Regulation of the Transcription Factor EB-PGC1α Axis by Beclin-1 Controls Mitochondrial Quality

21 Pages·2015·10.75 MB·English

(cid:2)
Regulation of the Transcription Factor EB-PGC1 Axis by Beclin-1
Controls Mitochondrial Quality and Cardiomyocyte Death under
Stress
XiucuiMa,a,cHaiyanLiu,a,cJohnT.Murphy,aSarahR.Foyil,a,cRebeccaJ.Godar,a,cHaedarAbuirqeba,aCarlaJ.Weinheimer,a
PhilipM.Barger,aAbhinavDiwana,b,c
DivisionofCardiologyandCenterforCardiovascularResearch,DepartmentofInternalMedicine,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,USAa;
DepartmentofCellBiologyandPhysiology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,USAb;JohnCochranVAMedicalCenter,St.Louis,Missouri,
USAc
Incardiacischemia-reperfusioninjury,reactiveoxygenspecies(ROS)generationandupregulationofthehypoxia-induc-
D
ibleproteinBNIP3resultinmitochondrialpermeabilization,butimpairmentinautophagicremovalofdamagedmito- o
chondriaprovokesprogrammedcardiomyocytedeath.BNIP3expressionandROSgenerationresultinupregulationof w
n
beclin-1,aproteinassociatedwithtranscriptionalsuppressionofautophagy-lysosomeproteinsandreducedactivationof lo
transcriptionfactorEB(TFEB),amasterregulatoroftheautophagy-lysosomemachinery.Partialbeclin-1knockdown a
d
transcriptionallystimulateslysosomebiogenesisandautophagyviamTORinhibitionandactivationofTFEB,enhancing e
removalofdepolarizedmitochondria.TFEBactivationconcomitantlystimulatesmitochondrialbiogenesisviaPGC1(cid:2)in- d
f
ductiontorestorenormallypolarizedmitochondriaandattenuateBNIP3-andhypoxia-reoxygenation-inducedcelldeath. ro
Conversely,overexpressionofbeclin-1activatesmTORtoinhibitTFEB,resultingindeclinesinlysosomenumbersand m
suppressionofPGC1(cid:2)transcription.Importantly,knockdownofendogenousTFEBorPGC1(cid:2)resultsinacompleteor h
t
partialloss,respectively,ofthecytoprotectiveeffectsofpartialbeclin-1knockdown,indicatingacriticalroleforbothmi- tp
:
tochondrialautophagyandbiogenesisinensuringcellularviability.Thesestudiesuncoveratranscriptionalfeedbackloop //
m
forbeclin-1-mediatedregulationofTFEBactivationandimplicateacentralroleforTFEBincoordinatingmitochondrial
c
autophagywithbiogenesistorestorenormallypolarizedmitochondriaandpreventischemia-reperfusion-inducedcardio- b
.
myocytedeath. a
s
m
.
o
Preservation of healthy mitochondria is essential for energy todrivereformationofnewlysosomes(18–20).Thisisfacili- rg
/
generationandmaintenanceofcontractilefunctionincar- tated via a transcriptional induction of autophagy-lysosome o
n
diacmyocytes(1).Incardiacischemia-reperfusion(IR)injury, machinery proteins orchestrated by nuclear translocation of
A
mitochondrial permeabilization results in activation of pro- the basic helix-loop-helix (bHLH) transcription factor EB p
grammed cell death pathways and cardiomyocyte loss (2). (TFEB)(21–25),amasterinduceroftheautophagy-lysosome ril
Removal of damaged mitochondria by macroautophagy, a machinery (23), thereby sustaining autophagic flux. In con- 3
,
lysosomaldegradativepathway,isessentialtopreventcardiomy- trast,lysosomenumbersprogressivelydeclinewithBNIP3-in- 2
ocytedeathandlimitmyocardialinfarctsize(3,4).Cardiomyo- ducedautophagy,withoutreplenishment(15),suggestingthat 01
cyteautophagyisupregulatedwithIRinjury(5),butautophago- 9
animpairmentinthistranscriptionalresponseengenders“in-

