Alkalizing Drugs Induce Accumulation of Amyloid Precursor Protein By-products in Luminal ...

12 Pages·2007·1.5 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.282,NO.25,pp.18197–18205,June22,2007
©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Alkalizing Drugs Induce Accumulation of Amyloid Precursor
Protein By-products in Luminal Vesicles of
Multivesicular Bodies*□S
Receivedforpublication,October6,2006,andinrevisedform,April23,2007 Published,JBCPapersinPress,April27,2007,DOI10.1074/jbc.M609475200
Vale´rieVingtdeux‡§1,MalikaHamdane‡§,AnneLoyens‡§,PatrickGele´‡§,Herve´ Drobeck¶,Se´verineBe´gard‡§,
Marie-ChristineGalas‡§,Andre´ Delacourte‡§,Jean-ClaudeBeauvillain‡§,LucBue´e‡§,andNicolasSergeant‡§2
From‡INSERM,U837,NeurodegenerativeDisordersandNeuronalDeath,andthe§Faculte´deMe´decine,InstitutdeMe´decine
Pre´dictiveetdeRechercheThe´rapeutique,CentredeRecherchesJean-PierreAubert,Universite´Lille2,PlacedeVerdun,
F-59045Lille,Franceand¶CNRS,UMR8161,“LilleInstituteofBiology,”UniversityofLille1,PasteurInstituteofLille,
1,rueduProfesseurCalmette,F-59021LilleCedex,France
Amyloidprecursorprotein(APP)metabolismiscentraltothe processed by an (cid:2)- or a (cid:1)-secretase to produce C-terminal
pathogenesisofAlzheimerdisease.Weshowedrecentlythatthe fragments(CTFs)(forreview,seeRef.1).(cid:3)-Secretasefurther
amyloid intracellular domain (AICD), which is released by processes APP-CTFs (2, 3), releasing A(cid:1)from (cid:1)-CTF and
(cid:1)-secretase cleavage of APP C-terminal fragments (CTFs), is theamyloidintracellulardomain(AICDor(cid:4)-CTF)fromall
stronglyincreasedincellstreatedwithalkalizingdrugs(Vingt- APP-CTFs (2, 4–8). Several lines of evidence suggest that
deux,V.,Hamdane,M.,Be´gard,S.,Loyens,A.,Delacourte,A.,
AICDisatrans-regulatingfactorofgeneexpression(nepri-
Beauvillain, J.-C., Bue´e, L., Marambaud, P., and Sergeant, N. lysin,KAI1,APP,andglycogensynthasekinase-3(cid:1))(9–12).
(2007)Neurobiol.Dis.25,686–696).Herein,weaimedtodeter-
However, AICD is rapidly degraded and thus seldom
mine the cell compartment in which AICD accumulates. We
detected (13). We showed recently that AICD is strongly
show that APP-CTFs and AICD are present in multivesicular
increased upon treatment with alkalizing drugs, suggesting
structures.Multivesicularbodiescontainintraluminalvesicles
thattheendosomal/lysosomalpathwayregulatesAICDdeg-
(knownasexosomes)whenreleasedintheextracellularspace.
radation(14).
WedemonstratethatAPP,APP-CTFs,andAICDareintegrated
Theendosomal/lysosomalpathwayisessentialforA(cid:1)pro-
andsecretedwithinexosomesindifferentiatedneuroblastoma
ductionandAPPcatabolism.Forinstance,BACE-1(beta-site
andprimaryneuronalculturecells.Togetherwithrecentdata
showing that amyloid-(cid:2)is also found in exosomes, our data APP-cleavingenzyme1)resideswithinendosomes,andendo-
cytosisofBACE-1andAPPisaprerequisiteforgeneratingA(cid:1)
showthatmultivesicularbodiesareessentialorganellesforAPP
(15–17).AnacidicpHisnecessaryforoptimalBACE-1prote-
metabolismandthatallAPPmetabolitescanbesecretedinthe
extracellularspace. ase activity (18), and BACE-1 is degraded in lysosomes (19).
The(cid:3)-secretaseactivityhasbeenlocalizedattheendosomal/
lysosomalmembrane(20–23).Treatmentwithdrugsthatpre-
Amyloidprecursorprotein(APP)3metabolismiscentralto ventendosomal/lysosomalacidification(24–26)ordeletionof
Alzheimer disease etiopathogenesis. Extracellular amyloid theAPPinternalizationmotif(27,28)dramaticallyreducesA(cid:1)
deposits,aneuropathologicalhallmarkofAlzheimerdisease, secretion.
are composed of amyloid-(cid:1)(A(cid:1)) peptides that derive from The endosomal/lysosomal system is likely to be altered in
APPcatabolism.APPisatypeItransmembraneglycoprotein Alzheimerdisease(forreview,seeRef.29).SeveralAPPderiv-
atives accumulate in multivesicular bodies (MVBs), in trans-
genicanimalmodelsofamyloidosis(30,31),inAlzheimerdis-
*ThisworkwassupportedinpartbyINSERM,CNRS,theInstitutdeMe´decine ease(30),andincellmodels(32).MVBsbelongtotheendocytic
Pre´dictiveetdeRechercheThe´rapeutiqueofUniversityofLille2,andAPO-
pathway (33); are at the crossroad of several cellular mecha-
PISContractLSHM-CT-2003-503330.Thecostsofpublicationofthisarticle
weredefrayedinpartbythepaymentofpagecharges.Thisarticlemust nismssuchasmembranereceptorrecyclingandproteindegra-
thereforebeherebymarked“advertisement”inaccordancewith18U.S.C. dation; and can release their intraluminal vesicles, known as
Section1734solelytoindicatethisfact.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains exosomes (for review, see Refs. 34–36). More recently, exo-
supplementalFig.1. somesweredemonstratedtocontainA(cid:1)peptides(37).Taken
1RecipientofafellowshipfromAssociationFranceAlzheimer.
