The FASEB Journal • Research Communication

Inhibition of �-secretase causes increased secretion ofamyloid precursor protein C-terminal fragments inassociation with exosomes

Robyn A. Sharples*,†,1, Laura J. Vella,*,†,‡,§,1 Rebecca M. Nisbet,*,†,‡,§

Ryan Naylor,*,†,‡,§ Keyla Perez,†,‡,§ Kevin J. Barnham,†,‡,§ Colin L. Masters,‡,§

and Andrew F. Hill*,†,‡,§,2

*Department of Biochemistry and Molecular Biology, †Bio21 Molecular Science and BiotechnologyInstitute, and ‡Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia,and §The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia

ABSTRACT Alzheimer’s disease (AD) is the mostcommon form of dementia and is associated with thedeposition of the 39- to 43-amino acid �-amyloid pep-tide (A�) in the brain. C-terminal fragments (CTFs) ofamyloid precursor protein (APP) can accumulate inendosomally derived multivesicular bodies (MVBs).These intracellular structures contain intraluminal ves-icles that are released from the cell as exosomes whenthe MVB fuses with the plasma membrane. Here wehave investigated the role of exosomes in the process-ing of APP and show that these vesicles contain APP-CTFs, as well as A�. In addition, inhibition of �-secre-tase results in a significant increase in the amount of �-and �-secretase cleavage, further increasing the amountof APP-CTFs contained within these exosomes. Weidentify several key members of the secretase family ofproteases (BACE, PS1, PS2, and ADAM10) to be local-ized in exosomes, suggesting they may be a previouslyunidentified site of APP cleavage. These results pro-vide further evidence for a novel pathway in which APPfragments are released from cells and have implica-tions for the analysis of APP processing and diagnosticsfor Alzheimer’s disease.—Sharples, R. A., Vella, L. J.,Nisbet, R. M., Naylor, R., Perez, K., Barnham, K. J.,Masters, C. L., Hill, A. F. Inhibition of �-secretasecauses increased secretion of amyloid precursor pro-tein C-terminal fragments in association with exosomes.FASEB J. 22, 1469–1478 (2008)

Key Words: Alzheimer’s disease � protein processing � APP� secretases

The main component of Alzheimer’s disease (AD)amyloid plaques is an aggregated form of the �-amyloidpeptide (A�), a 39- to 43-amino acid peptide producedby proteolytic cleavage of the amyloid precursor pro-tein (APP) (1–7). APP is an integral membrane proteinwith a single membrane-spanning domain, a largeextracellular amino terminus and a short cytoplasmiccarboxyl terminus (8, 9). Mature APP molecules un-dergo proteolytic cleavage by at least three proteasestermed �-, �-, and �-secretases, which lead to thegeneration of a number of proteolytic fragments, in-

cluding A� when APP is cleaved by �- and �-secretases(10, 11). The identity of �-secretase remains unclearalthough the present candidates are three members ofthe ADAM family: ADAM9, ADAM10, and tumor necro-sis-� converting enzyme (TACE)/ADAM17 (12–15).�-Secretase has been identified as �-site APP-cleavingenzyme (BACE) 1, a novel type 1 transmembraneaspartyl protease (16–20), and �-secretase has beenidentified as a multimeric protein complex containingeither presenilin (PS) 1 or PS2 associated with nicas-trin, Aph-1, and Pen-2 (21–25).

The amyloidogenic pathway of APP processing in-volves sequential cleavage by �- and �-secretases.�-Secretase cleaves at the amino terminus of A� (26,27), resulting in the release of secreted APP (sAPP�)and leaves intact A� in a membrane-associated, 99-amino acid carboxyl-terminal fragment, �-CTF. �-CTFcan undergo endocytosis via clathrin-coated vesicles(28) and is trafficked to various endosomal compart-ments, including multivesicular bodies (MVBs) (29).The �-CTF is cleaved by �-secretase at the carboxyl-terminus of A� (1–3), predominantly at positions 40and 42, resulting in the release of intact A� peptidesand the 50-amino acid APP intracellular domain(AICD) (30–33).

