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First published online June 20, 2008
doi: 10.1242/10.1242/dev.016105
Review |
1 Department of Biology, Queens College, 65-30 Kissena Boulevard, Flushing, NY
11367, USA.
2 Department of Genetics, Cell Biology and Development, University of Minnesota,
6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
e-mails: alicia.melendez{at}qc.cuny.edu; neufe003{at}umn.edu
SUMMARY
The cell biological phenomenon of autophagy (or `self-eating') has attracted increasing attention in recent years. In this review, we first address the cell biological functions of autophagy, and then discuss recent insights into the role of autophagy in animal development, particularly in C. elegans, Drosophila and mouse. Work in these and other model systems has also provided evidence for the involvement of autophagy in disease processes, such as neurodegeneration, tumorigenesis, pathogenic infection and aging. Insights gained from investigating the functions of autophagy in normal development should increase our understanding of its roles in human disease and its potential as a target for therapeutic intervention.
Introduction
Autophagy is a ubiquitous catabolic process that involves the bulk degradation of cytoplasmic components through a lysosomal pathway. This process is characterized by the engulfment of part of the cytoplasm inside double-membrane vesicles called autophagosomes. Autophagosomes subsequently fuse with lysosomes to form an autophagolysosome in which the cytoplasmic cargo is degraded (Fig. 1).
Although this process was initially described over 40 years ago
(de Duve, 2005
), two
developments in the past decade have led to a substantial increase in interest
and activity in the field of autophagy research. The first of these
developments was a series of genetic screens in yeast that led to the
discovery of the autophagy-related (ATG) genes that control this
process (Harding et al., 1995;
Thumm et al., 1994;
Tsukada and Ohsumi, 1993).
Orthologs of most of these ATG genes have now been identified in
higher eukaryotes (Table 1),
and genetic analyses have begun to decipher the functions of autophagy in
multicellular organisms, such as nematodes, flies and mice.
|
).
Together, research into the basic cell biology of autophagy and its potential
disease functions has revealed a substantial influence of autophagy on
cellular physiology. For example, it is clear that autophagy plays an
essential role in ridding the cell of damaged or superfluous organelles and
proteins, in generating nutrients essential for cell survival under starvation
and other stressful conditions, and in some cases acting as a cell death
effector. Given its role in these crucial cellular functions, it would seem reasonable to expect that autophagy will have a significant impact on animal development, a process ultimately driven by changes in individual cell activity. Here, we discuss current concepts and recent findings regarding the cellular and developmental functions of autophagy. Although its role in development has yet to be fully defined, studies in model organisms have begun to describe a growing number of developmental processes that are influenced by autophagy, and are showing how this fundamental cellular process affects other cellular activities crucial for development.
The molecular machinery of autophagy
The autophagic process can be divided into several distinct steps:
signaling and induction; autophagosome nucleation; membrane expansion and
vesicle completion; autophagosome targeting, docking and fusion with the
lysosome; and, finally, degradation and re-export of materials to the
cytoplasm (Fig. 1). The
molecular cascade that regulates autophagy and the mechanisms by which
autophagy occurs have been the subject of recent comprehensive reviews
(Klionsky, 2007;
Maiuri et al., 2007b;
Mizushima and Klionsky, 2007;
Yorimitsu and Klionsky, 2005)
and thus are described only briefly below.
Autophagy occurs at basal levels in normal growing conditions, but can be
dramatically upregulated by a number of stimuli, including starvation,
hypoxia, intracellular stress, high temperature, high culture density,
hormones and growth factors deprivation. The best characterized of these
stimuli is nutrient starvation, which induces autophagy in part through the
inactivation of the protein kinase target of rapamycin (Tor)
(Fig. 2). In yeast, this
inductive step includes the Atg1, Atg13 and Atg17 proteins, the association of
which is regulated by TOR-dependent signaling
(Fig. 3). Atg1 also appears to
be a target of TOR signaling in higher eukaryotes
(Scott et al., 2007),
suggesting that this mechanism is widely conserved.
A second functional complex involved in the vesicle nucleation step
consists of the class III phosphatidylinositol 3 kinase Vps34, Atg6/Vps30 and
several associated factors (Fig.
3B). Two distinct Vps34/Atg6 complexes have been described in
yeast (Kametaka et al., 1998):
an autophagy-specific complex that is thought to localize other autophagy
proteins to the pre-autophagosomal structure or phagophore assembly site
(PAS); and a second complex involved in vacuolar protein sorting. Likewise in
metazoans, Vps34 participates in both autophagy and other vesicle trafficking
processes, most probably via its assembly into distinct complexes
(Zeng et al., 2006).
Interestingly, C. elegans bec-1 and the human beclin 1 gene, the
ATG6 orthologs, can complement the autophagy, but not the VPS,
function of ATG6 in yeast
(Meléndez et al.,
2003), suggesting that there are functional differences between
metazoan and yeast ATG6.
Two novel and highly conserved ubiquitin-like conjugation pathways, the
Atg12 conjugation system (consisting of a complex of Atg12p, Atg5p, and
Atg16p), and the Atg8 lipidation system (consisting of Atg8p, Atg3p, and
Atg7p) (Fig. 3C) mediate
vesicle expansion and vesicle completion
(Mizushima, 2007;
Ohsumi, 2001;
Suzuki and Ohsumi, 2007).