some processing is impaired early after reperfusion, which b
sufficient”cytoprotectiveautophagy. y
preventsautophagicremovalofdamagedmitochondria(6).The
Relevant to this discussion is our observation that upon g
hypoxicinsultalsoprovokestranscriptionalinductionofBNIP3 u
reperfusion/reoxygenation, a rapid reactive oxygen species e
(Bcl2andnineteen-kilodaltoninteractingprotein3),aprodeath s
(ROS)-induced increase in beclin-1 abundance paradoxically t
Bcl2familyprotein(7,8)whichistargetedtoandpermeabilizes
impairs autophagic flux in cardiomyocytes (6). Interestingly,
mitochondria(9–11)andtriggerscardiomyocytedeathinIRin-
whilebasalbeclin-1levelsplaycriticalrolesinautophagosome
jury(12).WhileBNIP3hasbeensuggestedtofacilitatemitochon-
drialautophagybyfunctioningasanadaptortosequesterdam-
aged mitochondria within autophagosomes (13, 14), increased
BNIP3expressionprovokesdeclinesinlysosomenumbers,with Received25August2014 Returnedformodification15September2014
Accepted30December2014
impairedautophagicflux,resultinginaccumulationofdamaged
Acceptedmanuscriptpostedonline5January2015
mitochondriaandcardiomyocytedeath(15).Theseobservations
CitationMaX,LiuH,MurphyJT,FoyilSR,GodarRJ,AbuirqebaH,WeinheimerCJ,
implicateafailureoftheautophagy-lysosomemachinerytoclear BargerPM,DiwanA.2015.RegulationofthetranscriptionfactorEB-PGC1(cid:2)axisby
damagedmitochondriaasacauseofcelldeathwithIRinjury,but beclin-1controlsmitochondrialqualityandcardiomyocytedeathunderstress.
theunderlyingmechanismsremaintobedefined. MolCellBiol35:956–976.doi:10.1128/MCB.01091-14.
Mitochondria are also targeted for degradation by starva- AddresscorrespondencetoAbhinavDiwan,[email protected].
tion, wherein autophagy is critical for cell survival (16, 17). Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
Interestingly,withstarvation,lysosomenumbersrapidlyplum- doi:10.1128/MCB.01091-14
met(18),butendogenousmechanismsarepromptlyrecruited
956 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
formation(26)andprotectionagainstcardiomyocytedeath(6),we Generationofadenoviralconstructs.Lentiviralparticlescodingfor
observedthatincreasedbeclin-1abundanceissufficienttosuppress mCherry-GFP-LC3 expression have been described (6). Adenoviral
transcriptionofautophagy-lysosomemachinerygenes(6).Taken delivery of constructs was employed to achieve a high efficiency of
together with the in vivo observation that haploinsufficiency of transductionandpermitevaluationofdose-dependenteffectsinpri-
beclin-1bytargeteddisruptionofaBECN1alleleconferscytopro- maryNRCMs,aspreviouslydescribed(6,15).Adenoviralparticlesfor
expression of short hairpin RNAs (shRNAs) targeting rat TFEB,
tectionincardiacIRinjury(5),thesedatasuggestthehypothesis
BECN1,andPPARGC1(cid:2)weregeneratedwiththeBLOCKiTadenoviral
thatROS-inducedupregulationofbeclin-1transcriptionallyim-
system (Invitrogen). Specific oligonucleotide sequences employed
pairs the lysosomal machinery to prevent removal of damaged
were as follows (with targeted coding sequences underlined): (i)
mitochondria and cause cell death with BNIP3 expression and
shRNAtargetingratTFEB,5=-CACCGAATCAAGGAGCTGGGAATG
hypoxia-reoxygenationinjury.Inthisstudy,weuncoveredanau- CTGATCGAAATCAGCATTCCCAGCTCCTTGATTC-3=(top-strand
toregulatoryloopwherebybeclin-1levelsregulateTFEBactivity, oligonucleotide) and 5=-AAAAGAATCAAGGAGCTGGGAATGC
which coordinates mitochondrial autophagy with biogenesis to TGATTTCGATCAGCATTCCCAGCTCCTTGATTC-3= (bottom-strand
controlmitochondrialqualityandregulatestress-inducedcardi- oligonucleotide);(ii)shRNAtargetingBECN1(annotatedshBECN1-2),
omyocytedeath. 5=-CACCGCTCAGTACCAGCGAGAATATCGAAATATTCTCGCTGG
TACTGAGC-3= (top-strand oligonucleotide) and 5=-AAAAGCT
D
MATERIALSANDMETHODS CAGTACCAGCGAGAATATTTCGATATTCTCGCTGGTACTGA o
GC-3= (bottom-strand oligonucleotide); and (iii) shRNA targeting rat w
Invivoischemia-reperfusionmodeling.BECN1heterozygousnullmice PPARGC1(cid:2), 5=-CACCGAGCAAGTATGACTCTCTGCGAACAGAGAG n
(BECN1(cid:3)/(cid:4);micewithhaploinsufficiencyofbeclin-1)(26)andwild- TCATACTTGCTC-3= (top-strand oligonucleotide) and 5=- lo
type (WT) mice of the C57BL/6 strain were obtained from Jackson a
AAAAGAGCAAGTATGACTCTCTGTTCGCAGAGAGTCAT d
Laboratories and subjected to reversible left anterior descending e
ACTTGCTC-3=(bottom-strandoligonucleotide).
(LAD)coronaryarteryocclusiontoinduceischemiafor30min,fol- d
Adenoviral particles for shRNAs targeting BECN1 (annotated
lowedby4hofreperfusion,asdescribedpreviously(6).Briefly,mice fr
shBECN1-1) (5) and LacZ, as a nontargeting control for studies with o
were anesthetized with a mixture of ketamine (80 mg/kg of body m
shRNA-mediatedknockdown,andforoverexpressionofLacZ(asacon-
weight) and xylazine (5 mg/kg), surgically prepped, and ventilated.
trol), green fluorescent protein (GFP)-tagged LC3, FLAG-hBNIP3, h
Afterthoracotomy,theLADarterywasidentified,anda9-0polypro- t
mBECLIN-1, and hemagglutinin (HA)-tagged hTFEB have been de- t
pylenesuturewaspassedundertheLADartery.Aknotwastiedovera p
scribed previously (15). Adenoviral particles for expression of FLAG- :
1-mmsectionofPE-10tubingplaceddirectlyoverthevesseltocreate PPARGC1(cid:2)wereengineeredwithataggingcodingsequenceforhuman //m
theocclusion.Ischemiawasconfirmedbyanabsenceofbloodflow,
PPARGC1(cid:2)withaFLAGtagattheNterminus,usingGatewaycloning c
verifiedvisually,andbythepresenceofSTelevationsonelectrocar- b
diogram (EKG). Reperfusion was induced to release the occlusion, technology(Invitrogen).Viralparticlesweregeneratedperthemanufac- .a
whichwasconfirmedbyresolutionofSTsegmentelevation.Atsacri- turer’sinstructions.Inallexperiments,controladenoviralparticleswere s
added to groups as necessary to ensure equal viral particle doses, i.e., m
fice,theventriclesweresectionedintothreepieces;theapical1/3was
multiplicitiesofinfection(MOIs),betweengroups. .o
subjectedtobiochemicalanalysisasthe“injured”region,andthebasal
1/3wasprocessedasthe“remote”region.Sham-treatedmiceunder- Assessmentofcelldeath.Celldeathassayswereperformedin96-well rg
wenttheentiresurgicalprocedurewithoutocclusionoftheLADar- plates(Nunc,Fisher)withaLive-Deadcytotoxicityviabilitykitformam- o/
tery. Autophagic flux was assessed in comparison to that of mice malian cells (Invitrogen), using a Tecan Infinite M200 Pro microplate n
pretreatedwithchloroquine(CQ;40mg/kg,administeredintraperi- reader(Tecan)followingthemanufacturer’sinstructions,asdescribed A
previously(6). p
toneally[i.p.])30minpriortoIRmodeling,aspreviouslydescribed(6, r
27).AllanimalstudieswereapprovedbytheAnimalStudiesCommit- AssessmentofTUNELpositivity.Terminaldeoxynucleotidyltrans- il
ferase-mediateddUTP-biotinnickendlabeling(TUNEL)wasperformed 3
teeattheWashingtonUniversitySchoolofMedicineandtheInstitu- ,
tionalAnimalCareandUseCommitteeattheJohnCochranVAMed- onfixedandpermeabilizedNRCMsbyusingtheDead-Endfluorometric 2
TUNEL system (Promega) following the manufacturer’s directions, as 0
icalCenter. 1
Cellculture.Isolationofneonatalratcardiacmyocytes(NRCMs) previouslydescribed(15). 9
wasperformedasdescribedpreviously(6).Heartswereremovedfrom CellularATPcontentanalysis.NRCMsweretransducedwithadeno- b
1-day-old Sprague-Dawley rats, the atria and great vessels were viralparticlesfor24handthentrypsinized,and5(cid:7)104cellswereplated y
g
trimmedoff,andtissuewasfinelyminced,followedbysequentialdi- ineachwellofa96-wellplate.CellularATPlevelswerequantifiedusinga u
gestionwith0.5mg/mlcollagenase(WorthingtonBiochemical,NJ). luciferasereaction-basedassaykit(CellTiter-Gloreagent;Promega),and e
s
Ventricularcardiomyocyteswereseparatedfromfibroblastsbydiffer- readingswerenormalizedtothoseofviablecellsassessedbythetrypan t
entialplatinginmediumcontainingDulbecco’smodifiedEagle’sme- blueexclusionmethodaspreviouslydescribed(28).
dium(DMEM),10%horseserum,5%fetalcalfserum,100(cid:5)mol/liter Immunofluorescenceimaging.ImagingformCherry-GFP-LC3and
bromodeoxyuridine, penicillin, streptomycin, and L-glutamine, fol- LysoTrackerRedwasperformedwithliveNRCMs,andimagingforGFP-
lowedbytissuecultureingelatin-coatedplatesinmediumcontaining LC3,LAMP1,andTFEB(withimmunofluorescenceoftheHAtag)was
modifiedDMEM,100(cid:5)Mbromodeoxyuridine,penicillin,streptomy- performedwithNRCMsfixedwith4%paraformaldehyde(20min)and
cin, L-glutamine, 10 ng/ml insulin, 10 (cid:5)g/ml transferrin, and 0.5 thenpermeabilizedwith0.1%TritonX-100(5min),usinganAxioscap
mg/mlbovineserumalbumin. uprightmicroscopefittedwithanAxioCamHRCcameraandPlanNeo-
Nutrient deprivation was induced by culture in serum-free Hanks fluarobjectives(20(cid:7)[numericalaperture{NA}(cid:8)0.60],40(cid:7)[NA(cid:8)
balancedsaltsolution(HBSS;Invitrogen).HEK293cellswereculturedas 0.75],and63(cid:7)[oil][NA(cid:8)1.25][Zeiss]),alongwithappropriatefilter
describedpreviously(15). cubes,aspreviouslydescribed(6).NuclearlocalizationofTFEBwasas-
Hypoxia modeling. Cells were subjected to hypoxia in vitro in an sessedin60cells/group.LAMP1-positivepunctain50cells/groupwere
oxygen control cabinet (Coy Laboratories, Grass Lake, MI) mounted countedusingImageJsoftwareasdescribedpreviously(15),andresults
withinanincubatorandequippedwithanoxygensensorforcontinuous are expressed as numbers of dots/nucleus. Imaging for BNIP3 and
oxygenlevelmonitoring.Amixtureof95%nitrogenand5%CO was COX-IVwasperformedonfixedandpermeabilizedNRCMswithaZeiss
2
infusedtocreatehypoxia,andoxygenlevelsinthechamberweremoni- confocalLSM700laserscanningconfocalmicroscopeusing63(cid:7)Zeiss
toredandmaintainedat(cid:6)1%,asdescribedpreviously(6). Plan-Neofluar 40(cid:7)/1.3 and 63(cid:7)/1.4 oil immersion objectives. Nuclei
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 957
Maetal.
werestainedbluewithHoechstdye(forlive-cellimaging)(H3750;In- HEK293cellsweretransfectedwiththePGL3.Lucvector,drivenbythe
vitrogen) or DAPI (4=,6-diamidino-2-phenylindole) (for fixed cells) humanPPARGC1(cid:2)promoterwithout(i.e.,wild-typepromoter)orwith
(H1200;VectorLaboratories).Epifluorescenceandconfocalimageswere mutationsinthe“CLEARsequence,”located69bpupstreamofthetran-
acquired and analyzed using Zeiss Axiovision and Zen 2010 software, scriptionalstartsite(i.e.,(cid:4)18CLEARsitemutant),togetherwithpRL-
respectively. SV40forRenillaluciferaseexpression(ataratioof100:1).Inaddition,
Flow cytometric assessment. NRCMs were incubated with Lyso- cellswerecotransfectedwithexpressionvectorscodingfortheconstitu-
TrackerRed(1mmol/literfor15min)toassesslysosomeabundance,with tivelyactivetranscriptionfactorCREB,theCREBactivatorTORC2(i.e.,
nonyl-acridineorange(NAO)(acardiolipin-bindingdye;10nmol/liter CRTC2)(seebelowfordetails),TFEB(pAd-CMV/V5-hTFEB),orLacZ
for15min)andMitotrackerGreen(200nmol/literfor45min)toassess (pAd-CMV/V5-LacZ)(asacontrol),andluciferaseactivitiesweremea-
mitochondrialmass,withJC-1(10mg/mlfor10min)ortetramethylrho- sured48haftertransfection.Fireflyluciferasereadingswerenormalized
damine,ethylester(TMRE;50nmol/literfor30min)toassessmitochon- tothoseforRenillaluciferasetocontrolfortransfectionefficiency.
drialpolarization,andwithcarboxy-H DCFDA(acell-permeatingROS Subcellular fractionation. NRCMs were subjected to biochemical
2
indicator;10(cid:5)mol/literfor30min)toassessthelevelsofROS.Following fractionationtorecovernucleus-enrichedandcytoplasmicfractions,
incubation with these compounds at 37°C in 5% CO , the cells were aspreviouslydescribed(23).Heartswerefractionatedintonucleus-
2
trypsinizedandsubjectedtoflowcytometryonaFACScaninstrument enrichedandcytoplasmicsamplesbyusingaCelLyticNuCLEARex-
(Becton-Dickinson)aspreviouslydescribed(15).Cyflogicsoftware(Cy- tractionkit(Nxtract;Sigma).Expressionofproteinslocalizedtothe
D
Flo)wasemployedtoanalyze20,000eventsperrun. nucleus(histoneH3)andcytoplasm(glyceraldehyde-3-phosphatede- o
Assessment of mitochondrial DNA content. Mitochondrial DNA hydrogenase [GAPDH]) was examined to confirm relative enrich- w
contentwasassessedbyquantitativePCRanalysisaspreviouslydescribed ment. n
lo
(29).Briefly,DNAwaspreparedfromNRCMpellets,andreal-timequan- Coimmunoprecipitation studies. Murine embryonic fibroblasts
a
titativePCRwasperformedforthemitochondrialgeneNADH2(with (MEFs)fromC57BL/6wild-typemicewereadenovirallytransducedwith d
primers5=-ATCACCACCATTCTCGCAAT-3=and5=-TCCTATGTGGG beclin-1–HA(MOI(cid:8)100)ortransfectedwithN-terminalHA-tagged e
d
CAATTGATG-3=)andthenucleargeneRCAN1(withprimers5=-GGTT TSC1(Addgene)(32).Threehundredmicrogramsofproteinwasincu-
f
r
TGCTGAGCCTCGAAG-3=and5=-CTTCATCCCTCCTTTGTAAC-3=). batedwithanti-HA(H3663;Sigma)ornormalrabbitIgG,andimmuno- o
m
Theratioofcopiesofmitochondrialtonucleargenesrepresentstherela- precipitationwasperformedusingDynaBeads(Invitrogen).

