Fig. 1

15 Pages·2015·3.22 MB·English

TheJournalofNeuroscience,September2,2015•35(35):12137–12151•12137
NeurobiologyofDisease
Neuronal-Targeted TFEB Accelerates Lysosomal
(cid:2)
Degradation of APP, Reducing A Generation and Amyloid
Plaque Pathogenesis
QingliXiao,1PingYan,1XiucuiMa,2,4HaiyanLiu,2,4RonaldoPerez,1AlecZhu,1ErnestoGonzales,1
XDanielleL.Tripoli,1XLeahCzerniewski,1AndreaBallabio,5JohnR.Cirrito,1XAbhinavDiwan,2,3,4*and
XJin-MooLee1*
1DepartmentofNeurologyandtheHopeCenterforNeurologicalDisorders,2CenterforCardiovascularResearchinDepartmentofMedicine,and
3DepartmentofCellBiologyandPhysiology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri63110,4JohnCochranVAMedicalCenter,St.
Louis,Missouri63108,and5TelethonInstituteofGeneticsandMedicine,Pozzuoli,Naples,Italy80078
InAD,animbalancebetweenA(cid:2)productionandremovaldriveselevatedbrainA(cid:2)levelsandeventualamyloidplaquedeposition.APP
undergoesnonamyloidogenicprocessingvia(cid:3)-cleavageattheplasmamembrane,amyloidogenic(cid:2)-and(cid:4)-cleavagewithinendosomesto
generateA(cid:2),orlysosomaldegradationinneurons.Consideringmultiplereportsimplicatingimpairedlysosomefunctionasadriverof
increasedamyloidogenicprocessingofAPP,weexploredtheefficacyoftargetingtranscriptionfactorEB(TFEB),amasterregulatorof
lysosomalpathways,toreduceA(cid:2)levels.CMVpromoter-drivenTFEB,transducedviastereotactichippocampalinjectionsofadeno-
associatedvirusparticlesinAPP/PS1mice,localizedprimarilytoneuronalnucleiandupregulatedlysosomebiogenesis.Thisresultedin
reductionofAPPprotein,the(cid:3)and(cid:2)C-terminalAPPfragments(CTFs),andinthesteady-stateA(cid:2)levelsinthebraininterstitialfluid.
Inagedmice,totalA(cid:2)levelsandamyloidplaqueloadwereselectivelyreducedintheTFEB-transducedhippocampi.TFEBtransfectionin
N2acellsstablyexpressingAPP695,stimulatedlysosomebiogenesis,reducedsteady-statelevelsofAPPand(cid:3)-and(cid:2)-CTFs,andatten-
uatedA(cid:2)generationbyacceleratingfluxthroughtheendosome-lysosomepathway.Cycloheximidechaseassaysrevealedashorteningof
APPhalf-lifewithexogenousTFEBexpression,whichwaspreventedbyconcomitantinhibitionoflysosomalacidification.Thesedata
indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce A(cid:2)generation.
ActivationofTFEBinneuronsisaneffectivestrategytoattenuateA(cid:2)generationandattenuateamyloidplaquedepositioninAD.
Keywords: Alzheimer’sdisease;amyloid;amyloidprecursorprotein;lysosome;TFEB
SignificanceStatement
AkeydriverforADpathogenesisisthenetbalancebetweenproductionandclearanceofA(cid:2),themajorcomponentofamyloid
plaques.Herewedemonstratethatlysosomaldegradationofholo-APPinfluencesA(cid:2)productionbylimitingtheavailabilityof
APPforamyloidogenicprocessing.UsingviralgenetransferoftranscriptionfactorEB(TFEB),amasterregulatoroflysosome
biogenesisinneuronsofAPP/PS1mice,steady-statelevelsofAPPwerereduced,resultingindecreasedinterstitialfluidA(cid:2)levels
andattenuatedamyloiddeposits.TheseeffectswerecausedbyacceleratedlysosomaldegradationofendocytosedAPP,reflected
byreducedAPPhalf-lifeandsteady-statelevelsinTFEB-expressingcells,withresultantdecreaseinA(cid:2)productionandrelease.
Additionalstudiesareneededtoexplorethetherapeuticpotentialofthisapproach.
Introduction
InAD,increasedgenerationoftheA(cid:2)peptideinneuronssyner-
gizeswithimpairedA(cid:2)removalfromtheinterstitialfluid(ISF)in toJ.R.C.WethankWimAnnaert,VIB(FlandersInstituteforBiotechnology),Leuven,Belgium,forprovidingthedualfluorescentAPP
constructandStuartKornfeld,WashingtonUniversity,forscientificinput.
Theauthorsdeclarenocompetingfinancialinterests.
*A.D.andJ.-M.L.contributedequallytothiswork.
ReceivedFeb.19,2015;revisedJuly2,2015;acceptedJuly24,2015. Correspondenceshouldbeaddressedtoeitherofthefollowing:Jin-MooLee,ProfessorofNeurology,660South
Authorcontributions:Q.X.,J.R.C.,A.D.,andJ.-M.L.designedresearch;Q.X.,P.Y.,X.M.,H.L.,R.P.,A.Z.,E.G.,D.L.T., EuclidAvenue,CB8111,St.Louis,MO63110.E-mail:[email protected];orAbhinavDiwan,AssistantProfessorof
L.C.,J.R.C.,andA.D.performedresearch;A.B.contributedunpublishedreagents/analytictools;Q.X.,P.Y.,X.M.,H.L., Medicine, Cell Biology and Physiology, 4940 Parkview Place, CSRB 827, St. Louis, MO 63110. E-mail:
A.Z.,D.L.T.,L.C.,J.R.C.,A.D.,andJ.-M.L.analyzeddata;Q.X.,J.R.C.,A.D.,andJ.-M.L.wrotethepaper. [email protected].
ThisstudywassupportedbygrantsfromtheAlzheimer’sAssociation(NIRG12-242588)toA.D.,BrightfocusFoundation DOI:10.1523/JNEUROSCI.0705-15.2015
(A2012151)toJ.R.C.,andfromtheNationalInstitutesofHealth(R21NS082529)toJ.-M.Land(R01AG042513andR21AG045691) Copyright©2015theauthors 0270-6474/15/3512137-15$15.00/0
12138•J.Neurosci.,September2,2015•35(35):12137–12151 Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation
driving extracellular amyloid plaque formation (Haass et al., milliliterintobilateralhippocampiat6monthsofage,asdescribedpre-
2012)andtheresultantneuropathogenesis(Bloom,2014).After viously(Xiaoetal.,2012).Wild-typemiceofeithersexwerealsoinjected
synthesisintheendoplasmicreticulum,APPistraffickedfrom withAAV8-CMV-FLAG-TFEBorAAV8-CMV-GFPat4monthsofage
the Golgi to the plasma membrane, where it is cleaved by toassesstheeffectofTFEBtransductiononendogenousAPPprocessing
(cid:3)-secretaseswithintheA(cid:2)peptidefragmentregion,inamanner after4months.Beforeinjection,AAVparticlesweresubjectedtotwo
thatprecludesA(cid:2)generation(i.e.,nonamyloidogenicor(cid:3)-cleav- roundsofcesiumchloridegradientpurificationandtiterswerequanti-
fiedbyassessmentofviralgenomes.Forplaqueloadstudies,TFEBor
age;RajendranandAnnaert,2012).Alternately,APPisendocy-
control-transduced APP/PS1 mice (six males and eight females per
tosed, and sequentially cleaved by a (cid:2)-secretase (BACE1) and
group) were killed 4 months later. One hemisphere was fixed and
then the (cid:4)-secretase complex (comprised of presenilins, nicas- coronal sections (50 (cid:5)m) were cut for histological analysis (X-34
trin,APH-1,andPEN2)withinendosomes,togenerateA(cid:2)pep- stainingandHJ3.4immunostaining).Theotherhemispherewassnap
tides (amyloidogenic cleavage; Rajendran and Annaert, 2012). frozen on dry ice and stored at (cid:4)80°C for biochemical analysis
Accumulatingevidencesuggeststhatdysregulationofendocyto- (ELISAandimmunoblotting).ToassesstheabundanceofA(cid:2)species
sis(Haroldetal.,2009;Ginsbergetal.,2010)withaccumulation inpredepositingmice,theabovementionedAAVviralvectorswere
of large endosomes within neurons, as observed in preclinical injected into the hippocampi of APP/PS1 mice of either sex at 2
monthsofageandtheanimalswerekilled1monthlater.Allanimal
stages of AD pathology (Cataldo et al., 2000) and in neurons
care and surgical procedures were approved by the Animal Studies
transdifferentiatedfromfibroblastsofpatientswithAD(Israelet
CommitteeofWashingtonUniversitySchoolofMedicineinaccor-
al.,2012),increasestheproclivityforamyloidogenicprocessing
dancewithguidelinesoftheNIH.
