Apoptosis Autophagy

Classification of cell death
Cell death
receptor-caspase 8
mitochondria-caspase 9
• Apoptosis (1972)
– Greek word “falling off”
• Built-in (programmed)
• or self-destructionsuicide
• Type of programmed
cell death based upon
morphological features
• Apoptosis is programmed cell death, which prevents damage
to neighboring cells by controlling how the affected cell dies
• During apoptosis, the cell's cytoskeleton is broken down,
causing multiple bulges in the membrane. This is called
• These blebs (a.k.a. apoptotic bodies) can separate from the
cell, taking a portion of cytoplasm with them. Phagocytic cells
eventually consume these fragments.
• Hence, apoptosis keeps damaged cellular contents from
spilling out and damaging other cells.
Two lymphocytes
Staurosporin-treated HeLa cells
s Apoptosi
Weinberg Fig 9.18 Different parts of the
apoptotic programme. Markers for the
Fragmentation of DNA
Phagocytosis of apoptotic bodies
Fragmentation of Golgi bodies
Phosphorylation of Histone 2B
Nature Reviews Cancer 2; 647-656 (2002)
Death Receptordependent
Physiological Relevance of Apoptosis
Embryonic Development
Regulation of/by Immune System
Negative Selection
CTL Killing (eg. Immune surveillance, viral infections)
Terminating Active Immune Response
Tight Regulation of Cell Number (eg. BM, GI, Uterus, Skin)
Compensatory Response to Cell Stress
Intrinsic Pathway (e.g. GF removal, XRT, Chemo.)
Extrinsic Pathway (e.g. FAS.Ag, TNF-R Activation)
Senescence (ageing)
• Necrosis is traumatic cell death caused by
injury. Necrosis of cells will lyse and damage
neighboring cells by spilling all the
intracellular contents. This causes
inflammation of neighboring tissues and
further trauma.
Morphologic appearance of necrosis
• Increased eosinophilia:
Loss of RNA in the cytoplasm
Increased binding of eosin to denatured
cytoplasmic protein
• More glassy homogeneous appearance
Loss of glycogen particles
• Vacuolated and moth-eaten cytoplasm
• Calcification of necrotic cells
Principle Sites of Damage in Cell Injury
Necrosis vs. Apoptosis
• Apoptosis – programmed cell death
• Necrosis – un-programmed cell death
Necrotic cells
Necrotic cell with multiple lesions
Apoptotic cell with blebbing
Contrast of Apoptosis and necrosis
Death by apoptosis is a neat, orderly process
The Roles of Therapy-Induced Autophagy and Necrosis in Cancer Treatment
• Inhibiting autophagy for cancer
therapy. A, surgery,
chemotherapy, targeted
therapies, and radiation can all
activate autophagy. Treatment
of cancer cells with chloroquine
(CQ) derivatives leads to
deacidification of lysosomes
followed by an accumulation of
ineffective autophagic vesicles.
In cells dependent on
autophagy for survival,
autophagy inhibition with
chloroquine leads to cell death.
B and C, electron micrographs of
PC3 prostate cancer cells either
untreated (B) or treated with
chloroquine (C). Bar, 2 μm.
The Roles of Therapy-Induced Autophagy and Necrosis in
Cancer Treatment
Regulation of autophagy and necrosis. A, autophagy. Multiple stresses, including
interrupted growth factor signaling, activate autophagy. Fluctuations in small molecules,
such as increased ROS, may be the link between signal transduction events and the
enzymatic activation of autophagy genes. Once initiated, autophagic vesicles form around
damaged mitochondria and proteins. This double-membrane structure fuses with the
lysosome resulting in the degradation of contents. PI3K, phosphatidylinositol 3-kinase;
mTOR, mammalian target of rapamycin. B, necrosis. Initiators of necrosis include the NAD+depleting PARP and the death receptor adaptors RIPK1 and TRAF2. Activated RIPK1
translocates to the mitochondria, disrupting the association of cyclophilin D (Cyc. D) and
adenosine nucleotide translocator (ANT). The subsequent depletion of NAD+ and ATP and
accumulation of calcium and ROS lead to activation of calpain proteases and lipid
peroxidation followed by widespread membrane disruption.
Clin Cancer Res 2007;13:7271-7279. The Roles of Therapy-Induced Autophagy and
Necrosis in Cancer Treatment
Mechanistic interplay between apoptosis and autophagy
Research in the Lane Lab focuses of mechanisms of cell death, with a particular interest in the molecular
relationships between apoptosis and autophagy. Autophagy is activated during many forms of cell stress,
and is upregulated during terminal differentiation. It is generally regarded as a protective mechanism in
cells undergoing starvation and oxidative stress, but has been proposed to switch to a cell death mechanism
in its own right when other death pathways (such as apoptosis) are prevented. Evidence suggests that
apoptosis and autophagy are linked by common regulatory factors (see below), so identifying and
characterising molecules and pathways that couple apoptosis and autophagy may lead to novel therapeutic
mechanisms for cancer and neurodegeneration - important diseases that feature both of these cellular
- Molecules identified todate that play dual roles in apoptosis and autophagy include Beclin-1 (a Bcl-2/BclXL interactor that forms the core of a multi-component, type III PI3-kinase involved in autophagosome
membrane supply) and Atg5 (a factor required for autophagosomal membrane biogenesis and Atg8
recruitment which is cleaved by calpains becoming highly cytotoxic). We have reported that Atg4D (a
member of the Atg4 endopeptidase family required for Atg8 processing) is cleaved by caspases, whereupon
it gains Atg8 processing activity but acquires apoptosis-inducing capabilities. Autophagy (macroautophagy) is characterised by the formation of double membrane-bound organelles that
sequester regions of cytoplasm including misfolded protein aggregates and organelles.The autophagosome
membrane is decorated with a protein known as Atg8 (mammalian orthologues include LC3, Gate-16 and
GABARAP), enabling quantitation of autophagy upregulation by fluorescence microscopy of Atg8 puncta
combined with automated microscopy. EM is also routinely used to identify/quantitate autophagosomes.
Light microscopy - dynamic assessment
Electron microscopy - high resolution assessment
• Molecules identified todate that play dual roles in apoptosis and autophagy
include Beclin-1 (a Bcl-2/Bcl-XL interactor that forms the core of a multicomponent, type III PI3-kinase involved in autophagosome membrane supply)
and Atg5 (a factor required for autophagosomal membrane biogenesis and Atg8
recruitment which is cleaved by calpains becoming highly cytotoxic). We have
reported that Atg4D (a member of the Atg4 endopeptidase family required for
Atg8 processing) is cleaved by caspases, whereupon it gains Atg8 processing
activity but acquires apoptosis-inducing capabilities.
Autophagy and its inhibitors
From the following article:
Self-eating and self-killing: crosstalk between autophagy and apoptosis
M. Chiara Maiuri, Einat Zalckvar, Adi Kimchi & Guido Kroemer
Nature Reviews Molecular Cell Biology 8, 741-752 (September 2007)
Autophagy starts with the stepwise engulfment of cytoplasmic material (cytosol and/or organelles) by the phagophore (also
called isolation membrane), which sequesters material in double-membraned vesicles named autophagosomes (also called
autophagic vacuoles). In many cellular settings, the first regulatory process (see figure, step 1) involves the de-repression of
the mTOR Ser/Thr kinase, which inhibits autophagy by phosphorylating autophagy protein-13 (Atg13). This phosphorylation
leads to the dissociation of Atg13 from a protein complex that contains Atg1 kinase and Atg17, and thus attenuates the Atg1
kinase activity. When mTOR is inhibited, re-association of dephosphorylated Atg13 with Atg1 stimulates its catalytic activity
and induces autophagy. Notably, the mammalian orthologue of the yeast Atg13 has not been identified to date. Among the
initial steps of vesicle nucleation is the activation of mammalian Vps34, a class III phosphatidylinositol 3-kinase (PI3K), to
generate phosphatidylinositol-3-phosphate (PtdIns3P) (step 2). Vps34 activation depends on the formation of a multiprotein
complex in which beclin-1 (Becn1; the mammalian orthologue of Atg6), UVRAG (UV irradiation resistance-associated tumour
suppressor gene) and a myristylated kinase (Vps15, or p150 in humans) participate.
Two ubiquitin-like conjugation systems are part of the vesicle elongation process (step 3). One pathway involves the
covalent conjugation of Atg12 to Atg5, with the help of the E1-like enzyme Atg7 and the E2-like enzyme Atg10. The second
pathway involves the conjugation of phosphatidylethanolamine (PE) to LC3/Atg8 (LC3 is one of the mammalian homologues
of Atg8) by the sequential action of the protease Atg4, the E1-like enzyme Atg7 and the E2-like enzyme Atg3. Lipid
conjugation leads to the conversion of the soluble form of LC3 (named LC3-I) to the autophagic-vesicle-associated form
(LC3-II). LC3-II is used as a marker of autophagy because its lipidation and specific recruitment to autophagosomes provides
a shift from diffuse to punctate staining of the protein and increases its electrophoretic mobility on gels compared with LC3I. Moreover, green fluorescent protein–LC3 fusion proteins can be used to visualize autophagosomes by fluorescence
videomicroscopy91. The mechanism of retrieval in which the Atg9 complex participates is poorly studied (step 4).
Autophagosomes undergo maturation by fusion with lysosomes to create autolysosomes (steps 5 and 6). In the
autolysosomes, the inner membrane as well as the luminal content of the autophagic vacuoles is degraded by lysosomal
enzymes that act optimally within this acidic compartment4, 92. Pharmacological inhibitors and small interfering RNAs that
are capable of inhibiting distinct steps of this process are shown (red blocking arrows). Bcl2 and Bcl-XL are regulators of
beclin-1. Lamp2, lysosome-associated membrane glycoprotein-2.
Caspase-dependent and caspase-independent routes to cell death
From the following article:
Self-eating and self-killing: crosstalk between autophagy and apoptosis
M. Chiara Maiuri, Einat Zalckvar, Adi Kimchi & Guido Kroemer
Nature Reviews Molecular Cell Biology 8, 741-752 (September 2007)
Two main pathways lead to caspase-dependent apoptosis (see figure). The extrinsic pathway involves
stimulation of members of the tumour necrosis factor receptor (TNFR) superfamily, such CD95/Fas, TNFR
or TRAILR (death receptors). The intrinsic pathway is characterized by mitochondrial outer membrane
permeabilization (MOMP) and the release of mitochondrial cytochrome c, which results in assembly of a
caspase-activating complex between caspase-9 and APAF1 (the apoptosome). Death-receptor stimulation
typically results in the recruitment and activation of caspase-8 by the Fas-associated via death domain
(FADD)/TNFR1-associated death domain protein (TRADD) to form a death-inducing signalling complex
(DISC) that can further propagate death signals in three ways: via proteolysis of the BCL2 homology-3
(BH3)-only protein BID, which provokes translocation of truncated BID to mitochondria and consequent
MOMP; by direct proteolytic activation of downstream effector caspases; or via activation of the kinase
RIP93. In the intrinsic pathway, a range of BH3-only proteins act as sentinels for cell stress, organellespecific damage or infection, and can be mobilized via post-translational modification (such as proteolysis
or phosphorylation) or subcellular relocalization to initiate MOMP. The BH3-only proteins stimulate MOMP
by triggering the oligomerization of BAX and/or BAK in the outer mitochondrial membrane, thereby
forming channels that permit the escape of multiple proteins from the mitochondrial intermembrane
space26, 56, 57. In the context of DNA damage, stabilization of the p53 tumour-suppressor protein can result
in transcriptional activation of the BH3-only proteins (such as PUMA and NOXA) that can promote MOMP
via the BAX/BAK channel52. Alternatively, DNA damage can activate caspase-2 in a multinuclear complex
that involves the p53-induced protein with a death domain (PIDD) and the RIP-associated protein with a
death domain (RAIDD) (together known as the piddosome)94. Caspase-2 may then induce MOMP and/or
direct caspase activation. Several factors among the mitochondrial proteins that are released as a result of
MOMP (apoptosis-inducing factor (AIF), Omi, EndoG) can promote caspase-independent cell death95,
which can also result from stimuli that cause lysosomal membrane permeabilization (LMP), resulting in the
release of cathepsin proteases into the cytosol9. Such cathepsins can also trigger MOMP, thereby
stimulating the mitochondrial pathway of apoptosis. JNK, c-Jun N-terminal kinase; LMP, lysosomal
membrane permeabilization; ROS, reactive oxygen species.
One protein, multiple functions: against molecular reductionism
From the following article:
Self-eating and self-killing: crosstalk between autophagy and apoptosis
M. Chiara Maiuri, Einat Zalckvar, Adi Kimchi & Guido Kroemer
Nature Reviews Molecular Cell Biology 8, 741-752
Many cell-death researchers assumed that the sole function of a whole series of
proteins — including the BCL2-family proteins, caspases and their activators — was the
regulation of apoptosis. However, recent work has revealed that caspases regulate
normal activation and differentiation processes96, 97 and that BCL2 proteins may
regulate neural plasticity98. Even in Caenorhabditis elegans, it was found that the sole
BCL2 homology-3 (BH3)-only protein EGL-1, which is required for developmental cell
death, is also required for non-lethal autophagy induced by starvation58. Similarly, an
essential component of the caspase activation complex, CED-4 (the nematode
equivalent of APAF1), has a non-apoptotic role in the DNA-damage checkpoint99.
Some of the Atg proteins, which are required for autophagy, may also have unsuspected
roles outside of the autophagic process. One prominent example is ATG5, which, under
certain circumstances, is proteolytically activated to become a pro-apoptotic molecule
that translocates to mitochondria and triggers mitochondrial outer membrane
permeabilization (MOMP)51. Another example is beclin-1, which bears a BH3 domain58,
62 that might favour apoptosis, at least in some circumstances. As a result, it may be an
oversimplification to assume that the inhibition of cell death in response to DNA
damage or the activation of apoptosis/necrosis during nutrient deprivation (which both
result from the knockout or knockdown of a single Atg gene) is due to suppression of
the autophagic process. Several genes from different modules of autophagy should be
knocked down independently to find out whether the resulting phenotype is similar
before final conclusions are drawn. The same holds for experiments in which apoptotic
pathways have been interrupted by knocking out or knocking down single genes.
A model for the roles of apoptosis and autophagy in tumorigenesis.