tivemitochondrialDNAcontent. Immunoblotting. Immunoblotting was performed on cellular ex- h
t
Assessmentofmitochondrialfragmentation.Mitochondrialmor- tracts by use of previously described techniques (15). Antibodies em- t
p
phologywasascertainedtobefragmentedortubularbyvisualinspection ployedwereasfollows:antibodiesforFLAG(F3165;Sigma),HA(H6908; :
/
/
ofindividualcellsatahighmagnification,asdescribedpreviously(30). Sigma),LAMP1(AB24170;Abcam),beclin-1(ab16998[Abcam]andsc- m
Fifty cells were evaluated per experimental group, and results are ex- 11427[SantaCruz]),p70S6K(2708;CellSignaling),p-p70S6K(9234;Cell c
b
pressedaspercentagesofcellsshowingpredominantlyfragmentedmito- Signaling),4-EBP1(9644;CellSignaling),p-4EBP1(2855;CellSignaling), .
a
chondria. p-mTOR(2974;CellSignaling),mTOR(2983;CellSignaling),histoneH3 s
Assessmentofmitochondrialpolarization.Mitochondrialdepolar- (9715;CellSignaling),TFEB(MBS120432;employedtodetectadenovi- m
izationwasassessedbyexpressionofTMREorwiththeratiometricdye rallytransducedHA-taggedhumanTFEBwhenHAdetectionwaspre- .o
JC-1,whichaggregatesintoredfluorescentpolymersinnormallypolar- cludedforspecificity),GAPDH(ab22555;Abcam),peroxisomeprolifera- rg
izedmitochondriabutispredominantlyobservedasgreenfluorescent tor-activated receptor gamma coactivator 1(cid:2) (PGC1(cid:2)) (ab106814; /
o
monomers in cells with depolarized mitochondria, as previously de- Abcam),tuberoussclerosiscomplex1(TSC1)(6935;CellSignaling),and n
scribed(6,15). tuberoussclerosiscomplex2(TSC2)(3612;CellSignaling).MurineTFEB A
TEM.NRCMswerefixedinmodifiedKarnovsky’sfixative(3%glu- was detected with a Bethyl Labs antibody (A303-673A). The multiple p
r
taraldehyde,1%paraformaldehydein0.1Msodiumcacodylatebuffer), immunoreactiveTFEBbandsobserved(seeFig.12CandD)likelyrepre- il
followedbypostfixationin2%osmiumtetroxidein0.1Msodiumcaco- sent various TFEB isoforms (NP_001155194.1 and NP_035679.3 ) or 3
,
dylatebufferfor1h.Thecellswerethenstainedwith2%aqueousuranyl posttranslationally modified forms of the protein in the myocardium. 2
acetatefor30min,dehydratedinaseriesofgradedethanolconcentra- Immunoblotting for (cid:2)-sarcomeric actin ((cid:2)SA) (ab52219; Abcam) was 0
1
tions,andembeddedinPolyBed(Polysciences).Blocksweresectionedat employedasaloadingcontrolunlessotherwiseindicated.ImageJsoftware 9
a90-nmthickness,poststainedwithVenable’sleadcitrate,andviewed wasemployedforquantitativeanalysis.Proteinabundancewasnormal- b
with a JEOL model 1200EX transmission electron microscope (TEM) izedto(cid:2)-sarcomericactinproteinexpressionandreportedasthefold y