These conjugation systems are widely conserved in eukaryotes and have an
essential role in autophagy.
|
Cellular functions of autophagy
Starvation survival
One of the best understood and perhaps most fundamental cellular roles of
autophagy is to provide an internal source of nutrients under starvation
conditions. In most cell types, nutrient withdrawal elicits a robust
stimulation of autophagy, and this can significantly extend the survival time
of yeast and cultured mammalian cells in the absence of nutrients. Indeed,
many yeast ATG genes were first identified through screens for
starvation sensitivity (Tsukada and
Ohsumi, 1993). Apoptosis-deficient Bax-/-
Bak-/- mouse bone marrow cells can remain viable for several
weeks in the absence of IL3, which is essential for nutrient uptake in these
cells; disruption of autophagy under these conditions results in rapid cell
death (Lum et al., 2005a). In
mammalian cells with an intact apoptotic system, such as in mouse embryonic
fibroblasts or HeLa cells, the genetic or pharmacological inhibition of
autophagy also significantly accelerates starvation-induced death
(Boya et al., 2005). Under
normal conditions in vivo, autophagy probably acts as a buffer against
fluctuations in exogenous nutrient availability, maintaining intracellular
nutrient concentrations at a relatively constant level.
How do the nutrients liberated by autophagy promote survival during
starvation? The death of autophagy-deficient IL3-deprived
Bax-/- Bak-/- mouse cells can be prevented by
the addition of methylpyruvate (Lum et
al., 2005b), a cell-permeable substrate of the tricarboxylic acid
(TCA) cycle in mitochondria, indicating that autophagy-derived nutrients have
a crucial role in ATP production. In addition, in starved ATG mutant
yeast cells, the intracellular level of free amino acids drops significantly,
becoming limiting for protein synthesis
(Onodera and Ohsumi, 2005).
Thus, nutrients derived from autophagy can be essential for both energetic and
biosynthetic functions.
In addition to providing these functions cell autonomously,
autophagy-derived nutrients can be exported out of the cell to support
peripheral tissues. Accordingly, fasting induces a more severe reduction of
plasma amino acid concentrations in Atg5-/- mice than in
controls (Kuma et al., 2004).
Thus, in multicellular organisms, autophagy helps to maintain extracellular
nutrient levels within a narrow limit compared with the wider range faced by
single celled organisms or cells grown in culture. Nonetheless, the finding
that fasting leads to the induction of autophagy in most of the tissues of
transgenic mice that express the autophagosome marker GFP-Atg8 (a notable
exception being cells of the CNS)
(Mizushima et al., 2004)
indicates that, despite the buffering effects of autophagy, the magnitude of
extracellular nutrient fluctuation in animals can be considerable.
Organelle turnover and cellular renewal
Although autophagy is most evident following starvation, a basal level of
constitutive autophagy appears to be a universal feature of nearly all
eukaryotic cells. One important function of basal autophagy is to rid the cell
of defective or superfluous organelles, and autophagy would appear to be the
sole cellular process by which this occurs. Mitochondria accumulate oxidative
damage with age, and, in cells with defective autophagy, such damaged
mitochondria fail to be eliminated and accumulate to high levels
(Kim and Sun, 2007;
Zhang et al., 2007).
Defective mitochondria also appear to be a significant source of reactive
oxygen species, leading to genotoxic damage in atg mutant cells,
which may partially explain the potential tumor-suppressive effects of
autophagy (Mathew et al.,
2007). Both the random and selective incorporation of mitochondria
into autophagosomes can contribute to this process
(Kissova et al., 2007),
sometimes referred to as mitophagy. Conditions that lead to mitochondrial
damage cause a strong induction of mitophagy, and, indeed, mitophagy might
have a confounding effect on chemotherapeutic agents that function through
mitochondria-dependent damage in metabolically active cells
(Amaravadi et al., 2007).
|
). This autophagic response is crucial for surviving ER
stress, although sequestration of the ER into autophagosomes without
subsequent lysosomal degradation might be sufficient for protection in some
cases (Bernales et al., 2006).
Selective autophagy also plays a key role in eliminating excess peroxisomes in
yeast and mammalian cells following removal of peroxisome-proliferating drugs
or media (Iwata et al., 2006;
Sakai et al., 2006).
In addition to these effects on organelles, basal autophagy also plays a
key role in eliminating misfolded, aggregated or otherwise damaged proteins,
which may be resistant to degradation by the ubiquitin-proteosome system.
Indeed, ubiquitinated protein aggregates accumulate in atg mutant
mouse hepatocytes and neuronal cells (Hara
et al., 2006; Juhász et
al., 2007; Komatsu et al.,
2005), and autophagy can suppress neurodegenerative phenotypes
caused by the expression of aggregate-prone proteins in various fly, nematode
and mouse neurodegeneration models
(Florez-McClure et al., 2007;
Jia et al., 2007;
Ravikumar et al., 2004).
Interestingly, the inhibition of proteosome function can lead to an increased
rate of autophagy, indicating that communication occurs between these two,
major, degradative pathways (Ding et al.,
2007). Together, the turnover of organelles and macromolecules
through basal autophagy plays a major role in cell renewal and viability, and
the upregulation of this process is crucial for surviving cellular insults and
stresses. The activation of autophagy in response to fasting may further boost
this renewal effect, and may contribute to the anti-aging effects of caloric
restriction (Bergamini et al.,
2004).
Cell death
Seemingly at odds with its role in promoting cell survival, autophagy is
often observed at high levels in dying cells, and in some cases might actively
contribute to cell death. Also referred to as type II programmed cell death,
autophagic cell death is distinguished from type I (apoptotic) cell death by
the presence of abundant autophagic structures in the dying cell, by a lack of
phagocyte recruitment and, in some instances, by caspase independence
(Baehrecke, 2005;
Schweichel and Merker, 1973).
