TEÓRICO AUTOFAGIA
REGULATION OF MAMMALIAN AUTOPHAGY IN PHYSIOLOGY AND PATHOPHYSIOLOGY
(Macro)autophagy is a bulk degradation
process that mediates the clearance of
long-lived proteins and organelles.
Autophagy is initiated by double-
membraned structures, which engulf
portions of cytoplasm. The resulting
autophagosomes ultimately fuse with
lysosomes, where their contents are
degraded. Although the
term autophagy was first used in 1963, the
field has witnessed dramatic growth in the
last 5 years, partly as a consequence of the
discovery of key components of its cellular
machinery. In this review we focus on
mammalian autophagy, and we give an
overview of the understanding of its
machinery and the signaling cascades that
regulate it. As recent studies have also
shown that autophagy is critical in a range
of normal human physiological processes,
and defective autophagy is associated with
diverse diseases, including
neurodegeneration, lysosomal storage
diseases, cancers, and Crohn's disease, we
discuss the roles of autophagy in health
and disease, while trying to critically
evaluate if the coincidence between
autophagy and these conditions is causal
or an epiphenomenon. Finally, we consider
the possibility of autophagy upregulation
as a therapeutic approach for various
conditions.
I. INTRODUCTION
The word autophagy is derived from the
Greek roots “auto” (self) and “phagy”
(eating) and broadly refers to the cellular
catabolic processes in which cytoplasmic
target material is transported to lysosomes
for degradation. Christian de Duve, who
was awarded the Nobel Prize for his work
on lysosomes, first used the
1
term autophagy in 1963. He used the word
to describe the phenomenon associated
with single- or double-membraned vesicles
that contained cytoplasm, including
organelles, at various stages of digestion.
In this way, autophagy can be
distinguished from heterophagy, where
cells degrade extracellular material. De
Duve's “autophagy” likely described
macroautophagy, which we will call
autophagy, the focus of this review.
Autophagy, which is highly conserved from
yeast to humans, is a bulk degradation
process involved in the clearance of long-
lived proteins and organelles. During
autophagy, phagophores [also called pre-
autophagosomal structures (PAS)]
elongate and fuse while engulfing a portion
of cytoplasm within double-membraned
vesicles, called autophagosomes. The
autophagosomes first fuse with
endosomes to form hybrid organelles
called amphisomes that later fuse with
acidic lysosomes where the entrapped
cytosolic contents are degraded (Fig. 1).
There are two other forms of autophagy,
which we will not review, but will briefly
mention. The first, chaperone-mediated
autophagy (CMA), involves selective
translocation of the cytosolic proteins that
are marked by a pentapeptide motif with a
consensus sequence similar to KFERQ
across the lysosomal membrane. Cytosolic
chaperones aid in the target recognition
and unfolding, and a multimer comprising
the lysosomal protein LAMP-2a
(lysosomal-associated membrane protein-
2a) subunits is thought to be rate-limiting
for target translocation into lysosomes.
The second is microautophagy, a poorly
understood phenomenon in mammalian
cells, where the cytosolic contents are
engulfed by direct invagination of the
lysosomal membranes into tubulovesicular
structures.
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TEÓRICO AUTOFAGIA 3

Fig. 1.Mammalian macroautophagy (autophagy). Autophagy involves the degradation of

cytosolic proteins and organelles in the lysosomes via double-membraned structures called
autophagosomes. Pre-autophagosomal structures (PAS) or phagophores elongate and fuse to
form autophagosomes. The membrane source involved in autophagosome biogenesis may
involve contributions from the ER, mitochondria, and the plasma membrane. Two
macromolecular complexes regulate the initiation of PAS formation: mTOR-Atg13-ULK1
complex and Beclin-Vps34 complex. 3-Methyladenine (3-MA) and wortmannin inhibit Vps34.
Several Beclin 1 binding proteins that regulate mammalian autophagy have been identified
(red, inhibit autophagy; green, induce autophagy). Phosphorylation of Bcl-2 results in its
dissociation from Beclin 1 to induce autophagy. The Beclin 1-Bcl-2 interaction was diminished
by the autophagy-inducing agent xestospongin B that however did not affect the IP3R-Bcl-2
interaction. NAF-1 interacts with Bcl-2 at the ER and stabilizes the Bcl-2-Beclin 1 interaction.
Two ubiquitin-like conjugation systems [Atg5-Atg12 conjugation and LC3-phosphatidyl
ethanolamine (PE) conjugation] are involved in the elongation of PAS. The Atg5-Atg12
congugation involves Atg7 (E1-like) and Atg10 (E2-like), while Atg7 and Atg3 act as the E1-like
and E2-like, respectively, in LC3-PE conjugation. The Atg12-Atg5 is noncovalently conjugated
to Atg16L1 (Atg12-Atg5.Atg16L1), resulting in an 800-kDa complex containing Atg12-
Atg5.Atg16L1 tetramers that are linked via the coiled-coil domain of Atg16L1. The Atg12-
Atg5.Atg16L1 complex exhibits an E3-like activity towards LC3-PE conjugation. Cross-talk
between the two ubiquitin-like systems has been implicated. Beclin 1 complex containing
Rubicon and UVRAG is involved in the fusion step of autophagy.
Until the early 1990s, autophagy was from macromolecules and by assisting the
predominantly studied using clearance of misfolded proteins and
morphological and biochemical methods. damaged organelles, autophagy is vital in a
In 1992, Ohsumi's group demonstrated range of physiological and pathological
that autophagy occurred in Saccharomyces situations, including during early
cerevisiae (baker's yeast). Soon thereafter, embryonic development and neonatal
Ohsumi's lab and others, like Klionsky and starvation, for the degradation of disease-
Thumm, published data using this tractable causing aggregate-prone proteins and in
organism to identify autophagy genes (Atg the clearance of pathogenic bacteria.
genes). The discovery of yeast autophagy Dysfunction in the autophagy pathway has
genes was followed by the identification of been implicated in an increasing number of
mammalian orthologs with similar roles human diseases, from infectious diseases
and provided a series of reagents for to cancer and neurodegeneration (341).
characterizing the molecular machinery of
the system as well as its roles in normal In this review, we aim to broadly outline
physiology and disease (240). the biology of mammalian autophagy and
the key genes involved in the process. We
Autophagy in mammalian systems occurs will consider how autophagy is important
under basal conditions and can be for normal mammalian physiology and its
stimulated by stresses like starvation, roles in a range of disease. Finally, we will
various pathologies, or by treatment with consider the possibility that autophagy
pharmacological agents like rapamycin. In upregulation may be beneficial for certain
addition to its roles in maintaining normal human diseases.
cellular homeostasis by liberating nutrients
TEÓRICO AUTOFAGIA
II. AUTOPHAGY MACHINERY
More than 30 different genes regulating
autophagy have been identified in yeast,
and many of these have mammalian
orthologs. In addition, many genes with
multiple functions, including roles in
autophagy that were not identified in the
relatively specific yeast screens, have been
identified. These genes can be grouped
according to their functions at key stages
of the autophagy pathway: initiation,
elongation, maturation, and fusion with
the lysosomes.
A. Initiation of Autophagosome Formation
The membrane source from which
autophagosomes arise is still a matter of
debate, and there may be multiple
sources. It has been hypothesized that
autophagosomes can either be generated
de novo from preexisting intracellular
precursor molecules, or could arise from
other intracellular membrane structures
like the endoplasmic reticulum (ER) (8).
The latter hypothesis has recently been
supported by more evidence suggesting
that ER could contribute to
autophagosome formation (168, 582).
Autophagosomes have frequently been
reported to form in the vicinity of ER.
Furthermore, rough ER was found to
surround both the outer and inner surfaces
of the cup-shaped pre-autophagosomal
structures. Accumulation of these ER-
associated early autophagic structures was
seen in cells that had a block in maturation
of pre-autophagosomal structures. ER
association with the outer surface was,
however, not observed when the pre-
autophagosomal structures matured into
autophagosomes. Thus the ER may be one
of the membrane sources contributing to
the maturation of pre-autophagosomal
structures (168, 582). It has recently been
suggested that mitochondria may be a
4
membrane source to autophagosomes
during starvation (150a). Recently, we
showed that plasma membrane is a key
contributor for autophagosome precursors
(415a). The formation of new
autophagosomes requires the activity of
the class III phosphatidylinositol 3-kinase
(PI3K), Vps34. Phosphatidylinositol-3-
phosphate (PI-3-P), the product of Vps34
activity, plays an essential role in the early
stages of the autophagy pathway. Recent
studies have identified strong
colocalization of early autophagosome
markers in PI-3-P-enriched structures that
were formed upon starvation (8). The
precise role of PI-3-P is still unclear;
however, this raises the importance of PI-
3-P effector proteins in autophagosome
formation. Indeed, the recently identified
mammalian Atg18 homolog WIPI-1 binds
to PI-3-P and is recruited to
autophagosomal membranes, especially
upon autophagy induction (401). The small
GTPase Rab5, an early endocytic protein,
interacts with and activates Vps34
(58, 483). Inhibition of Vps34 activity by 3-
methyladenine (3-MA) or wortmannin
leads to inhibition of autophagosome
formation (26, 391, 472).
Vps34 is part of the autophagy-regulating
macromolecular complex (PI3K complex)
consisting of Beclin 1/Atg6, Atg14/barkor,
and p150/Vps15 (Fig. 1) (189, 227, 500).
The activity of Vps34 is enhanced by its
interaction with Beclin 1 (123). Several
Beclin 1 binding proteins have been
identified, and disruption of their
interaction with Beclin 1 affects
autophagosome formation. The Beclin 1
binding partners that induce autophagy
include ambra-1 (110), UVRAG (298), and
bif-1 (506). On the other hand, the binding
of the anti-apoptotic proteins Bcl-2 or Bcl-
XL to Beclin 1 inhibits autophagy (389).
Furthermore, the inositol 1,4,5-
trisphosphate receptor (IP3R) that can bind
TEÓRICO AUTOFAGIA
to Bcl-2 can also interact with Beclin 1. The
IP3R antagonist xestospongin B decreases
this interaction and induces autophagy
(552). The Beclin 1-Bcl-2 interaction was
also diminished by xestospongin B, even
though this drug does not affect the IP3R-
Bcl-2 interaction (67). Another component
in the IP3R complex, NAF-1 (nutrient-
deprivation autophagy factor-1), has
recently been identified and acts at the ER
(45). NAF-1 interacts with Bcl-2 at the ER
and stabilizes the Bcl-2-Beclin 1
interaction, while knockdown of NAF-1
decreases Bcl-2-Beclin 1 interaction and
promotes autophagy (45). Activation of c-
Jun NH2-terminal kinase-1 (JNK1) during
starvation results in phosphorylation of
Bcl-2 and Bcl-XL that releases their binding
to Beclin 1, thus inducing autophagosome
formation (562).
A second macromolecular complex
implicated in the initiation step of
autophagosome formation is the FIP200
(163)-ULK1/Atg1 complex
(44, 46, 126, 178, 209). Atg13 binds ULK1
or its homolog ULK2 and mediates their
interaction with FIP200. Under nutrient
deprivation conditions, Atg13 and ULK1/2
are dephosphorylated (see section V for
details), thereby activating ULK1/2, which
phosphorylates FIP200 to induce
autophagosome formation
(46, 126, 178, 209).
B. Elongation
Elongation of phagophores probably
requires membrane input from other
organelles. Atg9 is one protein that may
play this role. Atg9 is a transmembrane
protein that cycles between the trans-
Golgi network and endosomes, probably
carrying membrane for expansion of
phagophore (584). Other components of
Atg9 sorting remain to be identified.
5
Two ubiquitin-like reactions are involved in
the elongation of the pre-autophagosomal
structures. In the first of the reactions, the
ubiquitin-like protein Atg12 is covalently
tagged to Atg5 (342). Atg12 is first
activated by Atg7 (E1 ubiquitin activating
enzyme-like) and then transferred to Atg10
(E2 ubiquitin conjugating enzyme-like).
Atg12 is finally linked by its COOH-terminal
glycine to an internal lysine (K130) residue
of Atg5 (342). The Atg12-Atg5 then forms a
conjugate with Atg16L1 (Atg12-
Atg5.Atg16L1), resulting in an 800-kDa
complex containing Atg12-Atg5.Atg16L1
tetramers that are linked via the coiled-coil
domain of Atg16L1 (340). This complex is
essential for the elongation of the pre-
autophagosomal membrane, but
dissociates from fully formed
autophagosomes.
The second ubiquitin-like reaction involves
the protein microtubule-associated
protein 1 light chain 3 (MAP1-
LC3/LC3/Atg8). LC3 is synthesized as a
precursor form and is cleaved at its COOH
terminus by the protease Atg4B (170, 516),
resulting in the cytosolic isoform LC3-I.
LC3-I is conjugated to
phosphatidylethanolamine (PE) in a
reaction involving Atg7 (E1-like) and Atg3
(E2-like) to form LC3-II (211, 518). LC3-II is
specifically targeted to the elongating
autophagosome membrane and, unlike
the Atg12-Atg5.Atg16L1 complex, remains
on completed autophagosomes until
fusion with the lysosomes, after which LC3-
II on the cytoplasmic face of
autolysosomes can be delipidated by Atg4
and recycled (516). (LC3 is also found on
the internal surface of autophagosomes,
and this is degraded in the autolysosomes.)
The relatively specific association of LC3-II
with autophagosomes makes it an
excellent marker for studying autophagy
(241).
TEÓRICO AUTOFAGIA
Cross-talk between the two ubiquitin-like
systems has been reported. The Atg12-
Atg5.Atg16L1 complex can function in an
E3 ubiquitin ligase-like manner to facilitate
LC3-I conjugation to PE (157). The Atg16L1
complex is believed to bring LC3 to the site
of lipidation for the final conjugation with
PE, but the mechanism by which LC3 is
targeted to specific membranes remains to
be identified (121). Atg10 can interact with
LC3 and facilitate LC3 conjugation to PE
(358). Similarly, Atg3
coimmunoprecipitates with Atg12, and
overexpression of Atg3 increases Atg5-
Atg12 conjugation (517). LC3 can mediate
membrane tethering and hemifusion, and
these functions may be crucial for the
expansion and/or assisting the final fusion
of the double-membrane cups into fused
vesicles (355). Consistent with this
hypothesis, loss of Atg3 or a loss-of-
function mutation in Atg4B leads to
decreased closure of isolation membranes
(120, 494).
C. Maturation and Fusion
Unlike in yeast cells where
autophagosomes are formed at the single
phagophore assembly site next to the
vacuole, mammalian autophagosomes are
formed randomly in the cytoplasm.
Autophagosomes move bidirectionally
along microtubules with a bias towards the
microtubule organising center (MTOC),
where the lysosomes are enriched. This
transport towards MTOC requires the
function of dynein motor proteins
(233, 413, 566). Depolymerization of
microtubules or inhibition of dynein-
dependent transport results in inhibition of
autophagy (245, 560). Autophagosomes
first fuse with endosomes and then with
lysosomes where the fate of the
autophagosomes ends. The fusion step
involves proteins such as ESCRT, SNAREs,
Rab7, and the class C Vps proteins
6
(7, 109, 147, 199, 282, 428). Mutation or
loss of proteins important for formation of
multivesicular bodies (MVBs) (e.g., ESCRT)
leads to an inhibition of autophagosome
maturation (109, 282, 428). UVRAG, a
Beclin 1 interacting protein, is also involved
in the maturation step by recruiting the
fusion machinery on the autophagosomes.
This function is independent of its
interaction with Beclin 1 (299). UVRAG
recruits the class C Vps proteins and via this
interaction activates Rab7, thereby
promoting fusion with late endosomes and
lysosomes (299). A recently identified
Beclin 1 interacting protein, Rubicon, also
functions in the maturation of
autophagosomes. Rubicon is thought to be
a part of a distinct Beclin 1 complex
containing hVps34, hVps15, and UVRAG
that suppresses autophagosome
maturation (329, 603). More work is
required to exactly characterize the
different Beclin 1 complexes and where in
the autophagy pathway they act.
In addition to the fusion machinery, proper
lysosomal function is also essential for
fusion to be successful. Inhibition of the
lysosomal H+-ATPase by chemicals like
bafilomycin A1 inhibits the fusion of
autophagosomes with
endosomes/lysosomes (577). This might
be because lysosomal acidification is
required for normal fusion. However, the
possible mechanisms responsible are
unclear.
A recent study has identified an alternative
autophagy pathway that seems to be
activated when mouse cells lacking Atg5 or
Atg7 were treated with stressors like
etoposide. This Atg5/Atg7-independent
autophagy requires certain other key
autophagy proteins, like Ulk1 and Beclin 1,
and the progression of this autophagy
process occurs in a Rab9-dependent
manner (362).
TEÓRICO AUTOFAGIA
III. SELECTIVE AUTOPHAGY
Autophagy has long been considered to be
a nonselective bulk degradation pathway.
However, in the last few years, several
forms of selective autophagy have been
identified which lead to degradation of
specific organelles, proteins, and
pathogens in yeast, flies, and mammals.
Whether nonselective autophagy really
occurs in a random manner is an
interesting question. Recently, a study of
the dynamics of 1,500 proteins revealed
they were degraded in an ordered fashion
during starvation, implying selectivity
under conditions typically associated with
nonselective autophagy (260). Cytosolic
proteins were degraded rapidly, whereas
proteins annotated to various complexes
and organelles were degraded later at
different time periods. Selective
autophagy may be involved, possibly via
positive selection for incorporation into
autophagosomes [e.g., by
posttranslational modifications like
ubiquitination or acetylation (202, 238)]
and/or negative selection (e.g., due to
decreased accessibility of certain
proteins/organelles for autophagic
engulfment). However, some of these
observations may also be unrelated to any
selection and may be the consequence of
multiple different pathways contributing
to degradation of a given substrate; many
proteins are degraded both by the
ubiquitin-proteasome system (where
substrates are “primed” for degradation by
E3 ligases) and autophagy.
A variety of organelles are cleared by
autophagy. Homeostatic levels of ER,
peroxisomes, mitochondria, and
ribosomes are maintained by ER-phagy,
pexophagy, mitophagy, and ribophagy,
respectively (20, 259, 432, 530). Lipid
degradation by autophagy
(macrolipophagy) and degradation of
7
pathogens (xenophagy) have also been
described as specific phenomena
(289, 563). Mitochondria are a major
source of reactive oxygen species (ROS)
and are therefore especially prone to ROS
damage. Maintaining a healthy population
of mitochondria is essential for the well-
being of cells. The major degradative
pathway in mitochondrial turnover is
mitophagy (230). Mitophagy has been
directly visualized in hepatocytes from
transgenic mice expressing green
fluorescent protein (GFP) fused with LC3
(230, 343). GFP-LC3 formed small green
dotted structures that became associated
with mitochondria following starvation.
These structures became crescent-like in
shape and went on to completely
sequester individual mitochondria.
Mitophagy can be induced by nutrient
deprivation and photodamage. Both these
stimuli can induce the mitochondrial
permeability transition (MPT) (286). Unlike
in yeast cells, a common feature of
mammalian mitophagy is loss of
membrane potential. During MPT, the
permeability transition pore, a
nonselective large-conductance inner
mitochondrial membrane channel, opens,
resulting in depolarization and
mitochondrial outer membrane rupture.
Cyclosporin A, an MPT blocker, can prevent
mitophagy occurring in mitochondria
undergoing MPT (230). In HeLa cells,
inhibitor factor 1 (IF1), a nuclear DNA
encoded protein that regulates F1F0-ATP
synthase, is involved in mitochondrial
clearance by autophagy (39). Mitophagy is
also induced during normal developmental
processes like reticulocyte maturation or
development of sperm cells. Mitochondrial
clearance in reticulocytes required the
Bcl2-related protein Nix along with Ulk1
activity (267, 469). Nix has recently been
shown to bind to LC3/GABARAP and thus
may target mitochondria to
TEÓRICO AUTOFAGIA
autophagosomes (366, 468). During
hypoxia, BNIP3, a protein whose
expression is suppressed in many forms of
cancer, is involved in mitochondrial
clearance as a survival mechanism to
control generation of ROS (13, 598).
One way that mitophagy may be beneficial
would be if it could selectively enhance
removal of dysfunctional mitochondria.
This may occur via the protein Parkin,
which is mutated in certain cases of
autosomal recessive Parkinson's disease.
Parkin is selectively recruited to
dysfunctional mitochondria with low
membrane potential. After recruitment,
Parkin facilitates the engulfment of
mitochondria by autophagosomes and
thus allows selective removal of damaged
mitochondria (357). The relocation of
Parkin to mitochondria relies on the
expression of phosphatase and tensin
homolog (PTEN)-induced putative kinase 1
(PINK1), another protein mutated in forms
of autosomal recessive Parkinson's
disease. Overexpression of the wild-type,
but not the mutant, PINK1 causes
translocation of Parkin to mitochondria
(554). Loss of PINK1 induces oxidative
stress and subsequently leads to the
clearance of depolarized mitochondrial by
autophagy requiring beclin-1 (55, 76).
Another way that one may be able to
mediate selective clearance of
mitochondria appears to be related to the
increased likelihood that a small
mitochondrion could be engulfed by an
autophagosome, compared with a larger
mitochondrion. Mitochondria undergo
multiple cycles of fusion and fission in a
“kiss and run” pattern. Fission events often
generate unlike daughter mitochondria:
one daughter has increased membrane
potential [delta psi(m)] and a high
probability of subsequent fusion, while the
other has decreased membrane potential
8
and a reduced probability for a fusion
event. This predicts the formation of a
subpopulation of nonfusing mitochondria
that also have reduced delta psi(m) and
decreased levels of the mitochondrial
fusion protein OPA1. Inhibition of fission
decreases mitochondrial autophagy and
results in the accumulation of oxidized
mitochondrial proteins, reduced
respiration, and impaired insulin secretion.
These studies suggest a fusion-fission-
related process that enables selective
removal of damaged mitochondria by
autophagy (540).
Peroxisome abundance is increased in cells
in response to peroxisome proliferators, a
process that is counterbalanced by
degradation of peroxisomes by pexophagy
(432). Evidence for the contribution of the
autophagic machinery in peroxisomal
degradation in mammals comes from
conditional knockout mice for Atg7, an
essential autophagy gene. In control
hepatocytes, excess peroxisomes were
surrounded by autophagosomes,
suggesting a selective process. Atg7-
deficient hepatocytes had impaired
removal of excess peroxisomes, and these
excess peroxisomes were not surrounded
by autophagosomes (194). It is possible
that one way that peroxisomes may be
targeted to autophagosomes is via
ubiquitination of cytosolic-facing proteins.
Such proteins may be recognized by the
ubiquitin-binding protein p62 that is
targeted to autophagosomes by binding to
Atg8 (231).
ER volume is increased in response to ER
stress where an excess of unfolded
proteins is sensed by the unfolded protein
response (UPR). When the UPR becomes
inactivated, the ER reestablishes its
homeostatic volume by ER-phagy (20).
Electron microscopic studies in yeast have
revealed large vesicles, bound by double
TEÓRICO AUTOFAGIA
membranes, densely filled with stacked
membrane cisternae and no other
organelles, indicating a selective process
(20). Autophagy may also play a role during
the UPR by eliminating areas of ER that are
enriched in potentially dangerous unfolded
proteins. In mammalian cells, autophagy
occurs in response to ER stress and ER
protein aggregates (545), but whether or
not this is a selective process is yet to be
demonstrated.
Regulation of selective autophagy has
been well studied in yeast where elegant
genetic experiments have elucidated
genes that are specifically involved in the
different selective forms of autophagy, for
example, Atg19 and Atg20 in ER-phagy,
Uth1, Aup1, Atg32 in mitophagy, PEX3 and
PEX14 in pexophagy, and Ubp3p/Bre5p in
ribophagy (216, 259, 376, 545). Much less
is known about the molecular targets and
signaling pathways responsible for
selective pexophagy and ER-phagy in
mammals. Indeed, it is frequently very
difficult to make a strong claim for
selective autophagy without identifying
candidate modulators that can influence
autophagic degradation of the specific
organelle/protein in question, without
influencing autophagic clearance of other
substrates.
Selective autophagy is not limited to
degradation of organelles like
mitochondria and the ER. Glycogen
autophagy occurs in mammals under
conditions of demand for the massive
hepatic production of glucose, such as in
the postnatal period. Glycogen autophagy
appears to be a selective process, since
autophagic vacuoles are predominantly
distributed at the borders of glycogen
stores and contain various quantities of
glycogen (256). Glycogen autophagy is
induced by glucagon, which is secreted
after birth as a result of the normal
9
postnatal hypoglycemia. Glucagon
elevates cAMP levels resulting in the
formation of hepatic autophagic vacuoles,
activation of the glycogen-hydrolyzing acid
glucosidase, and degradation of glycogen
to glucose (256).
IV. CROSS-TALK BETWEEN THE
UBIQUITIN-PROTEASOME SYSTEM,
CHAPERONE-MEDIATED AUTOPHAGY,
AND AUTOPHAGY
A. Ubiquitin-Proteasome System and
Autophagy
Besides autophagy, the other major route
for intracellular protein degradation is the
ubiquitin-proteasome system (UPS). In
contrast to macroautophagy, this process
is highly specific. In mammalian cells,
about a thousand E3 enzymes are
responsible for the specific detection and
binding of their substrates, which include
misfolded proteins and short half-life
regulatory proteins. In this process, the
ubiquitously expressed, 76-amino acid
protein ubiquitin is first activated by an E1
enzyme and subsequently transferred onto
an E2 ubiquitin-conjugating enzyme. The
interaction between an E2 enzyme
(carrying the activated ubiquitin molecule)
and an E3 enzyme (carrying the
degradation substrate) leads to the
transfer of the ubiquitin onto the target
substrate. In this reaction, a covalent
isopeptide bond is formed between the
COOH-terminal glycine of the ubiquitin
molecule and the lysine residues in the
target protein (171). The ubiquitin
molecule itself contains seven internal
lysines at positions 6, 11, 27, 31, 33, 48,
and 63. In a second cycle, the cascade of
E1, -2, and -3 enzymes adds another
ubiquitin molecule to one of these seven
internal lysines. Depending on which of
these lysines link the ubiquitin molecules
to a chain, the tagged protein is destined
TEÓRICO AUTOFAGIA
for different pathways. A protein that
carries a chain of at least four K48-linked
ubiquitins can be recognized by the
machineries that allow transfer to the
proteasome. At least in some
circumstances, this appears to be
mediated by ubiquitin-binding proteins,
like p97 (152).
The proteasome consists of a 20S central
complex and two 19S lid complexes. The
barrel-shaped central complex comprises
four stacked rings. The two identical outer
rings are each made up of seven α-subunits
(termed α 1–7), and the two identical inner
rings each consist of seven β-subunits
(termed β 1–7). The proteolytic activity
resides in the inner two β-rings. The two
19S lid complexes that bind to either side
of the central complex restrict access to
the proteolytic sites. Subunits of these lid
complexes bind to ubiquitin chains on
degradation substrates, release the
ubiquitin chains, and allow the substrate to
enter the central, proteolytic complex.
Proteasomal degradation of target
substrates results in short polypeptides
that are released into the cytoplasm or the
nucleus, which are then further degraded
into amino acids by peptidases (171).
The UPS and autophagy have long been
regarded as two separate cellular
pathways with distinct functions. It was
previously thought that autophagy
predominantly degraded long half-life
proteins and that the clearance of the
short half-life proteins that were typical
UPS substrates would not be affected by a
compromise in autophagy. However,
recent findings suggest that the two
systems do “communicate,” at least under
certain circumstances, and might even
have compensatory functions in some
cases. Indeed, short half-life proteins
normally degraded by the UPS can be
degraded by autophagy under certain
10
conditions (117, 294), and long-lived
proteins can also be degraded by the UPS
(116). It is now clear that a number of
proteins can be degraded by both
autophagy and the proteasome
(71, 81, 225, 559). Furthermore, mutations
that interfere with the proteasomal
degradation of a protein may increase the
dependency of such proteins on autophagy
for their degradation, as this will then
become the default clearance route. This is
especially true for mutated proteins with
an increased aggregation tendency, as
oligomeric and higher-order structures will
become inaccessible to the narrow
proteasome barrel.
Numerous studies in yeast and mammalian
cells, including primary neurons, have
reported that proteasome inhibition leads
to the upregulation of autophagy
(49, 88, 89, 193, 418). This cross-talk may
be mediated by JNK1 activation following
proteasome inhibition, which would be
predicted to induce autophagy via Bcl-2
phosphorylation, thereby reducing its
ability to bind and inhibit the function of
Beclin 1 (337, 389, 562). In vivo data
further support these findings: impairment
of the proteasome leads to
neurodegeneration in the fly eye. This
phenotype is enhanced when essential
autophagy genes are knocked down, and
rescued by autophagy induction with
rapamycin (380).
While proteasome inhibition may induce
autophagy, the converse does not occur.
Indeed, inhibition of autophagy leads to
impaired degradation of substrates
destined for the proteasome (255). This is
dependent on the ubiquitin-binding
protein p62, which is an autophagy
substrate that accumulates in autophagy-
deficient cells (25, 250). The resulting
elevated levels of p62 appear to
compromise the UPS by delaying the
TEÓRICO AUTOFAGIA
delivery of ubiquitinated substrates to the
proteasome, by sequestering them away
from other ubiquitin-binding proteins (e.g.,
p97). Therefore, prolonged inhibition of
autophagy would also result in the reduced
flux through UPS; this may be relevant to
the interpretation of studies examining the
effects of autophagy knockout in various
tissues, since some of the effects may be
secondary consequences of UPS
compromise (e.g., p53 elevation and
apoptosis) (306, 310).
An interesting coordinate regulation of
autophagy and the UPS appears to occur
during muscle atrophy. In this condition,
both proteasomal and autophagic protein
degradation are upregulated, and this
upregulation is coordinated by the
transcription factor FoxO3 (600). In this
case, FoxO3 appears to mediate
independent effects on autophagy and the
UPS (600).
Recently, a growing amount of data have
drawn attention to p62 and its possible
role in connecting autophagy with the UPS.
p62 is cleared by both the UPS and
autophagy (250, 255) and is commonly
detected in ubiquitin-containing protein
aggregates associated with various
neurodegenerative diseases (25, 91, 268–
270, 352). p62 polymerizes via its NH2-
terminal PB1 domain and binds to
polyubiquitin chains via its COOH-terminal
UBA domain. In vitro data reveal that p62
interacts with LC3, and it has been
suggested that this may facilitate the
autophagic clearance of ubiquitin-positive
protein aggregates (25, 250, 381). The
strongest support for this hypothesis
comes from studies using an artificially
ubquitinated peroxisomal integral
membrane protein, where it was
demonstrated that a single ubiquitin
molecule is sufficient as a degradation
signal, in contrast to the tetra-ubiquitin
11
chain that is necessary to signal for the
degradation of proteasome clients (231).
However, definitive data supporting this
mechanism for the clearance of
endogenous proteins are still awaited. p62
is unlikely to be necessary for the clearance
of most autophagic substrates, as there
does not appear to be a defect in bulk
autophagy in p62 knockout mice (250).
Another LC3 interactor with many
functional similarities to p62 has recently
been identified [NBR1 protein (neighbor of
BRCA1 gene 1)], and it is possible that
there may be redundancy for their putative
roles in autophagy (236–238, 273). Also,
HDAC6, which also binds ubiquitin and
interacts directly with microtubules, is
involved in autophagic degradation of
mutant proteins during proteasomal
inhibition (193, 380). A recent study
reported that degradation of
polyubiquitinated proteins by the
proteasome or autophagy is mediated by
BAG1 and BAG3, respectively. A switch
from BAG1 to BAG3 in aged cells suggests
that autophagy is used more extensively
for the turnover of polyubiquitinated
proteins (125). Although ubiquitinated
proteins are autophagy substrates, more
work is needed to understand the exact
role of ubiquitination as a signal for
selective autophagy.
B. ERAD and Autophagy
Most secreted and transmembrane
proteins in eukaryotic cells are folded and
modified in the ER before they are further
transported towards their destination.
Proteins that fail to fold properly in the ER
initially receive aid by an intricate system
of molecular chaperones. However,
proteins that do not fold at this stage are
retrotranslocated to the cytoplasm and
targeted for proteasomal degradation via
cytosolic polyubiquitination, a process
called ER-associated degradation (ERAD).
TEÓRICO AUTOFAGIA
When misfolded proteins are not exported
efficiently to the cytoplasm and
accumulate in the ER, the UPR is often
induced. Three main pathways are
activated to alleviate the accumulation of
proteins in the ER. 1) Inositol-requiring
protein-1 (IRE1) oligomerizes in the
stressed ER and via X-box binding protein-
1 (XBP1) activates the transcription of UPR
genes, whose products are involved in lipid
synthesis, or ERAD, or code for
chaperones. 2) ER stress leads to the
processing of activating transcription
factor-6 (ATF6). The resulting ATF6
fragment translocates to the nucleus and
activates UPR genes. 3) Protein kinase RNA
(PKR)-like ER kinase (PERK) oligomerizes
under ER stress and phosphorylates the α-
subunit of eukaryotic translation initiation
factor-2 (eIF2α), thus inhibiting translation,
thereby decreasing the ER protein load.
Additionally, PERK activates UPR gene
transcription (423, 548).
Many studies have reported that ER
stressors, like DTT, tunicamycin, or
thapsigargin, or proteasome inhibitors
induce the formation of autophagosomes
(19, 89, 180, 257, 367, 433, 583). The
activation of autophagy under ER stress
may have a cytoprotective effect, as
genetic or chemical inhibition of
autophagy leads to a higher susceptibility
towards cell death (19, 89, 367). The
molecular pathway that is responsible for
the induction of autophagy after ER stress
is not entirely clear. Different data suggest
an involvement of IRE1, signaling via JNK-1
but not XBP1, and independence of PERK1
and ATF6 (89, 367). The involvement of
JNK-1 is particularly interesting, as
phosphorylation of Beclin 1 by this kinase
is essential for the induction of autophagy
under starvation conditions (562). Another
mechanism may involve Death-associated
protein kinase 1 (DAPK1) activation by ER
stress leading to Beclin 1 phosphorylation,
12
which induces autophagy by reducing
Beclin 1 binding to Bcl-xL (142, 592). On the
other hand, Kouroku et al. (257)
demonstrated that ER stress caused
activation of autophagy via PERK1 and
eIF2α. Calcium release due to ER stress has
also been implicated in the pathway
leading to autophagy induction (180, 433).
However, some of these calcium studies
were based on experiments using
thapsigargin, which may have incorrectly
interpreted that the increased numbers of
autophagosomes occurring after
thapsigargin treatment were the result of
increased autophagosome synthesis. It
appears that thapsigargin causes a calpain-
dependent reduction in autophagosome
synthesis along with a block in
autophagome-lysosome fusion. So,
although this compound increases
autophagosome numbers (due the block in
autophagosome-lysosome fusion), its
overall effects are to impede autophagic
flux and the clearance of autophagic
substrates (566).
C. CMA and Autophagy
In addition to the relationships with the
UPS, (macro)autophagy is also modulated
by the activity of CMA. Deletion of the
lysosomal receptor LAMP-2a, a key
component of CMA, induces
macroautophagy (321, 322). Whereas
macroautophagy is a relatively unspecific
degradation pathway, CMA is highly
specific. Thus it is somewhat surprising
that autophagy-incompetent cells activate
CMA (221), as this pathway requires a
specific pentapeptide signal in its
substrates. While only 30% of the
cytoplasmic proteins carry the required
signal (56), enhanced degradation of these
proteins may be sufficient to reduce the
overall load of proteins and thereby
alleviate cell stress.
TEÓRICO AUTOFAGIA
V. SIGNALING PATHWAYS REGULATING
MAMMALIAN AUTOPHAGY
Several signaling pathways seem to
regulate autophagy in mammalian cells.
Similar to yeast, the classical pathway
involves the serine/threonine kinase,
mammalian target of rapamycin (mTOR).
The induction of autophagy by mTOR
inhibition under starvation conditions is a
well-known phenomenon in mammalian
cells (509). Various pathways and small
molecules regulating autophagy via mTOR
and mTOR-independent mechanisms,
which have been identified in recent years,
are described below.
A. mTOR Pathway
The mTOR pathway is the most studied
pathway regulating mammalian autophagy
(Fig. 2). mTOR [also known as rapamycin
and FKBP12 target-1 (RAFT1), rapamycin
target-1 (RAPT1), and FKBP12-rapamycin
associated protein (FRAP)] is an important
13
signaling molecule that regulates diverse
cellular functions, such as initiation of
mRNA translation, cell growth and
proliferation, ribosome biogenesis,
transcription, cytoskeletal reorganization,
long-term potentiation, and autophagy
(445). mTOR is the mammalian ortholog of
the yeast protein kinase TOR that
negatively regulates autophagy (291). The
mTOR pathway involves two functional
complexes: a rapamycin-sensitive mTOR
complex 1 (mTORC1), which regulates
autophagy, consisting of the mTOR
catalytic subunit, raptor (regulatory
associated protein of mTOR), GβL (G
protein β-subunit-like protein; also known
as mLST8), and PRAS40 (proline-rich Akt
substrate of 40 kDa); and mTOR complex 2
(mTORC2) consisting of mTOR, rictor
(rapamycin-sensitive companion of
mTOR), GβL, SIN1 (SAPK-interacting
protein 1), and PROTOR (protein observed
with rictor) that is not a direct autophagy
regulator (145, 581).
TEÓRICO AUTOFAGIA 14

