Stroke 43, 199–204
(2012).
42. Sheng, R. et al. Preconditioning stimuli induce
autophagy via sphingosine kinase 2 in mouse
cortical neurons. J. Biol. Chem. 289, 20845–20857
(2014).
43. Duran, J., Gruart, A., Garcia-Rocha, M.,
Delgado-Garcia, J. M. & Guinovart, J. J. Glycogen
accumulation underlies neurodegeneration and
autophagy impairment in Lafora disease. Hum. Mol.
Genet. 23, 3147–3156 (2014).
44. Sumanont, Y. et al. Effects of manganese complexes of
curcumin and diacetylcurcumin on kainic acid-induced
neurotoxic responses in the rat hippocampus. Biol.
Pharm. Bull. 30, 1732–1739 (2007).
45. McMahon, J. et al. Impaired autophagy in neurons
after disinhibition of mammalian target of rapamycin
and its contribution to epileptogenesis. J. Neurosci.
32, 15704–15714 (2012).
46. Zeng, L. H., Xu, L., Gutmann, D. H. & Wong, M.
Rapamycin prevents epilepsy in a mouse model of
tuberous sclerosis complex. Ann. Neurol. 63,
444–453 (2008).
47. Zhou, J. et al. Pharmacological inhibition of mTORC1
suppresses anatomical, cellular, and behavioral
©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS
abnormalities in neural-specific Pten knock-out mice.
J. Neurosci. 29, 1773–1783 (2009).
48. Zeng, L. H. et al. Tsc2 gene inactivation causes a more
severe epilepsy phenotype than Tsc1 inactivation in a
mouse model of tuberous sclerosis complex. Hum.
Mol. Genet. 20, 445–454 (2011).
49. Pun, R. Y. et al. Excessive activation of mTOR in
postnatally generated granule cells is sufficient to
cause epilepsy. Neuron 75, 1022–1034 (2012).
50. Dong, X. X. et al. p53 mediates autophagy activation
and mitochondria dysfunction in kainic acid-induced
excitotoxicity in primary striatal neurons.
Neuroscience 207, 52–64 (2012).
51. Sadasivan, S. et al. Acute NMDA toxicity in cultured
rat cerebellar granule neurons is accompanied by
autophagy induction and late onset autophagic cell
death phenotype. BMC Neurosci. 11, 21 (2010).
52. Wang, Y. et al. IGF‑1 alleviates NMDA-induced
excitotoxicity in cultured hippocampal neurons against
autophagy via the NR2B/PI3K–AKT–mTOR pathway.
J. Cell. Physiol. 229, 1618–1629 (2014).
53. Ginet, V. et al. Involvement of autophagy in hypoxic–
excitotoxic neuronal death. Autophagy 10, 846–860
(2014).
This study uses the stereotaxic intrastriatal
delivery of a lentiviral vector encoding a
Becn1‑specific shRNA to investigate the effect of
autophagic responses in newborn rats subjected to
severe hypoxia–ischaemia.
54. Baek, S. H. et al. Modulation of mitochondrial function
and autophagy mediates carnosine neuroprotection
against ischemic brain damage. Stroke 45,
2438–2443 (2014).
55. Martorell-Riera, A. et al. Mfn2 downregulation in
excitotoxicity causes mitochondrial dysfunction and
delayed neuronal death. EMBO J. 33, 2388–2407
(2014).
56. Wang, W. et al. MFN2 couples glutamate excitotoxicity
and mitochondrial dysfunction in motor neurons.
J. Biol. Chem. 290, 168–182 (2015).
57. Van Laar, V. S. et al. Glutamate excitotoxicity in
neurons triggers mitochondrial and endoplasmic
reticulum accumulation of Parkin, and, in the presence
of N‑acetyl cysteine, mitophagy. Neurobiol. Dis. 74,
180–193 (2015).
58. Alexander, A. et al. ATM signals to TSC2 in the
cytoplasm to regulate mTORC1 in response to ROS.
Proc. Natl Acad. Sci. USA 107, 4153–4158 (2010).
59. Kim, T. S. et al. The ZFHX3 (ATBF1) transcription
factor induces PDGFRB, which activates ATM in the
cytoplasm to protect cerebellar neurons from
oxidative stress. Dis. Model. Mech. 3, 752–762
(2010).
60. Hou, Y. S. et al. Sestrin2 protects dopaminergic cells
against rotenone toxicity through AMPK-dependent
autophagy activation. Mol. Cell. Biol. 35, 2740–2751
(2015).
61. Sai, Y. et al. 14‑3‑3 proteins in the regulation of
rotenone-induced neurotoxicity might be via its
isoform 14‑3‑3ε’s involvement in autophagy. Cell. Mol.
Neurobiol. 33, 1109–1121 (2013).