ofAPP.
In vivo microdialysis. AAV particles driving expression of TFEB
EndocytosedAPPcanbesortedbacktotheGolgi(Morelet
(AAV8-CMV-FLAG-TFEB) or GFP as control (AAV8-CMV-GFP)
al., 2013) or trafficked to lysosomes along with other endo- weretransducedbystereotacticallyguidedinjectioninthehippocam-
somal cargo (The´ry et al., 2002). Indeed, a large fraction of pusof2-month-oldAPP/PS1transgenicmiceofeithersexfollowed
full-lengthAPPisdegradedwithinlysosomes(Caporasoetal., byinvivomicrodialysis,1monthlater,aspreviouslydescribed(Cir-
1992,1994;Goldeetal.,1992;Haassetal.,1992).Experimen- ritoetal.,2003,2011).Allstudieswereinitiatedatthesametimeof
tal inhibition of lysosome function markedly prolongs APP day. Briefly, a guide cannula (BR-style; Bioanalytical Systems) was
half-life(Caporasoetal.,1992)andimpairsendocytoticflux implanted and cemented with the tip at coordinates: bregma (cid:4)3.1
mm,midline(cid:4)2.5mm,1.2mmbelowduraata12°angle.A2mm
withaccumulationofenlargedvesiclesindystrophicneurites
microdialysis probe was then inserted into the hippocampus that
mimicking AD pathology (Lee et al., 2011), with increased
containeda38kDaMWCOsemipermeablemembrane(Bioanalytical
BACE1(Chiaetal.,2013)and(cid:2)-C-terminalfragment(Golde
Systems)allowingmoleculessmallerthanthiscutofftodiffuseinto
et al., 1992; Sannerud et al., 2011) resulting in markedly in- theprobe.A(cid:2)capableofenteringtheprobeisdubbed“exchangeable
creasedA(cid:2)generation.Relevanttothisdiscussionisthatim- A(cid:2)” (eA(cid:2); Cirrito et al., 2003). The probe was flushed with 0.15%
pairedlysosomalfunctionisobservedwithaging(Katoetal., bovineserumalbumin(Sigma)inanartificialCSFperfusionbufferat
1998;CuervoandDice,2000;Wolfeetal.,2013)andwithloss aconstantrate(1.0(cid:5)l/min).Theeffluatewascollectedintoarefrig-
ofpresenilins(duetomutationscausingfamilialAD;Leeetal., eratedfractioncollectorandassayedbysandwichELISAforA(cid:2)
x-40
2010; Neely et al., 2011; Coen et al., 2012), and is suspected peptidesattheendofeachexperiment.Duringmicrodialysis,animals
withpolymorphismsingenescodingforlysosomalproteins, werehousedinspeciallydesignedcagestopermitfreemovementand
adlibitumfoodandwaterwhileISFA(cid:2)wassampled.Baselinelevelsof
whichconferincreasedriskforsporadicAD,includingphos-
ISFA(cid:2)weresampledevery90minbetweenhours9and16(afterthe
pholipase D3 (Cruchaga et al., 2014), cystatin C (Hua et al.,
microdialysisprobeisinserted)andaveragedtodeterminethe“base-
2012), and Cathepsin D (Schuur et al., 2011). Also, levels of lineISFA(cid:2)level”ineachmouse.Absoluteinvivoconcentrationof
APP, its (cid:2)-CTF, and A(cid:2)are increased in mouse models of ISFeA(cid:2)foreachmousewasdeterminedbycorrectingforthe20.8%
lysosome storage disorders, namely, Neimann-Pick disease recovery (1.0 (cid:5)l/min) as obtained by the interpolated zero flow
(Kodametal.,2010),mucolipidoses(Keilanietal.,2012),and methoddescribedpreviously(Menacherryetal.,1992;Cirritoetal.,
sphingolipidoses (Tamboli et al., 2011). These observations 2003).Athour16(t(cid:5)0),a(cid:4)-secretaseinhibitor,CompoundE(200
incriminate lysosomal dysfunction as the common cellular nM reverse microdialysis; synthesized by AsisChem), was adminis-
mechanism in provoking increased A(cid:2)generation and im- tereddirectlytothehippocampusbyaddingthedrugtothemicrodi-
pairedA(cid:2)removal(FunkandKuret,2012;PericandAnnaert, alysisperfusionbuffer.ISFA(cid:2)wasthensampledevery60minforan
2015). additional8h.Thisenabledmeasurementoftheeliminationhalf-life
ofendogenousISFA(cid:2)invivoasdescribedpreviously(Cirritoetal.,
Asatherapeuticstrategytoaddresslysosomalinsufficiencyin
2003).
astrocytes, we previously evaluated activation of transcription
Immunohistochemistry.Frozenbrainsectionswereincubatedin0.3%
factor EB (TFEB), a master inducer of lysosomal degradative
H O inTBSfor10minandblockedwith3%drymilkinTBS-X(0.25%
pathways(Settembreetal.,2013a),anddemonstratedthatastro- 2 2
TritonX-100inTBS)for1h,followedbyincubationwithHJ3.4antibody
cytic TFEB expression stimulates A(cid:2)uptake and its lysosomal (anti-A(cid:2)-1-13;Rohetal.,2012)overnight.ThereaftersolutionAfrom
degradationtoreduceISFA(cid:2)(Xiaoetal.,2014).Inthisstudy,we VectastainABCkit(1:400)wasappliedfor1h,followedby0.025%DAB
evaluatedtheefficacyofneuronalTFEBactivationinenhancing in0.25%NiCl and0.05%H O for10–15min.Thesliceswereplaced
2 2 2
lysosomaldegradationofAPPtoreduceA(cid:2)levelsandattenuate onglassslides,driedovernight,dehydratedandmounted,andimaged
amyloidplaquepathogenesis. withconfocalmicroscopy(ZeissLSM).
X-34plaquestaining.BrainslicesweremountedonSuperFrostPlus
slides,permeabilizedwith0.25%TritonX-100for30min,andstained
MaterialsandMethods
withX-34(dissolvedin40%ethanol,60%water,pH10.0;agenerousgift
Studiesinanimalmodels.APP/PS1transgenicmice(B6C3-Tg(APPswe/ fromRobertMach,WashingtonUniversity,St.Louis,MO)for20min.