Fig. 2.The phosphatidylinositol 3-kinase (PI3K)-mammalian target of rapamycin (mTOR)

pathway regulating autophagy. The PI3K pathway is triggered by the binding of insulin (or
growth factors) to insulin receptor (IR), thereby activating PI3K. Activated PI3K converts PIP 2 to
PIP3, which then recruits phosphoinositide-dependent kinase 1 (PDK1) and Akt to the cell
membrane. Akt is then phosphorylated and activated, which in turn phosphorylates and
inactivates tuberous sclerosis complex (TSC) 1/2, leading to activation of Rheb and
consequently mTORC1. Akt can also be phosphorylated and activated by mTORC2. AMPK, a
cellular energy sensor of changes in the intracellular ATP/AMP ratio, directly phosphorylates
TSC2, thereby providing the priming phosphorylation for subsequent phosphorylation of TSC2
by glycogen synthase kinase 3 (GSK3) to inhibit mTORC1 signaling. The mTOR pathway
involves two functional complexes: a rapamycin-sensitive mTORC1 that regulates autophagy,
consisting of the mTOR catalytic subunit, raptor, mLST8, and PRAS40; and mTORC2 comprising
of mTOR, rictor, mLST8, SIN1, and PROTOR. Amino acids activate mTORC1 via Rag GTPases
and suppress autophagy. The rate-limiting factor of amino acid signaling is L-glutamine, which
is initially taken up by its high-affinity transporter SLC1A5, followed by its efflux by the
SLC7A5/SLC3A2 bidirectional transporter in exchange for uptake of essential amino acids,
which subsequently activates mTORC1. The ULK1-Atg13-FIP200 complex acts as an integrator
of the autophagy signals downstream of mTORC1. Under nutrient-rich conditions, mTORC1
suppresses autophagy by interacting with this complex and mediating phosphorylation-
dependent inhibition of Atg13 and ULK1. Under starvation conditions or rapamycin treatment,
mTOR dissociates from the complex, resulting in dephosphorylation-dependent activation of
ULK1 and ULK1-mediated phosphorylations of Atg13, FIP200, and ULK1 itself, which triggers
autophagy. The mTORC1 pathway regulates cell growth mainly through 4E-BP1 and p70S6K.
Phosphorylation-dependent activation of p70S6K can also inhibit IRS1, thereby exerting a
feedback loop mechanism.

Many diverse signals, such as growth
factors, amino acids, glucose, energy
status, and different forms of stress,
regulate the raptor-mTOR (mTORC1)
pathway (445). The activity of mTORC1 can
be inhibited by rapamycin (sirolimus), a
lipophilic macrolide antibiotic first isolated
from Streptomyces
hygroscopicus (145, 365). Rapamycin is a
potent autophagy inducer in various cell
lines from yeast to mammalian cells,
including neurons (28, 365, 415, 416, 427).
In mammalian cells, rapamycin forms a
complex with the immunophilin FK506-
binding protein of 12 kDa (FKBP12), which
then stabilizes the raptor-mTOR
association and inhibits the kinase activity
of mTOR (228). The binding of GβL to
mTOR stimulates its kinase activity, and
GβL is necessary for the formation of a
rapamycin-sensitive interaction between
raptor and mTOR (229). Inhibition of
mTORC1 and induction of autophagy by
rapamycin is associated with reduced
phosphorylation of two of its downstream
effectors, ribosomal protein S6 kinase-1
(S6K1, also known as p70S6K) and
translation initiation factor 4E binding
protein-1 (4E-BP1, also known as PHAS-1)
at Thr389/Thr421/Ser424 and
Thr37/Thr46, respectively (242, 445, 464).
Interestingly, a study by Scott et al. (471)
showed that p70S6K is a positive regulator
of autophagy. When TOR is inactivated
during starvation-induced autophagy in
the Drosophila fat body, p70S6K needs to
be activated for some time to allow
maximal autophagy, after which loss of
p70S6K activity prevents excessive
autophagy in TOR-inactive state, probably
by inhibiting the PI3K pathway through a
negative-feedback loop involving
TEÓRICO AUTOFAGIA
downregulation of the insulin receptor
substrates (IRS) (243, 471). Furthermore,
p70S6K activation appears to be necessary
but not sufficient to induce autophagy, as
it activates autophagy in TOR mutants but
not in wild-type Drosophila (471). Whether
these effects of p70S6K on autophagy are
seen in mammals is unclear.
A recent study indicates that rapamycin
does not inhibit mTORC1 completely, due
to rapamycin-resistant functions of
mTORC1. Instead, a selective ATP-
competitive small molecule mTOR
inhibitor, Torin1, was found to induce
autophagy to a much greater extent than
rapamycin (528). Although Torin1 inhibits
both mTORC1 and mTORC2 directly, its
effects on autophagy are mTORC2
independent but mTORC1 dependent due
to suppression of rapamycin-resistant
functions of mTORC1 that are required for
inhibition of autophagy (528).
Other conditions, such as nutrient
deprivation, also stabilize the raptor-mTOR
complex, thereby inhibiting mTORC1 and
inducing autophagy (228, 242, 332). Under
conditions of limited extracellular amino
acids, autophagy recycles intracellular
constituents to provide an alternative
source of amino acids (242). Conversely,
diverse extracellular signals, such as amino
acids and growth factors, activate mTORC1
and suppress autophagy (145, 242, 332).
Recently, the rate-limiting factor that
enables essential amino acids to inhibit
mTORC1 has been identified as L-
glutamine (360). The influx of L-glutamine
by its high-affinity transporter SLC1A5
initially increases its intracellular
concentration. The heterodimeric
SLC7A5/SLC3A2 bidirectional transporter
then uses L-glutamine as an efflux
substrate in exchange for the cellular
uptake of essential amino acids, which
subsequently activates mTORC1 (360).
15
However, the role of glutamine may be
tissue specific, since this amino acid
stimulates autophagy in intestinal cells
(434). The Rag GTPases mediate amino
acid signaling to mTORC1 activation by
binding to raptor and further redistributing
mTOR to a subcellular compartment
containing its activator rheb (440). A
recent report suggests that amino acids
induce the translocation of mTORC1 to
lysosomal membranes where the Rag
GTPases reside. This recruitment of
mTORC1 requires a trimeric Ragulator
complex containing the proteins encoded
by the MAPKSP1, ROBLD3, and c11orf59
genes. Thus, in the presence of amino
acids, the Rag GTPases that are tethered to
lysosomal surface by the Ragulator act as a
docking site for mTORC1, which is then
activated by Rheb (439).
The other mTOR complex, mTORC2, which
phosphorylates (Ser-473) and activates
Akt, is known to be rapamycin insensitive
(195, 444). However, recent studies have
shown that prolonged treatment with
rapamycin inhibits mTORC2 activity and
Akt signaling in certain mammalian cell
types (446, 595). Rapamycin derivatives
such as temsirolimus (CCI-779) and
everolimus (RAD001) inhibit the activity of
mTORC1, thereby reducing the
phosphorylation of p70S6K and 4E-BP1
(595).
Many of the molecular mechanisms linking
TOR signaling to the autophagic machinery
have been dissected in yeast, where the
Atg1 kinase and the Atg1-Atg13-Atg17
complex are thought to act downstream of
TOR regulating autophagosome formation
(212, 573). Recent studies have identified
some of the molecular components
downstream of mTORC1 in mammalian
autophagy. Atg13 binds to the mammalian
Atg1 homologs ULK1 or ULK2 and mediates
the interaction of ULK1/2 with FIP200,
TEÓRICO AUTOFAGIA
thereby forming a ULK1/2-Atg13-FIP200
stable complex that signals to the
autophagic machinery downstream of
mTOR (126, 178, 209). Under nutrient-rich
conditions, mTORC1 suppresses
autophagy through direct interaction with
this complex and mediates
phosphorylation-dependent inhibition of
the kinase activities of Atg13 and ULK1.
Under starvation conditions or rapamycin
treatment, mTOR dissociates from the
complex, resulting in the inhibition of
mTOR-mediated phosphorylation of Atg13
and ULK1. This leads to
dephosphorylation-dependent activation
of ULK1 (and ULK2) and ULK1-mediated
phosphorylations of Atg13, FIP200, and
ULK1 itself, which triggers autophagy
(126, 178, 209). Therefore, the ULK1-
Atg13-FIP200 complex acts as an
integrator of the autophagy signals
downstream of mTORC1. However, it is not
clear yet how phosphorylation of these
proteins regulates their activities.
B. PI3K Pathway
A major signaling cascade controlling
mTORC1 is the PI3K pathway (Fig. 2) (242).
The binding of growth factors or insulin to
cell surface receptors activates the class Ia
PI3K, wherein its regulatory subunit
mediates activation of its p110 catalytic
subunit by the direct interaction with
phosphotyrosine residues of the activated
receptors or adaptor proteins. Activated
PI3K then converts the plasma membrane
lipid phosphatidylinositol-4,5-
bisphosphate (PIP2) to
phosphatidylinositol-3,4,5-trisphosphate
(PIP3), which in turn recruits pleckstrin
homology (PH) domain proteins, such as
the serine/threonine kinases
phosphoinositide-dependent kinase 1
(PDK1) and Akt/PKB, to the plasma
membrane (40). Akt is activated by its
phosphorylation on two different sites. The
16
rictor-mTOR complex phosphorylates Akt
on Ser-473, which may facilitate the
phosphorylation by PDK1 of the activation
loop of Akt on Thr-308. Akt has been
shown to modulate autophagic activity
regulated by the PI3K pathway. Mutations
causing Akt activation or inactivation cause
suppression and induction of autophagy,
respectively (80).
The different classes of PI3K have distinct
effects on autophagy (334). Increases in
the class I PI3K product, PIP3, inhibit
autophagy by inducing Akt activation
(391), whereas hyperactivity of the
phosphoinositide phosphatase PTEN
(phosphatase and tensin homologue
deleted from chromosome 10), a tumor
suppressor, stimulates autophagy by
inhibiting the PI3K pathway (6). In contrast,
increasing the levels of the class III PI3K
product, PI-3-P, stimulates autophagy
(391). Autophagy can be downregulated by
inhibitors of PI3K activity, such as 3-MA,
wortmannin. and 2-(4-morpholinyl)-8-
phenylchromone (LY294002)
(26, 391, 472).
The phosphorylation-dependent Akt
activation results in the phosphorylation of
a host of other proteins, including the
tumor suppressor proteins mutated in
tuberous sclerosis which form a complex
called the tuberous sclerosis complex
(TSC), consisting of a heterodimer of TSC1
(hamartin) and TSC2 (tuberin). TSC1/2
affect cell growth and survival (40, 445).
TSC1/2, an upstream integrator of various
signals regulating the mTORC1 pathway, is
the GTPase-activating protein (GAP) for
the Ras family GTP-binding protein rheb,
which directly binds and activates the
raptor-mTOR complex (130, 315, 521).
Several other kinases, including AMP-
activated kinase (AMPK), ribosomal S6
kinase 1 (RSK1), and extracellular signal
regulated kinase 1/2 (ERK1/2), signal to
TEÓRICO AUTOFAGIA
mTORC1 by phosphorylating TSC2 and
inhibiting the activity of the TSC1/2
heterodimer (445).
Apart from amino acids, mTORC1 can be a
sensor of changes in the cellular energy
state via AMPK (334). AMPK, which senses
changes in the intracellular ATP/AMP ratio,
directly phosphorylates TSC2, thereby
providing the priming phosphorylation for
subsequent phosphorylation of TSC2 by
glycogen synthase kinase 3 (GSK-3) to
inhibit mTOR signaling (186). We have
recently shown that inhibition of GSK-3β
suppress autophagy by activating mTOR
(449). While AMPK activation inhibits TOR
and stimulates autophagy in yeast, the
AMPK activator AICAR inhibits autophagy
in mammalian cells, but this is probably not
due to direct effects on AMPK (333).
Recent evidence indicates that AMPK is
required for mammalian autophagy, as
inhibition of AMPK activity by compound C
or a dominant negative construct strongly
suppresses autophagy (333, 336). AMPK
also mediates hypoxia-induced autophagy
by downregulating mTOR (382). Both LKB1
and Ca2+/calmodulin-dependent protein
kinase kinase-β (CaMKKβ) play important
roles in the phosphorylation and activation
of AMPK. In addition, AMPK can be
activated by other upstream kinases, but
the physiological significance of this is
currently unclear.
C. Other Protein Kinases
Apart from mTOR and certain protein
kinases like AMPK discussed above, several
other kinases have been reported to
regulate autophagy. Physiological or
pharmacological stimulation of IKK (IκB
kinase) induces autophagy via AMPK
activation and mTOR inhibition, but in an
NFκB-independent manner (68).
Eukaryotic translation initiation factor 2α
(eIF2α) kinases, which are serine threonine
17
kinases that regulate stress-induced
translational control programs, are also
involved in the regulation of stress-induced
autophagy. The mammalian eIF2α kinase
signaling pathway is essential for both
virus- and starvation-induced autophagy,
which is antagonized by the herpes simplex
virus (HSV)-encoded neurovirulence gene
product ICP34.5 (511). Eukaryotic
elongation factor-2 (eEF-2) kinase, also
referred to as Ca2+/calmodulin-dependent
kinase III, also regulates autophagy via the
mTOR pathway. Inhibition of eEF-2 kinase
inhibits autophagy. The activity of eEF-2
kinase is enhanced in glioblastoma and
other malignancies. Nutrient deprivation,
which decreases the activity of S6 kinase,
was shown to enhance the activity of eEF-
2 kinase activity (569).
The mitogen-activated protein kinase
(MAPK)/extracellular signal-regulated
kinase (ERK) pathway, which is commonly
activated in cancers, regulates autophagy.
Carcinogens such as lindane, which
activates MAPK/ERK pathway, impair
autophagy (62). However, starvation-
induced autophagy activates the ERK1/2
MAPK pathway, and inhibition of the
pathway by a MAPK kinase (MAPKK)
MEK1/2 inhibitor (PD-98059) suppresses
starvation-induced autophagy in human
colon cancer HT-29 cell line (371). A recent
study shows that sustained MEK/ERK
activation results in complete disassembly
of both mTORC1 and mTORC2, strongly
enhancing Beclin 1 activity, resulting in
cytodestruction, while moderately
enhanced Beclin 1 activity, resulting in
cytoprotection, is mediated via transient
activation of MEK/ERK (556). Codogno and
colleagues (369) have shown that a
cytoplasmic heterotrimeric Gi3 protein
regulates autophagy in a human colon
cancer HT-29 cell line. Autophagy is
minimal when the Gαi3 protein is in the
GTP-bound form and becomes stimulated
TEÓRICO AUTOFAGIA
when GDP is bound to the Gαi3 protein
(370). A similar function of Gαi3 protein in
the regulation of the anti-autophagic
activity of insulin in the liver has also been
reported (138). Likewise, Gα-interacting
protein (GAIP), which is a regulator of G
protein signaling (RGS) protein and
activates the hydrolysis of GTP by the
Gαi3 protein, increases autophagy (372).
Activation of ERK1/2 stimulates autophagy
by phosphorylating GAIP, which stimulates
its GTPase activity towards the GTP-bound
conformation of the Gαi3 protein.
Phosphorylation of GAIP by the ERK1/2
MAPK-dependent pathway is sensitive to
amino acids, since the inhibition of
autophagy by amino acids correlates with
the inhibition of the ERK1/2 MAP kinases
and a low level of GAIP phosphorylation
(371). Amino acids interfere with ERK1/2-
dependent autophagy by inhibiting the
activation of the kinase Raf-1 (388).
Autophagy is also modulated by cell
hydration, which is sensitive to a p38 MAPK
THE ROLE OF AUTOPHAGY IN NEURODEGENERATIVE DISEASE
Autophagy is a lysosomal degradative
process used to recycle obsolete cellular
constituents and eliminate damaged
organelles and protein aggregates. These
substrates reach lysosomes by several
distinct mechanisms, including delivery
within endosomes as well as
autophagosomes. Completion of digestion
involves dynamic interactions among
compartments of the autophagic and
endocytic pathways. Neurons are
particularly vulnerable to disruptions of
these interactions, especially as the brain
ages. Not surprisingly, mutations of genes
regulating autophagy cause
18
inhibitor (SB203580) that prevented
autophagy inhibition, implicating a role of
p38 MAPK in autophagy (167, 555).
Accumulation of mutant glial fibrillary
acidic protein (GFAP) in Alexander disease
induces autophagy by activating p38 MAPK
and inhibiting mTOR signaling pathways
(514). Recent studies show that the
negative regulation of autophagy by p38α
was due to a direct competition with
mAtg9 for binding to p38-interacting
protein (p38IP) (561).
Recently, the stress-activated signaling
molecule, c-Jun NH2-terminal kinase 1
(JNK1), but not JNK2, was shown to be an
important mediator of starvation-induced
autophagy in mammalian cells. Starvation
signal activates JNK1, which in turn
phosphorylates Bcl-2 at multiple sites (Thr-
69, Ser-70, and Ser-87) in the unstructured
loop, thereby disrupting the Bcl-2-Beclin 1
complex and triggering autophagy (562).
neurodegenerative diseases across the age
spectrum with exceptional frequency. In
late-onset disorders such as Alzheimer’s
disease, amyotrophic lateral sclerosis and
familial Parkinson’s disease, defects arise
at different stages of the autophagy
pathway and have different implications
for pathogenesis and therapy. This Review
provides an overview of the role of
autophagy in neurodegenerative disease,
focusing particularly on less frequently
considered lysosomal clearance
mechanisms and their considerable impact
on disease. Various therapeutic strategies
for modulating specific stages of
TEÓRICO AUTOFAGIA
autophagy and the current state of drug
development for this purpose are also
evaluated.
Close on the heels of his discovery of
lysosomes in 1956, Christian DeDuve and
his colleagues described a process by
which cells digest their own cytoplasmic
constituents within lysosomes, which they
termed autophagy (auto-self; phagy-
eating)1. Subtypes of autophagy have
since been identified, each involving
different mechanisms of substrate delivery
to the lysosome, but lysosomal digestion
remains the common cardinal feature.
Until recently, lysosomes were viewed
merely as chambers for the terminal
degradation of nonessential cell
components, but they are now also
becoming appreciated as vital biosensors
of cell homeostasis that report on the
general nutritional status of a cell through
the release of its digestion products.
Lysosomes serve as a docking platform for
an amino acid–sensing protein complex
and for mammalian target of rapamycin
(mTOR) kinase, a master regulator of
autophagy induction, and thus lysosome
activity influences early and late events in
autophagy by modulating rates of
substrate sequestration and delivery to
lysosomes as well as the transcription of
genes involved in autophagosome and
lysosome biogenesis (Fig. 1). Lysosomes
also receive plasma membrane
components and internalized materials via
endocytosis. Endosomes carrying these
cargoes frequently reach the lysosome by
fusing first with autophagosomes during
autophagy (Fig. 1). The active
communication between endocytic and
autophagic degradative compartments
and the numerous regulatory mechanisms
shared between these pathways
constitutes the greater ‘lysosomal
network’ that controls the elimination of
obsolete cellular constituents, the
19
recycling of basic metabolites for new
synthesis and energy stores and the
reporting of information about the cell’s
health status and external environment.
The heavy reliance of neurons on
autophagy is evident from observations
that the brain is often the most severely
affected organ in primary lysosomal
disorders and that mutations in genes
involved in the global lysosomal network
(autophagy and endosomal pathway) are
causatively linked to neurodegenerative
disorders with exceptional frequency.
Because neurons have unusually large
expanses of dendritic and axonal
cytoplasm, they face particular hurdles in
preventing dysfunctional organelles and
cellular waste from accumulating over a
lifetime without the aid of cell division,
which mitotic cells can rely on to dilute
these waste burdens. Young neurons
achieve this task by clearing autophagic
substrates very efficiently2, despite the
long distances that the many autophagic
vacuoles generated in axons must travel to
reach lysosomes, which are concentrated
mainly near the cell body3. Not
surprisingly, neurons are particularly
vulnerable to slowdowns in the proteolytic
clearance of autolysosomal substrates3.
Without competent autophagy, neurons
accumulate ubiquitinated protein
aggregates and degenerate 4,5. In
neurodegenerative diseases, autophagy
goes awry at various points along the
pathway, giving rise to distinct pathologic
patterns with different implications for
modulating autophagy as a therapy. To
review these issues, I will consider
autophagy as three major stages—
selection, sequestration and lysosomal
digestion of substrates—and discuss how a
defect in one or more stages contributes to
pathogenesis in different
neurodegenerative disorders. I then
consider the merits and caveats of
modulating autophagy
TEÓRICO AUTOFAGIA 20