62. Filomeni, G. et al. Neuroprotection of kaempferol by
autophagy in models of rotenone-mediated acute
toxicity: possible implications for Parkinson’s disease.
Neurobiol. Aging 33, 767–785 (2012).
63. Pan, T. et al. Rapamycin protects against rotenone-
induced apoptosis through autophagy induction.
Neuroscience 164, 541–551 (2009).
64. Wu, Y. et al. Neuroprotection of deferoxamine on
rotenone-induced injury via accumulation of HIF‑1α
and induction of autophagy in SH‑SY5Y cells.
Neurochem. Int. 57, 198–205 (2010).
65. Liang, S., Figtree, G., Aiqun, M. & Ping, Z. GAPDH-
knockdown reduce rotenone-induced H9C2 cells death
via autophagy and anti-oxidative stress pathway.
Toxicol. Lett. 234, 162–171 (2015).
66. Colell, A. et al. GAPDH and autophagy preserve
survival after apoptotic cytochrome c release in the
absence of caspase activation. Cell 129, 983–997
(2007).
67. Wang, R. C. et al. Akt-mediated regulation of
autophagy and tumorigenesis through Beclin 1
phosphorylation. Science 338, 956–959 (2012).
These authors demonstrate that BECN1 can be
directly phosphorylated by AKT1, resulting in
suppressed autophagic responses upon the
sequestration of BECN1 by 14‑3‑3ε.
68. Su, L. Y. et al. Melatonin attenuates MPTP-induced
neurotoxicity via preventing CDK5‑mediated
autophagy and SNCA/α-synuclein aggregation.
Autophagy 11, 1745–1759 (2015).
ADVANCE ONLINE PUBLICATION | 15
REVIEWS

69. Nopparat, C., Porter, J. E., Ebadi, M. &
Govitrapong, P. 1‑Methyl‑4‑phenylpyridinium-induced
cell death via autophagy through a Bcl‑2/Beclin 1
complex-dependent pathway. Neurochem. Res. 39,
225–232 (2014).
70. Liu, K., Shi, N., Sun, Y., Zhang, T. & Sun, X.
Therapeutic effects of rapamycin on MPTP-induced
Parkinsonism in mice. Neurochem. Res. 38, 201–207
(2013).
71. Li, X. Z. et al. Therapeutic effects of valproate
combined with lithium carbonate on MPTP-induced
parkinsonism in mice: possible mediation through
enhanced autophagy. Int. J. Neurosci. 123, 73–79
(2013).
72. Park, H. J., Shin, J. Y., Kim, H. N., Oh, S. H. &
Lee, P. H. Neuroprotective effects of mesenchymal
stem cells through autophagy modulation in a
parkinsonian model. Neurobiol. Aging 35,
1920–1928 (2014).
73. Li, L. et al. Parkinson’s disease involves autophagy and
abnormal distribution of cathepsin L. Neurosci. Lett.
489, 62–67 (2011).
74. Gonzalez-Polo, R. A. et al. Inhibition of paraquat-
induced autophagy accelerates the apoptotic cell
death in neuroblastoma SH‑SY5Y cells. Toxicol. Sci.
97, 448–458 (2007).
75. Gonzalez-Polo, R. et al. Silencing DJ‑1 reveals its
contribution in paraquat-induced autophagy.
J. Neurochem. 109, 889–898 (2009).
76. Pan, T. et al. Neuroprotection of rapamycin in
lactacystin-induced neurodegeneration via autophagy
enhancement. Neurobiol. Dis. 32, 16–25 (2008).
77. Guo, M. L. et al. Cocaine-mediated microglial
activation involves the ER stress–autophagy axis.
Autophagy 11, 995–1009 (2015).
78. Cao, L. et al. Cocaine-mediated autophagy in
astrocytes involves sigma 1 receptor, PI3K, mTOR,
Atg5/7, Beclin‑1 and induces type II programmed cell
death. Mol. Neurobiol. http://dx.doi.org/10.1007/
s12035-015-9377-x (2015).
79. Guha, P., Harraz, M. M. & Snyder, S. H. Cocaine elicits
autophagic cytotoxicity via a nitric oxide–GAPDH
signaling cascade. Proc. Natl Acad. Sci. USA 113,
1417–1422 (2016).
80. Sutton, L. P. & Caron, M. G. Essential role of D1R in
the regulation of mTOR complex1 signaling induced
by cocaine. Neuropharmacology 99, 610–619
(2015).
81. Wu, J., McCallum, S. E., Glick, S. D. & Huang, Y.
Inhibition of the mammalian target of rapamycin
pathway by rapamycin blocks cocaine-induced
locomotor sensitization. Neuroscience 172, 104–109
(2011).