g
(JEOL,Tokyo,Japan).DigitalimageswereacquiredusinganAMTAd- changeversusthecontrol.Chemicalsemployedwereasfollows:3-methyl-
u
vantageHR(AdvancedMicroscopyTechniques,Danvers,MA)high-def- adenine(3MA;EMD4Biosciences),torin-1(Tocris),MG-132(Cayman e
s
initioncharge-coupleddevice(CCD)1.3-megapixelTEMcamera. ChemicalCompany),andMnTMPyP(Calbiochem).Equivalentquanti- t
Assessmentofpromoter-drivenluciferaseactivity.Atotalof3(cid:7)104 ties of appropriate diluent were employed as controls in all studies in
cells were plated per well of a 96-well plate and transfected with the whichthesechemicalswereemployed.
PGL3.Lucvector,carryinga2,760-bpfragmentofthehumanPPARGC1(cid:2) QuantitativePCRanalysisformRNA.Real-timePCRwasperformed
promoter(forfireflyluciferaseexpression),andthepRL-SV40vector(for asdescribedpreviously(6).Briefly,totalRNAwaspreparedfromNRCMs
Renillaluciferaseexpression[31])ata100:1ratio,usingLipofectamine byuseofanRNA-easyminikit(Qiagen),andcDNAwassynthesizedwith
2000 (Invitrogen). Constructs coding for the human PPARGC1(cid:2) pro- 1(cid:5)goftotalRNAbyusingtheSuperScriptIIIfirst-strandsynthesissystem
moter(GenBankaccessionnumberNG_028250.1)withindividualmu- (Invitrogen).OnemicroliterofcDNAtemplatewasmixedwith12.5(cid:5)lof
tationsinthethreeCLEARsitesdetectedwithin(asdepictedinFig.9D) 2(cid:7)SYBRgreenPCRmastermix(Invitrogen)andsubjectedtoquantita-
weregeneratedbysite-directedmutagenesisusingaQuikChangeLight- tivePCRintriplicateunderthefollowingconditions:50°Cfor2minand
ningsite-directedmutagenesiskit(Stratagene)andverifiedbysequenc- 95°Cfor10minfollowedby40cyclesof95°Cfor15sand60°Cfor1min
ing.Luciferaseactivitywasmeasuredwithadual-luciferaseassaykit(Pro- inanABI7500Fastreal-timePCRsystem.TheGAPDHgenewasalso
mega) and a Tecan Infinite M200 Pro microplate reader (Tecan) amplifiedinparallelasareferenceforthequantificationoftranscripts.
followingthemanufacturers’instructions.Fireflyluciferasevalueswere PrimersetsemployedareshowninTable1.Resultswerecalculatedusing
normalizedtothoseforRenillaluciferasetocontrolfortransfectioneffi- the(cid:9)(cid:9)C methodandexpressedaspreviouslydescribed(6).
T
ciency.Toassesswhetherthebp(cid:4)18sitemutantconstructretainedac- ChIP assays. Chromatin immunoprecipitation (ChIP) was per-
tivitytodrivetranscriptionalactivation(otherthanthatdrivenbyTFEB), formedaspreviouslyreported(25,33).Briefly,HEK293Acellsweretrans-
958 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
TABLE1PrimersequencesemployedforqPCRanalysisoftheindicatedgenes
Primersequence(5=–3=)
Targetgene Forward Reverse
Ratgenes
MAP1LC3B TTTGTAAGGGCGGTTCTGAC CAGGTAGCAGGAAGCAGAGG
SQSTM1 GCTGCCCTGTACCCACATCT CGCCTTCATCCGAGAAAC
LAMP1 TCTTCAGCGTGCAAGTCCAG ATGAGGACGATGAGGACCAG
PPARGC1(cid:2) ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC
BECN1 TTCAAGATCCTGGACCGAGTGAC AGACACCATCCTGGCGAGTTTC
TFEB GTCCAGCAGCCACCTGAACGT ACCAGGGAAGCCGTGACCTG
LAMP2A CCAAATTGGGATCCTAACCTAA TGGTGAAGCAGTGTTTATTAATTCC
LAMP2B GGTGCTGGTCTTTCAGGCTTGATT ACCACCCAATCTAAGAGCAGGACT
CTSB AAATCAGGCGTATACAAGCATGAA AGCCCAGAATGCGGATGG
CTSD CTGAGTGGCTTCATGGGGAT CCTGACAGTGAAGAAGGAGC
RAB7 TTACTTCGAGACCAGTGCCAAGGA TGTCCAGTTTGATGGGTTCAGGGA
D
GAPDH GGCCGAGGGCCCACTA TGTTGAAGTCACAGGAGACAACCT o
RPS12 AAGGCATAGCTGCTGGAGGTGTAA AGTTGGATGCGAGCACACACAGAT w
RPL32 CCTCTGGTGAAGCCCAAGATC TCTGGGTTTCCGCCAGTTT n
lo
a
Mousegenes d
MAP1LC3B CGTCCTGGACAAGACCAAGT ATTGCTGTCCCGAATGTCTC e
d
LAMP1 TCTTCAGTGTGCAGGTCCAG ATGAGGACGATGAGGACCAG
f
PPARGC1(cid:2) ATGAATGCAGCGGTCTTAGC AACAATGGCAGGGTTTGTTC ro
BECN1 AATCTAAGGAGTTGCCGTTATAC CCAGTGTCTTCAATCTTGCC m
TFEB GTCTAGCAGCCACCTGAACGT ACCATGGAGGCTGTGACCTG h
LAMP2A CCAAATTGGGATCCTAACCTAA TGGTCAAGCAGTGTTTATTAATTCC tt
p
LAMP2B GGTGCTGGTCTTTCAGGCTTGATT ACCACCCAATCTAAGAGCAGGACT :
/
RAB7 TTACTTCGAGACCAGTGCCAAGGA TGTCCAGTTTGATGGGTTCAGGGA /m
CTSB AAATCAGGAGTATACAAGCATGA GCCCAGGGATGCGGATGG c
CTSD CTGAGTGGCTTCATGGGAAT CCTGACAGTGGAGAAGGAGC b
.
SQSTM1 GCTGCCCTATACCCACATCT CGCCTTCATCCGAGAAAC a
s
GAPDH ACTCCCACTCTTCCACCTTC TCTTGCTCAGTGTCCTTGC m
RPS12 AAGACCGCCCTCATCCACGATG TTGGTGCTCAGCACAAAGTGCC .
o
RPL32 CCTCTGGTGAAGCCCAAGATC TCTGGGTTTCCGCCAGTTT r
g
/

o
n

fectedwiththeemptypAAV-CMV-TFEB-IRES-GFPconstruct,described verified, following which statistical differences were assessed with un- A
p
earlier(34),orwithemptyvector(asacontrol)for48h.Cellswerecol- paired2-tailedStudent’sttestfortwoexperimentalgroups,one-wayanal- r
lected,fixedwith1%formaldehydefor10min,andlysedonicewithChIP ysisofvariance(ANOVA)formultiplegroups,andtwo-wayANOVAfor il
3
lysisbuffer(50mMTris-HCl,pH7.5,100mMNaCl,1%TritonX-100, testingoftwovariablesacrossmultiplegroups,usingSPSSsoftware.Bon- ,