• A common cellular response to metabolic stress
is cell death mediated by apoptosis, which limits
tumour growth. Tumours may trigger autophagymediated cell survival in certain metabolicstressed tumour regions. In apoptotic-defective,
metabolic-stressed tumour cells, activation of
autophagy prevents death from necrosis,
whereas defects in autophagy lead to
accumulation of p62, damaged mitochondria,
ROS and protein aggregates, resulting in genome
damage and tumorigenesis. For additional
information, see refs 68 and 119.
The relationship between apoptosis and autophagy
Self-eating and self-killing: crosstalk between autophagy and apoptosis
M. Chiara Maiuri, Einat Zalckvar, Adi Kimchi & Guido Kroemer
Nature Reviews Molecular Cell Biology 8, 741-752 (September 2007)
• Similar stressors can induce either apoptosis or autophagy in a contextdependent fashion. It is possible that different sensitivity thresholds, the exact
nature of which remain to be determined, can dictate whether autophagy or
apoptosis will develop. Alternatively, the choice between apoptosis and
autophagy is influenced by the fact that the two catabolic processes exhibit some
degree of mutual inhibition. In some cases, a mixed phenotype of apoptosis and
autophagy can be detected at the single-cell level. Although autophagy mostly
allows cells to adapt to stress, massive autophagy can also kill cells.
Regulation of autophagy and apoptosis by p53 and
Oncogenic stress can activate two different isoforms of murine p19ARF (and of the human orthologue p14ARF). One isoform
corresponds to the full-length nucleolar p19ARF/p14ARF and the second one corresponds to a shorter form, smARF, which results
from internal initiation of translation. Whereas smARF translocates to mitochondria, reduces the mitochondrial transmembrane
potential (Dm) and stimulates autophagy and caspase-independent cell death, the nucleolar p19ARF/p14ARF activates p53 by
binding and inhibiting the negative regulator of p53, MDM2. p53 induces caspase-dependent apoptotic cell death via the
induction of multiple pro-apoptotic proteins, most of which function in the intrinsic pathway. Whereas the nucleolar pathway is
inhibited by Z-VAD, smARF-induced cell death is blocked by knocking down beclin-1 (Becn1) or Atg5. In some cellular settings,
activated p53 induces a transcriptional programme that favours the accumulation of autophagic vacuoles via the induction of the
lysosomal protein DRAM (damaged-regulated autophagy modulator). p53-elicited DRAM may be essential, both for autophagic
vacuolization and for apoptosis.
BH3 proteins and mimetics act on the beclin-1–BCL2
• Proteins that contain BCL2 homology-3 (BH3) domains or small molecules that
mimic BH3 domains can bind to the BH3-receptor domain of BCL2 or BCL-XL and,
hence, competitively disrupt the interaction between BCL2 or BCL-XL and beclin-1.
This probably leads to the activation of the lipid kinase activity of the class III
phosphatidylinositol 3-kinase VPS34 (which depends on the interaction with
UVRAG (UV irradiation resistance-associated tumour suppressor gene), thereby
provoking the production of phosphatidylinositol-3-phosphate (PtdIns3P). In turn,
this leads to vesicle nucleation in a manner that probably involves WIPI-1a (WDrepeat protein interacting with phosphoinositides).
Molecular switches between apoptosis and autophagy
a | Dual function of autophagy protein-5 (ATG5)
in autophagy. Full-length ATG5 can participate
in the initial stages of autophagy with the help
of a ubiquitin-like conjugation system. ATG12
(activated by ATG7 and then transferred to
ATG10) is conjugated with ATG5 at a lysine-amino group, allowing it to bind to ATG16. The
resulting ATG12–ATG5–ATG16 complex imposes
curvature on the crescent phagophore and
recruits activated LC3 to the elongating
membrane (LC3 is one of the mammalian
homologues of Atg8, which is conjugated to
phosphatidylethanolamine by the action of
ATG4, ATG7 and ATG3) (Box 1). Proteolysis by a
calpain results in a truncated ATG5, which can
translocate to mitochondria and induce MOMP.
b | BCL2 homology-3 (BH3)-only proteins as
inducers of apoptosis and autophagy.
Depending on their specificity (for pro- or antiapoptotic and autophagy-inhibitory proteins
that contain BH3 receptors), as well as their
preferential subcellular localization
(mitochondria or endoplasmic reticulum), BH3only proteins can preferentially activate
apoptosis or autophagy. A specific class of
inactivator BH3-only protein interacts with antiapoptotic and anti-autophagic multidomain
proteins of the BCL2 family (red blocking
arrows symbolize preferential affinities),
thereby releasing activator BH3-only proteins
that allosterically activate BAX and BAK (for the
induction of apoptosis). Alternatively, they
release beclin-1 from its inhibitory interaction
with multidomain proteins of the BCL2 family,
allowing it to activate the lipid kinase activity of
VPS34 and, hence, to activate autophagy.
Several steps in this working model still need
further experimental validation.
Scenarios for the interaction between apoptosis and
a | Schematically, a particular set of insults induces apoptosis (part 1), which, if
inhibited, can switch to autophagy. At least in some cellular settings, autophagy
serves as a defence mechanism that prevents or retards necrosis (parts 2,3). b |
Some conditions can trigger a lethal autophagic response that is responsible for
cell death, for example, in naïve cells (parts 1,2) or in cells in which the apoptotic
pathways have been interrupted. c | Another set of stimuli (or perhaps simply a
lower dose of insults) provokes a protective autophagic response (part 1), which is
required for adaptation of the cell and the avoidance of apoptosis (part 2). d |
Frequently, lethal conditions trigger an autophagic response that, independently
of the autophagic response, is followed by apoptosis (part 1). In this case,
inhibition of apoptosis causes either cell survival (part 2) or necrosis (part 3). In
this scenario, the order of events (autophagy, then apoptosis) is chronological, not
hierarchical, meaning that inhibition of autophagy does not prevent apoptosis. e |
Autophagy can be indispensable for sustaining the high ATP levels that are
required for cells to emit signals to phagocytic cells that engulf the apoptotic
bodies (part 1). Inhibition of autophagy does not affect apoptotic cell death, yet it
abolishes the heterophagic removal of apoptotic material (part 2). LPC,
lysophosphatidylcholine; PS, phosphatidylserine.
• Eukaryotes have two major protein degradation systems within cells. One is
the ubiquitin-proteasome system, which accounts for the selective
degradation of most short-lived proteins. The other is the lysosomal system.
Autophagy (Macroautophagy) is the primary means for the degradation of
cytoplasmic constituents in the lysosome.
Intracellular protein degradation systems
(Upper) Macroautophagy. A small volume of cytoplasm is enclosed by the
autophagic isolation membrane, which eventually results in the formation of
an autophagosome. The outer membrane of the autophagosome then fuses
with the lysosome where the cytoplasm-derived materials are degraded.
(Lower) The ubiquitin-proteasome pathway. Polyubiquitinated proteins are
degraded by the 26S proteasome. This is a selective process.
Cellular events in autophagy
The cellular events during digestion of self constituents or intracellular pathogens follow three distinct
stages: initiation (formation of the phagophore), elongation (growth and closure) and maturation of a
double membrane autophagosome into an autolysosome. a | Autophagy sequesters and removes cellular
constituents from the cytosol, including surplus or damaged organelles from the cytosol. b | Autophagy can
eliminate bacteria (free in the cytosol or inside a phagosome), viruses and protozoan parasites in a manner
similar to the elimination of self constituents.
The cellular process of autophagy.
Conditions such as nutrient starvation, pathogen infection and other
environmental stressors, can induce autophagy. Autophagy begins with the
isolation of double-membrane-bound structures inside an intact cell. Previously,
these structures were believed to be derived from the ribosome-free region of the
rough endoplasmic reticulum82, 83, but recent studies indicate that they might
originate from a pre-existing membrane structure called a phagophore84, or could
be formed de novo80, 85. These membrane structures elongate and mature, and
microtubule-associated protein 1 light chain 3 (LC3) is recruited to the membrane.
The elongated double membranes form autophagosomes, which sequester
cytoplasmic proteins and organelles such as mitochondria. The formation of the
pre-autophagosomal structure can be inhibited by the phosphatidylinositol 3phosphate kinase (PI3K) inhibitor 3-methyladenine (3-MA). Sequestration requires
ATP and is regulated mainly by class III PI3K46. The autophagosomes mature with
acidification by the H+-ATPase71 and fuse with lysosomes to become
autolysosomes (also known as the degradative autophagic vacuoles). Microtubules
are important mediators of this fusion process. This process is inhibited by the H+ATPase inhibitor bafilomycin A1, or by microtubule inhibitors such as vinblastine
and nocodazole86, 87. Eventually, the sequestered contents are degraded by
lysosomal hydrolases for recycling. One assay for autophagic cells is to detect the
presence of membrane-bound LC3, which accumulates on autophagosomes.
Autophagy, meaning ‘to eat oneself’, is one of the main mechanisms for maintaining
cellular homeostasis. This pathway is not directly a death pathway, rather a selfcannibalisation pathway. Mediated via the lysosomal degradation pathway,
autophagy is responsible for degrading cellular proteins and is currently the only
known process for degrading cellular organelles, recycling them to ensure cell survival.
Research on autophagy has been on-going for over 40 years, but has been restricted
by lack of knowledge about the molecular machinery behind this process. Over the
last decade, huge advances have been made and genetic screens in yeast (s.
cerevisiae) have led to the identification of over ~30 autophagy-related genes (ATGgenes), many of which have identified mammalian homologues (see table below)
Autophagy is essential in helping to maintain the balance between the increase and
decrease in the number of a cell population. It is undoubtedly active at a basal level
in most cells and contributes to the routine turnover of cytoplasmic components (1, 21).
Table of identified autophagy-related genes (ATG-genes)
TABLE 1 | Autophagy genes in mammals
Protein function
Atg1 is a serine/threonine protein kinase; it may be involved in regulation and vesicle formation
Atg3 functions as an ubiquitin-conjugating-like enzyme that covalently attaches Atg/LC3 to
Atg4 is a cysteine protease that cleaves the C-terminus of Atg8/LC3 to expose a glycine residue
for subsequent conjugation
Atg5 is covalently attached to Atg12, and binds Atg16 as part of a tetrameric complex of
unknown function
ATG6, Beclin 1‡
Atg6 is a component of the class III phosphotidylinositol-3-kinase complex that is required for
Atg7 is a homologue of the ubiquitin-activating enzyme; it activates both Atg8/LC3 and Atg12
before conjugation
Atg8/LC3 has structural similarity to ubiquitin; it is conjugated to phosphatidylethanolamine,
and is part of the autophagosome, but its function is not known
Atg9 is a transmembrane protein that may be involved in delivering membrane to the forming
Atg10 functions as an ubiquitin-conjugating-like enzyme that covalently attaches Atg12 to Atg5
Atg12 has some structural similarity to ubiquitin; it is conjugated to an internal lysine of Atg5
through its C-terminal glycine
Atg16 binds Atg5 and homo-oligomerizes to form a tetrameric complex
this table we have only listed certain key genes in which the gene product has been confirmed to play a role in
autophagy in higher eukaryotes.
the autophagy-related gene (ATG) name and number is listed except when that name is not used in higher
eukaryotes. However, many of the autophagy genes are present as multiple isoforms, usually denoted as a, b and so
TABLE 2 | Known small molecules that influence autophagy
Target of rapamycin
Induces autophagy
Lithium, sodium
Enzymes that ultimately
affect myo-inositol-1,4,5triphosphate levels, such as
inositol monophosphatase
Lowers myo-inositol-1,4,5triphosphate levels, induces
Class III
Inhibits autophagy
Class III
Inhibits autophagy
Bafilomycin A1
Inhibits autophagy
Lysosomal pH
Inhibits autophagy
Lysosomal pH
Inhibits autophagy
Beclin 1
Increases Beclin 1, induces
• Figure 1: Steps of autophagy and associated genes. Shown are the three
steps of autophagy accompanied by the genes associated with each step.
Genes listed in green have already been assessed by Ray Choi, genes listed
in red will be the target of assessment in this paper and genes listed in
black remain to be assessed in the future.
• The term ‘Autophagy’ covers three processes;
microautophagy, macroautophagy and chaperonemediated autophagy (1):
• Microautophagy is the transfer of cytosolic components into the
lysosome by direct invagination of the lysosomal membrane and
subsequent budding of vesicles into the lysosomal lumen (17).
• Macroautophagy involves formation of a double-membrane
structure called the autophagosome which sequesters cytosolic
material and delivers it to the lysosome for degradation (18).
Although this degradation can be selective (i.e. specific removal
of damaged mitochondria sparing normal functioning ones) (19),
degradation of soluble cytosolic proteins is non-selective.
• Chaperone-mediated autophagy (CMA) is characterised by its
selectivity regarding the specific substrates (cytosolic proteins)
degraded (20).
Autophagy - the lysosomal system
In general terms, 'autophagy' refers to any intracellular process that involves the degradation of cytosolic components by the
lysosome. There are at least three distinct autophagic pathways:
Macroautophagy, Microautophagy and Chaperone-mediated autophagy. The terms autophagy and macroautophagy are used
Autophagy is involved in physiological and pathological processes
Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development, and homeostasis.
Under certain circumstances, it is considered as a non-apoptotic cell death pathway. Autophagy is involved in diverse
pathologies, including infections, cancer, neurodegeneration and aging as well as in heart, liver and kidney diseases.
The morphological hallmark of autophagy
Macroautophagy is a multistep process by which portions of cytoplasm, damaged proteins and/or organelles are sequestered in
a double or multimembrane structure (autophagosome). Upon fusion of the autophagosome with the lysosome
(autophagolysosome), sequestered materials are digested by lysosomal hydrolases. The recycling of these intracellular
constituents serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability.
