FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-9357com.
 (The FASEB Journal. 2008;22:1469-1478.)
© 2008 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-9357comv1
fj.07-9357comv2
22/5/1469    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sharples, R. A.
Right arrow Articles by Hill, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sharples, R. A.
Right arrow Articles by Hill, A. F.

Inhibition of {gamma}-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes

Robyn A. Sharples*,{dagger},1, Laura J. Vella*,{dagger},{ddagger},§,1, Rebecca M. Nisbet*,{dagger},{ddagger},§, Ryan Naylor*,{dagger},{ddagger},§, Keyla Perez{dagger},{ddagger},§, Kevin J. Barnham{dagger},{ddagger},§, Colin L. Masters{ddagger},§ and Andrew F. Hill*,{dagger},{ddagger},§,2

* Department of Biochemistry and Molecular Biology,

{dagger} Bio21 Molecular Science and Biotechnology Institute, and

{ddagger} Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia, and

§ The Mental Health Research Institute of Victoria, Parkville, Victoria, Australia

2Correspondence: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville Victoria 3010, Australia. E-mail: a.hill{at}unimelb.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer’s disease (AD) is the most common form of dementia and is associated with the deposition of the 39- to 43-amino acid β-amyloid peptide (Aβ) in the brain. C-terminal fragments (CTFs) of amyloid precursor protein (APP) can accumulate in endosomally derived multivesicular bodies (MVBs). These intracellular structures contain intraluminal vesicles that are released from the cell as exosomes when the MVB fuses with the plasma membrane. Here we have investigated the role of exosomes in the processing of APP and show that these vesicles contain APP-CTFs, as well as Aβ. In addition, inhibition of {gamma}-secretase results in a significant increase in the amount of {alpha}- and β-secretase cleavage, further increasing the amount of APP-CTFs contained within these exosomes. We identify several key members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) to be localized in exosomes, suggesting they may be a previously unidentified site of APP cleavage. These results provide further evidence for a novel pathway in which APP fragments are released from cells and have implications for the analysis of APP processing and diagnostics for 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 {gamma}-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes.


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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE MAIN COMPONENT of Alzheimer’s disease (AD) amyloid plaques is an aggregated form of the β-amyloid peptide (Aβ), a 39- to 43-amino acid peptide produced by proteolytic cleavage of the amyloid precursor protein (APP) (1 2 3 4 5 6 7) . APP is an integral membrane protein with a single membrane-spanning domain, a large extracellular amino terminus and a short cytoplasmic carboxyl terminus (8 , 9) . Mature APP molecules undergo proteolytic cleavage by at least three proteases termed {alpha}-, β-, and {gamma}-secretases, which lead to the generation of a number of proteolytic fragments, including Aβ when APP is cleaved by β- and {gamma}-secretases (10 , 11) . The identity of {alpha}-secretase remains unclear although the present candidates are three members of the ADAM family: ADAM9, ADAM10, and tumor necrosis-{alpha} converting enzyme (TACE)/ADAM17 (12 13 14 15) . β-Secretase has been identified as β-site APP-cleaving enzyme (BACE) 1, a novel type 1 transmembrane aspartyl protease (16 17 18 19 20) , and {gamma}-secretase has been identified as a multimeric protein complex containing either presenilin (PS) 1 or PS2 associated with nicastrin, Aph-1, and Pen-2 (21 22 23 24 25) .

The amyloidogenic pathway of APP processing involves sequential cleavage by β- and {gamma}-secretases. β-Secretase cleaves at the amino terminus of Aβ (26 , 27) , resulting in the release of secreted APP (sAPPβ) and leaves intact Aβ in a membrane-associated, 99-amino acid carboxyl-terminal fragment, β-CTF. β-CTF can undergo endocytosis via clathrin-coated vesicles (28) and is trafficked to various endosomal compartments, including multivesicular bodies (MVBs) (29) . The β-CTF is cleaved by {gamma}-secretase at the carboxyl-terminus of Aβ (1 2 3) , predominantly at positions 40 and 42, resulting in the release of intact Aβ peptides and the 50-amino acid APP intracellular domain (AICD) (30 31 32 33) .

Aβ is associated with small membrane vesicles known as exosomes (34) , which are secreted into the culture medium by many cell types, suggesting that APP processing may occur through this specialized pathway. Exosomes are small membrane vesicles (50–100 nm in diameter) that correspond to internal vesicles formed by invagination of the membrane of MVBs (35) . Exosome secretion into the extracellular environment occurs on fusion of MVBs with the cell membrane. The physiological relevance of exosomes has been established by the identification of exosomes in vivo, in association with follicular dendritic cells (36) , urine (37) , and malignant tumor effusions (38) . The function of exosomes appears to be much more important than the simple removal of unwanted cellular proteins, and a role for exosomes in mediating intercellular communication has been identified (39 40 41) .

As {gamma}-secretase activity is essential for the release of intact Aβ, {gamma}-secretase inhibitors have been suggested in the treatment of AD. These inhibitors have been shown to decrease Aβ production after administration to transgenic mice overexpressing human APP (42) . At the cellular level, APP-CTFs have been shown to accumulate in endosomal compartments after {gamma}-secretase inhibition (43) . As exosomes derive from these endocytic compartments, we analyzed the effect of {gamma}-secretase inhibitors on processing and packaging of APP cleavage products in exosomes. Here we demonstrate that APP, Aβ, and other proteolytic fragments of APP formed during processing by {alpha}-, β-, and {gamma}-secretase are associated with exosomes derived from cultured cells expressing wild-type human APP. On treatment of cells with {gamma}-secretase inhibitors we observed a previously unreported effect, whereby {alpha}-cleavage of APP was increased, resulting in a significant increase in sAPP{alpha} into the extracellular environment. Analysis of exosomes from cells treated with these inhibitors revealed a concomitant accumulation of carboxyl-terminal fragments of APP in exosomes as identified by 1) immunoblotting using antibodies against C-terminal epitopes of APP and 2) surface enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS). To determine whether cleavage of APP could occur within exosomes we screened these vesicles for the presence of secretase components and identified {alpha}- and β-secretase (ADAM10 and BACE, respectively) to be localized in exosomes. In contrast, not all components of the {gamma}-secretase complex were detected, suggesting that this activity is a minor event. These data provide firm evidence for a novel pathway of APP processing in exosomes. The identification of proteolytic fragments of APP in association with exosomes has implications for the diagnostic and therapeutic assessment of APP processing in the pathogenesis of Alzheimer’s disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Anti-APP antibody 22C11 was obtained from Millipore (Temecula, CA, USA); 369 (50-mer raised against residues 645–694 of the C terminus of APP 695 isoform) was a gift from Professor Sam Gandy (Thomas Jefferson University, Philadelphia, PA, USA); WO2 (Aβ 5–8) has been described previously (44) ; APP C-terminal (CT) (raised against residues 751–770 of APP 770 isoform) and BACE (46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65) antibodies were from Merck Biosciences (Darmstadt, Germany); anti-Aβ (residues 17–24) antibody 4G8 was from Covance (Princeton, NJ, USA); ADAM10, TACE, and Tsg101 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); flotillin-1, nucleoporin, GM130, and Bcl2 were obtained from BD Biosciences (San Jose, CA, USA); anti-nicastrin was from Sigma-Aldrich (St. Louis, MO, USA), and the anti-PS1 and -PS2 antibodies were a gift from Dr. Janetta Culvenor and have been previously described (45) .