1%Tween20)for20min.Thelysatewasdigestedwithmicrococcalnu- ferroni’sandDunnett’sT3posthoctestswereemployedafterANOVAfor 2
0
clease(Sigma)for15minat37°C,andthereactionwasterminatedusing testingforsignificantdifferencesbetweengroupsfordatawithequaland 1
EDTA(2mM)andsodiumdodecylsulfate(SDS;1%).SDS-Out(Pierce) unequalvariances,respectively.Nonparametrictestswereemployedfor 9

wasemployedtoprecipitatetheunboundSDS,andlysatesdiluted1:1 datathatwerenotnormallydistributed.Atwo-tailedPvalueof(cid:6)0.05was b
y
withChIPdilutionbuffer(50mMTris-HCl,pH7.5,100mMNaCl,0.5% consideredstatisticallysignificant.
g
TritonX-100,2mMEDTA)wereincubatedwithhigh-capacityNeutr- u
Avidinagarose(Pierce).Biotinylatedanti-FLAGantibody(F9291;Sigma) RESULTS es
or isotype control IgG1 (M9035; Sigma) was precoupled with Neutr- t
BNIP3expressionincreasesbeclin-1abundancewithtranscrip-
Avidinbeadsfor30minat4°C,andtheprotein-DNAcomplexeswere
tionalsuppressionofautophagy-lysosomemachinery.BNIP3,a
immunoprecipitated overnight at 4°C. After six washes, the DNA was
elutedbyadditionof8mMbiotin,1%SDSinTris-EDTA(TE)bufferand Bcl-2familyprodeathprotein,istranscriptionallyinducedincar-
precipitatedafterreversingthecross-linkagebyuseof200mMNaClat diacIRinjuryandprovokesmitochondrialpermeabilization(8,
65°Covernight.PCRwasperformedusingprimersflankingtheCLEAR 11,12).WehaveobservedthatBNIP3expressionprovokespro-
site18bpupstreamofthetranscriptionalstartsite(forwardprimer,5=-C gressivedeclinesinlysosomenumbers,withaccumulationoffrag-
GTCACGAGTTAGAGCAGCA-3=;andreverseprimer,5=-TCGCCCTTA mentedanddepolarizedmitochondria,leadingtocelldeath(15),
AGCTTTTTCAA-3=)(204-bpamplicon).Anadditionalprimersetwithin and that restoring lysosome biogenesis by exogenous TFEB ex-
an adjacent segment located between the CLEAR sites at bp (cid:4)18 and
pressionreestablishesmitochondrialautophagytorescueBNIP3-
(cid:4)469 (forward primer, 5=-CTGTCATGAAACAGGGAGCTTT-3=; and
inducedcelldeath(15).Toexaminethemechanismsforimpaired
reverseprimer,5=-GCTTTGAATGCCACAGACTCTA-3=)(125-bpam-
autophagicremovalofBNIP3-damagedmitochondria,weevalu-
plicon)didnotgenerateaproduct,demonstratingspecificity(datanot
atedtheregulationoftheautophagy-lysosomemachinerybyad-
shown).
Statisticalanalysis.Resultsareexpressedasmeans(cid:10)standarderrors enoviraltransductionofNRCMswithBNIP3.Wehypothesized
ofthemeans(SEM),withthesamplesize(n)pergroupindicatedinthe thatthedeclineinlysosomenumberisdrivenbyBNIP3-induced
figurelegends.Assumptionsofnormalityandequalityofvariancewere autophagy,asalsoobservedearlyduringstarvation-inducedau-
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 959
Maetal.
D
o
w
n
FIG 1BNIP3 expression results in transcriptional downregulation of autophagy-lysosome machinery. (A) Confocal images demonstrating LysoTracker lo
fluorescence(top)andLAMP1expression(bottom)inNRCMstransducedwithBNIP3(green)orLacZ(24h),withorwithout3MA(7mmol/liter)andwith a
quantitation of the number of LAMP1-positive dots/nucleus. *, #, and $, P (cid:6) 0.05 versus LacZ, BNIP3, and BNIP3 plus 3MA, respectively. (B and C) de
Representativeflowcytometrictracings(B)andquantitation(C)ofLysoTrackerfluorescence(n(cid:8)3).*,P(cid:6)0.05versuscontrol(con)(whitebar).(DandE) d
Representativeimmunoblot(D)andquantitation(E)forLAMP1inNRCMstreatedasdescribedforpanelA(n(cid:8)3).*,P(cid:6)0.05versuscontrol(whitebar).(F f
r
toH)Representativeimmunoblot(aspreviouslydescribed[15])(F)andquantitationofLAMP1protein(n(cid:8)3)(G)andLAMP1transcripts(n(cid:8)4to6)(H)in o
m
NRCMs transduced with BNIP3 or LacZ for the indicated durations. (I) Levels of representative autophagy-lysosome machinery transcripts in NRCMs
transducedwithBNIP3orLacZ(24h;n(cid:8)8).AlltransductionswereperformedatanMOIof100/sample. h
t
t
p
:
/
/
tophagy (18). However, BNIP3-induced declines in lysosome anism for induction of autophagosome formation (14), we m
abundance (assessed with the acidophilic dye LysoTracker Red examinedtheeffectofBNIP3expressiononbeclin-1.Beclin-1 c
b
[Fig.1AtoC]andbyimmunostainingforLAMP1[acandidate protein abundance was increased early (at 12 and 24 h) after .a
lysosomalproteinwhoselevelsareusedtotracklysosomeabun- BNIP3 transduction (Fig. 2A and B), and this was not due to s
m
dance{35}][Fig.1A])andinLAMP1proteinabundance(Fig.1D transcriptionalinduction(Fig.2C).Infact,BECN1transcript .
o
andE)wereonlypartiallyrestoredwith3MA(atypeIIIphospha- levelsweresignificantlyreducedcomparedwiththoseofcon- r
g
tidylinositol 3-kinase inhibitor which inhibits autophagosome trols at 24 h, coinciding with the suppression of other au- /

formation)(Fig.1AtoE),to65%,59%,and72%,respectively,of tophagy-lysosome machinery transcripts (Fig. 1I), and only on
thelevelsforsimilarlytreatedcontrols.Thisindicatesthatlyso- increased subsequently, in parallel with a decline in beclin-1 A
some consumption in BNIP3-induced autophagy is only partly proteinabundance(Fig.2BandC)secondarytoconsumption p
r
responsiblefortheobserveddeclineinlysosomeabundance(15). withpersistentautophagy(datanotshown).Thissuggeststhat il
Importantly, we observed progressive declines in LAMP1 tran- 3
a stabilization of beclin-1 protein results in its accumulation ,