Steps in macroautophagy and chaperone-mediated autophagy (CMA). Macroautophagy: 1.) Nucleation. An unidentified membrane source delivers lipid bi-layers for
the formation of the phagophore. In yeast this early structure is termed pre-autophagosomal structure (PAS), its identity in mammalian cells is uncertain. A class III
PI3K complex consisting of at least BECN1, PIK3C3, PIK3R4, UVRAG, and AMBRA1 is required for PAS formation and MAP1LC3 is anchored to the membrane via a
phosphoethanolamine (PE) anchor (LC3-II). 2.) Expansion. The PAS or a comparable structure in mammals sequesters cytosolic cargo (either specifically via SQSTM1
[p62] or nonspecifically) by invagination, forming a double-membranous vesicle. This stage is also called "isolation membrane". More membrane and LC3-II is being
recruited to the developing vacuole. 3.) Maturation. The completed autophagosome undergoes multiple maturation steps and fusion events with multi-vesicular
bodies (MVB) or endosomes. The exact nature and sequence of this maturation, and whether these steps are always required is currently unknown. The
autophagosomal lumen becomes more acidified during this maturation. 4.) Docking and fusion. During docking and fusion the inner membrane compartment together
with its content gets released into the lysosome/autolysosome and is being degraded by lysosomal hydrolases. The components of the outer membrane are available
for re-usage. Chaperone-mediated autophagy: 5.) Recognition and binding. The HSC70 chaperone complex (consisting of HSC70, HSP90 and maybe other proteins)
recognizes unfolded proteins with the KFERQ sequence and moves them to the lysosome. 6.) Translocation. LAMP2A and a lysosomal form of HSC70 (l-HSC70)
translocate the substrate protein across the lysosomal membrane into the lumen for degradation. The autophagy delivered substrates get degraded inside the
lysosomes and their macromolecular components are made available to the cell's metabolism via permeases that allow their transport back into the cytosol.
Jaeger and Wyss-Coray Molecular Neurodegeneration 2009 4:16 doi:10.1186/1750-1326-4-16
Macroautophagy is a nearly universal process that eukaryotic cells employ to reutilize the constituents of cytoplasm and
organelles. We have used the genetics of Dictyostelium to isolate mutants in genes that are essential for macroautophagy.
These mutants have taught us how to design a screen and a selection for other genes that regulate macroautophagy and also
provide material for studying autophagosome formation. A collection of mutants has been used to show that macroautophagy,
previously thought to be essential for replication of the intracellular pathogen Legionella pneumophila, is not required for
growth of this parasite. We now are asking how autophagy is linked to development and whether there are additional genes
that are essential for autophagy.
Macroautophagy: Macroautophagy as a cell biological and structural phenomenon has been known for many years to manage
the bulk degradation of cytoplasm and organelles. One of its interesting features is that it promotes the transfer of material
from one topologically distinct compartment to another - from the cytosol to the vacuole in baker yeast or to lysosomes in
other eukaryotic cells. An initiating structure called the Pre-autophagosomal Structure or PAS has been defined, but its origins,
once thought to be from the endoplasmic reticulum, are not clear. The favorable vacuolar structure of Saccharomyces
cerevisiae has been married to the genetics of that organism to understand the biochemical reactions involved in the
production of autophagosomes (for reviews see (Abeliovich and Klionsky, 2001; Klionsky and Emr, 2000; Thumm, 2000). S.
cerevisiae contains a single large central vacuole, which serves as lysosome, among other functions. When autophagy is
induced, double-membrane autophagosomes are produced, and these fuse with the vacuole and release single membrane
autophagic bodies that are degraded by resident hydrolases and proteases. When vacuolar proteases are inhibited by PMSF,
one observes an accumulation of small spheres (autophagic bodies) within a larger vacuole. Using several screening methods
to create mutants in which an accumulation of the small autophagic bodies is not observed, several laboratories, notably
those of Ohsumi, Thumm, and Klionsky, have isolated autophagy mutants (called atg). The affected genes have been used to
define a series of phosphorylation and ubiquitination-like reactions that are necessary for cytoplasm to vesicle transport (CVT)
and macroautophagy. CVT encapsulates specific targets during growth, while autophagy envelopes bulk cytoplasm and
organelles during starvation. Many of the molecules that mediate autophagy reside in the Pre-autophagosomal structure (PAS,
also called the perivacuolar compartment (PVC)), which in budding yeast lies next to the vacuole (Suzuki et al., 2001) (Kim et
al., 2002) (Noda et al., 2002). Gradient and immuno-purification studies suggest that the PAS does not contain markers from
other compartments of the cell (Kim et al., 2002). The creation of an autophagosome is thought to occur by extension of an
enveloping membrane, called an isolation membrane, which then closes around cytoplasmic constituents, as shown in Figure
1 (Kim et al., 2002). A similar situation may occur in mammalian cells (Mizushima et al., 2001). Autophagosomes then fuse
with late endosomes or lysosomes to create autophagolysosomes and provide constituents and energy for the cell. Although
there has been progress in understanding this process, especially in determining the genes involved and mapping known gene
products onto physical structures, a number of questions remain. What is the origin of the PAS and of what is it composed,
beyond the known molecules? Does it assemble de novo or does it derive from pre-existing membranes? How does the PAS
convert into an isolation membrane and how does that membrane seal around its cargo? What is the source of the lipid that
forms the membrane? How is size determined? Is there selectivity of cargo? What are the molecules that mediate fusion with
the endosomal system?
Schematic overview of macroautophagy.
Potential therapeutic applications of autophagy
David C. Rubinsztein, Jason E. Gestwicki, Leon O. Murphy & Daniel J. Klionsky
Nature Reviews Drug Discovery 6, 304-312 (April 2007)
A membrane of unknown origin forms the initial phagophore or isolation membrane.
The phagophore expands, sequestering cytoplasm and on completion forms a doublemembrane autophagosome. The autophagosome fuses with a lysosome, containing
acid hydrolases (AH), which can now gain access to the inner vesicle, termed an
autophagic body. The fused compartment where the autophagic body and its contents
are degraded is called an autophagolysosome or autolysosome. Following breakdown,
the resulting macromolecules are released back into the cytosol through permeases for
reuse in metabolic processes (during starvation). Alternatively, the cargo might be
inactivated or killed (when macroautophagy acts as part of the immune response to
eliminate microbial pathogens), or the removal of cytoplasm might result in cell death.
See main text for details.
It is a conceptually simple, intellectually appealing, and experimentally testable hypothesis. Since joining the faculty of the
Pharmacology Department of RWJMS in 2003, my laboratory has conducted extensive research around this basic theme.
We are working in four interrelated research areas and have been making significant progress. First, we are seeking
experimental proof that a defect in autophagy compromises mitochondrial functions, a leading cause of aging. Second, we
are determining the direct cause of cancer development as a result of reduced autophagy. Third, we are trying to
understand the regulation of autophagy. Fourth, we are exploring the potential of targeting autophagy as a therapeutic
adjuvant to cancer therapy. In addition to the Arnold Levine Laboratory, where I was trained as a postdoc, we have made
several UMDNJ laboratories our scientific allies along the way, including the labs of Eileen White, William Hait, and Leroy Liu.
These various collaborations have helped move the science forward far more quickly.
Three hundred years ago, most people died before the age of 50. Few suffered from cancer, dementia, or type II diabetes.
Since the Industrial Revolution, the average lifespan of human beings has increased dramatically. Alas, so has the suffering
associated with these aging-related diseases. If autophagy is indeed involved in aging control and preventing those agingrelated diseases, understanding autophagy might reveal the secret that prolongs the human lifespan without increasing
suffering. Are we looking for the “Holy Grail”? Yes, that is our dream.
It turned out that this strain of mouse, which contains a deletion in a gene called beclin1, has a much higher chance
of getting cancer when it is old. In other words, this result demonstrates that the normal function of the beclin1
gene is to prevent tumor development in old age.
Interestingly, the human version of the beclin1 gene is also deleted in sporadic human cancers, tumors affecting only
older people. Approximately 50% of sporadic breast cancers, 75% of sporadic ovarian cancers, and 40% of sporadic
prostate cancers contain a deletion of the human version of the beclin1 gene. What is even more interesting is that
the gene is not related to any known processes that cause cancer. Instead, this gene participates in autophagy, an
evolutionarily conserved process that is involved in recycling cytoplasmic components. As shown in Figure 1,
autophagy starts with the emergence of a crescent, double membrane structure, which grows and engulfs a portion
of cytoplasm, including mitochondria, ribosomes, and proteins. The structure then matures to a closed, double
membrane vesicle with a diameter between 0.3 and 0.9 micrometers, which is called an autophagosome or
autophagic vacuole. The autophagosome then travels toward the lysosome, a garbage depot and recycling center in
the cell. The outer membrane of the autophagosome is then fused with the lysosome membrane, leading to the
release of its cargo, which is wrapped by the inner membrane into the lysosome. The cargos are then degraded by
the various enzymes in the lysosome. Basically, it is believed that one function of autophagy is to serve as sanitation
workers busily collecting and recycling garbage in the cells.
Strikingly, the same autophagy process has been demonstrated to be important in regulating the life-spans of the
flatworm C. elegans, a common and convenient animal model used in the biological laboratory. The laboratory of
Beth Levine, MD, at Southwest Medical Center, first cloned the beclin1 gene and independently demonstrated that a
mouse strain lacking the beclin1 gene has higher cancer rates, and Leroy Liu, PhD, chair of the Pharmacology
Department at UMDNJ-Robert Wood Johnson Medical School (RWJMS), demonstrated that inactivation of
autophagy genes shortens the worms’ lives. In the meantime, other scientists, including Zhenyu Yue, PhD, who is
now an assistant professor at Mount Sinai School of Medicine in New York and works on the neurobiological aspects
of autophagy, found that some aging-related neurodegenerative diseases caused by the abnormal accumulation of
protein aggregates in neurons, such as Huntington’s disease, are associated with autophagy reduction. The
pharmacological induction of autophagy can help clear the disease-causing aggregates in cells and alleviate the
symptoms of these diseases.
An exciting theme then emerged: Autophagy is a critical garbage collecting and recycling process in healthy cells. As
we get older, the process slows down or becomes less discriminating. Consequently, haphazard agents are
accumulated in cells, which damage various parts of cells and tissues, leading to some aging-related diseases. For
example, failure to clear protein aggregates in neurons of the central nervous system causes dementia; failure to
clear ROS (reactive oxygen species)-producing mitochondria leads to nuclear DNA mutations and cancer. Collectively,
these pathological conditions contribute to, or even define, the process of aging.
Figure 1
Specific and nonspecific autophagy
From the following article:
Autophagy: from phenomenology to molecular understanding in less than a decade
Daniel J. Klionsky
Nature Reviews Molecular Cell Biology 8, 931-937 (November 2007)
During nonspecific autophagy (see figure part a), sequestration begins with the formation of a
phagophore that expands into a double-membrane autophagosome while surrounding a portion of
the cytoplasm. The autophagosome may fuse with an endosome (the product of endocytosis),
which is a form of heterophagy (in a heterophagic process, the cell internalizes and degrades
material that originates outside of the cell, in contrast to autophagy, in which the cell consumes
part of itself). The product of endosome–autophagosome fusion is known as an amphisome. The
completed autophagosome or amphisome fuses with a lysosome, which supplies acid hydrolases.
The enzymes in the resulting compartment, an autolysosome, break down the inner membrane
from the autophagosome and degrade the cargo. The resulting macromolecules are released
through permeases and recycled in the cytosol.
The yeast cytoplasm-to-vacuole targeting (Cvt) pathway is one example of specific autophagy (see
figure part b), and the only example of a biosynthetic autophagy-related pathway. The overall
morphology is identical to nonspecific autophagy; however, the sequestering Cvt vesicles are
smaller than autophagosomes and they appear to exclude bulk cytoplasm. The phagophore
assembly site (PAS) either becomes the sequestering vesicle or generates it. The precursor form of
aminopeptidase I (prApe1) forms oligomers in the cytosol, and is targeted through the action of a
receptor, Atg19, and the adaptor or scaffold protein Atg11 to allow selective cargo recognition and
packaging. The completed vesicle fuses with the vacuole, the yeast analogue of the mammalian
lysosome. The larger size of the vacuole allows the release of the inner single-membrane
compartment of the Cvt vesicle, which is now termed a Cvt body; during nonspecific autophagy in
yeast, this structure is termed an autophagic body. The Cvt body is lysed and prApe1 is matured by
proteolytic removal of a propeptide to generate the active, resident hydrolase mApe1.
Regulation of autophagy in mammalian cells
From the following article:
Autophagy: from phenomenology to molecular understanding in less than a decade
Daniel J. Klionsky
Nature Reviews Molecular Cell Biology 8, 931-937 (November 2007)
• In the figure, the green circles represent components that stimulate
autophagy, whereas the purple boxes correspond to inhibitory
factors. 3-methyladenine (3-MA) and wortmannin (Wm) also inhibit
class I phosphatidylinositol 3-kinases (PI3K), but the overall effect of
these compounds is a block in autophagy (because they inhibit the
downstream class III enzyme that produces phosphatidylinositol-3phosphate (PtdIns(3)P), which is needed for autophagy). The
regulation of autophagy is complex and far from understood.
Historically, TOR (target of rapamycin) has been considered to be
the central regulator of autophagy, because TOR inhibition with
rapamycin (Rap) induces autophagy. However, it is now clear that
there are also TOR-independent types of regulation. For example,
beclin-1 and Atg4 might be regulated by the c-Jun N-terminal kinase
(JNK) and reactive oxygen species (ROS), respectively. Additional
information is available in recent reviews (for example, see Refs 74,
• Perspectives
• Nature Reviews Molecular Cell Biology 8, 931-937
(November 2007) Autophagy: from phenomenology to
molecular understanding in less than a decade
• Daniel J. Klionsky
• Abstract
• In 2000, it was suggested to me that “Autophagy will be
the wave of the future; it will become the new apoptosis.”
Few people would have agreed at the time, but this
statement turned out to be prophetic, and this process of
'self-eating' rapidly exploded as a research field, as
scientists discovered connections to cancer,
neurodegeneration and even lifespan extension.
Amazingly, the molecular breakthroughs in autophagy
have taken place during only the past decade.
Autophagy, the process by which proteins, organelles and invading pathogens are
sequestered in autophagosomal vesicles and delivered to the lysosome for degradation,
provides a primary route for turnover of stable, defective and unwanted cellular
components. Defects in this cellular homeostasis system are linked with numerous human
diseases. This process involves a series of dynamic membrane-rearrangement reactions
mediated by a core set of autophagy (Atg) proteins. Although conserved protein kinase, lipid
kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome
formation and cargo recruitment have been defined, several gaps remain in our
understanding of autophagy. For instance, it has been unclear how the distinct protein
complexes involved in autophagy talk to each other or to other cellular machinery involved
in membrane formation and membrane trafficking. And how do key signals that trigger
autophagy talk to the Atg proteins?
Autophagy networks. (Top) Overview of macroautophagy. (Bottom) Major signal
transduction systems in autophagy.
