Review series

Autophagy in cell death: an innocent convict?

Beth Levine1 and Junying Yuan2
1Departments

of Internal Medicine and Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA. 2Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

The visualization of autophagosomes in dying cells has led to the belief that autophagy is a nonapoptotic form of programmed cell death. This concept has now been evaluated using cells and organisms deficient in autophagy genes. Most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro-survival rather than a pro-death mechanism. This review summarizes the evidence linking autophagy to cell survival and cell death, the complex interplay between autophagy and apoptosis pathways, and the role of autophagy-dependent survival and death pathways in clinical diseases.
Cellbiologistshavelongrecognizedthepossibilitythateukaryotic cellsmayundergononapoptoticformsofprogrammedcelldeath. Autophagy,alysosomalpathwayinvolvingthebulkdegradation ofcytoplasmiccontents,hasbeenidentifiedasaprimesuspect insuchdeath,andrecentstudieshaveimplicatedtheautophagy pathwayasacauseofnonapoptoticcellulardemise.However, mostevidencelinkingautophagytocelldeathiscircumstantial. Now,withnewtoolstoassesscausality,itisanopportunetimeto revisitthecaseofautophagyincelldeath.Isautophagyaninnocentbystander,adirectdeathexecutionpathway,adefensemechanismthatultimatelyfailsinitsmissiontopreservecellviability, and/oragarbagedisposalmechanismthatcleansupremnantsof acellalreadycommittedtodie? Autophagy is a regulated lysosomal degradation pathway Thetermautophagy(Greek,toeatoneself)doesnotrefertoadeath process;itdenotestheprocessofself-cannibalizationthroughalysosomaldegradationpathway.Autophagyisthecellsmajorregulated mechanismfordegradinglong-livedproteinsandtheonlyknown pathwayfordegradingorganelles(reviewedinrefs.1,2).During autophagy,anisolationmembraneforms,presumablyarisingfrom avesicularcompartmentknownasthepreautophagosomalstructure,invaginates,andsequesterscytoplasmicconstituentsincluding mitochondria,endoplasmicreticulum,andribosomes(Figure1). Theedgesofthemembranefusetoformadoubleormultimembranousstructure,knownastheautophagosomeorautophagic vacuole.Theoutermembraneoftheautophagosomefuseswiththe lysosome(inmammaliancells)orvacuole(inyeastandplants)to delivertheinnermembranousvesicletothelumenofthedegradativecompartment.Degradationofthesequesteredmaterialgeneratesnucleotides,aminoacids,andfreefattyacidsthatarerecycled formacromolecularsynthesisandATPgeneration. Autophagyoccursatlowbasallevelsinallcellstoperformhomeostaticfunctions(e.g.,cytoplasmicandorganelleturnover)butisrapidlyupregulatedwhencellsneedtogenerateintracellularnutrientsand energy(e.g.,duringstarvationortrophicfactorwithdrawal),undergo architecturalremodeling(e.g.,duringdevelopmentaltransitions),
Nonstandard abbreviations used:Htt,Huntingtin;3-MA,3-methyladenine;MEF, murineembryonicfibroblast;MPT,mitochondrialpermeabilitytransition;polyQ, polyglutamine;RNAi,RNAinterference;TOR,targetofrapamycin;TRAIL,TNFrelatedapoptosis-inducingligand. Conflict of interest:Theauthorshavedeclaredthatnoconflictofinterestexists. Citation for this article:J. Clin. Invest.115:26792688(2005). doi:10.1172/JCI26390.

orridthemselvesofdamagingcytoplasmiccomponents(e.g.,during oxidativestress,infection,andaccumulationofproteinaggregates). Nutritionalstatus,hormonalfactors,andothercuesliketemperature,oxygenconcentrations,andcelldensityareimportantinthe controlofautophagy.Twoevolutionarilyconservednutrientsensors playrolesinautophagyregulation:(a)thetargetofrapamycin(TOR) kinaseisthemajorinhibitorysignalthatshutsoffautophagyduringnutrientabundance(reviewedinref.3),and(b)theeukaryotic initiationfactor2(eIF2)kinaseGcn2anditsdownstreamtarget Gcn4,atranscriptionaltransactivatorofautophagygenes,turnon autophagyduringnutrientdepletion(4).TheclassIPI3K/AktsignalingmoleculeslinkreceptortyrosinekinasestoTORactivation andtherebyrepressautophagyinresponsetoinsulin-likeandother growthfactorsignals(reviewedinref.3). DownstreamofTORkinase,thereareapproximately17gene productsessentialforautophagyandrelatedpathwaysinyeast, referredtoastheATGgenes(5),andmostyeastATGgeneshave orthologsinhighereukaryotes(reviewedinref.2).TheATGgenes encodeproteinsneededfortheinductionofautophagy,andthe generation,maturation,andrecyclingofautophagosomes.These proteinsarecomposedof4functionalgroups,includingaproteinserine/threoninekinasecomplexthatrespondstoupstream signalssuchasTORkinase(Atg1,Atg13,Atg17),alipidkinase signalingcomplexthatmediatesvesiclenucleation(Atg6,Atg14, Vps34,andVps15),2novelubiquitin-likeconjugationpathways thatmediatevesicleexpansion(theAtg8andAtg12systems),and arecyclingpathwaythatmediatesthedisassemblyofAtgproteins frommaturedautophagosomes(Atg2,Atg9,Atg18)(Figure2). Autophagy as a cell death mechanism Thetermautophagiccelldeathdescribesaformofprogrammed celldeathmorphologicallydistinctfromapoptosisandpresumed toresultfromexcessivelevelsofcellularautophagy(6).Inclassical apoptosis,ortypeIprogrammedcelldeath,thereisearlycollapse ofcytoskeletalelementsbutpreservationoforganellesuntillate intheprocess.Incontrast,inautophagic,ortypeII,programmed celldeath,thereisearlydegradationoforganellesbutpreservation ofcytoskeletalelementsuntillatestages.Whereasapoptoticcell deathiscaspase-dependentandcharacterizedbyinternucleosomal DNAcleavage,caspaseactivationandDNAfragmentationoccur verylate(ifatall)inautophagiccelldeath(Figure3).Incontrast withnecrosis,bothapoptoticandautophagiccelldeatharecharacterizedbythelackofatissueinflammatoryresponse. Largenumbersofautophagicvacuoleshavebeenobservedin dyingcellsofanimalsofdiversetaxa(reviewedinrefs.69)(Table1).
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Figure 1
The autophagy pathway and its role in cellular adaptation to nutrient deprivation. Starvation or growth factor deprivation results in a decrease in intracellular nutrients and activation of nutrient-sensing signaling pathways (reviewed in ref. 97) that stimulate autophagy. Autophagy involves the sequestration of cytoplasmic material by an isolation membrane (derived from the preautophagosomal structure) to form a double-membrane vacuole, the autophagosome. The autophagosome undergoes fusion with a late endosome or lysosome, to form an autolysosome, in which the sequestered material is degraded. Degradation of membrane lipids and proteins by the autolysosome generates free fatty acids and amino acids that can be reused by the cell to maintain mitochondrial ATP energy production and protein synthesis and thereby promote cell survival. Disruption of this pathway by autophagy gene inactivation prevents cell survival in diverse organisms (Table 2). The same molecular machinery and overlapping dynamic membrane rearrangement events that occur during starvation may also be used in other settings to degrade unwanted cytoplasmic contents, including damaged mitochondria, protein aggregates, and intracellular pathogens. See text for discussion. TCA cycle, tricarboxylic acid cycle.