It is unclear whether autophagy provides the killing mechanism in such cells,
or whether it represents a failed attempt at survival or the elimination of
cell debris. Nonetheless, the disruption of autophagy can block the death of
cultured cells with disabled apoptotic machinery
(Shimizu et al., 2004;
Yu et al., 2004), and can
impair or delay developmental cell death in Drosophila
(Berry and Baehrecke, 2007;
Juhász et al., 2007),
as discussed further below. In addition, the induction of high levels of
autophagy can be sufficient to cause cell death
(Kang et al., 2007;
Pattingre et al., 2005;
Scott et al., 2007). Although
the mechanisms by which autophagy leads to cell death are unclear in most
cases, they may involve the targeted destruction of factors required for cell
survival (Yu et al., 2006).
Future studies are required to understand the different contexts under which
autophagy can promote either cell survival or destruction.
Cell growth
It has long been noted that there exists an inverse correlation between
rates of autophagy and cellular growth. For example, most conditions that
stimulate autophagy, such as starvation, growth factor withdrawal, and
rapamycin treatment, result in reduced cell growth
(Neufeld, 2004). Furthermore,
many negative regulators of autophagy are growth-promoting oncoproteins,
whereas positive regulators are often tumor suppressors
(Botti et al., 2006). Although
these correlative data may simply reflect regulatory pathways that are common
to autophagy and cell growth, they are also consistent with a mechanism by
which autophagy inhibits cell growth under growth-suppressive conditions.
Consistent with this model, in studies of cultured mouse embryonic fibroblasts
and Drosophila larvae, it has been recently found that Atg
mutant cells do not undergo a normal reduction in cell growth rate in response
to starvation or rapamycin (Hosokawa et
al., 2006; Scott et al.,
2007). It was also shown that the overexpression of Atg1 leads to
the induction of autophagy and to sharply reduced cell growth. These results
indicate that autophagy probably contributes to the reduced rate of cell
growth in response to specific growth inhibitory signals. Whether this results
from the general catabolic nature of autophagy or reflects the selective
degradation of growth-promoting proteins or organelles has not been
determined.
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Is autophagy essential for normal development?
It is clear that autophagy has many important cellular functions, many of
which are involved in development. What is the evidence that autophagy plays a
role in development and through which cellular function does autophagy
influence a given developmental process? Surprisingly, genetic studies in
mice, flies and worms have found that although some mutations in genes
essential for autophagy result in embryonic lethality, other mutants with
strong autophagy phenotypes remain viable throughout embryogenesis, with
defects becoming apparent only postnatally or in adulthood.
In C. elegans, bec-1 homozygous animals, derived from
bec-1 heterozygous parents, exhibit a highly penetrant lethal
phenotype; they arrest at various stages of development, exhibiting the
formation of cellular vacuoles, uncoordinated phenotypes and molting defects
(Takacs-Vellai et al., 2005).
Mutant bec-1 animals that reach adulthood are sterile. Thus,
bec-1 activity is required for viability and fertility. A lethal
phenotype is also observed in worms carrying a mutation in let-512,
which encodes the C. elegans ortholog of the BEC-1 partner Vps34
(Roggo et al., 2002). In
addition, RNAi-mediated knockdown of two other C. elegans atg
orthologs (Atg8/lgg-1 and Atg18/F41E6.13) result in early
developmental arrest, during or before the first larval stage
(Meléndez et al.,
2003). By contrast, the C. elegans ATG1 ortholog
unc-51 is dispensable for embryonic development, despite playing an
essential role in autophagy. Mutations in unc-51 result in adults
with an uncoordinated phenotype, and axons of the mutant animals display
abnormal vesicles and membrane cisternae
(Hedgecock et al., 1985;
McIntire et al., 1992). These
results indicate that different autophagy genes may play distinct roles during
embryonic development or that some genes display partial redundancy.
A similar conclusion can be drawn from genetic studies in flies and mice.
In Drosophila, null mutations in Atg1 result in fully
penetrant lethality late in the pupal stage, just prior to eclosion
(Scott et al., 2004). Earlier
lethality at the larval stage is observed in animals carrying mutations in the
Drosophila Atg18 or Atg6 genes. Mutations in the beclin 1
gene also disrupt embryonic development in the mouse, resulting in severely
reduced growth at embryonic day (E) 7.5 and death by E8.5
(Yue et al., 2003). By
contrast, flies with mutations in Drosophila Atg7 or Atg8a
develop to adulthood, and mice with mutations in Atg4C, Atg5, Atg7 or
Bif-1 progress through embryonic development in normal numbers,
although in each of these cases, serious defects are observed postnatally (see
below) (Juhász et al.,
2007; Komatsu et al.,
2005; Kuma et al.,
2004; Marino et al.,
2007; Scott et al.,
2007; Simonsen et al.,
2008; Takahashi et al.,
2007).
The mechanistic basis for the broad range of Atg mutant phenotypes
in these genetic model organisms is poorly understood, in part due to our
incomplete understanding of the cellular focus of the lethality. We also do
not understand to what extent potential embryonic phenotypes may be masked by
maternally contributed mRNA or protein, nor at what stages of development
these maternal supplies may be depleted in each case. Overall, these findings
suggest that autophagy plays somewhat subtle or redundant roles during
development, or that defects in autophagy can be compensated for by the
activation of other cellular processes. In addition, the range of mutant
phenotypes indicates that some Atg genes have additional roles in
other essential cellular functions. For example, Atg1/UNC-51 is involved in
endocytic processes in the neurons of mice and worms, and mammalian beclin 1
protein interacts with members of the Bcl2 family, suggesting a potential role
in apoptosis (Liang et al.,
1998; Tomoda et al.,
2004; Zhou et al.,
2007).