PS1(cid:2)E9)85Dbo/Mmjax)ofeithersexwereobtainedfromTheJackson ThereafterthetissuewasthoroughlyrinsedinPBSandmountedwith
Laboratory(Jankowskyetal.,2004)andinjectedwithAAV8viralparti- Fluoromountmountingmedia.
cles(generatedbytheHopeCenterViralCoreatWashingtonUniversity) Amyloidplaquequantification.Fiftymicrometerthickbrainsections
drivingexpressionofTFEB(AAV8-CMV-FLAG-TFEB)orGFPascon- werecollectedevery300(cid:5)msfromtherostralanteriorcommissureto
trol(AAV8-CMV-GFP),eachwith2(cid:5)lof1.5(cid:3)1012viralgenomesper caudal hippocampus. For plaque imaging, sections were stained with
Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation J.Neurosci.,September2,2015•35(35):12137–12151•12139
X-34orimmunostainedwithHJ3.4anti-A(cid:2)antibodies.High-resolution agedmice(10monthsold,whenplaquesareabundant),hippocampal
digitalimagesofthestainedbrainsliceswereobtainedwiththeNanoZo- tissuesweresequentiallyhomogenizedinPBSfollowedby5Mguanidine
omerDigitalScanner(HamamatsuPhotonics).Thetotalareaofplaque in TBS, pH 8.0 (to extract fibrillar and membrane-bound A(cid:2)). For
coveragewasmeasuredusingNIHImageJintheregionofthehippocam- ELISA assays, A(cid:2) and A(cid:2) peptides were captured with mouse
x-40 x-42
pusorpiriformcortex(devoidofviraltransduction)andexpressedas monoclonal-coating antibodies HJ2 (anti-A(cid:2)35-40) and HJ7.4 (anti-
percentagetotalareaforeachslice.Resultsfromn(cid:5)4sectionswere A(cid:2)37-42)orHJ5.1(anti-A(cid:2)13-28),respectively,aspreviouslydescribed
averagedtorepresenteachanimal. (Kimetal.,2009;Xiaoetal.,2014).HJ5.1(anti-A(cid:2)13-28),abiotinylated
Immunofluorescence.Paraffin-embeddedbrainsectionswerewashed antibodytargetingthecentraldomain,orHJ3.5(anti-A(cid:2)1-13,provided
withPBSandpermeabilizedwith0.3%TritonX-100inPBSfor20min byDr.DavidHoltzman,WashingtonUniversitySchoolofMedicine,St.
followedbyblocking.Fordoublelabeling,amixtureofthefollowing Louis,MO),whichtargetstheN-terminalaminoacids,wasusedasthe
antibodies was used: mouse anti-TFEB (MyBioSource, 1:100), rabbit detecting antibody, followed by streptavidin-poly-HRP-40 (Fitzgerald
anti-NeuN(Abcam;1:100),rabbitanti-GFAP(Sigma;1:100),andrab- Industries).AllELISAassaysweredevelopedusingSuperSlowELISA
bitanti-Iba1(WakoChemicals;1:1000).Asecondaryantibodymixture TMB(Sigma)andabsorbancereadonaBio-TekEpochplatereaderat
of Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 650nm.StandardcurvesweregeneratedfromsynthetichumanA(cid:2)
1–40
594-conjugatedgoatanti-rabbitIgG(Invitrogen)wasapplied.Sections orA(cid:2) peptides(AmericanPeptide).
1–42
wereimagedbyconfocalmicroscopy(ZeissLSM). QuantitativePCR.Real-timePCRwasperformedasdescribedprevi-
Neuronalcounts.Stereologicalanalysisforneuronalcountswasper- ously(Maetal.,2012).Briefly,totalRNAwaspreparedfromtransfected
formedonsectionsstainedwith0.05%cresylvioletsolution,asprevi- N2a-APP695cellsorAAV-transducedhippocampususinganRNA-easy
ouslydescribed(Xiaoetal.,2014).Briefly,cresylviolet-stainedcellswith minikit(Qiagen),andcDNAwassynthesizedwith1(cid:5)goftotalRNA
nuclei imaged within the inclusive zone of each dissector frame were usingtheSuperScriptIIIfirst-strandsynthesissystem(Invitrogen).One
counted in four equally spaced sections (50 (cid:5)m apart) in the dorsal microliterofcDNAtemplatewasmixedwith12.5(cid:5)lof2(cid:3)SYBRGreen
hippocampus of strata pyramidale CA1 and CA3, using a computer- PCRMasterMix(Invitrogen)andsubjectedtoquantitativePCRintrip-
basedstereologysystem(StereoInvestigator;MicroBrightField)withthe licateunderthefollowingconditions:50°C,2min;95°C,10min;fol-
opticalfractionator(Westetal.,1991). lowed by 40 cycles of 95°C, 15 s; 60°C, 1 min in the ABI7500 Fast
In vitro studies. N2a cells stably expressing the human APP695 RealTimePCRsystem.ThehousekeepinggeneGAPDHwasalsoampli-
construct (N2a-APP695) were grown in DMEM/Opti-MEM (50:50) fiedinparallelasareferenceforthequantificationoftranscripts.Primer
supplementedwith5%FBSand200(cid:5)g/mlG418andtransfectedwith sequencesusedhavebeenpreviouslydescribed(Xiaoetal.,2014).
FLAG-tagged murine TFEB or empty vector, as described previously Immunoblotting.Proteinsampleswererunon4–12%Bis-Trisgels.
(Xiao et al., 2014). We observed (cid:6)90% efficiency for transfection of Blotswereprobedwiththefollowingantibodies:FLAG(Sigma;1:1000),
exogenousTFEB(orGFPascontrol)intheseinvitrostudies.Forassess- TFEB (MyBioSource; 1:500), LAMP1 (Santa Cruz Biotechnology;
mentofAPPhalf-lifebycycloheximidechaseassay,N2a-APP695cells 1:500),CathepsinB(Abcam;1:500),CathepsinD(agenerousgiftfrom
transfectedwithTFEB(orcontrol)weretreatedwith50(cid:5)g/mlcyclohex- StuartKornfeld,WashingtonUniversity,St.Louis,MO;1:2000),APP
imidefollowedbypreparationofcellularextractsatvarioustimeinter- CT695(todetectbothfull-lengthAPPandC-terminalfragments;Invit-
valsforimmunoblottingforAPP.Toinhibitlysosomeacidificationand rogen;1:500),6E10(todetectsAPP(cid:3);Covance;1:1000),Adam10(Santa
function,studieswereperformedinthepresenceofbafilomycinA1(100 CruzBiotechnology;1:100),Adam17(Millipore;1:1000),BACE1(Cell
nM),aspreviouslydescribed(Xiaoetal.,2014).Studieswithcell-surface Signaling Technology; 1:1000), Psen1 (Santa Cruz Biotechnology;
biotinylation to assess internalization of APP were performed as de- 1:200),nicastrin(ThermoScientific;1:1000),IDE(Abcam;1:2000),ne-
scribed previously (Xiao et al., 2012). Briefly, cells cultured in 6-well prilysin (Millipore; 1:500), MMP2 (Abcam; 1:1000), MMP9 (Abcam;
plateswerewashedwithcoldPBSandsurfaceproteinswerelabeledwith 1:1000), HRP-conjugated biotin (Cell Signaling Technology; 1:1000),
the nonmembrane-permeant, cleavable biotin derivative, sulfo-NHS- and actin (Sigma; 1:2000). Normalized band intensity was quantified
SS-biotin(1mg/mlinPBS).Cellswerekeptat4°Cfor30mininthedark usingImageJsoftware.