Generation of the Chinese hamster ovary (CHO)-APP695 cell 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) as described previously (46) . Cells were transfected using Lipofectamine 2000 and cultured in RPMI 1640 medium supplemented with 1 mM glutamine and 5% fetal bovine serum (Invitrogen, Mount Waverley, Victoria, Australia). Transfected cells were selected and maintained using 7.5 µg/ml puromycin (Sigma-Aldrich).

Treatment of cells with {gamma}-secretase inhibitors
Stably transfected CHO-APP695 cells were diluted 1:10 in RPMI 1640 plus supplements and plated in six-well plates (Nalge Nunc International, Rochester, NY, USA) 2 h before the addition of the inhibitor; 1 µM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) (Merck Biosciences) or L685,458 (Merck Biosciences) dissolved in dimethyl sulfoxide (DMSO) was added directly to the culture medium of the cells. DMSO (0.1% v/v) was used as a negative control. Cells were maintained at 37°C in a 5% CO2 atmosphere for 16 h after which the media were collected. Conditioned media posttreatment were centrifuged at 3000 rpm (5415 D centrifuge; Eppendorf-5 Prime, Inc., Boulder, CO) to remove cell debris before analysis for sAPP and Aβ secretion by immunoblotting.

Immunoblotting
Samples were diluted in Tricine sample buffer (8% sodium dodecyl sulfate, 30% glycerol, 100 mM Tris, 100 mM Tricine, and 0.01% phenol red) containing 10% 2-mercaptoethanol and boiled for 5 min at 100°C. Samples were electrophoresed using 10–20% Tricine acrylamide gels (Invitrogen). Proteins were transferred to a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and then blocked in 5% nonfat milk powder in PBST (PBS+0.5% Tween-20) for 1 h. Membranes were washed in PBST and incubated with primary antibody overnight at room temperature. Membranes were again washed in PBST and probed with secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:10,000) (Amersham Biosciences Corp., Piscataway, NJ, USA), for 1 h at room temperature. Specific binding was determined using ECL Plus (GE Healthcare, Chalfont St. Giles, UK). Blots undergoing quantification were scanned using a charge-coupled device camera system (SciTech, Preston, VIC, Australia). Data were 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 calf serum before its use in cell culture media by overnight (16 h) ultracentrifugation at 100,000 g (45Ti rotor, Beckman Coulter Inc., Fullerton, CA, USA). Cells (5x107) were cultured for 4–5 days in this bovine-exosome-depleted media before exosome isolation by differential centrifugation as described previously with minor modifications (47 , 48) . Briefly, cellular debris was removed by centrifugation at 2000 g for 20 min, 0.22-µm filtration, and ultracentrifugation at 100,000 g for 1 h at 4°C. Exosomes were pooled, washed in PBS, repelleted, and resuspended in 100 µl of Tricine sample buffer or PBS, boiled at 100°C for 5 min, and either used immediately or stored at –20°C.

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

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

SELDI-TOF MS
Exosome fractions were analyzed using PS20 ProteinChip arrays (Ciphergen Biosystems, Fremont, CA, USA). The PS20 arrays were first washed with 4 µl of PBS before antibody solutions (2 µl, 0.25 mg/ml in PBS) were placed on the chip and incubated at 4°C overnight. The antibody array was washed with 10 µl of deactivation buffer (0.5 M ethanolamine in PBS, pH 8.0; Ciphergen Biosystems) for 30 min, followed by 5-min washes with 5 µl of PBS + 0.5% Triton X-100 (wash buffer) and then 5 µl of PBS. All washes and incubations were performed on a shaking table.

Exosome samples were resuspended in PBS and loaded onto the PS20 antibody array and incubated for 3 h at room temperature. The sample was then removed, and each array was washed twice with wash buffer, PBS, and 1 mM HEPES (pH 7.4). The arrays were air dried, and then 1 µl of {alpha}-cyano-4-hydroxycinnamic acid (50% saturated in 50% acetonitrile and 0.5% trifluoroacetic acid; Bio-Rad Laboratories) was applied to each spot twice. The arrays were air dried before analysis.

Arrays were analyzed using a PBSIIC SELDI-TOF MS ProteinChip reader, and the data were analyzed with ProteinChip Software 3.1 (Ciphergen Biosystems). A protein profile was generated in which individual proteins were displayed within spectra as unique peaks based on their mass-to-charge ratio.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conditioned media were harvested from CHO-APP695 cells and assayed by immunoblotting to test the expression levels of sAPP and Aβ. A stably expressing CHO-APP695 cell line was used whereby sAPP{alpha} and Aβ were reliably visualized by immunoblotting with monoclonal antibody WO2 (Aβ 5–8) without the need for immunoprecipitation (Fig. 1 A). Levels of Aβ were significantly reduced to undetectable levels when the cells were treated with {gamma}-secretase inhibitors DAPT or L685,458 at 1.0 µM, suggesting that the CHO-APP695 cells represent a model for studying wild-type APP processing (Fig. 1B ).


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Figure 1. A) Western blot analysis of APP/Aβ expression in CHO-APP695 cells. Conditioned media from CHO cells transfected with vector only or APP695 were separated on 10–20% Tricine gel, and APP/Aβ expression was analyzed by immunoblotting with WO2. B) Treatment of CHO-APP695 cells with {gamma}-secretase inhibitors. CHO-APP695 cells were incubated in the presence (+) and absence (–) of {gamma}-secretase inhibitors DAPT and L685,458 at 1.0 µM 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 immunoblotting with WO2. C) Increase in sAPP after treatment with {gamma}-secretase inhibitor. CHO-APP695 cells were treated separately with increasing concentrations of DAPT to a final concentration of 1 µM for 16 h. Treatment with DMSO was used as a negative control. Posttreatment, conditioned media were separated on 8% SDS-PAGE and analyzed by immunoblotting with WO2; quantitation of the increase in sAPP{alpha} secretion is shown graphically.

We observed a significant increase in the amount of sAPP{alpha} in the conditioned media of the CHO-APP695 cells after inhibition with DAPT. This increase occurred in a dose-dependant manner, and the secretion of sAPP{alpha} was greatest at 1 µM, the highest concentration tested (Fig. 1C ). Quantitation of sAPP{alpha} observed by immunoblotting revealed a greater than 3-fold increase in sAPP{alpha} over the vehicle-only control when treated with 1 µM DAPT (Fig. 1C ). Levels of Aβ were undetectable by immunoblotting of conditioned cell media treated with concentrations as low as 0.02 µM DAPT (data not shown).