scripts(Fig.1H)parallelingthedeclineinLAMP1proteinabun- 2
earlyafterBNIP3expression. 0
dance(Fig.1FandG).Thistranscriptionalsuppressionalsoaf- We next examined if ROS mediate the BNIP3-induced in- 1
fected multiple proteins (Fig. 1I) that participate in autophagy 9
creaseinbeclin-1abundanceobservedwithreperfusioninjury
b
(LC3, p62, and RAB7), constitute the lysosomes (LAMP2A,
(6).BNIP3triggeredROSgenerationearlyafteritsexpression y
LAMP2B,cathepsinB,andcathepsinD),andfunctionasthemas- g
(at 12 h) (Fig. 2D and E), likely as a result of mitochondrial
tertranscriptionalregulatoroftheautophagy-lysosomemachin- u
depolarization (Fig. 2F and G). Scavenging of ROS with e
ery(TFEB)butdidnotalterexpressionoftheunrelatedribosomal s
MnTMPyP,acell-permeatingsuperoxidedismutasemimetic, t
proteinsRPL32andRPS12(changesintranscript/GAPDHabun-
dancesof0.95-(cid:10)0.02-fold[versus1.01-(cid:10)0.03-foldforLacZ] did not affect BNIP3-induced mitochondrial depolarization
and0.95-(cid:10)0.03-fold[versus1.04-(cid:10)0.03-foldforLacZ],respec- (Fig. 2F and G) but prevented the increase in beclin-1 abun-
tively; the P value was nonsignificant [NS]; n (cid:8) 8). Taken to- dance (Fig. 2H and I), indicating that BNIP3-induced mito-
chondrialpermeabilizationisthecauseratherthantheconse-
gether, these observations indicate that in contrast to the well-
quenceofROSgeneration,whichprovokesincreasedbeclin-1
describedtranscriptionalstimulationoftheautophagy-lysosome
machinery observed with starvation (21–24), BNIP3 expression abundance.TreatmentwithMnTMPyPinducedreductionsin
resultsintranscriptionalsuppressionofthismachinerytoprevent LAMP1transcripts(Fig.2J)andproteinlevels(Fig.2KandL),
itsreplenishment,whileautophagyisrobustlyinducedtoremove consistent with prior reports indicating that basal ROS levels
BNIP3-damagedmitochondria(15). maydrivetranscriptionoflysosomalgenes(36).Importantly,
We and others have observed a rapid increase in beclin-1 treatmentwithMnTMPyPpreventedBNIP3-induceddeclines
abundance with reperfusion/reoxygenation injury (5, 6), and inLAMP1transcripts(Fig.2J)andprotein(Fig.2KandL)and
elevated beclin-1 levels transcriptionally suppress autophagy- inlysosomes(Fig.2MandN)andattenuatedBNIP3-induced
lysosomemachinerygenes(6).SinceBNIP3expressionispos- cardiomyocytedeath(Fig.2O).Takentogether,theseobserva-
tulatedtodisplacebeclin-1fromitsbindingtoBcl2asamech- tions indicate that BNIP3-induced ROS mediate increased
960 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
c
b
.
a
s
m
.
o
r
g
FIG2BNIP3-inducedROSdriveincreasedbeclin-1proteinlevelsandsuppressionoflysosomeabundance.(AtoC)Representativeimmunoblot(A)and /
quantitationofbeclin-1protein(n(cid:8)3)(B)andBECN1transcripts(n(cid:8)3or4)(C)inNRCMstransducedwithBNIP3orLacZfortheindicateddurations.(D o
andE)Representativeflowcytometrictracings(D)andquantitation(E)ofROS(n(cid:8)3or4)inNRCMstransducedwithBNIP3orLacZfor12h.HO (500 n
mmol/liter;4h)wasusedasthepositivecontrol.(FandG)Representativeflowcytometrictracings(F)andquantitation(G)ofTMREfluorescence(n2(cid:8)23)in A
NRCMstreatedasdescribedforpanelD.(HandI)Representativeimmunoblot(H)andquantitation(I)ofbeclin-1(n(cid:8)3or4)inNRCMstransducedwith p
[BnN(cid:8)IP43]o)rabLuacnZdapnlucessMinnNTMRCPMyPs(t5r0ea(cid:5)temdoals/lditeesrc;r2ib4ehd)f.o(rJptoanLe)lLHA.M(MP1antrdanNsc)rRipetp(rnes(cid:8)ent6a)ti(vJe)aflnodwpcryottoemine(trriecptrreasceinntgasti(vMe)imanmduqnuoabnlotitta[tKio]nan(Nd)qoufanLtyistoatTioranck[Le]r ril 3
fluorescence(n(cid:8)3).(O)CelldeathinNRCMstreatedasdescribedforpanelH(n(cid:8)7or8).AlltransductionswereperformedatanMOIof100/sample.*,P(cid:6) ,
2
0.05versuscontrol(whitebars). 0
1
9

b
y
beclin-1abundanceandtheobservedtranscriptionalsuppres- therelativeabundanceofautolysosomesandreducedthelevelof g
sionofthelysosomemachinery. autophagosomes (versus the shLacZ control) in BNIP3-trans- u
e
Partialbeclin-1knockdowntranscriptionallystimulatesau- ducedNRCMs(Fig.3GandH),pointingtoenhancedautophagic s
t
tophagic flux to attenuate BNIP3-induced cardiomyocyte flux. Partial beclin-1 knockdown also resulted in an increased
death. We have observed that experimental modulation of abundanceofautophagyproteins(totalLC3andp62)(Fig.3I),
beclin-1levelstranscriptionallyaffectsautophagy-lysosomema- which was transcriptional as we described earlier (6; data not
chinery proteins in a reciprocal fashion (6). To determine if shown),andinLAMP1(Fig.3IandJ),parallelingtheincreased
BNIP3-induced upregulation of beclin-1 contributes to the ob- lysosome abundance. Importantly, partial beclin-1 knockdown
serveddeclineinLAMP1,weperformedapartialknockdownof attenuatedtheBNIP3-induceddeclineinLAMP1protein(Fig.3I
BECN1 transcripts (with two different shRNA constructs) in andJ),reducedBNIP3-inducedTUNELpositivity(Fig.3KandL),
BNIP3(andLacZ)-treatedNRCMs.Partialbeclin-1knockdown and prevented BNIP3-induced cell death (Fig. 3M), mimicking
(reductioninBECN1transcriptsto(cid:11)69%ofcontrollevelandin the observations seen with exogenous expression of TFEB (15).
beclin-1proteinto(cid:11)45to50%ofcontrollevel)(Fig.3AtoC) Interestingly, partial beclin-1 knockdown resulted in reduced
stimulated LAMP1 transcription (Fig. 3D) and increased lyso- BNIP3protein,whichwaspreventedbyconcomitantinhibition
someabundance(Fig.3EandF)topreventBNIP3-inducedde- ofautophagosomeformationwith3MA(Fig.3N)butnotbypro-
clines in lysosome numbers (Fig. 3E and F). BNIP3 expression teasome inhibition with MG-132, suggesting an enhanced au-
resulted in autophagosome accumulation, indicating impaired tophagicdegradationofBNIP3togetherwiththemitochondria,
autophagicflux(15),andpartialbeclin-1knockdownincreased whereupon it is localized. Partial beclin-1 knockdown with the
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 961
Maetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
c
b
.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
3
,

2
0
1
9

b
y

g
u
e
s
t
962 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
FIG4Nearlycompletebeclin-1knockdowninhibitsautophagytoincreaseBNIP3-inducedcelldeath.(A)BECN1transcriptlevelsinNRCMstransducedwith c
shBECN1-1,shBECN1-2,orshLacZ(MOI(cid:8)10;72h).(B)Representativeimmunoblot(top)andquantitation(bottom)ofbeclin-1proteinabundancein b
.
NRCMstreatedasdescribedforpanelA(n(cid:8)4).*,P(cid:6)0.05versusshLacZ.(CandD)GFP-taggedLC3localization(magnification,(cid:7)630;representativeof3 a
experiments)(C)andquantitationofpunctateGFP(D)inNRCMstreatedasdescribedforpanelAandcotransducedwithBNIP3orLacZ(24h)(n(cid:8)15to20 sm
cells/group).*and#,P(cid:6)0.05versuscontrol(whitebars)andtheBNIP3-plus-shLacZgroup,respectively.(E)CelldeathinNRCMstreatedasdescribedforpanel .
C(n(cid:8)7to15).*,P(cid:6)0.05versuscontrol(whitebar). o
r
g
/