andgentlyrockedduringtheincubationperiod.Thebiotinreagentwas Statistical analysis. All data are shown as Mean (cid:7) SEM. Data were
quenchedbytreatingthecellswithtwo15minwashesof0.1Mglycinein analyzedbytwo-tailedStudent’sttestfordetectingsignificantdiffer-
PBS.CellswererinsedwithPBSandlysedinRIPAbuffercontaining50 encesbetweentwogroups.Differencesamongmultiplegroupswerean-
mMTris-HCl,pH7.5;150mMNaCl;1%(v/v)NonidetP-40;0.5%(w/v) alyzedbyone-wayandtwo-wayANOVAforoneandtwoindependent
deoxycholate; and 1(cid:3) protease inhibitor mixture. Lysates were incu- variables,respectively,followedbyposthocTukey’stest.Statisticalsignif-
batedwithDynabeadsMyOneStreptavidinT1(Invitrogen)for2hina icancewaspeggedatp(cid:8)0.05(*p(cid:8)0.05and**p(cid:8)0.01).
rotarymixertoisolatebiotin-labeledproteins.Acohortofsurfacebiotin-
labeledcellswasincubatedfor0,1,2,3,5,7.5,10,20,and30minat37°Cto Results
allowinternalization,followedbyrapidcoolingonicetostopendocytosis. AAV-CMV-TFEBtransducesTFEBspecificallyinneuronsof
Tocleavebiotinexposedatthecellsurface,thesecellswereincubatedthree
APP/PS1micewithupregulatedlysosomeabundance
timesfor20minat4°Cwith50mM2-mercaptoethanesulfonicacid(Sigma) Lysosomaldysfunctionisspeculatedtoplayacentralroleinen-
in50mMTris-HCl,pH8.7;100mMNaCl;and2.5mMCaCl2.Afterthorough hancedA(cid:2)generationinneurons(Leeetal.,2010;Neelyetal.,
rinsingwithPBScontaining20mMHEPES,cellswerelysedinRIPAbuffer,
2011; Coen et al., 2012; Funk and Kuret, 2012; McBrayer and
and internalized biotinylated proteins were immunoprecipitated with
streptavidinfollowedbyimmunoblottingforAPP(CT695;Invitrogen).Up- Nixon,2013;Wolfeetal.,2013)oritsimpairedclearance(Wyss-
takeofbiotin-labeledtransferrin(T3915;Sigma)wasassessedinparallelin Corayetal.,2003;Majumdaretal.,2011;Leeetal.,2012;Kane-
TFEB and vector-transfected cells to determine the effect of TFEB on kiyo et al., 2013). In previous studies, we demonstrated that
clathrin-mediated endocytosis. Immunofluorescence was performed on targetingexpressionofTFEB,amasterinduceroflysosomalbio-
C17.2cellscotransfectedwithCer-APP-YFPandTFEBorcontrolvectorand genesis(Sardielloetal.,2009;Settembreetal.,2011,2013b),spe-
incubatedwithLysoTrackerRed(L7526;Invitrogen)tolabelthelysosome cifically to astrocytes, stimulated lysosome biogenesis and
compartment,andlivecellswereexaminedbyconfocalmicroscope(Zeiss function resulting in accelerated A(cid:2)uptake and degradation
LSM).
(Xiaoetal.,2014).Inthecurrentstudy,weexploredthehypoth-
A(cid:2)ELISA.A(cid:2)speciesincelllysatesandmediafromtransfectedN2a-
esisthatenhancinglysosomalbiogenesisinneuronswithexoge-
APP695weredetectedbysandwichELISA,aspreviouslydescribed(Xiao
et al., 2014). To detect total A(cid:2)in the hippocampus in 3-month-old nousexpressionofTFEBwillstimulatelysosomaldegradationof
mice,dissectedtissuewassequentiallyhomogenizedinPBSfollowedby holo-APP to attenuate A(cid:2)production. Accordingly, we per-
RIPAbuffertoobtaindetergent-solubleA(cid:2)atanagewhenplaquesare formedstereotacticallyguidedinjectionsofAAV8particlesen-
notobserved(Yanetal.,2009),andsampleswerepooledforanalysis.In coding a CMV promoter-driven construct to transduce TFEB
12140•J.Neurosci.,September2,2015•35(35):12137–12151 Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation
(AAV8-CMV-FLAG-TFEB; and AAV8-
CMV-GFP as control) in the hippocam-
pus of 6-month-old APP/PS1 mice, at a
stage of early plaque deposition and
growth (Yan et al., 2009). Stereotactic
AAV injections consistently resulted in
widespreadtransductioninthehippocam-
pus(datanotshown).Asweobservedpre-
viously (Xiao et al., 2012), the CMV
promoter-driven construct specifically re-
sultedinTFEBexpressioninneuronswitha
neuron-specificmarker,NeuN,andnot
inastrocytesormicroglia(Fig.1A).Im-
portantly,exogenousTFEBlocalizedto
the neuronal nuclei (Fig. 1A) and hip-
pocampal extracts from AAV8-TFEB-
transduced mice demonstrate robust
overexpression with TFEB (11-fold in-
creaseovercontrol,p(cid:8)0.01;N(cid:5)3per
group)withincreasedabundanceoflys-
osomal proteins, LAMP1, and Cathep-
sinBandCathepsinD(Fig.1B,C),two
lysosomalproteasesthathavebeenim-
plicated in APP degradation (Higaki et
al.,1996;Mueller-Steineretal.,2006).
NeuronalTFEBexpressiondrives
reducedAPPabundance,invivo
APP695issynthesizedandprocessedpri-
marily in neurons, where its sequential
cleavageviathe(cid:2)-and(cid:4)-secretasesresults
in generation of amyloidogenic A(cid:2)pep-
tides(Hartmannetal.,1997;Kamenetzet
al.,2003).Alternately,APPprocessingcan
proceedvia(cid:3)-secretasesinamannerthat Figure1. AAV8-CMV-TFEBdriveslysosomebiogenesisinneurons.A,Representativeconfocalimagesdemonstratingexpres-
precludes A(cid:2) generation (Haass et al., sionofTFEB(green)invariousCNScelltypesmarkedbytheexpressionofNeuN(neurons;red,top),GFAP(astrocytes;red,middle),
andIba-1(microglia;red,bottom)inthehippocampusofAPP/PS1miceinjectedwithAAV8-CMV-FLAG-TFEBparticles.Theboxed
2012). Studies have demonstrated that a
inserts(upperleftcorners)demonstratemagnifiedimagesofTFEB-expressingcells.B,C,Immunoblots(B)withquantification(C)
largefractionoffull-lengthAPPproteinis
oflysosomalproteinsinextractsfromhippocampitransducedwithAAV8-CMV-FLAG-TFEB(TFEB)orAAV8-CMV-GFP(GFP).N(cid:5)3
also degraded within lysosomes, which
pergroup;**p(cid:8)0.01.
maylimitthepoolofAPPavailablefor(cid:3)-,
(cid:2)-,and(cid:4)-cleavage(Caporasoetal.,1992,
thesedatasuggestthatTFEBexpressionfacilitatesholo-APPdegra-
1994;Goldeetal.,1992).GiventhatTFEBdrivesupregulationof
dation via pathways that do not involve APP processing by
the lysosomal machinery, we examined the effect of neuronal (cid:3)-cleavageorsequential(cid:2)-and(cid:4)-cleavage.