In some cases, the connection between a specific cellular function of
autophagy and a given developmental process is straightforward. For example,
the final stages of erythrocyte maturation occur through direct autophagic
elimination of mitochondria and other organelles
(Fader and Colombo, 2006;
Schweers et al., 2007). Other
less extreme examples of this type of cellular remodeling are likely to
contribute widely to cell differentiation. However, in many cases, the
mechanisms by which autophagy contributes to development are less clear and
may be indirect, and more than one cellular function of autophagy may be at
play. Below, we discuss recent studies of development-related phenotypes of
autophagy mutants in three specific areas - stress adaptation, cell death and
neuronal development - emphasizing the underlying cellular mechanisms where
known.
Adaptation to starvation and stress
A role for autophagy in C. elegans development was first provided
by studies of dauer morphogenesis
(Meléndez et al.,
2003). C. elegans larvae respond to unfavorable
environmental conditions (e.g. starvation, high population density, increased
temperature) by arresting in an alternative third larval stage, referred to as
the dauer diapause (Cassada and Russell,
1975; Riddle and Albert,
1997). Dauer larvae are characterized by radial constriction and
elongation of the body and pharynx, resistance to detergent (sodium dodecyl
sulfate or SDS) treatment, hyperpigmentation of the intestinal granules,
increased fat accumulation, long lifespan and the ability to exit the dauer
stage if the environmental conditions improve. The control of dauer
development in C. elegans shares similarities with the induction of
autophagy in other eukaryotes. Environmental cues, such as temperature,
starvation, and high population, are potent inducers of autophagy in yeast,
Dictostyelium and mammals, and also induce dauer formation in C.
elegans. Dauer development is regulated by different signaling pathways
that also regulate fat storage, longevity, reproduction and stress responses,
including the insulin/IGF-1, the transforming growth factor β
(TGF-β) and the cyclic guanosine monophosphate (cGMP) pathways
(Barbieri et al., 2003;
Patterson and Padgett, 2000;
Raizen et al., 2006). In
dauer constitutive daf-2 (the insulin/IGF-1 receptor) mutants, an
increase in autophagy occurs as noted by the localization of GFP::LGG-1 into
discrete punctate structures in hypodermal seam cells (a cell required for
multiple aspects of dauer morphogenesis)
(Meléndez et al.,
2003). This autophagy induction is required for dauer
morphogenesis, as most daf-2 mutant animals that are grown at
dauer-inducing temperature and in which bec-1, unc-51/Atg1, atgr-7,
lgg-1/Atg8, and atgr-18 were individually knocked down by RNAi
fail to complete normal dauer development
(Meléndez et al.,
2003). These animals lack the characteristics associated with
dauer: they do not undergo pharyngeal and total body constriction and
elongation, they have less fat accumulation, they are not resistant to SDS,
they die within a few days, and they fail to resume normal development when
transferred to the non-dauer-inducing temperature. Autophagy is also required
for dauer formation that is triggered by the lack of daf-7/TGF-β
(Meléndez et al.,
2003). Together, these data suggest that autophagy genes act
downstream of insulin/IGF-1 and TGF-β signaling, that autophagy is
required for the normal process of dauer morphogenesis, and that autophagy may
be required for the cellular and tissue remodeling that permits C.
elegans to adapt successfully to environmental stress. How autophagy is
regulated during dauer formation is not known. Whether autophagy, in addition
to its role in dauer morphogenesis, is required for dauer survival and/or
dauer recovery is also unknown.
The autophagic machinery has been shown to promote organismal survival
directly during starvation. Using animals that express the autophagy marker
GFP::LGG-1 in the pharynx (see Box
1), Kang et al. have shown that starvation activates autophagy in
the pharyngeal muscle (Kang et al.,
2007). Interestingly, a deficiency in the levels of autophagy,
caused by either a partial knockdown of bec-1 or of atgr-7,
promotes the death of starved animals. The decrease in pharyngeal pumping that
occurs in knockdown bec-1 animals can be attenuated by giving them
food. A correlation between pumping efficiency and survival suggests that
autophagy may provide the energy that is essential to maintain pharyngeal
pumping efficiency and to ensure organismal survival during starvation.
Whether autophagy is induced in other tissues in response to starvation is not
yet known. In mammals, other muscle cells may also undergo autophagic
catabolism to generate nutrients in order to maintain the health of neurons
and other vital tissues.
Although the developmental response to starvation in Drosophila is
quite distinct from that of C. elegans, autophagy has an important
role in the survival of stress and starvation in the fly, and at least some of
the genetic circuitry appears to be conserved. The larval fat body is a
nutrient storage organ that contains large deposits of lipid and glycogen,
somewhat analogous to the vertebrate liver. Drosophila larvae can
survive for several days in the complete absence of nutrients, and two or more
weeks in the absence of amino acids
(Britton and Edgar, 1998).