The increased secretion of sAPP{alpha} into the cell media after treatment of cells with DAPT led us to examine the {alpha}-secretase, "nonamyloidogenic" pathway of APP cleavage in more detail. The observed increase in sAPP{alpha} in CHO-APP695 cells treated with DAPT suggests an increase in the corresponding CTF arising from the inhibition of {gamma}-secretase. We reasoned that the plasma membrane-localized CTF remaining after {alpha}-secretase cleavage of APP may be endocytosed and subsequently incorporated into membrane-bound vesicles. Low levels of Aβ have been reported to be associated with small, secreted membrane vesicles known as exosomes in cultured cell lines overexpressing mutant APP constructs (34) . As CHO cells have been reported to secrete exosomes (50) , we therefore investigated whether APP CTFs were associated with exosomes from CHO-APP695 cells.

Culture media were removed from CHO-APP695 cells after 5 days and subjected to filtration and ultracentrifugation as described previously (47 , 48 , 51) . The purified extracellular particles from the conditioned media were examined by electron microscopy (EM). Negative staining EM revealed that the conditioned media contained microvesicles that were membrane bound and "cup shaped" and had a size similar (50–80 nm diameter) to that of exosomes described previously (Fig. 2 A) (47 , 48) . Little to no cell debris was visualized by EM, confirming the purity of the exosome preparation. To further confirm the identity of these microvesicles as exosomes, continuous sucrose density gradient centrifugation was performed. Immunoblot analysis of gradient fractions showed that CHO-APP695 microvesicles possessed a buoyancy consistent with exosomes for previously reported cultured cell lines, including the CHO cell line (Fig. 2B ) (47 , 48 , 52) .


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		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 to equilibrium followed by immunoblotting with WO2. C) Whole cell lysates or exosomes from CHO-APP695 cells were separated on 10–20% Tricine gel and analyzed by immunoblotting using antibodies to flotillin, Tsg101, GM130, nucleoporin, or Bcl2.

Exosomal protein composition is limited to a subset of cellular proteins, as proteins of nuclear, mitochondrial, endoplasmic reticulum, or Golgi apparatus origin are absent. Exosomes are enriched in raft associated proteins, membrane transport proteins, and adhesion molecules (53) . Equivalent amounts of CHO-APP695 cell lysate or microvesicles were analyzed for protein composition with a panel of organelle marker antibodies (Fig. 2C ). Flotillin is enriched in exosomes as is the protein Tsg101 (35) . Tsg101 is a component of the endosomal sorting complex, and flotillin is a constituent of lipid subdomains in recycling endosomes. Antibodies against GM130 (Golgi), nucleoporin (nucleus), and Bcl2 (mitochondria) were also used to test for microvesicle purity and to ensure the absence of cell debris contamination and apoptotic blebs (54) . The presence of flotillin and Tsg101 and the absence of GM130, nucleoporin, and Bcl2 in our microvesicles fulfill the criteria for them to be described as exosomes (54) .

Immunoblotting with a series of antibodies was used to determine whether APP and/or Aβ was associated with exosomes. The antibodies WO2, 369, and APP CT all demonstrated a specific enrichment of APP fragments in the exosome fractions compared with conditioned media (Fig. 3 ). A small amount of Aβ was found to be associated with exosomes in addition to a series of APP CTFs. Further analysis of the exosome preparation with APP CT and 369 antibodies (both antibodies raised against the C terminus of APP) revealed that full-length APP (fl-APP) is contained within these vesicles. To ensure that this fl-APP was actually an exosomal protein rather than bound nonspecifically to the outside of these vesicles, the exosome purification was modified to include an additional PBS wash and further centrifugation of the exosome pellet at 100,000 g. Minimal APP signal was detected in this additional PBS wash fraction, and the fl-APP signal was consistently enriched in the exosome preparation, confirming its true association with exosomes (data not shown).


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Figure 3. APP and Aβ contained within exosomes. Conditioned media from CHO cells transfected with vector only or APP695 and exosomes from CHO-APP695 cells were separated on 10–20% Tricine gel followed by immunoblotting with WO2, APP CT, and 369 antibodies.

After establishing that CHO-APP695 cells treated with {gamma}-secretase inhibitors completely reduced detectable levels of Aβ in conditioned media with a concomitant increase in sAPP{alpha}, we sought to assess whether these inhibitors had any effect on other processing products of APP that are associated with exosomes. Thus, cells were incubated in the presence of 1 µM DAPT for a period of 5 days after which exosomes were isolated from the culture media. Although we have shown DAPT to have an effect within 16 h, a longer incubation time was required to isolate exosomes from the conditioned media. We observed no differences in cell growth, and previous studies (55) have reported DAPT to be nontoxic for incubation periods of 5 days. Using antibodies to different regions of APP, we found that APP CTFs associated with exosomes and increased in the presence of the {gamma}-secretase inhibitor compared with untreated cells. In contrast, the majority of APP N-terminal fragments (NTFs) (identified with antibody 22C11) were found in the conditioned media and not associated with exosomes (Fig. 4 ).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Figure 4. Increase in CTFs in exosomes from {gamma}-secretase-treated cells. Exosomes were isolated from vector only, and CHO-APP695 cells in the presence (+) or absence (–) of {gamma}-secretase inhibitor DAPT at 1 µM. Conditioned media and exosomes were separated on 10–20% Tricine gel and immunoblotted with 369 and 22C11. M = conditioned media; E = exosome preparation.

SELDI-TOF MS was used to further characterize CTFs detected via immunoblotting and to differentiate between {alpha}- and β-CTFs. Exosomes treated with DAPT demonstrated an increase in peak size by SELDI-TOF MS at the predicted molecular weight of β-CTF (11,314 Da) compared with untreated cells. An additional peak at 9717 Da also increased on treatment of cells and may represent {alpha}-cleaved CTF (Fig. 5 A). These results confirm an increase in both {alpha}- and β-CTF in the presence of the {gamma}-secretase inhibitor DAPT, and this effect was observed using two independent APP C-terminal antibodies.


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Figure 5. Effect of {gamma}-secretase inhibitors on accumulation of APP C-terminal fragments in exosomes. A) CHO-APP695 cells were incubated in the presence and absence of 1.0 µM DAPT. Exosomes were isolated and subject to SELDI-TOF MS using 4G8 (Aβ residues 17–24) and APP CT (APP 751–770 of APP 770 isoform) antibodies. {alpha}-CTFs m/z = 9,717 Da, and β-CTFs m/z = 11,314 Da; * represents the major peaks in each trace. B) CHO-APP695 cells were incubated for 16 h in the presence and absence of two different {gamma}-secretase inhibitors at 1.0 µM. 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.