82. Cubells, J. F., Rayport, S., Rajendran, G. & Sulzer, D.
Methamphetamine neurotoxicity involves vacuolation
of endocytic organelles and dopamine-dependent
intracellular oxidative stress. J. Neurosci. 14,
2260–2271 (1994).
83. Castino, R. et al. Suppression of autophagy
precipitates neuronal cell death following low doses of
methamphetamine. J. Neurochem. 106, 1426–1439
(2008).
84. Pasquali, L. et al. Role of autophagy during
methamphetamine neurotoxicity. Ann. NY Acad. Sci.
1139, 191–196 (2008).
85. Lenzi, P. et al. A subcellular analysis of genetic
modulation of PINK1 on mitochondrial alterations,
autophagy and cell death. Arch. Ital. Biol. 150,
194–217 (2012).
86. Lin, M. et al. Methamphetamine-induced neurotoxicity
linked to ubiquitin–proteasome system dysfunction
and autophagy-related changes that can be
modulated by protein kinase Cδ in dopaminergic
neuronal cells. Neuroscience 210, 308–332 (2012).
87. Ma, J. et al. Methamphetamine induces autophagy as
a pro-survival response against apoptotic endothelial
cell death through the Kappa opioid receptor. Cell
Death Dis. 5, e1099 (2014).
88. Sinchai, T. et al. Caffeine potentiates
methamphetamine-induced toxicity both in vitro and
in vivo. Neurosci. Lett. 502, 65–69 (2011).
89. Pitaksalee, R. et al. Autophagy inhibition by caffeine
increases toxicity of methamphetamine in SH‑SY5Y
neuroblastoma cell line. Neurotox. Res. 27, 421–429
(2015).
90. Moon, J. H. et al. Caffeine prevents human prion
protein-mediated neurotoxicity through the induction
of autophagy. Int. J. Mol. Med. 34, 553–558 (2014).
91. Sanchez, A. B. et al. Antiretrovirals,
methamphetamine, and HIV‑1 envelope protein
gp120 compromise neuronal energy homeostasis in
association with various degrees of synaptic and
neuritic damage. Antimicrob. Agents Chemother. 60,
168–179 (2015).
92. Chandramani Shivalingappa, P., Jin, H.,
Anantharam, V., Kanthasamy, A. & Kanthasamy, A.
N‑acetyl cysteine protects against methamphetamine-
induced dopaminergic neurodegeneration via
modulation of redox status and autophagy in
dopaminergic cells. Parkinsons Dis. 2012, 424285
(2012).
93. Li, Y. et al. Taurine attenuates methamphetamine-
induced autophagy and apoptosis in PC12 cells
through mTOR signaling pathway. Toxicol. Lett. 215,
1–7 (2012).
94. Nopparat, C., Porter, J. E., Ebadi, M. &
Govitrapong, P. The mechanism for the
neuroprotective effect of melatonin against
methamphetamine-induced autophagy. J. Pineal Res.
49, 382–389 (2010).
95. Li, I. H. et al. Autophagy activation is involved in
3,4‑methylenedioxymethamphetamine (‘ecstasy’)-
induced neurotoxicity in cultured cortical neurons.
PLoS ONE 9, e116565 (2014).
96. Chen, G. et al. Autophagy is a protective response to
ethanol neurotoxicity. Autophagy 8, 1577–1589
(2012).
97. Wang, H., Bower, K. A., Frank, J. A., Xu, M. & Luo, J.
Hypoxic preconditioning alleviates ethanol
neurotoxicity: the involvement of autophagy.
Neurotox. Res. 24, 472–477 (2013).
98. Pla, A., Pascual, M., Renau-Piqueras, J. & Guerri, C.
TLR4 mediates the impairment of ubiquitin–
proteasome and autophagy–lysosome pathways
induced by ethanol treatment in brain. Cell Death Dis.
5, e1066 (2014).
99. Kroemer, G., Galluzzi, L., Kepp, O. & Zitvogel, L.
Immunogenic cell death in cancer therapy. Annu. Rev.
Immunol. 31, 51–72 (2013).
This is an exhaustive review of the molecular and
cellular mechanisms whereby RCD can be perceived
as immunogenic by the immune system and hence
initiate an adaptive immune response.
100. Vucicevic, L. et al. Autophagy inhibition uncovers the
neurotoxic action of the antipsychotic drug olanzapine.
Autophagy 10, 2362–2378 (2014).
101. Fabrizi, C. et al. Role of autophagy inhibitors and
inducers in modulating the toxicity of trimethyltin in
neuronal cell cultures. J. Neural Transm. (Vienna) 119,
1295–1305 (2012).
102. Fabrizi, C. et al. Impairment of the autophagic flux in
astrocytes intoxicated by trimethyltin.