During these periods of starvation, nutrients are mobilized from the fat body
to support the imaginal tissues, which are destined to give rise to adult
structures of the fly. This starvation-induced mobilization occurs in large
part through autophagy, and indeed autophagy-defective Drosophila
mutants die more rapidly under starvation conditions
(Juhász et al., 2007;
Scott et al., 2004). Like
dauer development in C. elegans, this starvation response occurs
through the downregulation of insulin/IGF and TOR signaling, and constitutive
activation of these pathways prevents the induction of autophagy by starvation
and leads to starvation hypersensitivity
(Britton et al., 2002;
Scott et al., 2004).
Paradoxically, the downregulation of TOR signaling in response to starvation
leads both to the induction of autophagy and to the reduced activity of S6K, a
substrate of TOR that is required for autophagy
(Scott et al., 2004). This
self-limiting feature of the TOR pathway may prevent autophagy from reaching
excessive levels during chronic starvation. Autophagy also appears to promote
Drosophila survival against a number of additional stressors,
including oxidative stress, chill-induced coma and CO2 anesthesia
(Juhász et al.,
2007).
Box 1. Detection of autophagy in vivo
Several methods are used to monitor the progression of autophagy in vivo.
Autophagy is classically detected by electron microscopy. In electron
micrographs, autophagosomes are characterized by a double membrane that
surrounds sequestered cytoplasmic material. The discovery that Atg8 and its
metazoan orthologs become stably associated with the autophagosomal membrane
led to the development of green fluorescent protein (GFP)-tagged Atg8
molecules as effective and reliable ways of monitoring autophagy in nematodes,
flies and mammals (Kabeya et al.,
2000
GFP-LC3 has been used to detect autophagosomes through direct fluorescence
microscopy both in mammalian cultured cells and in mice in vivo. Panel A shows
GFP-LC3-labeled autophagosomes between myofibrils in the muscle tissues from
mice after 48 hours of starvation (asterisks indicate nuclei; modified, with
permission, from Mizushima et al.,
2004
In C. elegans, GFP fusions to the Atg8 ortholog LGG-1 have been
used to detect autophagy in most developing tissues
(Meléndez et al.,
2003
In Drosophila, GFP-Atg8 can be used to monitor both
starvation-induced autophagy and the developmental induction of autophagy in
the larval fat body towards the end of the larval period
(Rusten et al., 2004
An important point when detecting autophagy in vivo is that autophagy is a
dynamic process that can be positively or negatively regulated. Thus,
accumulation of autophagosomes may reflect the induction of autophagy or
reduced turnover of autophagosomes. Other strategies for detecting autophagy
biochemically include: determining the static or flux-dependent lipidation
state of Atg8 (Atg8-II/Atg8-I) by western blot analysis; measuring the
degradation of long-lived protein by pulse-chase labeling; and determining the
levels of selective autophagy substrates. [See review by Klionsky et al.
(Klionsky et al., 2008
|
Autophagy also plays a crucial role in stress responses in mammals, and is
required for starvation survival during postnatal development in the mouse
(Komatsu et al., 2005;
Kuma et al., 2004). Newborn
mice experience an early period of starvation just after birth, owing to the
lack of placental nutrient supply. This early starvation induces autophagy in
various neonatal tissues, such as the heart, lung and pancreas. In the absence
of Atg5 or Atg7 function, mice do not survive the period of
neonatal starvation and die within a day of birth
(Komatsu et al., 2005;
Kuma et al., 2004). Mutant
mice deficient for the Atg4C gene progress to adulthood, possibly
owing to the activity of other Atg4 gene family members, but show a
reduction in locomotor activity under prolonged starvation, and an increased
susceptibility to chemical carcinogens
(Marino et al., 2007).
Similarly, in mice carrying a disruption of the Bif1 gene
(Zbtb24 - Mouse Genome Informatics), which encodes a crucial
component of the beclin 1-Vps34 protein complex, early development appears
normal, but adult mutant mice display enlarged spleens and an increased
incidence of spontaneous tumors
(Takahashi et al., 2007).
In each of these examples, we have only a dim appreciation of the cellular
functions and cell-specific requirements that are supported by autophagy. In
autophagy-defective newborn mice, plasma concentrations of amino acids are
significantly reduced, and these animals display electrocardiograms consistent
with a shortage of respiratory substrates
(Kuma et al., 2004). Thus,
general defects in energy metabolism may be the immediate cause of the
premature death of Atg5- and Atg7-null mice, although other
abnormalities observed in these mutants, such as suckling defects and
ubiquitin-positive cytoplasmic inclusions
(Komatsu et al., 2005), are
consistent with their having additional problems earlier in development. The
role of autophagy in dauer formation in C. elegans is likely to be
more complicated, given the involvement of multiple cell types and
physiological responses in this process. Autophagy is likely to serve as a
crucial source of the nutrients and energy that are necessary for the
extensive morphogenetic changes that occur during dauer development. In
addition, autophagy may directly contribute to cellular remodeling by
eliminating superfluous cytoplasmic components, and may influence the survival
of specific cell types. Mosaic analysis of autophagy mutants may help to
identify cell types with special requirements for autophagy in these
developmental processes.
Neuronal development
An exclusive role for autophagy in mouse neuronal development has been
reported for the Ambra1 (activating molecule in beclin 1-regulated autophagy)
protein (Fimia et al., 2007).
Ambra1 was identified in a gene-trap approach in mice to find genes expressed
in the developing nervous system. Cecconi and colleagues have shown that
Ambra1 interacts with beclin 1 protein in vivo, and regulates Vps34-dependent
autophagy. As in beclin 1 in vitro studies, overexpression of Ambra1 in human
fibrosarcoma cells leads to constitutively high levels of autophagy and to
decreased cell proliferation. Furthermore, downregulation of Ambra1 impairs
the interaction between beclin 1 and Vps34 proteins. Ambra1-null
mutant mice display embryonic lethality (starting from E14.5) that is
characterized by severe neural tube defects associated with an impairment in
the autophagy pathway, an excess in programmed cell death, an increase in cell
proliferation and an accumulation of ubiquitinated proteins. However, no
defects in neuronal specification were detected
(Fimia et al., 2007).