Recently, we have performed a systematic proteomic analysis of the human autophagy
system with the long-term goal of providing molecular insight into some of the unanswered
questions in the field. This effort has provided a glimpse into the global architecture of the
autophagy interaction network (AIN). Briefly, 65 autophagy-related proteins have been
analyzed proteomically by IP-coupled LC-MS/MS, revealing a dense interaction network with
extensive cross-talk among functional signaling modules. The current AIN contains many
new components linked to vesicle trafficking, ATG8 conjugation, protein phosphorylation,
protein ubiquitination, and lipid modification, and together with genetic (RNAi) and
localization studies performed in parallel, provides a framework for understanding the
molecular architecture of the pathway and how individual sub-networks communicate with
each other. Complementary, we carried out detailed analyses of ATG8 interaction proteins to
delineate regulators and cargo of selective autophagy. Together, our work provides a
resource for detailed mechanistic and further systematic analysis of this critical protein
homeostasis pathway.
• Three predominant cellular functions can be
assigned to autophagy:
• Autophagy is a response to nutrient starvation. Decreased
levels of amino acids can induce the autophagic response in
numerous cell types and situations e.g. the neonatal period,
when the supply of nutrients via the milk has not yet
replaced the nutrients via the placenta (6,7)
• Autophagy is a housekeeping process whereby long-lived
proteins and organelles are recycled e.g. Mitochondria (22)
• Autophagy has tissue-specific roles e.g. during erythrocyte
development, following nucleus expulsion, autophagy is
required to degrade the remaining organelles. Degradation
of the autophagic vesicle results in the functional biconcave
shape (23, 24).
The Four Stages of Autophagy Induction: Following external/internal stimuli (e.g. nutrient depletion or
ischemia), mTOR is inhibited, leading to induction of autophagy.
Autophagosome formation: Cytosolic proteins and organelles are sequestered by a double membrane
vesicle, the origin of which is uncertain, but may arise from the endoplasmic reticulum. Formation of this
vesicle is co-ordinated by complexes of Atg proteins, which encode for the “start codons,” which encode
for the amino acid methionine.
Docking and fusion with the lysosome.
Breakdown of the autophagic vesicle. The molecular mechanism behind the fusion with the lysosome and
subsequent breakdown of the autophagic vesicle are poorly understood.
Schematic diagram of the steps of autophagy. Autophagy begins with the formation of the
phagophore or isolation membrane (vesicle nucleation step). The concerted action of the
autophagy core machinery proteins at the phagophore assembly site (PAS) is thought to lead to
the expansion of the phagophore into an autophagosome (vesicle elongation). The
autophagosome can engulf bulk cytoplasm nonspecifically, including entire organelles, or target
cargos specifically. When the outer membrane of the autophagosome fuses with an endosome
(forming an amphisome before fusing with the lysosome) or directly with a lysosome (docking
and fusion steps), it forms an autophagolysosome. Finally, the sequestered material is degraded
inside the autophagolyosome (vesicle breakdown and degradation) and recycled.
Human autophagy
Signalling regulation of mammalian autophagy.
In the figure, the blue components represent the factors that stimulate autophagy,
whereas the red ones correspond to inhibitory factors. Autophagy is regulated by a
complex signalling network of various stimulatory (blue arrows) and inhibitory (red
bars) inputs. TOR plays a central role in autophagy by integrating the class I
PtdIns3K signalling and amino acid-dependent signalling pathways. Activation of
insulin receptors stimulates the class I PtdIns3K complex and small GTPase Ras,
leading to activation of the PtdIns3K–PKB–TOR pathway. PKB phosphorylates and
inhibits the tuberous sclerosis complex 1/2 (TSC1–TSC2), leading to the
stabilization of Rheb GTPase, which in turn activates TOR, causing inhibition of
autophagy. Amino acids inhibit the Raf-1–MEK1/2–ERK1/2 signalling cascade,
leading to inhibition of autophagy. Energy depletion causes the AMP-activated
protein kinase (AMPK) to be phosphorylated and activated by LKB1. AMPK
phosphorylates and activates TSC1–TSC2, leading to inactivation of TOR and
autophagy induction. p70S6K kinase is a substrate of TOR that may negatively feed
back on TOR activity, ensuring basal levels of autophagy that are important for
homeostasis. JNK1 and DAPK phosphorylate and disrupt the association of antiapoptotic proteins, Bcl-2 and Bcl-XL, with Beclin 1, leading to the activation of the
Beclin 1-associated class III PtdIns3K complex and stimulation of autophagy. Beclin
1 is shown bound to the phagophore membrane.
Schematic depiction of autophagy.
From Eaten alive: a history of macroautophagy
Zhifen Yang1
Daniel J. Klionsky1
Journal name: Nature Cell Biology Volume: 12, Pages: 814–822 (2010)
(a) In yeast, both autophagy and the Cvt pathway engulf cargoes within distinct double-membrane vesicles,
which are thought to originate from the phagophore assembly site (PAS). The PAS is defined as the initial
site for autophagy-related (Atg) protein recruitment. The Cvt pathway is one example of selective autophagy,
and the only example of a biosynthetic autophagy-related pathway. The Cvt vesicle (140–160 nm in
diameter) appears to closely enwrap the specific cargo — the Cvt complex (consisting of the precursor form
of aminopeptidase I — prApe1 — and the Atg19 receptor), and exclude bulk cytoplasm. The
autophagosome (300–900 nm in diameter) engulfs cytoplasm, including organelles, and also the Cvt
complex. The completed vesicles then fuse with the vacuole, the yeast analogue of the mammalian
lysosome, and release the inner single-membrane vesicle (autophagic or Cvt body) into the lumen.
Subsequent breakdown of the inner vesicles allows the maturation of prApe1 and the degradation of
cytoplasm, and hence the recycling of the resulting macromolecules through vacuolar permeases. (b)
Mammalian autophagy is initiated by the formation of the phagophore, followed by a series of steps,
including the elongation and expansion of the phagophore, closure and completion of a double-membrane
autophagosome (which surrounds a portion of the cytoplasm), autophagosome maturation through docking
and fusion with an endosome (the product of fusion is known as an amphisome) and/or lysosome (the
product of fusion is known as an autolysosome), breakdown and degradation of the autophagosome inner
membrane and cargo through acid hydrolases inside the autolysosome, and recycling of the resulting
macromolecules through permeases. So far, there is no evidence for a PAS that exists in mammalian cells,
and so the mammalian phagophore could be equivalent to the yeast PAS, or derived from the PAS. The core
molecular machinery is also depicted, such as the ULK1 and ULK2 complexes that are required for
autophagy induction, class III PtdIns3K complexes that are involved in autophagosome formation,
mammalian Atg9 (mAtg9) that potentially contributes to the delivery of membrane to the forming
autophagosome and two conjugation systems, the LC3-II and Atg12–Atg5–Atg16L complex, which are
proposed to function during elongation and expansion of the phagophore membrane.
Schematic diagram of the presumed role f
or C. elegans autophagy proteins involved in the formation of an autophagosome.
A) Regulation of induction: In yeast, the Tor kinase and its effectors regulate the induction of autophagy. UNC-51 is the C.
elegans Atg1 ortholog, however, it is not clear if there are similar regulatory proteins to Atg17 or Atg13.
B) Vesicle nucleation requires a lipid kinase complex, which includes the class III phosphatidylinositol 3-kinase (PI3K),
Vps34 (in C. elegans LET-512). In yeast, Vps34 activation depends on its binding partners, Atg6, Atg14, and Vps15 (Kihara
et al., 2001). In C. elegans, the ATG6 ortholog is bec-1, however, orthologs to ATG14 and VPS15 appear to be missing in the
C. elegans genome. The interaction between the antiapoptotic protein CED-9 and BEC-1 is conserved in C. elegans (TakácsVellai et al., 2005). A similar interaction between the mammalian proteins Bcl-2 and Beclin 1, inhibits autophagy (Maiuri et
al., 2007a; Maiuri et al., 2007b; Pattingre et al., 2005).
C) Two novel ubiquitin-like conjugation pathways: the Atg12 conjugation system (Atg5, Atg12, and Atg16), and the Atg8
lipidation system (Atg8, Atg3, and Atg7) mediate vesicle expansion, and vesicle completion. Orthologs to all the members of
these two complexes have been found in C. elegans, where they are named ATG-3, ATG-4, ATG-5, ATG-7, ATG-16, LGG-1/Atg8
and LGG-3/Atg12. In yeast, Atg8 undergoes two posttranslational processing events resulting in conjugation to
phosphatidylethanolamine (PE) and recruitment to the PAS membrane. ATG-7 is an E1 ubiquitin activating enzyme required
for the activation of of LGG-1. LGG-3-ATG-5 oligomerize with ATG-16 to allow for the formation of the multimeric complex.
ATG-3 and ATG-10 are E2-like ubiquitin conjugating enzymes and ATG-4 is a cysteine protease. As for its yeast ortholog, LGG1 appears to remain in the completed autophagosome and thus is an excellent marker for early and late autophagosomal
D) The retrieval of the integral membrane protein ATG-9 from the phagophore assembly site (PAS) involves ATG-2 and ATG18, two interacting peripheral proteins
The four stages of Autophagy
1) Induction: Following external/internal stimuli (e.g. nutrient depletion or ischemia) mTOR is
inhibited, leading to induction of Autophagy. Key genes in yeast are Atg1 and Atg13, for which the
mammalian homologues are yet to be identified.
2) Autophagosome formation: Cytosolic proteins and organelles are sequestered by a double
membrane vesicle, the origin of which is uncertain, but may arise from the endoplasmic
reticulum. Formation of this vesicle is co-ordinated by complexes of Atg proteins, in particular
Atg5 and Atg12, that are conjugated enabling the recruitment of LC3 (Atg8). Beclin-1 forms a
complex with Atg14.
3) Docking and fusion with the lysosome
4) Breakdown of the autophagic vesicle. The molecular mechanism behind the fusion with the
lysosome and subsequent breakdown of the autophagic vesicle are poorly understood, although
Lamp-2 is thought to play a key role.
Induction – Autophagy can be induced by both internal and external stimuli. Autophagy is inhibited under
nutrient-rich conditions and therefore simulated by starvation (25, 26). A key regulator or gatekeeper of autophagic
induction is Tor/mTOR (14, 27, 28). In nutrient-rich conditions, mTOR has an inhibitory effect on autophagy, under
starvation conditions, mTOR is inactivated – leading to the inhibition of autophagy being released. Tor inactivation
leads to downstream dephosphorylation events resulting in transcriptional activation of autophagy genes (1, 28).
Autophagosome formation – Following induction, a double membrane vesicle forms in the cytosol, sequestering
those cytoplasmic components for autophagic degradation. The mechanism for the formation of this membranous
vesicle is not well defined although it is known that most of the ATG genes identified to date participate in the
formation of this vesicle (29). It has been postulated that the endoplasmic reticulum is the origin of this membrane
(30, 31)
Autophagosome fusion - During this phase, the autophagosome fuses with the lysosome, as a result the contents
of the autophagosome are released into the lysosome for degradation by lysosomal proteases (1, 32).
Autophagosome breakdown – Following fusion of these two vesicle bodies, the autophagosome membrane is the
broken down by the lysosomal proteases (1, 31).
Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ and Rubinsztein DC. (2005) Lithium induces
autophagy by inhibiting inositol monophosphatase. Journal of Cell Biology 170(7):1101-1111. Access article
Supplementary information
Abstract: Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the
only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which
inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel
mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby,
enhances the clearance of autophagy substrates, like mutant huntingtin and alpha-synucleins. This effect is not
mediated by glycogen synthase kinase 3beta inhibition. The autophagy-enhancing properties of lithium were
mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased
myo-inositol-1,4,5-triphosphate (IP3) levels. Our data suggest that the autophagy effect is mediated at the level of
(or downstream of) lowered IP3, because it was abrogated by pharmacologic treatments that increased IP3. This
novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of
neurodegenerative diseases, like Huntington‘s disease, where the toxic protein is an autophagy substrate.
Sci. STKE, 4 October 2005
Vol. 2005, Issue 304, p. tw349
NEURODEGENERATIVE DISEASE Lithium Decreases IP3 to Promote Autophagy
Lithium is widely known for its application in the treatment of bipolar and unipolar mood disorders. Sarkar et al.
provide evidence that lithium may also be effective in the treatment of neurodegenerative disorders arising
from toxicity of the accumulation of aggregate-prone proteins, such as mutant huntingtin and mutant synuclein, which are implicated in Huntington's disease and certain forms of Parkinson's disease. Stimulation of
autophagy may be a mechanism to prevent the accumulation of these aggegrate-prone proteins, and Sarkar et
al. show that lithium promotes autophagy, detected as an increase in the formation of autophagic vesicles and
a decrease in the abundance of known autophagic substrates. Furthermore, lithium decreased the
accumulation of mutant -synuclein or huntingtin aggregates and the toxicity (cell death) of these proteins in
inducible PC12 cell lines or COS-7 cell lines, an effect that was blocked by the autophagic inhibitor 3methyladenine (3-MA). Lithium is a nonspecific inhibitor of several enzymes. The effect on autophagy appeared
to be mediated by the inhibition of inositol monophosphatase (IMPase), because autophagy and clearance of
mutant huntingtin and -synuclein was also stimulated by inhibition of IMPase with a specific pharmacologic
agent (L-690,330), whereas selective inhibition of glycogen synthase kinase-3ß (GSK-3ß, another lithium target)
did not promote clearance of the aggregate-prone proteins. Both L-690,330 and lithium decreased inositol1,4,5-trisphosphate (IP3) concentrations. That lithium exerted its effects on autophagy through decreased IP3
concentrations was validated by experiments in which IP3 concentrations were increased before lithium
treatment by addition of myo-inositol or prolyl endopeptidase inhibitor 2 (PEI). Increased IP3 concentration
promoted the accumulation of mutant huntingtin and cell death and blocked the effects of lithium to reduce
these phenomena. Autophagy is also stimulated by inhibition of mTOR (mammalian target of rapamycin);
however, lithium appeared to act independently from the pathway regulated by mTOR, because inhibition of
mTOR with rapamycin combined with lithium had an additive effect on clearance of the aggregate-prone
proteins and cell survival. The ability of lithium to stimulate autophagy and clearance of aggregate-prone
proteins was also observed for other mood-stabilizing drugs that promote inositol depletion. Thus, a pathway
involving IP3 and inositol appears to be one mechanism regulating autophagy that may be exploited for
therapeutic benefit in certain neurodegenerative diseases.
S. Sarkar, A. Floto, Z. Berger, S. Imarisio, A. Cordenier, M. Pasco, L. J. Cook, D. C. Rubinsztein, Lithium induces
autophage by inhibiting inositol monophosphate. J. Cell Biol. 170, 1101-1111 (2005). [Abstract] [Full Text]
Citation: Lithium Decreases IP3 to Promote Autophagy. Sci. STKE 2005, tw349 (2005).