Theconsensusviewhasbeenthatautophagiccelldeathoccurs primarilywhenthedevelopmentalprogram(e.g.,insectmetamorphosis)orhomeostaticprocessesinadulthood(e.g.,mammary glandpostlactationalinvolution,prostateinvolutionfollowing castration)requiremassivecellelimination.Recently,studieshave alsodescribedautophagiccelldeathindiseasedmammaliantissuesandintumorcelllinestreatedwithchemotherapeuticagents (Table1).Inmanyofthesecases,morphologicfeaturesofautophagicandapoptoticcelldeathorofautophagicandnecroticcell deathareobservedinthesamecell. Whatistheevidencethatautophagyisadeathexecutionmechanisminautophagiccelldeath?Ifcelldeathistrulyduetoautophagy,thenpharmacologicorgeneticinhibitionofautophagyshould preventthedeath.Yet,formostofthedevelopmental,disease-associated,andtoxicstimulus-induceddeathsthatarepresumedtobe autophagic(Table1),theevidenceforitsroleisonlycorrelative. Moreover,incertaincasesofautophagiccelldeath,theavailableevidencecallsintoquestionacausativeroleofautophagy.Forexam2680

ple,inDrosophila,autophagiccelldeathbutnotautophagyobserved duringsalivaryglandregressionispreventedbymutationsinthe ecdysone-regulatedtranscriptionfactorsBR-CandE74A(10).Inthe slimemoldDictyostelium,anullmutationintheautophagygene atg1blocksvacuolizationbutnotcelldeathinaninvitromodelof autophagiccelldeath(11).Thus,inthesemodelsystems,autophagyperseisneithersufficientnorrequiredforautophagiccelldeath. Furthermore,thecaspaseinhibitorp35blocksmetamorphiccell deathinDrosophilawithoutcompleteinhibitionofautophagy,suggestingthatitiscaspase-mediatedapoptosis,ratherthanautophagy,thatplaysakeyroleinthisdeathprocess(10). Thereis,however,someevidenceincertaininvitrosettingsthat pharmacologicorgeneticinhibitionofautophagycanpreventcell death.Thepharmacologicinhibitorofautophagy3-methyladenine (3-MA),anucleotidederivativethatblocksclassIIIPI3Kactivity (1214),delaysorpartiallyinhibitsdeathinstarvedhepatocytes fromcarcinogen-treatedrats(15),inanti-estrogentreatedhuman mammarycarcinomacells(16),inchloroquine-treatedcorticalneu-

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Figure 2
The molecular mechanisms of autophagy. The autophagy (Atg) proteins can be divided into 4 functional groups, including (A) a protein kinase autophagy regulatory complex that responds to upstream signals, including nutrient limitation; (B) a lipid kinase signaling complex that mediates vesicle nucleation; (C) ubiquitin-like protein conjugation pathways that are required for vesicle expansion and completion; and (D) a retrieval pathway required for the disassembly of Atg protein complexes from matured autophagosomes. Shown are the yeast Atg proteins that participate in each functional group. Yeast Atg proteins with known orthologs in higher eukaryotes are underlined. PI, phosphatidylinositol; PI3-P, phosphatidylinositol 3-phosphate; PE, phosphatidylethanolamine.

