FEBS Letters 584 (2010) 1342–1349

journal homepage: www.FEBSLetters .org

Review

Autophagy takes flight in Drosophila

Yu-Yun Chang, Thomas P. Neufeld *

University of Minnesota, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 December 2009Revised 6 January 2010Accepted 7 January 2010Available online 13 January 2010

Edited by Noboru Mizushima

Keywords:Autophagy-related gene 1Unc-51 like kinase 1Vps34Jun-N-terminal kinaseTarget of rapamycin

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.01.006

Abbreviations: Atg1, autophagy-related gene 1; TOphosphoinositide 3-kinase; Ulk1, Unc-51 like kinaskinase; JNK, Jun-N-terminal kinase

* Corresponding author. Fax: +1 612 625 5158.E-mail addresses: chang220@umn.edu (Y.-Y. Chan

Neufeld).

Drosophila has been shown to be a powerful model to study autophagy, whose regulation involves acore machinery consisting of Atg proteins and upstream signaling regulators similar to those inyeast and mammals. The conserved role in degrading proteins and organelles gives autophagy animportant function in coordinating several cellular processes as well as in a number of pathologicalconditions. This review summarizes key studies in Drosophila autophagy research and discussespotential questions that may lead to better understanding of the roles and regulation of autophagyin higher eukaryotes.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction mechanism involving a series of Atg proteins, first identified by

Autophagy is a conserved process in which cytoplasmic pro-teins or organelles are non-selectively packaged into lysosomes(or vacuoles in yeast) for degradation. Autophagic substrates arebroken down to small molecules that are recycled for macromolec-ular synthesis or used for generating energy, and therefore autoph-agy is considered as an adaptive system that allows cells to survivestarvation. In addition to this non-selective form of autophagy,studies from the last decade have identified subsets of selectiveautophagic processes that specifically degrade intracellular organ-elles, such as mitochondria (mitophagy), endoplasmic reticulum(reticulophagy) or peroxisomes (pexophagy) [1]. These specificforms of autophagy provide an alternative way to clear damagedorganelles, which can be toxic if accumulated to high levels. Inmammals, autophagy has been implicated in several pathologicalconditions, such as neurodegenerative diseases, tumors and patho-genic infections. Collectively, autophagy is an important cellularprocess with multiple functions across species.

The delivery of autophagic substrates occurs through special-ized double-membraned vesicles called autophagosomes. The for-mation of autophagosomes requires a tightly controlled

chemical Societies. Published by E

R, target of rapamycin; PI3K,e 1; S6K, RPS6-p70-protein

g), neufe003@umn.edu (T.P.

systematic screens in yeast [2]. The core proteins for autophagy in-clude three major groups, whose functions correspond to the stepsof autophagosome formation [1]. The induction signal is trans-duced through an autophagy-related gene 1 (Atg1) kinase com-plex; this directs the membrane nucleation of autophagosomesthrough a second protein complex containing the PI(3)-kinaseVps34; finally, vesicle expansion is mediated by two ubiquitin-likegroups, Atg8 and Atg5–Atg12–Atg16. The matured autophago-somes then fuse with lysosomes with the help of a set of generaldocking proteins to degrade components inside autolysosomes. To-gether with the target of rapamycin (TOR), a conserved regulatorykinase that inhibits autophagy, these molecules form a complexnetwork for the regulation of autophagy [1].

The short life cycle and powerful genetics of Drosophila, alongwith a physiology comparable to mammals, has made this organ-ism a handy model system for a wide variety of experimental ques-tions. Together with yeast and mammalian cultured cells whereautophagy is extensively studied, Drosophila has provided a usefulmodel to dissect the molecular mechanisms and the physiologicalroles of autophagy in vivo. Autophagy is inducible by starvation inthe Drosophila larval fat body, an analogous organ to mammalianliver, and studies of this response have contributed to our under-standing of nutrient-dependent regulation of autophagy. In addi-tion, high levels of autophagy are observed in certain dying cellsduring metamorphosis and oogenesis in Drosophila, and appearto act in concert with the apoptotic machinery in these contextsto promote cell elimination. The roles of autophagy in aging,neurodegeneration and oxidative stress have also been effectively

lsevier B.V. All rights reserved.

Y.-Y. Chang, T.P. Neufeld / FEBS Letters 584 (2010) 1342–1349 1343

addressed in this system. Through these studies, several Drosophilagenes have been identified for their roles in regulating autophagy,including a group of upstream signaling molecules and the essen-tial Atg homologs (Table 1). These genes all share evolutionary con-servation across species and together they depict the molecularmechanism of autophagy, forming the basis for the applicationsof autophagy in human diseases in the Drosophila model. There-fore, studies in Drosophila can contribute considerably to ourunderstanding of the autophagic process. Here, we summarize re-cent advances in our knowledge of autophagy function and regula-tion from experiments in the Drosophila system.