Regulation of membrane traffic by phosphoinositide 3-kinases
• Regulation of autophagy in Drosophila by PI 3-kinases. Autophagy is known to be
regulated by both the class I and the class III PI 3-kinases. The class III PI 3-kinase is
required for autophagy through producing PtdIns(3)P via the autophagy-specific
complex containing Beclin-1, Vps15 and hVps34. (In yeast, an additional accessory
protein, Vps14, has been shown to be part of the complex.) By contrast, the class I
PI 3-kinase inhibits autophagy by activating the Akt/PKB pathway. The insect
hormone ecdysone acts through nuclear receptors and triggers programmed
autophagy in the Drosophila fat body by downregulating the class I PI 3-kinase
pathway through an unknown mechanism. The overexpression of the PI 3phosphatase PTEN mimics this effect.
Sarkar S, Davies JE, Huang Z, Tunnacliffe A and Rubinsztein DC. (2007) Trehalose, a novel mTOR-independent
autophagy inducer, accelerates clearance of mutant huntingtin and alpha-synuclein. Journal of Biological
Chemistry 282(8):5641-5652. Access article Supplementary information
Abstract: Trehalose, a disaccharide present in many non-mammalian species, protects cells against various
environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone
properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTORindependent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy
substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with
Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition
by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because
of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against
subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as
an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be
relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.
• Model of autophagosome formation in mammalian cells
The Atg12-Atg5 conjugate and Apg16L localize to the isolation membrane
throughout its elongation process. LC3 (Atg8 homolog) is recruited to the
membrane in the Apg5-dependent manner. Apg12-Apg5 and Apg16L
dissociate from the membrane upon completion of autophagosome
formation, while LC3(-II) remains on the autophagosome membrane. LC3
dissociates from the autolysosomal membrane.
• We generated Atg5-/- mouse embryonic stem (ES) cells and demonstrated
that mammalian Atg5 is also required for autophagy. Atg5-/- ES cells can grow
normally but bulk protein degradation was greatly reduced in these cells,
suggesting that autophagy is actually a major degradation process.
Atg5-/-EScells exhibit a block in the autophagic pathway
Cells were cultured in Hanks' solution for 2 hours to induce autophagy. Bar, 1
um. (by Dr. Akitsugu Yamamoto at Nagahama Institute of Bio-Science and
• In the mammalian Apg12 system, Apg12 is attached to Lys130 of Apg5. When the
Atg5K130R mutant, in which Lys130 is replaced with Apg, is expressed in Atg5-/ES cells, Apg5 is no longer conjugated with Apg12. Even in such cells, Apg5K130R is
able to localize to the small autophagosome precursors with Apg16L, suggesting
that the covalent modification of Apg5 with Apg12 is not required for membrane
targeting of Apg5 and Apg16L. However, the membrane does not elongate to form
cup-shaped isolation membrane and autophagosomes. Thus, the Apg12-Apg5Apg16L complex is essential for elongation of the isolation membranes, not for the
generation of the precursor structures.
Atg12 conjugation is not required for membrane targeting of Atg5, but it is
essential for maturation of the isolation membrane into autophagosome and
recruitment of LC3 to the membrane.
Development of a transgenic mouse model with a fluorescent autophagosome marker
Although the possible involvement of autophagy in homeostasis, development, cell death and pathogenesis has
been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly due to a
limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated
transgenic mice systemically expressing GFP fused to LC3, which serves as a marker protein for
autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient
starvation in most tissues. This transgenic mouse model is a useful tool to study mammalian autophagy.
In vivo analysis of autophagy using GFP-LC3 transgenic mice
(Upper) Gastrocnemius muscle samples were prepared from GFP-LC3 transgenic mice before (left) or after 24-h
starvation (right). Small dots represent autophagosomes.
(Lower) Embryonic fibroblasts from GFP-LC3 mice were cultured in Hanks' solution for 2 hours. Bar, 10 um.
Molecular Mechanism of Autophagy
In the yeast, Saccharomyces cerevisiae, at least 16 of ATG genes have been identified to be
required for autophagosome formation. The most surprising findings are discoveries of the
two novel ubiquitylation-like conjugation systems: one mediates conjugation of Atg12 to
Atg5, and the other mediates a covalent linkage between Atg8 (LC3 in mammals) and
phosphatidylethanolamine (PE) (Suppl. Fig. 1)B
We have dissected the autophagic process in mammalian cells using Atg homologs. We
proposed a model in which the cup-shaped isolation membrane is developed from a small
crescent-shaped compartment. We have also examined the localization and function of
mammalian Atg proteins during this process.
(A) Punctate signals of GFP-Apg5 increase under starvation conditions in ES cells. (B)
Starved ES cells co-expressing CFP-Atg5 and YFP-LC3.
Molecular mechanism of autophagy
Autophagy has been recently implicated in various human pathological and physiological
conditions, such as neurodegeneration, immunity, cancer, development, myopathies,
heart diseases, liver diseases and longevity. Basal autophagy is essential for removing
misfolded proteins and damaged organelles, and therefore, plays a vital role in
maintaining cellular homeostasis in all tissues.
Autophagy and Disease
The autophagic response has been described in various pathophysiological situations, including
neurobiology, cancer and more recently cardiovascular disease, however its role has yet to be
teased out; the crux of the problem being, is the response a cell-protective one, or a mechanism of
cell death?
3.1 Neurodegenerative diseases
Studies have shown that autophagy plays a role in the removal of misfolded proteins which
accumulate in the cell, such as the polyglutamine aggregates formed in Huntington’s chorea (33, 34).
It could be the case though, that a disproportionate autophagic response could contribute to the
pathology seen in such disorders.
3.2 Cancer
A number of tumour-suppressor proteins control autophagy (e.g. Beclin-1 and PTEN), so it would
seem reasonable to assume that a decrease in autophagy would lead to tumour progression (28, 35,
36). Indeed experimental evidence backs up this hypothesis - over expression of Beclin-1 induces
autophagy in cultured breast cancer cells (9) and stimulation of autophagy was observed in cancer
cells following anti-cancer treatments (37). Is this up-regulation of autophagy really a cell-protective
mechanism, or in fact an alternative cell-death mechanism given that in cancer cells the apoptosis
pathway is commonly altered (28)? It must also be considered that whilst during tumour
establishment, autophagy maybe a mechanism through which to target tumour cells, once the
tumour is established autophagy may in fact provide a way for the cancer-cells to overcome
nutrient-limiting conditions especially within the internal mass of the tumour which is poorly
vascularised (28, 38, 39)
• Nature Reviews Drug Discovery 7, 476-477 (June
• Neurodegenerative disease: A new pathway to
• Charlotte Harrison
• Abstract
• Stimulating autophagy — a major cellular
clearance route for intracellular protein
aggregates — could represent a therapeutic
strategy for Huntington's disease. However, the
only drug that has been shown to induce
autophagy in the brain is rapamycin, a mammalian
target of rapamycin (mTOR) kinase inhibitor that
modulates several cellular processes besides
Neurodegenerative disorders: An aid to digestion
SOURCE: home | subscribe A high-throughput screening strategy to identify novel modulators of mammalian autophagy may
provide drug development leads for Huntington's disease.
Autophagy, the lysosomal digestion of cytoplasmic proteins and organelles, may play a protective role in certain
neurodegenerative and infectious diseases. It may also have an inhibitory role in certain cancers. Currently, the only small
molecule known to regulate autophagy in mammalian brains is rapamycin. However, owing to the involvement of mTOR
(mammalian target of rapamycin) proteins in many cellular processes, the long-term use of rapamycin is associated with many
complications. Writing in Nature Chemical Biology, Sarkar and colleagues describe a new approach to identify novel modulators
of mammalian autophagy, which might provide leads for the development of drugs for Huntington's disease.
With the aim of finding safer methods of modulating autophagy, the authors performed high-throughput screening of 50,729
compounds in yeast to identify small molecules that either enhanced or suppressed the effects of rapamycin on cell growth. This
resulted in the identification of a structurally non-redundant set of 21 small-molecule inhibitors of rapamycin (SMIRs) and 12
small-molecule enhancers of rapamycin (SMERs).
To test the capacity of these compounds to modulate mammalian autophagy, independent of rapamycin, they assessed their
ability to induce clearance of the autophagy substrate A53T -synuclein, which is associated with a form of familial Parkinson's
disease. Thirteen SMIRs were shown to slow the clearance of this autophagy substrate, whereas four SMERs enhanced it.
The authors then focused on a subset of autophagy-inducing SMERs that lacked toxicity in various cell lines. These might have
therapeutic potential in neurodegenerative disorders such as Huntington's disease, the underlying cause of which is an expansion
of a polyglutamine (polyQ) tract in the huntingtin protein. In cell models expressing the mutant huntingtin protein — another
substrate for autophagy — each of the SMERs enhanced clearance of this substrate, reducing mutant protein aggregation and cell
death. In a Drosophila model of Huntington's disease, the SMERs protected against neurodegeneration as assessed by the
reduction in the number of visible rhabdomeres in the ommatidia of the eye over time.
Interestingly, unlike rapamycin, these compounds exerted no reduction in the phosphorylation of substrates of mTOR kinase,
which suggests that their induction of autophagy is mediated through an mTOR-independent mechanism, or through an
unknown component of the mTOR autophagy pathway downstream of mTOR. In addition, treatment of cell models with SMERs
together with rapamycin resulted in an additive effect on the clearance of A53T -synuclein and the reduction of mutant
huntingtin aggregation.
This study illustrates the use of a high-throughput screening strategy to identify small molecule modulators of mammalian
autophagy. Structure–activity relationship analysis of the identified SMERs has highlighted functional groups that are required for
their specific activity, and additional candidates for therapeutic development.
Sarah Crunkhorn
Sarkar, S. et al. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nature Chem. Biol. 3,
331–338 (2007). | Article | PubMed |
Huang, J. et al. Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and
proteome chips. Proc. Natl Acad. Sci. USA 101, 16594–16599 (2004) | Article | PubMed |
Rubinzstein, D. C. et al. Potential therapeutic applications of autophagy. Nature Rev. Drug Discov. 6, 304–312 (2007)
| Article | PubMed |
Implication of autophagy deregulation in Parkinson's disease (PD).
There are 3 types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Deregulation of
both macroautophagy and CMA are implicated in the pathogenesis of PD. CMA is involved in the degradation of soluble
wildtype alpha-synuclein, the major constituent of Lewy bodies. Nonetheless, once oligomerized or aggregated, alpha-synuclein
is likely degraded by macroautophagy instead of CMA. Interestingly, A53T and A30P mutants of alpha-synuclein are poorly
degraded by CMA, but are instead degraded by macroautophagy. Furthermore, the alpha-synuclein mutants inhibit CMA,
reducing CMA-mediated degradation of alpha-synuclein and survival factor MEF2D. Concomitant with the inhibition of CMA,
overexpression of alpha-synuclein mutants results in a compensatory activation of macroautophagy. Activation of
macroautophagy is also evident in cells treated with neurotoxin MPP+, a well-established model for Parkinsonism, or following
overexpression of GPR37, another protein that is present in Lewy bodies. Finally, recent studies reveal that macroautophagy
also plays a role in the turnover of fragmented mitochondria. These observations highlight the potential involvement of the
autophagic pathways in the pathogenesis of PD.
• Nature Reviews Neurology 4, 60 (February 2008)
• Rationale for treating Huntington's with a
sirolimus and lithium combination
• Abstract
• Sarkar S et al. (2007) A rational mechanism for
combination treatment of Huntington's disease
using lithium and rapamycin. Hum Mol Genet 17:
170–178 PubMed
• A promising treatment approach to Huntington's
disease is promotion of autophagy to dispose of
aggregate-prone proteins such as mutant
Sarkar S*, Perlstein EO*, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O’Kane CJ,
Schreiber SL and Rubinsztein DC. (2007) Small molecules enhance autophagy and reduce toxicity in Huntington’s
disease models. Nature Chemical Biology 3(6):331-338. Access article Supplementary information Chemical
compounds Cover highlight *Equal contribution
Abstract: The target of rapamycin proteins regulate various cellular processes including autophagy, which may play
a protective role in certain neurodegenerative and infectious diseases. Here we show that a primary smallmolecule screen in yeast yields novel small-molecule modulators of mammalian autophagy. We first identified new
small-molecule enhancers (SMER) and inhibitors (SMIR) of the cytostatic effects of rapamycin in Saccharomyces
cerevisiae. Three SMERs induced autophagy independently of rapamycin in mammalian cells, enhancing the
clearance of autophagy substrates such as mutant huntingtin and A53T alpha-synuclein, which are associated with
Huntington's disease and familial Parkinson's disease, respectively. These SMERs, which seem to act either
independently or downstream of the target of rapamycin, attenuated mutant huntingtin-fragment toxicity in
Huntington's disease cell and Drosophila melanogaster models, which suggests therapeutic potential. We also
screened structural analogs of these SMERs and identified additional candidate drugs that enhanced autophagy
substrate clearance. Thus, we have demonstrated proof of principle for a new approach for discovery of smallmolecule modulators of mammalian autophagy.
Sarkar S, Krishna G, Imarisio S, Saiki S, O’Kane CJ and Rubinsztein DC. (2008) A rational mechanism for combination treatment of
Huntington's disease using lithium and rapamycin. Human Molecular Genetics 17(2):170-178. Access article Supplementary
Abstract: Huntington's disease (HD) is caused by a polyglutamine expansion mutation in the huntingtin protein that confers a toxic
gain-of-function and causes the protein to become aggregate-prone. Aggregate-prone proteins are cleared by macroautophagy, and
upregulating this process by rapamycin, which inhibits the mammalian target of rapamycin (mTOR), attenuates their toxicity in
various HD models. Recently we demonstrated that lithium induces mTOR-independent autophagy by inhibiting inositol
monophosphatase (IMPase) and reducing inositol and IP(3) levels. Here we show that glycogen synthase kinase-3beta (GSK-3beta),
another enzyme inhibited by lithium, has opposite effects. In contrast to IMPase inhibition that enhances autophagy, GSK-3beta
inhibition attenuates autophagy and mutant huntingtin clearance by activating mTOR. In order to counteract the autophagy
inhibitory effects of mTOR activation resulting from lithium treatment, we have used the mTOR inhibitor rapamycin in combination
with lithium. This combination enhances macroautophagy by mTOR-independent (IMPase inhibition by lithium) and mTORdependent (mTOR inhibition by rapamycin) pathways. We provide proof-of-principle for this rational combination treatment
approach in vivo by showing greater protection against neurodegeneration in an HD fly model with TOR inhibition and lithium, or in
HD flies treated with rapamycin and lithium, compared to either pathway alone.