Figure 1 An overview of
macroautophagy.
Macroautophagy is responsible
for the bulk turnover of
cytoplasm and is the cell’s only
mechanism for organelle
turnover, which can occur
selectively or nonselectively.
Other subtypes of autophagy
deliver intracellular substrates
into lysosomes by different
mechanisms (see Fig. 3). The
principal stages of
macroautophagy include highly
regulated formation of an
isolation membrane (or
phagophore) under direction of
multiple signaling and protein
modification assemblies;
elongation of the isolation
membrane around a region of
cytoplasm or selected
substrate; closure of the inner
and outer bilayers of the
isolation membrane to form a
double membraned
autophagosome; fusion of the
autophagosome with a
lysosome, or, alternatively, sequential fusion of the autophagosome with an endosome
followed by a lysosome to form an autolysosome; and digestion of the autophagosome
contents by hydrolytic enzymes, yielding basic metabolites that are released into the
cytoplasm for new synthesis or as sources for energy. The process is depicted as a cycle,
emphasizing that amino acids released during lysosomal digestion are one mechanism by
which TORC1 on lysosomes may be modulated and influence signaling pathways controlling
autophagy induction. MVB, multivesicular body; PAS, preautophagosomal structure.

at a specific stage to treat these diseases membranelimited vacuole, the digestion of

and review the current state of drug the substrate by lysosomal hydrolases and
development for these purposes. the release of digestion products, some of
Substrate sequestration and which then influence signaling
autophagosome formation mechanisms that control autophagy
Macroautophagy, the major subtype of induction (Fig. 1). Macroautophagy is
autophagy, is a cycle involving the initiated through changes in the
enwrapping of selected substrates within a phosphorylation states of individual
TEÓRICO AUTOFAGIA 21

components of a stable autophagy-related of autophagy, influences neuronal

2 (Atg1)–Unc51-like kinase (ULK) complex
(ULK1, ULK2, Atg13, FIP200 and
Atg101)6,7. ULK1 phosphorylation is
mainly regulated by the mTOR complex 1
(TORC1) but may be activated by
AMPactivated protein kinase (AMPK)
independently of TORC1 (ref. 8), which
therefore suggests at least two potential
routes for modulating autophagy induction
therapeutically. Various cellular stress
signals, including lowered concentrations
of specific amino acids, growth factors or
ATP, hypoxia, certain types of protein
aggregates, and endoplasmic reticulum
stress9, suppress TORC1, thus activating
ULK1 and ULK2 and turning on autophagy.
In neurons, toggling between inactivation
of TORC1 and activation of this complex,
which promotes protein synthesis instead
functions as diverse as synaptic
plasticity10, dendritic arborization11 and
myelination12. The phosphorylation of
ULK1 triggers translocation of a
multiprotein complex containing beclin-1
(also known as Atg6) and class III
phosphoinositide 3-kinase (PI3K CIII, also
known as Vps34) from the
cytoskeleton13,14 to a
preautophagosomal structure15 to initiate
autophagosome formation (Fig. 2).
Elongation of the isolation membrane and
its closure around a cytoplasmic cargo
requires membrane components from
several possible sources as well as two
complexes containing ubiquitin-like
proteins and their respective conjugation
machineries17 and several SNARE
proteins18 (Fig. 2).

Figure 2 Autophagy
induction and
autophagosome
biogenesis.
Autophagosome
formation can be
initiated via mTOR
inhibition or AMPK
activation. This results in
the phosphorylation of
ULK1 at sites that
activate it and catalyze
phosphorylation of
other components of the
Atg1–ULK complex,
composed of ULK1,
ULK2, Atg13, FIP200 and
Atg101. ULK1 also
phosphorylates AMBRA,
a component of the PI3K CIII complex I (Vps34, Vps15, Atg14, and beclin-1), enabling it to
relocate from the cytoskeleton to the isolation membrane. Phosphatidylinositol 3-phosphate
(PI3P), generated by Vps34 activity, specifically binds the PI3P effectors WD repeat domain
phosphoinositide-interacting 1 (WIPI1) and WIPI2 and catalyzes the first of two types of
ubiquitination-like reactions that regulate isolation membrane elongation. In this first
TEÓRICO AUTOFAGIA 22

reaction, Atg5 and Atg12 are conjugated to each other in the presence of Atg7 and Atg10.
Attachment of the fully formed complex containing Atg5, Atg12 and Atg16L on the isolation
membrane induces the second complex to covalently conjugate phosphatidylethanolamine to
LC3 (ref. 183), which facilitates closure of the isolation membrane184. Atg9 (the Atg9–Atg2–
Atg18 complex), another factor essential for this event, cycles between endosomes, the Golgi
and the phagophore, possibly carrying lipid components for membrane expansion. Atg4
removes LC3-II from the outer surface of newly formed autophagosomes, and LC3 on the inner
surface is eventually degraded when the autophagosome fuses with lysosomes. The steps
known to be affected in neurodegenerative diseases are indicated in red. HD, Huntington’s
disease; PD, Parkinson’s disease; PE, phosphatidylethanolamine.

Figure 3 Substrate
recognition and
selective autophagy.
Misfolded proteins and
protein aggregates are
selectively targeted for
macroautophagy by
specific autophagy
receptors. Initial
recognition by a
chaperone complex
(Bag3, HspB8 and
Hsc70) and E3 ligase–
mediated
ubiquitination recruits
p62, resulting in the
assembly of the
ubiquitin (Ub)- labeled
proteins into p62
bodies. Autophagic
degradation occurs
when p62 and adaptor
proteins (for example,
NBR1 and ALFY)
interact with LC3 on
the forming
autophagosome.
Mitophagy refers to
the selective targeting
of a damaged
mitochondrion for
macroautophagy.
Reduced PINK1
turnover on the injured depolarized mitochondrion increases the abundance of PINK1, which
recruits and phosphorylates the E3 ubiquitin ligase parkin. Voltage-dependent anion channel
TEÓRICO AUTOFAGIA 23

1 (VDAC1) is polyubiquitinated by parkin, recruiting p62, which binds LC3 to initiate

mitochondrion sequestration in the forming autophagosome. In CMA, the Hsc70 chaperone
complex delivers cytosolic proteins containing a KFERQ motif to the lysosomal lumen for
degradation through the binding to a LAMP2-containing protein complex located on the
lysosomal membrane. Microautophagy (not shown) delivers small quantities of proteins or
organelles directly into late endosomes and possibly lysosomes by invagination of the surface
membrane of the vesicle. HD, Huntington’s disease; MFN2, mitofusin-2; PD, Parkinson’s
disease; PDB, Paget’s disease of bone.

It is worth noting for the later discussion
of drug discovery that unconventional
forms of autophagy under certain stress
conditions may occur independently of
some components of this machinery19 and
that some Atg proteins have functions
unrelated to autophagy20. Despite its
complexity, autophagosome formation has
only infrequently been reported to be
impaired in chronic neurodegenerative
diseases and, in some like Alzheimer’s
disease and amyotrophic lateral sclerosis
(ALS), autophagy induction may actually be
higher than normal, as discussed later.
Autophagy is disrupted at multiple stages
in Parkinson’s disease, a disorder
characterized by inclusions containing α-
synuclein (‘Lewy bodies’) in affected
neurons. One effect of characteristically
high intracellular α-synuclein
concentrations is inhibition of the GTPase
Rab1A, believed to interfere with
omegasome formation through Atg9
mislocalization21 (Fig. 2). In models of
Huntington’s disease, autophagosomes
form properly and are cleared despite
slower-than-normal macroautophagic
protein turnover. A possible explanation
lies in the observation that
autophagosomes seem to be unusually
devoid of cargoes in these models and in
brain samples from people with
Huntington’s disease, suggesting that
autophagosome membranes fail to engage
substrates properly during sequestration.
The underlying mechanism may involve
adventitious binding of beclin-1 by
aggregates of mutant huntingtin leading to
its depletion, which then interferes with
autophagosome membrane nucleation
and sequestration. In Lafora’s disease, a
form of myoclonus epilepsy associated
with progressive neurodegeneration, loss-
of-function mutations in the protein
phosphatase laforin abnormally activate
mTOR24 and thereby reduce the rate,
although not necessarily the competence,
of autophagosome formation, leading to
the cytoplasmic accumulation of
ubiquitinated proteins. Substrate
recognition and selective autophagy
Autophagy was originally recognized as a
nonselective process, but several forms of
selective autophagy that target specific
subgroups of substrates have now been
identified (Fig. 3). Newly discovered
autophagic receptors respond to focal
cellular insults, such as organelle injury,
pathogen invasion or abnormally altered
or aggregated proteins, and then target
these altered structures for selective
autophagy. Autophagic receptors and
other selectivity adaptor proteins tether
the targeted substrate to core autophagic
machinery, such as microtubule-associated
protein light chain 3 (LC3) and Atg12– Atg5,
effecting elimination of the substrate but
minimally altering overall
macroautophagy25. The autophagic
receptors p62 (also known as SQSTM1),
NBR1, NDP52, optineurin (OPTN), histone
deacetylase 6 (HDAC6) and NIX26
recognize and facilitate elimination
ofubiquitinated proteins, and some of
these receptors also target peroxisomes
and internalized pathogens27,28.
TEÓRICO AUTOFAGIA
Additional molecular chaperones and
adaptor proteins with a predilection for
misfolded proteins aid this process25,28.
Parkin (also known as PARK2) and PINK1
(also known as PARK6), two proteins
involved in mitochondrial fusionfission
events, target damaged mitochondria for
selective autophagy (‘mitophagy’). PINK1,
a serine-threonine kinase on the outer
mitochondrial membrane, is activated
when mitochondrial function is
compromised and the membrane
depolarized. This enables parkin, a
cytoplasmic E3 ubiquitin ligase, to bind and
ubiquitinate exposed membrane proteins,
thereby recruiting p62, LC3 and HDAC6,
which initiate mitophagy29–31. The
recognition of cargoes for sequestration or
delivery into the lysosomal lumen is
relatively susceptible to disruption in
neurodegenerative diseases. Mutations in
SQSTM1 initially identified in patients with
Paget’s disease of bone have now been
found in familial and sporadic ALS32,
disorders that typically feature p62-
positive inclusions in affected neurons.
Mutant p62 may promote pathogenesis in
several ways, including overactivating
nuclear factor-κB (NF-κB)33 and disrupting
autophagic clearance of ubiquitinated
proteins and protein aggregates34,
although the latter mechanism has not
been shown directly. Rare ALS-associated
mutations of another autophagy receptor,
OPTN, also impair NF-κB signaling35 while
causing the protein to accumulate in TAR
DNA-binding protein 43–positive neuronal
inclusions36. OPTN may also have
ubiquitin-independent roles in protein
aggregate clearance by autophagy37,
although, again, disruption of this function
by disease mutations has not been directly
demonstrated. Selective autophagy is also
disrupted by mutations in valosin-
containing protein (VCP, also known as
p97), an endosomal member of the AAA+
family of ATPase proteins that lead to
24
inclusionbody myopathy, Paget’s disease
of the bone and frontotemporal dementia
(IBMPFD). VCP regulates endolysosomal
sorting of endocytosed ubiquitinated
cargoes38, and its depletion or the
expression of IBMPFD-linked mutants in
cell models causes immature
autophagosomes containing strongly
ubiquitin-positive substrates to
accumulate, which suggests a role for VCP
in selective clearance of ubiquitinated
substrates and possibly autophagosome
maturation39,40. Mutations in the genes
encoding PINK1 and parkin, which account
for the majority of autosomal-recessive
cases of Parkinson’s disease, impede
mitophagy, causing damaged
mitochondria to accumulate and possibly
initiate apoptosis through the intrinsic
pathway or via reactive oxygen species–
mediated oxidation of membrane lipids
and lysosome membrane
destabilization41. Interestingly, another
function of VCP, which is abrogated by
pathogenic mutations, is the clearance of
damaged mitochondria via the PINK1–
parkin pathway42. Chaperone-mediated
autophagy (CMA), another form of
selective (and constitutive) autophagy,
mediates the lysosomal degradation of a
sizeable family of proteins containing a
consensus pentapeptide motif, (for
example, KFERQ) in their primary
sequence, which includes a substantial
number of disease-causing aggregate-
prone proteins. In CMA, a cytosolic
chaperone protein, Hsc70, escorts
substrate proteins to lysosome-associated
membrane protein type 2a (LAMP2A), the
CMA receptor on a subpopulation of
lysosomes. The protein substrate is then
unfolded, translocated across the
lysosomal membrane and degraded in
lysosomes43. As might be imagined, CMA
is especially vulnerable in
neurodegenerative diseases involving
misfolded and aggregated pathogenic
TEÓRICO AUTOFAGIA
proteins (for example, tau in
frontotemporal lobar dementia with
ubiquitin-positive inclusions (FTLD-U) and
leucine-rich repeat kinase 2 (LRRK2) and α-
synuclein in Parkinson’s disease)44–46.
These altered proteins bind abnormally
tightly to LAMP2A, which blocks their
translocation across the lysosomal
membrane. As importantly, the loss of
function of LAMP2A, believed to be the
rate-limiting factor for CMA, impedes
access of other CMA substrates to the
lysosomal lumen46. Possibly relevant to
various late-age onset neurodegenerative
diseases, CMA activity declines with age
owing to falling LAMP2A amounts on
lysosomes47. The decrease in the amounts
of LAMP2A and Hsc70 in nigral
dopaminergic neurons of people with
Parkinson’s disease outpaces the decline
during normal brain aging and is also seen
in transgenic mice expressing mutant α-
synuclein48,49. Lysosomal digestion of
autophagic substrates Autophagosome-
lysosome fusion. Autophagosomes fuse
either with lysosomes generating an
autolysosome or initially with late
endosomes to form amphisomes that later
fuse with lysosomes (Fig. 4a). Cells increase
opportunities for lysosomal fusion by
directing autophagosomes to move along
microtubules toward the perinuclear
microtubule-organizing center (MTOC) of
the neuron, where lysosomes are most
abundant3. Under conditions of stress,
lysosomes also move toward the MTOC to
engage autophagosomes even more
quickly50. Disassembling microtubules
with vinblastine or deleting dynein, the
motor component of the dynein–dynactin
complex mediating retrograde transport,
provokes a massive buildup of
autophagosomes in neuronal
processes2,51, which highlights the
importance of microtubule-based vesicle
transport for autophagy. Neurons are
particularly vulnerable to disruption at this
25
stage of autophagy because the many
autophagosomes generated in axons and
nerve terminals depend heavily on
retrograde transport to complete the
digestion process. During this retrograde
journey, nearly all autophagosomes first
fuse with late endosomes abundant in the
neuritic processes3,52,53. The formation
of these amphisomes requires some of the
same proteins that direct formation of late
endosomes (also referred to as
multivesicular bodies), including Rab11,
the HOPS complex and components of
multiple endosomal sorting complex
required for transport (ESCRT) complexes
that mediate cargo sorting into
intraluminal vesicles of the multivesicular
bodies25,54,55. Autolysosome formation
also requires late endosome proteins,
including notably Rab7 (ref. 56) and
syntaxin 5 (ref. 57), and is strongly
influenced by changes in intracellular lipid
content58. Rab7 and LC3 form a complex
with FYVE and coiled-coil domain
containing 1 (FYCO1) that promotes
microtubule plus end-directed transport of
autophagic vacuoles59. Interactions of
Rab7 with other endolysosome proteins
influence lysosome positioning60,61.
These interrelationships closely link
endocytic and autophagic dysfunction in
various neurodegenerative disorders62.
The dynein–dynactin complex is the target
of mutations causing lower motor neuron
disease (with mutations in the dynactin
p150 subunit) and axonal Charcot-Marie-
Tooth hereditary neuropathy type 2
(CMT2, with mutations in the dynein motor
complex), and its dysregulation may also
underlie the motor neuron disease spinal
and bulbar muscular atrophy63. Decreased
activity of the dynein complex as a result of
dynactin mutations is also believed to
cause autophagosomes to appear in motor
neurons of people with ALS before clinical
symptoms, as well as autolysosomes and
p62-positive inclusions to accumulate as
TEÓRICO AUTOFAGIA 26

these neurons degenerate at clinical stages several protein-misfolding diseases65.

of disease64. Loss of dynein complex Amphisome formation may be impaired in
function increases autophagosome familial ALS and frontotemporal dementia
number and the amounts of aggregation- linked to chromosome 3.
prone proteins in models of ALS and