blockedcelldeathinmouseL929cellstreatedwiththecaspase inhibitorzVAD(23).Further,RNAiagainstautophagygenesatg5 andbeclin 1blockeddeathofbax/,bak/murineembryonicfibroblasts(MEFs)treatedwithstaurosporineoretoposide(24).Notably, inbothofthesestudies,atggeneRNAiblockedthedeathofcells whoseapoptoticpathwayhadbeencrippled.Althoughthesefindings excludethepossibilitythatautophagyistriggeringdeaththrough apoptosisinduction,theyraisethequestionofwhetherautophagyis adeathmechanismincellswhoseapoptoticmachineryisintact. Interestingly,inetoposide-treatedwild-typeMEFs(whichdieby apoptosis),onlyminimalautophagicactivityandnoinhibitionof deathby3-MAisseen,indicatingthatautophagyisnotinvolved inthedeathprocessunlessapoptosisisblocked(24).Thesedata areconsistentwiththetheorypreviouslyproposedbyLockshin andZakerithatcellspreferentiallydiebyapoptosisbutwilldieby anyalternativeavailableroute,includingautophagy,ifexposedto harshenoughstimuli(9).Arelatedpossibilityisthatapoptotic deathisfasterthanautophagicdeathand,therefore,autophagyis onlywitnessedplayingaroleincelldeathinapoptotic-deficient cells.Thishypothesisisconsistentwithrecentdataindicatingthat growthfactordeprivedwild-typecellsundergoarapidapoptotic death,whereasgrowthfactordeprivedbax/,bak/cellsundergoa slowdemisecharacterizedbyprogressiveself-cannibalization(25). Giventheuncertainphysiologicrelevanceofautophagygene dependentcelldeathinzVAD-treatedcellsorinbax/,bak/cells, itseemsprematuretoconcludethatautophagyisaphysiologicallyimportantcauseofcelldeath.Toprovethatautophagyisan importantcelldeathpathwayinnormalcells,itwillbenecessary todemonstratecelldeathresistancephenotypesinapoptotic-competentcellslackingautophagygenes. Autophagy as a cell survival mechanism Acontradictory,butequallyplausible,explanationforthepresenceofautophagyindyingcellsisthatactivationofautophagyis acellularsurvivalstrategy.Thisconceptwasfirstproposedin1977 (26)andconsideredradical(8)butnowissupportedbystudies demonstratingincreaseddeathincellsororganismslackinggene productsessentialforautophagy(Table2).Thepro-survivalfunctionofautophagyisanevolutionarilyancientprocess,conserved fromyeasttomammals,andbestcharacterizedinnutrientdeficiency.Duringnutrientdeficiency,degradationofmembranelipidsandproteinsbytheautolysosomegeneratesfreefattyacidsand aminoacidsthatcanbereusedtofuelmitochondrialATPenergy productionandmaintainproteinsynthesis(Figure1).Presumably, thisrecyclingfunctionofautophagyislinkedmechanisticallyto itsabilitytosustainlifeduringstarvation.
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rons(17),innervegrowthfactordeprivedsympatheticneurons(18), inserum-andpotassium-deprivedcerebellargranulecells(19),in serum-deprivedPC12cells(20),andinTNF-treatedhumanTlymphoblasticleukemiacells(21).However,inseveralofthesestudies, autophagyoccurredincellsthoughttodiebyapoptosis,anditwas presumedthatautophagytriggeredapoptosis,ratherthanplayinga directroleinthedeathprocess.Moreover,3-MAcaninhibitkinases otherthanclassIIIPI3K(18),someofwhichmayindependently affectdeathsignaling,aswellasinhibitthepermeabilitytransition inmitochondria(22).Thus,itisnotpossibletodirectlyimplicate autophagyindeathexecutionfromthese3-MAinhibitorstudies. Tworecentstudiesprovidethefirstgeneticevidencethatthe autophagypathwayiscapableofkillingcells(Table2).RNAinterference(RNAi)directedagainst2autophagygenes,atg7andbeclin 1,

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Likethepro-deathfunctionofautophagygenesinetoposidetreatedbax/,bak/knockoutcells,itwillbeimportanttodetermine whetherthispro-survivalfunctionofautophagygenesduringgrowth factordeprivationinbax/,bak/cellsisconservedincellswithintact apoptoticmachinery.Itwillalsobeinterestingtoexaminewhether autophagygenesplayasimilarcytoprotectiveroleduringwithdrawal ofhormonalsupportorgrowthfactorsbesidesIL-3.Perhaps,rather thancontributingtodeathexecution,autophagydelaysinitiation oftheapoptoticdeathpathwayincellsdeprivedoftrophicsupport. Althoughstudieshavenotbeenperformedinapoptosis-competent cellsdeprivedoftrophicfactors,autophagygenespreventtheonset ofapoptosisduringnutrientdeprivation.RNAiagainstbeclin 1,atg5, atg10,andatg12enhancesstarvation-induced,butnotstaurosporineinduced,apoptoticcelldeath(36).Thus,themechanismbywhich autophagygenespromotesurvivalduringnutrientdeprivationmay involvesuppressionofthecanonicalapoptoticdeathpathway. Themechanismsbywhichautophagypromotescellsurvivalare notrestrictedtoitsroleinmaintainingcellularenergyhomeostasisduringstarvation.Autophagyisalsoinvolvedinremovingdamagedmitochondriaandotherorganelles,indegrading intracellularpathogens,andindegradingproteinaggregatestoo largetoberemovedbytheubiquitin-proteasomalsystem.These functionsofautophagycouldpromotecellularsurvivalduring aging,infectiousdiseases,andneurodegenerativeprocesses.In additiontoacell-autonomousroleforautophagyinpromoting survival,autophagymayregulateprogrammedcelldeathduring physiologicprocessesinvivo.Forexample,duringtheplantinnate immuneresponse,silencingofautophagygenesbeclin 1,vps34, atg3,andatg7doesnotalterthedeathofinfectedcellsorpathogenspreadbutresultsinuncontrolledspreadofprogrammed celldeathbeyondsitesofpathogeninfection(32).Thissuggests thatautophagylimitscelldeathtothesiteofinfection,allowing plantinnateimmunitytocontainpathogenspreadwithoutdeath ofinnocentbystandercells.Itisnotknownwhetherautophagy alterstheproductionofdeath-promotingsignals,preventsthe movementofdeath-promotingsignalsintouninfectedtissues,or protectsuninfectedtissuesagainstdeathinducedbythesesignals, orwhetherasimilarfunctionofautophagyplaysaroleinthespatialrestrictionofdevelopmentandstress-inducedprogrammed celldeathinothereukaryoticorganisms. Autophagy as a self-clearance mechanism Athirdexplanationforhighlevelsofautophagyindyingcellsis thatitisaclean-uporself-clearancemechanismincellscommittedtodiebyapoptosisornecrosis.Thistheorymightexplainwhy onlyselectedpopulationsofdyingapoptoticcellshavemorphologicfeaturesofautophagy.Thedogmaisthatmostapoptotic cellsareengulfedbyphagocytes,withthelysosomesofthephagocyteresponsibleforthefinaldegradationofdeadcellbodies.However,insomeformsofdevelopmentalprogrammedcelldeath(e.g., embryogenesis,insectmetamorphosis),theavailabilityofengulfmentcellsmaybeinsufficientforclearanceofdeadcells.Insuch cases,dyingcellsmayactivateautophagytotargetthecellscontentsfordegradationbyitsownlysosomes. Thisneedmightcontributetotheoverlapbetweensignaling pathwaysthatactivateapoptosisandautophagy.Ithasbeenshown thattheproapoptoticsignalingmoleculeTNF-relatedapoptosisinducingligand(TRAIL)regulatesautophagyinaninvitromodel ofmammaryglandformation(37).Here,TRAIL-dependentinductionofautophagyoccursinparallelwithapoptosis.Suppression