together,agrowingbodyofevidencesuggeststhatAPPproc-
2To whom correspondence and reprint requests should be addressed:
INSERM,U837,NeurodegenerativeDisordersandNeuronalDeath,Jean- essingtakesplacemainlybetweentheplasmamembraneand
PierreAubertResearchCentre,1,PlacedeVerdun,F-59045Lille,France. late endosomal compartments such as multivesicular endo-
Tel.:[phone];Fax:[phone];E-mail:[email protected].
somes.Herein,westudiedthelocalizationofAPPanditsderiv-
3Theabbreviationsusedare:APP,amyloidprecursorprotein;A(cid:1),amyloid-(cid:1);
CTFs, C-terminal fragments; AICD, amyloid intracellular domain; MVBs, atives in SY5Y neuroblastoma cells stably overexpressing
multivesicular bodies; BafA1, bafilomycin A1; PBS, phosphate-buffered humanAPPanddemonstratethatAPP,APP-CTFs,andAICD
saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES,
accumulateintheluminalvesiclesofmultivesicularendosomes
4-morpholineethanesulfonic acid; APPWT, wild-type amyloid precursor
protein. andarealsofoundinexosomes.
This is an Open Access article under the CC BY license.
JUNE22,2007•VOLUME282•NUMBER25 JOURNALOFBIOLOGICALCHEMISTRY 18197
APP-CTFsAreSecretedinExosomeVesicles
EXPERIMENTALPROCEDURES Science,Meylan,France).Thecellhomogenatewassonicated
andheat-treatedfor5minat100°C.Theproteinconcentration
Antibodies—APPCter-C17 is a well characterized rabbit
wasdeterminedusingaPlusOneTM2DQuantkit(Amersham
antibody raised against the last 17 amino acids of the human
Biosciences,Orsay,France),andsampleswerekeptat(cid:1)80°C
APPsequence(7,38–40).Anti-HSP70(heatshockproteinof
untilused.
70 kDa) antibody was purchased from Euromedex (Mundol-
WesternBlottingandMassSpectrometry—Anequalquantity
sheim, France), Anti-flotillin antibody was from Translab
of total proteins (20 (cid:5)g/lane) was loaded onto a 16.5% Tris/
(Erembodegem,Belgium);anti-LAMP-2andanti-Tsg101anti-
Tricine- or 8–16% Tris/glycine-polyacrylamide gel. Tris/
bodies were from Santa Cruz Biotechnology, Inc.); and anti-
Tricine/SDS-PAGEwasperformedfollowingtheprocedureof
EEA1 antibody (early endosome antigen 1) was from Upstate
Scha¨ggerandvonJagow(41)withaPROTEANIIxicell(Bio-
(LakePlacid,NY).Themousemonoclonalantibody8E5(raised
Rad,Marnes-la-Coquette,France).Followingthefinalsucrose
against residues 444–592 of human APP770) was a generous
giftfromDr.PeterSeubert(ElanPharmaceuticals).Anti-(cid:1)-tu- densitygradient(seebelow),proteinswereloadedontoCrite-
rionXT/MESgels(Bio-Rad)asdescribedpreviously(14).Pro-
bulinantibodyandhorseradishperoxidase-coupledsecondary
teins were transferred to nitrocellulose membrane (Hybond,
antibodywerepurchasedfromSigma(Saint-Quentin-Fallavier,
Amersham Biosciences) at 2.5 mA/cm2/gel using a semidry
France).
NovaBlot transfer system (Amersham Biosciences) according
Cell Culture of SKNSH-SY5Y Neuroblastoma Cells and
to the manufacturer’s instructions. Proteins were reversibly
Differentiation—ThehumanneuroblastomacelllineSKNSH-
stainedwithPonceauredtocheckthequalityofproteintrans-
SY5Y(referredtoasSY5Y)wasmaintainedinDulbecco’smod-
fer.Membraneswereblockedin25mMTris-HCl(pH8.0),150
ifiedEagle’smedium(Invitrogen,Cergy-Pontoise,France)sup-
mMNaCl,0.1%(v/v)Tween20,and5%(w/v)skimmilkfor30
plementedwith10%fetalcalfserum,2mML-glutamine,1mM
min. Membranes were then incubated overnight at 4°C with
nonessential amino acids, and 50 units/ml penicillin/strepto-
the appropriate dilutions of the primary antibody and incu-
mycin(Invitrogen)ina5%CO humidifiedincubatorat37°C.
2 batedfor1hatroomtemperaturewiththesecondaryantibody.