2. Autophagy regulation: TOR and the Atg1 kinase complex

With its multiple functions, autophagy is a tightly regulatedprocess under the control of several intracellular signaling net-works. The highly conserved TOR pathway is an important compo-nent of these networks, integrating multiple cellular responses togrowth factors, nutrients and energy levels (Fig. 1A). Recent workin a number of systems have identified the Ser/Thr protein kinaseAtg1 as a central target of TOR in directing the formation of auto-phagosomes [1]. Loss of Atg1 blocks the formation of autophago-somes, and consensus observations across species have placedAtg1 downstream of TOR [3–5]. The ability of Atg1 to regulateautophagy relies on a group of interacting proteins without enzy-matic activities. In yeast, Atg13 and Atg17 are two major compo-nents of a multi-protein Atg1 complex [5]. Atg1 activity isdepleted in atg13 or atg17 mutant cells and autophagosome forma-tion is greatly impaired in these lines. Whereas clear homologs ofAtg17 have not been identified in Drosophila and other highereukaryotes, Atg13 is indispensable for autophagy in both yeast

Table 1The core genes for autophagy in Drosophila.

Name Functions

Insulin-TOR signaling pathwayInR Insulin-like receptorPI3K PI3K class IRheb Ras small GTPase familyTSC1/TSC2 GTPase-activating protein (GAP)TOR PIK-family Ser/Thr protein kinase

Autophagosome inductionAtg1 Ser/Thr protein kinaseAtg13 Atg1 complexFIP200 (CG1347) Atg1 complexAtg101 (CG7053) Atg1 complex

Autophagosome nucleationAtg6 Vps34 complex

Ser/Thr protein kinaseVps15 Vps34 complex

PI3K class IIIVps34 Vps34 complexAtg14 (CG11877) Vps34 complexUVRAG (CG6116) Vps34 complexRubicon (CG12772) Vps34 complex

Autophagosome expansionAtg3 E2-like enzyme for Atg8-PEAtg4 Cysteine protease cleaves Atg8 C-terminusAtg5 Atg5-Atg12-Atg16 complexAtg7 E1-like enzyme for Atg8 and Atg12

Ubiquitin-like,Atg8a/ Atg8b Conjugated to PEAtg10 (CG12821) E2-like enzyme for Atg12

Ubiquitin-like,Atg12 Atg5-Atg12-Atg16 complexAtg16 (CG31033) Atg5-Atg12-Atg16 complex

RecyclingAtg2 Atg2-Atg9 complexAtg9 Atg2-Atg9 complexAtg18 PI3P binding

and metazoans. The well-established yeast model has shown thatphosphorylation of Atg13 by TOR signaling disrupts the interactionof Atg1 and Atg13. Upon starvation, Atg13 is dephosphorylated andquickly binds Atg1 to turn on autophagy [5]. In contrast to thisyeast model, in which the interaction of Atg1 and Atg13 is limitedto starved cells, Drosophila Atg1 and Atg13 interact constitutivelyregardless of nutrition conditions [6]. Similarly, the mammalianAtg1 homolog Unc-51 like kinase 1 (Ulk1) forms a 3MDa complexwith Atg13, Atg101 and FIP200 that is stable under both fed andstarved conditions [7,8]. These observations indicate a regulatorydiscrepancy in yeast and higher eukaryotes, in which the basalautophagy is constantly maintained (Fig. 2).

Whereas the yeast Atg1 complex contains at least eight proteinsand mammalian Ulk1 can form a 3MDa complex, the number ofDrosophila Atg1-interacting proteins for autophagy regulation re-mains to be determined. Among 18 Drosophila proteins that havebeen identified as potential Atg1-interactors by yeast two-hybrid(http://www.thebiogrid.org/), thus far only Atg13 has been shownto play a role in autophagy [6]. Drosophila Atg1 has also beenshown to form a complex with the kinesin heavy chain adaptorprotein Unc-76, which has an important function in axonal trans-port that is distinct from the role of Atg1 in autophagy [9]. Collec-tively, Drosophila Atg1 may exert distinct functions by recruitingdifferent partners, and in order to fully understand the role ofAtg1 in autophagy control, discovering Atg1-interacting proteinsspecific to autophagy regulation will be a critical task.

Given that Atg1 is a protein kinase, how the kinase activity ofAtg1 is involved in autophagy is important to address. Atg1 kinaseactivity increases after starvation both in yeast and mammaliancells, suggesting this activity is regulated by nutrition cues andcontributes to autophagosome formation [5,10]. In addition, Atg1

Confirmed Role in Autophagy References

Yes (inhibition) [3,38,45]Yes (inhibition) [3,27–28]Yes (inhibition) [3]Yes [3]Yes (inhibition) [3]

Yes [3,6,12,27]Yes [6]??

Yes [3,27]

Yes [16]

Yes [16]???