Figure 4 Clearance of autophagic substrates. (a) Fusion of an autophagosome with an

endosome or a lysosome is shown. LC3-II– associated autophagosomes move along
microtubule tracks, the principal binding sites for LC3. Autophagosomes generated actively in
axons fuse quickly with Rab7-positive late endosomes, and the amphisomes move nearly
exclusively in the retrograde direction via the dynein–dynactin complex. Microtubule
acetylation, regulated in part by HDAC6, recruits dynein and kinesin-1 to microtubules and is
essential for fusion of autophagosomes with lysosomes. Amphisome formation requires some
of the same proteins that direct formation and maturation of multivesicular bodies (MVBs)
and late endosomes, including Rab11, the HOPS complex, and components of multiple ESCRT
complexes that mediate cargo sorting into intraluminal vesicles of the multivesicular bodies.
Autolysosome formation also requires late endosome proteins, including Rab7, syntaxin 5,
several SNARES, LAMP1 and LAMP2 and endosomal Vps34 PI3K CIII complex. Following
TEÓRICO AUTOFAGIA 27

autophagosome-lysosome fusion, the inner membrane LC3-II is degraded and the outer
membrane LC3-II is deconjugated and recycled by Atg4. (b) For lysosomal digestion, lysosomal
acidification depends on vATPase, a multimeric enzyme complex that moves protons across
the lysosome membrane. The electrical gradient created is counterbalanced by parallel influx
of anions mediated by chloride proton antiporters. Cations, including calcium, can efflux
through distinct channels or transporters, including TPC2 and TRPML (mucolipin), which may
also influence pH. Proton leak pathways (dashed lines) require continued vATPase activity to
maintain a steady-state pH. Acidification kinetics are also contingent on the luminal buffering
power (not depicted). The more than 60 intralumenal acid hydrolases and the major lysosomal
integral membrane proteins are not depicted. The functions of many of the 25 lysosomal
transmembrane proteins are not yet clarified. Following substrate digestion, autophagy
regulation continues through interactions of the generated amino acids and a protein
complex, the ragulator, composed of multiple amino acid–sensing small GTPases, including
Rheb and Rag, which reversibly dock TORC1 and TFEB, depending on the amino acid
concentration. At low amino acid concentrations, as occurs during starvation, TORC1 activity
and phosphorylation of its substrate TFEB are inhibited, which releases TFEB to translocate to
the nucleus and induce5 transcription of genes that support increased autophagy. In a final
mTOR-dependent event, membranes from the maturing autolysosome are recovered for
lysosome biogenesis by a process involving the pinching off of evaginated surface membrane
(bottom drawing). AD, Alzheimer’s disease; FTD3, frontotemporal dementia 3; MND, motor
neuron disease; ML IV, mucolipidosis type IV; MPS IIIA, mucopolysaccharidosis type IIIA; MSD,
multiple sulfatase deficiency; NCL, neuronal ceroid lipofuscinosis; PD, Parkinson’s disease;
SBMA, spinal-bulbar muscular atrophy

In both disorders, mutations of CHMP2B superoxide dismutase (SOD1), the rate of

(loss of function charged multivesicular
body protein 2B) disrupt the ESCRT
machinery and cause ubiquitin-positive
aggregates to accumulate in neurons66,67.
Rab7, another effector of endosome
maturation essential for complete
autophagy flux68, is mutated in forms of
CMT2, resulting in Rab7
overactivation69,70. Rab7 affects
functions of another lateendosome
protein PLEKHM1, and mutations in the
gene encoding this protein cause
osteopetrosis71, a disease primarily of
lysosomes in osteoclasts but that
frequently has a neurodegenerative
component (Table 1). A mutant form of
ALS2, an activator of Rab5, impedes ALS2-
mediated Rab5 activation on newly
forming amphisomes in this familial form
of ALS72, and, when it is expressed in a
mouse model of ALS that expresses
lysosome protein degradation slows,
inducing autophagosome accumulation
accompanied by neurodegeneration73.
Lysosomal mechanisms. Completion of
autophagy requires lysosomal digestion of
autophagic cargoes and the release of
metabolites for reuse and signaling
functions (Fig. 4b). Upon fusion with an
autophagosome, lysosomes introduce the
necessary degradative machinery,
including a proton pump—the vacuolar
ATPase (vATPase)—and several dozen
hydrolytic enzymes of all types (for
example, proteases, lipases and
nucleases). These hydrolases, most of
which have acidic pH optima, are activated
when the autolysosomal lumen becomes
acidified to pH ~4.5–5.0 by the vATPase, a
large protein complex on the lysosomal
membrane that imports hydrogen ions74.
The ionic milieu of the lysosome is
TEÓRICO AUTOFAGIA
controlled by anion and cation channels
that regulate ion fluxes (Ca2+, Cl−, K+ and
others) and may also modulate H+ content
(that is, lysosomal pH) and possibly
interactions of lysosomes with the plasma
membrane and other vesicles74. Heavily
glycosylated membrane proteins, such as
LAMP1 and LAMP2, protect lysosomal
membranes from self-digestion74, but
lysosome integrity is susceptible to
disruption in disease states by a host of
modified products of lysosomal digestion
or by internalized exogenous agents, and
the stability of these lysosomal
membranes may be a potential
therapeutic target75. Although crucial for
proteolytic clearance during autophagy,
lysosomes also influence the initial stages
of autophagy. Notably, this occurs through
a signaling complex anchored to the
vATPase, which reversibly docks TORC1
and the transcription factor EB (TFEB) (Fig.
4b). When translocated to the nucleus,
TFEB coordinately upregulates expression
of most genes involved in lysosome
biogenesis and additional genes required
for autophagosome formation. TORC1
prevents this nuclear translocation76,77
and preserves its tether to protein 14-3-3
on lysosomes. Negative regulation of TFEB
by TORC1 is mediated through the amino
acid–Rag GTPase pathway78,79, a
component of a multiprotein signaling
complex (the ‘ragulator’) containing
several small GTPases, including RHEB, that
serve as amino acid–sensing activators of
TORC1 (ref. 80). TORC1 activation and TFEB
phosphorylation on lysosomes are
maintained in part by amino acids
accumulating within the lysosome or
released during lysosomal proteolysis and
by a coupled glutamine–leucine shuttle
system regulating intracellular leucine81.
When amino acid supplies are lowered, as
occurs during starvation, the inhibited
TORC1 relocates away from lysosomes,
which induces autophagy and allows
28
nuclear translocation of dephosphorylated
TFEB and the increased expression of
lysosomal network genes. A sustained
period of induction reactivates TORC1 and
returns it to the lysosome, thus
terminating autophagy50,76. Lysosomal
membrane, but not intralumenal contents,
is recaptured from expanded
autolysosomes by pinching off regions of
the autolysosomal membrane76, which is
also a TORC1-dependent process. These
lysosome–TORC1 inter-relationships have
thus far been mainly demonstrated under
nutrient starvation conditions that are
normally not seen in neurons in vivo
because the brain has unique mechanisms
to buffer against such extreme nutritional
challenges82. Nevertheless, it seems
reasonable to suspect that the
pathophysiological conditions in
neurodegenerative disease states can elicit
similar lysosome–TORC1 cross talk. For
example, loss of the lysosomal proton
gradient caused by energy depletion or
pathological conditions may depress
TORC1 activity by blocking proton-coupled
exchange of nutrients or by directly
affecting the vATPase83. Similarly,
lysosomal membrane permeabilization
could promote nutrient leakage and
suppression of TORC1 (refs. 84,85). The
very close connection between lysosomal
dysfunction and neurodegeneration is
illustrated by the lysosomal storage
disorders (LSDs), the majority of which are
characterized by severe
neurodegenerative phenotypes. In many
LSDs, a defect in any one of several
functionally dissimilar lysosomal enzymes
leads to impaired autophagic turnover of
proteins, causing particularly extensive
autophagy pathology in the brain86. For
example, the lysosomal protease cathepsin
D (CatD) is ubiquitously expressed, yet
loss-of-function mutations of CatD cause
neuronal ceroid lipofuscinoses (NCLs; also
known as Batten’s disease), a severe
TEÓRICO AUTOFAGIA
neurodegenerative disorder associated
with congenital mental retardation, or a
juvenile neurodegenerative NCL subtype
associated with dementia in humans and
other mammalian species87. Deleting CatD
or CatB and CatL causes similar phenotypes
in mice88. In Niemann-Pick type C disease
(NPC), defective trafficking of cholesterol
and other lipids caused by mutations in
either of two related endosomal genes,
NPC1 and NPC2, leads to robust
autophagic vacuole accumulation
preceding neuronal loss89. Notably, NPC is
one of a very few disorders in which
neuropathological lesions typical of
Alzheimer’s disease, such as neurofibrillary
tangles, endosome anomalies and
intracellular amyloid β-peptide (Aβ42),
develop in the absence of mutations in
Alzheimer’s-related genes90. Dystrophic
neurites filled mainly with autophagic
vacuoles, another identifying feature of
Alzheimer’s disease pathologies that is also
seen in NPC, are caused by an inhibition of
lysosomal proteases caused by stored
lipids and exacerbated by excessive
autophagy induction89. Defective
autophagy may also be the basis for
neurodegeneration in mucolipidosis type
IV, a congenital disorder that
disproportionately affects the brain. Loss-
of-function mutations in TRPML1, an ion
channel on late endosomes and lysosomes,
abnormally acidifies lysosomal pH91 and
delays the clearance but not the fusion of
autophagic vacuoles92. Autophagosome
clearance may be impaired in
osteopetrosis associated with
neurodegeneration93,94. In one form of
the disease, the vATPase A3 subunit is
mutated, leading to defective lysosomal
acidification95. Autophagosome-lysosome
fusion is also impaired in several additional
lysosomal storage disorders95,96 (Table
1). Disruption of autolysosomal proteolysis
in Alzheimer’s disease causes a striking
neuronal autophagy pathology similar to
29
that in the LSDs and more robust than in
any other late-onset brain disease.
Autophagic vacuoles and especially
autolysosomes are the principal organelles
contained within characteristic giant
neuritic swellings97, which suggests that
autophagosomes can form and fuse with
lysosomes but that elimination of
substrates from these autolysosomes is
defective. Inhibiting lysosomal proteolysis
produces similar neuropathology in wild-
type mice and exacerbates amyloid and
autophagy pathology in mouse models of
Alzheimer’s disease98. Key genetic risk
factors and proteins involved in
Alzheimer’s disease pathogenesis directly
impair lysosome function98. The
Alzheimer’s disease–linked protein
presenilin 1 is required for lysosome
acidification and thus for protease
activation99. Presenilin 1 mutations, the
most common cause of early-onset familial
Alzheimer’s disease, disrupt these
lysosomal functions99 and markedly
accelerate disease onset and
neuropathological severity100. The
strongest genetic risk factor for late-onset
Alzheimer’s disease is an allele of APOE
(which encodes a neuronal cholesterol
transport protein) that encodes a protein
variant called ApoE4 that destabilizes
lysosomal membranes101 in an allele-
specific manner. Other factors contributing
to Alzheimer’s disease pathogenesis such
as reactive oxygen species and
accumulated Aβ peptide, oxidized lipids
and lipoproteins similarly impede
lysosomal proteolysis, damage lysosomal
membranes and disrupt lysosomal
integrity, releasing proteases that can
mediate the mixed necrosis and apoptosis
pattern of neuronal cell death seen in this
disease102–104. Abnormal upregulation
of Rab5 and associated endocytosis
effectors, one of the earliest disease-
specific responses of neurons in
Alzheimer’s disease brains105, interferes
TEÓRICO AUTOFAGIA
with lysosome proteolysis by markedly
accelerating endocytosis and backing up
protein and lipid cargoes in swollen late
endosomes106. Many factors that increase
risk of Alzheimer’s disease also promote
Rab5 upregulation, including mutant forms
of β-amyloid precursor protein (APP) found
in familial Alzheimer’s disease, APP
duplication, the ApoE4 variant of
apolipoprotein and elevated amounts of
dietary cholesterol98. In sporadic and
familial Parkinson’s disease, the affected
neurons develop inclusions containing α-
synuclein. Elevated amounts of this
protein, such as those resulting from
triplication of the gene encoding α-
synuclein, are sufficient to cause
Parkinson’s disease107. Much of the α-
synuclein, especially overexpressed and
misfolded aggregate-prone forms, is
degraded by macroautophagy, which is
upregulated as CMA becomes
compromised in this disease108–110.
Autophagic vacuoles accumulate when
mutant or even wild-type α-synuclein is
overexpressed in transgenic mice109,110,
and, in rat PC12 cells, expression of mutant
α-synuclein decreases lysosomal
acidification111 and slows lysosomal
protein turnover46. Lysosomal pH is also
high in cells from patients with Parkinson’s
disease caused by loss-of-function
mutations of lysosomal ATPase, ATP13A2,
leading to proteolytic failure112. The most
common autosomal-dominant form of
Parkinson’s disease and a familial variant
that closely resembles sporadic
Parkinson’s disease clinically113 involves
LRRK2 mutation. Specific inhibitors of the
kinase encoded by this gene exist114,
making it a particularly interesting
therapeutic target. The range of LRRK2
functions in different diseases is
expanding115, and roles in autophagy and
endosomal trafficking (via GTPase
regulation) have been proposed. Altered
LRRK2 activity due to Parkinson’s disease–
30
related mutations has been linked to
defects in endosomal-lysosomal
trafficking116, lysosomal pH and calcium
regulation117 and CMA45. A mouse model
of Parkinson’s disease resulting from
exposure to the neurotoxin 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP)
shows accumulation of autophagosomes
and an early decrease in lysosome number
in dopaminergic neurons as a result of
lysosomal membrane destabilization and
cytosolic release of cathepsins84. Finally,
mutations in lysosomal acid β-glucosidase
(GCase) that cause Gaucher’s disease also
increase the risk of Parkinson’s disease in
carriers who are not affected develop-
mentally by Gaucher’s disease118. The
compromised lysosomal degradation and
neurodegeneration seen after deletion of
the gene encoding GCase in mice can be
largely prevented by overexpressing wild-
type, but not mutant, forms of GCase119.
Elevated autophagy induction in
neurodegenerative states Increased
induction of autophagy is relatively
frequent in neurodegenerative diseases,
including in some disorders in which
autolysosome clearance mechanisms are
impaired. What this greater induction rate
signifies for disease development or
implies for autophagy-related therapies,
however, is not straightforward. In
experimental models in which a disease-
related protein (for example, α-synuclein,
or SOD1) is overexpressed120–124, the
observed autophagy induction may in part
be a response to increased protein burden
independently of disease-specific factors,
and assessing this contribution may
require future investigations of additional
model systems (such as knock-in mice or
fibroblasts and stem cells from patients).
Autophagy induction in response to
mutant or damaged proteins and
aggregates could be compensatory and
neuroprotective at early stages of disease,
becoming counterproductive only if
TEÓRICO AUTOFAGIA
lysosomal clearance becomes
compromised as in some overexpression
models120–123. Compromised
autolysosome function could also
secondarily elevate autophagy induction
when the concentrations of amino acids
are decreased and TORC1 activity is
suppressed. A pathological level of
autophagy induction has been inferred in a
few neuropathological states from the
cytoprotection that is seen when
autophagy is blocked genetically or
pharmacologically, as in hypoxic ischemic
brain injury125 and two Parkinson’s
disease models: 6-hydroxydopamine–
injured nigral neurons and N-methyl-4-
phenyl pyridium (MPP) neurotoxicity126.
Although autophagy induction responses
are generally viewed as neuroprotective,
possibly in these acute injury situations the
response may be overexuberant. In NPC1-
null cells89 and possibly in the mutant
ESCRT-III mouse model of frontotemporal
dementia54, in both of which inhibition of
autophagy had ameliorative effects, the
observed benefit may reflect a lowered risk
for lysosomal membrane destabilization
due to reduced delivery of autophagic
substrates to compromised lysosomes95.
Therapeutic modulation in such situations
is clearly complex; however, in certain
disorders in which autophagy clearance
mechanisms are not known to be
substantially compromised, such as
Huntington’s disease, the further
promotion of autophagy induction might
be a logical interventional strategy.
Autophagy modulation as a therapy
Autophagy is an attractive therapeutic
target for a diverse range of diseases127.
For most of the neurodegenerative
disorders discussed earlier, the available
evidence favors a strategy of enhancing
the efficacy of autophagy by targeting the
stages that are specifically disrupted in
each disease. The expected benefits of
such modulation might include lowered
31
amounts of toxic protein aggregates and
incompletely digested autolysosomal
metabolites, more effective responses to
stress from redeploying non-essential
constituents for energy and adaptive
protein synthesis, and suppression of
apoptotic cascades or necrosis by
prevention of lysosomal membrane
destabilization103,123. Stimulating the
induction of autophagy has received the
greatest attention so far, although
preventing or reversing impairments in
autolysosomal clearance of substrates is an
equally promising therapeutic approach
and may prove to be the strategy of choice
or necessity for certain neurodegenerative
disorders (Table 2). At least in cell
models2,89,128, when lysosomal
clearance is impaired, inducing autophagy
exacerbates pathology, which implies that,
in a disease such as Alzheimer’s disease,
the success of any autophagy induction
intervention may depend on first relieving
the block in lysosomal clearance or
possibly preempting this block by
stimulating autophagy induction very early
in the disease. Enhancing autophagy
induction and sequestration. Substantial
benefits of therapy with autophagy-
inducing agents have been observed in
various mouse models of different
neurodegenerative diseases. Rapamycin, a
relatively selective inhibitor of TORC1,
ameliorates neuropathology and
neurodegeneration in transgenic mouse
models of Huntington’s disease (mutant
huntingtin)129, Alzheimer’s disease
(mutant APP)130,131, prion disease132,
spinocerebellar ataxia type 3 (SCA type 3;
ref. 133) and Parkinson’s disease (mutant
α-synuclein)134. Notably, with a few
exceptions, these studies have relied on
models in which a mutant pathogenic
protein is overexpressed, and how an
increased protein burden contributes to
the observed phenotype or its rescue by
stimulation of autophagy is currently
TEÓRICO AUTOFAGIA
unclear. Moreover, in nearly all of these
studies, contributions of possible
autophagyindependent salutary effects of
mTOR inhibition on neural cells and mouse
behavior135 have not been excluded. Also,
in a few models— SOD1 (G93A) transgenic
mice136 and MPTP neurotoxin models of
Parkinson’s disease137— rapamycin
worsens autophagy functions and
neurotoxicity, possibly for reasons
discussed in the previous section. TORC1
inhibitors with greater specificity and
fewer pharmacological limitations or side
effects are now under investigation,
including agents that inhibit both TORC1
and TORC2 (ref. 138). For all of these
agents, a key caveat to their use is the
possibility of off-target effects given the
diverse physiological roles of mTOR
signaling. Autophagy induction can be
upregulated independently of mTOR by
activating the ULK1 kinase AMPK, through
the stimulation of cAMP– inositol 1,4,5-
trisphosphate (IP3) or calpain–G-
stimulatory protein α (Gsα) pathways139.
Not surprisingly, drugs influencing these
signaling pathways affect multiple
processes besides autophagy, some of
which may amplify or limit therapeutic
efficacy140. The mood stabilizer lithium is
one of a group of clinically used agents (for
example, valproate and carbamazepine)
that induce mTOR-independent autophagy
by inhibiting inositol monophosphatase
and the phosphoinositol cycle141. Lithium
enhances the cellular clearance of
aggregate-prone forms of huntingtin and
α-synuclein141. It has similar benefits in
SOD1 G93A–expressing mice that respond
poorly to rapamycin142, although,
interestingly, lithium yields mixed results in
several ALS models that do respond to
rapamycin143,144, which suggests that
the disease mechanisms in the different
ALS models may not be identical. Mainly
because of its additional inhibitory effect
on glycogen synthase kinase 3β (GSK-3β)-
32
mediated tau phosphorylation, lithium has
been studied in relatively small numbers of
individuals with prodromal Alzheimer’s
disease (mild cognitive impairment), and
positive outcomes on some cognitive and
tau measures have been observed145.
Negative results have been reported in
several small short-term trials of lithium in
people with Alzheimer’s disease, although
these may reflect trial design
limitations145 as well as the lower efficacy
expected of therapeutics applied late in
this disease. Among a group of other US
Food and Drug Administration–approved
compounds that also inhibit the
phosphoinositol cycle139, the
hypertensive agent rilmenidine showed
promising effects in a study of a transgenic
mouse model of Huntington’s disease that
expresses mutant huntingtin, and it is in
clinical trials for this disease127. Trehalose,
a disaccharide with pharmacological
chaperone activity, which possibly acts
through AMPK activation, enhances
clearance of mutant forms of huntingtin, α-
synuclein and tau while conferring
cytoprotective effects in cell and
transgenic mouse models146. Plant-based
polyphenols have a wide range of
molecular actions, which for some include
activation of AMPK. Polyphenols, such as
resveratrol, quercetin and catechins, and
plant alkaloids, such as berberine and
curcumin, share this property and a range
of neuroprotective actions in models of
neurodegenerative disease, particularly in
models of Alzheimer’s disease—a disorder
in which the link to altered AMPK signaling
is relatively strong140. That some of these
compounds extend lifespan in part by
promoting autophagy via sirtuin 1 (SIRT1),
a molecule that influences longevity147,
indicates how closely cellular aging
mechanisms are tied to autophagy failure
in late-onset neurodegenerative diseases.
Combining an agent that lowers IP3
concentrations (for example, lithium,
TEÓRICO AUTOFAGIA
trehalose, small-molecule enhancers of
rapamycin, L-690,330 and calpastatin) with
rapamycin may have additive
benefits143,144 but may also possibly
multiply off-target effects—good and bad.
Several compounds, such as the natural
flavone finsetin, have dual actions on
TORC1 and AMPK activities, both favoring
autophagy induction, and are
neuroprotective in several disease
models148. Protein phosphatase 2A
(PP2A) agonists, which are currently in
clinical trials for Alzheimer’s disease
because of their inhibitory effects on tau
hyperphosphorylation, also promote
induction via TORC1 and AMPK149.
Interestingly, the clinically used
antidiabetic agent metformin, which
attenuates disease development in some
but not all Alzheimer’s disease mouse
studies140, is a strong AMPK activator and
also increases PP2A activity among other
metabolic effects. Targeting a different
early stage of autophagy, the beclin-1–PI3K
CIII complex, when modulated, can
enhance autophagosome formation.
Intracerebral delivery of virally packaged
Becn1 accelerates autophagy, reduces
accumulation of pathogenic proteins and
slows the development of
neurodegenerative changes in transgenic
mouse models of Parkinson’s disease110
and SCA type 3 (ref. 150). Smallmolecule
beclin-1 mimetics151 could, therefore,
hold clinical promise for
neurodegenerative disease. Enhancing
selective autophagy. Selectively
eliminating specific autophagic targets
while preserving basic housekeeping
functions of macroautophagy has
considerable conceptual appeal as a
therapeutic intervention.
Pharmacologically mobilizing an
autophagic receptor that targets specific
substrates could conceivably be achieved
by altering the receptor’s expression or
phosphorylation state152 or by modifying
33
the substrate target. For example,
acetylation of mutant huntingtin
selectively targets it to
autophagosomes153 and might be
accomplished using histone deacetylase
inhibitors, already known to be
neuroprotective in Huntington’s disease
models. Aging-related declines in LAMP2A
function could be targeted to enhance
CMA, as suggested by the increased
huntingtin clearance seen in cells
overexpressing LAMP2A or Hsc70 (ref.
154). Membrane-penetrable synthetic
‘minichaperones’ mimicking the actions of
Hsc70 or other co-chaperones might be
modeled after earlier prototypes155.
Enhancing lysosome efficiency.
Autolysosomal dysfunction is relatively
unexplored as a therapeutic target given its
frequent occurrence in brain diseases.
Numerous therapeutic directions are
possible, and those investigated so far
show promise in disease models, strongly
supporting this approach and underscoring
the pathogenic importance of lysosomal
dysfunction in Alzheimer’s disease,
Parkinson’s disease and possibly other
neurodegenerative disorders. Stimulation
of sluggish cathepsin activities, achieved by
genetically deleting the endogenous
lysosomal cysteine protease inhibitor
cystatin B, substantially accentuated
autophagy and attenuated β-amyloid
pathology as well as cognitive decline in a
transgenic mouse model of mutant APP
expression156. Similar functional benefits
and improved synaptic functions have
been seen in other APP mouse models
after either deleting cystatin C157,
overexpressing CatB158 or
pharmacologically raising the activities of
cysteine proteases159. The latter strategy,
which involves the chronic administration
of a cysteine protease inhibitor, suggests a
possible chaperone mechanism by which
more active protease is stabilized and
delivered from the endoplasmic reticulum
TEÓRICO AUTOFAGIA
to lysosomes. Indeed, reversible cell-
penetrant small-molecule inhibitors that
act as pharmacological chaperones for
deficient or defective lysosomal hydrolases
have proven effective in treating models of
several LSDs160 and are in early-stage
clinical trials for Gaucher’s disease161.
Pharmacological chaperones might be
used to promote folding and delivery of
other lysosomal components essential for
proteolysis, such as vATPase in Alzheimer’s
disease99 and ATPase ATP13A2 in
Parkinson’s disease112, or to clear
accumulated lipids that impede
proteolysis89,162,163. Unclogging
lysosomes by clearing lipids is how 2-
hydroxypropylβ-cyclodextrin is suspected
to work. This molecule is currently in early
clinical trials for NPC on the basis of its
efficacy in lowering autophagic substrate
burden and rescuing neurological deficits
in an NPC mouse model164. Accumulation
of the lipopigment lipofuscin during brain
aging and in disease may also lower
lysosomal system efficiency165, and its
removal in several neuropathological
settings, including clinical macular
degeneration—a disorder with prominent
lipofuscinosis166—is cytoprotective. The
venerable lipofuscinolytic agent
centrophenoxine (meclofenoxate), a well-
tolerated ‘antiaging’ antioxidant that had
equivocal effects for late-stage dementia
in small early clinical trials167, may serve
as a prototype for more effective
lipofuscinolytic agents. Additional
approaches to clear storage materials or
other interfering factors from lysosomes
by pharmacologically reducing the
synthesis of precursors to the offending
substrates or by replacing the missing or
defective hydrolase using direct
administration or viral or stem cell delivery
are in clinical use for LSDs or are being
actively investigated in clinical
trials161,168. Although this is an area of
active and promising investigation, obvious
34
limitations are imposed by the blood-brain
barrier in treating central nervous system
disease. Additional lysosomal clearance
enhancement approaches exploit TFEB-
mediated upregulation of the gene
transcription that supports
autophagosome formation and lysosomal
biogenesis, which can be modulated by
phosphorylation. TFEB expression
attenuates storage phenotypes in
neuronal stem cells isolated from the
cerebral cortex of mice modeling multiple
sulfatase deficiency and
mucopolysaccharidosis type IIIA and in
fibroblasts from people with Batten’s
disease by a mechanism that also involves
the activation of lysosome exocytosis169.
In this regard, non-neuronal cells are
known to jettison some incompletely
digested lysosomal substrates via calcium-
regulated lysosomal exocytosis, a property
shared by glial cells and negatively
regulated by neutral sphingomyelinase 2
and neuraminidase170,171. This process is
impaired in mucolipidosis type IV172.
Mature neurons are not known to
exocytose lysosomal contents, but they
can release cargoes via exosomes—a
regulated and potentially druggable exit
pathway from multivesicular bodies. It has
also been proposed that amphisome
cargoes such as neuronal Aβ, might be
disposed of in part by this alternative
route173. A potential caveat to this
approach is the recent discovery that the
release and reuptake of pathogenic
proteins such as tau and synuclein may
promote neuron-to-neuron propagation of
disease via a prion-like templating of the
misfolded state of the protein174.
Lysosomal stability. The neuronal cell
death that is tightly linked to autophagy
clearance failure involves lysosomal
membrane permeabilization (LMP) as a
major triggering or amplifying factor103.
Despite the knowledge that many
pathogenic factors in neurodegenerative
TEÓRICO AUTOFAGIA
disease promote LMP as previously
discussed, there have been few attempts
to mitigate this process pharmacologically
so far. Calpains, which are abnormally
activated in Alzheimer’s disease and
several other major neuropathological
states, negatively regulate autophagy
induction and are important mediators of
LMP in neuronal cell death. They act in part
by cleaving Hsp70, an established
protectant against LMP175. Synthetic
calpain inhibitors that may potentially act
against varied targets have striking
therapeutic effects in mouse models of
Alzheimer’s disease and other
tauopathies176. Their action in both
promoting autophagy induction and
protecting against LMP further strengthens
a previously strong rationale to develop
highly specific calpain inhibitors to treat
neurodegenerative disease. Lipids that
accumulate abnormally in lysosomes in
Alzheimer’s disease and some LSDs,
including gangliosides and sphingosine,
promote LMP162,163,177 and provide
another rationale for pharmacologically
reducing their amounts using approaches
previously mentioned. Finally, elevated
lysosomal pH has been linked to autophagy
failure and disease pathogenesis in a
growing number of neurodegenerative
disorders, and its correction is an obvious
therapeutic target and could be potentially
druggable through the multiple pathways
that regulate lysosomal pH178,179.
Concluding considerations The evidence
supporting a pathogenic role for
autophagy impairment in several major
neurodegenerative diseases is becoming
persuasive and provides a strong rationale
for developing therapeutics to modulate
autophagy in these disorders. As promising
as these beginnings are, the surface has
barely been cracked on the understanding
of regulation of autophagy, especially in
the broader context of proteostasis and
general metabolic regulation. The recent
35
identification of over 400 candidate
interacting proteins that support the basal
autophagy network180 underscores the
complexity of autophagy but, more
importantly, hints at the potential for
manipulating a range of new control points
along the autophagy pathway in
increasingly selective ways. These more
strategic interventions of the autophagic
pathway may ultimately replace the
current first generation of drug targets
such as mTOR with its many possible off-
target effects on cell signaling. Concurrent
programs to identify autophagy inhibitors
or enhancers for cancer, infectious disease
and other major disorders, which
complement the drug discovery programs
for neurodegenerative diseases, should
yield a steady pipeline of candidate
compounds. A major roadblock in moving
these agents closer to the clinic, however,
is the dearth of tools to measure
autophagy activity in tissues and to
establish target engagement or drug
efficacy in vivo. Available histological and
biochemical readouts in tissues mainly
assess autophagy induction and not overall
autophagic substrate turnover, which is
often blocked by disease at sites
downstream of autophagy induction.
Methods are urgently needed to monitor
autophagic turnover rates in vivo directly
or through surrogate biomarkers and
ideally to quantify changes in activity at
each stage of the autophagy pathway,
including the later lysosomal proteolytic
stages, where static measurements of
protease activities in tissues are
notoriously uninformative of actual
activities in situ. Evaluating the in vivo
efficacy of autophagy drugs in the brain is
further challenged by obstacles such as
blood-brain barrier penetration of the
agents and cellular heterogeneity of brain
tissue. How neurons and glia contribute
differentially to the overall autophagy
activity in brain and the possibility that
TEÓRICO AUTOFAGIA
different, or even reciprocal, responses to
a drug may occur in different neural cell
types are a few of the issues that will need
to be considered in these in vivo
evaluations. Functional assays that enable
autophagy fluxes in specific brain cell
populations to be quantified in situ are
essential for current drug validation
studies in mouse models and for later use
in clinical trials. Fluorescent reporters of
CMA, autophagosome maturation and
lysosomal proteolysis have recently been
developed for cell studies, and the
expression of such constructs in neuronal
populations in mice would be an important
first step forward in preclinical testing of
new compounds. The recent major clinical
trials of Alzheimer’s disease drugs have
emphasized the importance of establishing
target engagement to interpret outcomes,
and the ongoing trials now rely heavily on
surrogate biomarkers measured in
cerebrospinal fluid or by neuroimaging in
addition to cognitive measures. Clinically
useful biomarkers of autophagy, a
particularly high priority, have yet to be
investigated. Successes at preventing
disease onset in mouse models with
autophagy inducers and the more limited
success so far in reversing preexisting
MECHANISMS AND REGULATION OF AUTOPHAGOSOME FORMATION
Autophagy is an intracellular pathway for
the bulk degradation of cytoplasmic
substances such as cytosol, protein
aggregates and organelles. Autophagy is
characterized by the formation of double-
membrane bound vesicles called
autophagosomes, which engulf the cargo
and transport it to the vacuole/lysosome
for breakdown and recycling. Even though
several proteins in this pathway have been
identified, little is known about the
36
disease suggest that early drug
administration and thus very high margins
of drug safety are necessary. In this regard,
therapies involving dietary products or
manipulations represent a promising
direction for future investigation given that
autophagy is, above all, a homeostatic
response to changing supplies of nutrients,
growth factors or energy. More complete
characterization of nutrientsensing
mechanisms and their regulation by
dietary factors and caloric intake may
ultimately enable basal autophagy to be
enhanced safely over long periods of time.
Indeed, lifestyle practices, including diets
and exercise regimens to prevent the
metabolic syndrome of obesity, diabetes
and cardiovascular dysfunction are already
linked to a lowered risk for Alzheimer’s
disease181, and failure of autophagy is
strongly suspected to have an important
role in promoting this syndrome182. The
diversity of possible ways that autophagy
might be targeted therapeutically provides
optimism that autophagy modulation may
ultimately prove to be an effective
therapeutic approach for neuro-
degenerative diseases.
mechanism of action of these proteins
during autophagosome biogenesis. In this
review we briefly discuss recent findings on
the molecular players and mechanisms
involved in autophagosome formation. In
particular, we will focus on the
mechanisms regulating membrane
recruitment as well as membrane
remodeling during autophagosome
formation.
Introduction
TEÓRICO AUTOFAGIA
Macroautophagy (hereafter autophagy) is
an important intracellular degradation
pathway conserved among eukaryotes. It
mediates intracellular homeostasis by the
degradation of long-lived proteins and
entire organelles. Autophagy also
contributes to the adaptive response to
starvation and various other extrinsic and
intrinsic stresses. Not surprisingly, defects
in autophagy have been associated with
several human diseases, including
increased susceptibility to infectious
diseases, neurodegenerative disorders and
cancer. Despite its various important roles,
the regulation and mechanisms of
autophagy induction and autophagosome
formation remain poorly understood.
During autophagy cytoplasmic cargo is
engulfed by double-membrane organelles
called autophagosomes. After their
formation autophagosomes fuse with
lysosomes (or the vacuole in yeast), within
which the inner membrane and the cargo
are degraded. Both non-selective ‘bulk’
autophagy as well as the selective
autophagy of specific proteins or
organelles have been described [1]. During
non-selective autophagy, bulk cytosol and
other cytoplasmic components are
thought to be non-specifically sequestered
into autophagosomes. By contrast, during
selective autophagy cargo is selectively
sequestered into autophagosomes. These
latter structures tend to contain little
cytoplasm and their shape reflects that of
their cargo. Selective types of autophagy
mediate the degradation of mitochondria
(mitophagy) [2,3], the elimination of
peroxisomes (pexophagy) [4], the selective
removal of ribosomes (ribophagy) [5] as
well as the elimination of invading bacteria
and viruses (xenophagy) [6,7]. In addition,
another selective and highly related
pathway called the cytoplasm-to-vacuole
37
targeting (Cvt) pathway exists in yeast
cells. This pathway fulfilling a biosynthetic
function selectively delivers three cytosolic
enzymes – aminopeptidase 1 (Ape1), a-
mannosidase (Ams1), and aspartyl
aminopeptidase (Ape4) – to the vacuole
[8–10]. In the following sections we will
briefly discuss recent progress in our
understanding of autophagy with
emphasis on the regulation and
mechanisms of autophagosome
formation.
Autophagy proceeds through a series of
steps
Autophagy pathways can be broken down
into the following series of steps:
Induction, cargo recognition,
autophagosome nucleation,
autophagosome expansion and
completion, autophagosome fusion with
the vacuole/lysosome and breakdown, and
recycling of the resulting macromolecules
(Figure 1, [11]). Genetic analyses in yeast
have identified over 35 genes, termed Atg1
to Atg35 that are required at one or more
of these steps during autophagy [12]. A
subset of these Atg proteins is required for
autophagosome formation in all types of
autophagy. These proteins are referred to
as the ‘core’ autophagic machinery [13]
and can be divided into three different
functional groups: Firstly, the Atg1-ULK1
kinase complex is required for the
induction of autophagy and regulates
together with Atg2 and Atg18 the cycling of
Atg9 to an from the site of autophagosome
formation. Secondly, Atg6 and Atg14 are
part of a phosphatidylinositol 3-kinase
complex required for vesicle nucleation.
Finally, two conjugation systems that are
built around the ubiquitin-like proteins
Atg8 and Atg12 are essential for
autophagosome formation. The first
TEÓRICO AUTOFAGIA 38