Sarkar S, Ravikumar B, Floto RA and Rubinsztein DC. (2009) Rapamycin and mTOR-independent autophagy
inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death
and Differentiation 16(1):46-56. Access article
Abstract: The formation of intra-neuronal mutant protein aggregates is a characteristic of several human
neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders,
including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant
huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins.
Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all
mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical
mTOR-independent pathway, which has multiple drug targets, involving links between Ca2+–calpain–Gsa and
cAMP–Epac–PLC-e–IP3 signalling. Both pathways enhance the clearance of mutant huntingtin fragments and
attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are
autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the
expression of different autophagy genes is reduced, implicating that its effects are not mediated by
autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy
enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we
describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for
HD and related conditions.
Sarkar S, Korolchuk V, Renna M, Winslow A and Rubinsztein DC. (2009) Methodological considerations for
assessing autophagy modulators: A study with calcium phosphate precipitates. Autophagy 5(3):307-313. Access
Abstract: Autophagy has been implicated in various physiological and disease conditions in recent years. A
number of small molecule modulators have been identified, both as tools and as potential therapeutics. Despite
extensive characterization of autophagy in yeast, mammalian autophagy pathways are not fully understood.
Recently, calcium phosphate precipitates (CPP), which are used to transfect DNA into cells, were reported to
induce autophagy, when assayed up to 6 h after treatment. Because of the widespread use of this reagent, we
attempted to confirm these findings. Consistent with the previous study, we showed that CPP induces
autophagosome synthesis at early time-points, such as 4 h and 6 h. However, at 24 h after treatment, we were
surprised to see that autophagy flux was reduced, due to impaired autophagosome-lysosome fusion. At this time
point, there was an accumulation of autophagy substrates and the formation of abnormally large
autophagosomes. Thus, one may need to consider assaying autophagy modulators at different time-points with a
range of assays in order to understand their effects. Finally, the complex consequences of CPP on autophagy
suggest that it is best avoided as a transfection reagent in studies aiming to analyse autophagy itself, or processes
that are modulated by autophagy, like apoptosis.
Sarkar S, Ravikumar B and Rubinsztein DC. (2009) Autophagic clearance of aggregate-prone proteins
associated with neurodegeneration. Methods in Enzymology Klionsky D.J., editor, Autophagy in disease and
clinical applications, Academic Press 453C:83-110. Access article
Abstract: Autophagy has emerged as a field of rapidly growing interest with implications in several disease
conditions, such as cancer, infectious diseases, and neurodegenerative diseases. Autophagy is a major
degradation pathway for aggregate-prone, intracytosolic proteins causing neurodegenerative disorders, such as
Huntington's disease and forms of Parkinson's disease. Up-regulating autophagy may be a tractable therapeutic
intervention for clearing these disease-causing proteins. The identification of autophagy-enhancing compounds
would be beneficial not only in neurodegenerative diseasesautophagy may act as a protective pathway.
Furthermore, small molecule modulators of autophagy may also be useful in dissecting pathways governing
mammalian autophagy. In this chapter, we highlight assays that can be used for the identification of autophagy
regulators, such as measuring the clearance of mutant aggregate-prone proteins or of autophagic flux with
bafilomycin A1. Using these methods, we recently described several mTOR-independent autophagy-enhancing
compounds that have protective effects in various models of Huntington's disease.
Aguado C*, Sarkar S*, Korolchuk VI, Criado O, Vernia S, Boya P, Sanz P, de Cordoba SR, Knecht E and
Rubinsztein DC. (2010) Laforin, the most common protein mutated in Lafora disease, regulates
autophagy. Human Molecular Genetics 19(14):2867-2876. Access article Supplementary information
Abstract: Lafora disease (LD) is an autosomal recessive, progressive myoclonus epilepsy, which is characterized
by the accumulation of polyglucosan inclusion bodies, called Lafora bodies, in the cytoplasm of cells in the
central nervous system and in many other organs. However, it is unclear at the moment whether Lafora bodies
are the cause of the disease, or whether they are secondary consequences of a primary metabolic alteration.
Here we describe that the major genetic lesion that causes LD, loss-of-function of the protein laforin, impairs
autophagy. This phenomenon is confirmed in cell lines from human patients, mouse embryonic fibroblasts from
laforin knockout mice and in tissues from such mice. Conversely, laforin expression stimulates autophagy.
Laforin regulates autophagy via the mammalian target of rapamycin kinase-dependent pathway. The changes in
autophagy mediated by laforin regulate the accumulation of diverse autophagy substrates and would be
predicted to impact on the Lafora body accumulation and the cell stress seen in this disease that may
eventually contribute to cell death.
Laforin in autophagy: A possible link between carbohydrate and protein in Lafora
Volume 6, Issue 8 2010 Pages 1229 - 1231
Rajat Puri and Subramaniam Ganesh
View affiliations Hide affiliations Rajat Puri Department of Biological Sciences and
Bioengineering; Indian Institute of Technology; Kanpur, India Subramaniam Ganesh
Department of Biological Sciences and Bioengineering; Indian Institute of Technology;
Kanpur, India
The progressive myoclonus epilepsy of Lafora disease (LD) is a fatal form of
neurodegenerative disorder associated with progressive intellectual decline and ataxia in
addition to epilepsy. The disease can be caused by defects in the EPM2A gene encoding
laforin phosphatase or the NHLRC1 gene encoding malin ubiquitin ligase. Laforin and malin
function together as a complex in the ubiquitin-proteasome system, and hence defects
in proteolytic processes are thought to underlie some of the symptoms in LD. One of the
pathological hallmarks of LD is the presence of cytoplasmic polyglucosan inclusions, the
Lafora bodies. While Lafora bodies are known as a lesser branched form of glycogen with
high phosphate content, a physiological basis for their genesis in the cytoplasm was not
well understood. Recently it was shown in a mouse model for LD that loss of laforin
inhibits autophagosome formation, suggesting that laforin plays a critical role in
autophagosome biogenesis. The polyglucosan inclusions could be one of the substrates of
autophagy, and loss of laforin might affect their sequestration into autophagosomes
leading to their aggregation as Lafora bodies. Thus, laforin’s proposed role in autophagy
suggests a possible link between the proteolytic system and the polyglucosan inclusions in
Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells
María Salazar, Arkaitz Carracedo, Íñigo J. Salanueva, Sonia Hernández-Tiedra, Mar Lorente, Ainara Egia, Patricia
Vázquez, Cristina Blázquez, Sofía Torres, Stephane García, Jonathan Nowak, Gian María Fimia, Mauro Piacentini,
Francesco Cecconi, Pier Paolo Pandolfi, Luis González-Feria, Juan L. Iovanna, Manuel Guzmán, Patricia Boya, Guillermo
J Clin Invest. 2009; 119(5):1359–1372
THC induces autophagy via ER stress–evoked p8 and TRB3 upregulation.
(A and B) Effect of ISP-1 (1 μM) on THC-induced eIF2α phosphorylation (A; 3 h; n = 3)
and LC3 immunostaining (B, left panels; 18 h; percentage of cells with LC3 dots
relative to the total cell number, mean ± SD; n = 3; scale bar: 20 μm) in U87MG cells.
sip8, p8-selective siRNA; siTRB3, TRB3-selective siRNA. (C) Effect of THC on p8, ATF4,
CHOP, and TRB3 mRNA levels of eIF2α WT and eIF2α S51A MEFs as determined by
real-time quantitative PCR (8 h; n = 3). Numbers indicate the mean fold increase ±
SD relative to vehicle-treated eIF2α WT MEFs. (D) Top: Analysis of p8 and TRB3
mRNA levels. Results from a representative RT-PCR experiment are shown. The
numbers indicate gene expression levels as determined by real-time quantitative
PCR (mean fold change ± SD relative to siC-transfected cells; n = 5). Bottom: Effect of
THC on LC3 immunostaining (green) of U87MG cells transfected with siC, sip8, or
siTRB3 (18 h; n = 4). The percentage of cells with LC3 dots relative to cells
cotransfected with a red fluorescent control siRNA is shown in each panel (mean ±
SD). Scale bar: 20 μm. (E) Effect of THC on LC3 lipidation in U87MG cells transfected
with siC, sip8, or siTRB3 (18 h; n = 6). (F) Effect of THC on LC3 lipidation (top; 18 h; n
= 5) and LC3 immunostaining (bottom; 18 h; percentage of cells with LC3 dots
relative to the total cell number, mean ± SD; n = 4; scale bar: 40 μm) in p8+/+ or p8–/–
MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (B), eIF2α WT
(C), or p8+/+ (F) cells and compared with siC-transfected, THC-treated U87MG cells
The ubiquitin proteasome system in neuropathology
by Lehman, Norman L.
Acta Neuropathologica Vol. 118 Issue 3: 2009-07-27
• Cbl family E3 ligases and UBE3A E3 ligase are single proteins that associate with
an E2 Ubc and the target substrate. The SCF and APC/C E3 ubiquitin ligases are
multimeric complexes. The APC/C has only two known adapter subunits, Cdc20
and Cdh1 (not to be confused with E-cadherin, which is sometimes also
referred to as Cdh1). The SCF can associate with several different substrate
adapter proteins known as F-box proteins
• The functions of neddylation are less understood; however, neddylation of
cullin proteins, essential subunits of certain E3 ubiquitin ligase complexes,
appears to be required for normal ligase function (Fig. 2 ). Both HECT and RING
types may possess E3 ligase function as single proteins, for example the HECTtype ligase UBE3A and the RING-type ligase Cbl (Fig. 2 ). Examples include the
Skp/cullin/F-box (SCF) and anaphase promoting complex/cyclosome (APC/C) E3
ubiquitin ligases that exhibit specificity for different substrates depending on
the adaptor protein bound to the complex (Fig. 2 ). Familial juvenile onset
Parkinson’s disease is caused by a defect in an SCF E3 ubiquitin ligase
component known as parkin (PARK2) (Fig. 2 ). pVHL protein is the substrate
recognition component of the ubiquitin ligase that ubiquitinates HIF-1α [ 17 ]
(Fig. 2 ). The APC/C is associated with two different activator subunits that
impart general substrate specificity, Cdh1 and Cdc20 (Fig. 2 ).
Gene product and function
Parkinson disease
Autosomal dominant (early onset)α-Synuclein (SNCA) (PARK1), aggregates in Lewy bodies
Autosomal dominant (late onset)
Leucine-rich repeat kinase 2 (LRRK2) (PARK8), a CHIP ubiquitin ligase
substrate, contains a Roc domain as found in SCF ligases
Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) (PARK5), a DUB, acts
Autosomal dominant (late onset) as an E3 ligase when dimerized, polymorphisms linked to rare forms of
familial disease
Autosomal recessive (juvenile
Autosomal recessive (early
Autosomal recessive (early
Parkin (PARK2), a subunit of a SCF E3 ubiquitin ligase
PINK1 (PARK6), promotes parkin ubiquitin ligase activity
DJ-1 (PARK7) chaperone, promotes parkin ubiquitin ligase activity
Spinocerebellar ataxias
Ataxin-1, a UBE3A E3 ligase, mutation blocks its ubiquitination and
association with the ubiquitin receptor A1Up and the DUB enzyme
Ataxin-2, associates with c-Cbl E3 ligase and is involved in membrane
protein endocytosis, and is a parkin E3 ligase substrate
Ataxin-3, a deubiquitinating enzyme (DUB)
Prion diseases
Prions may block normal function of the proteasome, HECT2D E3
ubiquitin ligase haplotypes are associated with vCJD and Kuru
Autosomal recessive ALS
ALS2, an endosomal membrane associated protein involved in
endosome membrane fusion and trafficking, mutation decreases ALS2
protein stability
Angelman syndrome
Loss or mutation of UBE3A E3 ligase at Angelman/Prader–Willi locus
Rett syndrome
Decreased UBE3A E3 ligase due to MECP2 mutations
Copy number alterations of UBE3A and other UPS genes
Giant axon neuropathy
Mutation of gigaxonin, an E3 ubiquitin ligase
Mutation of valosin-containing protein (VCP), involved in ubiquitinmediated processing of membrane and cytosolic proteins.
Sporadic IBM
VCP and ubiquitin are found in inclusion bodies.
von Hippel–Lindau disease
pVHL, substrate-binding subunit of ubiquitin ligase targeting HIF1-α
Overexpression of several signaling pathway genes that are
ubiquitinated by the SCF: c-myc, β-catenin, Gli, stabilizing mutations of
Gli and β-catenin prevent their ubiquitin-dependent proteolysis
Stabilizing β-catenin mutations preventing its ubiquitin-dependent
Misregulation and mutation of cell cycle control proteins regulated by
the UPS: CDKs, CDK inhibitors, p53, altered expression of APC/C E3
ubiquitin ligase regulators Emi1 and RASSF1A
3.3 Cardiac myopathies
Cardiac myocytes are terminally differentiated cells, so correct functioning of the autophagic pathway is
essential for maintenance of myocyte homeostasis. Unsurprisingly, therefore, autophagic deficiencies have
been associated with a variety of cardiac pathologies (reviewed in 40).
One such example which has helped understanding of autophagy in the cardiac system is Danon disease.
Deficiency of the autophagy gene LAMP-2 was shown to cause this disease. Patients have an increase of
autophagic vacuoles leading to cardiomyopathy (15), a phenotype seen in mice with inactivation of the
LAMP-2 protein (16). Whether the increase in autophagic vacuoles is due to up-regulation of autophagosome
formation or due to a decrease in autophagosome – lysosome fusion is currently unclear (reviewed in 36).
Autophagic vacuoles have been described in the heart tissue of patients with idiopathic dilated
cardiomyopathy (41). Studies looking at models of ischaemic heart disease have also shown a role of
autophagy. In chronically stunned porcine myocardium, autophagic vacuoles were seen after three episodes
of ischaemia/reperfusion (I/R) and were maximal after 6 episodes, associated with increasing expression
levels of autophagy-related molecules like Beclin-1. This correlated with declining levels of apoptosis
between the 3-6 I/R episodes, suggesting that the autophagic response seen could be the result of a
cardioprotective effect against I/R injury (42).