Figure 3
Ultrastructural examples of apoptotic and autophagic cell death. Electron micrographs of a FasL-treated Jurkat cell undergoing cell death with apoptotic features (A) and of a tamoxifen-treated MCF7 human breast carcinoma cell undergoing cell death with autophagic features (B). In A, note chromatin condensation (cell in center) and cytoplasmic vacuolization (cell in upper right). In B, note absence of chromatin condensation and presence of numerous autophagosomes. Images in A and B reproduced with permission from Nature Cell Biology (98) and Landes Bioscience (90), respectively.

Unicellularorganismswithnullmutationsinautophagygenes areviableinnormalgrowthconditions;however,unliketheirwildtypecounterparts,theydierapidlyduringstarvation(Table2) (2729). In plants, deletion of autophagy genes (e.g., ATG6, ATG7,andATG9)resultsinlossofchlorophyllandaccelerated senescencefollowingnutrientdeprivation(3032).MicelackingAtg5,anacceptormoleculefortheubiquitin-likemolecule Atg12,dieduringtheneonatalperiod,whentheplacentalblood supplyisinterruptedandtheyundergoaformofstarvation(33). Atg5/micehavedecreasedaminoacidlevels,decreasedcardiac ATPproduction,andmyocardialdamage.Althoughthedeath ofindividualcellshasnotyetbeenassessedinatg5/mice,itis predictedthattherecyclingfunctionofautophagyiscriticalto maintaincellularenergyhomeostasisandcellularsurvivalduringtheneonatalperiod.Thisisparticularlylikelyintissuessuch astheheartanddiaphragmthathavesuddenincreasesinenergy needsandexhibitincreasedautophagyimmediatelyfollowing birth.Similarly,theinabilityofCaenorhabditis eleganswithRNAisilencedautophagygenes(e.g.,unc-51,bec-1,atg8,andatg18)to surviveduringdauerdiapause(34)likelyreflectsaninabilityto recyclenutrientsattheorganismallevel. Autophagygenesmayalsobecriticalformaintainingcellularbioenergeticsandsurvivalwhencellsareunabletotakeup externalnutrients(i.e.,duringgrowthfactordeprivation).Inthe absenceofgrowthfactorssuchasIL-3,thereisdecreasedsurface expressionofnutrienttransporters,decreasednutrientuptake, andanintracellulardeficiencyofnutrients(35).Growthfactor withdrawalusuallyresultsinrapidapoptoticcelldeath,butrecent studiesinapoptotic-deficientbax/,bak/cellshaveunraveledan essentialroleforautophagygenes(e.g.,atg5,atg7)inmaintaining cellularsurvivalfollowingIL-3deprivation(25).Asinnutrient starvationinyeast,autophagyisaself-limitedsurvivalstrategy duringgrowthfactordeprivation.IL-3deprivedbax/,bak/cells eventuallydie,presumablybecauseofexcessiveself-consumption andbioenergeticfailure.However,atanypointbeforedeath,the additionofgrowthfactorreversesthecatabolicprocessandmaintainscellviability.Theseobservationsareconsistentwiththeconceptthatautophagyisaself-limitedsurvivalstrategy,ratherthan aprimaryorirreversibledeathexecutionprogram.
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Table 1 Examples of cell death with morphologic features of autophagyA
Species Tissue/cell type Setting References

Developmental programmed cell death Insects Chironomus tentans Manduca sexta Gryllus bimaculatus Calliphora vomitoria Drosophila melanogaster Orgyia leucostigma Salivary gland Intersegmental muscle, labial gland, prothoracic gland, larval fat body, motoneuron Prothoracic gland Salivary gland Salivary gland, midgut cells, larval fat body Epithelial wing Limb bud, neurons, mesonephros, mllerian duct epithelium, feather melanocytes, heart Ovarian follicle Tail nerve cord Gills, neurons Palatal epithelium Mammary epithelial cells Metamorphosis Metamorphosis Metamorphosis Metamorphosis Metamorphosis Adult reduction in females Embryonic development Atresia Metamorphosis Larval development Embryonic development Lumen formation (8) (37, 106) (reviewed in refs. 7, 8) (7) (8) (8) (99, 100; reviewed in ref. 7) (7, 101) (103) (10, 103105) (reviewed in ref. 7)