o
n
secondshRNAconstruct(shBECN1-2)(Fig.3AtoD)recapitu- Partial beclin-1 knockdown activates TFEB to attenuate
A
latedthesefindings(datanotshown).Takentogether,thesedata BNIP3-inducedcardiomyocytedeath.Intriguingly,theeffectsof p
indicatethatpartialbeclin-1knockdowntranscriptionallystimu- partial beclin-1 knockdown (Fig. 3) mimicked our observa- ril
latesautophagicfluxtorestoreimpairedautophagosomeprocess- tions with TFEB activation (15). Therefore, we evaluated the 3
,
ingwithBNIP3expression. effect of partial beclin-1 knockdown on TFEB activation. Re- 2
Notably, as we observed previously (6), a more complete centstudiesuncoveredatightlyregulatedmechanismforTFEB 0
1
knockdownofBECN1(with10-foldhigheradenoviralshBECN1 activation, whereby TFEB is phosphorylated and sequestered 9
transduction,to(cid:11)13%oftranscriptlevelswithshLacZ,resulting in the cytoplasm, in the unstressed state, and starvation-in- b
inareductionofbeclin-1proteinabundanceto(cid:11)7%ofthecon- duced mTOR inactivation provokes dephosphorylation and y
g
trollevel)(Fig.4AandB)resultedininhibitionofautophagosome nuclear translocation of TFEB (21, 22, 24). Given the lack of u
e
formation (Fig. 4C and D) and increased BNIP3-induced cell suitableantibodiestodetectendogenousTFEBinratcells(24), s
death (Fig. 4E), consistent with an obligate role for beclin-1 in weexaminedtheeffectofstarvationonsubcellulardistribution t
initiationofautophagy(26). ofexogenousTFEBinNRCMs.ExogenousTFEBexpressedat
FIG3Partialbeclin-1knockdownstimulateslysosomebiogenesisandinducesautophagicfluxtoattenuateBNIP3-inducedcelldeath.(A)BECN1transcript
levelsinNRCMstransducedwithtwoshBECN1constructsorshLacZ(MOI(cid:8)1;72h).(BandC)Representativeimmunoblot(B)andquantitation(C)of
beclin-1protein(n(cid:8)4)inNRCMstreatedasdescribedforpanelA.*,P(cid:6)0.05versusshLacZ.(D)LAMP1transcriptlevelsinNRCMstreatedasdescribed
forpanelAandcotransducedwithBNIP3orLacZ(MOI(cid:8)100;24h)(n(cid:8)4to12).*and#,P(cid:6)0.05versuscontrol(whitebar)andtherespective
shBECN1-treatedgroup,respectively.(EandF)Representativeflowcytometrictracings(E)andquantitation(F)ofLysoTrackerfluorescenceinNRCMs
transducedwithshBECN1-1orshLacZandcotransducedwithBNIP3orLacZasdescribedforpanelD(n(cid:8)3).(GandH)mCherry-GFP-LC3localization
(magnification,(cid:7)630;representativeof3experiments)(G)andquantitationofautophagosomes,autolysosomes,andboth(H)inNRCMstreatedasdescribed
forpanelB(n(cid:8)20to40nuclei/group).*,#,and$,P(cid:6)0.05forautophagosomes,autolysosomes,andboth,respectively,versusshLacZ-plus-LacZcontrol.(Iand
J)Immunoblotdepictingautophagy-lysosomeproteins(I)andquantitationofLAMP1(n(cid:8)3)(J)inNRCMstreatedasdescribedforpanelE.*,P(cid:6)0.05versus
control(whitebar).(KandL)Representativeimages(magnification,(cid:7)200)(K)andquantitationofTUNELpositivity(greeninpanelK)(n(cid:8)4)(L).(M)Cell
deathinNRCMstreatedasdescribedforpanelE(n(cid:8)20to24).*,P(cid:6)0.05versuscontrol(whitebar).(N)BNIP3expressioninNRCMstransducedwith
shBECN1-1orshLacZ(MOI(cid:8)1;72h)andcotransducedwithBNIP3orLacZ(MOI(cid:8)100;24h),withorwithout3MA(7mmol/liter)orMG-132(10
(cid:5)mol/liter).
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 963
Maetal.
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
m
c
b
.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
3
,

2
0
1
9

b
FIG5Partialbeclin-1knockdownactivatesTFEBtoattenuateBNIP3-inducedcelldeath.(A)SubcellulardistributionofTFEB(magnification,(cid:7)630)in y
NRCMstransducedwithHA-TFEB(MOI(cid:8)1;24h),withorwithouttorin-1(250nmol/liter;1h),subjectedtostarvation(4h),ortransducedwithshBECN1-1, g
shBECN1-2,orshLacZ(MOI(cid:8)1;72h),withquantitationofcellswithnuclearTFEB(numbersbelowthepanels).*and#,P(cid:6)0.05versusdiluentandshLacZ, u
e
respectively.(BtoD)ImmunoblotsdepictingTFEBandphosphorylationofS6K,4EBP-1,andmTORinNRCMstreatedasdescribedforpanelA.(EtoG) s
ImmunoblotsdepictingTFEBinNRCMstreatedwithtorin-1,shBECN1-1,andshBECN1-2asdescribedforpanelA,followedbybiochemicalfractionationinto t
cytoplasmic(GAPDH-expressing)andnucleus-enriched(histoneH3-expressing)fractions.Whitelinesseparatepartsofthesamegel.ThedataforpanelsAto
Garerepresentativeof2experiments.(HandI)TFEB(H)andLAMP1(I)transcriptlevelsinNRCMstransducedwithshBECN1constructs(orshLacZ),with
orwithoutshTFEB(MOI(cid:8)1;72h)(n(cid:8)4).(J)CelldeathinNRCMstransducedwithshBECN1-1(orshLacZ),withorwithoutshTFEB(MOI(cid:8)1;72h),and
cotransducedwithBNIP3orLacZ(MOI(cid:8)100;24h)(n(cid:8)7or8).*,P(cid:6)0.05versuscontrol(whitebars).
lowlevelslocalizedtothecytoplasminrestingcardiomyocytes nucleus-enrichedfractionincardiacmyocytes(Fig.5E),asob-
and rapidly translocated to the nucleus with starvation and servedinothercelltypes(21,22,24).
torin-1treatment(Fig.5A),viainhibitionofmTORactivity(as Partialbeclin-1knockdownalsoresultedinactivationofTFEB
evidenced by a reduction in Thr-389 phosphorylation of S6 withincreasednuclearlocalization(Fig.5A)andinincreasesin
kinase [S6K], Thr-37/46 phosphorylation of 4E-binding pro- faster-migratingforms(Fig.5C,D,F,andG)andnuclearTFEB
tein1[4EBP-1],andSer-2481autophosphorylationofmTOR) levelscomparedtothoseofthecontrol(Fig.5FandG).Todeter-
(Fig.5BtoD).Torin-1-inducedmTORinhibitionprovokedan minethemechanismforTFEBactivation,weexaminedmTOR
increaseinfaster-migrating(i.e.,dephosphorylated)formsof activitywithpartialbeclin-1knockdown,whichrevealedreduc-
TFEB(Fig.5B)andmarkedlyincreasedTFEBabundanceinthe tions in 4EBP-1 phosphorylation and mTOR autophosphoryla-
964 mcb.asm.org MolecularandCellularBiology March2015 Volume35 Number6
Beclin-1RegulatesMitochondrialQualityviaTFEB
tion,withoutachange(orevenanincrease)inS6Kphosphoryla- knockdownrestoresnormalmitochondriainBNIP3-expressing
tion(Fig.5CandD),suggestingthatmTORinactivationmaybe NRCMs.
partial and/or S6K phosphorylation may be affected simultane- PartialBECLIN1knockdownconcomitantlystimulatesmi-
ouslybymTOR-independentmechanisms(37).Indeed,complete tochondrialbiogenesisviaTFEBtorescueBNIP3-inducedcell
inhibitionofmTORwithtorin-1resultedinfurtherTFEBactiva- death. The effects of partial beclin-1 knockdown (Fig. 7A to F)
tioninshBECN1-treatedcells,asevidencedbytheappearanceof mimicked those of exogenous TFEB in BNIP3-expressing cells
faster-migrating (dephosphorylated) TFEB bands (Fig. 5C and (15) and suggested a concomitant induction of mitochondrial
D).Interestingly,TFEBproteinabundancewasmodestlyreduced biogenesis (Fig. 7E to G), as observed with TFEB activation in
withpartialbeclin-1knockdownandstarvation-inducedactiva- noncardiomyocytes(25,42).Indeed,exogenousTFEBlocalized
tion(butnotwithtorin-1)(Fig.5BtoG),suggestinganenhanced tothenucleiofNRCMsatincreasingdosesirrespectiveofanother
degradation of activated TFEB, as observed previously (22, 38). stimulus,suchasstarvation(Fig.8A),resultinginincreasedau-
Partialbeclin-1knockdownalsoresultedinamarkedincreasein tophagic structures and lysosomes, indicating stimulation of
endogenousTFEBtranscripts(Fig.5H),consistentwiththeob- autophagy and lysosome biogenesis (Fig. 8B), and in increased
servationthatactivatedTFEBinducesitsowntranscription(25). mitochondrialmass(Fig.8CtoF),indicatingthatTFEBissuffi-
Importantly,simultaneousknockdownofendogenousTFEB(Fig. cienttostimulatemitochondrialbiogenesisinNRCMs. D
5H) prevented the transcriptional upregulation of LAMP1 (Fig. We observed that TFEB regulated the abundance of tran- o
w
5I)andnegatedthesalutaryeffectsofpartialbeclin-1knockdown scripts for PGC1(cid:2) (a modulator of mitochondrial biogenesis n
onBNIP3-inducedcardiomyocytedeath(Fig.5J),indicatingthat [1, 43]) in both resting and starved NRCMs (Fig. 9A to C) lo
a
theeffectsofpartialbeclin-1knockdownaretransducedviaacti- and that exogenous TFEB activated transcription from a d
vationofendogenousTFEB. PPARGC1(cid:2) promoter-luciferasereporterconstruct(Fig.9D). e
d
Conversely, consistent with the observation that exogenous WeproceededtoindividuallymutatethethreeTFEB-binding
f
r
beclin-1issufficienttosuppresstranscriptionofautophagy-lyso- E-boxes(termed“CLEAR”sites,withonlyonebasepairmis- o
m
someproteinsandinhibitautophagicflux(6),exogenousbeclin-1 match at a degenerate location compared with the ideal site