To confirm the specificity of {gamma}-secretase inhibition, we treated cells with an alternative {gamma}-secretase inhibitor, L685,458. The same effect was observed—complete ablation of Aβ production and an increase in both {alpha}- and β-CTFs in association with exosomes (Fig. 5B )—confirming the effect as a direct result from {gamma}-secretase inhibition and not a nonspecific effect of the inhibitor. Although the SELDI-TOF MS data indicate that β-CTF predominates over {alpha}-CTF (Fig. 5A ), these results should be interpreted as changes in the relative abundance of each fragment, not as a comparison between fragments. Immunoblot analysis using WO2 and APP CT antibodies demonstrates that the dominant CTF band present in exosomes as detected by APP CT antibody is {alpha}-CTF, as a corresponding band of equivalent size is not detected by WO2 (Fig. 5B ).

Because fl-APP, Aβ, and other processing products of APP were found to be associated with exosomes and {gamma}-secretase inhibitors demonstrated a specific effect on the CTFs associated with exosomes, we surmised that these microvesicles may provide a possible site (albeit minor) for active processing of APP. BACE has been shown to localize in endosomal compartments where it can actively cleave APP (16 , 34 , 56) , and exosomes have previously been shown to contain the metalloproteases ADAM10 and ADAM17 (49) , which are both candidates for the {alpha}-secretase cleavage of APP. We therefore examined exosomes isolated from CHO-APP695 cells for the presence of secretase components. First, by comparing cell lysate and exosome fractions, we were able to detect BACE in exosomes from the CHO-APP695 cells using two independent antibodies (Fig. 6 ). Immunoblotting of exosomes derived from CHO-APP695 cells with an ADAM10 antibody (Fig. 6) confirmed previous findings that ADAM10 is present in exosomes (49) . {gamma}-Secretase is a membrane protein complex comprising the presenilin proteins, nicastrin, Aph-1, and Pen-2. Our studies demonstrated that PS1 was abundant in the exosome preparation, whereas PS2 is present in minor amounts (Fig. 6) .


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Figure 6. Secretases are located within exosomes. Whole cell lysates or exosomes from CHO-APP695 cells were separated on 10–20% 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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results from our studies demonstrate that in cells expressing wild-type APP (695-amino acid isoform), a fraction of Aβ is secreted in association with exosomes. We provide evidence that APP CTFs are also found associated with exosomes, and on treatment of the cells with {gamma}-secretase inhibitors we observe an increase in CTFs in addition to increased sAPP{alpha} in the conditioned media from CHO-APP695 cells (Fig. 7 A). In the absence of a reliable sAPPβ specific antibody, it was difficult to determine the direct effect of {gamma}-secretase inhibitors on β-secretase cleavage via immunoblotting; however, SELDI-TOF MS combined with Western immunoblotting analysis identified an increase in both {alpha}- and β-CTFs posttreatment, which would imply a corresponding increase in sAPPβ. This finding is in agreement with previous studies in which inactivation of PS1 (and thus {gamma}-secretase) results in accumulation of APP CTFs in both mouse models (57 58 59) and cell-based systems (30 , 60 , 61) . Furthermore, we have identified members of the secretase family of proteases associated with exosomes, suggesting that cleavage of APP may occur within these vesicles (Fig. 7B ). Together, these data suggest that an alternative pathway exists for APP processing through the secretion of exosomes.


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Figure 7. The fate of APP fragments in exosomes after {gamma}-secretase inhibition. A) Effect of {gamma}-secretase inhibitors on APP and proteolytic fragments associated with exosomes. An exosome is represented by the oval shape. B) Secretases localized in exosomes. Figure 7 = detected; X = not detected; n.t. = not tested.

Whereas CHO cells are a well-established system for studying APP processing, the majority of studies have involved the expression of APP mutants representing familial AD-associated mutations. An example of this is the APP V717F mutant, which, when expressed in CHO cells, produces secreted oligomeric forms of Aβ that have neurotoxic properties and are able to inhibit long-term potentiation, a marker of memory (62) . This result suggests that CHO cells produce Aβ species that may have toxic properties similar to that found in AD brain tissue. Whereas the secretion of a minor fraction of Aβ associated with exosomes was reported in a study using mouse neuroblastoma (N2a) cells expressing the APP-Swedish familial AD mutation reported, the authors were unable to detect any other APP-derived fragments (34) . It is widely reported that APP familial mutations can preferentially alter the site of {gamma}-secretase cleavage and cause an increase in Aβ production and secretion, particularly Aβ1–42 species (63 64 65) . As the majority of AD cases are sporadic, involving wild-type and not mutant APP, we used CHO cells stably transfected with wild-type APP as a more relevant system to investigate APP processing and secretion.

Exosomes from the CHO-APP695 cells contain detectable levels of fl-APP, a number of APP CTFs, and Aβ. Under the conditions used, NTFs of APP were only identified in the conditioned media and were not enriched in exosomes, suggesting they are secreted on their initial cleavage at the plasma membrane. This finding is consistent with a previous report showing that Aβ and APP CTFs accumulate in MVBs from the brains of APP transgenic mice, whereas APP NTFs are absent (66 , 67) . Confirmation of the presence of APP CTFs identified in the purified exosomes was made using two different antibodies raised against the C-terminal region of APP. Stringent washing during the ultracentrifugation steps did not reduce the fl-APP signal observed on the immunoblots, confirming that fl-APP was an exosomal protein and not nonspecifically bound to the periphery. Previously it has been difficult to detect evidence of APP CTFs in the culture medium after secretase cleavage. We propose that these fragments are specifically targeted to secretion in exosomes, which are a minor fraction of the conditioned media. On purification of exosomes from the cultured media, these fragments become readily detectable. Our results support those of a concurrent study (60) that demonstrated accumulation of APP CTFs in exosomes using SY5Y cells transfected with wild-type APP. This result has implications for the analysis of APP processing, for which this pathway of APP fragment secretion has not previously been considered.

Although we were able to detect the candidate {alpha}-secretase ADAM10 and BACE in the CHO-APP695 exosomes, we were able to detect only some {gamma}-secretase complex components, namely PS1 and PS2, in our exosome preparations. This is the first report to localize these molecules in exosomes and adds to the number of proteins identified that can be components of these vesicles. The presence of the secretase components in exosomes suggests that APP could undergo endocytosis and become encapsulated in MVBs where it can then undergo {alpha}- or β-secretase cleavage within the vesicle itself to generate APP CTFs. Protein cleavage in exosomes is not unprecedented and has been reported previously for other unrelated proteins (68 69 70) . Because of the limited expression of some {gamma}-secretase complex components, only a minor proportion of these CTFs then undergo {gamma}-secretase cleavage to release Aβ or p3 fragments, with the remainder being secreted as CTFs in association with exosomes. This could be an explanation for the low levels of Aβ found in exosomes compared with other APP fragments.