Birds Chicken Quail Amphibians Frog Mammals Mouse Human Disease-associated cell death Mammals Mouse

Rat Hamster Gerbil Human

Brain (cerebellar Purkinje cells) Striatal neuronal cell line Macrophages PC12 neuronal cell line Brain Hippocampus Heart Dopaminergic neurons

Lurcher mutation Mutant Huntingtin expression Salmonella infection Mutant -synuclein expression Experimentally induced transmissible spongiform Ischemic injury Dilated cardiomyopathy Aortic stenosis Parkinson disease

(67) (107) (83) (108) (65) (109) (110) (111) (63)

Drug-, toxin-, or stress stimulusassociated cell death Protozoans Leishmania donovani Tetrahymena thermophila Dictyostelium discoideum Mammals Mouse Heart Cortical neurons Neural precursor cells Sympathetic neurons Cultured cerebellar granule cells Retinal explant Hippocampal neurons PC12 pheochromocytoma cells Oral keratinocytes Ovarian carcinoma cell lines Mammary carcinoma cell line Glioma cell lines Endothelial cells
AIncludes

Antimicrobial peptide treatment Staurosporine treatment Dual exposure to starvation and differentiation-induced factor Diphtheria toxin treatment Chloroquine treatment FGF withdrawal Nerve growth factor withdrawal Serum and potassium deprivation Anisomycin treatment N-methyl-d-aspartate treatment Serum deprivation 5-Fluorouracil treatment Resveratrol treatment Anti-estrogen treatment Arsenic trioxide treatment Ceramide treatment Endostatin treatment

(112) (113) (114)

Rat

Human

(115) (17) (50) (18) (19) (116) (117) (118) (119) (120) (16, 121) (95, 122) (43) (123)

only those references in which (a) autophagy is observed at the ultrastructural level, and (b) autophagy is postulated by the authors to be a cause of death; excludes references in which (a) autophagy is assessed only at the light microscopic or biochemical level (e.g., by monodansylcadaverine staining, GFP-Atg8/LC3 staining, or LC3I-to-LC3II conversion), or (b) autophagy is not postulated to be mechanistically important in death.

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Table 2 Examples of autophagy genedependent cell death, cell survival, and metazoan survival
Species Cell death Mammals Mouse Human Cell survival Yeast Saccharomyces cerevisiae Protozoans Dictyostelium discoideum Plants Arabidopsis thaliana Nicotiana benthamiana Mammals Mouse Human Metazoan survival Caenorhabditis elegans Drosophila Mouse
ASetting

Tissue/cell type

ATG gene

SettingA

References

bax/, bak/ embryonic fibroblasts L929 fibroblast cells, U937 monocytoid cells

atg5, beclin 1 atg7, beclin 1

Etoposide or staurosporine treatment Caspase inhibition

(24) (23)

All ATG genes ATG1, ATG5, ATG6, ATG8, ATG12 Leaves Leaves ATG7, ATG9 ATG3, ATG7, beclin 1, vps34

Starvation Starvation Starvation Pathogen infection/innate immune response IL-3 withdrawal Alphavirus encephalitis Starvation Dauer development Larval development Larval/pupal development Embryonic development Early postnatal starvation

(27) (28, 29)

(30, 31) (32)

bax/, bak/ hematopoietic cells Brain HeLa cells

atg5, atg7 beclin 1B atg5, beclin 1, atg10, atg12 unc-51, bec-1, atg7, atg8, atg18 bec-1, atg8, atg18 ATG1, ATG3 beclin 1 atg5

(25) (79) (36) (34) (34) (85, 124) (88, 89) (33)

indicates conditions in which mutation in the indicated autophagy gene blocks cell death, cell survival, or metazoan survival. BThe role of this gene was assessed by overexpression; in all other studies referenced in the table, the role of each gene was assessed by RNAi silencing or loss-of-function mutation.