Neurotoxicology 52, 12–22 (2015).
103. Ginet, V. et al. Dying neurons in thalamus of
asphyxiated term newborns and rats are autophagic.
Ann. Neurol. 76, 695–711 (2014).
104. Xie, C. et al. Neuroprotection by selective neuronal
deletion of Atg7 in neonatal brain injury. Autophagy
12, 410–423 (2016).
105. Ginet, V., Puyal, J., Clarke, P. G. & Truttmann, A. C.
Enhancement of autophagic flux after neonatal
cerebral hypoxia–ischemia and its region-specific
relationship to apoptotic mechanisms. Am. J. Pathol.
175, 1962–1974 (2009).
106. Koike, M. et al. Inhibition of autophagy prevents
hippocampal pyramidal neuron death after hypoxic–
ischemic injury. Am. J. Pathol. 172, 454–469
(2008).
107. Zhu, C. et al. The influence of age on apoptotic and
other mechanisms of cell death after cerebral
hypoxia–ischemia. Cell Death Differ. 12, 162–176
(2005).
This study is the first to report the lipidation of LC3
in the brains of mice subjected to unilateral
hypoxia–ischaemia.
108. Takada, S. H. et al. Neonatal anoxia in rats:
hippocampal cellular and subcellular changes related
to cell death and spatial memory. Neuroscience 284,
247–259 (2015).
109. Carloni, S., Buonocore, G. & Balduini, W. Protective
role of autophagy in neonatal hypoxia–ischemia
induced brain injury. Neurobiol. Dis. 32, 329–339
(2008).
110. Carloni, S., Buonocore, G., Longini, M., Proietti, F. &
Balduini, W. Inhibition of rapamycin-induced
autophagy causes necrotic cell death associated with
Bax/Bad mitochondrial translocation. Neuroscience
203, 160–169 (2012).
111. Carloni, S. et al. Activation of autophagy and Akt/
CREB signaling play an equivalent role in the
neuroprotective effect of rapamycin in neonatal
hypoxia–ischemia. Autophagy 6, 366–377 (2010).
112. Li, H. et al. Lithium-mediated long-term
neuroprotection in neonatal rat hypoxia–ischemia is
associated with anti-inflammatory effects and
enhanced proliferation and survival of neural stem/
progenitor cells. J. Cereb. Blood Flow Metab. 31,
2106–2115 (2011).
113. Li, Q. et al. Lithium reduces apoptosis and autophagy
after neonatal hypoxia–ischemia. Cell Death Dis. 1,
e56 (2010).
114. Shi, R. et al. Excessive autophagy contributes to
neuron death in cerebral ischemia. CNS Neurosci.
Ther. 18, 250–260 (2012).
115. Lu, Q., Harris, V. A., Kumar, S., Mansour, H. M. &
Black, S. M. Autophagy in neonatal hypoxia ischemic
brain is associated with oxidative stress. Redox Biol.
6, 516–523 (2015).
116. Puyal, J., Vaslin, A., Mottier, V. & Clarke, P. G.
Postischemic treatment of neonatal cerebral ischemia
should target autophagy. Ann. Neurol. 66, 378–389
(2009).
117. Shi, R. Y. et al. BNIP3 interacting with LC3 triggers
excessive mitophagy in delayed neuronal death in
stroke. CNS Neurosci. Ther. 20, 1045–1055 (2014).
118. De Angelis, C. & Haupert, G. T. Hypoxia triggers
release of an endogenous inhibitor of Na+-K+-ATPase
from midbrain and adrenal. Am. J. Physiol. 274,
F182–F188 (1998).
119. Li, W. L. et al. The regulatory role of NF‑κB in
autophagy-like cell death after focal cerebral ischemia
in mice. Neuroscience 244, 16–30 (2013).
120. Liu, N., Shang, J., Tian, F., Nishi, H. & Abe, K. In vivo
optical imaging for evaluating the efficacy of
edaravone after transient cerebral ischemia in mice.
Brain Res. 1397, 66–75 (2011).
121. Mehta, S. L. et al. Manganese superoxide dismutase
deficiency exacerbates ischemic brain damage under
hyperglycemic conditions by altering autophagy.
Transl. Stroke Res. 2, 42–50 (2011).
122. Li, L. et al. Transmembrane protein 166 regulates
autophagic and apoptotic activities following focal
cerebral ischemic injury in rats. Exp. Neurol. 234,
181–190 (2012).
123. Wen, Y. D. et al. Neuronal injury in rat model of
permanent focal cerebral ischemia is associated with
activation of autophagic and lysosomal pathways.
Autophagy 4, 762–769 (2008).
124. Xin, X. Y. et al. 2‑Methoxyestradiol attenuates
autophagy activation after global ischemia. Can.