How the multiprotein {gamma}-secretase complex cleaves APP within the plasma membrane has been a subject of much discussion and debate. As a target for therapy, several inhibitors of {gamma}-secretase that can effectively reduce levels of Aβ have been developed; however, the target molecule within the {gamma}-secretase complex and mode of action for many of these inhibitors remain unknown. Measurements of Aβ levels are usually performed on conditioned media from cultured cell lines expressing APP that have been exposed to these inhibitors. Treatment of CHO-APP695 cells with {gamma}-secretase inhibitors produced the novel finding that sAPP{alpha} secretion was increased in addition to the excretion of APP CTFs in exosomes. SELDI-TOF MS analysis, together with immunoblotting, identified the presence of both {alpha}- and β-CTFs in exosomes, and these fragments were increased on treatment with {gamma}-secretase inhibitors. By blocking {gamma}-secretase cleavage, not only do we get accumulation of the CTFs as expected, but we also induce {alpha}-secretase cleavage, increasing the level of sAPP{alpha}, which indicates a possible feedback mechanism linking the secretase pathways. {alpha}-Secretase cleavage of APP occurs predominantly at the plasma membrane, and any uncleaved APP is then recycled through the endocytic pathway. {gamma}-Secretase inhibitors may affect the trafficking of APP, resulting in an increase of APP being reinternalized and recycled back to the plasma membrane such that it has increased exposure to {alpha}-secretase. sAPP{alpha} has been reported as being neuroprotective (71 72 73) , suggesting a positive side effect of blocking {gamma}-secretase cleavage and a possible drug target for treatment of AD. However, the current {gamma}-secretase inhibitors are not specific for cleavage of APP, resulting in inhibition of {gamma}-secretase cleavage of other substrates such as Notch (74 , 75) , which is important in embryonic development and cell-to-cell signaling.

To confirm that the presence of CTFs was not an artifact of APP overexpressing cell lines in non-neuronal cell lines we examined exosomes isolated from SY5Y human neuroblastoma cells endogenously expressing APP. Significantly, fl-APP and CTFs were present, although the level of expression was too low to be able to detect Aβ (data not shown).

As soluble, oligomeric forms of Aβ are now gaining support as being the toxic species responsible for the neurotoxicity in AD (62 , 76) , further studies on the processing and secretion of APP and associated fragments through the exosome pathway are warranted. In CHO-APP695 cells, the majority of Aβ is soluble with only a small percentage remaining membrane bound and released into the extracellular environment in association with exosomes. Previous reports have localized the longer Aβ42 (increased in familial AD) predominantly to MVBs of neurons in normal mouse, rat, and human brain, and these levels are increased in APP transgenic mice and AD human brain (67) . In addition, exosomal proteins have been found to accumulate in the plaques of brains of patients with AD (34) ; therefore, exosomes provide a mechanism of Aβ and APP-CTF trafficking around the body, possibly contributing to amyloid deposition in the brain. More importantly, APP and Aβ have been found to circulate in extracellular fluids, including cerebrospinal fluid and plasma (3 , 10 , 77 , 78) , and recent work has confirmed the presence of exosomes in both plasma (79) and in association with neuronal cells (51 , 80) , providing a possible diagnostic tool for AD. We therefore propose that exosomes should be considered as a diagnostic target for the complete characterization of APP processing after therapeutic interventions with secretase inhibitors.


   ACKNOWLEDGMENTS
 
We thank Dr. Janetta Culvenor for the gift of the antibodies against presenilin-1 and presenilin-2, Professor Sam Gandy for antibody 369, and Genevieve Evin for critical comments on the manuscript. We thank Anna Friedhuber for technical assistance with electron microscopy. This work was supported by 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 Melbourne Postgraduate Research Scholarships, R.N. is the recipient of an Australian Postgraduate Award, K.J.B. is an NHMRC Senior Research Fellow, and A.F.H. is the recipient of an NHMRC RD Wright Career Development Award.