APP695 cDNA was subcloned into the eukaryotic expression
TheimmunoreactivecomplexeswererevealedusinganECLTM
vector pcDNA3 (Invitrogen), allowing for G418 (Invitrogen)
WesternblottingkitandHyperfilm(AmershamBiosciences).
selectionofclones.ThisAPPcDNAwastransfectedintoSY5Y
For mass spectrometry analyses, proteins were resolved with
cellsusingtheethyleneiminepolymerExGen500(Euromedex)
CriterionXTgels(Bio-Rad)andstainedwithCoomassieBril-
accordingtothemanufacturer’sinstructions.Cellsexpressing
liant Blue G-250 (Sigma). In-gel trypsin digestion and mass
APPwereselectedbytheadditionof200(cid:5)g/mlG418tothecell
spectrometryanalyseswereperformedasdescribedpreviously
medium.Fordifferentiation,SY5Ycellsweremaintainedfor15
(42).
daysinDulbecco’smodifiedEagle’smedium/nutrientmixture
ExosomeIsolationandSucroseDensityGradients—Thepro-
F-12supplementedwith2mML-glutamine,50units/mlpeni- tocolusedwasderivedfromthatofTheryetal.(43).Thecell
cillin/streptomycin, 7 mg/ml progesterone, 1% insulin/trans- culturemediawerecentrifugedfor10minat1200(cid:2)g,andthe
ferrin/selenium (Invitrogen), and 1 mM retinoic acid (Sigma). supernatant was filtered on a 0.22-(cid:5)m filter (Millex(cid:2) GP,
Themediumwasreplenishedevery3days.
hydrophilic membrane of polyethersulfone; Millipore, Mol-
PrimaryEmbryonicNeuronalCulture—Ratprimarycortical sheim,France)andcentrifugedfor30minat12,000(cid:2)gbefore
neuronswerepreparedfrom17–18-dayWistarratembryosas ultracentrifugationat100,000(cid:2)gfor1hat4°C.Thepelletwas
follows.Briefly,thebrainandmeningeswereremoved,andthe
addedoncewithalargevolumeofPBSandfurthercentrifuged
cortexwascarefullydissectedoutandmechanicallydissociated at100,000(cid:2)gfor1h.Forpurificationofexosomesonasucrose
in culture medium by triturating with a polished Pasteur densitygradient,the100,000(cid:2)gpelletwasresuspendedin0.25
pipette.Oncedissociatedandafterbluetrypancounting,cells MsucroseinHEPES/NaOHbuffer(pH7.4).Astepgradientof
were plated in 25-cm2 flasks at a density of 900 cells/mm2 in sucroseat2.25,2.00,1.75,1.5,1.25,1.00,0.75,0.5,and0.25M
poly-D-lysine-andlaminin-coatedplates.Fordissociation,plat- waslayeredwith0.25Mexosome-containingsolution.Thegra-
ing, and maintenance, we used Neurobasal medium supple- dientwasspunat200,000(cid:2)gfor18husinganSW41Tirotor
mentedwith2%B27containing200mMglutamineand1%anti- (BeckmanCoulter).Fractionsof1mlweredilutedwith3mlof
biotic/antimycotic agent (Invitrogen). The medium was HEPES/NaOHbuffer(pH7.4)andspunat200,000(cid:2)gfor1h
replenishedevery3daysand24hbeforecelltreatment. usinganSW60Tirotor.PelletswereusedforWesternblotting
Drug Treatments—N-[N-(3,5-Difluorophenacetyl)-L-ala- orelectronmicroscopy.
nyl)]-3-(S)-amino-1-methyl-5-phenyl-1,3-dihydrobenzo[e]- Immunofluorescence—SY5Y cells expressing wild-type APP
(1,4)-diazepin-2-one (compound E, (cid:3)-secretase inhibitor (APPWT;referredtoasSY5Y-APPWTcells)weredifferentiated
XVIII;usedatafinalconcentrationof30nM)andbafilomycin onpoly-L-lysine-coatedglasscoverslips.Afterdrugtreatment,
A1(BafA1;usedatafinalconcentrationof100nM)werepur- cellswerefixedin0.1MPBScontaining4%paraformaldehyde
chasedfromCalbiochem(Fontenay-sous-Bois,France).Chlo- for30minatroomtemperatureandfurtherpermeabilizedwith
roquine(10(cid:5)M)waspurchasedfromSigma.Cellswereplated 0.25% (v/v) Triton X-100 in PBS. After blocking in 1% (w/v)
in6-wellplates24hbeforedrugexposure.Cellswerewashed bovineserumalbumin,thefixedmaterialswereincubatedfor
with warmed phosphate-buffered saline (PBS) and scraped 2 h at room temperature with the primary antibody in PBS
with a policeman rubber in 70 (cid:5)l of Laemmli sample buffer containing1%(w/v)bovineserumalbuminand0.25%Triton
containingproteaseinhibitors(Completemini,RocheApplied X-100. After washing, the secondary antibody (Alexa Fluor
18198 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER25•JUNE22,2007
APP-CTFsAreSecretedinExosomeVesicles
488-conjugatedanti-rabbitIgGorAlexaFluor568-conjugated
anti-mouseIgG;MolecularProbes)wasused.Coverslipswere
mountedonslideswithVECTASHIELD(VectorLaboratories).
ImageswereacquiredwithaLeicaTCSNTlaserscanningcon-
focalmicroscope.
For APP internalization analysis, cells were incubated with
the8E5antibodyfor1hat4°Cinice-coldDulbecco’smodified
Eagle’s medium supplemented with 1% (w/v) bovine serum
albuminandthenwashedandincubatedat37°Cforthetimes
indicated.Cellswerefixedasdescribedabove,permeabilizedby
incubation in 0.3% (v/v) Triton X-100 in PBS, and incubated
withAlexaFluor568-conjugatedanti-mouseIgGantibody.