conjugation system entails the conjugation phosphatidylethanolamine (PE) [14]. Atg8–

of Atg8 (or its orthologs in complex PE conjugation is mediated by Atg3 and
eukaryotes) to the membrane lipid Atg7.

The second conjugation system entails the Signaling in autophagy

conjugation of Atg12 to an internal lysine
The level of autophagy in a cell must be
of Atg5 [15]. The Atg5–Atg12 conjugate
finely balanced, as too little can be
subsequently forms a non-covalent
deleterious, but excessive levels of
complex with the coiled-coil protein Atg16
autophagy may also be harmful. The
[16]. Orthologs of most of these yeast Atg
cellular signaling events inducing or
proteins exist in complex eukaryotes [13].
restricting autophagy, as well as the
mechanisms of membrane formation and
TEÓRICO AUTOFAGIA
cargo selection, remain poorly understood.
Epistasis analyses in yeast revealed that
the conserved Atg1/ULK1 kinase functions
upstream of cargo selection and
autophagosome induction. Atg1 is a
conserved serine-/threonine protein
kinase and a key regulator of autophagy
and the Cvt pathway [17]. It is found in a
large complex composed of several
proteins including Atg13, another Atg
protein essential for autophagy function
[18]. Atg1/ULK1 and Atg13 are highly
phosphorylated. Their phosphorylation
status changes depending on the nutrient
availability, suggesting a phospho-
dependent regulatory mechanism for
autophagy induction [18–20]. Indeed,
Atg1/ULK1 and Atg13 were found to be
phosphorylated by the target of rapamycin
(TOR) complex 1 under nutrient-rich
conditions in mammals and yeast, and
additional regulated phosphorylation
events were observed for yeast Atg1 [21],
indicating that extra kinases are involved in
regulating the Atg1 complex. Atg13
phosphorylation in yeast is believed to
reduce its binding affinity for Atg1, thereby
preventing Atg1 kinase activation under
nutrient rich conditions. In flies and
mammals, conversely Atg1/ULK1 and
Atg13 constitutively bind to each other
independent of the nutrient availability.
This indicates mmthat the Atg1 complex in
these organisms is unlikely to be regulated
by its association with Atg13, but rather by
phosphorylation or the nutrient-
dependent interaction of additional factors
[22,23]. While it seems likely that
Atg1/ULK1 plays an essential role in
autophagy regulation from yeast to
humans, the precise regulation and
components of the Atg1/ULK1 kinase
complex as well as Atg1 kinase targets
39
important for autophagy function remain
to be investigated in more detail.
Atg9: the membrane-recruiting factor?
In yeast autophagosomes are generated at
the pre-autophagosomal structure (PAS),
which is localized close to the vacuole.
Most Atg proteins are cytosolic and only
transiently associate with the PAS [24].
Atg9 is the only conserved transmembrane
protein essential for autophagosome
formation [25]. In yeast, Atg9 is one of the
first components associating with the PAS
and most other Atg proteins require Atg9
for their localization at this perivacuolar
site [24], suggesting a role for Atg9 in
assembling the core autophagic
machinery. Atg9 (mAtg9 in mammals)
forms a conserved complex together with
Atg2 and Atg18 (WIPI-1 in mammals).
Owing to its early localization to the PAS
and it being an integral membrane protein,
Atg9 has been the focus of many studies
aimed at understanding the nature of the
PAS and the origin of the autophagosomal
membranes. Autophagosomes have been
proposed to either form from preexisting
membranes or to assemble de novo by
vesicular transport. Various membrane
sources have been suggested to supply
lipids during autophagosome formation
(see below). In contrast to other Atg
proteins, Atg9 in yeast is found in
peripheral cytoplasmic structures in
addition to the PAS, and has been
proposed to cycle between these two
locations [26]. This cycling depends on the
Atg1 kinase complex [26] as well as on the
ability of Atg9 to multimerize [27]. Both
cycling and multimerization have been
suggested to contribute to the recruitment
of autophagosomal membranes, as
mutants defective in cycling and
multimerization fail to support
TEÓRICO AUTOFAGIA
autophagosome formation [26,27]. In
mammalian cells mAtg9 also shows a dual
localization. One pool of mAtg9 localizes to
the perinuclear region where it colocalizes
with proteins from the trans-Golgi
network. A second pool has been shown to
partially overlap with endosomal markers
[28]. The endosomal pool increases
following nutrient starvation and ULK1 has
been found to regulate this redistribution
event similar to yeast Atg1 [28]. The
precise role of Atg1/ULK1 in regulating the
localization of Atg9/mAtg9 remains to be
further investigated. However, if Atg9
cycling results in a net transport of lipids to
the autophagosomal membrane, the
density of Atg9 on the membrane
structures leaving the PAS must be higher
than on the structures reaching the PAS.
Importantly, Atg9 is believed to be absent
from the completed autophagosome [25],
implying that a retrieval mechanism from
these structures must also exist. Atg1
40
signaling might be involved in regulating
this process. Several factors involved in
Atg9 trafficking have been described in
yeast and mammals and a picture is slowly
emerging (Figure 2). Nevertheless, further
analysis of thenature of the cycling events
taken by Atg9, as well as the regulation of
the retrieval mechanisms by Atg1/ULK1
and other factors, is required to
understand the role of Atg9 in
autophagosomal membrane recruitment.
Membrane dynamics during
autophagosome formation
In mammalian cells several sources for the
autophagosomal membrane have been
suggested. The endoplasmic reticulum
[29–31], the Golgi apparatus [32 ,33 ],
mitochondria [34] as well as the plasma
membrane [35,36 ] have been implicated
in contributing lipids to isolation
membranes. As discussed in a number of
recent reviews it now seems likely that
TEÓRICO AUTOFAGIA
mammalian cells have the ability to utilize
different compartments as a lipid source
for autophagosome formation [37,38].
However, it is not clear whether a common
mechanism exists that mediates the
formation of isolation membranes and
ultimately autophagosomes from these
various donor membranes. It has been
observed in electron microscopy studies
that isolation membranes are connected to
the ER by small necklike continuities
[30,31]. Supporting evidence for continuity
between the isolation membrane and the
ER comes from light microscopy
experiments where it was observed that an
ER-resident transmembrane protein has
access to isolation membranes [29].
Similarly, it has been postulated from
advanced light microscopy studies that
isolation membranes grow out of the outer
mitochondrial membrane [34]. The
mechanism by which the isolation
membranes keep their unique identity
while connected to their donor
membranes(ER and mitochondria,
respectively) remains unclear.
Alternatively, it has been proposed that
isolation membranes are formed by the
fusion of vesicular carriers that are derived
from the plasma membrane [35,36 ] or the
Golgi apparatus [32 ,33 ]. The fusion of
these vesicular carriers could mediate the
formation of isolation membranes and
eventually autophagosomes.In these
vesicular fusion models there is no need
for any diffusion barrier separating the
isolation membrane from the donor
membrane, since autophagosome
formation occurs far from the donor
membrane. Recent evidence lends support
to the vesicular precursor hypothesis. By
following Atg9 in Atg9 overexpressing cells
using electron microscopy, it was observed
that after induction of autophagy a post-
41
Golgi tubulo-vesicular compartment
positive for Atg9 moves en bloc to the
vacuole [39].It was further proposed that
this tubulo-vesicular compartment
undergoes homotypic fusion and
remodeling in order to give rise to isolation
membranes [39]. It remains unclear how
this remodeling occurs. The mechanism by
which vesicular precursors fuse to mediate
isolation membrane formation is not fully
understood. Yeast Atg8 when conjugated
to PE has been proposed to mediate vesicle
tethering and hemifusion [40]. Hemifusion
is an intermediate step towards full fusion
where the contacting but not the distal
monolayers of the membranes are fused
[41]. Recently the tethering activity of
yeast Atg8 has been confirmed; however,
the hemifusion activity of Atg8 is only
observed when very high PE
concentrations are present in the vesicles
[42]. These high PE concentrations are
unlikely to represent the physiological
conditions in vivo. Thus, unless the tubulo-
vesicular precursors have an unusual and
highly fusogenic lipid composition, Atg8 is
unlikely to mediate fusion. In the same
paper the authors presented evidence for
the involvement of SNARE proteins in
membrane fusion events that give rise to
the Atg9-positive tubulovesicular
precursors [42]. It therefore seems likely
that these SNAREs are localized on the
precursors when they undergo homotypic
fusion during isolation membrane
formation and could thus mediate their
fusion. As in yeast, the lipidated forms of
the mammalian Atg8 homologues LC3 and
GATE-16 have been shown to mediate
membrane tethering [43 ]. In addition, in
the same study both proteins were shown
to mediate full fusion of vesicles at
relatively low and thus physiologically
relevant PE concentrations [43 ]. The
TEÓRICO AUTOFAGIA
reason for the higher apparent fusogenic
activity of LC3 and GATE-16 compared to
yeast Atg8 is unclear. However, in
agreement with findings in yeast where
SNAREs promote the fusion of
autophagosomes [42], it has recently been
shown that SNARE-dependent homotypic
fusion of Atg16L positive precursors is
required for autophagosome formation in
mammalian cells [36 ]. Cells may therefore
employ the conventional fusion machinery
composed of SNAREs and their accessory
molecules to form autophagosomes.
Concluding remarks
In recent years tremendous progress has
been made regarding the identification of
MAMMALIAN AUTOPHAGY: CORE MOLECULAR MACHINERY AND SIGNALING
REGULATION
Autophagy, a cellular catabolic pathway, is
evolutionarily conserved from yeast to
mammals. Central to this process is the
formation of autophagosomes, double-
membrane vesicles responsible for
delivering long-lived proteins and excess or
damaged organelle into the lysosome for
degradation and reuse of the resulting
macromolecules. In addition to the
hallmark discovery of core molecular
machinery components involved in
autophagosome formation, complex
signaling cascades controlling autophagy
have also begun to emerge, with mTOR as
a central but far from exclusive player.
Malfunction of autophagy has been linked
to a wide range of human pathologies,
including cancer, neurodegeneration, and
pathogen infection. Here we highlight the
recent advances in identifying and
understanding the core molecular
42
the core molecular machinery required for
autophagy. The challenge for the future is
to understand the mechanisms of action as
well as the regulation of the autophagic
protein machinery. For example, how does
Atg1 induce autophagosome formation
(i.e. what are its relevant targets)? What is
the role of Atg9 during autophagosome
formation? Does it indeed supply lipids to
the isolation membrane? How are
membranes sculpted into isolation
membranes and finally autophagosomes?
What are the roles of the Atg8 and Atg12
conjugation systems during these
processes? Answering these questions will
bring us closer to a mechanistic
understanding of the autophagic process.
machinery and signaling pathways that are
involved in mammalian autophagy.
Introduction
Autophagy, literally meaning ‘self-eating’,
embraces three major intracellular
pathways in eukaryotic cells,
macroautophagy, microautophagy, and
chaperonemediated autophagy (CMA),
which share a common destiny of
lysosomal degradation, but are
mechanistically different from one another
[1,2]. During macroautophagy, intact
organelles (such as mitochondria) and
portions of the cytosol are sequestered
into a double-membrane vesicle, termed
an autophagosome. Subsequently, the
completed autophagosome matures by
fusing with an endosome and/or lysosome,
thereby forming an autolysosome. This
latter step exposes the cargo to lysosomal
TEÓRICO AUTOFAGIA
hydrolases to allow its breakdown, and the
resulting macromolecules are transported
back into the cytosol through membrane
permeases for reuse (Figure 1). By
contrast, microautophagy involves the
direct engulfment of cytoplasm at the
lysosome surface, whereas CMA
translocates unfolded, soluble proteins
directly across the limiting membrane of
the lysosome. In this review, we will focus
on mammalian macroautophagy
(hereafter referred to as autophagy),
which plays important physiological roles
in human health and disease. The basal,
constitutive level of autophagy plays an
important role in cellular homeostasis
through the elimination of damaged/old
organelles as well as the turnover of long-
lived proteins and protein aggregates, and
thus maintains quality control of essential
cellular components. On the other hand,
when cells encounter environmental
stresses, such as nutrient starvation,
hypoxia, oxidative stress, pathogen
infection, radiation, or anticancer drug
treatment, the level of autophagy can be
dramatically augmented as a the recent advances in mammalian
cytoprotective response, resulting in
adaptation and survival; however,
dysregulated or excessive autophagy may
lead to cell death. Thus, defective
autophagy has been implicated in the
pathogenesis of diverse diseases, such as
certain types of neuronal degeneration
and cancer, and also in aging [3]. Although
43
autophagy was first identified in
mammalian cells approximately 50 years
ago, our molecular understanding of it only
started in the past decade, largely based on
the discovery of autophagy-related (ATG)
genes initially in yeast followed by the
identification of homologs in higher
eukaryotes [4]. Among these Atg proteins,
one subset is essential for autophagosome
formation, and is referred to as the ‘core’
molecular machinery [5]. These core Atg
proteins are composed of four subgroups:
first, the Atg1/unc-51-like kinase (ULK)
complex; second, two ubiquitin-like
protein (Atg12 and Atg8/LC3) conjugation
systems; third, the class III
phosphatidylinositol 3-kinase
(PtdIns3K)/Vps34 complex I; and fourth,
two transmembrane proteins, Atg9/mAtg9
(and associated proteins involved in its
movement such as Atg18/WIPI-1) and
VMP1. The proposed site for
autophagosome formation, to which most
of the core Atg proteins are recruited, is
termed the phagophore assembly site
(PAS). In this review, we mainly highlight
autophagy in terms of the molecular
machinery involved in the formation and
maturation of autophagosomes and the
signaling cascades needed for the
regulation of autophagy. The clarification
of how autophagy is modulated in
response to intracellular and
TEÓRICO AUTOFAGIA 44
TEÓRICO AUTOFAGIA
extracellular stresses relies largely on the
elucidation of the signaling network
upstream of the Atg machinery. Core
molecular machinery ULK complexes The
yeast serine/threonine kinase Atg1 plays a
key role in the induction of autophagy,
acting downstream of the target of
rapamycin (TOR) complex 1 (TORC1). A
family of mammalian Atg1 proteins has
been identified; among these, unc-51-like
kinases 1 (ULK1) and 2 have the highest
similarity with yeast Atg1 and appear to be
closely related. siRNA knockdown of ULK1
or ULK2 blocks autophagy in HEK293 cells
[6 ]. However, ULK1/ mice display normal
autophagy in response to nutrient
deprivation, but delay mitochondrial
clearance during reticulocyte maturation
[7]. The basis for these differences is not
known. It is possible that in some tissues,
ULK2 can compensate for the deficiency of
ULK1. Furthermore, a role of ULK3 in
autophagy induction in oncogene-induced
cell senescence has been described
recently [8]. Thus, at least three ULKs are
involved in mammalian autophagy
regulation and they have mechanistically
different roles in vivo. Yeast Atg1 exists in
a complex with Atg13 and Atg17. Atg13 is
phosphorylated in a TORC1-dependent
manner and the phosphorylation state of
Atg13 modulates its binding to Atg1 and
Atg17; inactivation of TORC1 leads to
dephosphorylation of Atg13, increasing
Atg1–Atg13– Atg17 complex formation
and activating autophagy [4,9]. ULK1 and
ULK2 are also in a large complex that
includes the mammalian homolog of Atg13
(mAtg13) and the scaffold protein FIP200
(an ortholog of yeast Atg17) [6 ,10,11 ].
mAtg13 is essential for autophagy, and it
directly interacts with ULK1, ULK2, and
FIP200 independent of its phosphorylation
state [6 ,11 ]. FIP200 is also required for
45
autophagy and binds to ULK1 and ULK2
independent of nutrient status [12 ], in
contrast to the yeast Atg1–Atg17
interaction. In addition, under nutrient-
rich conditions, the large ULK1–Atg13–
FIP200 complex contains mammalian
TORC1 (mTORC1); conversely, following
nutrient deprivation, mTORC1 is quickly
dissociated from the ULK1 complex [11 ].
There are several phosphorylation events
within this complex, including
phosphorylation of mAtg13 by ULK1, ULK2,
and mTORC1, phosphorylation of FIP200
by ULK1 and ULK2, and phosphorylation of
ULK1 and ULK2 by mTORC1 (Figure 1) [6 ,11
]. Under conditions that induce autophagy,
a decrease in mTORC1 activity leads to
dephosphorylation of ULK1, ULK2, and
mAtg13, activation of ULK1 and ULK2, and
phosphorylation of mAtg13 and FIP200 by
ULK1 and ULK2 [6 ,11 ]. Further studies are
required to characterize the functional
significance of these phosphorylation
events. Recently, a new, mAtg13-
interacting protein, Atg101, was found to
interact with ULK1 in a mAtg13-dependent
manner, and is essential for autophagy
[13]. However, the role of the ULK1–
Atg13–Atg101 complex in autophagy
regulation remains unclear.
Two ubiquitin-like proteins, Atg12 and
Atg8/LC3, and their conjugation systems
Studies in yeast and mammals have
identified two ubiquitin-like proteins,
Atg12 and Atg8/LC3, and their respective,
partially overlapping, conjugation systems,
which are proposed to act during
elongation and expansion of the
phagophore membrane. Atg12 is
conjugated to Atg5 in a reaction that
requires Atg7 and Atg10 (E1 and E2-like
enzymes, respectively). The Atg12–Atg5
conjugate then interacts noncovalently
TEÓRICO AUTOFAGIA
with Atg16L, which oligomerizes to form a
large multimeric complex called the Atg16L
complex. Atg8/LC3 is cleaved at its C
terminus by Atg4 to generate the cytosolic
LC3-I with a Cterminal glycine residue,
which is conjugated to
phosphatidylethanolamine (PE) in a
reaction that requires Atg7 and the E2-like
enzyme Atg3. The lipidated form of LC3
(LC3-II) is attached to both faces of the
phagophore membrane, but is ultimately
removed from the autophagosome outer
membrane, which is followed by fusion of
the autophagosome with a late
endosome/lysosome [4]. Recent work
suggests that these two ubiquitination-like
systems are closely connected. On the one
hand, the Atg16L complex is localized to
the phagophore and it can act as a novel
E3-like enzyme, determining the sites of
Atg8/LC3 lipidation [14,15]. On the other
hand, the Atg8/LC3 conjugation machinery
seems to be essential for the formation of
the Atg16L complex. In Atg3- deficient
mice, where no LC3-II can be detected,
Atg12–Atg5 conjugation is markedly
reduced, and dissociation of the Atg16L
complex from the phagophore is delayed;
autophagosomes are smaller than in the
wild type and appear either open-ended or
multilamellar [16], indicating a role for the
Atg16L complex and LC3 lipidation for the
elongation and closure of the phagophore.
This hypothesis is further supported by the
observation that overexpression of an
inactive mutant of Atg4 inhibits the
lipidation of LC3, and in these cells a
significant number of nearly complete
autophagosomes are not closed [17].
Class III phosphatidylinositol 3-kinase
complex