Recently, studies investigating the role of autophagy in increasing the level of myocyte loss studied the role
of Beclin-1 following a single I/R episode. These experiments showed that levels of Beclin-1 are upregulated following a single I/R episode and that this up-regulation correlates to increased autophagy.
Indeed, the use of the cardioprotective agent Urocortin reduced levels of Beclin-1 and concomitantly
autophagic levels. Suggesting that in order to provide protection from myocyte loss following I/R, therapies
which target the autophagic response alongside the apoptotic and necrotic response should be investigated
Clearly, the most fundamental question for autophagy is whether its role is harmful or protective and this
remains to be answered. Indeed it would appear that autophagy could have a role during disease
progression, moving from a cell protective mechanism to one contributing to cell pathology. The
generation of new reagents, especially, antibodies with which to study the autophagic pathway will lead to
increased understanding of the regulation of this critical homeostatic pathway. Future cellular and clinical
studies will be identified which will determine whether this pathway can be manipulated to provide
therapeutic potential for cardiac disease.
Autophagy in heart disease
By Joseph Hill *
Autophagy poster and related antibodies
Autophagy is an evolutionarily conserved, lysosomal
pathway of engulfment, degradation and recycling of
cellular contents, including long-lived proteins and
organelles. This process promotes cell survival and
maintains cellular homeostasis, under both resting and
stress conditions. In addition, it plays a critical role in a
number of clinical disorders, including heart disease.
Autophagy is a complex process occurring in a stepwise
manner of several stages; nucleation, expansion, and
Williams A*, Sarkar S*, Cuddon P*, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D, Goldsmith
P, O’Kane CJ, Floto RA and Rubinsztein DC. (2008) Novel targets for Huntington's disease in an mTORindependent autophagy pathway. Nature Chemical Biology 4(5):295-305. Access article Supplementary
information Chemical compounds *Equal contribution
Abstract: Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases
such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance
of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to
identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel
opener minoxidil, and the Gi signaling activator clonidine induce autophagy. These drugs revealed a cyclical
mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain
activity, which completes the cycle by cleaving and activating Gsa, which regulates cAMP levels. This pathway
has numerous potential points where autophagy can be induced, and we provide proof of principle for
therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also
suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding
clearance of aggregate-prone proteins.
• Autophagy-Targeted Therapies
• Autophagy is an important topic in modern cell biology. It is a dynamic process
for the maintenance of cellular and metabolic homeostasis. Scientists’ interest in
autophagy-related mechanisms involved in the pathogenesis of cancer and
neurodegenerative diseases (Alzheimer’s disease, Huntington’s disease) and an
array of other disorders is rapidly increasing. Reflecting this interest, the number
of publications related to autophagy is growing exponentially. Prous Institute has
initiated a research program focused on the mechanisms driving autophagy to
discover new targets for the design of new small-molecule modulators of
autophagy as therapeutic agents.
• Several studies have shown that links may exist between autophagy and
apoptosis, a subtype of programmed cell death. For this reason our Institute has
a special interest in considering apoptosis in its research program
Autophagy eliminates intracellular microorganisms
• a | Group A Streptococci captured within an autophagosome. Image kindly
provided by Tamotsu Yoshimori, Osaka University, Japan. b |
Mycobacterium bovis bacillus Calmette–Guérin (BCG) present in a
mycobacterial autophagosome (MAP) that is fusing with a multivesicular
body (MVB). Image reproduced with permission from Ref. 42 © (2004) Cell
Press. c | Herpes simplex virus type I (HSV-1) virion(s) in the process of
being surrounded by an isolation membrane (left panel), engulfed inside
an autophagosome (middle panel) or degraded inside an autolysosome
(right panel). Image reproduced with permission from Ref. 77 © (2006)
Landes Bioscience.
Inhibition of host autophagy by virus
Autophagy is an evolutionary conserved mechanism for the sequestration and subsequent
lysosomal degradation of discrete intracellular portions of eukaryotic cells, facilitating the
removal of materials not degraded through the ubiquitin-proteasome pathway. In addition,
autophagy plays important roles in innate and adaptive immune responses to pathogens.
Several viruses have develop ways to subvert the pathway for their own benefit in order to
avoid the immune response or to increase their viral replication. Beclin-1 is an essential
autophagy protein and constitutes the major target for manipulation of autophagy by
viruses. For example, HHV-1 ICP34.5 binds to Beclin-1 and inhibits autophagosome
formation. HIV and Influenza A virus use the same mechanism through Nef and M2
respectively, that also interact with and inhibit host Beclin-1
Activation of host autophagy by virus
Autophagy is an evolutionary conserved mechanism for the sequestration and subsequent lysosomal degradation of discrete
intracellular portions of eukaryotic cells, facilitating the removal of materials not degraded through the ubiquitin-proteasome
pathway. In addition, autophagy plays important roles in innate and adaptive immune responses to pathogens
Several viruses are able to activate host autophagy as a cellular survival mechanism. Indeed, viruses can activate programmed
cell death during infection that prevent them from spreading to healthy tissue. By activating autophagy, viruses delay or inhibit
apoptosis. For example, SV40 ST antigen protects cancer cells under glucose deprivation by triggering autophagy. KSHV Rta is
able to enhance the autophagic process in order to facilitate viral lytic replication.
Application of autophagy modulation to cancer therapy.
From the following article:
Role of autophagy in cancer
Robin Mathew, Vassiliki Karantza-Wadsworth & Eileen White
Nature Reviews Cancer 7, 961-967 (December 2007)
• a | In apoptosis-defective tumours that are reliant on autophagy to
survive metabolic stress, autophagy inhibitors can be used to induce acute
necrotic cell death that may be facilitated by proteasome inhibition,
enabling tumour eradication. b | In the adjuvant setting, and after
elimination of a large proportion of the tumour by radiation and
chemotherapy, the remaining cells can reside in a disrupted and stressed
environment, susceptible to inhibition of the autophagy survival
mechanism. Tumour cells in the process of metastasis can be similarly
vulnerable. c | Autophagy stimulators may be therapeutically useful to
either promote autophagic cell death or to prevent the damaging effects
of autophagy deficiency and mismanagement of metabolic stress leading
to DNA damage and tumour progression. By limiting protein, organelle
and ultimately DNA damage, autophagy stimulators may suppress tumour
progression. In human breast, ovarian and prostate cancers, where allelic
loss of BECN1 occurs with high frequency, correction of the autophagy
deficiency with autophagy stimulators may delay tumour progression by
reducing the rate at which tumour-promoting mutations accumulate.
Role of apoptosis and autophagy in
a | Tumour-initiating mutational events such as oncogene activation promote cell
proliferation, but also apoptosis, which limits tumour growth. Following the
acquisition of defects in apoptosis, tumour proliferation is sustained in the
absence of apoptotic cell death. b | Tumour growth is initially limited by the
absence of a blood supply, which can trigger autophagy-mediated survival in the
most metabolically stressed tumour regions, commonly the hypoxic center19, 20, 23.
The eventual recruitment of a blood supply cures the tumour of hypoxia and
metabolic stress, and the tumour cells formerly surviving through autophagy can
emerge to contribute to tumour growth30. c | In tumours formed by cells with
defects in both apoptosis and autophagy, necrotic cell death is stimulated in
metabolically stressed tumour regions and this necrosis is associated with the
activation of an inflammatory response, DNA damage and tumour progression19, 20,
23. Analogous to a wound-healing response, chronic necrosis and inflammation can
stimulate angiogenesis and tumour growth24, 25, 26.
The molecular regulation of autophagy.
From the following article:
The role of autophagy in cancer development and response to therapy
Yasuko Kondo, Takao Kanzawa, Raymond Sawaya & Seiji Kondo
Nature Reviews Cancer 5, 726-734 (September 2005)
In the presence of growth factors, growth factor receptor signalling activates class I
phosphatidylinositol 3-phosphate kinase (PI3K) at the plasma membrane to keep
cells from undergoing autophagy42. PI3K activates the downstream target AKT,
leading to activation of mammalian target of rapamycin (mTOR), which results in
inhibition of autophagy. p70S6 kinase (p70S6K) might be a good candidate for the
control of autophagy downstream of mTOR. Overexpression of the phosphatase
and tensin homologue (PTEN) gene, by an inducible promoter, antagonizes class I
PI3K47 to induce autophagy. RAS has a dual effect on autophagy — when it
activates class I PI3K25, autophagy is inhibited, but when it selectively activates the
RAF1–mitogen-activated protein kinase kinase (MEK)–extracellular signalregulated kinase (ERK) cascade, autophagy is stimulated55. Rapamycin, an inhibitor
of mTOR, induces autophagy39. A complex of class III PI3K and beclin 1 (BECN1) at
the trans-Golgi network acts to induce autophagy49. This pathway is inhibited by 3methyladenine (3-MA)42. Downregulation of BCL2, or upregulation of BCL2–
adenovirus E1B 19-kD-interacting protein 3 (BNIP3) or HSPIN1 at the mitochondria,
also induces autophagy, indicating that BCL2 protects against autophagy52, BNIP3
(Refs 36,53,57) and HSPIN1 (Ref. 54) trigger autophagy. Autophagy is also induced
by the cell death-associated protein kinase (DAPK) and the death-associated
related protein kinase 1 (DRP1)51.
Potential strategies for treating cancer by
manipulating the autophagic process
• a | Cancer cells that have defects in the autophagic
pathway might be treated by replacing the autophagic
signal through expression of beclin 1 (BECN1) or the
phosphatase and tensin homologue (PTEN) tumour
suppressor, resulting in induction of autophagy and cell
death, or inhibition of proliferation. b | Cancer cells that
are capable of undergoing autophagy in response to
anticancer therapies might be treated with autophagy
inducers, such as rapamycin, to promote autophagyinduced cell death. c | Cancer cells that undergo autophagy,
to protect themselves from the effects of anticancer
therapies, might be treated with autophagy inhibitors, such
as bafilomycin A1 or short interfering RNAs specific for the
autophagy-related genes, to induce apoptosis.
A Matter of Life or Death (or Both): Understanding
Autophagy in Cancer
A, in the presence of nutrients (glucose, amino acids, and growth factors), protein synthesis is stimulated
and autophagy is inhibited. This is mediated through activation of mTOR (via activation of PI3K and Akt and
inactivation of the tuberous sclerosis complex TSC1 and TSC2). mTOR phosphorylates S6 kinase and
increases the translation of mRNAs that encode ribosomal and other proteins involved in translation. This
initiates translation by phosphorylating 4EBP1, an inhibitor of initiation, causing its disassociation from
eIF4E. Active eIF4 promotes cell proliferation by increasing translation of cyclin D1, c-Myc, and vascular
endothelial growth factor. mTOR and S6 kinase also release the cellular check on peptide elongation by
phosphorylating and inactivating eEF-2 kinase. eEF-2 kinase phosphorylates eEF-2, a 100-kDa protein that
mediates the translocation step in peptide-chain elongation by inducing the transfer of peptidyl-tRNA from
the ribosomal A to P site. Phosphorylation of eEF-2 at Thr56 by eEF-2 kinase decreases the affinity of the
elongation factor for ribosomes and terminates elongation. Activation of TOR in yeast inhibits induction of
autophagy via phosphorylation of the APG1-APG-13 complex, a process inhibited by rapamycin in both
yeast and mammalian cells. B, formation of the autophagosome. This process requires the coordinated
efforts of a series of gene products that respond to nutrient deprivation (autophagy regulatory complex),
lipid kinase signaling molecules that participate in the formation of vesicles, ubiquitin-like proteins that
complete vesicle formation, and a complex of proteins that mediate the disassembly of the
autophagosome. The initial step is the envelopment of cytoplasmic materials into a phagophore or isolating
membrane. This leads to the sequestration of cytoplasm into the autophagosome, which are characterized
by a double membrane decorated with microtubule-associated protein 1 light chain 3 (LC3). The fusion of
autophagosomes with lysosomes to for the autolysosome leads to the acidification and degradation of
cytoplasmic components for recycling of amino acids and fatty acids for energy production.
Clin Cancer Res 2006;12:1961-1965. William N. Hait, Shengkan Jin and Jin-Ming Yang
Autophagy in Cancer
A Matter of Life or Death (or Both): Understanding
The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human
ovarian cancer cells
Zhen Lu, Robert Z. Luo, Yiling Lu, Xuhui Zhang, Qinghua Yu, Shilpi Khare, Seiji Kondo, Yasuko
Kondo, Yinhua Yu, Gordon B. Mills, Warren S.-L. Liao, Robert C. Bast
J Clin Invest. 2008; 118(12):3917–3929
• ARHI is required for autophagy.
• (A–J) ARHI and rapamycin (RM) induce autophagy in ovarian cancer cells. (A–D)
SKOv3-ARHI cells were transfected with GFP-LC3 and treated with or without DOX
to induce ARHI expression or with or without rapamycin to inhibit mTOR activity.
Scale bar: 1 μm. (E–J) ES2 and OC316 cells were not transfected or were
transfected with ARHI expression vector and ARHI siRNA or control siRNA for 24
hours before they were transfected with GFP-LC3. Cells were treated with 50 nM
rapamycin at the time of siRNA transfection and examined for autophagy by
fluorescence microscopy 48 hours later. Scale bar: 1 μm. (K) ARHI expression is
necessary for rapamycin-induced autophagy in ovarian cancer cells. ES2 and OC316
ovarian cancer cells were not transfected or were transfected with ARHI or control
siRNA for 48 hours. Expression of ARHI and the control GAPDH were examined by
RT-PCR. (L–U) NOSE cells undergo spontaneous autophagy. (L–O) Two NOSE cell
lines were transfected with GFP-LC3 and treated with or without rapamycin. Scale
bar: 1 μm. (P–U) GFP-LC3 plasmid was transfected into OSE106 cells alone or was
cotransfected with ARHI siRNA or control siRNA. Transfected cells were treated
with or without rapamycin. Scale bar: 1 μm.