Yes [12,27]Yes [55]Yes [3,12]Yes [3,27,36]

Yes [3,12,27,35]?

Yes [3]?

Yes [3,27]? [3]Yes [3,27]

Fig. 1. Three major autophagy induction routs in Drosophila. (A) Upon starvation or removal of growth factors, autophagy is induced through inhibition of TOR, whichnegatively regulates autophagy in well-fed organisms. The activity of TOR is under the control of Rheb and TSC1/2, both acting downstream of PI3K and AKT but withopposing effects on TOR. (B) Developmental autophagy is triggered by ecdysone signaling to eliminate tissues or organs, accompanied by the activation of cell death genes.The involvement of caspases during developmental autophagy is cell-specific. (C) JNK can direct the induction of autophagy in response to oxidative stress. Atg1 and other Atggenes are transcriptionally activated by JNK signaling, possibly through the dFOXO transcriptional factor.

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kinase activity is decreased in yeast atg13 mutants, and coexpres-sion of Atg13 enhances Atg1 kinase activity in both Drosophila andmammalian cells [5,6,11]. Together with the failure of kinase-defective Atg1 to rescue the lethality and autophagy defect of Dro-sophila Atg1 mutants [12], these findings support the notion thatkinase activity of Atg1 is required for autophagy. Klionsky andco-workers further demonstrated two distinct functions of yeastAtg1: assembly of the pre-autophagosomal structure (PAS)requires a kinase-independent structural role of Atg1 in associationwith Atg13 and Atg17, whereas dissociation of Atg proteinsrequires Atg1 kinase activity [13]. This finding separates Atg1kinase activity from the initiation of autophagy in yeast and raisesthe possibility that Atg/Ulk1 kinase activity may be required at oneor more steps following the induction of autophagosome formationin higher eukaryotes.

Coexpression of Atg1 and Atg13 in Drosophila increases thephosphorylation of both of these proteins in a TOR- and Atg1kinase-dependent manner [6]. This suggests that Atg13 and Atg1itself are substrates of Atg1 kinase, although indirect phosphoryla-tion by other kinases has not been excluded. Similar hyper-phos-phorylation of Atg1 and Atg13 by TOR and Atg1 are alsoobserved in mammals in vivo and in vitro [7,11]. A global,in vitro analysis of peptide phosphorylation identified 188 proteinsas potential substrates of Atg1 kinase, including Atg8, Atg18 andAtg21 [14]. Identification of the direct substrates of Atg1 forautophagy regulation will be an important line of futureinvestigation.

Overexpression of Drosophila Atg1 is sufficient to induceautophagy; in contrast, high levels of Ulk1 expression blocksstarvation-induced autophagy in mammalian cells [4,12]. A com-

parable inhibitory effect on autophagy induction also occurs inresponse to Drosophila Atg13 overexpression [6]. These observa-tions suggest that the Atg1–Atg13 complex can have both posi-tive and negative roles in autophagy regulation. Considering thatyeast Atg1 functions as a scaffold protein to initiate autophagy,it is possible that overexpression of either Atg1 or Atg13 makesmolecules essential for autophagy unavailable by sequesteringthem away from their normal loci. Alternatively, autophagyinduction may require a strictly balanced ratio of Atg1 andAtg13, and disruption of this balance by overexpression of eitherprotein may lead to autophagic deficiency. This hypothesis isfurther supported by the observation that coexpression of Atg1and Atg13 at low levels leads to autophagy induction underfed conditions [6].

In addition to its direct role in autophagosome formation, Atg1induces autophagy partly through a negative feedback loop to TOR.The activity of TOR signaling is down-regulated in a dose-depen-dent manner when Atg1 is overexpressed, evident by reducedTOR-dependent phosphorylation of RPS6-p70-protein kinase(S6K) [12]. Coexpression of low levels of Atg1 and Atg13 altersthe intracellular localization of TOR from a diffuse perinuclearcompartment to large cytoplasmic vesicles, which may indicate adisruption of the normal nutrient-dependent trafficking of TOR[6]. In addition, the sequestering of TOR from its normal loci mayrely on the physical binding of TOR and Atg1 [6]. A similar dynamicinteraction of TOR and Ulk1 complex is also evident in mammaliancells [7]. Taken together, the interaction of TOR and Atg1/Ulk1complexes appear to involve several different levels, and the ulti-mate decision of autophagy induction likely reflects the balanceof TOR and Atg1/Ulk1 activity.