TFEBexpressiononabundanceofAPPanditsvariouscleavage
fragments. TFEB-transduced hippocampal extracts demon-
strateda28%reductioninAPPproteinabundance,accompanied NeuronalTFEBexpressionreducesISFA(cid:2)levels,invivo
byamatching31and36%reductionin(cid:3)-CTFand(cid:2)-CTF(Fig. A(cid:2)peptides are released into the ISF and removed via cellular
2A,B).Givenpreviousobservationsthatamyloidpathogenesisis uptake and degradation, extracellular proteolysis, or transport
acceleratedinfemalemice(Callahanetal.,2001),weexamined across the blood–brain barrier. Consequently, the steady-state
theeffectsofTFEBtransductioninmaleandfemalemicesepa- levelofA(cid:2)intheISFisdeterminedbythebalancebetweenitsrate
rately. We observed comparable reductions in APP in the two of production and degradation. Considering that exogenous
sexes(by29%inmalesvs30%infemales;datanotshown).These TFEBexpressionresultsinareductionintotalAPPlevelswitha
datashowthattheeffectofneuronalTFEBexpressiononAPP parallel reduction in abundance of its cleaved fragments (Fig.
abundanceisindependentofgender. 2A,B), we hypothesized that TFEB transduction will attenuate
ThereductioninAPPabundancewasnotcausedbyadifference A(cid:2)productionresultinginreducedA(cid:2)steady-statelevelsinthe
in the transcript levels for the transgenically expressed human ISF. Accordingly, we transduced 2-month-old APP/PS1 mice
APP695protein(Fig.2C)orintheexpressionof(cid:3)-secretasesand with AAV8-CMV-FLAG-TFEB and control AAV8-CMV-GFP
componentsofthe(cid:2)-and(cid:4)-secretasemachinerybetweentheTFEB viralparticlesandperformedinvivomicrodialysis1monthlater
and control-transduced hippocampi (Fig. 2C–E). Importantly, todeterminewhetherneuronalTFEBexpressionimpactsISFA(cid:2)
TFEB-transducedhippocampididnotdemonstrateadifferencein atatimepointbeforedepositionofplaques(Yanetal.,2009)to
neuronalcountscomparedwithcontrol(Fig.2F),rulingoutneuro- avoid a potential confounding influence from exchangeable
naltoxicityastheunderlyingcausefortheseobservations.Together plaque-boundA(cid:2)pool.TFEB-transducedmicedemonstrateda
withtheproportionatelycomparablereductionsinvariousCTFs, 57%reductioninsteady-stateISFA(cid:2)levelscomparedwithcon-
Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation J.Neurosci.,September2,2015•35(35):12137–12151•12141
Figure2. NeuronalTFEBtransductionresultsinreducedAPPabundance.A,B,Immunoblot(A)withquantitation(B)ofAPPandits(cid:3)-and(cid:2)-CTFsinAPP/PS1mousehippocampitransducedwith
AAV8-CMV-FLAG-TFEB(TFEB)orAAV8-CMV-GFP(GFP).N(cid:5)6pergroup(n(cid:5)3malesandn(cid:5)3females);**p(cid:8)0.01.Representativeimmunoblotisfrommalemousetissues.C,Expressionof
transcriptscodingforvariouscomponentsoftheAPP-processingmachineryinhippocampaltissuestransducedwithAAV8-CMV-FLAG-TFEB(TFEB)orAAV8-CMV-GFP(GFP).N(cid:5)5pergroup.No
statisticallysignificantdifferenceswerenotedforanyofthetranscripts.D,E,Immunoblot(D)andquantitation(E)ofAPP-processingmachineryproteinsinhippocampitransducedwithTFEB(orGFP
ascontrol,asinA).N(cid:5)3pergroup.F,NeuronalcountsintheCA1andCA3layersofthehippocampifromAAV8-CMV-FLAG-TFEB(TFEB)andAAV8-CMV-GFP(GFP)-transducedAPP/PS1mice.N(cid:5)
4pergroup.
trols(Fig.3A).Aftersteady-statemeasuresofISFA(cid:2)wereob- Importantly, total immunodetectable A(cid:2)levels were also
tained, a potent (cid:4)-secretase inhibitor, Compound E, was reduced in the hippocampal extracts from TFEB-transduced
administeredtoinhibitAPPcleavagetoA(cid:2)(Cirritoetal.,2011), mice at this early stage of amyloid pathogenesis, compared
andISFwassampledforanadditionalperiodof8htodetermine with controls (Fig. 3F,G), confirming that the reduction in
theeliminationrate(half-life)ofpre-existingA(cid:2)(Fig.3A),which steady-state levels of ISF A(cid:2)was not due to intracellular A(cid:2)
was observed to follow first-order kinetics in both control and accumulation.TogetherwiththereductioninAPPabundance
TFEB-transduced mice (Fig. 3B). We observed a modest 24% and that of its various cleaved fragments (Fig. 2A,B), these
reductioninhalf-lifeofISFA(cid:2)inCMV-TFEB-transducedhip- datapointtoapredominanteffectofneuronalTFEBexpres-
pocampi,indicatingthatneuronalTFEBtransductionalsoaccel- sion on attenuating A(cid:2)generation with a modest effect on
eratesA(cid:2)removalfromISF(Fig.3B).Thatsteady-stateISFA(cid:2) accelerating A(cid:2) removal as the primary driver of reduced
levelsarereducedby57%,buthalf-lifeisreducedbyonly24%, steady-stateISFA(cid:2)levels.
stronglysuggeststhatTFEBhasonlyaminorroleinenhancing
ISFA(cid:2)clearancewiththeprimaryeffectinsteadonreducingA(cid:2) NeuronalTFEBexpressionreducesA(cid:2)levelsandamyloid
production. Indeed, previous studies have demonstrated that plaquesinAPP/PS1transgenicmice
neuronstakeupA(cid:2)viaendocytosisandmacropinocytosis,andit ISFA(cid:2)levelscorrelatewithamyloidplaquepathology(Beroet
isconceivablethatTFEBtransductionstimulatesA(cid:2)uptakein al.,2011),supportingthehypothesisthatA(cid:2)aggregatesintoam-
neurons via the latter similar to our studies with astrocyte- yloidplaquesinaconcentration-dependentmanner,invivo.In-
targetedTFEBexpression(Xiaoetal.,2014).Importantly,the deed, studies from our laboratory as well as others have
effectofexogenousTFEBonreductioninISFA(cid:2)levelsandA(cid:2) demonstratedthatstrategiestoreduceISFA(cid:2)levelsdriveare-
half-lifewascomparableinbothsexes(datanotshown).We ductioninamyloidplaquedeposition(Crameretal.,2012;She-
also explored the possibility that TFEB transduction could lineetal.,2014;Xiaoetal.,2014).Accordingly,wepositedthat
enhanceextracellulardegradationofA(cid:2),butdidnotobserve neuronalTFEBtransductionwilldrivereductioninfibrillarA(cid:2)
any alteration in the transcript levels for known A(cid:2)- aggregatesandattenuatedepositionofamyloidplaquesinaging
metabolizingenzymes(MMP2,MMP9,IDE,orneprilysin)in APP/PS1mice.Toinvestigatethispremise,wesubjectedTFEB-
TFEB-transduced hippocampi (Fig. 3C). Analogously, the transduced(andcontrol)hippocampaltissuetosequentialho-
proteinabundancefortheseenzymeswasunchanged(MMP2, mogenization in PBS followed by guanidine to quantify A(cid:2)in
MMP9,andIDE)ormodestlyreduced(neprilysin;Fig.3D,E), PBS-soluble and PBS-insoluble fractions. In addition, we per-
suggestingthatregulationofextracellularproteasesisunlikely formedA(cid:2)immunohistochemistryandX-34stainingtoassess
to contribute to a reduction in ISF A(cid:2)levels with neuronal A(cid:2)andamyloidload,respectively.Asshown(Fig.4A–C),TFEB
TFEBtransduction. transductionresultedina40and50%reductioninsolubleA(cid:2)40
12142•J.Neurosci.,September2,2015•35(35):12137–12151 Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation
Figure3. NeuronalTFEBtransductionreducesISFA(cid:2)levels,invivo.A,AssessmentofA(cid:2)levelsbyinvivomicrodialysisin3-month-oldAPP/PS1micetransducedwithAAV8-CMV-FLAG-TFEB
(TFEB)andAAV8-CMV-GFP(GFP)withserialhourlymeasurements.N(cid:5)8miceinTFEBgroup,n(cid:5)9miceinGFPgroup.Att(cid:5)0,micewerecontinuallyadministeredCompoundEdirectlytothe
hippocampus(200nM,reversemicrodialysis)followedbyhourlysamplingforA(cid:2).Insetshowsmeanabsoluteinvivo“exchangeable”A(cid:2)(eA(cid:2)x-40)concentrationaveragedovera9hperiodbefore
drugadministration;*p(cid:8)0.05.B,SemilogplotofdeclineinpercentagebasalISFA(cid:2)levelsduringadministrationofCompoundEinanimalsinA.InsetshowsA(cid:2)half-lifeinthetwogroups;*p(cid:8)
0.05.C–E,Transcriptlevels(C)andproteinabundance[immunoblot(D)withquantitation(E)]forenzymesimplicatedinextracellularA(cid:2)metabolisminhippocampaltissuefromAAV8-CMV-FLAG-
TFEB(TFEB)andAAV8-CMV-GFP(GFP)-transducedAPP/PS1mice.N(cid:5)5pergroup;**p(cid:8)0.01.F,G,A(cid:2)40andA(cid:2)42levelsindissectedhippocampaltissuesfromAAV8-CMV-FLAG-TFEB(TFEB)and
AAV8-CMV-GFP(GFP)-transducedAPP/PS1miceat3monthsofage.TissuewashomogenizedfirstinPBS(D)theninRIPA(E)quantifiedwithELISAassay.HJ2andHJ7.4antibodieswereusedfor
captureA(cid:2)40andA(cid:2)42,respectively,andHJ5.1antibodywasusedfordetection.N(cid:5)6micepergroup;**p(cid:8)0.01.