In yeast, the only PtdIns3K is Vps34, and it
exists in two different complexes,
46
complexes I and II. Complex I, consisting of
Vps34, Vps15, Atg6, and Atg14, is required
for the induction of autophagy, and the
lipid kinase activity of Vps34 is essential for
generating phosphatidylinositol (3)-
phosphate (PtdIns(3)P) at the PAS to allow
the recruitment of other Atg proteins.
Complex II, consisting of Vps34, Vps15,
Atg6, and Vps38, is required for the
vacuolar sorting of carboxypeptidase Y. In
mammals, there are two types of PtdIns3K:
classes I and III. Formation of the
mammalian class III PtdIns3K complex,
including hVps34, Beclin 1 (a homolog of
Atg6), and p150 (a homolog of Vps15), is
conserved. The orthologs of Atg14 and
Vps38 have recently been identified and
are called Atg14-like protein (Atg14L or
Barkor) and ultraviolet irradiation
resistance-associated gene (UVRAG),
respectively [18–20]. Atg14L plays an
important role in mammalian autophagy.
Under nutrient-rich conditions, a
subpopulation of Atg14L localizes to the
ER; upon starvation, Atg14L localizes to
Atg16L-positive and LC3-positive
structures, indicating the phagophore and
autophagosome, respectively,
independently of the interaction of Atg14L
with hVps34 and Beclin 1 [18,21].
Importantly, depletion ofAtg14L reduces
Atg16L and LC3 puncta formation [21].
Overexpression of Atg14L stimulates the
kinase activity of hVps34, and induces
autophagy, whereas Atg14L knockdown
reduces PtdIns(3)P production, and inhibits
autophagy [19,22]. Thus, a possible role of
Atg14L is to direct the class III PtdIns3K
complex to the phagophore to initiate the
recruitment of Atg machinery. Recent
studies suggest that UVRAG participates in
at least four different mechanisms to
regulate autophagy. First, UVRAG
competes with Atg14L for binding to Beclin
TEÓRICO AUTOFAGIA
1; the interactions of Atg14L and UVRAG
with the Beclin 1–hVps34–p150 complex
are mutually exclusive [18,19]. Second,
UVRAG interacts with Bif-1 (Baxinteracting
factor 1); Bif-1 is required for autophagy
and colocalizes with Atg5, LC3, and mAtg9
during starvation [23]. It is proposed that
the recruitment of Bif-1 via UVRAG may
provide the machinery to deform
membranes, as Bif-1 has an N-BAR domain
and shows membrane binding and bending
activities [24]. Third, UVRAG interacts with
the class C Vps/HOPS proteins, promoting
autophagosome fusion with the late
endosome/lysosome, thereby accelerating
delivery and degradation of autophagic
cargo [25 ]. Fourth, the recently identified
Rubicon (RUN domain and cysteine-rich
domain containing, Beclin 1-interacting)
protein forms a complex with UVRAG–
Beclin 1–hVps34–p150; this complex
localizes to the late endosome/lysosome
and negatively regulates autophagosome
maturation [21,22]. Rubicon reduces
hVps34 activity and inhibits autophagy. In
addition to hVps34, Atg14L, and UVRAG,
Beclin 1 also interacts with Ambra1
(activating molecule in Beclin 1- regulated
autophagy). Ambra1 functions as a positive
regulator of autophagy and the mechanism
remains unclear [26]. Collectively, there
exist multiple mammalian hVps34–Beclin 1
complexes that may participate in distinct
steps of autophagy regulation (Figure 1),
either at the early stage to promote
autophagosome formation or at the later
stage to promote autophagosome
maturation.
Transmembrane proteins in mammalian
autophagy
Mammalian Atg9 (mAtg9) and vacuole
membrane protein 1 (VMP1) are the two
transmembrane proteins so far identified
47
that are required for mammalian
autophagy. mAtg9, with both the N and C
termini in the cytosol, spans the
membrane six times. It is located in the
transGolgi network and late endosomes,
and upon starvation or rapamycin
treatment, redistributes to peripheral
sites, overlapping with GFP-LC3-positive
autophagosomes. The cycling of mAtg9
after starvation is ULK1-dependent, and
also requires the kinase activity of hVps34
[27], which is similar to the yeast protein
[28]. Although its functions remain
unclear, based on the existing data from
yeast Atg9, mAtg9 potentially contributes
to the delivery of membrane to the
forming autophagosome, an attractive
model that needs to be experimentally
tested in mammalian cells. In contrast to
mAtg9, VMP1 has no known homologs in
yeast. The localization of VMP1 is
controversial: in mammalian cells it is
localized to the plasma membrane and also
colocalizes with LC3 and Beclin 1 upon
autophagy induction [29], whereas the
VMP1 homolog in Dictyostelium
discoideum localizes to the ER [30]. In
mammalian cells, ectopical overexpression
of VMP1 triggers autophagy even under
nutrient-rich conditions, whereas
depletion of VMP1 blocks starvation-
induced and rapamycin-induced
autophagy [29]. Importantly, VMP1
interacts with Beclin 1, and this interaction
is essential for autophagy induced by
VMP1 overexpression [29]. VMP1 might
function as a transmembrane protein that
recruits Beclin 1 and other components in
the class III PtdIns3K complex to the
phagophore. This is supported by a recent
finding that a novel VMP1-interacting
protein, TP53INP2 (tumor protein 53-
induced nuclear protein 2), is essential for
the translocation of Beclin 1 and LC3 to
TEÓRICO AUTOFAGIA
autophagosomes upon autophagy
stimulation, potentially through its
interaction with VMP1 [31 ]. TP53INP2 is
essential for autophagy. It translocates
from the nucleus to autophagosomes upon
autophagy induction, where it interacts
with LC3 as well as VMP1, but not Beclin 1.
SIGNALING PATHWAYS REGULATING
AUTOPHAGY
PtdIns3K–Akt–mTORC1
TOR is a highly conserved serine/threonine
protein kinase that acts as a central sensor
of growth factors, nutrient signals, and
energy status. TOR serves as a master
regulator of autophagy [32]. TOR exists in
two distinct complexes, TORC1 and TORC2
that are conserved from yeast to
mammals, and TORC1 has a primary
function in regulating autophagy. In yeast,
inhibiting the TORC1 complex during
nitrogen starvation or by rapamycin
stimulates autophagy [4]. The mTORC1 is
48
also sensitive to rapamycin, which in many
settings stimulates autophagy. However, a
recent report challenged this view by
showing that rapamycin and siRNA
knockdown of one of the key downstream
effectors of mTORC1, S6 kinase 1 (S6K1),
inhibit autophagy in cancer cells [33], and
a more recent finding shows that mTORC1
regulates autophagy through an unknown
mechanism that is essentially insensitive to
rapamycin [34]. mTORC1 integrates
upstream activating signals that inhibit
autophagy through the class I PtdIns3K-
protein kinase B (PKB, also known as Akt)
pathway (Figure 2). Upon association with
growth factor, receptor tyrosine kinases
undergo autophosphorylation and become
activated, leading to the stimulation of two
key signal-transducing components: the
small GTPase Ras and class I PtdIns3K. Class
I PtdIns3K catalyzes the production of
PtdIns(3)P at the plasma membrane, which
increases
TEÓRICO AUTOFAGIA
membrane recruitment of both PKB and its
activator PDK1 (phosphoinositide-
dependent protein kinase 1), leading to the
activation of PKB. PtdIns3K activity can be
opposed by PTEN, a 30 -phosphoinositide
phosphatase, subsequently decreasing
PKB activity, and inhibiting mTOR.
PtdIns3K–PKB activation suppresses
autophagy in mammalian cells. PKB further
activates mTORC1 through inhibiting a
49
downstream protein complex, the
tuberous sclerosis complex 1/2
(TSC1/TSC2). The TSC1/TSC2 heterodimer,
which is a stable complex, senses the
upstream inputs from various kinases,
including PKB and ERK1/2 [35,36].
Phosphorylation of TSC2 by PKB or ERK1/2
leads to the disruption of its complex with
TSC1, and results in mTOR activation.
TSC1/TSC2 acts as the GTPase-activating
TEÓRICO AUTOFAGIA
protein for Rheb, a small GTP-binding
protein that binds to and activates mTOR
in its GTP-bound form. Ras has opposing
roles in autophagy regulation: it inhibits
autophagy by activating the PtdIns3K–
PKB–mTORC1 pathway, and at the same
time, it may induce autophagy via the Raf-
1–MEK1/2–ERK1/2 pathway [37,38].
Finally, the mTORC2 complex is also
involved in autophagy regulation. Full
activation of PKB requires mTORC2 [39],
and inhibition of PKB, caused by mTORC2
depletion, reduces the phosphorylation of,
and therefore activates, the forkhead box
O (FoxO3) transcription factor, which
stimulates autophagy in muscle cells
independent of the activity of mTORC1
[40].
AMPK
The AMP-activated protein kinase (AMPK)
is another sensor of cellular bioenergetics,
specifically in response to energy stress.
During nutrient and energy depletion,
AMPK is activated by a decreased
ATP/AMP ratio through the upstream LKB1
kinase (encoded by the Peutz–Jeghers
syndrome gene). Active AMPK leads to
phosphorylation and activation of
TSC1/TSC2 and inhibition of mTORC1
activity. Thus, the phosphorylation of
TSC1/TSC2 by AMPK and PKB has opposite
effects on mTORC1 and connects mTORC1
with energy and growth factor signaling,
respectively (Figure 2). Recently, it is
reported that AMPK regulates mTORC1
signaling through an alternative
mechanism, whereby AMPK directly
phosphorylates Raptor, a subunit of
mTORC1, and this Raptor phosphorylation
is important for the inhibition of mTORC1
signaling by AMPK [41]. Thus, AMPK serves
as a positive regulator of autophagy. Under
stress conditions, the LKB1–AMPK
50
pathway phosphorylates and stabilizes
p27kip1, a cell cycle inhibitor, and
stabilized p27kip1 induces autophagy [42].
An increase in the cytosolic free Ca2+
concentration and cytokines (such as
TRAIL) activates AMPK via activation of the
Ca2+/ calmodulin-dependent kinase
kinase-b (CaMKKb) and transforming
growth factor-b-activating kinase 1 (TAK1),
respectively, and these pathways are
required for Ca2+- induced or TRAIL-
induced autophagy [43,44 ]. Moreover,
AMPK activity contributes to the induction
of autophagy during hypoxia [45].
p53
The p53 tumor suppressor, the ‘guardian
of the cellular genome’, has dual positive
and negative regulatory roles in autophagy
induction (Figure 2) [46]. Upon genotoxic
stress or oncogenic activation, the
activation of p53 induces autophagy; p53
activates AMPK, which in turn, activates
the TSC1/TSC2 complex, leading to the
inhibition of the mTORC1 pathway [47].
p53 can also induce autophagy through
upregulation of the damage-regulated
autophagy modulator (DRAM) [48].
Remarkably, chemical inhibition of p53,
knockdown of p53 with siRNA, or deletion
of the p53 gene can trigger the onset of
autophagy [49]. Several stimuli, including
starvation or ER stress, can induce HDM2-
dependent proteasomal degradation of
p53 to favor autophagy induction,
positioning p53 as a negative regulator of
autophagy. HDM2, the p53-specific E3
ubiquitin ligase, targets p53 to
proteasome-mediated destruction. The
inhibition of HDM2 blocks the depletion of
p53 and also prevents the activation of
autophagy [49]. More importantly, it is the
cytoplasmic p53 that exerts its inhibitory
function toward autophagy, in contrast to
TEÓRICO AUTOFAGIA
the transcriptionally active nuclear p53
that promotes autophagy. Upon
reintroduction into p53/ cancer cells,
mutants of p53 that are restricted to the
cytosol effectively inhibit autophagy,
whereas mutants of p53 that accumulate
within the nucleus fail to block autophagy
[49]. The inhibitory role of cytosolic p53 in
autophagy may contribute to the strong
oncogenic action of certain p53 mutants
that are preferentially localized to the
cytosol [50 ].
Bcl-2 protein family
In mammals, the Bcl-2 protein family plays
a dual role in autophagy regulation.
Antiapoptotic proteins, such as Bcl2, Bcl-
XL, Bcl-w, and Mcl-1, can inhibit
autophagy, whereas proapoptotic BH3-
only proteins, such as BNIP3, Bad, Bik,
Noxa, Puma, and BimEL, can induce
autophagy [51]. The binding of Bcl-2 to
Beclin 1 disrupts the association of Beclin 1
with hVps34, decreases Beclin 1-
associated hVps34 PtdIns3K activity and
thereby inhibits autophagy. There are at
least three distinct mechanisms that may
account for the release of Beclin 1 from its
inhibitory interaction with Bcl-2/Bcl-XL
(Figure 2). One model depicts that the BH3
domain of BH3-only proteins such as Bad,
may competitively disrupt the inhibitory
interaction of Beclin 1 and Bcl-2/Bcl-XL [52
]. A second mechanism for the dissociation
of Beclin 1 from its inhibitory interaction
with Bcl-2 involves the phosphorylation of
Bcl-2 by the stress-activated c-Jun N-
terminal kinase 1 (JNK1). Starvation
induces the phosphorylation of Bcl-2 at
residues T69, S70, and S87 of the
nonstructured loop; expression of a
nonphosphorylatable Bcl-2 mutant (T69A,
S70A, and S87A) or inhibition of JNK1
abolishes the starvation-triggered
51
dissociation of Bcl-2 from Beclin 1, and
inhibits autophagy; expression of a
constitutively active JNK1 results in
constitutive Bcl-2 multisite
phosphorylation, dissociation of Bcl-2 from
Beclin 1 and stimulation of autophagy [53
]. Third, a recent finding shows that the
activation of Beclin 1 to induce autophagy
involves the phosphorylation of Beclin 1 by
the death-associated protein kinase
(DAPK). DAPK physically interacts with
Beclin 1, and phosphorylates Beclin 1 on
Thr119 located at a crucial position within
the BH3 domain of Beclin 1, and thus
promotes the dissociation of Beclin 1 from
its inhibitor, Bcl-XL, and autophagy
induction [54 ].
Concluding remarks
In the past decade there has been a
tremendous advance in our understanding
of the molecular machinery involved in
mammalian autophagy. Nonetheless,
many outstanding questions remain to be
answered, including the mystery of the
membrane source for autophagosome
formation. By comparison, our knowledge
about the signaling regulation of
autophagy is relatively limited, in
particular, with regard to the complex
coordination between autophagy
machinery and signaling inputs. As an
intracellular self-destructive system,
autophagy must be tightly regulated in
order to adapt to different intracellular and
extracellular stresses. This raises a
fundamental question: How does the cell
determine the specificity and magnitude of
autophagy based on the inputs from a
variety of signaling mechanisms?
Mammalian autophagy has gained
tremendous attention because of its
implications in a wide range of
physiological processes and diseases in
TEÓRICO AUTOFAGIA
humans. Our current understanding of this
process and continued examination of its
mechanism and regulation hold the
potential for practical modulation of
AUTOPHAGY AND THE INTEGRATED STRESS RESPONSE
Abstract
Autophagy is a tightly regulated pathway
involving the lysosomal degradation of
cytoplasmic organelles or cytosolic
components. This pathway can be
stimulated by multiple forms of cellular
stress, including nutrient or growth factor
deprivation, hypoxia, reactive oxygen
species, DNA damage, protein aggregates,
damaged organelles or intracellular
pathogens. Both specific, stimulus-
dependent and more general stimulus-
independent signaling pathways are
activated to coordinate different phases of
autophagy. Autophagy can be integrated
with other cellular stress responses
through parallel stimulation of autophagy
and other stress responses by specific
stress stimuli, through dual regulation of
autophagy and other stress responses by
multi-functional stress signaling molecules,
and/or through mutual control of
autophagy and other stress responses.
Thus, autophagy is a cell biological process
that is a central component of the
integrated stress response.
Introduction
Eukaryotic cells must adapt continuously
to fluctuations in external conditions,
including physical parameters, such as
temperature and ultraviolet light; chemical
cues such as ion concentrations, pH,
oxygen tension, redox potentials and
metabolite concentrations; extracellular
52
autophagy and its use as a therapeutic
intervention.
signals such as contact-dependent signals,
hormones, cytokines and
neurotransmitters; and microbial
pathogens. Beyond a certain threshold,
such fluctuations are considered ‘stresses’,
meaning that the cell's response to this
stress determines whether it can function
properly and survive.
During the response to sublethal stress,
cells undergo rapid changes to adapt their
metabolism and protect themselves
against potential damage. This is
orchestrated through a multifaceted
cellular program, which involves the
concerted action of diverse stress response
pathways. One of the key pathways that
mediates stress-induced metabolic
adaptation and damage control is
macroautophagy, referred to simply as
“autophagy”. During autophagy, cells form
double-membraned vesicles,
autophagosomes, that sequester
organelles, proteins, or portions of the
cytoplasm, for delivery to the lysosome (He
and Klionsky, 2009). The sequestered
contents are degraded in the lysosome,
allowing cells to eliminate damaged or
harmful components through catabolism
and recycling to maintain nutrient and
energy homeostasis. Autophagy
constitutes a major protective mechanism
that allows cells to survive in response to
multiple stressors and that helps defend
organisms against degenerative,
inflammatory, infectious, and neoplastic
TEÓRICO AUTOFAGIA 53

diseases (Levine and Kroemer, The Core Pathway of Mammalian

2008; Mizushima et al., 2008).
Besides autophagy, the cellular response
to stress involves numerous other
pathways including those that regulate
nutrient uptake, intermediary metabolism,
cell cycle and growth control, cell fate and
lineage decisions, and cellular
survival/death programs. Therefore, it is
not surprising that there is a close
integration between signals that regulate
these cellular processes and those that
regulate autophagy. In this review, we will
summarize recent advances in
understanding how different cellular stress
signals and stress stimuli regulate
autophagy.
Autophagy
The core pathway of mammalian
autophagy (Fig. 1) begins with the
formation of an isolation membrane (also
called a phagophore) and involves at least
five molecular components, including: (1)
the Atg1/unc-51-like kinase (ULK) complex;
(2) the Beclin 1/class III
phosphatidylinositol 3-kinase (PI3K)
complex; (3) two transmembrane proteins,
Atg9 and vacuole membrane protein 1
(VMP1); (4) two ubiquitin-like protein
(Atg12 and Atg8/LC3) conjugation systems;
and (5) proteins that mediate fusion
between autophagosomes and lysosomes
(Yang and Klionsky, 2010). Some of these
core autophagy pathway components are
directly controlled by cellular stress signals
(Fig. 1).
TEÓRICO AUTOFAGIA 54

FIG. 1 Overview of the major components of the core pathway of mammalian autophagy
Several key molecular components participate in the initiation, execution and completion of
autophagy. Autophagy inducers such as starvation modulate the inhibitory interaction of
TORC1 with the ULK1/2 complex. Through phosphorylation of Ambra1, and maybe through
other putative interactions, ULK1/2 complex (A) also regulates the activity of Beclin 1/ class III
phosphatidylinositol 3-kinase (PI3K) complex (B). Beclin 1 interacts with several enhancing
(blue) or inhibitory (grey) factors that modulate its binding to Vps34, the catalytic unit of the
PI3K, whose lipid kinase activity is essential for autophagy. In addition to these two complexes,
autophagosome formation requires the participation of two ubiquitin-like protein (Atg12 and
Atg8/LC3) conjugation systems and two transmembrane proteins (Atg9 and VMP-1) (C).
Whereas the roles of Atg9 and VMP-1 are currently not completely understood, both
conjugation systems are essential for the biogenesis of the isolation membrane, also called
‘phagophore’. In addition, the Atg8/LC3 system is required for autophagosome transport and
maturation, as well as for the selection of autophagic cargo. Fully mature autophagosomes
can fuse with Rab7-positive late endosomes to form amphisomes. Finally, autophagosomes or
amphisomes fuse their external membranes with those from acidic lysosomes to acquire
hydrolytic activity, degrade their cargo and recycle essential biomolecules to the
cytoplasm (D).