Fig. 2. Comparison of Atg1 complexes in yeast, Drosophila and mammals. (A) Inyeast, the phosphorylation of Atg13 by TOR signaling prevents Atg13 from forminga complex with Atg1. No autophagy occurs until the phosphorylation on Atg13 isremoved in response to starvation. (B) Drosophila Atg1–Atg13 complex is presentconstitutively in fed and starved conditions. Atg1 and Atg13 are both phosphor-ylated by Atg1 and TOR signaling; however, Atg1 is more sensitive to TOR signalingin fed animals while phosphorylation of Atg13 is highest under starved condition,where Atg1 activity is elevated. (C) Similar to Drosophila, mammalian Atg1complexes show little change in composition in response to nutrient status, exceptthat mTOR has higher affinity for the Atg1 complex under fed conditions. AlthoughAtg1 and Atg13 are both substrates of mTOR and Atg1, similar to their Drosophilacounterparts, starvation leads to decreased phosphorylation of Atg13 due to lowermTOR activity as well as higher Atg1-dependent phosphorylation of FIP200.

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3. Type III phosphoinositide 3-kinase (PI3K) complex

The double membrane of autophagosomes is a unique feature,making autophagosomes distinct from other vesicles. However,the origin of this double membrane is still debatable, and differentorigin sources have been suggested, such as ER or mitochondria[15]. A phosphatidylinositol-3-phosphate (PI3P)-enriched struc-ture seems to be the site at which autophagosomes form. PI3P isthe product of PI3Ks and is known to play a critical role in autoph-agy. Treatment with Wortmannin or 3-methyladenine, generalinhibitors of PI3Ks, potently blocks autophagy in mammalian cells,supporting the involvement of PI3P in autophagy formation.Although there is only one PI3K in yeast, three classes of PI3K havebeen characterized in Drosophila and mammals, and mutations inVps34, the type III PI3K that generates PI3P, block the formationof autophagosomes in Drosophila [15,16]. These genetic resultsdemonstrate the requirement of PI3K for autophagy, consistentwith the effects of PI3K inhibitors in mammals. Interestingly,although overexpression of Drosophila Vps34 can increase theintensity of autophagy in starved animals, this is insufficient to in-duce autophagy under fed conditions [16]. These results indicatethat generation of PI3P is not enough to induce autophagy without

the coordinated effects of other Atg proteins or TOR-dependentsignals.

In yeast, Vps34 regulates autophagy through a complex con-taining Atg6, Atg14 and Vps15 [1]. Both Drosophila Atg6 andVps15 are required for autophagy and can interact with Vps34,suggesting that this conserved machinery is utilized in Drosophila[16]. Interestingly, a number of different Vps34 complexes havebeen observed in mammals, each containing the core proteinsAtg6/Beclin 1, Vps15/p150 and Vps34, and different combinationsof Atg14L, Ambra1, UVRAG or Rubicon [15]. Orthologs of Atg14L,UVRAG and Rubicon are also present in the Drosophila genome,indicating that Drosophila Vps34 may similarly form different com-plexes with specific functions in directing autophagosomeformation.

4. Endocytic pathway and autophagy

The observation that Vps34 functions both in autophagy andendocytosis raises the question whether other components of theendocytic pathway are also involved in autophagy [16]. The endo-somal sorting complex required for transport (ESCRT) complexcontains 4 subgroups, including ESCRT-0, ESCRT-I, ESCRT-II andESCRT-III. These subgroups function stepwise to control the deliv-ery of ubiquitinated receptors to multivesicular bodies (MVB) [15].Mutations in Drosophila vps28 (ESCRT-I), vps25 (ESCRT-II), vps32(ESCRT-III), or vps4 (an AAA ATPase required for ESCRT-III function)each show increased levels of Atg8 punctate structures in fat bodyand ovarian follicle cells [16,17]. Observation of these mutants byelectron microscopy reveals the accumulation of autophagosomesbut lack of autolysosomes or amphisomes, which result from fu-sion of autophagosomes and endosomal compartments. These re-sults indicate that ESCRT components are required for anessential step in the maturation and fusion of autophagosomeswith the endosomal compartment. Similar accumulations of auto-phagosomes in ESCRT mutations are evident in mammalian andnematode cells [18]. Interestingly, ESCRT components are not re-quired for autophagy in yeast, as autophagosomes are apparentlyable to fuse directly with the yeast vacuole, without prior inputfrom the endocytic pathway [15].

The fusion of autophagosomes with lysosomes requires a groupof docking proteins acting on both sides of autophagosomes andlysosomes. These docking proteins include components of thehomotypic fusion and protein sorting (HOPS) complex, consistingof the Vps–C complex (Vps18, Vps11, Vps33 and Vps16) togetherwith Vps41 and Vps39. Mutation in Drosophila deep orange (dor),encoding a Vps18 homolog, causes accumulation of endosomes,suggesting a conserved role in endocytic trafficking. As observedin ESCRT mutants, autophagosomes accumulate in dor mutants inlarval fat body cells, where developmental autophagy is inducedto degrade fat bodies for pupation [19]. Similar accumulation ofautophagosomes in mutants of dvps16A, one of two vps16 in Dro-sophila genome, supports the idea that the full HOPS complex isessential for autophagy in multicellular organisms, as in the yeastmodel [20]. Interestingly, UVRAG is able to associate with theHOPS complex, and overexpression of UVRAG enhances auto-phagosome fusion and autophagic flux in a Beclin 1-independentmanner in mammals [21]. The function of this Vps34 componentat a late step of autophagosome formation raises the question ofhow these dynamic endocytic proteins are regulated and inte-grated in autophagy regulation. For proteins with functions in boththe endocytic pathway and autophagy, it is necessary to clarifywhether and how these two functions overlap as well as their pre-cise roles in autophagosome formation. As mentioned above, thefunction of Drosophila UVRAG has not yet been studied, and it willbe interesting to determine whether Drosophila UVRAG has similar