J. Neurol. Sci. 38, 631–638 (2011).
125. Liu, L. et al. β-asarone attenuates ischemia–
reperfusion-induced autophagy in rat brains via
modulating JNK, p‑JNK, Bcl‑2 and Beclin 1. Eur.
J. Pharmacol. 680, 34–40 (2012).
126. Shang, J. et al. Antiapoptotic and antiautophagic
effects of glial cell line-derived neurotrophic factor and
hepatocyte growth factor after transient middle
cerebral artery occlusion in rats. J. Neurosci. Res. 88,
2197–2206 (2010).
127. Zheng, Y. et al. Inhibition of autophagy contributes to
melatonin-mediated neuroprotection against transient
focal cerebral ischemia in rats. J. Pharmacol. Sci. 124,
354–364 (2014).
128. Qin, A. P. et al. Autophagy was activated in injured
astrocytes and mildly decreased cell survival following
glucose and oxygen deprivation and focal cerebral
ischemia. Autophagy 6, 738–753 (2010).
129. Cui, D. R. et al. Propofol prevents cerebral ischemia-
triggered autophagy activation and cell death in the
rat hippocampus through the NF‑κB/p53 signaling
pathway. Neuroscience 246, 117–132 (2013).
130. Zhou, H. et al. N‑acetyl-serotonin offers
neuroprotection through inhibiting mitochondrial
death pathways and autophagic activation in
experimental models of ischemic injury. J. Neurosci.
34, 2967–2978 (2014).
131. Dohi, E. et al. Hypoxic stress activates chaperone-
mediated autophagy and modulates neuronal cell
survival. Neurochem. Int. 60, 431–442 (2012).
132. Sheng, R. et al. Autophagy activation is associated
with neuroprotection in a rat model of focal cerebral
ischemic preconditioning. Autophagy 6, 482–494
(2010).
133. Jiang, T. et al. Acute metformin preconditioning
confers neuroprotection against focal cerebral
ischaemia by pre-activation of AMPK-dependent
autophagy. Br. J. Pharmacol. 171, 3146–3157
(2014).
134. Wang, P. et al. Induction of autophagy contributes to
the neuroprotection of nicotinamide
phosphoribosyltransferase in cerebral ischemia.
Autophagy 8, 77–87 (2012).

16 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.

135. Sheng, R. et al. Autophagy regulates endoplasmic
reticulum stress in ischemic preconditioning.
Autophagy 8, 310–325 (2012).
136. Fan, J. et al. Ischemic preconditioning enhances
autophagy but suppresses autophagic cell death in rat
spinal neurons following ischemia–reperfusion. Brain
Res. 1562, 76–86 (2014).
137. Yan, W. et al. Autophagy activation is involved in
neuroprotection induced by hyperbaric oxygen
preconditioning against focal cerebral ischemia in rats.
Brain Res. 1402, 109–121 (2011).
138. Gao, B. et al. The endoplasmic reticulum stress
inhibitor salubrinal inhibits the activation of
autophagy and neuroprotection induced by brain
ischemic preconditioning. Acta Pharmacol. Sin. 34,
657–666 (2013).
139. Jiang, T. et al. Ischemic preconditioning provides
neuroprotection by induction of AMP-activated
protein kinase-dependent autophagy in a rat model of
ischemic stroke. Mol. Neurobiol. 51, 220–229
(2015).
140. Jiang, Z. et al. The effects of methylene blue on
autophagy and apoptosis in MRI-defined normal
tissue, ischemic penumbra and ischemic core. PLoS
ONE 10, e0131929 (2015).
141. Zhao, G., Zhang, W., Li, L., Wu, S. & Du, G.
Pinocembrin protects the brain against ischemia–
reperfusion injury and reverses the autophagy
dysfunction in the penumbra area. Molecules 19,
15786–15798 (2014).
142. Wang, P. et al. ARRB1/β-arrestin‑1 mediates
neuroprotection through coordination of
BECN1‑dependent autophagy in cerebral ischemia.
Autophagy 10, 1535–1548 (2014).
143. Papadakis, M. et al. Tsc1 (hamartin) confers
neuroprotection against ischemia by inducing
autophagy. Nat. Med. 19, 351–357 (2013).
These authors elegantly demonstrate that the
inhibition of autophagy by the stereotaxic delivery
of a lentiviral vector encoding a Tsc1‑targeting
shRNA aggravates the neurotoxicity of tMCAO in
adult rats.
144. Wang, P. et al. Nicotinamide
phosphoribosyltransferase protects against ischemic
stroke through SIRT1‑dependent adenosine
monophosphate-activated kinase pathway. Ann.
Neurol. 69, 360–374 (2011).