Origin of the isolation membrane The Atg1/ULK Complex

The isolation membrane (also called
‘phagophore’) can be generated from
multiple sources that include the ER (Axe
et al., 2008; Hayashi-Nishino et al.,
2009; Yla-Anttila et al., 2009), the outer
mitochondrial membrane (Hailey et al.,
2010), and the plasma membrane
(Ravikumar et al., 2010). PI3P is required
for the formation of ‘omegasomes’, the Ω-
shaped structures that bud from the ER
during the initial steps of vesicle
nucleation/autophagosome formation
(Axe et al., 2008). ATG16L1 directly
interacts with clathrin, which connects the
endocytic pathway to autophagy
(Ravikumar et al., 2010). While it appears
that general autophagy inducers such as
starvation stimulate lipid recruitment from
all known sources, it is not known whether
specific stimuli (such as ER stress,
mitochondrial damage or signals
emanating from the plasma membrane)
cause autophagosomes to be
preferentially formed from specific
membrane sources.
The mammalian orthologs of yeast Atg1,
ULK1 and ULK2, play a key role in
autophagy induction, acting downstream
of the mammalian target of rapamycin
(mTOR) complex 1 (mTORC1, a polyprotein
complex that contains mTOR, Raptor,
mLST8/GßL, Deptor and PRAS40 (Efeyan
and Sabatini, 2010)). In normal (nutrient-
replete) conditions, mTORC1 possesses
kinase activity and interacts with a
complex that contains ULK1, Atg13, FIP200
and Atg101 (Fig. 1A). Upon mTORC1
inhibition, for example by starvation (see
below), mTORC1 dissociates from the ULK
complex, leading to dephosphorylation of
specific residues within ULK1 (or ULK2) and
Atg13 (which are normally phosphorylated
by mTORC1), the catalytic activation of
ULK1 (or ULK2) and the ULK1 (or ULK2)-
mediated phosphorylation of other
residues in Atg13 and FIP200 (Fig. 1A).
Thus, mTORC1 inhibition is probably
coupled to ULK1 (or ULK2) activation
through a process that involves
dissociation of a large protein complex and
(de)phosphorylation events (Mizushima,
TEÓRICO AUTOFAGIA 55

2010). An intriguing, but not yet tested,
possibility, is that the ULK1/ULK2 complex
might also be positively regulated in low
energy conditions by its recently described
interactions with several subunits of the
energy-sensing kinase, AMP-activated
protein kinase (AMPK) (Behrends et al.,
2010). It is not entirely known how ULK1/2
activates downstream components of the
autophagic machinery. However, ULK1 can
phosphorylate Ambra1 (Di Bartolomeo et
al., 2010), a component of the Beclin
1/Class III PI3K complex (He and Levine,
2010) (Fig. 1A).
There are multiple circuits of positive and
negative feedback in mTORC1-mediated
autophagy regulation (Neufeld, 2010) (Fig.
2A). Feed-forward mechanisms involve
Deptor, PRAS40 and ULK1/2, which are
each inhibited by mTORC1-mediated
phosphorylation and, in turn, inhibit mTOR
activity. This may lead to amplification of
initially modest changes in TOR activity.
Negative feedback control is achieved via
the products of autophagy including amino
acids as well as via inhibition of S6 kinase,
a protein that has mTORCI-dependent
activity and which may be necessary for
sustained autophagy (Neufeld, 2010).
Thus, mTORC1 is part of a rheostat that is
either switched off (to inhibit autophagy at
the level of ULK1/2) or on (to induce
autophagy by ULK1/2 activation, as a result
of a positive amplification loop). When on,
these effects are self-limited due to the
presence of negative feedback loops (Fig.
2A).

FIGURE 2
Overview of selected signal transduction pathways that regulate autophagy components
that function in vesicle nucleation/phagophore formation
Selected signals that converge on ULK1/2 (A) and the Beclin 1 complex (B) are depicted. Note
the multiple positive and negative feedback loops depicted in A. For details see text.
phosphate (PI3P) which recruits effectors
Beclin 1 and its Interactome
such as the double FYVE domain-
The “Beclin 1 core complex” involves Beclin containing protein 1 (DFCP1) (Axe et al.,
1, Vps15, Vps34 and likely, Ambra1 (He and 2008) and WD-repeat protein interacting
Levine, 2010). This multiprotein complex with phosphoinositides (WIPI) family
must be formed for the allosteric proteins (Polson et al., 2010) to mediate
activation of the class III PI3K Vps34 to the initial stages of vesicle
generate phosphatidylinositol-3- nucleation/autophagosome formation.
TEÓRICO AUTOFAGIA
Numerous proteins that interact with
Beclin 1 induce or inhibit autophagy (Fig.
1B). Atg14 (also called Atg14L or Barkor,
for Beclin 1-associated autophagy-related
key regulator) is essential for PI3K activity
and autophagy induction. UVRAG (UV
radiation resistance-associated gene)
competes with Atg14 for binding to Beclin
1 and may promote PI3K activity in a cell
type-specific fashion, and through
interactions with class C Vps/HOPS
complexes, promotes autophagosome
fusion with the late endosome/lysosome.
Bif-1/endophilin B1 interacts with Beclin 1
via UVRAG to function as a positive
regulator of the PI3K complex, and has an
N-BAR domain that may promote
membrane curvature (Fig. 1B). Rubicon
(RUN domain protein as Beclin 1
interacting and cysteine-rich containing)
negatively regulates autophagy (as well as
endocytic trafficking) through its
interaction with Beclin 1/PI3K complexes
(Fig. 1B).
Anti-apoptotic family members (such as
Bcl-2, Bcl-XL and Mcl-1) are also important
negative regulators of autophagy through
an inhibitory interaction of their BH3-
binding groove with the BH3 domain of
Beclin 1 (Maiuri et al., 2007; Pattingre et
al., 2005) (Fig. 2B). There are several
distinct mechanisms through which
autophagy-inducing signals can disrupt this
inhibitory interaction, including
competition by pro-apoptotic BH3-only
proteins (such as BNIP3, Bad, Bik, Noxa,
Puma and BimEL) (Maiuri et al., 2007),
phosphorylation of the BH3 domain of
Beclin 1 by death-associated protein kinase
(DAPK) (Zalckvar et al., 2009) or
phosphorylation of Bcl-2 by c-Jun N-
teminal kinase-1 (JNK1) (Pattingre et al.,
2009; Wei et al., 2008). Accordingly, BH3-
only proteins (such as BAD in starvation or
BNIP3 in hypoxia) (Bellot et al.,
2009; Maiuri et al., 2007) as well as Beclin
1/Bcl-2 dissociating kinases (such as JNK1
56
in starvation conditions) (Wei et al., 2008)
are required for autophagy induction in
response to specific forms of stress.
The inositol-1,4,5 trisphosphate (IP3)
receptor, which is an IP3-activated calcium
channel at the endoplasmic reticulum (ER),
interacts with Beclin 1 indirectly, via Bcl-2
(Fig. 2B). Upon cellular reduction of
IP3 levels (or antagonist binding) and
starvation, this interaction is disrupted
(Vicencio et al., 2009). This mechanism
may explain how agents that cause a
reduction of IP3 levels cause autophagy in
an mTOR-independent fashion (Sarkar et
al., 2007). The toll-like receptor (TLR)
adaptor molecules MyD88 and TRIF also
may interact with Beclin 1, thereby
reducing the binding of Beclin 1 to Bcl-2
and promoting autophagy (Shi and Kehrl,
2008). During TLR4-induced autophagy,
tumor necrosis factor receptor (TNFR)-
associated factor 6, TRAF6, interacts with
Beclin 1 and mediates K63-linked
ubiquitination of Beclin 1, which enhances
Class III PI3K activity (Shi and Kehrl, 2010).
The protein PINK1, which has been studied
as a serine-threonine kinase that interacts
with Parkin to stimulate mitophagy, also
interacts with Beclin 1 (Michiorri et al.,
2010). Although it is not clear how all these
proteins affect the overall composition and
function of the multiprotein Beclin 1-
containing complex, an attractive model is
that multiple proteins directly or indirectly
interact with Beclin 1 to relay extracellular
and intracellular stress signals to the
autophagic machinery.
Atg9 and VMP1
Atg9 may provide lipids to the phagophore
membrane by cycling between distinct
subcellular compartments (Fig. 1C). The
cycling of Atg9 requires Atg1/ULK1 and the
kinase activity of Vps34. Another
possibility is that Atg9 cycling involves the
UVRAG/Bif-1-containing Beclin 1 complex
TEÓRICO AUTOFAGIA
since Bif-1 transiently associates with Atg9
after starvation (Orsi et al., 2010). VMP1,
which interacts with Beclin 1, may function
as a transmembrane protein that recruits
Beclin 1 (and other components of the
Beclin 1 complex) to the phagophore (Fig.
1C). It also interacts with TP53INP2 (tumor
protein 53-induced nuclear protein 2),
which like VMP1 is essential for autophagy
and the translocation of Beclin 1 and LC3 to
autophagosomes (Yang and Klionsky,
2010).
Conjugation Systems
Two ubiquitin-like conjugation systems are
part of the vesicle elongation process (Fig.
1C). The first pathway involves the
covalent conjugation of Atg12 to Atg5,
with the help of the E1-like enzyme Atg7
and the E2-like enzyme Atg10. This
conjugate is organized into a complex by
associating with Atg16 in a non-covalent
fashion to form the multimeric Atg12-
Atg5-Atg16 complex, which functions as
the E3 ligase of LC3 (Yang and Klionsky,
2010). The abundance of Atg5 may be
regulated by calcium-dependent activation
of the cysteine protease, calpain, which
cleaves and inactivates Atg5, at least in
cultured cells. Thus, the reduction of
cytosolic Ca2+ (or calpain inactivation) may
prevent Atg5 cleavage (Fig. 1C), resulting in
increased cellular levels of full-length Atg5
and the pro-autophagic Atg12-Atg5
conjugate (Xia et al., 2010).
The second pathway involves the
conjugation of phosphatidylethanolamine
(PE) to a glycine (Gly) residue of yeast
Atg8/mammalian LC3 by the sequential
action of the protease Atg4, the E1-like
enzyme Atg7, and the E2-like enzyme Atg3
(Fig. 1B). This lipid conjugation leads to the
conversion of the soluble form of LC3
(named LC3-I) to the autophagic vesicle-
associated form, LC3-II (Yang and Klionsky,
2010). The lipidated form of LC3 is stably
57
associated with the autophagosome
membrane, and its biochemical and
microscopic detection is widely used to
measure cellular autophagy (Mizushima et
al., 2010) (Fig. 1C). In mammals, four Atg4
(Atg4A-B) and at least six orthologues of
Atg8 exist, among which LC3B (hereafter
referred to simply as LC3), GABARAP and
GATE16 have been most studied
(Weidberg et al., 2010).
Several stress signals regulate autophagy
at the level of the Atg8/LC3 conjugation
system. For example, the death-associated
protein kinase, DAPK, positively regulates
autophagy by associating with the LC3-
interacting cytoskeleton molecule MAP1B
(Harrison et al., 2008) (in addition to
phosphorylating Beclin 1, discussed above
(Zalckvar et al. 2009)). The cellular FLICE-
like inhibitor protein, c-FLIP, negatively
regulates autophagy by preventing Atg3
from binding and activating LC3 (Lee et al.,
2009). Moreover, reactive oxygen species
may regulate the active cysteine site of
Atg4 (Fig. 1C) (Scherz-Shouval R et al.
2007). The enrichment of proteins with
lipid kinase, WD40, and GTPase regulatory
domains in the mammalian Atg8-
interaction network (Behrends et al., 2010)
may provide additional clues as to how
stress signals interface with autophagy
control at the level of Atg8/LC3 regulation.
Several proteins that possess an LC3-
interacting region (LIR) and interact with
LC3 (and its paralogs) serve as adaptors to
target defined structures such as
ubiquitinated proteins or mitochondria to
the autophagic machinery (Fig. 1D). The
best-characterized examples are p62 (also
known as sequestosome1, SQSTM1) and
NBR1 (Neighbor of BRCA1), which both
recognize ubiquitinated proteins (Kirkin et
al., 2009) as well as BNIP3L (also known as
NIX), which binds to mitochondrial
membranes (Novak et al., 2010). The
regulation of these autophagy adaptor
TEÓRICO AUTOFAGIA
proteins is not yet well understood, but will
likely be key to understanding how specific
stress stimuli trigger selective autophagy.
Go to:
Autophagy, Stress Stimuli, and Stress
Signals
Autophagy is induced by a variety of stress
stimuli, including nutrient and energy
stress, ER stress, pathogen-associated
molecular patterns (PAMPs) and danger-
associated molecular patterns (DAMPs),
hypoxia, redox stress, and mitochondrial
damage. The stimulation of autophagy by
these stimuli involves diverse signals that
have overlapping functions in autophagy
and the control of other cellular stress
responses.
FIGURE 3
Overview of the major signal transduction pathways that regulate autophagy in response
to starvation
58
Autophagy Induced by Nutrient Stress
Nutrient depletion is the most potent
known physiological inducer of autophagy.
In the majority of cultured mammalian
cells, nutrient depletion induces
autophagy within minutes, with maximal
levels observed when cells are cultured in
the simultaneous absence of nutrients
(such as amino acids and glucose) and
growth factors (such as those contained in
serum) (Boya et al., 2005). In mice,
following starvation for 24-48 hours, most
cells in most tissues display increased
numbers of autophagosomes (Mizushima,
2010). Several critical molecules regulate
starvation-induced autophagy (Fig. 3A); of
these, mTOR and AMPK have been best
characterized, and recent studies also
suggest a crucial role for sirtuins.
TEÓRICO AUTOFAGIA
A summary of starvation-induced pro-autophagic signaling (A) is followed by a schematic
overview of the signaling cascades involving sirtuin-1 and Foxo 3a (B), AMPK (C) and mTORC1
(D).
Sirtuins and protein (de)acetylation
Sirtuins are a family of NAD-dependent
deacetylases that sense environmental
stress (Haigis and Sinclair, 2010). The
induction of autophagy by starvation (but
not by mTORC1 inhibition or ER stress)
requires Sirt1 (Lee et al., 2008; Morselli et
al., 2010). Accordingly, Sirt1−/− mice display
a phenotype consistent with impaired
autophagy, including increased levels of
the autophagy substrate p62,
accumulation of damaged organelles,
disruption of energy homeostasis, and
early perinatal lethality (Lee et al., 2008).
The transfection of cells with sirtuin 1
(Sirt1) with intact deacetylase activity is
sufficient to stimulate autophagy in
cultured mammalian cells (Lee et al.,
2008). In this context, it is intriguing that
p300 acetyltransferase knockdown (Lee
and Finkel, 2009), as well as histone
acetylase inhibition by spermidine, a
natural polyamine, potently induce
autophagy (Eisenberg et al., 2009),
suggesting that protein acetylation may
play a general role in autophagy
regulation.
Sirt1 can deacetylate Atg5, Atg7 and LC3
(Lee et al., 2008) (Fig. 3A), while p300 can
acetylate Atg5, Atg7, LC3 and Atg12 (Lee
and Finkel, 2009). Furthermore, Sirt1
deacetylates the transcription factor
forkhead box O3a, FOXO3a, yet another
hub of autophagy regulation, leading to
enhanced expression of pro-autophagic
Bnip3 (Kume et al., 2010). Akt inhibition
resulting from growth factor depletion also
causes FOXO3 activation, although via a
different mechanism. Following its
dephosphorylation, FOXO3 translocates
into the nucleus (Fig. 3A) and upregulates
multiple autophagy-related genes such as
59
ULK2, Beclin 1, VPS34, BNIP3 and BNIP3L,
ATG12, ATG4B, LC3,
and GABARAPL1 (Mammucari et al., 2007).
Another sirtuin, Sirt2, dissociates from
FOXO1 upon serum starvation, thus
causing the acetylation of FOXO1, favoring
its interaction with Atg7 in the cytoplasm
and stimulating autophagy (Zhao et al.,
2010). Thus, protein (de)acetylation
reactions influenced by sirtuins and other
enzymes may control autophagy at
multiple levels.
AMPK in starvation, energy depletion,
and beyond
AMPK acts as a central node that
integrates several stress stimuli with
autophagy initiation (Fig. 3C). AMPK
monitors the energy status of the cell by
sensing its AMP:ATP ratio. Several
upstream kinases, including liver kinase B1
(LKB1, which is activated by energy
depletion), calcium/calmodulin kinase
kinase-ß (CaMKKß, which is activated by
cytosolic Ca2+), and TGFß-activated kinase-
1 (TAK-1, which is also involved in IKK
activation), can activate AMPK by
phosphorylating a threonine residue on its
catalytic α-subunit (Ruderman et al.,
2010). In addition, Sirt1 and AMPK engage
in a coordinated positive amplification
loop (the “Sirt1/AMPK cycle”) (Ruderman
et al., 2010), that acts to initiate autophagy
in nutrient deprivation conditions. Sirt1-
mediated deacetylation of LKB1 increases
its serine-threonine kinase activity and
stimulates its translocation from the
nucleus to the cytoplasm where it activates
AMPK. AMPK can also indirectly stimulate
Sirt1 activation by the reduction of
nicotinamide (NAM) and an increase in
NAD+ that may be catalyzed by NAM
phosphoribosyltransferase upregulation,
TEÓRICO AUTOFAGIA
thus completing the feed forward circuitry
(Lan et al., 2008; Ruderman et al., 2010).
The best-studied mechanism by which
AMPK induces autophagy is through
mTORC1 inhibition (see below), via
phosphorylation of the tuberous sclerosis
complex 2 (TSC2) and the regulatory
associated protein of mTOR, Raptor (Fig.
3D). The recent identification of an
interaction between AMPK and ULK1
(Behrends et al., 2010) raises the
speculative possibility that AMPK may also
act more directly on core components of
the autophagy machinery to initiate
autophagy.
mTORC1 inhibition in starvation-induced
autophagy
Besides inhibition by AMPK, mTORC1 is
also inhibited upon withdrawal of growth
factors such as insulin or insulin-like
growth factor (Fig. 3C,D). In response to
growth factors, Akt becomes catalytically
active (due to the sequential stimulation of
growth factor-activated class I PI3K and
PI3K-dependent protein kinase 1, PDK1,
which phosphorylates Akt). Moreover,
growth factors activate Ras, which
stimulates a cascade involving Raf-1,
MEK1/2 and ERK1/2. Both Akt and ERK1/2
can phosphorylate one of two subunits of
the tuberous sclerosis complex 1/2
(TSC1/TSC2), and Akt can phosphorylate
Raptor, thus causing the activation of
mTOR (Fig. 3D) (Neufeld, 2010). Moreover,
amino acids activate mTORC1
independently of the Akt-TSC1/TSC2 axis,
through the Rag family of GTPases, which
directly interact with Raptor and recruit
mTORC1 to the lysosomal surface (Efeyan
and Sabatini, 2010) (Fig. 3D). Thus, mTOR is
inhibited through multiple mechanisms
during starvation conditions (Sengupta et
al., 2010).
60
Additional kinases involved in starvation-
induced autophagy
Several kinases besides AMPK function in
starvation-induced upregulation of
autophagy. This includes JNK1, which both
phosphorylates Bcl-2, reducing its affinity
for the BH3 domain of Beclin 1 (Wei et al.,
2008), and phosphorylates Sirt1,
promoting its enzymatic activity (Nasrin et
al., 2009) (Fig. 3A). Moreover, as noted
below, the phosphorylation of eIF2α
(Kouroku et al., 2007; Talloczy et al., 2002)
and the activation of IKK (Criollo et al.,
2010) are essential for starvation-induced
autophagy. Other kinases such as p38α
mitogen-activated protein kinase (p38α
MAPK, also known as MAPK14) potently
inhibit starvation-induced autophagy.
p38α MAPK acts on p38IP, which is
required for starvation-induced mAtg9
trafficking and autophagosome formation
(Webber and Tooze, 2010). Thus, the
coordinated activation (and inhibition) of
several kinases is essential for the
induction of autophagy by nutrient
depletion. The precise details of such
coordination, however, remain poorly
understood.
ER Stress and Autophagy
The ER is not only involved in protein
synthesis and maturation (including
correct folding), but may also constitute a
major source/scaffold of the autophagic
isolation membrane (Hayashi-Nishino et
al., 2009; Yla-Anttila et al., 2009). The
unfolded protein response (UPR), the
major ER stress pathway (Buchberger et
al., 2010), is a potent stimulus of
autophagy. The three canonical branches
of the UPR are mediated by three ER
membrane-associated proteins, PERK
(PKR-like eIF2α kinase, also known as
EIF2AK3), ATF6 (activating transcription
factor-6), and IRE1 (inositol requiring
TEÓRICO AUTOFAGIA
enzyme 1) (Fig. 4A), all of which are
normally bound to and inactivated by the
chaperone BiP/GRP78. The occupancy of
BiP/GRP78 by misfolded proteins releases
FIGURE 4
Summary of the major signal transduction pathways that connect autophagy to ER stress
(A), eIF2α phosphorylation (B) and IKK activation (C)
For details see text.
PERK and eIF2α phosphorylation
PERK mediates the transcriptional
activation of the proteins LC3 and Atg5 in
hypoxic responses through the action of
the transcription factors ATF4 and CHOP,
respectively (Rouschop et al., 2010). PERK
may also reduce the translation of IkBα
(Deng et al., 2004), thereby activating NF-
κB, which also could contribute to
autophagy (Fig. 4B). PERK phosphorylates
the eukaryotic initiation factor 2α (eIF2α)
on residue serine 51, which then initiates a
cascade of events that decreases the
overload of misfolded proteins, thereby
alleviating ER stress (Harding et al., 2003).
61
PERK, IRE1 and ATF6 from their inhibition.
Among these, PERK and ATF6 acts as
autophagy inducers, while IRE1 acts a
negative regulator of autophagy.
eIF2α phosphorylation initiates both a
general inhibition of protein translation, as
well as the selective translation of some
stress-responsive transcripts including that
of the ATF4 transcription factor and certain
autophagy genes (Hotamisligil, 2010).
Cells that carry a non-phosphorylatable
mutant of eIF2α (S51A) fail to induce
autophagy in response to starvation,
suggesting that eIF2α phosphorylation on
serine 51 (and by extension eIF2α kinases)
plays a major role in autophagy regulation
(Kouroku et al., 2007; Talloczy et al., 2002).
This phosphorylation event integrates
various types of environmental and
TEÓRICO AUTOFAGIA
endogenous stress signals beyond ER
stress, such as amino acid deprivation,
exposure to double-stranded viral RNA,
osmotic stress, UV light exposure, heme
deficiency, hypoxia and oxidative stress
(Harding et al., 2003). These divergent
signals activate four different eIF2α kinases
including PERK (which is activated by ER
stress, radiation or hypoxia), general
control non-derepressible-2 (GCN2, which
is activated by uncharged tRNAs in amino
acid-starved cells), heme-regulated
inhibitor (HRI, which is activated by heme
deficiency in erythroid precursor cells), and
PKR (which is activated by double-stranded
RNA and in some contexts, ER stress
(Nakamura et al., 2010)) (Fig. 4B). Yeast
GCN2 and mammalian PKR and PERK have
been shown to be required for autophagy
induced by starvation, viral infection, and
ER stress, respectively (He and Klionsky,
2009). These findings illustrate the
importance of eIF2α kinases in autophagy
control, not only in the UPR but also in
other conditions of stress.
How eIF2α phosphorylation contributes to
autophagy initiation is not known. One
highly speculative possibility is that eIF2α
phosphorylation might somehow affect
the ER in a manner that promotes the
physical formation of the isolation
membrane. Another possibility is that
eIF2α phosphorylation stimulates
autophagy via its effects on the
transactivation of autophagy genes. eIF2α
phosphorylation stimulates the selective
translation of the ATF4 transcription factor
(although general translation is shut off),
which stimulates LC3 expression (which is
necessary for sustained autophagy) (Milani
et al., 2009) (Fig. 4B). Moreover, there are
direct interactions between eIF2α subunits
and core autophagy proteins, although it is
not yet known whether these interactions
are biologically significant (Behrends et al.,
2010).
62
IRE1
IRE1 is a serine/threonine kinase (which
activates JNK1) and also an
endoribonuclease, which catalyzes the
splicing of the mRNA encoding form of the
transcription factor XBP1, which then
transactivates UPR genes involved in ERAD,
protein folding and protein quality control
(Hetz and Glimcher, 2009; Hotamisligil,
2010). Perhaps unexpectedly, the
inhibition of IRE1 and its downstream
effector XBP1 enhances autophagy
induction. Mice lacking XBP1 in neurons
exhibit increased levels of baseline
autophagy, which leads to increased
turnover of an autophagic substrate,
mutant superoxide dismutase-1 (SOD1),
and protection against mutant SOD1-
induced amyotrophic lateral sclerosis (Hetz
et al., 2009). Since the predominant
outcome of ER stress is the induction
(rather than inhibition) of autophagy, it is
possible that IRE1/XBP1 dependent signals
have the function to dampen excessive
autophagy triggered via the PERK/eIF2α
pathway (Fig. 4A).
Immune Signals
Infection (or exposure of cells to microbial
products) constitutes a specialized form of
cellular stress that, in many cases, results
in autophagy induction (Sumpter and
Levine, 2010). Autophagy induction during
infection is regulated by cytokines such as
IFNγ (and downstream immunity-related
GTPases) as well as pathogen recognition
receptors (PRRs) that recognize conserved
components of pathogens or products of
their replication (PAMPs). The PRRs include
families of Toll-like receptors (TLRs) that
recognize PAMPS to activate pro-
inflammatory cytokines and type I
interferon production via NF-κB-, MAPK-
and interferon-regulatory pathways; NOD-
like receptors (NLRs) that primarily activate
signaling via NF-κB and MAPK and
TEÓRICO AUTOFAGIA
components of the inflammasome; RIG-I
like receptors (RLRs) that recognize
cytoplasmic viral RNA and dA:dT rich
dsDNA; C-type lectins, and the double-
stranded RNA binding protein kinase PKR
(Sumpter and Levine, 2010). Since these
PRRs recognize not only PAMPS, but also
DAMPs (which include products of necrotic
cells, hypoxia, abnormal intracellular ion
gradients, reactive oxygen species, and
accumulation of misfolded proteins), this
family of “immune signals” that regulates
autophagy may represent a more
generalized system that cells use to elicit
autophagy in response to different forms
of stress.
TAK1, IKK and NF-κB
The transcription factor, NF-κB, and some
of its upstream regulators, function to
integrate diverse stress signals including
immune signals with the autophagy
pathway. NF-κB is activated when the
inhibitor of NF-κB (IκB) is phosphorylated
by the IκB kinase (IKK), a kinase that is
composed of one regulatory subunit (IKKγ,
also known as NEMO) and two catalytic
subunits (IKKα, IKKβ). The IKK complex is
activated in response to multiple different
stressors such as reactive oxygen species,
DNA damage, and ligation of death
receptors and PRRs. Frequently, this IKK
activation is mediated by another
upstream kinase, TAK1 (Fig. 4C). (Baud and
Karin, 2009). In murine and human cells,
knockout and/or knockdown of Tak1 or
any of the Ikk subunits (but not that of
the Nf-κb subunit p65) prevents the
induction of autophagy in response to
diverse stimuli including starvation,
rapamycin, p53 inhibition, or ER stress
(Criollo et al., 2010; Herrero-Martin et al.,
2009). Conversely, expression of
constitutively active IKK subunits stimulate
autophagy through an NF-κB-independent
pathway that relies on the activation of
AMPK and JNK1 (Criollo et al., 2010).
63
However, the NF-κB family member
p65/RelA can upregulate beclin
1 expression and autophagy during T cell
activation (Copetti et al., 2009) and NF-κB-
incompetent cells are deficient in heat
shock-induced autophagy (Nivon et al.,
2009). Thus, there may be both IKK-
dependent, NF-κB-independent as well as
NF-κB-dependent mechanisms for
autophagy induction. In addition, the p105
subunit of NF-κB can interact with certain
autophagy proteins, including Beclin 1
(Behrends et al., 2010), but the significance
of these interactions has yet to be
explored.
Aside from the regulation of autophagy by
NF-κB, autophagy may, in turn, regulate
NF-κB signaling. For example, NF-κB
signaling may be negatively regulated by
targeting IKK and the NF-κB inducing
kinase, NIK, for autophagic degradation
(Qing et al., 2007). Furthermore, when
autophagy is inhibited, p62 accumulates
and activates the NF-κB signaling pathway
(Mathew et al., 2009), suggesting that
normal levels of autophagy may provide a
“break” on NF-κB signaling.
PKR and the “meta-inflammasome”
An interesting recent study uncovers a
potential relationship between an
autophagy-regulatory eIF2α kinase, PKR,
previously believed to respond primarily to
virus infection (Talloczy et al.,
2002; Talloczy et al., 2006), and the
coordinate regulation of JNK1 and insulin
signaling with ER stress and fatty acid
exposure. PKR was found to respond to ER
stress and fatty acid exposure, and be
required for the JNK1 phosphorylation,
serine phosphorylation of the insulin
receptor substrate 1 (IRS1) and
consequent insulin resistance that occurs
in response to these stimuli (Nakamura et
al., 2010). Based on these findings and a
previous report that PKR activates the
TEÓRICO AUTOFAGIA
IKKβ-NF-κB pathway (Bonnet et al., 2000),
Hotamisligil et al. (Hotamisligil, 2010)
proposed the existence of a ‘meta-
inflammasome’ composed of PKR, eIF2α,
JNK, IRS, IKK and other components that
link ER stress to more global stress
responses, including inflammatory
signaling and metabolic dysfunction. In the
postulated “meta-inflammasome”, PKR
phosphorylates eIF2α, (which induces
autophagy), activates the ‘inflammatory
kinases’ IKKß and JNK1 (which both induce
autophagy), and phosphorylates (and
inhibits) IRS1 (which also would induce
autophagy) (Nakamura et al., 2010). Thus,
this hypothetical molecular platform might
explain the functional overlap and
crosstalk between the JNK1, IKK and eIF2α
pathways in the induction of autophagy.
It is, however, unclear whether this
autophagy-promoting activity, rather than
other downstream effects of the signaling
molecules, would explain the pro-
inflammatory and adverse metabolic
effects of the “metainflammasome”, since
genetic deletions of autophagy genes
generally activates inflammatory signaling
(Sumpter and Levine, 2010; Virgin and
Levine, 2009) and a recent study found
that hepatic suppression of
the Atg7 autophagy gene results in
increased ER stress and insulin resistance
(Yang et al., 2010). Perhaps, the activation
of autophagy by the metainflammasome
serves as a negative feedback mechanism
64
to limit ER stress; this hypothesis would
reconcile the speculated role of the meta-
inflammasome in autophagy activation
with the cytoprotective roles of autophagy.
Hypoxia and Anoxia
Hypoxia and anoxia (with oxygen
concentrations <3% and <0.1%,
respectively) both cause autophagy
through a variety of different mechanisms.
Hypoxia-induced autophagy depends on
hypoxia-inducible factor, HIF, while anoxia-
induced autophagy is HIF-independent
(Majmundar et al., 2010; Mazure and
Pouyssegur, 2010). HIF is a heterodimer of
a constitutive ß subunit and an oxygen-
regulated α subunit that only becomes
stabilized (and hence expressed) when
oxygen concentration declines below a
threshold of ~5%. Upon moderate hypoxia
(1-3% oxygen), HIF activates the
transcription of BNIP3 and BNIP3L(NIX),
two BH3-only proteins that can disrupt the
inhibitory interaction between Beclin 1 and
Bcl-2 (Bellot et al., 2009) (Fig. 5A).
Moreover, BNIP3L, which often is present
at the outer surface of mitochondria,
possesses a WXXL motif that binds to LC3
and its homolog GABARAP (Novak et al.,
2010), thereby targeting mitochondria for
autophagic destruction. The transcription
of BNIP3 is also upregulated by the
transcription factor FOXO3, provided that
it is deacetylated by Sirt1 (Kume et al.,
2010).
TEÓRICO AUTOFAGIA 65