Altered capacity for apoptosis and autophagy can dictate cell fate in response to metabolic stress.
a | Apoptosis is a common response to metabolic stress in which cells activate
caspases and die efficiently. For immortal epithelial cells, metabolic stress triggers
apoptosis within 24 to 48 hours19, 20, 23. Execution of apoptosis occurs in less than
an hour and cell viability loss is five to six orders of magnitude19. b | Defective
autophagy (through loss of BECN1 or ATG5, for example) can increase apoptotic
cell death in some cells in response to metabolic stress56. In human mammary cells
in 3D culture in vitro, this accelerated apoptosis manifests as increased lumen
formation in mammary acini20. Therefore, preservation of cell metabolism through
autophagy might increase the threshold for apoptosis activation. c | In cells with
defects in apoptosis, survival in metabolic stress is dependent on autophagy and is
prolonged for weeks19, 20, 22, 23. During the maintenance phase, activities such as
cell division and motility are sustained. Prolonged starvation and progressive
autophagy causes cells to gradually shrink in size but restoration of nutrients
allows recovery. In the preservation phase, cell division and motility decrease,
presumably as a bioenergenic conservation effort, creating the minimal cell that is
capable of recovery (MCCR). Eventually, restoration of nutrients fails to allow
recovery. In this way, autophagy can be viewed as an interruptible path to cell
death. d | Cells with defects in apoptosis and autophagy fail to tolerate metabolic
stress, undergo metabolic catastrophe and die by necrosis36.
Molecular events in autophagy.
From the following article:
Unveiling the roles of autophagy in innate and adaptive immunity
Beth Levine & Vojo Deretic
Nature Reviews Immunology 7, 767-777 (October 2007)
Autophagy is regulated by a set of autophagy-related proteins (ATG proteins). In the absence of amino
acids or in response to other stimuli, ATG1 and a complex of the class III PI3K (phosphoinositide 3kinase) VPS34 and beclin 1 lead to the activation of downstream ATG factors that are involved in the
initiation (a), elongation (b) and maturation (c) of autophagy. a | In amino-acid-rich conditions, VPS34
contributes to mTOR (mammalian target of rapamycin) activation and inhibition of ATG1 and autophagy.
The sources of membrane for autophagosome initiation and elongation may include those containing
the only known membrane integral ATG protein ATG9, redistributing between a resting location to
autophagosomes in an ATG1- and PI3K-dependent manner. ATG9 redistribution may depend on ATG18,
which binds phosphatidylinositol-3-phosphate (PtdIns3P). b | The elongation and shape of the
autophagosome are controlled by two protein (and lipid) conjugation systems, similar to the
ubiquitylation systems: the ATG12 and LC3 (also known as ATG8)–phosphatidylethanolamine (PE)
conjugation pathways, which include E1-activating and E2-conjugating enzymes. ATG12 is initially
conjugated to ATG7 (an E1-activating enzyme) and then is transferred to the E2-like conjugating
enzyme ATG10. This intermediate presents ATG12 for conjugation to an ATG5 lysine residue. The ATG5–
ATG12 conjugate, stabilized non-covalently by ATG16, triggers oligomerization on the outside
membrane of the growing autophagosome, and enhances LC3 carboxy-terminal lipidation through the
LC3 conjugation system. Upon autophagosome closure, ATG5–ATG12–ATG16 and LC3 (delipidated by
ATG4) are recycled. c | LC3 associated with the lumenal membrane remains trapped in the
autophagosome and is degraded during maturation into the autolysosome, which involves fusion of
autophagosomes with late endosomes, including endosomal multivesicular bodies and lysosomal
organelles, and dissolution of the internal membrane. VPS34 has a role in the formation of late
endosomal multivesicular bodies and lysosomal organelles contributing to the maturation stages of
Functions of
autophagy in
innate and
infection with
• a | Intracellular pathogens (bacteria, parasites and viruses) that are
either free inside the cytosol, inside phagosomes or inside pathogencontaining vacuoles are surrounded by isolation membranes, engulfed
into autophagosomes, which fuse with lysosomes, and then degraded
inside autolysosomes. b | Viral nucleic acids are transferred by
autophagy from the cytoplasm to intracellular compartments
containing Toll-like receptor 7 (TLR7), which signals the induction of
type I interferon (IFN) production. c | Viral antigens (and potentially
other endogenously synthesized microbial antigens and self antigens)
are engulfed into autophagosomes that fuse with MHC-class-IIcontaining late endosomes (MIICs), and then loaded onto MHC class II
molecules for presentation to CD4+ T cells. Cytosolic antigens that
contain a KFERQ recognition motif may also be directly imported into
MIICs by chaperone-mediated autophagy. CLIP, class II-associated
invariant chain peptide.
Autophagy Regulators
Table 1: Selected small-molecule inducers of autophagy.
Cell Line
Inhibits (mTOR)
Activates autophagy
HeLa, HepG2, Jurkat
Inhibits (mTOR)
Activates autophagy
ATP-competitive inhibitor of mTOR
Activates autophagy
Activates autophagy
Unknown, mTOR-independent
Activates autophagy
Bafilomycin A1
Inhibits Vacuolar-ATPase
Inhibits lysosome function
Alkalinizes Lysosomal pH
Inhibits lysosome function
Abolishes the inhibitory effect of PI3K
Activates autophagy & inhibits lysosome
HeLa, HepG2, Jurkat
Alkalinizes Lysosomal pH
Activates autophagy
Alkalinizes Lysosomal pH
Activates autophagy
Activates autophagy
Activates autophagy
Selective proteasome inhibitor
Activates autophagy
HeLa, Jurkat
Alkalinizes Lysosomal pH
Inhibits lysosome function
How to Live Long and Prosper: Autophagy, Mitochondria, and Aging
FIGURE 1. Schematic diagram of selective and nonselective autophagy
Three fundamentally different modes of autophagy are macroautophagy, microautophagy, and chaperonemediated autophagy. Depending on the specificity of the cargos, autophagy can be a selective or a
nonselective process. During nonselective autophagy, a portion of the cytoplasm is sequestered into a doublemembrane autophagosome, which then fuses with the lysosome/vacuole. In contrast, the specific
degradation of peroxisomes in certain conditions can be achieved by either a macro- or microautophagy-like
mode, termed macropexophagy and micropexophagy, respectively. Piecemeal microautophagy of the nucleus
allows the degradation of a portion of the nucleus. The specific degradation of mitochondria, termed
mitophagy also takes place. A biosynthetic cytoplasm to vacuole targeting (Cvt) pathway in yeast also shares
similar morphological features. Note that this schematic illustrates aspects of autophagy in both yeast cells
and higher eukaryotes.
Physiology, Vol. 23, No. 5, 248-262, October 2008
Int. Union Physiol. Sci./Am. Physiol. Soc.
REVIEW :How to Live Long and Prosper: Autophagy, Mitochondria, and Aging
Wei-Lien Yen and Daniel J. Klionsky
Schematic diagram of selective and
nonselective autophagy
Three fundamentally different modes of
autophagy are macroautophagy,
microautophagy, and chaperone-mediated
autophagy. Depending on the specificity of
the cargos, autophagy can be a selective or
a nonselective process. During nonselective
autophagy, a portion of the cytoplasm is
sequestered into a double-membrane
autophagosome, which then fuses with the
lysosome/vacuole. In contrast, the specific
degradation of peroxisomes in certain
conditions can be achieved by either a
macro- or microautophagy-like mode,
termed macropexophagy and
micropexophagy, respectively. Piecemeal
microautophagy of the nucleus allows the
degradation of a portion of the nucleus. The
specific degradation of mitochondria,
termed mitophagy also takes place. A
biosynthetic cytoplasm to vacuole targeting
(Cvt) pathway in yeast also shares similar
morphological features. Note that this
schematic illustrates aspects of autophagy in
both yeast cells and higher eukaryotes.
Control of autophagy. Autophagy is a major housekeeping pathway and under the control of many different signaling cascades.
Mammalian Target of rapamycin (mTOR) plays a central role in the regulation of autophagic activity as it integrates signaling from
different sensors of cellular homeostasis. When mTOR is active in yeast it keeps an important ULK1 binding partner (ATG13)
phosphorylated, thus inhibiting the induction of autophagy. While signals indicating abundant nutritional and trophic support
activate mTOR (and deactivate autophagy), signals of starvation or other stressors inhibit mTOR (and activate autophagy).
Autophagy can be directly stimulated by intracellular debris (such as unfolded proteins and damaged organelles) or by indicators
of an overwhelmed ubiquitin-proteasome system (UPS). Also certain pathogens activate autophagy. Autophagy can be directly
inhibited by genetic ablation of important Atg genes, inhibitors of the class III PI3K-complex (WM, 3-MA), high nutrient levels,
and inositol signaling. More recently screenings of small compound libraries have yielded inducers and inhibitors of autophagy,
both mTOR dependent and independent. And last, transcriptional regulators, such as p53, eIF2α, E2F4, or FOXO3 regulate
autophagy by controlling the expression levels of many Atg genes. For further details. Jaeger and Wyss-Coray Molecular
Neurodegeneration 2009 4:16
The molecular machinery of autophagy: unanswered questions
Regulation of induction and vesicle nucleation. The regulation of autophagy
has been characterized in studies of yeast and mammalian cells. (A) In yeast,
a class III PI 3-K is required for autophagic activity and may function at the
pre-autophagosomal membrane. A putative complex consisting of Atg1
kinase and several other proteins characterized as being required primarily
for autophagy (in purple) or the Cvt pathway (in green) may be a
downstream effector of Tor kinase to regulate the type of pathway that
operates, depending on the nutritional conditions or other signals.
Autophagy in yeast is primarily a starvation response; Tor, along with other
regulatory components not shown (including PKA), responds to nutrient
levels. In nutrient-rich conditions, Atg1 and Atg13 are more highly
phosphorylated and have a lower affinity for each other; during starvation
the two proteins are partially dephosphorylated. The PI 3-K complex I,
consisting of Vps15, Vps34, Atg6/Vps30 and Atg14, is required for both the
Cvt pathway and autophagy. (B) In mammalian cells, a class I PI 3-K is
stimulated in response to the binding of a ligand to a receptor such as the
insulin receptor (InR). PtdIns(3,4)P2 and PtdIns(3,4,5)P3 generated at the
plasma membrane allow the binding and activation of 3-phosphoinositidedependent protein kinase 1 (PDK1) and Akt/PKB, whereas PTEN antagonizes
this pathway through its 3′-phosphoinositide phosphatase activity. Akt
inhibits the GTPase-activating protein complex TSC1-TSC2, resulting in the
stabilization of RhebGTP, which activates Tor, resulting in the inhibition of
autophagy. Both Tor and PDK1 stimulate p70S6 kinase (p70S6k). The
downregulation of p70S6k activity in starvation conditions (when Tor is
inhibited) might prevent excessive autophagy (Scott et al., 2004). It is also
possible that p70S6k indirectly inhibits Tor by interfering with activation of
the class I PI 3-K, as suggested by studies in mammalian cells (Um et al.,
2004). In nutrient-rich conditions, activation of p70S6k should inhibit PI 3-K,
allowing a low level of autophagy for homeostatic purposes, whereas in
starvation conditions the eventual inactivation of p70S6k should allow
activation of PI 3-K to prevent excessive autophagy. The class III PI 3-K serves
a stimulatory role possibly similar to that of the yeast enzyme complex.
The regulation of autophagy – unanswered questions
Dynamics of Atg1 complexes upon autophagy induction in different eukaryotes. (A) In yeast,
under nutrient-rich conditions, the active TOR complex 1 (TORC1) hyperphosphorylates
Atg13 (Kamada et al., 2010). This prevents the association of Atg1 with Atg13, which is
bound to Atg17, Atg31 and Atg29, leading to inhibition of autophagy induction. Under
starvation conditions when TORC1 is inactivated, Atg13 is no longer phosphorylated by
TORC1, whereas Atg1 is autophosphorylated, leading to the association of Atg1 with the
complex between Atg13, Atg17, Atg31 and Atg29, and subsequent autophagy induction
(Cebollero and Reggiori, 2009; Chang and Neufeld, 2010; Kamada et al., 2010; Nakatogawa
et al., 2009). (B) In contrast to yeast, mammalian ULK (ULK1 or ULK2, the homologs of yeast
Atg1) forms a stable complex with mammalian Atg13, FIP200 (a putative counterpart of
yeast Atg17) and Atg101 (an Atg13-binding protein), irrespective of TORC1 activation. Under
nutrient-rich conditions, the active TORC1 associates with the ULK complex (ULK1 (or ULK2)–
Atg13–FIP200-Atg101), phosphorylates ULK1 (or ULK2) and hyperphosphorylates Atg13,
which inhibits the kinase activity of ULK1 (or ULK2) and thus blocks autophagy induction.
Under starvation conditions when TORC1 is inactivated, TORC1 dissociates from the ULK
complex, preventing phosphorylation of Atg13 and ULK1 (or ULK2) by TORC1 and leading to
autophagy induction, whereas ULK1 (or ULK2) still phosphorylates Atg13 and itself, and
hyperphosphorylates FIP200 (Chang and Neufeld, 2010; Mizushima, 2010; Yang and Klionsky,
2010). (C) Similar to the situation in mammals, in Drosophila Atg1 forms a complex with
Atg13 irrespective of TORC1 activation (Chang and Neufeld, 2010). Under nutrient-rich
conditions, the active TORC1 phosphorylates Atg13 and hyperphosphorylates Atg1, leading
to the inhibition of autophagy induction. Under starvation conditions, when TORC1 is
inactivated, Atg1 and Atg13 are no longer phosphorylated by TORC1, whereas Atg1 still
phosphorylates itself and hyperphosphorylates Atg13, leading to autophagy induction.
Figure modified from Chang and Neufeld (Chang and Neufeld, 2010) with permission.
Linking different lifespan-prolonging treatments to
• Summary of the genetic and pharmacological manipulations of autophagy
that cause lifespan extension. Pharmacological treatment with spermidine,
resveratrol or rapamycin, caloric restriction, depletion of p53 or
overexpression of sirtuin 1 prolong lif…
Can autophagy promote longevity?
Frank Madeo,1 Nektarios Tavernarakis2 & Guido Kroemer3
Journal name: Nature Cell Biology Volume: 12, Pages: 842–
846 (2010)
• Abstract
• Organismal lifespan can be extended by genetic
manipulation of cellular processes such as histone
acetylation, the insulin/IGF-1 (insulin-like growth factor 1)
pathway or the p53 system. Longevity-promoting regimens,
including caloric restriction and inhibition of TOR with
rapamycin, resveratrol or the natural polyamine spermidine,
have been associated with autophagy (a cytoprotective
self-digestive process) and in some cases were reported to
require autophagy for their effects. We summarize recent
developments that outline these links and hypothesize that
clearing cellular damage by autophagy is a common
denominator of many lifespan-extending manipulations.
Linking autophagy-mediated lifespan extension
to cytoprotection.close
• Putative mechanisms linking autophagy to the inhibition of cell death
(either apoptosis or necrosis) and to the induction of longevity.

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