145. Zhang, X. et al. Cerebral ischemia–reperfusion-
induced autophagy protects against neuronal injury by
mitochondrial clearance. Autophagy 9, 1321–1333
(2013).
146. Zhang, X. et al. Endoplasmic reticulum stress induced
by tunicamycin and thapsigargin protects against
transient ischemic brain injury: involvement of
PARK2‑dependent mitophagy. Autophagy 10,
1801–1813 (2014).
147. Su, J., Zhang, T., Wang, K., Zhu, T. & Li, X. Autophagy
activation contributes to the neuroprotection of
remote ischemic perconditioning against focal cerebral
ischemia in rats. Neurochem. Res. 39, 2068–2077
(2014).
148. Zhou, X. et al. GSK‑3β inhibitors suppressed
neuroinflammation in rat cortex by activating
autophagy in ischemic brain injury. Biochem. Biophys.
Res. Commun. 411, 271–275 (2011).
149. Buckley, K. M. et al. Rapamycin up‑regulation of
autophagy reduces infarct size and improves
outcomes in both permanent MCAL, and embolic
MCAO, murine models of stroke. Exp. Transl Stroke
Med. 6, 8 (2014).
150. Zheng, C. et al. NAD+ administration decreases
ischemic brain damage partially by blocking
autophagy in a mouse model of brain ischemia.
Neurosci. Lett. 512, 67–71 (2012).
151. Kubota, C. et al. Constitutive reactive oxygen species
generation from autophagosome/lysosome in neuronal
oxidative toxicity. J. Biol. Chem. 285, 667–674 (2010).
152. Zheng, Y. Q., Liu, J. X., Li, X. Z., Xu, L. & Xu, Y. G.
RNA interference-mediated downregulation of Beclin1
attenuates cerebral ischemic injury in rats. Acta
Pharmacol. Sin. 30, 919–927 (2009).
This work demonstrates that a lentiviral vector
encoding a Becn1‑specific shRNA stereotaxically
injected into the ipsilateral lateral ventricle
decreases infarct volume, histological injury and
neurological deficits in adult rats subjected to
tMCAO.
153. Clark, R. S. et al. Autophagy is increased in mice after
traumatic brain injury and is detectable in human
brain after trauma and critical illness. Autophagy 4,
88–90 (2008).
NATURE REVIEWS | NEUROSCIENCE
154. Sarkar, C. et al. Impaired autophagy flux is associated
with neuronal cell death after traumatic brain injury.
Autophagy 10, 2208–2222 (2014).
155. Liu, C. L., Chen, S., Dietrich, D. & Hu, B. R. Changes in
autophagy after traumatic brain injury. J. Cereb. Blood
Flow Metab. 28, 674–683 (2008).
156. Diskin, T. et al. Closed head injury induces
upregulation of Beclin 1 at the cortical site of injury.
J. Neurotrauma 22, 750–762 (2005).
157. Park, Y. et al. Chaperone-mediated autophagy after
traumatic brain injury. J. Neurotrauma 32,
1449–1457 (2015).
158. Kanno, H., Ozawa, H., Sekiguchi, A. & Itoi, E. Spinal
cord injury induces upregulation of Beclin 1 and
promotes autophagic cell death. Neurobiol. Dis. 33,
143–148 (2009).
159. Liu, S. et al. Disrupted autophagy after spinal cord
injury is associated with ER stress and neuronal cell
death. Cell Death Dis. 6, e1582 (2015).
160. Chen, S., Wu, H., Tang, J., Zhang, J. & Zhang, J. H.
Neurovascular events after subarachnoid hemorrhage:
focusing on subcellular organelles. Acta Neurochir.
Suppl. 120, 39–46 (2015).
161. Wang, Z. Y., Liu, W. G., Muharram, A., Wu, Z. Y. &
Lin, J. H. Neuroprotective effects of autophagy
induced by rapamycin in rat acute spinal cord injury
model. Neuroimmunomodulation 21, 257–267
(2014).
162. Goldshmit, Y. et al. Rapamycin increases neuronal
survival, reduces inflammation and astrocyte
proliferation after spinal cord injury. Mol. Cell
Neurosci. 68, 82–91 (2015).
163. Wang, H. et al. VEGF inhibits the inflammation in
spinal cord injury through activation of autophagy.
Biochem. Biophys. Res. Commun. 464, 453–458
(2015).
164. Zhou, Y. et al. Retinoic acid prevents disruption of
blood–spinal cord barrier by inducing autophagic flux
after spinal cord injury. Neurochem. Res. 41,
813–825 (2015).
165. Gao, K. et al. Neuroprotective effect of simvastatin via
inducing the autophagy on spinal cord injury in the rat
model. Biomed. Res. Int. 2015, 260161 (2015).