   FOOTNOTES
 
1 These authors contributed equally to this work.

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


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Estus, S., Golde, T. E., Kunishita, T., Blades, D., Lowery, D., Eisen, M., Usiak, M., Qu, X., Tabira, T., Greenberg, B. D., Younkin, S. G. (1992) Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid protein precursor. Science 255,726-728[Abstract/Free Full Text]
  2. Golde, T. E., Estus, S., Younkin, L. H., Selkoe, D. J., Younkin, S. G. (1992) Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 255,728-730[Abstract/Free Full Text]
  3. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., Selkoe, D. L. (1992) Amyloid β-peptide is produced by cultured cells during normal metabolism. Nature 359,322-325[CrossRef][Medline]
  4. Murakami, K., Irie, K., Morimoto, A., Ohigashi, H., Shindo, M., Nagao, M., Shimizu, T., Shirasawa, T. (2002) Synthesis, aggregation, neurotoxicity, and secondary structure of various Aβ1–42 mutants of familial Alzheimer’s disease at positions 21–23. Biochem. Biophys. Res. Commun. 294,5-10[CrossRef][Medline]
  5. Cai, X., Golde, T. E., Younkin, S. G. (1993) Release of excess amyloid β protein from a mutant amyloid β protein precursor. Science 259,514-516[Abstract/Free Full Text]
  6. Serpell, L. C., Smith, J. M. (2000) Direct visualisation of the β-sheet structure of synthetic Alzheimer’s amyloid. J. Mol. Biol. 299,225-231[CrossRef][Medline]
  7. Findeis, M. A. (2000) Approaches to discovery and characterization of inhibitors of amyloid β-peptide polymerization. Biochim. Biophys. Acta 1502,76-84[Medline]
  8. Kang, J., Lemaire, H., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K., Multhaup, G., Beyreuther, K., Müller-Hill, B. (1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325,733-736[CrossRef][Medline]
  9. Selkoe, D. J. (1994) Cell biology of the amyloid β-protein precursor and the mechanism of Alzheimer’s disease. Annu. Rev. Cell Biol. 10,373-403[CrossRef][Medline]
  10. Busciglio, J., Gabuzda, D. H., Matsudaira, P., Yankner, B. A. (1993) Generation of β-amyloid in the secretory pathway in neuronal and nonneuronal cells. Proc. Natl. Acad. Sci. U. S. A. 90,2092-2096[Abstract/Free Full Text]
  11. Haass, C., Hung, A. Y., Schlossmacher, M. G., Teplow, D. B., Selkoe, D. J. (1993) β-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms. J. Biol. Chem. 268,3021-3024[Abstract/Free Full Text]
  12. Kowalska, A. (2003) Amyloid precursor protein gene mutations responsible for early-onset autosomal dominant Alzheimer’s disease. Folia Neuropathol. 41,35-40[Medline]
  13. Asai, M., Hattori, C., Szabo, B., Sasagawa, N., Maruyama, K., Tanuma, S., Ishiura, S. (2003) Putative function of ADAM9, ADAM10, and ADAM17 as APP {alpha}-secretase. Biochem. Biophys. Res. Commun. 301,231-235[CrossRef][Medline]
  14. Buxbaum, J. D., Liu, K. N., Luo, Y. X., Slack, J. L., Stocking, K. L., Peschon, J. J., Johnson, R. S., Castner, B. J., Cerretti, D. P., Black, R. A. (1998) Evidence that tumor necrosis factor {alpha} converting enzyme is involved in regulated {alpha}-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273,27765-27767[Abstract/Free Full Text]
  15. Lammich, S., Kojro, E., Postina, R., Gilbert, S., Pfeiffer, R., Jasionowski, M., Haass, C., Fahrenholz, F. (1999) Constitutive and regulated {alpha}-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Acad. Sci. U. S. A. 96,3922-3927[Abstract/Free Full Text]
  16. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., Citron, M. (1999) β-Secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286,735-741[Abstract/Free Full Text]
  17. Yan, R., Bienkowski, M. J., Shuck, M. E., Miao, H., Tory, M. C., Pauley, A. M., Brashier, J. R., Stratman, N. C., Mathews, W. R., Buhl, A. E., Carter, D. B., Tomasselli, A. G., Parodi, L. A., Heinrikson, R. L., Gurney, M. E. (1999) Membrane-anchored aspartyl protease with Alzheimer’s disease β-secretase activity. Nature 402,533-537[CrossRef][Medline]
  18. Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., Jacobson-Croak, K., Jewett, N., Keim, P., Knops, J., Lieberburg, I., Power, M., Tan, H., Tatsuno, G., Tung, J., Schenk, D., Seubert, P., Suomensaari, S. M., Wang, S., Walker, D., Zhao, J., McConlogue, L., Varghese, J. (1999) Purification and cloning of amyloid precursor protein β-secretase from human brain. Nature 402,537-540[CrossRef][Medline]
  19. Hussain, I., Powell, D., Howlett, D. R., Tew, D. G., Meek, T. D., Chapman, C., Gloger, I. S., Murphy, K. E., Southan, C. D., Ryan, D. M., Smith, T. S., Simmons, D. L., Walsh, F. S., Dingwall, C., Christie, G. (1999) Identification of a novel aspartic protease (Asp 2) as β-secretase. Mol. Cell. Neurosci. 14,419-427[CrossRef][Medline]
  20. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A., Tang, J. (2000) Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 97,1456-1460[Abstract/Free Full Text]
  21. Shi, X. P., Tugusheva, K., Bruce, J. E., Lucka, A., Wu, G. X., Chen-Dodson, E., Price, E., Li, Y., Xu, M., Huang, Q., Sardana, M. K., Hazuda, D. J. (2003) β-Secretase cleavage at amino acid residue 34 in the amyloid β peptide is dependent upon {gamma}-secretase activity. J. Biol. Chem. 278,21286-21294[Abstract/Free Full Text]
  22. Evin, G., Weidemann, A. (2002) Biogenesis and metabolism of Alzheimer’s disease Aβ amyloid peptides. Peptides 23,1285-1297[CrossRef][Medline]
  23. Goutte, C., Tsunozaki, M., Hale, V. A., Priess, J. R. (2002) APH-1 is a multipass membrane protein essential for the Notch signaling pathway in Caenorhabditis elegans embryos. Proc. Natl. Acad. Sci. U. S. A. 99,775-779[Abstract/Free Full Text]
  24. Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song, Y. Q., Rogaeva, E., Chen, F., Kawarai, T., Supala, A., Levesque, L., Yu, H., Yang, D. S., Holmes, E., Milman, P., Liang, Y., Zhang, D. M., Xu, D. H., Sato, C., Rogaev, E., Smith, M., Janus, C., Zhang, Y., Aebersold, R., Farrer, L. S., Sorbi, S., Bruni, A., Fraser, P., St. George-Hyslop, P. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and βAPP processing. Nature 407,48-54[CrossRef][Medline]
  25. Francis, R., McGrath, G., Zhang, J., Ruddy, D. A., Sym, M., Apfeld, J., Nicoll, M., Maxwell, M., Hai, B., Ellis, M. C., Parks, A. L., Xu, W., Li, J., Gurney, M., Myers, R. L., Himes, C. S., Hiebsch, R., Ruble, C., Nye, J. S., Curtis, D. (2002) aph-1 and pen-2 are required for Notch pathway signaling, {gamma}-secretase cleavage of betaAPP, and presenilin protein accumulation. Dev. Cell 3,85-97[CrossRef][Medline]
  26. Seubert, P., Oltersdorf, T., Lee, M. G., Barbour, R., Blomquist, C., Davis, D. L., Bryant, K., Fritz, L. C., Galasko, D., Thal, L. J., Lieberburg, I., Schenk, D. B. (1993) Secretion of β-amyloid precursor protein cleaved at the amino terminus of the β-amyloid peptide. Nature 361,260-263[CrossRef][Medline]
  27. Mattson, M. P. (1997) Cellular actions of β-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol. Rev. 77,1081-1132[Abstract/Free Full Text]