ofeitherapoptosisaloneorTRAILsignalingdoesnotprevent lumenformation,butsimultaneousinhibitionofapoptosisand TRAILsignalingpreventscellclearance. Thesefindingssuggestthatbothapoptosisandautophagymaybe involvedincavitationduringmammaryglandmorphogenesis.However,toconfirmaroleforautophagy,itwillbeimportanttoobserve whetherluminalfillingoccursifautophagygenesareinactivatedin cellswithintactTRAILsignaling.Itisnotclearwhetherautophagy isrequiredforluminalcelldeathorforremovalofcellscommitted todeathbyanapoptoticpathway.Similarly,itisnotknownwhether autophagyisrequiredforcaspase-dependentdeathand/orforthe clearanceofdyingcellsduringinsectmetamorphosis. Cross-talk between apoptotic signaling, autophagy, and mitochondria Severalproapoptoticsignalsinduceautophagye.g.,componentsoftheextrinsicapoptosispathway,TRAIL,TNF,andFADD (21,3740);thecalcium/calmodulinregulatedserine/threonine kinasesDRP-1andDAPk(41);andceramide(42,43).Conversely, antiapoptoticsignalingpathwayssuppressautophagye.g.,the classIPI3K/Akt/TORsignalingpathway(reviewedinrefs.3,44). Coordinatedregulationofapoptosisandautophagyisalsoreflectedintheresultsofgenome-wideanalysesoftranscriptionalchangesduringdevelopmentalprogrammedcelldeathoftheDrosophila salivarygland(45,46).
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Themitochondrionmayintegratecelldeathsignalsandautophagyactivation.Mitochondriagenerateapoptoticsignalsbutare removedwhendamagedbyautophagy;therefore,mitochondria representanexusatwhichautophagyandapoptosispathways mayinteract.Accordingly,genesinvolvedinmitochondrialphysiologyand/ormitochondrialregulationofapoptosisinteractwith theautophagypathway.Oneexampleistheyeastgene,UTH1,that encodesanoutermitochondrialmembraneproteininvolvedin mitochondrialbiogenesisandstressresponses.Uth1mutantsare defectiveindegradingmitochondriaduringautophagy(47)and surviveandproliferatewhenexpressingthemammalianproapoptoticcelldeathgenebaxorwhentreatedwiththeautophagyinducer rapamycin(48).ThesefindingsledCamougrandandcolleaguesto suggestthatUth1pmediatesmitochondrialautophagyandautophagicdeath.However,itisnotyetclearwhetherrapamycininduces celldeathversuscellcyclearrestinwild-typeyeast,andwhetherthe phenotypeofrapamycin-treateduth1mutantyeastsisduetodirect effectsofUTH1 andtheautophagypathwayindeathregulation. Inmammaliancells,Bcl-2familymembersintheoutermitochondrialmembranemodulateautophagy.Bcl-2downregulation increasesautophagyinacaspase-independentmannerinhuman leukemiccells(49),andBcl-2overexpressioninhibitsbothautophagyandcaspase-independentdeathingrowthfactordeprived neuralprogenitorcellsandinserum-andpotassium-deprived culturedcerebellargranulecells(19,50).Recentevidencesuggests

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thatBcl-2inhibitsautophagythroughadirectinteractionwiththe Beclin1autophagyproteinandthattheinteractionbetweenBcl-2 andBeclin1mayfunctionasarheostatthatmaintainsautophagyatlevelsthatarecompatiblewithcellsurvivalratherthancell death(51).Incontrast,Bcl-2orBcl-xLoverexpressionpotentiates autophagyandautophagygenedependentdeathinMEFstreated withtheproapoptoticstimulusetoposide(24).Thebasisforthe oppositeeffectsofBcl-2familymembersonautophagyindifferent settingsisunclear.Furthermore,itisnotyetclearthatBcl-2proteinsfunctionatthemitochondriontoregulateautophagy,since autophagyisinhibitedbyBcl-2targetedtoendoplasmicreticulum butnotbyBcl-2targetedtomitochondria(51). TheroleofproapoptoticBcl-2familymembersinautophagygene dependentlife-and-deathdecisionsisalsocontroversial.Asdiscussed earlier,bax/,bak/cellsundergoautophagygenedependentdeath whentreatedwithetoposide(24)butundergoautophagygene dependentsurvivalwhendeprivedoftrophicfactorsupport(25).It ispossiblethatinthesettingofbax/bakdeficiency,thestimulusplays acriticalroleindeterminingcellfate,andthatetoposide,butnot growthfactordeprivation,cantargetanintracellularpathwaythat turnsautophagyintoadeadlyprocess.SomeatypicalBcl-2family members,includingBNIP3andHspin,alsoactivateautophagyand nonapoptoticcelldeath(5254),butitisnotyetknownwhetherthis caspase-independentcelldeathrequiresautophagygenes. Anotherquestionishowtheautophagypathwayrecognizesdamagedmitochondria.Themitochondrialpermeabilitytransition (MPT)maytriggertheengulfmentofdepolarizedmitochondria byautophagy(55).However,itisnotknownwhetherinhibition ofautophagyincreasesthenumbersofdepolarizedmitochondria inmammaliancellsorhowadepolarizedmitochondrionmight betargetedtoautophagosomes.TheMPTmayrepresentapoint ofconvergenceofapoptoticandautophagypathways,sinceBcl-2 familymembersregulatetheMPT.TheproapoptoticfamilymemberBaxinteractswiththevoltage-dependentanionchannel(56) and/ortheadeninenucleotidetranslocator(57)toinducetheMPT incellsandisolatedmitochondriauponinductionofapoptosis.It iscurrentlynotclear,however,whethertheMPTregulatedbyBax triggersmitochondrialturnoverbyautophagy. Inmanycanonicalapoptosispathways,theMPTiscaspasedependent(58).Thus,iftheMPTisacriticalsignalformitochondrialdegradationthroughautophagy,inhibitionofcaspases shouldpreventthelossofmitochondria.However,Tolkovskyand coworkersreportedthat,althoughcaspaseinhibitorseffectively inhibitneuronalcelldeath,theyfailtopreventtheformationof autophagosomesorthedegradationofmitochondria.Infact,the long-termculturingofneuronsinthepresenceofproapoptotic stimuliandcaspaseinhibitorsleadstothelossofmitochondria (59,60).Thus,therelationshipamongMPT,caspase-dependent celldeath,andmitochondrialautophagyremainsunclear. Autophagy in neurodegenerative diseases Theaccumulationofmutantortoxicproteinsplaysamajorrole inchronicneurodegenerativediseases(61).Morphologicevidence ofautophagyhasbeenreportedinneurodegenerativediseases includingParkinson,Huntington,andAlzheimerdiseases,and transmissiblespongiformencephalopathies(6265).Itispossiblethatautophagyactivationcontributestoneurodegeneration (66,67),buttheevidenceiscorrelative.Acontrastingviewisthat autophagymaybeaprotectivemechanismtodegrademutantor toxicproteins.Accordingtothismodel,defectsinautophagy