166. Zhou, K. L. et al. Stimulation of autophagy promotes
functional recovery in diabetic rats with spinal cord
injury. Sci. Rep. 5, 17130 (2015).
167. Wang, Z. Y., Lin, J. H., Muharram, A. & Liu, W. G.
Beclin‑1‑mediated autophagy protects spinal cord
neurons against mechanical injury-induced apoptosis.
Apoptosis 19, 933–945 (2014).
168. Jing, C. H. et al. Autophagy activation is associated
with neuroprotection against apoptosis via a
mitochondrial pathway in a rat model of
subarachnoid hemorrhage. Neuroscience 213,
144–153 (2012).
169. Liu, Y. et al. Induction of autophagy by cystatin C:
a potential mechanism for prevention of cerebral
vasospasm after experimental subarachnoid
hemorrhage. Eur. J. Med. Res. 18, 21 (2013).
170. Zhao, H. et al. Role of autophagy in early brain injury
after subarachnoid hemorrhage in rats. Mol. Biol. Rep.
40, 819–827 (2013).
171. Chen, J. et al. Melatonin-enhanced autophagy
protects against neural apoptosis via a mitochondrial
pathway in early brain injury following a
subarachnoid hemorrhage. J. Pineal Res. 56, 12–19
(2014).
172. Shao, A. et al. Enhancement of autophagy by histone
deacetylase inhibitor trichostatin A ameliorates
neuronal apoptosis after subarachnoid hemorrhage in
rats. Mol. Neurobiol. 53, 18–27 (2016).
These authors provide robust pharmacological and
genetic evidence supporting the ability of
autophagy to limit neuronal loss following
hemicerebellectomy.
173. Viscomi, M. T. et al. Stimulation of autophagy by
rapamycin protects neurons from remote
degeneration after acute focal brain damage.
Autophagy 8, 222–235 (2012).
174. Erlich, S., Alexandrovich, A., Shohami, E. &
Pinkas-Kramarski, R. Rapamycin is a neuroprotective
treatment for traumatic brain injury. Neurobiol. Dis.
26, 86–93 (2007).
175. Song, Q., Xie, D., Pan, S. & Xu, W. Rapamycin protects
neurons from brain contusion-induced inflammatory
reaction via modulation of microglial activation. Mol.
Med. Rep. 12, 7203–7210 (2015).
176. Ding, K. et al. Melatonin protects the brain from
apoptosis by enhancement of autophagy after
traumatic brain injury in mice. Neurochem. Int. 91,
46–54 (2015).
©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS
177. Zhao, M., Liang, F., Xu, H., Yan, W. & Zhang, J.
Methylene blue exerts a neuroprotective effect against
traumatic brain injury by promoting autophagy and
inhibiting microglial activation. Mol. Med. Rep. 13,
13–20 (2016).
178. Xu, J. et al. Post-traumatic administration of luteolin
protects mice from traumatic brain injury: implication
of autophagy and inflammation. Brain Res. 1582,
237–246 (2014).
179. Ma, L. et al. 17AAG improves histological and
functional outcomes in a rat CCI model through
autophagy activation and apoptosis attenuation.
Neurosci. Lett. 599, 1–6 (2015).
180. Jin, Y., Lei, J., Lin, Y., Gao, G. Y. & Jiang, J. Y.
Autophagy inhibitor 3‑MA weakens neuroprotective
effects of posttraumatic brain injury moderate
hypothermia. World Neurosurg. 88, 433–446
(2016).
181. Cui, C. M. et al. Chloroquine exerts neuroprotection
following traumatic brain injury via suppression of
inflammation and neuronal autophagic death. Mol.
Med. Rep. 12, 2323–2328 (2015).
182. Luo, C. L. et al. Autophagy is involved in traumatic
brain injury-induced cell death and contributes to
functional outcome deficits in mice. Neuroscience
184, 54–63 (2011).
183. Bao, H. J., Zhang, L., Han, W. C. & Dai, D. K.
Apelin‑13 attenuates traumatic brain injury-induced
damage by suppressing autophagy. Neurochem. Res.
40, 89–97 (2015).
184. Zhang, M. et al. Hydrogen sulfide offers
neuroprotection on traumatic brain injury in parallel
with reduced apoptosis and autophagy in mice. PLoS
ONE 9, e87241 (2014).
185. Wang, Y. Q. et al. Necrostatin‑1 suppresses autophagy
and apoptosis in mice traumatic brain injury model.
Neurochem. Res. 37, 1849–1858 (2012).
186. Cui, C. et al. Neuroprotective effect of ceftriaxone in a
rat model of traumatic brain injury. Neurol. Sci. 35,
695–700 (2014).
187. Lai, Y. et al. Autophagy is increased after traumatic
brain injury in mice and is partially inhibited by the
antioxidant γ-glutamylcysteinyl ethyl ester. J. Cereb.