  28. Selkoe, D. J. (1996) Cell biology of the β-amyloid precursor protein and the genetics of Alzheimer’s disease. Cold Spring Harb. Symp. Quant. Biol. 61,587-596[Abstract/Free Full Text]
  29. Yamazaki, T., Koo, E. H., Selkoe, D. J. (1996) Trafficking of cell-surface amyloid β-protein precursor. 2. Endocytosis, recycling, and lysosomal targeting detected by immunolocalization. J. Cell Sci. 109,999-1008[Abstract]
  30. Sastre, M., Steiner, H., Fuchs, K., Capell, A., Multhaup, G., Condron, M. M., Teplow, D. B., Haass, C. (2001) Presenilin-dependent {gamma}-secretase processing of β-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch. EMBO Rep. 2,835-841[CrossRef][Medline]
  31. Weidemann, A., Eggert, S., Reinhard, F. B., Vogel, M., Paliga, K., Baier, G., Masters, C. L., Beyreuther, K., Evin, G. (2002) A novel {epsilon}-cleavage within the transmembrane domain of the Alzheimer amyloid precursor protein demonstrates homology with Notch processing. Biochemistry 41,2825-2835[CrossRef][Medline]
  32. Gu, Y., Misonou, H., Sato, T., Dohmae, N., Takio, K., Ihara, Y. (2001) Distinct intramembrane cleavage of the β-amyloid precursor protein family resembling {gamma}-secretase-like cleavage of Notch. J. Biol. Chem. 276,35235-35238[Abstract/Free Full Text]
  33. Yu, C., Kim, S. H., Ikeuchi, T., Xu, H., Gasparini, L., Wang, R., Sisodia, S. S. (2001) Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment {gamma}: evidence for distinct mechanisms involved in {gamma}-secretase processing of the APP and Notch1 transmembrane domains. J. Biol. Chem. 276,43756-43760[Abstract/Free Full Text]
  34. Rajendran, L., Honsho, M., Zahn, T. R., Keller, P., Geiger, K. D., Verkade, P., Simons, K. (2006) Alzheimer’s disease β-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. U. S. A. 103,11172-11177[Abstract/Free Full Text]
  35. Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., Raposo, G. (2002) The biogenesis and functions of exosomes. Traffic 3,321-330[CrossRef][Medline]
  36. Denzer, K., van Eijk, M., Kleijmeer, M. J., Jakobson, E., de Groot, C., Geuze, H. J. (2000) Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J. Immunol. 165,1259-1265[Abstract/Free Full Text]
  37. Pisitkun, T., Shen, R. F., Knepper, M. A. (2004) Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. U. S. A. 101,13368-13373[Abstract/Free Full Text]
  38. Andre, F., Schartz, N. E., Movassagh, M., Flament, C., Pautier, P., Morice, P., Pomel, C., Lhomme, C., Escudier, B., Le Chevalier, T., Tursz, T., Amigorena, S., Raposo, G., Angevin, E., Zitvogel, L. (2002) Malignant effusions and immunogenic tumour-derived exosomes. Lancet 360,295-305[CrossRef][Medline]
  39. Fevrier, B., Vilette, D., Archer, F., Loew, D., Faigle, W., Vidal, M., Laude, H., Raposo, G. (2004) Cells release prions in association with exosomes. Proc. Natl. Acad. Sci. U. S. A. 101,9683-9688[Abstract/Free Full Text]
  40. Gastpar, R., Gehrmann, M., Bausero, M. A., Asea, A., Gross, C., Schroeder, J. A., Multhoff, G. (2005) Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res. 65,5238-5247[Abstract/Free Full Text]
  41. Abusamra, A. J., Zhong, Z., Zheng, X., Li, M., Ichim, T. E., Chin, J. L., Min, W. P. (2005) Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood Cells Mol. Dis. 35,169-173[CrossRef][Medline]
  42. Dovey, H. F., John, V., Anderson, J. P., Chen, L. Z., de Saint Andrieu, P., Fang, L. Y., Freedman, S. B., Folmer, B., Goldbach, E., Holsztynska, E. J., Hu, K. L., Johnson-Wood, K. L., Kennedy, S. L., Kholodenko, D., Knops, J. E., Latimer, L. H., Lee, M., Liao, Z., Lieberburg, I. M., Motter, R. N., Mutter, L. C., Nietz, J., Quinn, K. P., Sacchi, K. L., Seubert, P. A., Shopp, G. M., Thorsett, E. D., Tung, J. S., Wu, J., Yang, S., Yin, C. T., Schenk, D. B., May, P. C., Altstiel, L. D., Bender, M. H., Boggs, L. N., Britton, T. C., Clemens, J. C., Czilli, D. L., Dieckman-McGinty, D. K., Droste, J. J., Fuson, K. S., Gitter, B. D., Hyslop, P. A., Johnstone, E. M., Li, W. Y., Little, S. P., Mabry, T. E., Miller, F. D., Audia, J. E. (2001) Functional {gamma}-secretase inhibitors reduce beta-amyloid peptide levels in brain. J. Neurochem. 76,173-181[CrossRef][Medline]
  43. Zhang, M., Haapasalo, A., Kim, D. Y., Ingano, L. A., Pettingell, W. H., Kovacs, D. M. (2006) Presenilin/{gamma}-secretase activity regulates protein clearance from the endocytic recycling compartment. FASEB J. 20,1176-1178[Abstract/Free Full Text]
  44. Ida, N., Hartmann, T., Pantel, J., Schroder, J., Zerfass, R., Forstl, H., Sandbrink, R., Masters, C. L., Beyreuther, K. (1996) Analysis of heterogeneous β-A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive western blot assay. J. Biol. Chem. 271,22908-22914[Abstract/Free Full Text]
  45. Culvenor, J. G., Ilaya, N. T., Ryan, M. T., Canterford, L., Hoke, D. E., Williamson, N. A., McLean, C. A., Masters, C. L., Evin, G. (2004) Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations. Eur. J. Biochem. 271,375-385[Medline]
  46. White, A. R., Du, T., Laughton, K. M., Volitakis, I., Sharples, R. A., Xilinas, M. E., Hoke, D. E., Holsinger, R. M., Evin, G., Cherny, R. A., Hill, A. F., Barnham, K. J., Li, Q. X., Bush, A. I., Masters, C. L. (2006) Degradation of the Alzheimer disease amyloid β-peptide by metal-dependent up-regulation of metalloprotease activity. J. Biol. Chem. 281,17670-17680[Abstract/Free Full Text]
  47. Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V., Melief, C. J., Geuze, H. J. (1996) B lymphocytes secrete antigen-presenting vesicles. J. Exp. Med. 183,1161-1172[Abstract/Free Full Text]
  48. Thery, C., Regnault, A., Garin, J., Wolfers, J., Zitvogel, L., Ricciardi-Castagnoli, P., Raposo, G., Amigorena, S. (1999) Molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73. J. Cell Biol. 147,599-610[Abstract/Free Full Text]
  49. Stoeck, A., Keller, S., Riedle, S., Sanderson, M. P., Runz, S., Le Naour, F., Gutwein, P., Ludwig, A., Rubinstein, E., Altevogt, P. (2006) A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44. Biochem. J. 393,609-618[CrossRef][Medline]
  50. Masciopinto, F., Giovani, C., Campagnoli, S., Galli-Stampino, L., Colombatto, P., Brunetto, M., Yen, T. S., Houghton, M., Pileri, P., Abrignani, S. (2004) Association of hepatitis C virus envelope proteins with exosomes. Eur. J. Immunol. 34,2834-2842[CrossRef][Medline]
  51. Vella, L. J., Sharples, R. A., Lawson, V. A., Masters, C. L., Cappai, R., Hill, A. F. (2007) Packaging of prions into exosomes is associated with a novel pathway of PrP processing. J. Pathol. 211,582-590[CrossRef][Medline]
  52. Van Niel, G., Heyman, M. (2002) The epithelial cell cytoskeleton and intracellular trafficking. II. Intestinal epithelial cell exosomes: perspectives on their structure and function. Am. J. Physiol. 283,G251-G255
  53. Thery, C., Duban, L., Segura, E., Veron, P., Lantz, O., Amigorena, S. (2002) Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nat. Immunol. 3,1156-1162[CrossRef][Medline]