FIGURE 5
Overview of selected stress pathways that induce autophagy

The mechanisms involved in autophagy induction by hypoxia or anoxia (A); increased

oxidative damage (reactive oxygen species, ROS) (B); perturbation of the p53 system (C) or
mitochondrial dysfunction D) are represented schematically.
Additional pathways have been implicated
in hypoxia-induced autophagy, especially
in cases of severe hypoxia or anoxia (Fig.
5A). These include the protein DJ-1 (also
called CAP1/RS/PARK7, which regulates
autophagy by an unknown mechanism),
the autocrine stimulation of a PDGFR-
dependent pathway, the stimulation of
AMPK through metabolic stress, and the
UPR of the ER (Mazure and Pouyssegur,
2010). Maximal induction of autophagy by
hypoxia also requires PERK-mediated
phosphorylation of eIF2α (Rouschop et al.,
2010), further underscoring the
importance of eIF2α phosphorylation as a
general autophagy regulator. Hypoxia
increases the transcription of the essential
autophagy genes LC3 and Atg5 through
the transcription factors ATF4 and CHOP,
respectively, which are both regulated by
PERK (Rouschop et al., 2010).
Redox Stress and p53
Oxidative stress induces autophagy
through multiple mechanisms (Fig. 5B).
Exogenous hydrogen peroxide reportedly
can activate PERK (and thereby stimulate
eIF2α phosphorylation) (Liu et al., 2008),
directly oxidize and activate Atg4
proteases (and thereby accelerate the
production of proteolytically mature LC3)
(Scherz-Shouval et al., 2007), and inhibit
mTOR either directly (Liu et al., 2008) or
indirectly, via activation of PARP1 (as a
result of DNA damage) and/or via
activation of a cytoplasmic pool of ATM
TEÓRICO AUTOFAGIA
and the subsequent activation of LKB1 and
AMPK1(Alexander et al., 2010; Huang et
al., 2009). The cellular response to an
increase in ROS often involves the
activation of mitogen-activated protein
kinases (MAPKs), including JNK1, which can
activate autophagy (Wong et al., 2010).
Finally, DNA damage also stimulates the
expression of pro-autophagic p53-induced
target genes.
The tumor suppressor protein p53, which
can be activated by different types of stress
including redox stresss, has a dual effect on
autophagy (Fig. 5C), acting as a positive
regulator of autophagy via its
transcriptional activity and as a negative
regulator of autophagy via its cytoplasmic
functions (Green and Kroemer, 2009). As a
transcription factor, p53 transactivates
several autophagy inducers including
DRAM1 (which may operate through JNK1
activation) and Sestrin2 (which binds to the
ternary complex TSC1/TSC2-AMPK,
inducing phosphorylation and activation of
TSC2 by AMPK). One of the few p53-
induced genes that inhibits autophagy is
TIGAR, a fructose-2,6-bisphosphatase that
stimulates the pentose phosphate shunt
and helps to lower intracellular ROS
(Bensaad et al., 2009). When present in the
cytoplasm, p53 acts as a tonic inhibitor of
autophagy. In several contexts of
autophagy induction, p53 must be
depleted from the cytoplasm for optimal
autophagy induction to ensue. This may
involve the activation of the p53-specific
ubiquitin ligase, HDM2, which targets p53
to proteasome-mediated destruction
(Green and Kroemer, 2009). However, it is
not yet fully clear how cytoplasmic p53
inhibits autophagy. The only essential
autophagy protein that is known to
interact with p53 is FIP200, which is a
multifunctional protein that is present in
the ULK1 complex, yet also binds to Pyk2,
FAK, TSC2, ASK1 and TRAF2 and plays a role
in the control of cell adhesion, migration,
66
proliferation and cell death (Gan and Guan,
2008). Thus, an interesting question is
whether p53 somehow negatively
regulates the ULK1 complex through its
interaction with FIP200.
Mitochondrial Damage
Cells must remove damaged mitochondria
to prevent the accumulation of ROS. This
process of mitochondrial quality control is
mediated by mitophagy, the selective
autophagic removal of mitochondria.
Considerable advances have been made in
understanding the mechanisms by which
damaged mitochondria are targeted for
autophagy, as well as the functional
significance of mitochondrial quality
control in preventing aging,
neurodegenerative diseases, and other
pathologies.
In response to potentially lethal stress or
damage, mitochondrial membranes can be
permeabilized through multiple distinct
biochemical routes. Indeed, mitochondrial
membrane permeabilization (MMP)
constitutes one of the hallmarks of
imminent apoptotic or necrotic cell death
(Kroemer et al., 2007). However, if only a
fraction of mitochondria are
permeabilized, autophagic removal of the
damaged mitochondria can rescue the cell.
The autophagic recognition of depolarized
mitochondria is mediated by a refined
voltage sensor, involving the mitochondrial
kinase, PINK1. Under normal
circumstances, PINK1 is continuously
recruited to the mitochondrial outer
membrane, but subject to voltage-
dependent proteolysis, which leads to its
removal from mitochondria and to
proteasome-mediated degradation
(Narendra et al., 2010). Upon
mitochondrial depolarization, PINK1
rapidly accumulates on the mitochondrial
surface, facilitates the recruitment of the
E3 ubiquitin ligase Parkin (Narendra et al.,
TEÓRICO AUTOFAGIA
2010) which ubiquitinates mitochondrial
substrates including the outer membrane
protein VDAC1, recruits the autophagy
adaptor molecule, p62/SQSTM1, and
thereby targets mitochondria for
autophagic removal (Geisler et al., 2010)
(Fig. 5D). Both PINK1 and Parkin genes
were originally identified because loss-of-
function mutations affecting either of
them cause familial Parkinson's disease in
humans. Disease-causing mutations
in PINK1 and Parkin disrupt Parkin
recruitment and Parkin-induced
mitophagy (Geisler et al., 2010; Narendra
et al., 2010). At least in some cell types
(MEFs), depolarization-induced mitophagy
involves BNIP3L (also known as NIX)
(Novak et al., 2010) (Fig. 5D) and in mice,
BNIP3L/Nix is required for the removal of
mitochondria during erythroid maturation
(Sandoval et al., 2008). At present, the
functional relationship between NIX-
dependent mitophagy and the
PINK1/Parkin mitophagy pathway has not
been elucidated.
Interestingly, the maturation of
autophagosomes during mitophagy may
be controlled differently than during
starvation-induced autophagy. The
ubiquitin-binding deacetylase, histone
deacetylase-6 (HDAC6) promotes
autophagosomal maturation by recruiting
a cortactin-dependent, actin–remodelling
machinery to ubiquitinated structures,
where assembly of the F-actin network
facilitates autophagosome-lysosome
fusion. (Lee et al., 2010). However, HDAC6
deficiency leads to a failure of
autophagosomal maturation only in the
context of quality control autophagy
(mitophagy and protein aggregate
removal), but not in starvation-induced
autophagy (Lee et al., 2010). This specific
requirement for a ubiquitin-binding
deacetylase in quality control autophagy
suggests that ubiquitin modification may
67
be a key signal underlying distinct steps in
selective autophagy.
Autophagy Regulatory Pathways in the
General Stress Response
In the preceding section, we reviewed
specific stress stimuli and stress signals
that regulate autophagy. Certain general
concepts regarding the interrelationship
between autophagy regulatory pathways
and the general stress response are
beginning to emerge.
Integration of Autophagy and other
Cellular Stress Responses
The integration of autophagy and other
cellular stress responses can be
conceptualized in the framework of three
broad concepts. First, a single type of stress
stimulus elicits a variety of signals that
trigger distinct cellular responses (one of
which is autophagy) that cooperate for the
sake of optimal cellular repair and
adaptation. Second, distinct stress
responses are often integrated through the
ability of a single molecular event to
stimulate multiple adaptive pathways (one
of which is autophagy). Third, distinct
cellular stress response pathways,
including autophagy, mutually control
other stress response pathways.
Some representative examples of the first
concept (Fig. 6A) include (i) redox stress,
which induces transcriptional
reprogramming through HIF, NF-κB and
p53 activation, elicits the UPR, and
stimulates both general and selective
autophagy; (ii) hypoxia, which induces
adaptive responses including the
transcriptional activation of angiogenic
and cytoprotective cytokines in parallel
with autophagy stimulation; and (iii) DNA
damage, which elicits nuclear p53
translocation, cell cycle checkpoint
activation, cell cycle arrest, and autophagy.
TEÓRICO AUTOFAGIA 68

FIGURE 6
Hypothetical models of key cellular stress response networks

A. Particular stress stimuli (e.g. oxidative damage, hypoxia or anoxia, nutrient starvation, ER
stress) can elicit different responses that cooperate to achieve optimal cellular repair and
adaptation. A diverse range of stressors activate interconnected cytoprotective mechanisms
able to modulate autophagy at different levels, such as transcriptional reprogramming,
protein modifications (phosphorylation, acetylation, etc.) or cell cycle modulation. B.
Autophagy inhibition and stress. Autophagy impairment leads to the accumulation of
damaged proteins and organelles, which in turn can elicit cellular stress. Moreover, disabled
autophagy can increase the abundance of p62, resulting in an enhanced activity of NF-κB,
which leads to enhanced inflammation. By contrast, p62 accumulation leads to the activation
of Nrf2 transcription factor and in a consequent increase in the expression of stress response
enzymes. C. Mutual exclusion between autophagy and apoptosis. Autophagy, as a
cytoprotective pathway, eliminates potential sources of pro-apoptotic stimuli such as
damaged mitochondria, thereby setting a higher threshold against apoptosis induction. By
contrast, the apoptosis-associated activation of proteases such as calpain and caspase-3 may
destroy autophagy-specific factors (Atg4D, Beclin 1 or Atg5), thereby suppressing autophagy.
Many of the signals discussed in preceding autophagy (Criollo et al., 2010; Herrero-
sections illustrate the second concept. For Martin et al., 2009). Activation of LKB1-
example, eIF2α phosphorylation AMPK stimulates autophagy, as well as the
participates in the UPR in stress granule phosphorylation and activation of p27kip1, a
formation, in general translational control, cyclin-dependent kinase inhibitor that
and in autophagy induction (Kimball et al., leads to cell cycle arrest (Liang et al., 2007).
2003; Talloczy et al., 2002). Similarly, mTOR inhibition results in autophagy
activation of TAK1 and IKK coordinates the induction, as well as in the inhibition of
activation of NF-κB signaling and that of anabolic reactions including IRES-
TEÓRICO AUTOFAGIA
independent mRNA translation. FOXO3
activation induces the transcription of
genes whose products increase both
proteasomal and autophagic degradation
(Mammucari et al., 2007).
The control of other stress pathways by
autophagy was discussed briefly in
previous sections. For example, as noted
above, deficient autophagy leads to p62
accumulation, which activates NF-κB
signaling (Mathew et al., 2009). In addition,
p62 accumulation in the setting of
deficient autophagy activates the stress
responsive transcription factor Nrf2
(Komatsu et al., 2010). High levels of p62
disrupt the interaction between the
Cul3/Rbx1 ubiquitin ligase complex, Keap1
(which normally targets Nrf2 for
proteasomal degradation) and Nrf2,
resulting in enhanced Nrf2 activity and an
increase in expression of Nrf2-regulated
stress response enzymes (Komatsu et al.,
2010) (Fig. 6B). In addition, several
different autophagy genes regulate
different aspects of inflammatory
signaling, although, in some instances, it is
not known whether such regulation is a
consequence of autophagy or alternative
functions of the autophagy genes (Sumpter
and Levine, 2010; Virgin and Levine, 2009).
Autophagy genes negatively regulate RLR-
mediated induction of type I interferon
both via conjugation of Atg12-Atg5 to
CARD domains of RLR signaling molecules
and through elimination of dysfunctional
mitochondria. Atg16L1 and Atg7 negatively
regulate inflammatory signaling, including
IL-1β and IL-18 secretion. Moreover, Atg9,
but not Atg7, negatively regulates
activation of STING, a recently discovered
transmembrane protein that is required
for efficient activation of type I IFN and
pro-inflammatory cytokine production in
response to interferon stimulatory DNA
(Saitoh et al., 2009).
69
These examples underscore the mutual
control that exists between autophagy and
inflammatory signaling pathways. Another
important axis of mutual control of
different stress responses pathways is
between autophagy and cell cycle
regulation. Autophagy is blocked during G2
phase and mitosis, and a recent study
indicates that this may be mediated by
cyclin-dependent kinase-mediated
phosphorylation of Vps34, which
negatively regulates its interaction with
Beclin 1 during mitosis (Furuya et al.,
2010). Moreover, there is a strong
correlation between mitogenic signaling
and autophagy inhibition (Levine and
Kroemer, 2008). This correlation is further
strengthened by a recent genome-wide
screen, which found that a large
percentage of genes that negatively
regulate autophagy are positively involved
in cell growth and proliferation (Lipinski et
al., 2010). We speculate that this mutually
exclusive regulation of autophagy and cell
growth may constitute a fundamental
mechanism that cells use to appropriately
adapt their metabolism to stressful
environmental conditions.
Links among General Autophagy and
Quality Control Mechanisms
There is a growing consensus that
autophagy is stimulated either in response
to a central “command” of the cell (for
example in response to nutrient
depletion), in which case it is non-selective
and general, or as a result of ubiquitination
reactions that specifically target ‘garbage’,
i.e. protein aggregates or damaged
mitochondria or intracellular pathogens,
for autophagic degradation. Nonetheless,
these two types – general versus selective
– of autophagy may not be completely
separable from each other. Indeed,
stimulation of the general autophagic
pathway, either by inhibiting mTOR or by
TEÓRICO AUTOFAGIA
stimulating mTOR-independent autophagy
pathways, leads to reduced accumulation
of mutated, aggregation-prone proteins
(such as mutant α-synyclein- or huntingtin
protein) and damaged mitochondria
(Sarkar et al., 2007; Williams et al., 2008).
Similarly, starvation or mTOR inhibition
leads to increased autophagic targeting of
intracellular bacteria (Gutierrez et al.,
2004). One possible interpretation of these
findings is that a general increase in
protein and organelle turnover may be
sufficient to avoid protein aggregation and
organellar degeneration. Another
attractive interpretation is that general
autophagy is not completely non-selective
and may preferentially degrade proteins
and structures that are on the “verge” of
aggregation or damage. This yet unproven
concept predicts that an increase in
general autophagy would reset the
threshold of quality control so that
organelles that exhibit only minor
alterations (and that normally would not
be removed by autophagy) are subject to
autophagic turnover. This would explain
why an increase in general autophagy can
accelerate bacterial clearance, delay the
development of multiple
neurodegenerative diseases, and mediate
a general anti-aging effect (Madeo et al.,
2010).
Mutual Exclusion between Autophagy
and Apoptosis
Autophagy is usually a self-limited process
that protects cells from cell death by
multiple mechanisms that include, but are
not limited to, maintainance of
bioenergetic homeostasis, recycling of
misfolded and aggregate-prone proteins,
and removal of uncoupled or
permeabilized mitochondria. The intrinsic
pathway of apoptosis is initiated by
mitochondrial membrane
permeabilization (MMP) (Kroemer et al.,
70
2007). If MMP is limited to a fraction of
mitochondria, this will result in the
selective autophagic removal of
depolarized mitochondria and the
avoidance of cell death. Thus, the efficacy
of autophagy may set a higher threshold
for the ability of MMP to constitute an
irreparable, lethal event. In addition, it is
plausible that the liberation of Bcl-2 and
FLIP from activated autophagy protein
complexes may free up these molecules to
block the intrinsic and extrinsic pathways
of apoptosis, respectively (Lee et al.,
2009; Pattingre et al., 2005).
Given the predominantly cytoprotective
role of autophagy, it seems (teleo)logical
that induction of apoptosis would be
coupled to the inactivation of autophagy
(Fig. 6C). There are some examples of
molecular events that support this
concept. Caspase-3 cleaves Beclin 1,
thereby destroying its pro-autophagic
activity. The C-terminal fragment of Beclin
1 that results from this cleavage acquires a
new function and can amplify
mitochondrion-mediated apoptosis
(Wirawan et al., 2010). Caspase-3
activation also cleaves and activates
Atg4D, an enzyme that catalyzes the
delipidation of the LC3 paralog GABARPL1.
This proteolytic activation increases Atg4D
targeting to mitochondria via a putative
BH3 domain and enhances its cytotoxic
activity (Betin and Lane, 2009). Similarly,
the proteolytic activity of calpain can
destroy the pro-autophagic function of
Atg5 (Xia et al., 2010) while it generates an
Atg5-derived pro-apoptotic
mitochondrion-permeabilizing fragment of
Atg5 (Yousefi et al., 2006). All these results
underscore that concept that autophagy
and apoptosis are antagonistic events that
tend to inhibit each other.
TEÓRICO AUTOFAGIA 71

Concluding Remarks
In this review, we have described stimulus-
dependent, as well as common regulatory
and execution steps of autophagy, with a
focus on the close links that exist between
autophagy and different types of adaptive
and repair responses to stress. These links
consist of multiple and intricate
mechanisms that are intertwined in
complex intersecting pathways, which
often function in positive and negative
amplification loops, thus composing
molecular switches and homeostatic
devices. A more detailed molecular
comprehension of these regulatory
networks, as well as their biomathematical
integration using systems biology
approaches, may furnish testable models
for more refined experimental and
therapeutic manipulations of autophagy.
At present, the comprehension of
autophagic regulation has only just begun,
and its multiple links to cell growth,
proliferation, senescence and apoptosis
await further exploration. We expect that
additional major circuits of autophagy
regulation will emerge and perhaps
supersede in importance the pathways
that we currently know – or believe to
know.