and A(cid:2)42, and 36 and 35% reduction in insoluble A(cid:2)40 and immunostainedamyloidplaqueload(Fig.4G,H).Importantly,
A(cid:2)42,respectively,comparedwithcontrols,byanELISAassayto aswehaveobservedpreviouslywithastrocyticexpressionofex-
differentiallycapturethetwospeciesbyspecificC-terminalanti- ogenous TFEB (Xiao et al., 2014), the effect of neuronal TFEB
bodies.Toconfirmthesefindings,wealsousedanalternateA(cid:2) transduction on amyloid deposition was localized to the hip-
peptide capture and detection strategy using a combination of pocampus(seeareaoutlinedwithdottedlineinFig.4DandG
antibodies targeting distinct epitopes at the central domain andquantitationinEandH),whichwastargetedforTFEBex-
(HJ5.1)andNterminus(HJ3.5),aspreviouslydescribed(Xiaoet pressionbystereotacticinjectionandnotinthecorticalareasthat
al.,2014),andobserveda29and31%reductioninthesoluble aredistantfromthesiteofAAVtransduction.Itisinterestingto
and insoluble levels of all A(cid:2)species in TFEB-transduced hip- notethatthedegreeofreductioninplaqueloadmirroredthatof
pocampi(Fig.4A–C).ThissuggestsauniformreductioninA(cid:2) thereductioninA(cid:2)levels(Fig.4B,C)intheagedmiceandthatof
levelsratherthanareductioninspecifictruncatedfragmentswith thereductioninISFsteady-stateA(cid:2)(Fig.3A,B)andhippocam-
exogenousTFEBexpression. palA(cid:2)inthepredepositingmice(Fig.3F,G),attestingtoacon-
This was accompanied by a 35% reduction in X-34-stained sistenteffectofTFEBonA(cid:2)generationthroughoutthevarious
amyloid deposits (Fig. 4D,E) and a 40% reduction in A(cid:2)- stagesofamyloidpathogenesis.ItisalsonoteworthythatTFEB-
Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation J.Neurosci.,September2,2015•35(35):12137–12151•12143
transduction had a comparable effect on amyloid plaque load
reduction in either sex (Fig. 4F,I), as we have previously ob-
served (Xiao et al., 2014).These data suggest that TFEB
expressioninneuronsattenuatesamyloidplaquedepositionvia
reducingA(cid:2)generation.
NeuronalTFEBtransductionreducesAPPandA(cid:2)levelsin
wild-typemice
ToexcludethepossibilitythatTFEBprimarilyaffectstheprocess-
ing of the mutant APP695 protein, which is co-overexpressed
withafamilialADriskconferring(cid:2)E9mutantpresenilin-1pro-
teinintheAPP/PS1mice(Jankowskyetal.,2004),weexamined
theeffectofneuronallytransducedTFEBontheabundanceof
endogenous wild-type APP protein and its (cid:3)- and (cid:2)-CTFs in
hippocampiofwild-typemiceversusthoseinGFP-transduced
hippocampiascontrols.Remarkably,TFEBtransductionledtoa
comparabledegreeofreductioninabundanceoftotalendoge-
nousAPPandlevelsofthe(cid:3)-and(cid:2)-CTFs(Fig.5A,B)mimicking
itseffectinAPP/PS1transgenicmice(Fig.2A,B).Thistranslated
intoa27and21%reductioninA(cid:2)40andA(cid:2)42species(Fig.5C),
respectively,indicatingthatTFEBalsoaltersthemetabolismof
wild-typeAPPproteintoreduceA(cid:2)generation.
TFEBexpressionattenuatesA(cid:2)generation,invitro
ToconfirmthatTFEBexpressionresultsinreducedA(cid:2)genera-
tion, we transfected N2a cells stably expressing APP695 with
TFEBorvectorcontrol,andexaminedAPPprocessing.TFEB-
transfectedcellsdemonstratedincreasedlysosomeabundance,as
indicatedbyupregulationoflysosomalproteins(Figure6A,B)
drivenbyeightfoldoverexpressionofTFEB,asweobservedpre-
viouslyinC17.2cells(Xiaoetal.,2014).TFEB-transfectedcells
demonstratedareductioninAPPabundance,aswellasreduced
levelsof(cid:3)-and(cid:2)-CTFsandsAPP(cid:3)inthemedium(Fig.6C,D).
ThiswasnotassociatedwithachangeinAPPtranscript(TFEB/
GAPDH)mRNA1.04(cid:7)0.04-foldinTFEBtransfectedvs1.0(cid:7)
0.01-foldincontrol,p(cid:5)0.38,N(cid:5)3pergroup),indicatingthat
TFEBstimulatesincreaseddegradationofAPP,whichisconsis-
tentwiththeobservationsinTFEB-transducedhippocampi(Fig.
2).ConfocalimagingofadualfluorescentAPPconstruct(San-
nerudetal.,2011)alsodemonstratedaqualitativeupregulation
oflysosomeabundancewithreductioninfull-lengthAPPprotein
in TFEB-transfected cells, along with increased localization of
APP in lysosomes (Fig. 6E). Colocalization of full-length APP
(bearingbothblueandgreenfluorescenttags)withLysoTracker
Red-labeledlysosomes(seearrows)wasqualitativelyobservedto
beupregulatedinTFEB-expressingcells(Fig.6E,arrows).This
wasassociatedwithreducedA(cid:2)abundance,bothintracellularly
(Fig. 7A) and in the medium (Fig. 7B). Importantly, TFEB-
transfectedcellsdemonstratedareductioninA(cid:2)appearancein
themedium(Fig.7C,D),indicatingthatTFEBprimarilyattenu-
atesA(cid:2)generation,andreducedintracellularA(cid:2)levelsarenota
resultofincreasedA(cid:2)secretion.
Figure4. NeuronalTFEBtransductionreducesamyloidplaqueloadinAPP/PS1mice.A,
SchematicrepresentationofspecificantibodiesusedinELISA.B,C,A(cid:2)40andA(cid:2)42levelsin TFEBacceleratesAPPfluxdowntheendosome-
dissectedhippocampaltissuesfromAAV8-CMV-FLAG-TFEB(TFEB)andAAV8-CMV-GFP(GFP)- lysosomepathway
transducedmice(10monthsofage).TissuewashomogenizedfirstinPBS(solublelevels,B), The intracellular itinerary of APP follows a pathway from the
thenin5mMguanidine(insolublelevels,C)quantifiedwithELISAassay.HJ2andHJ7.4anti- Golgi (trans-Golgi network), wherein it is post-translationally
bodieswereusedforcaptureA(cid:2)40andA(cid:2)42,respectively,andHJ5.1antibodywasusedfor
detection.TotalA(cid:2)levelswerealsomeasuredwithacombinationofHJ5.1antibodyforcapture
4
andHJ3.5antibodyfordetection,asindicatedintheschematic.N(cid:5)8micepergroup;*p(cid:8)
0.05,**p(cid:8)0.01.D,RepresentativeX-34-stainedimagesfromAPP/PS1micetreatedasinA. G,RepresentativeA(cid:2)-immunostainedimagesfrommicetreatedasinB.H,I,Quantificationof
Theareaofthehippocampusisoutlinedwithadottedline.E,F,QuantificationofX-34-stained A(cid:2)-stainedplaqueburdeninthehippocampusinmicetreatedasinA(H)andplaqueburden
plaqueburdeninthehippocampus(HPC)inmicetreatedasinA(E)andplaqueburdenstratified stratifiedbysex(I).N(cid:5)14(6maleand8female)micepergroup;*p(cid:8)0.05,**p(cid:8)0.01.HPC,
by sex (F). N (cid:5) 14 (6 male and 8 female) mice per group;*p (cid:8) 0.05, **p (cid:8) 0.01. Hippocampus;CTX,cortex.
12144•J.Neurosci.,September2,2015•35(35):12137–12151 Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation
Figure5. NeuronalTFEBtransductionreduceslevelsofAPP,itsCTFs,andA(cid:2)speciesinwild-typemice.A,B,Immunoblot(A)withquantitation(B)ofAPP,andits(cid:3)-and(cid:2)-CTFsinwild-type
mousehippocampitransducedwithAAV8-CMV-FLAG-TFEB(TFEB)orAAV8-CMV-GFP(GFP)at8monthsofage.N(cid:5)3pergroup;*p(cid:8)0.05.C,A(cid:2)40andA(cid:2)42levelsindissectedhippocampal
tissueshomogenizedinRIPAbuffer,frommicetransducedasinA.HJ2andHJ7.4antibodieswereusedforcaptureA(cid:2)40andA(cid:2)42,respectively,andHJ5.1antibodywasusedfordetection.N(cid:5)
6pergroup;*p(cid:8)0.05.
Figure6. TFEBexpressionincreaseslysosomesandreduceslevelsofAPPanditscleavedCTFs,invitro.A,B,Immunoblots(A)withquantification(B)oflysosomalproteinsin
N2a-APP695cellstransfectedwithTFEBorvectorcontrol(for48h).C,D,Immunoblot(C)withquantitation(D)ofAPPandits(cid:3)-and(cid:2)-CTFsandofsAPP(cid:3)fragmentintheoverlying
medium,collectedoveradurationof6hincellstreatedasinA.N(cid:5)3pergroup;*p(cid:8)0.05,**p(cid:8)0.01.E,N2acellsweretransfectedwithadualfluorescentAPPconstruct(Sannerud
etal.,2011)withorwithouttheTFEBconstructandstainedwithLysoTrackerRed.Asshownintheseconfocalimages,colocalizationoffull-lengthAPP(bothblueandgreentags)with
LysoTrackerRed-labeledlysosomes(seearrows)wasqualitativelyincreasedinTFEB-expressingcells(seearrows)comparedwithemptyvector-expressingcells.
Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation J.Neurosci.,September2,2015•35(35):12137–12151•12145
al.,2012).Atvarioustimepointspostre-
warming, the cells were treated with
2-mercaptoethanesulfonicacidtoremove
cell-surface biotinylated APP molecules,
permitting assessment of kinetics of ap-
pearance and subsequent degradation of
total internalized biotin-tagged APP via
streptavidin capture and immunoblot-
ting.AsshowninFigure8,AandB,cells
transfected with TFEB demonstrated
reducedlevelsofbothsurfaceandinter-
nalizedAPP,suchthattheratioofinter-
nalized to surface APP was not altered
comparedwithcontrol.
To determine whether the reduced
steady-statelevelsofAPPareduetoaccel-
eratedendocytosisorenhancedfluxinto
thelysosomesfordegradation,wedeter-
mined the kinetics of intracellular APP
appearance in this assay (Fig. 8C,D). In-
terestingly,APPwasrapidlyendocytosed
and the rate of increase of intracellular
APPlevels(fromT(cid:5)1toT(cid:5)7.5min)
was not different between TFEB-expre-
Figure7. TFEBexpressionattenuatesA(cid:2)generation,invitro.A,B,AbundanceofA(cid:2)40andA(cid:2)42speciesinthecelllysates(A)
ssingcellsandcontrols(Fig.8C,D).These
andmedium(B)inN2a-APP695cellstransfectedwithTFEBorvectorcontrol.C,D,AccumulationofA(cid:2)40(C)andA(cid:2)42(D)species
dataareconsistentwithalackofeffectof
intheoverlyingmediumofcellstreatedasinA,overtheindicatedduration.N(cid:5)5pergroup;*p(cid:8)0.05,**p(cid:8)0.01.
TFEB on clathrin-mediated endocytosis,
consistentwithourpreviousobservations
modified after synthesis in the ER, to the plasma membrane (Xiaoetal.,2014).TodeterminewhetherTFEBexpressionaffects
whereitcanbecleavedviaanonamyloidogenic(cid:3)-cleavage.Al- the kinetics of other transmembrane proteins taken up by
ternatively,APPcanbeendocytosed,andmultiplestudieshave clathrin-mediatedendocytosis,weexaminedtheuptakeofexog-
demonstratedthatendocytosisofAPPisessentialforitscolocal- enouslyappliedbiotin-labeledtransferrin.Transferrinbindstothe
ization with (cid:2)- and (cid:4)-secretases within endosomes and mul- transferrinreceptorandisendocytosedfollowedbyrecycling
tivesicular bodies (formed by trafficking of endosomes into to the cell surface (Ciechanover et al., 1983). The kinetics of
intraluminalvesiclesviasequentialactivityofendosomal-sorting intracellular appearance of transferrin was not affected by
complexesrequiredfortransport,whicharetheprimarysitesfor TFEB expression (Fig. 8E), confirming that TFEB does not
A(cid:2)generationviaamyloidogeniccleavage;RajendranandAnn- accelerateclathrin-mediatedendocytosis.