relatedpathwayscontributetotheaccumulationofneurotoxic proteinsandtheensuingneuronalcelldeath.Althoughtheexact rolesofautophagyinneurodegenerativediseasesarenotfully defined,recentstudieshaveprovidedsomeinsights. heprotein-synucleinisamajorcomponentofneuronalcytoplasmicinclusionsthatcharacterizeParkinsonandotherneurodegenerativediseases(68).Althoughearlierstudiessuggested that-synucleinisdegradedthroughboththeproteasomeand classicalautophagypathways(69),arecentstudydemonstrated thattheturnoverof-synucleinisregulatedbychaperone-mediatedautophagy,whichinvolvesthedirectlysosomaltargetingof proteinscontainingspecificpentapeptiderecognitionmotifs(70). Interestingly,pathogenic-synucleinmutantsassociatedwith familial,autosomal-dominantformsofParkinsondisease(71,72) are inefficiently degraded by chaperone-mediated autophagy. Sincetheaccumulationofwild-type-synucleininneuronalinclusionsiscommoninadult-onsetneurodegenerativediseases,these experimentssuggestthatdefectsinautophagy-relatedpathways maycontributetomultipleneurodegenerativediseases. Consistentwiththishypothesis,autophagyhasalsobeenimplicatedinregulatingtheturnoverofHuntingtin(Htt),theprotein involvedinHuntingtondisease,anautosomal-dominantneurodegenerativedisordercausedbytheexpansionofapolyglutamine (polyQ)tractinHtt.Althoughthemechanismofneurotoxicity mediatedbyexpandedpolyQisstillcontroversial,expandedpolyQ provokesadominantgain-of-functionneurotoxicity,regardlessof thespecificproteincontextwithinwhichitresides.TheaccumulationofexpandedpolyQ-containingproteinsininsolubleaggregatesinaffectedneuronsisahallmarkfeatureofHuntingtonand otherpolyQexpansiondiseases(73). AlthoughneuronalHttproteinsininclusionsarehighlyubiquitinated,polyQisapoorsubstrateforproteasomes(74).Thus, thehighlyubiquitinatedstateofHttinclusionsmayindicatethe inabilityofproteasomesinaffectedneuronstoclearabnormal Httproteins.Incontrast,thereispharmacologicevidencetosuggestaroleforautophagyinthedegradationoftheN-terminus ofHtt.Forexample,3-MAincreasestheaggregationofHttwith expandedpolyQinclonalstriatalcells(62).Rapamycin,aninducer ofautophagy,reducestheaggregationofexpandedpolyQintransfectedcells(75),protectsagainstneurodegenerationinaflymodel ofHuntingtondisease,andimprovesperformanceonbehavioraltestsanddecreasesaggregateformationinamousemodelof Huntingtondisease(76).Theseresultssuggestapossibleroleof autophagyintheturnoverofexpandedpolyQproteinsandinprotectionofneuronsagainsttheirtoxicity. Autophagy and infectious diseases Theautophagicmachineryisusedtodegradeintracellularpathogens(reviewedinrefs.77,78)includingintracellularbacteria(e.g., Shigella flexneriandMycobacterium tuberculosis),mammalianviruses thatproduceencephalitis(e.g.,alphavirusesandherpessimplex virus),andplantviruses(32,7881).Italsomaybeusedtodegrade invadingextracellularpathogenssuchasgroupAStreptococcus (82). Itisreasonabletoproposethatautophagymightpromotecellular survivalduringpathogeninvasionbecauseofeitherenhanceddegradationofintracellularpathogensandconsequentdecreasesin microbialreplication;enhanceddegradationofspecificcytotoxic microbialvirulenceproducts;orpreservationofcellularnutrient statusduringaperiodofmicrobialparasitism(whichmimicsnutrientstarvation).However,withtheexceptionofthefindingthat
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forcedexpressionofthebeclin 1autophagygeneprotectsagainst Sindbisvirusinducedapoptosisinmousebrains(inparallelwith decreasingviralreplication)(79),directproofofacell-autonomous, pro-survivalroleofautophagyinpathogeninfectionislacking. Moreover,ithasbeenproposedthatavirulenceprotein,SipB,of theintracellularpathogenSalmonella entericacausesmacrophage deathbyinducingautophagy,perhapsbytriggeringmitochondrial fusionwithautophagosomes(83).Yet,inthisstudy,therewasno directevidencethatmacrophagecelldeathwascausedby,rather thansimplyassociatedwith,autophagy.Furtherstudiesinautophagy-deficienthostorganismsarerequiredtodeterminetheroleof autophagyinlife-and-deathdecisionsduringpathogeninfection. Autophagy and cancer Cancerresultsfromthedysregulationofpathwaysthatregulate celldifferentiation,cellproliferation,andcellsurvival.Autophagy mayprotectagainstcancerbysequesteringdamagedorganelles, permittingcellulardifferentiation,increasingproteincatabolism, and/orpromotingautophagicdeath.Alternatively,autophagy maycontributetocancerbypromotingthesurvivalofnutrientstarvedcells.Recentdataaremostconsistentwithamodelin whichautophagycontributestotumorsuppressionanddefects inautophagycontributetooncogenesis.Biochemicalevidencein mammaliancellsandgeneticevidenceinC. elegansandDrosophila indicatethatautophagyispositivelyregulatedbythePTENtumor suppressorgeneandnegativelyregulatedbytheoncogenicclassI PI3Ksignalingpathway(14,34,84,85).Furthermore,themammalianautophagygenebeclin 1hastumorsuppressoractivityin