Blood Flow Metab. 28, 540–550 (2008).
188. Yao, J., Zheng, K. & Zhang, X. Rosiglitazone exerts
neuroprotective effects via the suppression of
neuronal autophagy and apoptosis in the cortex
following traumatic brain injury. Mol. Med. Rep. 12,
6591–6597 (2015).
189. Subramani, S. & Malhotra, V. Non-autophagic roles of
autophagy-related proteins. EMBO Rep. 14,
143–151 (2013).
190. Witcher, K. G., Eiferman, D. S. & Godbout, J. P.
Priming the inflammatory pump of the CNS after
traumatic brain injury. Trends Neurosci. 38, 609–620
(2015).
191. Lee, S. J., Silverman, E. & Bargman, J. M. The role of
antimalarial agents in the treatment of SLE and lupus
nephritis. Nat. Rev. Nephrol. 7, 718–729 (2011).
192. Marino, G., Niso-Santano, M., Baehrecke, E. H. &
Kroemer, G. Self-consumption: the interplay of
autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 15,
81–94 (2014).
This article provides a comprehensive review of the
intricate crosstalk that exists between autophagy
and various forms of RCD.
193. Palikaras, K., Lionaki, E. & Tavernarakis, N.
Coordination of mitophagy and mitochondrial
biogenesis during ageing in C. elegans. Nature 521,
525–528 (2015).
194. Martins, I., Galluzzi, L. & Kroemer, G. Hormesis, cell
death and aging. Aging (Albany NY) 3, 821–828
(2011).
195. Madeo, F., Pietrocola, F., Eisenberg, T. & Kroemer, G.
Caloric restriction mimetics: towards a molecular
definition. Nat. Rev. Drug Discov. 13, 727–740
(2014).
196. Madeo, F., Tavernarakis, N. & Kroemer, G. Can
autophagy promote longevity? Nat. Cell Biol. 12,
842–846 (2010).
197. Kroemer, G. & Levine, B. Autophagic cell death:
the story of a misnomer. Nat. Rev. Mol. Cell Biol. 9,
1004–1010 (2008).
198. Berry, D. L. & Baehrecke, E. H. Growth arrest and
autophagy are required for salivary gland cell
degradation in Drosophila. Cell 131, 1137–1148
(2007).
This paper is one of the first to elegantly
demonstrate the causal contribution of autophagy
to the demise of salivary gland cells in developing
Drosophila melanogaster larvae.
ADVANCE ONLINE PUBLICATION | 17
REVIEWS

199. Menger, L. et al. Cardiac glycosides exert anticancer
effects by inducing immunogenic cell death. Sci. Transl
Med. 4, 143ra199 (2012).
200. Lin, M. H. & Akera, T. Increased (Na+,K+)-ATPase
concentrations in various tissues of rats caused by
thyroid hormone treatment. J. Biol. Chem. 253,
723–726 (1978).
Acknowledgements
G.K. is supported by the following agencies and institutes: the
French Ligue Contre le Cancer (Équipe Labellisée); the French
Agence National de la Recherche (ANR) — Projets Blancs; the
ANR under the framework of E‑Rare‑2 (the ERA-Net for
Research on Rare Diseases); the French Association pour la
Recherche sur le Cancer (ARC); Cancéropôle Ile‑de‑France; the
French Institut National du Cancer (INCa); the Fondation
Bettencourt Schueller; the Fondation de France; the French
Fondation pour la Recherche Médicale (FRM); the European
Commission (ArtForce); the European Research Council (ERC);
the LabEx Immuno-Oncology; the SIRIC (sites de recherche
intégrée sur le cancer) Stratified Oncology Cell DNA Repair and
Tumor Immune Elimination (SOCRATE); the SIRIC Cancer
Research and Personalized Medicine (CARPEM); the Swiss
Bridge Foundation; the Swiss Institute for Experimental Cancer
Research (ISREC); and the Paris Alliance of Cancer Research
Institutes (PACRI). K.B. is supported by the following agencies
and institutes: the Swedish Research Council; the Swedish
Childhood Cancer Foundation; the Swedish Cancer Foundation;
the Swedish Brain Foundation; the Swedish Board of Radiation
Safety; the Frimurare Barnhuset Foundation in Stockholm,
Sweden; and governmental grants from the Swedish Agreement
on Medical Education and Research (Avtal om läkarutbildning
och forskning (ALF)).
Competing interests statement
The authors declare no competing interests.
SUPPLEMENTARY INFORMATION
See online article: S1 (table) | S2 (table)
ALL LINKS ARE ACTIVE IN THE ONLINE PDF

18 | ADVANCE ONLINE PUBLICATION www.nature.com/nrn

©
2
0
1
6
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.