Hyperproliferation is the earliest detectable abnormality in the developing
neural tubes of these mutant embryos, followed by caspase-dependent apoptosis,
indicating that there is a complex interplay between autophagy, the regulation
of cell survival and the regulation of cell proliferation in mammals.
Interestingly, hyperproliferation is observed in response to the disruption of
any of several components of the beclin 1/Vps34 protein complex in mammalian
cells, including beclin 1, Ambra1, Uvrag or Bif1. This phenotype is not
observed in other autophagy mutants, indicating that the disruption of this
complex results in a distinct autophagy defect that leads to increased
proliferation, or that this complex has a growth suppressive function that is
separate from its role in autophagy. Once again, the cellular basis for the
developmental phenotypes of Ambra1 mutants is unclear, although the
increased rate of cell proliferation and death are consistent with defective
turnover of an oncogenic factor(s). The restriction of the Ambra1
phenotype to neurons suggests that other factors may supplant its role in
non-neuronal tissues.
A role for autophagy in the clustering of neurotransmitter receptors in
development has been reported in C. elegans
(Rowland et al., 2006). The
clustering of neurotransmitter receptors results from signaling events during
development that are initiated when presynaptic terminals are contacted by the
postsynaptic cell. In C. elegans, body-wall muscles are innervated by
both GABA and non-GABA neurons (White et
al., 1986). GABA terminals organize GABAA receptors
into synaptic clusters, which are internalized and degraded, as long as they
lack presynaptic input. This degradation of GABAA receptors is
specifically mediated by an autophagic pathway, whereas that of acetylcholine
receptors in the same cells is not
(Rowland et al., 2006).
Curiously, the mammalian GABAA-receptor-associated protein Gabarap
is an ortholog of the yeast autophagy protein Atg8p (two other mammalian
orthologs of Atg8p are LC3 and Gate16, see
Table 1), which may hint at a
potentially regulatory role of autophagy in balancing neuronal excitation and
inhibition (owing to selective GABAA receptor degradation). These
findings show an unexpected degree of specificity and a novel function for
autophagy in the degradation of neuronal cell surface receptors. Similar
mechanisms of selective receptor degradation by autophagy may also be at play
to regulate cellular growth, differentiation and development.
Programmed cell death
The developmental process of insect metamorphosis involves the wholesale
death and elimination of a substantial part of the larval body, providing both
space and nourishment for imaginal tissues as they are reorganized into their
adult form inside the pupal case. Destruction of larval structures such as the
Drosophila salivary gland and digestive tract is triggered by a sharp
rise in the steroid hormone 20-hydroxyecdysone, and is associated with a
dramatic upregulation of autophagy prior to and during cell death. This
process has thus served as a valuable model for studying developmentally
regulated autophagic cell death (Baehrecke,
2005). Interestingly, the death of these tissues displays
characteristics of both autophagy, such as abundant cytoplasmic vacuolization,
as well as apoptosis, including the upregulation of pro-apoptotic genes,
caspase activation and DNA cleavage (Lee
et al., 2002; Martin and
Baehrecke, 2004). Elimination of larval gut and salivary glands
can be partially suppressed or delayed by mutations in components of either
the apoptotic or autophagic machinery
(Berry and Baehrecke, 2007;
Juhász et al., 2007;
Muro et al., 2006).
Disruption of both autophagy and apoptosis results in a more complete
suppression (Berry and Baehrecke,
2007), indicating that these processes act cooperatively, and that
one process may be upregulated to compensate for the lack of the other. Pupal
development is delayed by 
4 hours in Atg7 mutant animals,
consistent with a reduction in the efficiency of cell elimination when
autophagy is defective (Juhász et
al., 2007). The ultimate viability of these mutants, however,
indicates that other death mechanisms are sufficient to compensate for this
defect. A similar combination of apoptotic and autophagic morphologies has
been described in the death of nurse and follicle cells during oogenesis in a
number of insects, including silkmoths, medflies and other Drosophila
species (Mpakou et al., 2006;
Nezis et al., 2006;
Velentzas et al., 2007),
although the requirement for these processes in cell death has yet to be
determined experimentally in these cases.
Under what conditions and in what cell types is autophagy likely to be used
as a means of cell death? Although phagocytes normally play an important role
in eliminating apoptotic corpses, few phagocytes are observed in dying
salivary glands. The extremely large size of polyploid larval cells, their
sequestration into inaccessible areas of the body cavity and the sheer number
of cells dying during metamorphosis may preclude their efficient engulfment by
phagocytes, necessitating their self-clearance by autophagy. However, although
the function of autophagy in the death of these cells would appear to be
direct, it is important to consider other mechanisms by which autophagy may
contribute to cell elimination. In an in vitro model of mouse embryonic
cavitation using embryoid bodies derived from ES cells, it was found that
disruption of autophagy prevented the clearance of the inner core of
ectodermal cells, which normally die by an apoptotic form of programmed cell
death (Qu et al., 2007). In
this case, however, autophagy was required not as an effector of death nor to
degrade dying cells, but rather as an energy source to facilitate signaling
from the dying cells to macrophages (Qu
et al., 2007). The absence of autophagy resulted in decreased
engulfment of apoptotic cells by macrophages, leading to accumulation of
apoptotic corpses within the embryoid bodies. These results suggest that
caution is warranted when interpreting effects of autophagy on apoptosis. For
example, the observed increase in apoptotic corpses in bec-1 mutant
embryos in C. elegans
(Takacs-Vellai et al., 2005)
may reflect either an increased rate of apoptosis or a decrease in the
clearance of dead cells.