breastcarcinomacells(86),iscommonlydeletedinhumanbreast ovarianandprostatecancer(87),andisahaploinsufficienttumor suppressorgeneinmice(88,89). Severaltheoriesregardingtheroleofautophagy-dependent deathandautophagy-dependentsurvivalincancerbiologyhave beenproposed(3,66,9094).Oneisthatautophagy-dependent deathisamechanismoftumorsuppression.However,thereare nodirectdatatosupportthishypothesis.Incontrast,studiesin cellsandanimalswithadeficiencyinbeclin 1suggestthatdeath inductionmaynotbeinvolvedinthetumorsuppressorfunctionofthisautophagygene.Beclin 1/EScellsarenotresistant todeathtriggeredbyUVirradiationorserumwithdrawal,and beclin 1/nullanimalsdieearlyduringembryogenesiswithmassivecelldeath(89).Moreover,inbeclin 1heterozygous-deficient mice(withreducedtissuelevelsofautophagy),thereishyperproliferationofmammaryepithelialcellsduringglandularmorphogenesisandincreasedantigen-drivenproliferationofBcells withoutdecreasedcelldeath(88).Together,theseobservations suggestthattheroleofthebeclin 1autophagygeneintumor suppressionisrelatednottocelldeathinduction,butratherto inhibitionofcellularproliferation. Itispossiblethatautophagyisinvolvedinthespontaneousor chemotherapy-induceddeathofexistingtumorcells.Although theroleofautophagyincelldeathinapoptosis-competentcells isunclear,autophagygenedependentdeathincellscrippledin apoptosis(e.g.,zVAD-treatedcells;bax/,bak/cells)mayhaverelevanceforcancerbiologyandtherapy,sincehumantumorcellsfrequentlycontainmutationsthatrenderthemresistanttoapoptosis.Onepredictionisthatsuchcellshaveanincreaseddependency onautophagypathwaysforself-destruction,andthattheimpact ofdecreasedautophagy-dependentcelldeathontumorprogressionmaybegreaterintumorcellsthatareresistanttoapoptosis.
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Anotherpredictionisthattheenhancedautophagy-dependent death potential of apoptosis-resistant tumor cells might be exploitedtherapeuticallybytheadministrationofautophagyinducingagents.Indeed,thereareexamplesofputativeautophagiccelldeathincancercelllinestreatedwithchemotherapeutic agents(Table1).Duringtamoxifen-induceddeathofMCF7cells(a celltypethatcontainsamutationincaspase-3),thereisamarked upregulationofBeclin1autophagyproteinexpression(42,90), and,insomeexamples,chemotherapy-inducedautophagiccell deathisinhibitedby3-MA(16,42,95).However,evidenceproving thatautophagyisabonafidedeathpathwayinchemotherapytreatedcancercellsislacking.Inaddition,rapamycin,aninhibitorofTORkinasethathaspromisingantitumoreffectsinhuman clinicaltrials(96),isoneofthemostpotentknowninducersof autophagybutisnotknowntoinduceautophagiccelldeath. Incontrasttopotentialpro-deatheffects,moreclearlyestablishedpro-survivaleffectsofautophagyduringnutrientstarvationmightfostertumorinitiationand/orprogression(3,91,93). Astumorcellsgrowbeyondtheirbloodsupply,theyareexposed tonutrient-limitingconditions,anditispossiblethattransformedcellsuseautophagyasasurvivalstrategyinthissetting. Ithasbeenproposedthatsuchaneedforautophagyintumor initiationmightexplaintheretentionofthewild-typeallele inalltumorsarisinginbeclin 1+/mice(92).However,therole ofautophagyintumorcellsurvivalinvivohasnotbeentested experimentally.Moreover,inconsideringtheneteffectofautophagyontumorigenesis,itisimportanttorecognizeitsotherfunctionsthatcouldcontributetorestrictingtumorigenesis(e.g.,the degradationofcertainproteinsororganellesrequiredforcell growthand/orthedegradationofdamagedmitochondriaand otherorganellesthatgenerategenotoxicstressandincreasethe likelihoodofoncogenicmutations). Conclusion Autophagyfunctionsacrossadiverserangeofspeciesasaprosurvivalpathwayduringnutrientdeprivationandotherforms ofcellularstress.Paradoxically,incellsthatcannotdiebyapoptosisand,morespeculatively,incellsthatcannotberemoved by engulfment cells, the autophagic machinery may also be usedforself-destruction.Thechallengeforscientistswillbeto understandthemolecularbasisofthisparadox.Thechallenge forclinicianswillbetoselectivelyturnonorturnoffautophagy genedependentsurvivalanddeathpathwaysinthetreatmentof differentclinicaldiseases. Acknowledgments TheworkfromtheauthorslaboratorieswassupportedbyNIH grantsRO1CA084254,RO1AI151367,andRO1CA109618(to B.Levine)andR37NIA12859(toJ.Yuan);AmericanCancer SocietygrantRSG98-339(toB.Levine);andanEllisonMedical Foundation Senior Scholar Award in Infectious Diseases (toB.Levine).WethankAlexiDegetrevforprovidingelectron micrographsandSophiePattingreandReneeTalleyforhelpwith manuscriptpreparation. Addresscorrespondenceto:BethLevine,DivisionofInfectious Diseases, University of Texas Southwestern Medical Center, 5323HarryHinesBoulevard,Dallas,Texas75390-9113,USA. Phone:(214)648-0493;Fax:(214)648-0284;E-mail:beth.levine@ utsouthwestern.edu.

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The Journal of Clinical Investigation http://www.jci.org Volume115 Number10 October2005