provokedadeclineinlysosomeabundance(Fig.6AandB),witha annotated for maximal activation [33]) in the human h
t
marked reduction in nuclear TFEB (Fig. 6C) as well as in the PPARGC1(cid:2) promoter (Fig. 9D, left panel). Only mutations tp
:
abundance of total TFEB (Fig. 6D and E), driven by enhanced withintheCLEARsequencelocated18bpupstreamfromthe //
m
proteasomal degradation. This was likely the result of the transcriptionalstartsitecompletelyablatedtheresponsiveness
c
beclin-1-inducedincreaseinmTORactivation(Fig.6F).beclin-1 toTFEB(Fig.9D,rightpanel),andweconfirmedTFEBbinding b
.
interactswithTSC1(Fig.6G),aspreviouslydescribed(39),with atthissitebyuseofaChIPassay(withaprimerpairflanking a
s
increasedTSC1andreducedTSC2abundance(Fig.6HtoJ),sug- thissite)(Fig.9E).Importantly,thebp(cid:4)18mutantconstruct m
gesting a mechanism whereby recruitment of TSC1 away from retained intact activity toward CREB (44) and TORC2 (45), .o
TSC2increasesitsdegradation,drivingincreasedmTORactiva- two known inducers of PPARGC1(cid:2) transcription (Fig. 9F), rg
tion via sustained Rheb activity (40, 41). Taken together, these confirming a specific effect of TFEB on PGC1(cid:2) transcription o/
findings indicate that beclin-1 levels reciprocally regulate TFEB viabindingtothissite. n

A
activity. MultipleobservationssuggestedanimpairmentofTFEBacti-
p
Partialbeclin-1knockdownrestoresnormallypolarizedmi- vationinthesettingofBNIP3-inducedautophagy,namely,reduc- r
tochondria in BNIP3-expressing cells. BNIP3 expression re- tionsinautophagy-lysosomegeneandTFEBtranscripts(Fig.1I) il 3
sulted in mitochondrial fragmentation, and partial beclin-1 and the accompanying impairment of autophagic flux (Fig. 3G ,
2
knockdown restored the filamentous appearance of the mito- andH)andarescueofBNIP3-inducedcelldeathwithexogenous 0
1
chondrial network in BNIP3-expressing cells (Fig. 7A). Ultra- TFEB(15)andwithpartialbeclin-1knockdown-mediatedTFEB 9

structuralexaminationofBNIP3-transducedcardiomyocytesre- activation(Fig.5).Indeed,weobservedareductioninthenuclear b
vealedwidespreadmitochondrialswellingandfragmentationand TFEBlevelinBNIP3-expressingcells(by33%;P(cid:8)0.005;n(cid:8)3) y
g
effacement of cristae, with a marked increase in prevalence of (Fig.10A),accompaniedbymTORactivation(foldchangeinp- u
autophagic structures, and concomitant partial beclin-1 knock- mTOR/mTORabundances,1.6(cid:10)0.1inBNIP3versus1.0(cid:10)0.1in es
down restored structurally normal-looking mitochondria in control;P(cid:8)0.005;n(cid:8)4),asthepossiblemechanism(Fig.10B). t
BNIP3-expressing cells (Fig. 7B). Concomitant partial beclin-1 WealsofoundamarkedreductionofPGC1(cid:2)proteinabun-
knockdownalsosignificantlyreducedtheprevalenceofBNIP3- danceinBNIP3-expressingNRCMs(Fig.10CandD),whichwas
depolarizedmitochondria(Fig.7CandD),consistentwiththeir drivenbyreducedPGC1(cid:2)transcription(Fig.10EandF)andres-
increased autophagic removal, as observed with exogenous ex- cuedbyconcomitantTFEBexpression(Fig.10CandD).Toex-
pressionofTFEB(15). amineifthebeclin-1proteinaffectsmitochondrialbiogenesisvia
BNIP3expressionresultedinreducedmitochondrialmass,as theTFEB-PGC1(cid:2)axis,weevaluatedtheeffectofmodulatingbe-
evidenced by reduced NAO fluorescence (Fig. 7E and F) and a clin-1levelsonPGC1(cid:2)transcriptionbyusinglossoffunctionand
reducedmitochondrialDNAcontent(Fig.7G).Interestingly,par- gainoffunctionofbeclin-1.Partialbeclin-1knockdowninduced
tialbeclin-1knockdownsignificantlyincreasedNAOfluorescence PPARGC1(cid:2) transcription in a TFEB-dependent manner (Fig.
(Fig. 7E and F), with a trend toward increased mitochondrial 10G), and conversely, beclin-1 overexpression suppressed
DNA(Fig.7G),inLacZ-treatedcontrols;restoredthemitochon- PPARGC1(cid:2)promoteractivity,whichcouldberescuedwithexog-
drialmassinBNIP3-expressingcells(Fig.7EtoG);andprevented enous TFEB (Fig. 10H). Taken together, these findings suggest
thedeclineincellularATPcontentwithBNIP3expression(Fig. thatBNIP3-inducedbeclin-1upregulationmayalsoimpairmito-
7H). Taken together, these data indicate that partial beclin-1 chondrial biogenesis via attenuation of TFEB activity and a de-
March2015 Volume35 Number6 MolecularandCellularBiology mcb.asm.org 965