aert, 2012). Studies have suggested that impairment in endo- Incontrast,internalizedAPPlevelsat30minwere32%lower
somalflux,likelysecondarytolysosomedysfunction,resultsin in TFEB-transfected cells versus 14% lower in control vector-
increasedtransittimewithinthisorganelle,whichincreasesthe transfectedcellsfromtheirrespectivepeaklevelsat7.5min(p(cid:5)
propensity for (cid:2)- and (cid:4)-cleavage and, hence, A(cid:2)generation 0.017,n(cid:5)3experimentaltrailspergroup),suggestingthatTFEB
(Cataldoetal.,2000;RajendranandAnnaert,2012;Moreletal., acceleratesdegradationofAPP,whichistargetedforlysosomal
2013;PericandAnnaert,2015).Indeed,afterendocytosis,studies degradation,incontrasttotransferrinthatgetsrecycledtothecell
have demonstrated that a significant proportion of APP is tar- surface (Ciechanover et al., 1983). Together with the observed
getedtothelysosomes(Benowitzetal.,1989;Haassetal.,1992; reductioninAPPanditsvariouscleavedfragments(Fig.6C,D)
Caporasoetal.,1994)andAPPlevelsrapidlybuildupinthecells and attenuated A(cid:2)generation (Fig. 7A–D), these observations
inthepresenceoflysosomalacidificationinhibitors(Goldeetal., suggest that TFEB expression does not affect APP endocytosis,
1992),suggestingthatlysosomaldegradationdrivesAPPprote- but stimulates increased flux of endocytosed APP down the
olysis to preclude formation of A(cid:2)peptides (Caporaso et al., endosome-lysosomepathwaywithoutbuildupofAPPproteinin
1992).GivenpreviousreportsthatTFEBexpressioncanstimu- intracellularcompartments,resultinginitslysosomaldegrada-
late endocytosis ( Pen˜a-Llopis et al., 2011), a potential adverse tionandreducedabundanceofAPPatsteadystate.
consequenceofTFEBactivationcouldbeincreasedAPPendocy-
tosisresultinginenhanced(cid:2)-and(cid:4)-cleavageandacceleratedA(cid:2) TFEBenhanceslysosomaldegradationofAPP
generation,asobservedwithotherexperimentalmanipulations ToassesstheroleoflysosomesinAPPdegradation,wetreated
tostimulateendocytosis(Grbovicetal.,2003;Schneideretal., TFEB-transfected(andcontrol)cellswithbafilomycinA1toin-
2008;Xiaoetal.,2012).Alternatively,TFEBmaystimulateflux hibit lysosome acidification and evaluated APP abundance. As
throughtheendosome-lysosomepathwaytopreventAPPaccu- showninFigure9A,bafilomycintreatmentresultedinincreased
mulation in endosomes and stimulate its proteolysis in lyso- APP levels (by 42% compared with control) and largely pre-
somes.Toevaluatethesepossibilities,weexaminedtheeffectof vented the TFEB-induced reduction in APP abundance (Fig.
TFEBonAPPinternalization.N2a-APP95cellsweretransfected 9A,B).Thiswasassociatedwithincreaseinboth(cid:3)-and(cid:2)-CTF
withTFEBorvectorcontrol,andcell-surfaceAPPwaslabeledby levels(Fig.9A,C,D)aswellasincreasedgenerationofA(cid:2)40(Fig.
biotinylation at 4°C (to prevent endocytosis), followed by re- 9E)andA(cid:2)42(Fig.9F),asinhibitinglysosomaldegradationof
warmingtostimulateitsinternalizationbyendocytosis(Xiaoet APPmakesitincreasinglyavailableasasubstrateforprocessing
12146•J.Neurosci.,September2,2015•35(35):12137–12151 Xiaoetal.•NeuronalTFEBExpressionAttenuatesA(cid:2)Generation
Figure8. TFEBexpressionreducessteady-statecell-surfaceandintracellularAPPlevelswithoutalteringitsendocytosis.A,B,Immunoblot(A)andquantitation(B)ofcell-surfaceand
internalizedAPPinN2a-APP695cellstransfectedwithTFEBorvectorcontrol,aftercell-surfaceAPPwaslabeledbybiotinylationat4°C(topreventendocytosis)followedbyrewarming(to37°C)to
stimulateitsinternalizationbyendocytosis(for10min).Cell-surfaceAPPwasassessedincellsmaintainedat4°C.ToassessinternalizedAPP,cellsweretreatedwith2-mercaptoethanesulfonicacid
toremovecell-surfacebiotinylatedAPPmolecules,followedbystreptavidincaptureandimmunoblotting(seeMaterialsandMethods).InsetshowsratioofinternalizedtosurfaceAPP;*p(cid:8)0.05.
C–E,Immunoblot(C)andquantitation(D)ofintracellularAPPasafractionofcell-surfaceAPPtodemonstratekineticsofintracellularA(cid:2)incellstransfectedasinAandkineticsofuptakeof
biotinylatedtransferrin[immunoblot(C)withquantitation(E)]incellstreatedasinA.N(cid:5)3pergroup.Nostatisticallysignificantdifferenceswereobservedbytwo-wayANOVA.
viathesecretases.WenextdeterminedAPPhalf-lifebytreating onstratethatTFEBexpressionacceleratesendogenouspathways
cellswithcycloheximidetoinhibitproteinsynthesisandfollow for holo-APP degradation via lysosomes, preventing its amy-
APPabundance.AsshowninFigure9,GandH,TFEBtransfec- loidogenicprocessingintoA(cid:2).
tionreducedAPPhalf-lifeby42%comparedwithcontrolindi-
cating enhanced APP degradation. Intriguingly, the levels of Discussion
exogenousTFEBalsodeclinedrapidly,consistentwiththeobser- ContemporaryapproachestoreducingA(cid:2)generationinneurons
vations that activated TFEB is degraded rapidly (Roczniak- havefocuseduponshiftingAPPprocessingtononamyloidogenic
Ferguson et al., 2012) possibly via the ubiquitin-proteasome pathwaysorsortingAPPawayfromtheendosomeswhereamy-
pathway(Maiaetal.,2015).Itwasalsointerestingtonotethatthe loidogenic cleavage takes place (Haass et al., 2012). Our study
abundanceofAPPinbafilomycin-treatedTFEB-expressingcells demonstratestheefficacyofacceleratingholo-APPdegradation
waslowerthantherespectivelevelsinvectorcontrols(Fig.9B), inlysosomesasastrategytoreduceA(cid:2)generation.Indeed,exog-
againpointingtothereductioninsteady-statelevelsofAPPwith enousTFEBexpressionstimulateslysosomebiogenesisinvitro,
TFEB expression, before application of bafilomycin to inhibit reducesfull-lengthAPPanditscleavedfragments,attenuatesA(cid:2)
lysosomeacidification.Expectedly,thisresultedinarelativede- generationandreleaseinthemedium(Figs.6,7),andmarkedly
clineintheCTFsandA(cid:2)peptidesinthissetting(Fig.9C–F). shortensAPPhalf-life(Fig.9)inalysosome-dependentmanner.
Asdescribedpreviously(Caporasoetal.,1992),inhibitionof These effects are likely secondary to acceleration of lysosomal
lysosomeacidificationwithbafilomycinA1prolongedAPPhalf- degradation of endocytosed APP, as suggested by reduction of
life,demonstratingthatasignificantfractionofAPPisdegraded steady-state APP at both the surface and within intracellular
viathelysosomes(Fig.9I–K).Importantly,APPhalf-lifewasalso compartments (Fig. 8), and reduced APP half-life with TFEB
markedlyprolongedby61%inthepresenceofbafilomycinA1in expression (Fig. 9). Mirroring these in vitro findings, AAV-
TFEB-transfectedcells(Fig.9L).Insummary,thesestudiesdem- mediated neuronal TFEB transduction in the hippocampus of