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separate functions in autophagosome induction and maturation.Another Drosophila protein with dual roles in autophagy and endo-cytosis is liquid facets (lqf), a homolog of vertebrate epsin, whosemutation impairs endocytosis and developmental autophagy[22]. The roles of lqf in autophagy and endocytosis are reminiscentof ESCRTs and Vps34, and the lack of accumulation of autophago-somes in lqf mutants implies that lqf may function at early stepof autophagy, similar to Vps34.

5. Developmental autophagy and apoptosis

Although both autophagy and apoptosis are capable of leadingcells to death as a final destiny, their relationship is still paradox-ical. Diverse approaches have been applied to answer this questionin different organisms, including yeast, Drosophila and mammals.The primary distinction of autophagy and apoptosis is based onthe morphology of cells undergoing either process. Whereas thedefining characteristic of autophagy is the formation of double-membrane vesicles containing organelles or cytoplasm, DNA frag-mentation and cytoplasmic blebbing serve as fundamental mor-phological indicators of apoptosis. In Drosophila, the steroidhormone ecdysone controls larval molting and metamorphosisduring the fruit fly life cycle. The level of ecdysone peaks beforeeach molting in larval stage, and disruption of normal ecdysonelevels can cause an arrest of larval growth [23]. A gradual rise inecdysone synthesis at the end of the larval period induces develop-mental autophagy, allowing cellular reorganization in response todevelopmental timing. A peak of ecdysone at the end of the larvalperiod triggers metamorphosis, the process to eliminate the larvaltissues which are no longer necessary for adults and to prepare thematuration of adult tissues. Several larval tissues that undergosuch elimination serve as excellent models to study the relation-ship between autophagy and apoptosis, and studies in Drosophilaare beginning to elucidate general mechanisms by which steroidhormones can control both autophagic and apoptotic responses(Fig. 1B).

Dying larval midgut cells display several markers of apoptosis,such as DNA fragmentation, acridine orange staining and activatedexpression of proapototic genes. Mutation of E93, an early-actingecdysone-regulated gene, blocks the destruction of the larval mid-gut; however, the surviving midgut cells still contain fragmentedDNA, suggesting that induction of apoptosis is not sufficient for lar-val midgut cell death [24]. Accordingly, midgut degradation is notdisrupted by expression of the pan-caspase inhibitor p35 nor bymutation of major caspases, further demonstrating that apoptosisis dispensable for developmental midgut degradation [25]. In con-trast, mutation of E93 does inhibit the accumulation of autophagicvesicles normally observed in dying midgut cells. In addition, mid-gut destruction is blocked in animals lacking Atg1, Atg2 or Atg18activity, directly implicating autophagy as a crucial process inecdysone-induced degradation of midgut cells [24,25]. Caspasedeficiency does not enhance the Atg mutant midgut phenotypes,indicating that autophagic cell death in the midgut is caspase-independent despite the high levels of caspase activity during thisprocess [25].

The larval salivary gland, another tissue that is degraded duringmetamorphosis, also utilizes autophagy for its destruction [26].The incomplete degradation of salivary glands in Atg mutant ani-mals clearly indicates that salivary gland cell death is autophagy-dependent [27]. Ecdysone-mediated induction of E93 is also criticalfor autophagy-dependent salivary gland destruction. Expression ofthe class I PI3K catalytic subunit, or its target, AKT, inhibits salivarygland degradation [27], reminiscent of the requirement for PI3Kdown-regulation by ecdysone signaling during developmentalautophagy in the larval fat body [28]. Caspase activity remains in-

tact in these glands with high PI3K activity, in contrast to the lowcaspase activity, lack of DNA fragmentation and persistent auto-phagic vacuoles in glands expressing p35 [26]. Caspase activity isapparently normal and DNA fragmentation is also clearly observedin the salivary glands of a number Atg mutants. The combination ofp35 expression with either elevated PI3K activity or Atg mutationenhances the malfunction of salivary gland destruction by eitherone, strongly suggesting a parallel regulation of salivary gland celldeath by PI3K/autophagy and caspases [27]. Atg1 overexpression issufficient to cause premature salivary gland degradation devoid ofDNA fragmentation, and this is not suppressed by p35 expression,supporting the proposal that autophagic death of salivary glandcells is caspase-independent. This parallel model differs fromobservations made in Drosophila aminoserosa, fat body and wingdisc cells, whose degradation induced by Atg1 is suppressed byp35 expression [12,29]. Further, DNA fragmentation is significantlyreduced in dying ovarian cells in Atg1 or Atg7 mutants, indicating astrong epistatic connection between autophagic cell death andcaspases [30,31]. It should be noted that caspases acts upstreamof autophagy to direct the starvation-induced ovarian cell death,while autophagy is required to activate caspases during develop-mental ovarian cell death [30,31]. Together with findings in mam-malian cells that autophagy can be induced as a backupmechanism when caspase activity is compromised [32], these dif-ferences in dependency on caspases of autophagic cell death mayreflect differences in development stages and cell types.

6. Oxidative stress and the Jun-N-terminal kinase (JNK) pathway

The versatile JNK pathway is best known for its role in apopto-sis. As a branch of the mitogen-activated protein kinase (MAPK)pathway, the activity of JNK is regulated via a kinase cascade. Dro-sophila JNK and its upstream kinase (JNKK) are both encoded bysingle genes, basket (bsk) and hemipterous (hep), respectively. Afteractivation by Hep, Bsk phosphorylates two transcriptional factors,Jun-related antigen (Jra) and Kayak (Kay) (Drosophila Jun and Fos,respectively). Jra and Kay facilitate the transcriptional inductionof an array of JNK target genes, including the phosphatase Puck-ered (Puc). Following activation by JNK, Puc down-regulates JNKsignaling through negative feedback to Bsk, which is dephospho-rylated and inactivated by Puc [33]. This feedback loop activatesJNK signaling in a precise timeframe, by which Drosophila JNK ishighly regulated and has been implicated in several cellular pro-cess, such as dorsal closure, wound healing and longevity [33].

Treating wild type larvae with H2O2 or paraquat, a chemical in-ducer of oxidative stress, simultaneously induces autophagosomeformation and activates JNK signaling, suggesting a connection be-tween autophagy and JNK (Fig. 1C) [34]. Accordingly, paraquat-in-duced autophagosome formation is suppressed in bsk mutantanimals, indicating that autophagy is a downstream effector ofJNK signaling. Flies with higher JNK activity have an increased sur-vival rate when challenged with paraquat, and this advantage islost when Atg1 and Atg6 levels are compromised, indicating thatthe anti-oxidative stress ability of JNK signaling requires intactautophagy machinery. Consistent with these findings, overexpres-sion of Atg6 or Atg8 increases the resistance of flies to oxidativestress, whereas flies bearing Atg7 or Atg8 mutations are more sen-sitive to oxidative stress [34–36].

Following paraquat treatment, expression of Atg1 and Atg18rises transiently in concert with the peak of JNK activation, imply-ing that Atg genes may be direct transcriptional targets of the JNKpathway [34]. Indeed, constitutive activation of JNK signaling byexpression of activated Hep leads to increased expression of Atg1and Atg8, and subsequent autophagy induction [34]. However,JNK signaling is dispensable for developmental or starvation-in-

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duced autophagy, evident by the observation that both autophagicprocesses proceed normally in the absence of JNK activity [34]. Incontrast to these results in Drosophila, JNK is activated by starva-tion in mammalian cells [37]. In fed cells, Bcl-2 is predominantlypartnered with Beclin 1. Upon the stimulus of starvation, phos-phorylation of Bcl-2 by JNK disrupts its association with Beclin 1,allowing Beclin 1 to interact with Vps34 and initiate autophago-some formation [37]. Together, these observations imply a uniquerole of Drosophila JNK in autophagy induction, and suggest theeffect of JNK on autophagy induction may be limited to non-nutri-tive stress in Drosophila.

Drosophila dFOXO is a member of the FOXO (Forkhead Box O)family of transcriptional factors, which are important for stressresistance. Genetic interaction experiments in Drosophila demon-strate a strong connection between JNK signaling and dFOXO. Tar-geted overexpression dFOXO in the developing eye results in asmall, rough eye phenotype, which is suppressed by reducingJNK activity; similarly, removing one copy of dFOXO suppressesan eye defect caused by expression of activated JNK [38]. HighJNK signaling up-regulates the expression of dFOXO target genes,including growth controlling effector eIF4E binding protein (4E-BP) and oxidative stress protective small heat shock proteins(sHsps) [38]. Thus, JNK positively regulates the activity of dFOXO,suggesting that the anti-oxidative stress effect of JNK may partlybe accounted for by the elevated expression of sHsps throughdFOXO. Recently, Juhasz et al. reported that dFOXO is essentialand sufficient for autophagy induction, establishing a direct con-nection between dFOXO and autophagy [39]. Given the connec-tions between FOXO and JNK pathways and their roles inautophagy regulation, it is reasonable to speculate that the effectsof JNK on autophagy are mediated through FOXO-dependent tran-scription of Atg genes. If so, it will be important to determine howthese signals are integrated with Fos/Jun-dependent outputs andnon-transcriptional branches of this pathway.

7. Effects of autophagy on lifespan

Aging is the ultimate path for all organisms, usually accompa-nied by signs of accumulation of cellular damage, increased sensi-tivity to stresses, and reduced fitness to the environment. The roleof autophagy in degrading defective cellular components andassisting cells against stresses suggests that this process may havebeneficial effects on lifespan. The expression levels of several Dro-sophila Atg genes, including Atg2, Atg8a and Atg18, decline as fliesage, consistent with a role of autophagy in anti-aging [35]. Simi-larly, Beclin 1 levels are reduced in elder human brains [40], andthe rate of autophagy has been suggested to decrease as organismsage.

Although flies bearing Atg8a or Atg7 mutations can survive toadult stage, they have a reduced lifespan, increased levels cellulardamage and sensitivity to oxidative stress, and perform poorly inaging-related mobility tests [35,36]. Mice lacking atg7 or atg5 pro-gress through embryogenesis with no apparent developmentalabnormality, but die soon after birth [41,42]. Similarly, mutationsin C. elegans atg7 and atg12 shorten lifespan, and down-regulationof bec-1 suppresses the extended lifespan caused by mutant daf-2,the C. elegans ortholog of insulin/IFG-1 receptor tyrosine kinase[43,44]. Interestingly, overexpression of Atg8a in the Drosophilacentral nervous system is sufficient to significantly increase life-span and reduce accumulation of ubiquitinated and oxidized pro-tein [35]. Pan-neuronal overexpression of Atg8a early indevelopment had no beneficial effect in this study. These resultssuggest that although Atg7 and Atg8a are largely dispensable forembryonic and larval development, survival during adulthood isclosely tied to the levels of autophagic proteins, and, presumably,

to autophagic capacity or rate. Thus, therapies aimed at maintain-ing autophagy at higher levels late in adult life may have a benefi-cial effect on lifespan.

The aging process is also controlled by insulin-like signaling inDrosophila. Reduced insulin-like signaling, through mutations ininsulin-like receptor (InR) or the InR substrate chico, is beneficialto longevity. dFOXO appears to be a critical factor downstream toinsulin-like signaling for longevity control. Phosphorylation ofdFOXO by insulin-like signaling causes its translocation from nu-cleus to cytosol, thereby inhibiting expression of dFOXO targetgenes. Specific expression of dFOXO in adult head fat body signif-icant prolongs lifespan [45]. More strikingly, this localized expres-sion of dFOXO induces systemic down-regulation of insulin-likesignaling throughout the organism, evident by the overall in-creased nuclear retention of dFOXO. The level of dFOXO is inver-sely correlated with the expression of Dilp2, one of seveninsulin-like molecules in Drosophila [45]. Together, these findingssuggest that the longevity effect of dFOXO is specific to adult headfat body and acts cell non-autonomously through Dilp2 [45].

As discussed above, JNK protects against oxidative stress in partthrough dFOXO-mediated transcription. Further, several lines ofevidence suggest that JNK may also promote longevity throughdFOXO-mediated inhibition of insulin-like signaling. Flies with in-creased JNK activity live longer, and this advantage is suppressedby loss of one copy of dFOXO. Activation of JNK signaling specifi-cally in insulin-like peptide-generating cells significantly extendslifespan and down-regulates the level of dilp-2 in a dFOXO-depen-dent manner [38]. As mention above, both JNK signaling anddFOXO are essential and sufficient for autophagy induction, raisingthe possibility that the beneficial effect of these factors on lifespanis via autophagy [34,39]. Further investigation of the JNK–FOXO–autophagy connection in Drosophila should address whether thelifespan effects of localized dFOXO and JNK expression reflect localbenefits of autophagy in the head or non-autonomous effects in theperipheral tissues.

8. Autophagy in Drosophila neurodegeneration models

Neurodegenerative diseases are progressive disorders that af-fect millions of people worldwide. The loss of specific neuronalpopulations is the classic pathology of neurodegeneration. A widerange of studies have converged toward the concept that the mis-folding and accumulation of specific proteins in neurons is the rootcause of neuronal cell degeneration and other symptoms of thesediseases such as uncontrolled movement [46]. For example, pa-tients with Huntington’s disease express a toxic form of huntingtinprotein with an expanded run of glutamine repeats, which formsaggregates in neurons, a typical pathological feature of this disease.The severity of neurodegenerative diseases usually correlates withthe expression levels of these specific mutant proteins. Thereforethe clearance mechanism of toxic proteins and aggregates in neu-ronal cells is of high clinical interest [46].

The short life cycle, powerful genetics, and visible morphologi-cal defects make Drosophila a useful system for studying neurode-generation. Several neurodegenerative disease models have beensuccessively developed in Drosophila, such as Huntington’s, Parkin-son’s and Alzheimer’s diseases. For example, age-dependent neuro-degeneration of the fly retina is observed in eyes expressingpathogenic versions of huntingtin, ataxin-1, or other aggregate-prone proteins carrying poly-glutamine or poly-alanine extensions[47,48]. Rapamycin treatment reduces the severity of these neuro-degeneration phenotypes, in an autophagy-dependent manner[48]. Similarly, inhibition of TOR in mouse models of Huntington’sdisease significantly increases the clearance of hungtingtin aggre-gates, whereas overexpression of Rheb increases huntingtin aggre-

1348 Y.-Y. Chang, T.P. Neufeld / FEBS Letters 584 (2010) 1342–1349

gation [47]. Interestingly, TOR protein is sequestered into patho-genic huntingtin aggregates, leading to decreased TOR signalingand induction of autophagy [47]. Sequestering effects on TOR pro-tein are also observed with intranuclear ataxin-1 and in brainsfrom patients with spinocerebellar ataxia type 2, 3 and 7 [47]. Anindependent study described a similar induction of autophagy byataxin-3 in Drosophila, suggesting that induction of autophagy bypathogenic aggregates is a common phenomenon in neurodegen-erative diseases [49]. Thus, aggregate-prone proteins appear toprotect cells from their own toxicity in part by recruiting andsequestering TOR into the aggregates, leading to autophagy induc-tion and increased protein clearance.

The autophagy-lysosomal pathway functions in parallel to theubiquitin-proteasome system, the other major pathway of cellulardegradation. In degenerative neuronal cells, ubiquitinated proteinsthat are marked for proteasomal degradation often accumulateand form aggregates. Accumulation of ubiquitinated protein aggre-gates is also a common observation in Drosophila and mice lackingAtg5, Atg7 or Atg8a [35,36,41,50], indicating an intriguing interac-tion between these two systems. A recent study showed that agingflies have increased expression of Ref(2)P, the Drosophila homologof P62, accompanied by an increased level of ubiquitinated protein[51]. Ref(2)P was shown to interact with ubiquitinated proteinaggregates through its ubiquitin-associated (UBA) forming deter-gent-insoluble aggregates. Similar to huntingtin aggregates, autoph-agy is required for the clearance of these p62 and ubiquitinatedprotein aggregates, which are also found in organisms with neurode-generative diseases. Disruption of either proteasomal or autophagyactivity significantly increases the level of these aggregates and en-hances their colocalization in young wild type flies. However, dele-tion of either the PBI multimerization domain or the UBA domainof p62 suppressed aggregate accumulation caused by Atg8a muta-tion, suggesting that binding of p62 to ubiquitin is crucial for aggre-gate formation. The ability of p62 to bind both Atg8/LC3 andubiquitin brings the autophagy machinery to p62-ubiquitinatedprotein aggregates for their degradation, which may exemplifyhow autophagy ameliorates neurodegeneration [52].

Another recent study further demonstrates the intersection ofthe autophagy and proteasome systems in controlling neurodegen-eration [53]. Inhibition of proteasomal activity by DTS7, a temper-ature-sensitive dominant-negative mutation of the beta2 subunitof the proteasome, causes a degenerative eye morphology. TheDTS7-induced eye phenotype is enhanced in Atg mutants andstrongly suppressed by rapamycin treatment. The suppression byrapamycin is impaired by loss of Atg12 or Atg6, indicating that defi-cient proteasomal activity causes neuronal degeneration in anautophagy-dependent manner.

9. Conclusions

The versatility of autophagy as a catabolic process with a vari-ety of substrates allows it to play unique roles in the control of celldeath, cell survival, organism development and disease control.These functions rely on a complex regulatory network, whose com-ponents are still being identified. The conserved morphology andregulation of autophagy allows researchers to study this processin different model organisms; among them, the advantages of Dro-sophila as a model to study the functions and mechanism ofautophagy are evident. A series of Drosophila proteins involved inthe autophagic process have been identified, including the coreproteins consisting of Atg proteins and TOR-related signaling regu-lators, as well as proteins with functions in other processes, such asthe endocytic pathway. An important future goal for researchersworking in Drosophila will be to use the powerful genetics of thissystem to identify new factors acting in autophagy through for-

ward genetic screens, and to piece together the mechanisms bywhich these components function together. Fruits from the firstof such screens are beginning to be realized, and suggest a widevariety of proteins impact this process through distinct mecha-nisms [23,49,54]. The evolutionary conservation of autophagy sug-gests that studies in Drosophila will provide useful resources tounderstanding the overall mechanism of autophagy across species.

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