So far, there is only limited evidence that autophagy functions as a death
mechanism in cells with an intact apoptotic pathway, apart from the studies
described above in Drosophila. In mammalian cells, most reports of
the involvement of autophagy in the death execution process are in cells that
are defective in the apoptotic pathways
(Levine and Yuan, 2005;
Maiuri et al., 2007b).
Furthermore, defects in autophagy, as in bec-1/ATG6 or
beclin 1 mouse knockouts, cause an increase, and not a decrease in cell death,
supporting a pro-survival role for autophagy during development.
Recent studies of EGL-1 in C. elegans, provide interesting
insights with regard to the connection between autophagy and apoptosis
(Maiuri et al., 2007a). EGL-1
encodes a novel protein that contains a Bcl2 homology 3 (BH3) domain. In
C. elegans, EGL-1 functions as a positive regulator of apoptosis by
interacting directly with the anti-apoptotic CED-9 to induce the release of
CED-4 from CED-4/CED-9 complexes, resulting in the activation of the caspase
CED-3 (Conradt and Horvitz,
1998; Conradt and Horvitz,
1999; del Peso et al.,
1998; Trent et al.,
1983). Although a deletion of egl-1 compromises
starvation-induced autophagy, a gain-of-function mutation of egl-1
induces autophagy (Maiuri et al.,
2007a). The interaction between mammalian Bcl2/Bcl-xL (Bcl2l1 -
Mouse Genome Informatics) and beclin 1 protein involves a BH3 domain within
beclin 1, and this interaction is competitively blocked by the overexpression
of BH3-only pro-apoptotic proteins or by pharmacological BH3 mimetics
(Maiuri et al., 2007b). Thus,
EGL-1 might not only positively regulate programmed cell death, but might also
positively regulate autophagy by interacting with CED-9 to induce BEC-1
release from CED-9/BEC-1 complexes. It is unclear whether the increase in
autophagy in egl-1 mutants contributes directly to cell death. Future
studies in C. elegans should clarify the molecular crosstalk between
the autophagic and apoptotic machinery during development.
In addition to these connections between autophagy and apoptosis, recent
studies suggest that autophagy may also play a role in necrotic cell death.
Gain-of-function mutations in C. elegans ion channel genes
(mec-4 or deg-1), the acetylcholine receptor channel subunit
gene (deg-3) and the Gs protein 
-subunit gene (gsa-1)
cause a necrotic-like cell degeneration in neurons
(Berger et al., 1998;
Chalfie and Wolinsky, 1990;
Driscoll and Chalfie, 1991;
Korswagen et al., 1997;
Treinin and Chalfie, 1995).
Studies of dying neurons in animals carrying the gain of function
mec-4(d) allele, display extensive degradation of cellular contents
during the process of necrosis (Hall et
al., 1997). This ultrastructural feature is reminiscent of
autophagy and does not require the caspase CED-3, which mediates programmed
cell-death, nor CSP-1, CSP-2 or CSP-3 (CED-3-related proteases)
(Chung et al., 2000),
indicating that a distinct and non-apoptotic mechanism may function in
neurodegeneration (Syntichaki et al.,
2002). The knockdown of three autophagy transcripts
(unc-51/ATG1, bec-1/ATG6 and lgg-1/ATG8) by RNAi suppresses
the degeneration of neurons with hyperactive toxic ion channels
(Toth et al., 2007),
suggesting a role for autophagy in cellular necrosis. Recently, Samara et al.
have also shown that excessive autophagosome formation is induced early during
necrotic cell death and that the autophagy pathway synergizes with lysosomal
proteolytic mechanisms to facilitate necrotic cell death in C.
elegans neurons (Samara et al.,
2008). Together these results suggest that the boundaries between
apoptotic, autophagic and necrotic forms of cell death are not sharply
defined, and aspects of each mechanism can be used together to achieve a cell
death process that is appropriate for a given developmental context.
Autophagy and disease
The recent surge in interest and activity in the field of autophagy
research is driven in part by the impact of this process on several aspects of
human health and disease, and by the potential opportunity to develop novel
therapies involving the manipulation of autophagy
(Box 2). In two areas in
particular, tumorigenesis and neurodegeneration, autophagy plays an important
role and developmental studies in model organisms are leading to new insights
into disease mechanism and potential therapeutic strategies.
| Box 2. Autophagy and human health
As a fundamental cellular process important for energy homeostasis and
renewal, it is perhaps not surprising that defects in autophagy are being
associated with an increasing assortment of human diseases. As in the case for
tumorigenesis and neurodegeneration described in the text, autophagy can
potentially play both beneficial and detrimental roles in these other contexts
as well. For example, autophagy affects immune system function at several
levels, acting to promote the degradation of a variety of pathogens, including
bacteria, viruses and intracellular parasites
(Andrade et al., 2006
Autophagy might also play an especially prominent role in the heart,
consistent with the high metabolic demand placed on contractile
cardiomyocytes. As observed in the nervous system, basal levels of autophagy
are crucial for normal cell function in the mouse heart
(Nakai et al., 2007
By contrast, autophagy might act more beneficially by promoting longevity.
Treatments that induce autophagy, such as caloric restriction and reduced
insulin or TOR signaling, increase lifespan across the animal kingdom and in
C. elegans these effects depend on autophagy
(Meléndez et al.,
2003
Together, these types of studies can point towards specific dietary
programs and pharmaceutical interventions that may provide effective therapy
for a number of medical conditions. Pharmacological inhibitors of autophagy
such as 3-methyladenine (3-MA) and the AMP-activated protein kinase activator,
5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), and
autophagy inducers such as rapamycin, an FDA-approved immunosuppressant, serve
as proof-of-principle that drugs targeting autophagy can be effectively
developed. Although these compounds are not selective for autophagy, recent
small-molecule screens have begun to identify additional autophagy modulators
(Sarkar et al., 2007
|
Although correlations between malignancy and defects in autophagy have long
been noted, the first indication of a mechanistic connection between autophagy
and cancer came from studies of the beclin 1 gene, which is monoallelically
deleted in a high percentage of human breast and prostrate cancer
(Aita et al., 1999;
Liang et al., 1999). The
heterozygous disruption of the beclin 1 gene in mice was found to increase the
rate of spontaneous and virally induced tumor formation, confirming the role
of beclin 1 protein as a haploinsufficient tumor suppressor
(Qu et al., 2003;
Yue et al., 2003). Other
components of the beclin 1/Vps34 protein complex, including Uvrag and Bif1,
also have tumor suppressive properties
(Liang et al., 2006;
Takahashi et al., 2007).
Recent studies by White and colleagues have demonstrated that a loss of
autophagy in immortalized mouse epithelial cells leads to a marked increase in
DNA damage, genomic instability and necrosis, all of which potentially
contribute to tumorigenesis (Degenhardt et
al., 2006; Mathew et al.,
2007). In contrast to this tumor suppressive function, autophagy
might promote tumorigenesis by supporting the growth and survival of solid
tumors at early stages of development, prior to vascularization. Thus,
autophagy appears to play both positive and negative roles at different stages
of cancer development. Studies of the normal functions of autophagy during
vasculogenesis in developing embryos may help to clarify these issues. Indeed,
observations of increased inflammatory responses in atg5 mutant mouse
embryos (Qu et al., 2007)
suggest additional mechanisms by which autophagy may contribute to
tumorigenesis.
An essential role for autophagy has also been found in the maintenance of
axonal homeostasis and prevention of neurodegeneration
(Rubinsztein, 2006). As in
the case of cancer, autophagy appears to play primarily a protective role
against neuropathy, probably stemming from the function of basal autophagy in
degrading damaged organelles and aggregate-prone proteins. This
neuroprotective role is exemplified by the neurodegeneration phenotypes
observed in fly and mouse autophagy mutants, and by the suppression of
polyglutamine-induced toxicity by autophagy in fly and worm models of
Huntington's and other neurodegenerative diseases
(Hara et al., 2006;
Jia et al., 2007;
Juhász et al., 2007;
Komatsu et al., 2006;
Sarkar et al., 2007).
However, in some cases, autophagy appears to play dual harmful and beneficial
roles in neuronal health. For example, autophagy contributes to the production
of amyloid β peptide through degradation of amyloid β precursor
protein (APP)-containing organelles. Normally this toxic peptide is degraded
following the fusion of autophagosomes with lysosomes; however, in Alzheimer's
disease, the fusion process of autophagosomes with lysosomes is defective,
leading to the massive accumulation of autophagic vacuoles and to increased
amyloid β levels in the degenerating neurons
(Nixon, 2007). A similar
accumulation of autophagic vacuoles has been described in an increasing number
of neurodegenerative conditions, but additional investigation is necessary to
determine whether this represents increased induction of autophagy or defects
in autophagosome turnover.
Conclusions
In the past few years, there has been a deluge of publications identifying many of the components of the protein machinery involved in autophagic function in yeast, and providing insights as to the molecular mechanism of autophagy. Although orthologs to many of these components exist in metazoans, in some cases formal proof of their involvement in autophagy is still lacking. Another area where our understanding is still limited is that of the regulatory pathways that control autophagy, and the level of crosstalk that probably exists between these pathways. Genetic model organisms may be able to shed light on this issue. Based on our current understanding of the physiological functions of autophagy, both basal levels and stress-induced levels of autophagy can promote mammalian health. Autophagy maintains energy homeostasis and provides nutrients under conditions of stress and nutrient deprivation. In addition, autophagy rids the cell of intracellular proteins and damaged organelles that may cause cellular degeneration, genomic instability, tumorigenesis and aging.
As our appreciation of the roles of autophagy in cancer, neurodegeneration and other diseases grows, it will become increasingly important to understand the normal range of autophagic functions and control mechanisms in the healthy state. Increased insight into the developmental roles of autophagy is likely to point towards ways in which this process can be exploited for therapeutic purposes. A challenge for the future will be to identify the signaling mechanisms and regulatory steps that can be targeted for intervention, and to determine the circumstances under which autophagy plays a net beneficial versus detrimental role. Developmental studies in nematodes, flies and mice have already begun to shed light onto the variety of uses to which autophagy has been put by evolution, and how this fascinating process influences a wide spectrum of developmental events.
ACKNOWLEDGMENTS
The work in the authors' laboratories is supported by the NIH, and by the PSC CUNY Research Award Program (A.M.). We thank Hannes Bülow for comments and helpful suggestions in the preparation of this manuscript.
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