A� is associated with small membrane vesicles knownas exosomes (34), which are secreted into the culturemedium by many cell types, suggesting that APP pro-cessing may occur through this specialized pathway.Exosomes are small membrane vesicles (50–100 nm indiameter) that correspond to internal vesicles formedby invagination of the membrane of MVBs (35). Exo-some secretion into the extracellular environment oc-curs on fusion of MVBs with the cell membrane. Thephysiological relevance of exosomes has been estab-lished by the identification of exosomes in vivo, in

1 These authors contributed equally to this work.2 Correspondence: Department of Biochemistry and Molec-

ular Biology, Bio21 Molecular Science and BiotechnologyInstitute, The University of Melbourne, Parkville Victoria3010, Australia. E-mail: a.hill@unimelb.edu.au

doi: 10.1096/fj.07-9357com

14690892-6638/08/0022-1469 © FASEB

association with follicular dendritic cells (36), urine(37), and malignant tumor effusions (38). The func-tion of exosomes appears to be much more importantthan the simple removal of unwanted cellular proteins,and a role for exosomes in mediating intercellularcommunication has been identified (39–41).

As �-secretase activity is essential for the release ofintact A�, �-secretase inhibitors have been suggested inthe treatment of AD. These inhibitors have been shownto decrease A� production after administration totransgenic mice overexpressing human APP (42). Atthe cellular level, APP-CTFs have been shown to accu-mulate in endosomal compartments after �-secretaseinhibition (43). As exosomes derive from these endo-cytic compartments, we analyzed the effect of �-secre-tase inhibitors on processing and packaging of APPcleavage products in exosomes. Here we demonstratethat APP, A�, and other proteolytic fragments of APPformed during processing by �-, �-, and �-secretase areassociated with exosomes derived from cultured cellsexpressing wild-type human APP. On treatment of cellswith �-secretase inhibitors we observed a previouslyunreported effect, whereby �-cleavage of APP was in-creased, resulting in a significant increase in sAPP�into the extracellular environment. Analysis of exo-somes from cells treated with these inhibitors revealeda concomitant accumulation of carboxyl-terminal frag-ments of APP in exosomes as identified by 1) immuno-blotting using antibodies against C-terminal epitopes ofAPP and 2) surface enhanced laser desorption ioniza-tion-time of flight mass spectrometry (SELDI-TOF MS).To determine whether cleavage of APP could occurwithin exosomes we screened these vesicles for thepresence of secretase components and identified �-and �-secretase (ADAM10 and BACE, respectively) tobe localized in exosomes. In contrast, not all compo-nents of the �-secretase complex were detected, sug-gesting that this activity is a minor event. These dataprovide firm evidence for a novel pathway of APPprocessing in exosomes. The identification of proteo-lytic fragments of APP in association with exosomes hasimplications for the diagnostic and therapeutic assess-ment of APP processing in the pathogenesis of Alzhei-mer’s disease.

MATERIALS AND METHODS

Antibodies

Anti-APP antibody 22C11 was obtained from Millipore (Te-mecula, CA, USA); 369 (50-mer raised against residues 645–694 of the C terminus of APP 695 isoform) was a gift fromProfessor Sam Gandy (Thomas Jefferson University, Philadel-phia, PA, USA); WO2 (A� 5–8) has been described previously(44); APP C-terminal (CT) (raised against residues 751–770of APP 770 isoform) and BACE (46–65) antibodies werefrom Merck Biosciences (Darmstadt, Germany); anti-A� (res-idues 17–24) antibody 4G8 was from Covance (Princeton, NJ,USA); ADAM10, TACE, and Tsg101 antibodies were fromSanta Cruz Biotechnology, Inc. (Santa Cruz, CA, USA);flotillin-1, nucleoporin, GM130, and Bcl2 were obtained fromBD Biosciences (San Jose, CA, USA); anti-nicastrin was fromSigma-Aldrich (St. Louis, MO, USA), and the anti-PS1 and

-PS2 antibodies were a gift from Dr. Janetta Culvenor andhave been previously described (45).

Generation of the Chinese hamster ovary (CHO)-APP695cell line

CHO-APP695 cells were generated by expressing the 695-amino acid APP cDNA in the pIRESpuro2 expression vector(Clontech Laboratories Inc., Mountain View, CA, USA) asdescribed previously (46). Cells were transfected using Lipo-fectamine 2000 and cultured in RPMI 1640 medium supple-mented with 1 mM glutamine and 5% fetal bovine serum(Invitrogen, Mount Waverley, Victoria, Australia). Trans-fected cells were selected and maintained using 7.5 �g/mlpuromycin (Sigma-Aldrich).

Treatment of cells with �-secretase inhibitors

Stably transfected CHO-APP695 cells were diluted 1:10 inRPMI 1640 plus supplements and plated in six-well plates(Nalge Nunc International, Rochester, NY, USA) 2 h beforethe addition of the inhibitor; 1 �M N-[N-(3,5-difluorophen-acetyl)-l-alanyl]-S-phenylglycine-t-butyl ester (DAPT) (MerckBiosciences) or L685,458 (Merck Biosciences) dissolved indimethyl sulfoxide (DMSO) was added directly to the culturemedium of the cells. DMSO (0.1% v/v) was used as a negativecontrol. Cells were maintained at 37°C in a 5% CO2 atmo-sphere for 16 h after which the media were collected.Conditioned media posttreatment were centrifuged at 3000rpm (5415 D centrifuge; Eppendorf-5 Prime, Inc., Boulder,CO) to remove cell debris before analysis for sAPP and A�secretion by immunoblotting.

Immunoblotting

Samples were diluted in Tricine sample buffer (8% sodiumdodecyl sulfate, 30% glycerol, 100 mM Tris, 100 mM Tricine,and 0.01% phenol red) containing 10% 2-mercaptoethanoland boiled for 5 min at 100°C. Samples were electrophoresedusing 10–20% Tricine acrylamide gels (Invitrogen). Proteinswere transferred to a 0.2-�m nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and then blocked in5% nonfat milk powder in PBST (PBS�0.5% Tween-20) for1 h. Membranes were washed in PBST and incubated withprimary antibody overnight at room temperature. Mem-branes were again washed in PBST and probed with second-ary antibody, horseradish peroxidase-conjugated anti-mouseIgG (1:10,000) or anti-rabbit IgG (1:10,000) (AmershamBiosciences Corp., Piscataway, NJ, USA), for 1 h at roomtemperature. Specific binding was determined using ECLPlus (GE Healthcare, Chalfont St. Giles, UK). Blots undergo-ing quantification were scanned using a charge-coupled de-vice camera system (SciTech, Preston, VIC, Australia). Datawere analyzed using Gel-Pro Analyzer 4.5 software.

Isolation of exosomes from conditioned cell culture media

Exosomes of bovine origin were removed from the fetal calfserum before its use in cell culture media by overnight (16 h)ultracentrifugation at 100,000 g (45Ti rotor, BeckmanCoulter Inc., Fullerton, CA, USA). Cells (5�107) were cul-tured for 4–5 days in this bovine-exosome-depleted mediabefore exosome isolation by differential centrifugation asdescribed previously with minor modifications (47, 48).Briefly, cellular debris was removed by centrifugation at 2000g for 20 min, 0.22-�m filtration, and ultracentrifugation at100,000 g for 1 h at 4°C. Exosomes were pooled, washed in

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PBS, repelleted, and resuspended in 100 �l of Tricine samplebuffer or PBS, boiled at 100°C for 5 min, and either usedimmediately or stored at �20°C.

Sucrose density gradient centrifugation

Isolated exosomes were resuspended in 5 ml of 2.5 M sucroseand 20 mM HEPES (pH 7.2), and a 6-ml linear sucrosegradient (2.0–0.25 M sucrose and 20 mM HEPES, pH 7.2)was layered on top of the exosome suspension. The samplewas centrifuged at 70,000 g for 16 h at 4°C (SW41 rotor;Beckman Coulter Inc.). Gradient fractions of 1 ml werecollected from the top of the tube (11 fractions) and dilutedin 10 ml of PBS, and each fraction was ultracentrifuged for1 h at 200,000 g. The pellets were solubilized in Tricinesample buffer and immunoblotted as described above.

Electron microscopy

Exosomes were fixed by suspension in 100 �l of 2.5% (w/v)glutaraldehyde in PBS, and 5 �l was applied to a 200-meshcopper grid supported with formvar/carbon (ProSciTech,Kirwan, QLD, Australia) and left to dry at room temperature.The grids were washed, and exosomes were negatively stainedwith 3% saturated aqueous uranyl acetate and viewed with atransmission electron microscope (Siemens Elmiskop 102).

SELDI-TOF MS

Exosome fractions were analyzed using PS20 ProteinChiparrays (Ciphergen Biosystems, Fremont, CA, USA). The PS20arrays were first washed with 4 �l of PBS before antibodysolutions (2 �l, 0.25 mg/ml in PBS) were placed on the chipand incubated at 4°C overnight. The antibody array waswashed with 10 �l of deactivation buffer (0.5 M ethanolaminein PBS, pH 8.0; Ciphergen Biosystems) for 30 min, followedby 5-min washes with 5 �l of PBS � 0.5% Triton X-100 (wash

buffer) and then 5 �l of PBS. All washes and incubations wereperformed on a shaking table.

Exosome samples were resuspended in PBS and loadedonto the PS20 antibody array and incubated for 3 h at roomtemperature. The sample was then removed, and each arraywas washed twice with wash buffer, PBS, and 1 mM HEPES(pH 7.4). The arrays were air dried, and then 1 �l of�-cyano-4-hydroxycinnamic acid (50% saturated in 50% ace-tonitrile and 0.5% trifluoroacetic acid; Bio-Rad Laboratories)was applied to each spot twice. The arrays were air driedbefore analysis.

Arrays were analyzed using a PBSIIC SELDI-TOF MSProteinChip reader, and the data were analyzed with Protein-Chip Software 3.1 (Ciphergen Biosystems). A protein profilewas generated in which individual proteins were displayedwithin spectra as unique peaks based on their mass-to-chargeratio.

RESULTS

Conditioned media were harvested from CHO-APP695cells and assayed by immunoblotting to test the expres-sion levels of sAPP and A�. A stably expressing CHO-APP695 cell line was used whereby sAPP� and A� werereliably visualized by immunoblotting with monoclonalantibody WO2 (A� 5–8) without the need for immu-noprecipitation (Fig. 1A). Levels of A� were signifi-cantly reduced to undetectable levels when the cellswere treated with �-secretase inhibitors DAPT orL685,458 at 1.0 �M, suggesting that the CHO-APP695cells represent a model for studying wild-type APPprocessing (Fig. 1B).

We observed a significant increase in the amount ofsAPP� in the conditioned media of the CHO-APP695cells after inhibition with DAPT. This increase occurred

Figure 1. A) Western blot analysis of APP/A� expression in CHO-APP695 cells. Conditioned media from CHO cells transfectedwith vector only or APP695 were separated on 10–20% Tricine gel, and APP/A� expression was analyzed by immunoblotting withWO2. B) Treatment of CHO-APP695 cells with �-secretase inhibitors. CHO-APP695 cells were incubated in the presence (�) andabsence (�) of �-secretase inhibitors DAPT and L685,458 at 1.0 �M for 16 h. DMSO (0.1% v/v) was used as a negative control(�). Conditioned media from these cells posttreatment were separated on 10–20% Tricine gel and analyzed by immunoblottingwith WO2. C) Increase in sAPP after treatment with �-secretase inhibitor. CHO-APP695 cells were treated separately withincreasing concentrations of DAPT to a final concentration of 1 �M for 16 h. Treatment with DMSO was used as a negativecontrol. Posttreatment, conditioned media were separated on 8% SDS-PAGE and analyzed by immunoblotting with WO2;quantitation of the increase in sAPP� secretion is shown graphically.

1471APP PROCESSING IN EXOSOMES

in a dose-dependant manner, and the secretion ofsAPP� was greatest at 1 �M, the highest concentrationtested (Fig. 1C). Quantitation of sAPP� observed byimmunoblotting revealed a greater than 3-fold increasein sAPP� over the vehicle-only control when treatedwith 1 �M DAPT (Fig. 1C). Levels of A� were undetect-able by immunoblotting of conditioned cell mediatreated with concentrations as low as 0.02 �M DAPT(data not shown).

The increased secretion of sAPP� into the cell mediaafter treatment of cells with DAPT led us to examinethe �-secretase, “nonamyloidogenic” pathway of APPcleavage in more detail. The observed increase insAPP� in CHO-APP695 cells treated with DAPT suggestsan increase in the corresponding CTF arising from theinhibition of �-secretase. We reasoned that the plasmamembrane-localized CTF remaining after �-secretasecleavage of APP may be endocytosed and subsequentlyincorporated into membrane-bound vesicles. Low lev-els of A� have been reported to be associated withsmall, secreted membrane vesicles known as exosomesin cultured cell lines overexpressing mutant APP con-structs (34). As CHO cells have been reported tosecrete exosomes (50), we therefore investigatedwhether APP CTFs were associated with exosomes fromCHO-APP695 cells.

Culture media were removed from CHO-APP695 cellsafter 5 days and subjected to filtration and ultracentrif-ugation as described previously (47, 48, 51). The puri-fied extracellular particles from the conditioned mediawere examined by electron microscopy (EM). Negativestaining EM revealed that the conditioned media con-tained microvesicles that were membrane bound and“cup shaped” and had a size similar (50–80 nm diam-eter) to that of exosomes described previously (Fig. 2A)(47, 48). Little to no cell debris was visualized by EM,confirming the purity of the exosome preparation. Tofurther confirm the identity of these microvesicles asexosomes, continuous sucrose density gradient centrif-ugation was performed. Immunoblot analysis of gradi-ent fractions showed that CHO-APP695 microvesicles

possessed a buoyancy consistent with exosomes forpreviously reported cultured cell lines, including theCHO cell line (Fig. 2B) (47, 48, 52).

Exosomal protein composition is limited to a subsetof cellular proteins, as proteins of nuclear, mitochon-drial, endoplasmic reticulum, or Golgi apparatus originare absent. Exosomes are enriched in raft associatedproteins, membrane transport proteins, and adhesionmolecules (53). Equivalent amounts of CHO-APP695cell lysate or microvesicles were analyzed for proteincomposition with a panel of organelle marker antibod-ies (Fig. 2C). Flotillin is enriched in exosomes as is theprotein Tsg101 (35). Tsg101 is a component of theendosomal sorting complex, and flotillin is a constitu-ent of lipid subdomains in recycling endosomes. Anti-bodies against GM130 (Golgi), nucleoporin (nucleus),and Bcl2 (mitochondria) were also used to test formicrovesicle purity and to ensure the absence of celldebris contamination and apoptotic blebs (54). Thepresence of flotillin and Tsg101 and the absence ofGM130, nucleoporin, and Bcl2 in our microvesiclesfulfill the criteria for them to be described as exosomes(54).

Immunoblotting with a series of antibodies was usedto determine whether APP and/or A� was associatedwith exosomes. The antibodies WO2, 369, and APP CTall demonstrated a specific enrichment of APP frag-ments in the exosome fractions compared with condi-tioned media (Fig. 3). A small amount of A� was foundto be associated with exosomes in addition to a series ofAPP CTFs. Further analysis of the exosome preparationwith APP CT and 369 antibodies (both antibodiesraised against the C terminus of APP) revealed thatfull-length APP (fl-APP) is contained within these vesi-cles. To ensure that this fl-APP was actually an exosomalprotein rather than bound nonspecifically to the out-side of these vesicles, the exosome purification wasmodified to include an additional PBS wash and furthercentrifugation of the exosome pellet at 100,000 g.Minimal APP signal was detected in this additional PBSwash fraction, and the fl-APP signal was consistently

Figure 2. Characterization of exosomes. A) Transmission electron micrographs of CHO-APP695 exosomes; scale bars � 100 nm.B) A continuous 0.25–2.5 M sucrose gradient was loaded on top of CHO-APP695 exosome preparations and ultracentrifuged toequilibrium followed by immunoblotting with WO2. C) Whole cell lysates or exosomes from CHO-APP695 cells were separatedon 10–20% Tricine gel and analyzed by immunoblotting using antibodies to flotillin, Tsg101, GM130, nucleoporin, or Bcl2.

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enriched in the exosome preparation, confirming itstrue association with exosomes (data not shown).

After establishing that CHO-APP695 cells treated with�-secretase inhibitors completely reduced detectablelevels of A� in conditioned media with a concomitantincrease in sAPP�, we sought to assess whether theseinhibitors had any effect on other processing productsof APP that are associated with exosomes. Thus, cellswere incubated in the presence of 1 �M DAPT for aperiod of 5 days after which exosomes were isolatedfrom the culture media. Although we have shownDAPT to have an effect within 16 h, a longer incubationtime was required to isolate exosomes from the condi-tioned media. We observed no differences in cellgrowth, and previous studies (55) have reported DAPTto be nontoxic for incubation periods of 5 days. Usingantibodies to different regions of APP, we found thatAPP CTFs associated with exosomes and increased inthe presence of the �-secretase inhibitor compared withuntreated cells. In contrast, the majority of APP N-terminal fragments (NTFs) (identified with antibody22C11) were found in the conditioned media and notassociated with exosomes (Fig. 4).

SELDI-TOF MS was used to further characterizeCTFs detected via immunoblotting and to differentiatebetween �- and �-CTFs. Exosomes treated with DAPTdemonstrated an increase in peak size by SELDI-TOFMS at the predicted molecular weight of �-CTF (11,314Da) compared with untreated cells. An additional peakat 9717 Da also increased on treatment of cells and mayrepresent �-cleaved CTF (Fig. 5A). These results con-firm an increase in both �- and �-CTF in the presenceof the �-secretase inhibitor DAPT, and this effect wasobserved using two independent APP C-terminal anti-bodies.

To confirm the specificity of �-secretase inhibition,we treated cells with an alternative �-secretase inhibitor,L685,458. The same effect was observed—completeablation of A� production and an increase in both �-and �-CTFs in association with exosomes (Fig. 5B)—confirming the effect as a direct result from �-secretaseinhibition and not a nonspecific effect of the inhibitor.Although the SELDI-TOF MS data indicate that �-CTFpredominates over �-CTF (Fig. 5A), these resultsshould be interpreted as changes in the relative abun-dance of each fragment, not as a comparison between

fragments. Immunoblot analysis using WO2 and APPCT antibodies demonstrates that the dominant CTFband present in exosomes as detected by APP CTantibody is �-CTF, as a corresponding band of equiva-lent size is not detected by WO2 (Fig. 5B).

Because fl-APP, A�, and other processing products ofAPP were found to be associated with exosomes and�-secretase inhibitors demonstrated a specific effect onthe CTFs associated with exosomes, we surmised thatthese microvesicles may provide a possible site (albeit

Figure 3. APP and A� contained within exo-somes. Conditioned media from CHO cellstransfected with vector only or APP695 and exo-somes from CHO-APP695 cells were separated on10–20% Tricine gel followed by immunoblottingwith WO2, APP CT, and 369 antibodies.

Figure 4. Increase in CTFs in exosomes from �-secretase-treated cells. Exosomes were isolated from vector only, andCHO-APP695 cells in the presence (�) or absence (�) of�-secretase inhibitor DAPT at 1 �M. Conditioned media andexosomes were separated on 10–20% Tricine gel and immu-noblotted with 369 and 22C11. M � conditioned media; E �exosome preparation.

1473APP PROCESSING IN EXOSOMES

minor) for active processing of APP. BACE has beenshown to localize in endosomal compartments where itcan actively cleave APP (16, 34, 56), and exosomes havepreviously been shown to contain the metalloproteasesADAM10 and ADAM17 (49), which are both candidatesfor the �-secretase cleavage of APP. We thereforeexamined exosomes isolated from CHO-APP695 cellsfor the presence of secretase components. First, bycomparing cell lysate and exosome fractions, we wereable to detect BACE in exosomes from the CHO-APP695cells using two independent antibodies (Fig. 6). Immu-noblotting of exosomes derived from CHO-APP695 cellswith an ADAM10 antibody (Fig. 6) confirmed previousfindings that ADAM10 is present in exosomes (49).�-Secretase is a membrane protein complex comprisingthe presenilin proteins, nicastrin, Aph-1, and Pen-2.Our studies demonstrated that PS1 was abundant in theexosome preparation, whereas PS2 is present in minoramounts (Fig. 6).

DISCUSSION

The results from our studies demonstrate that in cellsexpressing wild-type APP (695-amino acid isoform), a

fraction of A� is secreted in association with exosomes.We provide evidence that APP CTFs are also foundassociated with exosomes, and on treatment of the cellswith �-secretase inhibitors we observe an increase inCTFs in addition to increased sAPP� in the condi-tioned media from CHO-APP695 cells (Fig. 7A). In theabsence of a reliable sAPP� specific antibody, it wasdifficult to determine the direct effect of �-secretaseinhibitors on �-secretase cleavage via immunoblotting;however, SELDI-TOF MS combined with Western im-munoblotting analysis identified an increase in both �-and �-CTFs posttreatment, which would imply a corre-sponding increase in sAPP�. This finding is in agree-ment with previous studies in which inactivation of PS1(and thus �-secretase) results in accumulation of APPCTFs in both mouse models (57–59) and cell-basedsystems (30, 60, 61). Furthermore, we have identifiedmembers of the secretase family of proteases associatedwith exosomes, suggesting that cleavage of APP mayoccur within these vesicles (Fig. 7B). Together, thesedata suggest that an alternative pathway exists for APPprocessing through the secretion of exosomes.

Whereas CHO cells are a well-established system forstudying APP processing, the majority of studies haveinvolved the expression of APP mutants representing

Figure 5. Effect of �-secretase inhibitors on accumulation of APP C-terminal fragments in exosomes. A) CHO-APP695 cells wereincubated in the presence and absence of 1.0 �M DAPT. Exosomes were isolated and subject to SELDI-TOF MS using 4G8 (A�residues 17–24) and APP CT (APP 751–770 of APP 770 isoform) antibodies. �-CTFs m/z � 9,717 Da, and �-CTFs m/z � 11,314Da; � represents the major peaks in each trace. B) CHO-APP695 cells were incubated for 16 h in the presence and absence oftwo different �-secretase inhibitors at 1.0 �M. Exosomes were isolated from the conditioned media and separated on 10–20%Tricine gel, and the effects of inhibitors on CTFs were analyzed using WO2 and APP CT antibodies.

Figure 6. Secretases are located within exosomes. Whole cell lysates or exosomes from CHO-APP695 cells were separated on10–20% Tricine gel, and the presence of secretase components was analyzed by immunoblotting with ADAM10, BACE, PS1, PS2,and nicastrin antibodies. � � immunoreactive band representing antigen fragment; m � monomer; d � dimer.

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familial AD-associated mutations. An example of this isthe APP V717F mutant, which, when expressed in CHOcells, produces secreted oligomeric forms of A� thathave neurotoxic properties and are able to inhibitlong-term potentiation, a marker of memory (62). Thisresult suggests that CHO cells produce A� species thatmay have toxic properties similar to that found in ADbrain tissue. Whereas the secretion of a minor fractionof A� associated with exosomes was reported in a studyusing mouse neuroblastoma (N2a) cells expressing theAPP-Swedish familial AD mutation reported, the au-thors were unable to detect any other APP-derivedfragments (34). It is widely reported that APP familialmutations can preferentially alter the site of �-secretasecleavage and cause an increase in A� production andsecretion, particularly A�1–42 species (63–65). As themajority of AD cases are sporadic, involving wild-typeand not mutant APP, we used CHO cells stably trans-fected with wild-type APP as a more relevant system toinvestigate APP processing and secretion.

Exosomes from the CHO-APP695 cells contain detect-able levels of fl-APP, a number of APP CTFs, and A�.Under the conditions used, NTFs of APP were onlyidentified in the conditioned media and were notenriched in exosomes, suggesting they are secreted ontheir initial cleavage at the plasma membrane. This

finding is consistent with a previous report showing thatA� and APP CTFs accumulate in MVBs from the brainsof APP transgenic mice, whereas APP NTFs are absent(66, 67). Confirmation of the presence of APP CTFsidentified in the purified exosomes was made using twodifferent antibodies raised against the C-terminal re-gion of APP. Stringent washing during the ultracentrif-ugation steps did not reduce the fl-APP signal observedon the immunoblots, confirming that fl-APP was anexosomal protein and not nonspecifically bound to theperiphery. Previously it has been difficult to detectevidence of APP CTFs in the culture medium aftersecretase cleavage. We propose that these fragments arespecifically targeted to secretion in exosomes, whichare a minor fraction of the conditioned media. Onpurification of exosomes from the cultured media,these fragments become readily detectable. Our resultssupport those of a concurrent study (60) that demon-strated accumulation of APP CTFs in exosomes usingSY5Y cells transfected with wild-type APP. This resulthas implications for the analysis of APP processing, forwhich this pathway of APP fragment secretion has notpreviously been considered.

Although we were able to detect the candidate�-secretase ADAM10 and BACE in the CHO-APP695exosomes, we were able to detect only some �-secretasecomplex components, namely PS1 and PS2, in ourexosome preparations. This is the first report to localizethese molecules in exosomes and adds to the numberof proteins identified that can be components of thesevesicles. The presence of the secretase components inexosomes suggests that APP could undergo endocytosisand become encapsulated in MVBs where it can thenundergo �- or �-secretase cleavage within the vesicleitself to generate APP CTFs. Protein cleavage in exo-somes is not unprecedented and has been reportedpreviously for other unrelated proteins (68–70). Be-cause of the limited expression of some �-secretasecomplex components, only a minor proportion of theseCTFs then undergo �-secretase cleavage to release A�or p3 fragments, with the remainder being secreted asCTFs in association with exosomes. This could be anexplanation for the low levels of A� found in exosomescompared with other APP fragments.

How the multiprotein �-secretase complex cleavesAPP within the plasma membrane has been a subject ofmuch discussion and debate. As a target for therapy,several inhibitors of �-secretase that can effectivelyreduce levels of A� have been developed; however, thetarget molecule within the �-secretase complex andmode of action for many of these inhibitors remainunknown. Measurements of A� levels are usually per-formed on conditioned media from cultured cell linesexpressing APP that have been exposed to these inhib-itors. Treatment of CHO-APP695 cells with �-secretaseinhibitors produced the novel finding that sAPP� se-cretion was increased in addition to the excretion ofAPP CTFs in exosomes. SELDI-TOF MS analysis, to-gether with immunoblotting, identified the presence ofboth �- and �-CTFs in exosomes, and these fragmentswere increased on treatment with �-secretase inhibitors.By blocking �-secretase cleavage, not only do we getaccumulation of the CTFs as expected, but we also

Figure 7. The fate of APP fragments in exosomes after�-secretase inhibition. A) Effect of �-secretase inhibitors onAPP and proteolytic fragments associated with exosomes. Anexosome is represented by the oval shape. B) Secretaseslocalized in exosomes. � � detected; X � not detected; n.t. �not tested.

1475APP PROCESSING IN EXOSOMES

induce �-secretase cleavage, increasing the level ofsAPP�, which indicates a possible feedback mechanismlinking the secretase pathways. �-Secretase cleavage ofAPP occurs predominantly at the plasma membrane,and any uncleaved APP is then recycled through theendocytic pathway. �-Secretase inhibitors may affect thetrafficking of APP, resulting in an increase of APP beingreinternalized and recycled back to the plasma mem-brane such that it has increased exposure to �-secre-tase. sAPP� has been reported as being neuroprotective(71–73), suggesting a positive side effect of blocking�-secretase cleavage and a possible drug target fortreatment of AD. However, the current �-secretaseinhibitors are not specific for cleavage of APP, resultingin inhibition of �-secretase cleavage of other substratessuch as Notch (74, 75), which is important in embry-onic development and cell-to-cell signaling.

To confirm that the presence of CTFs was not anartifact of APP overexpressing cell lines in non-neuro-nal cell lines we examined exosomes isolated from SY5Yhuman neuroblastoma cells endogenously expressingAPP. Significantly, fl-APP and CTFs were present, al-though the level of expression was too low to be able todetect A� (data not shown).

As soluble, oligomeric forms of A� are now gainingsupport as being the toxic species responsible for theneurotoxicity in AD (62, 76), further studies on theprocessing and secretion of APP and associated frag-ments through the exosome pathway are warranted. InCHO-APP695 cells, the majority of A� is soluble withonly a small percentage remaining membrane boundand released into the extracellular environment inassociation with exosomes. Previous reports have local-ized the longer A�42 (increased in familial AD) pre-dominantly to MVBs of neurons in normal mouse, rat,and human brain, and these levels are increased in APPtransgenic mice and AD human brain (67). In addition,exosomal proteins have been found to accumulate inthe plaques of brains of patients with AD (34); there-fore, exosomes provide a mechanism of A� and APP-CTF trafficking around the body, possibly contributingto amyloid deposition in the brain. More importantly,APP and A� have been found to circulate in extracel-lular fluids, including cerebrospinal fluid and plasma(3, 10, 77, 78), and recent work has confirmed thepresence of exosomes in both plasma (79) and inassociation with neuronal cells (51, 80), providing apossible diagnostic tool for AD. We therefore proposethat exosomes should be considered as a diagnostictarget for the complete characterization of APP pro-cessing after therapeutic interventions with secretaseinhibitors.

We thank Dr. Janetta Culvenor for the gift of the antibodiesagainst presenilin-1 and presenilin-2, Professor Sam Gandyfor antibody 369, and Genevieve Evin for critical commentson the manuscript. We thank Anna Friedhuber for technicalassistance with electron microscopy. This work was supportedby an National Health and Medical Research Council(NHMRC) program grant (to K.J.B., C.L.M., and A.F.H.).L.J.V. and R.M.N. are recipients of University of MelbournePostgraduate Research Scholarships, R.N. is the recipient ofan Australian Postgraduate Award, K.J.B. is an NHMRC

Senior Research Fellow, and A.F.H. is the recipient of anNHMRC RD Wright Career Development Award.

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Received for publication July 23, 2007.Accepted for publication November 29, 2007.

1478 Vol. 22 May 2008 SHARPLES ET AL.The FASEB Journal