Electron Microscopy and Gold Immunolabeling—For con-
ventionalobservations,differentiatedSY5Ycellspretreatedor
notwith10%fetalcalfserumfor2minwerefixedin2%glutar-
aldehydein0.1MPBS(pH7.2)for10min.Afterwashing,they
werepost-fixedwith1%osmicacidinthesamebuffer.Thecells
werethenembeddedinAralditeafterdehydrationinethanol. FIGURE1.APPlocalizesinlargevesicularstructuresinSY5Y-APPWTcells
Sections were collected on Parlodion-coated nickel grids and treatedwithchloroquineandBafA1.Shownaretheresultsfromtheanaly-
sisofAPPlocalizationinSY5Y-APPWTcellstreatedornot(control(Ctrl))with
counterstained with uranyl acetate and lead citrate before
chloroquine(Chloro)orBafA .Cellswereprocessedforimmunofluorescence
1
observationonaZeiss9025electronmicroscope. andlabeledwiththeAPPCter-C17and8E5antibodies.Notetheintensestain-
For immunoelectron microscopy observations, the same ingofAPPinlargevesicularstructuresinchloroquine-andBafA1-treated
cells.
cells(treatedornot)werefixedin0.2%glutaraldehydeand4%
paraformaldehydeinPBSfor15minanddirectlyembeddedin kineticsofendocytosisof8E5-APPcomplexeswasfollowedby
LR White after dehydration. Polymerization occurred under indirect immunofluorescence (Fig. 2A). Following incubation
UVlightfor48h.Sectionswerecollectedoncoatednickelgrids ofthe8E5antibodyat4°CinbothcontrolandBafA -treated
1
andtreatedforgoldimmunolabeling.Briefly,thesectionswere cells,weobservedthatthemembranewasdelineatedbyimmu-
successivelyincubatedfor30mininbufferA(0.1MTris,0.15M nocomplexes,demonstratingthatendocytosiswasrepressedat
NaCl,1%bovineserumalbumin,and4%goatserum),antibody lowtemperatures.After5minat37°C,APPwasrapidlyinter-
dilutedinbufferA(for24h),andcolloidalgold(18nm)-labeled nalizedinsmallvesiclesunderbothconditions.Noteworthy,in
goatanti-rabbitIginbufferA(1.5h).Afterwashing,thesec- controlcells,theplasmamembranewasnotvisualized,whereas
tions were counterstained in 4% uranyl acetate in H O. For in BafA -treated cells, the plasma membrane was detected.
2 1
observationofisolatedexosomes,400meshcarbongridswere Althoughindirectimmunofluorescenceisnotquantitative,we
putfor5minonadropofexosomepelletdiluted1:50inPBS. observedthatAPPstainingstronglydisappearedundercontrol
Afterblotting,thegridsweresuccessivelyputinfixativesolu- conditions after 60 min at 37°C. In contrast, APP staining
tion (0.2% glutaraldehyde and 2% paraformaldehyde in PBS), remainedintenseinvesicularstructuresinBafA -treatedcells,
1
glycinesolution(0.2MinPBS),APPCter-C17antiserum(12h), andthesignalattheplasmamembranewasdiminished.BafA
1
andcolloidalgold-labeledgoatanti-rabbitIg(1.5h).Counter- induced the accumulation of the 8E5 antibody in vesicles
stainingwasperformedinuranylacetate. resemblingthoseobservedinFig.1,suggestingthatthesevesi-
cles belong to the endosomal/lysosomal pathway. To further
RESULTS
characterizethesevesicles,weusedantibodiesagainstmarkers
Accumulation of APP in Late Endosomes after Alkalizing oftheearlyandlateendosomalcompartments(Fig.2B).Incon-
DrugTreatments—Alkalizingdrugsinduceanaccumulationof trol and BafA -treated cells, antibody APPCter-C17 labeling
1
APP-CTFs and AICD as determined by biochemical analyses partially overlapped EEA1 early endosome marker labeling.
(14). Herein, we investigated the cellular compartments in However,EEA1stainingdidnotcorrespondtolargeengulfed
whichthisaccumulationmayarise.Weinvestigatedthecellular vesicles.Tsg101isamarkeroflateendosomalcompartments
localization of APP in chloroquine- and BafA -treated SY5Y- and particularly of multivesicular endosomes (46). In control
1
APPWTcellsbyimmunofluorescenceusing8E5andAPPCter- SY5Y-APPWT cells, few vesicles were stained by both anti-
C17antisera(Fig.1).Afterdrugtreatment,APPaccumulatedin Tsg101 and APPCter-C17 antibodies. In sharp contrast, all
numerousengulfedvesicles(Fig.1).Theselargeengulfedvesi- large vesicles were stained by anti-Tsg101 and APPCter-C17
cles were not seen under control conditions and could result antibodies in BafA -treated cells. We showed previously that
1
fromdefectiveendocytosisorroutingofAPP. theselargevesiclesarealsopartiallypositivefortheLAMP-2
BafA hasbeendemonstratedtoreduceslightlytherateof lysosomemarker(14).WesuggestthatBafA treatmentresults
1 1
internalization, recycling, and intracellular sorting of trans- intheaccumulationofAPPanditsderivativesinlateendoso-
membraneproteinandtocompletelyblockthetransportfrom malcompartments.
lateendosomestolysosomes(44,45).Weassessedtheinternal- APPandItsDerivativesAreFoundinMVBsinBafA -treated
1
izationofAPPincontrolandBafA -treatedcells.SY5Y-APPWT Cells—Wenextobservedcellularorganellesattheultrastruc-
1
cells were incubated with the 8E5 antibody at 4°C, and the turallevelincontrolandBafA -treatedcells.Numerouslarge
1
JUNE22,2007•VOLUME282•NUMBER25 JOURNALOFBIOLOGICALCHEMISTRY 18199
APP-CTFsAreSecretedinExosomeVesicles
We first investigated the pres-
enceorabsenceofexosomevesicles
in the medium of SY5Y-APPWT
cells (Fig. 4). SY5Y-APPWT cells
were differentiated with retinoic
acid for 15 days in a medium that
did not contain fetal calf serum,
whichisasourceofexogenousexo-
somes (43). To ascertain the pres-
enceofexosome-likevesiclesindif-
ferentiated SY5Y-APPWT cells, the
medium was collected after 24, 48,
or72hofcultureandprocessedas
described for the isolation of
secreted vesicles (43). Flotillin and
HSP70, which are often found in
exosomevesicles,wereusedasexo-
somemarkers(36,43,47).Wealso
usedelectronmicroscopytodeter-
mine the morphology of the
secretedvesicles.Followingtwolow
speed centrifugations, filtration,
and ultracentrifugation, the pellet
wasanalyzedforthepresenceofflo-
tillinandHSP70.Weobservedboth
flotillin and HSP70 by Western
blotting (Fig. 4B). Electron micros-
copy analysis of the same pellet
revealed the presence of numerous
membranevesicleswithameansize
of 50–90 nm and a typical “cup
shape”(Fig.4B),hencecorrespond-
ingtothemorphologicalcharacter-
istics of exosomes (35). We
excluded the possibility that the
FIGURE2.APPisinternalizedinthepresenceofalkalizingdrugsandcolocalizeswithlateendosome releaseofsuchvesiclesisacharac-
markers.A,thekineticsofAPPendocytosiswasindirectlyvisualizedbyinternalizationofthe8E5antibody.
teristicofourcellmodel,assecreted
CellswerepretreatedornotwithBafA for24h,placedat4°Cinacoldroom,andincubatedwiththe8E5
antibodyfor1h.Thetemperaturewasth1enshiftedto37°C,andcellswereprocessedforimmunofluorescence exosome vesicles were isolated in
at0,5,15,30,and60mintoallowAPPinternalization.B,untreatedSY5Y-APPWTcells(control(Ctrl))orcells mock SY5Y cells as well as in pri-
treatedwithBafA wereprocessedforimmunofluorescenceandlabeledwiththeAPPCter-C17,anti-EEA1,or
1 mary neuronal culture cells (Fig. 4,
anti-Tsg101antibody.
CandD).
To further demonstrate that
vesiclescontainingsmallerintraluminalvesicleswereobserved thesevesiclescorrespondtoexosomes,weanalyzedtheprotein
in BafA -treated SY5Y-APPWT cells, but not in control cells ina100,000(cid:2)gpellet.SY5Y-APPWTcellsweredifferentiated
1
(Fig.3AandsupplementalFig.1,AandD).Immunogoldlabel- with retinoic acid for 15 days, and the medium was collected
ingwithourAPPCter-C17antibodyshowedthatfewgoldpar- fromthelast72h.Exosomevesicleswereisolatedfollowingthe
ticleswerelocalizedinvesicularstructuresandMVBsincon- establishedprocedure,andthefinalpelletwashomogenizedin
trolcells(supplementalFig.1C).SY5Y-APPWTcellswerenot SDS lysis buffer. Proteins were resolved by SDS-PAGE and
stainedbythesecondaryantibodyalone(supplementalFig.1,A stained with Coomassie Blue (Fig. 5A). The identities of the
and D). In BafA -treated cells, intraluminal vesicles were proteinsweredeterminedbypeptidemappingafterin-geltryp-
1
stainedbyourAPPCter-C17antibody.Wehypothesizedthat sindigestionandmassspectrometryanalyses(42).Alowparts/
theselargevesiclescorrespondtoengulfedMVBs. millionerror(inferiorto30ppm)wasappliedtomatchapro-
MVBscanfusewiththeplasmamembranetoreleasetheir tein with a minimum of four matching peptides and a high
luminalcontent,includingthesmallvesiclestermedexosomes probability score (Fig. 5A, table). The proteome of exosomes
(33,36).Thelatteroriginatefromthebuddingoftheendosomal isolated from multiple biological sources has been described
membranetoformintraluminalvesicles.IfMVBsarethecell (43,48,49).Theproteinsthatwereidentifiedherein,suchasthe
compartments in which APP metabolites accumulate, then clathrin heavy chain, translation initiation factor-3, HSP90,
theseproductsshouldbeassociatedwithexosomes. pyruvatekinase,tubulin,actin,elongationfactor-1(cid:3),andRas-
18200 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER25•JUNE22,2007
APP-CTFsAreSecretedinExosomeVesicles
FIGURE3.MVBlocalizationofAPPinSY5Y-APPWTcells.A,ultrastructural
analysisofBafA -treatedSY5Y-APPWTcells;B,immunogoldlabelingofAPPin
1
SY5Y-APPWTcellstreatedwithBafA .18-nmgoldparticleswerelocalizedin
1
thesmallintraluminalvesiclesofalargervesicle.Notethattheshapeofthese
vesiclesisverysimilartothatofmultivesicularstructuresobservedinthe
samecellsunderthesameconditionsbyultrastructuralelectronmicroscopy
(A).Nu,nucleus.Scalebars(cid:4)1(cid:5)m.
relatednuclearprotein,werecharacterizedpreviouslyinexo-
somes(43,48,49).Ourdataindicatethattheisolatedvesicles
are SY5Y-derived exosomes. These exosomes are most likely FIGURE4.Secretionofexosome-likevesiclesbySY5Y-APPWTcells.SY5Y-
notcontaminatedbyproteinsofotherunrelatedorganelles,as APPWTcellsweredifferentiatedfor15dayswithretinoicacid,andthemedium
the vast majority of the proteins we have characterized were wascollectedafterthelast24,48or72h.Shownisasummaryoftheprotocol
usedtoisolateexosomesfromthecellmedia(A).The100,000(cid:2)gpellet
shownpreviously(43,48,49)inexosomesfromothersources. (exosomes)obtainedafterdifferentialcentrifugationfromthecrudecontrol
Moreover, we performed staining with anti-EEA1 antibody. (Ctrl)medium(B,firstlane)orfromthesupernatantafter24,48,and72hofcell
EEA1wasdetectedintheSY5Y-APPWTlysate,butnotinexo- culture(secondthroughfourthlanes)andthecelllysate(fifthlane)wereana-
lyzed by Western blotting with anti-flotillin and anti-HSP70 antibodies.
somesobtainedfrom10mlofculturemedium(Fig.5B). Shownaretheresultsfrommorphologicalanalysisbyelectronmicroscopyof
APP,APP-CTFs,andAICDAreSecretedinExosomeVesicles— the100,000(cid:2)gpelletobtainedfromthe72-hcellculturesupernatant.The
pellet was composed of 30–100-nm vesicles. Scale bar (cid:4) 100 nm. The
WenextaddressedthequestionofthepresenceofAPPmeta- 100,000(cid:2)gpelletobtainedafterdifferentialcentrifugationfromthecrude
bolicproductsinSY5Y-APPWTexosomes(Fig.6A).Immuno- medium(CandD,firstlanes)orfromthesupernatantafter24,48,and72hof
SY5Ycellculture(C,secondthroughfourthlanes)orafter72hofprimary
goldlabelingwithourAPPCter-C17antibodywasperformed
neuronalcellculture(D,secondlane)wasanalyzedbyWesternblottingwith
to localize APP metabolic products. Numerous vesicles of anti-flotillinandanti-HSP70antibodies.Shownaretheresultsfromelectron
50–90nmwerestainedwithgoldparticles18nmindiameter microscopyofthe100,000(cid:2)gpelletobtainedfromthe72-hcellculture
mediumofdifferentiatedSY5Ycells(C)orratprimaryneuronalculturecells
(Fig.6A).Thegoldparticleslocalizedinsidethevesicles.Using (D).Scalebars(cid:4)100nm.Notethecharacteristiccupshapeofthe50–90-nm
the same antibody, we next characterized the APP metabolic vesicles(insetsinB–D).
productsbyWesternblotting(Fig.6A).Thisanalysiswasper-
formed on exosome vesicles obtained from differentiated densitygradient.Aftercentrifugationat200,000(cid:2)gfor18h,
SY5Y-APPWT cells maintained in the culture medium for 24, 1-mlfractionswerecollectedandanalyzedbyWesternblotting
48,or72h.AnincreaseinHSP70stainingwasobserved(Fig. forthepresenceofEEA1,APP,APP-CTFs,AICD,HSP70,and
6A),suggestingthatexosomesaccumulatedinthecellmedium flotillin(Fig.6B).Exosomeswerefoundprincipallybetween1.0
withtime.TheAPPCter-C17antibodystainedfull-lengthAPP, and 1.25 M sucrose, corresponding to a density of 1.13–1.16
APP-CTFs,andAICD(Fig.6A).Whereasfull-lengthAPPstain- g/ml, as described previously (50, 51). APP, APP-CTFs, and
ing only slightly increased under the three conditions, APP- AICD were strongly detected in the 1.0–1.25 M fractions;
CTF staining strongly increased at 48 and 72 h. AICD was HSP70 and flotillin were strongly detected as well (Fig. 6B).
observedmainlyafter72h.TofurtherdemonstratethatAPP, Moreover, we verified the presence of exosomes in the 1.0 M
APP-CTFs, and AICD are associated with exosomes, the sucrosefractionbyelectronmicroscopy(Fig.6B)andshowed
100,000(cid:2)gpelletwasloadedonthetopofa0.25–2.5Msucrose alsothatEEA1wasnotdetectedinthisfraction,butwasslightly
JUNE22,2007•VOLUME282•NUMBER25 JOURNALOFBIOLOGICALCHEMISTRY 18201
APP-CTFsAreSecretedinExosomeVesicles
In the presence of the (cid:3)-secretase
inhibitor compound E, AICD was
detected neither in the cell lysate
nor in the exosomes. Conversely,
the amount of APP-CTFs was
stronglyincreasedinSY5Y-APPWT
cells and exosomes (Fig. 7B), dem-
onstratingthatAICDinexosomesis
producedby(cid:3)-secretasecleavageof
APP-CTFs. Moreover, we showed
that, in chloroquine-treated cells,
AICD was increased in the SY5Y-
APPWTlysateandinexosomes,sug-
gesting further that chloroquine
mightrepressAICDdegradationin
MVBs.Altogether,ourdatademon-
strate that APP, APP-CTFs, and
AICDarefoundinintraluminalves-
iclesofMVBs,wheretheyarelikely
degraded,asareothertypeItrans-
membraneproteins.
DISCUSSION
Inthisstudy,wehaveshownthat
differentiated SY5Y cells stably
expressing APP or not produced
andsecretedsmallvesiclesof50–90
nm in diameter, consistent with
morphological and biochemical
properties of exosomes. We have
also shown that proteins often
foundinexosomes,e.g.HSP70and
flotillin(36),weredetectedinthese
small vesicles secreted by SY5Y
cells. In contrast, a marker of early
endosomes (EEA1) was not found
associated with exosomes. Further
similarities were deduced from
characterization of the proteome
SY5Y-derivedvesicles.Fiftypercent
FIGURE5.ProteomiccharacterizationofexosomessecretedbySY5Y-APPWTcells.A,SDS-PAGEofthe
proteinsofexosomesobtainedfromthe72-hcellculturemediumandstainedwithCoomassieBrilliantBlue of the proteins characterized were
G-250.Thefirstlanecorrespondstothemolecularmassmarkers.CoomassieBlue-stainedbandswereexcised identified previously in exosomes
fromthegel,trypsinized,andanalyzedbymassspectrometryforpeptidemassfingerprints.Thenamesand
from other biological sources (43,
accessionnumberscorrespondtothoseprovidedbytheSwiss-ProtProteinKnowledgebase(ca.expasy.org/
sprot/).Thetheoreticalmass,numberofpeptidesmatched,andpercentcoverageareindicated.B,absenceof 48). Similar vesicles were also iso-
EEA1inexosomes.Exosomeswerepreparedfrom10mlofSY5Y-APPWTcellsdifferentiatedfor15daysaccord- latedfromprimaryneuronalculture
ingtotheprotocoldescribedinthelegendtoFig.4.Proteinsfromexosomesandfromthecelllysate(SY5Y-
APPWT)wereloadedontoSDS-polyacrylamidegelandWestern-blottedwithanti-EEA1,anti-HSP70,andanti- cells, demonstrating that the pro-
flotillinantibodies.NotethatEEA1wasdetectedonlyinSY5Y-APPWTcelllysate. ductionofexosomesisnotaspeci-
ficity of SY5Y neuroblastoma cells,
butispossiblyaphysiologicalproc-
detected in fractions below 2.0 M sucrose when the blot was ess of neuronal cells (51). More important, our data demon-
overexposed(Fig.6B).OurdatashowthatSY5Y-APPWTcells strate that APP derivatives, including full-length APP, APP-
producedexosomesinwhichAPPandAPPmetaboliteswere CTFs, and more surprisingly, AICD, are found in exosomes
co-sedimentedwithexosomemarkers.Finally,todemonstrate secretedbydifferentiatedSY5Ycells.TheseAPPfragmentsare
that the AICD we observed in exosomes was released by alsoco-sedimentedwithmarkersofexosomesatasucroseden-
(cid:3)-secretase and not by an unrelated proteolytic cleavage, we sitycorrespondingtothatdescribedforexosomes(50,51).The
used a specific (cid:3)-secretase inhibitor. SY5Y-APPWT cells were presenceofsolublefull-lengthAPPinthemediumwasdemon-
treatedfor24hwitheithercompoundEorchloroquine,and stratedpreviouslyincellstransfectedwithAPP,butremained
weanalyzedtheexosomecontentaftertreatment(Fig.7A). unexplained(52,53).SecretionofAPPwithinexosomesiscon-
18202 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER25•JUNE22,2007
APP-CTFsAreSecretedinExosomeVesicles
useofa(cid:3)-secretaseinhibitorabro-
gated the release AICD. Moreover,
AICDwasnotproducedduringthe
purification of exosomes. First, we
failed to detect presenilin in exo-
somes;andsecond,whenexosomes
were incubated at 37°C for 24 h,
APP-CTFswerenotprocessedfur-
ther to produce AICD or other
catabolites(datanotshown).How-
ever, our negative findings do not
excludethepossibilitythat(cid:3)-secre-
tase cleavage occurs prior to exo-
somesecretion.
Exosomesderivefromtheinward
buddingofthemembraneofendo-
somes en route for multivesicular
endosomes (54). Several proteins
are markers of this cellular com-
partment such as flotillin, HSP70,
and Tsg101 (36, 43). Interestingly,
flotillinwasshownrecentlytoasso-
ciate with AICD (55). We have
shownherethatAPPanditsderiva-
tives were found in exosomes and
multivesicular endosomes and
accumulated in engulfed vesicles
followingtreatmentswithalkalizing
drugs. Together with our recent
data,wesuggestthatAPPandAPP-
CTFs that are not processed by
(cid:3)-secretasearedirectedtothislate
endosomalcompartmentfordegra-
dationbyfusionofMVBswithlyso-
somes.A(cid:1)andAICD,whichresult
from the (cid:3)-secretase cleavage of
APP-CTFs,arealsodirectedtomul-
tivesicularendosomesandareinte-
grated in the luminal vesicles,
namely exosomes. The presence of
AICD in exosomes is consistent
with (cid:3)-secretase cleavage of APP-
CTFs that would occur during the
FIGURE6.APP,APP-CTFs,andAICDaresecretedandco-sedimentedwithexosomemarkers.A,shownare budding of the late endosomal
theresultsfromelectronmicroscopyofthe100,000(cid:2)gpelletobtainedfrom2mlofthe72-hcellculture membrane.However,thecontribu-
mediumimmunogold-labeledwiththeAPPCter-C17antibody.Scalebars(cid:4)0.1(cid:5)m.Exosomeswerealsoana-
tion of flotillin to this mechanism
lyzedbyWesternblottingwithanti-flotillin,anti-HSP70,andanti-APPCter-C17antibodies.Anequalvolume(3
ml)ofthenativeculturemedium(control(Ctrl))wasprocessedforexosomeisolationandloadedinthefirst remainstobeestablished.Theproc-
lane.Thelastlanecorrespondstothecelllysate.Tenmicrogramsofproteinwasloaded.B,exosomeswere
essofAPPfollowingendocytosisis
prepared from 65 ml of medium obtained after 72 h of culture of differentiated SY5Y-APPWT cells. The
100,000(cid:2)gpelletwasrecoveredin0.25Msucroseandlayeredoverasucrosedensitygradient.Tenfractions illustratedinFig.8.
of1mlwererecoveredaftercentrifugationat200,000(cid:2)gfor18handconcentrated.Theconcentrationsofthe Recentdatasuggestthatmultive-
sucrosefractionsareindicated.ThewholeproteinswereloadedontoSDS-polyacrylamidegel.Full-lengthAPP,
sicular endosomes are important
APP-CTFs,AICD,EEA1,HSP70,andflotillinweredetected.EEA1stainingwasperformedafterAPPlabeling.The
residualstainingofAPPisindicatedbytheasteriskbecauseaverylongexposuretimewasnecessaryto cellular compartments in neurode-
visualizeEEA1staining.AICDwasobservedafteralongerexposure(10min(mn))andisindicatedbythearrow. generativedisorders.Exosomesthat
Electronmicroscopywasperformedtoverifythepresenceofexosomesinthe1Msucrosefraction.Scalebar(cid:4)
100nm. are produced in these compart-
ments contain prion protein and
sistent with these observations and could be an explanation. have been suggested to mediate the diffusion of the scrapie
WehavealsodemonstratedthatAICDfoundinexosomeswas formoftheprotein(56).Morerecently,A(cid:1)hasbeenshownto
producedbycleavageofAPP-CTFsby(cid:3)-secretasebecausethe be a component of the exosomal membrane (37). Protease
JUNE22,2007•VOLUME282•NUMBER25 JOURNALOFBIOLOGICALCHEMISTRY 18203
APP-CTFsAreSecretedinExosomeVesicles
somes,buttheirpotentialrolesremainunclear(51).Herein,we
haveshownthatseveralmetabolitesofAPP,includingAICD,
arefoundinexosomes.
MVBsandexosomesareofgrowinginterestinneurodegen-
erativediseases.Wedonotknowwhetherthe“alkalizing”con-
ditionsusedhereinreflectanAlzheimerdisease-relatedsitua-
tion,althoughwehaveshownthatalkalizingdrugsinducedan
intracellular accumulation of APP catabolites. However, an
alteredroutingofAPPtolateendosomalcompartmentscould
be associated with an increase risk of developing Alzheimer
disease.ArecentworkbyRogaevaetal.(58)demonstratesthat
genetic polymorphisms of SORL1 are at risk for late onset
Alzheimer disease. SORL1 acts as a sorting receptor for full-
lengthAPPandfavorsitsrecycling.AlossoffunctionofSORL1
resultsinincreasedproductionofA(cid:1)inlateendosomalcom-
FIGURE7.Inhibitionof(cid:1)-secretaseortreatmentwithchloroquineabol- partments. Further work will be needed to study markers of
ishestheproductionorenhancesthequantityofAICDinexosomes,
respectively.A,SY5Y-APPWTcellsweredifferentiatedfor14daysandtreated exosomesinAlzheimerdiseaseandtherelationship,ifany,toa
thelast24hwithcompoundE(CompE)orchloroquine(Chloro);themedium potentialdefectoflateendosomalcompartmentssuchMVBs.
(3ml/condition)wascollected;andexosomeswereisolated.APP,APP-CTFs, Thereleaseofexosomesismostlikelyregulatedasdemon-
AICD,flotillin,andHSP70weredetectedbyWesternblottinginexosomesand
celllysates.Molecularmassesareindicatedtotheright.Ctrl,control.B,the stratedbyFaureetal.(51);thus,depolarizationstimulatesthe
ratioofAPP-CTFsdetectedinexosomestothosedetectedincellsisexpressed exosomes released. The release of full-length APP was also
inarbitraryunits.
demonstrated to be stimulated by cholinergic agonists (52).
Thesedata,togetherwiththedatafromthisstudy,showthat
APP and its catabolites can be released associated with exo-
somes and that this process is likely modulated by several
parameters.However,furtherworkwillbeneededtoinvesti-
gatethesehypotheses.
Inconclusion,wehavedemonstratedanovelsecretionpath-
wayofAPPmetabolites(includingAPP,APP-CTFs,andAICD)
thatismediatedbyexosomevesicles.Ourworkprovidesnew
researchperspectivestowardtheunderstandingofAPPbiolog-
ical function. A more complete characterization of the pro-
teomeofneuron-derivedexosomescouldalsobehelpfulindis-
coveringnewbiomarkersusefulfordiagnosticpurposesandin
developingtherapeuticdrugsforAlzheimerdisease.
Acknowledgment—The 8E5 antibody was kindly provided by Dr.
PeterSeubert.
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