  54. Thery, C., Zitvogel, L., Amigorena, S. (2002) Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2,569-579[Medline]
  55. Kienlen-Campard, P., Miolet, S., Tasiaux, B., Octave, J. N. (2002) Intracellular amyloid-β1–42, but not extracellular soluble amyloid-β peptides, induces neuronal apoptosis. J. Biol. Chem. 277,15666-15670[Abstract/Free Full Text]
  56. Huse, J. T., Pijak, D. S., Leslie, G. J., Lee, V. M., Doms, R. W. (2000) Maturation and endosomal targeting of β-site amyloid precursor protein-cleaving enzyme: the Alzheimer’s disease β-secretase. J. Biol. Chem. 275,33729-33737[Abstract/Free Full Text]
  57. Xia, W., Zhang, J., Ostaszewski, B. L., Kimberly, W. T., Seubert, P., Koo, E. H., Shen, J., Selkoe, D. J. (1998) Presenilin 1 regulates the processing of β-amyloid precursor protein C-terminal fragments and the generation of amyloid β-protein in endoplasmic reticulum and Golgi. Biochemistry 37,16465-16471[CrossRef][Medline]
  58. Yu, H., Saura, C. A., Choi, S. Y., Sun, L. D., Yang, X., Handler, M., Kawarabayashi, T., Younkin, L., Fedeles, B., Wilson, M. A., Younkin, S., Kandel, E. R., Kirkwood, A., Shen, J. (2001) APP processing and synaptic plasticity in presenilin-1 conditional knockout mice. Neuron 31,713-726[CrossRef][Medline]
  59. Saura, C. A., Chen, G., Malkani, S., Choi, S. Y., Takahashi, R. H., Zhang, D., Gouras, G. K., Kirkwood, A., Morris, R. G., Shen, J. (2005) Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice. J. Neurosci. 25,6755-6764[Abstract/Free Full Text]
  60. Vingtdeux, V., Hamdane, M., Loyens, A., Gele, P., Drobeck, H., Begard, S., Galas, M. C., Delacourte, A., Beauvillain, J. C., Buee, L., Sergeant, N. (2007) Alkalizing drugs induce accumulation of amyloid precursor protein byproducts in lumenal vesicles of multivesicular bodies. J. Biol. Chem. 282,18197-18205[Abstract/Free Full Text]
  61. Kim, S. H., Leem, J. Y., Lah, J. J., Slunt, H. H., Levey, A. I., Thinakaran, G., Sisodia, S. S. (2001) Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein. J. Biol. Chem. 276,43343-43350[Abstract/Free Full Text]
  62. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416,535-539[CrossRef][Medline]
  63. Kwok, J. B., Li, Q. X., Hallupp, M., Whyte, S., Ames, D., Beyreuther, K., Masters, C. L., Schofield, P. R. (2000) Novel Leu723Pro amyloid precursor protein mutation increases amyloid β42(43) peptide levels and induces apoptosis. Ann. Neurol. 47,249-253[CrossRef][Medline]
  64. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr, Eckman, C., Golde, T. E., Younkin, S. G. (1994) An increased percentage of long amyloid β protein secreted by familial amyloid β protein precursor (βAPP717) mutants. Science 264,1336-1340[Abstract/Free Full Text]
  65. Eckman, C. B., Mehta, N. D., Crook, R., Perez-Tur, J., Prihar, G., Pfeiffer, E., Graff-Radford, N., Hinder, P., Yager, D., Zenk, B., Refolo, L. M., Prada, C. M., Younkin, S. G., Hutton, M., Hardy, J. (1997) A new pathogenic mutation in the APP gene (1716V) increases the relative proportion of Aβ42(43). Hum. Mol. Genet. 6,2087-2089[Abstract/Free Full Text]
  66. Langui, D., Girardot, N., El Hachimi, K. H., Allinquant, B., Blanchard, V., Pradier, L., Duyckaerts, C. (2004) Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice. Am. J. Pathol. 165,1465-1477[Abstract/Free Full Text]
  67. Takahashi, R. H., Milner, T. A., Li, F., Nam, E. E., Edgar, M. A., Yamaguchi, H., Beal, M. F., Xu, H., Greengard, P., Gouras, G. K. (2002) Intraneuronal Alzheimer Aβ42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am. J. Pathol. 161,1869-1879[Abstract/Free Full Text]
  68. De Gassart, A., Geminard, C., Fevrier, B., Raposo, G., Vidal, M. (2003) Lipid raft-associated protein sorting in exosomes. Blood 102,4336-4344[Abstract/Free Full Text]
  69. Keller, S., Sanderson, M. P., Stoeck, A., Altevogt, P. (2006) Exosomes: from biogenesis and secretion to biological function. Immunol. Lett. 107,102-108[CrossRef][Medline]
  70. Potolicchio, I., Carven, G. J., Xu, X., Stipp, C., Riese, R. J., Stern, L. J., Santambrogio, L. (2005) Proteomic analysis of microglia-derived exosomes: metabolic role of the aminopeptidase CD13 in neuropeptide catabolism. J. Immunol. 175,2237-2243[Abstract/Free Full Text]
  71. Goodman, Y., Mattson, M. P. (1994) Secreted forms of β-amyloid precursor protein protect hippocampal neurons against amyloid β-peptide-induced oxidative injury. Exp. Neurol. 128,1-12[CrossRef][Medline]
  72. Mattson, M. P., Cheng, B., Culwell, A. R., Esch, F. S., Lieberburg, I., Rydel, R. E. (1993) Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β-amyloid precursor protein. Neuron 10,243-254[CrossRef][Medline]
  73. Schubert, D., Behl, C. (1993) The expression of amyloid β protein precursor protects nerve cells from β-amyloid and glutamate toxicity and alters their interaction with the extracellular matrix. Brain Res. 629,275-282[CrossRef][Medline]
  74. Wolfe, M. S. (2002) APP, Notch, and presenilin: molecular pieces in the puzzle of Alzheimer’s disease. Int. Immunopharmacol. 2,1919-1929[CrossRef][Medline]
  75. Kimberly, W. T., Esler, W. P., Ye, W., Ostaszewski, B. L., Gao, J., Diehl, T., Selkoe, D. J., Wolfe, M. S. (2003) Notch and the amyloid precursor protein are cleaved by similar {gamma}-secretase(s). Biochemistry 42,137-144[CrossRef][Medline]
  76. McLean, C. A., Cherny, R. A., Fraser, F. W., Fuller, S. J., Smith, M. J., Beyreuther, K., Bush, A. I., Masters, C. L. (1999) Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in Alzheimer’s disease. Ann. Neurol. 46,860-866[CrossRef][Medline]
  77. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindleshurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., Schenk, D. (1992) Isolation and quantification of soluble Alzheimer’s β-peptide from biological fluids. Nature 359,325-327[CrossRef][Medline]
  78. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X., McKay, D. M., Tintner, R., Frangione, B., Younkin, S. G. (1992) Production of the Alzheimer amyloid β protein by normal proteolytic processing. Science 258,126-129[Abstract/Free Full Text]
  79. Caby, M. P., Lankar, D., Vincendeau-Scherrer, C., Raposo, G., Bonnerot, C. (2005) Exosomal-like vesicles are present in human blood plasma. Int. Immunol. 17,879-887[Abstract/Free Full Text]
  80. Faure, J., Lachenal, G., Court, M., Hirrlinger, J., Chatellard-Causse, C., Blot, B., Grange, J., Schoehn, G., Goldberg, Y., Boyer, V., Kirchhoff, F., Raposo, G., Garin, J., Sadoul, R. (2006) Exosomes are released by cultured cortical neurones. Mol. Cell. Neurosci. 31,642-648[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-9357comv1
fj.07-9357comv2
22/5/1469    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sharples, R. A.
Right arrow Articles by Hill, A. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sharples, R. A.
Right arrow Articles by Hill, A. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS