Received: 6 April 2020 | Revised: 11 July 2020 | Accepted: 20 July 2020 DOI: 10.1096/fj.202000823R RESEARCH ARTICLE Extracellular vesicles: where the amyloid precursor protein carboxyl-terminal fragments accumulate and amyloid-β oligomerizes Rocío Pérez-González1,2 | Yohan Kim1,2 | Chelsea Miller1 | Javier Pacheco-Quinto3 | Elizabeth A. Eckman3 | Efrat Levy1,2,4,5 1 Center for Dementia Research, Nathan S. Kline Institute, Orangeburg, NY, USA 2 Department of Psychiatry, New York University School of Medicine, New York, NY, USA 3 Biomedical Research Institute of New Jersey, Cedar Knolls, and Atlantic Health Systems, Morristown, NJ, USA 4 Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA 5 Neuroscience Institute, New York University School of Medicine, New York, NY, USA Correspondence Rocío Pérez-González, Sant Pau Biomedical Research Institute (IIB-Sant Abstract Pau), Sant Quintí 77-79, 08041, Barcelona, Pleiotropic roles are proposed for brain extracellular vesicles (EVs) in the develop- Spain. ment of Alzheimer's disease (AD). Our previous studies have suggested a beneficial Email: rperezgo@santpau.cat role for EVs in AD, where the endosomal system in vulnerable neurons is compro- Present address mised, contributing to the removal of accumulated material from neurons. However, Rocío Pérez-González, Sant Pau the involvement of EVs in propagating AD amyloidosis throughout the brain has Biomedical Research Institute (IIB-Sant Pau), Center for Networked Biomedical been considered because the amyloid-β precursor protein (APP), APP metabolites, Research on Neurodegenerative Diseases and key APP cleaving enzymes were identified in association with EVs. Here, we (CIBERNED), and Department of undertook to determine whether the secretase machinery is actively processing APP Neurology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain in EVs isolated from the brains of wild-type and APP overexpressing Tg2576 mice. We found that full-length APP is cleaved in EVs incubated in the absence of cells. Funding information Alzheimer's Association, Grant/Award The resulting metabolites, both α- and β-APP carboxyl-terminal fragments and APP Number: NIRG-14-316622; National intracellular domain accumulate in EVs over time and amyloid-β dimerizes. Thus, Institute on Aging, Grant/Award Number: EVs contribute to the removal from neurons and transport of APP-derived neurotoxic AG017617 and AG056732 peptides. While this is potentially a venue for propagation of the pathology through- out the brain, it may contribute to efficient removal of neurotoxic peptides from the brain. Abbreviations: Aβ, amyloid-β; AD, Alzheimer's disease; AICD, APP intracellular domain; APP, Amyloid-β precursor protein; APP-CTFs, APP carboxyl- terminal fragments; DMEM, Dulbecco's Modified Eagle Medium; EVs, Extracellular vesicles; flAPP, full-length amyloid-β precursor protein; PS1, Presenilin-1; PS2, Presenilin-2. Rocío Pérez-González and Yohan Kim contributed equally to this study. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology 12922 |  wileyonlinelibrary.com/journal/fsb2 The FASEB Journal. 2020;34:12922–12931. | 15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License PÉREZ-GONZÁLEZ et al     12923 KEYWORDS Aβ oligomers, Alzheimer's disease, APP-CTFs, APP metabolism, exosomes 1 | IN T RO D U C T IO N deleterious effects. β-APP-CTFs are also a source of Aβ pep- tides that will assemble into oligomeric forms prior to the Extracellular vesicles (EVs) are highly stable membranous formation of senile plaques.29 These oligomeric forms of Aβ structures that are secreted by most cell types and can be are considered a major culprit in the synaptic dysfunction and subsequently taken up by recipient cells (reviewed in 1). neuronal loss that characterize AD.30-32 The process of oligo- The various species of EVs have different origins. While merization and subsequent aggregation of Aβ into fibrils can microvesicles bud directly from the plasma membrane, exo- be facilitated by EVs, as it has been shown that they can act somes have an endosomal origin because they are formed by as amyloid “seeding” sites.33,34 Further, the identification in invagination of the membrane of late endosomes (also called EVs of key enzymes involved in APP metabolism9,10,35,36 has multivesicular bodies).2 Given that sorting EVs derived from suggested that EVs are a potential site for the de novo gener- a particular biogenesis pathway remains difficult, the general ation of APP-CTFs and Aβ. Because EVs are oriented with term EVs will be used here to refer to small EVs enriched the cytoplasmic-side facing inward,37 the same orientation as with exosomes, following the International Society for EVs in the plasma membrane, it was hypothesized that cleavage recommendations.3 EV secretion was originally described as of flAPP would result in capture of APP-CTFs in the mem- a complementary process to the lysosomal and proteosomal brane of EVs and in the secretion of EV-generated Aβ into degradative pathways to discard obsolete membrane and cy- the extracellular space. Here, we investigated whether flAPP tosolic proteins in differentiating reticulocytes.4 Accordingly, is enzymatically cleaved in EVs in the absence of cells. our previous research has supported a beneficial role for EVs in vulnerable neurons with endosomal pathology, one of the earliest insults observed in Alzheimer's disease (AD),5 by 2 | M ATERIAL S AND M ETHOD S relieving the cells from toxic waste that was not efficiently degraded intracellularly.6,7 However, the identification of 2.1 | Mice full-length amyloid-β precursor protein (flAPP) and amy- loid-β precursor protein (APP) metabolites in EVs8-11 has Brains were derived from wild-type C57/B6 mice at the implicated EVs in AD amyloid pathology by carrying and age of 12 months and from transgenic mice overexpressing spreading pathology throughout the brain. The accumulation human APP with the K670N/M671L Swedish double muta- of the amyloid-β (Aβ) peptide in extracellular senile plaques tion (Tg2576)38 at 4 months of age. Both females and males is one of the pathological hallmarks of AD.12 Aβ is gener- were used for all analyses. All animal procedures were per- ated through sequential processing of flAPP, which under- formed following the National Institutes of Health guidelines goes proteolytic cleavage by α- and β-secretases to produce with approval from the Institutional Animal Care and Use α- and β-carboxyl-terminal fragments (APP-CTFs), respec- Committee at the Nathan S. Kline Institute for Psychiatric tively. β-APP-CTF (also referred to as C99) is the source of Research. Aβ following cleavage by γ-secretase,13 which also results in the generation of the APP intracellular domain (AICD).14 Processing of flAPP occurs in the plasma membrane and 2.2 | Brain EV isolation in endosomes15 and APP metabolites can be incorporated into EVs16 and released from the cells into the extracellu- Brain EVs were isolated and purified as we have previously lar environment. However, it was suggested that only a min- described.9 Briefly, frozen brain tissues were treated with ute fraction of total Aβ is associated with EVs,9,16 unlike 20 units/mL of papain (Worthington, Lakewood, NJ, USA) APP-CTFs, which are abundantly present within EVs.8-11,17 in Hibernate A solution (HA, 3.5 mL/sample; BrainBits, Accumulation of β-APP-CTFs in specific brain regions is a Springfield, IL, USA) for 15 minutes at 37°C. The brain tissue common early process in different AD mouse models that was gently dissociated in 6.5 mL of cold HA supplemented may contribute to AD pathology (reviewed in 18). Multiple with protease inhibitors, centrifuged at 300 g for 10 minutes studies have shown that β-APP-CTFs are neurotoxic, caus- at 4°C to discard the cells, and the supernatant was sequen- ing neuronal endosomal-lysosomal abnormalities19-21 lead- tially filtered through a 40 μm mesh filter (BD Biosciences, ing to neuronal cell death and memory loss.22-28 Therefore, San Jose, CA, USA) and a 0.2 μm syringe filter (Corning the transport of β-APP-CTF via EVs throughout the brain Life Sciences, Teterboro, NJ, USA). The filtrates were se- may result in the uptake of this peptide by neurons with quentially centrifuged at 4°C, at 2000 g for 10 minutes and |    15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12924 PÉREZ-GONZÁLEZ et al 10 000 g for 30 minutes to discard membranes and debris, the 10 µM of Aβ40 stock was diluted in DMEM to make a and at 100 000 g for 70 minutes to pellet the EVs. The EV 300 nM of Aβ40 solution, which was further diluted to the pellet was resuspended in 60 mL of cold PBS (Thermo Fisher final concentration of 10 nM in the solution used for the Scientific), and centrifuged at 100 000 g for 70 minutes at Western blot analysis. 4°C. The washed EV pellet was resuspended in 2 mL of 0.95 M sucrose solution and inserted inside a sucrose step gradient column (six 2-mL steps from 2.0 to 0.25 M sucrose). 2.5 | Western blot analysis The sucrose step gradient was centrifuged at 200 000 g for 16 hours and fractions were collected from the top of the gra- The same amount of EV proteins was separated by 4%-20% dient. The fractions were diluted in cold PBS and centrifuged Tris-HCl gel electrophoresis (Criterion precast gel, Bio-Rad, at 100 000 g for 70 minutes for pellet collection. Hercules, CA, USA) and transferred onto PVDF membranes (Immobilon, Millipore). Membranes were incubated with an- tibodies to HSC70 (1:1000, Cat# sc-7298, RRID:AB_62776; 2.3 | Incubation of isolated EVs at 37°C Santa Cruz Biotechnology), CD63 (1:1000, Cat# ab217345, RRID:AB_2754982; Abcam), APP and APP-CTFs Brain EV pellets from fractions C and D of the sucrose step (C1/6.1,39 1:1000), BACE1 (1:1000, Cat# 200-401-984, gradient9 were resuspended in 30 µL each of Dulbecco's RRID:AB_2243187; Rockland), ADAM10 (1:1000, Cat# Modified Eagle Medium (DMEM, Thermo Fisher Scientific) AB19026, RRID:AB_2242320; Millipore), Nicastrin and combined. The pooled EVs were divided into experi- (1:1000, Cat# MAB5556, RRID:AB_2235791; Millipore). mental groups incubated for the indicated times (0, 1, 4, 24, The antibodies to the subunits of the γ-secretase complex: 48, or 72 hours), in the absence or presence of either the PS1 (N-terminal, 1:1000), PS2 (N-terminal, 1:50), APH1a γ-secretase inhibitor L685,458, or Aβ-degrading enzyme in- (C-terminal, 1:1000), and APH1b (C-terminal, 1:1000) were hibitors. The same volume of DMEM containing 2X EDTA a kind gift from Dr Paul Fraser, University of Toronto. The (except in the experiments using inhibitors of Aβ-degrading PEN-2 antibody (N-terminal, 1:2500) was a kind gift from Dr enzymes), without or supplemented with γ-secretase inhibi- Thinakaran, University of Chicago. The secondary antibod- tor or an Aβ-degrading enzyme inhibitor, was added to the ies used were HRP-conjugated anti-rabbit or anti-mouse an- EV suspensions. The γ-secretase inhibitor, L685,458 (Tocris tibodies (Jackson ImmunoResearch, West Grove, PA, USA). Bioscience, Minneapolis, MN, USA) was used at the final The membranes were incubated in chemiluminescent fluid concentration of 10 µM. Inhibitors of Aβ-degrading enzymes (Pierce, Rockford, IL, USA) and chemiluminescence was (thiorphan [Cayman] and phosphoramidon [Sigma-Aldrich]) visualized on X-ray films. For identification of Aβ dimers, were added to the EV suspensions at the final concentrations the same amount of EV proteins was separated by 16.5% tris- of 10 and 100 µM, respectively. Though each can inhibit mul- tricine gels, blotted with a 1:1 mixture of the 6E10 (1:1000; tiple metalloproteases, at these concentrations thiorphan is Cat# 803001, RRID:AB_2564653; BioLegend) and 4G8 selective for neprilysin over endothelin-converting enzymes (1:1000; Cat# SIG-39220, RRID:AB_662812; Covance) an- (ECEs) and phosphoramidon inhibits neprilysin and ECEs. tibodies on PVDF. 6E10 and 4G8 antibodies are generated At time 0 hour EVs were immediately lysed in 2X RIPA against different epitopes within Aβ.40 For visualization of buffer (1% Triton-X, 1% Sodium deoxycholate, 0.1% SDS, Aβ monomers and AICD, EV proteins separated by 16.5% 150 mM NaCl, 50 mM Tris-HCl pH 7.4, and 1 mM EDTA) tris-tricine gels were transferred onto Nitrocellulose (Bio- supplemented with 2X Halt Protease inhibitors (Thermo Rad), which was subsequently boiled in PBS for 4 minutes, Fisher Scientific) and 1X EDTA (Thermo Fisher Scientific). followed by blotting in 6E10 for Aβ monomer or C1/6.1 for At times 1, 4, 24, 48, and 72 hours EVs were placed in a AICD detection. Protein bands were quantified using ImageJ 37°C bath incubator for the time periods indicated and subse- (National Institute of Health (NIH), Bethesda, MD, USA). quently lysed in 2X RIPA buffer. All lysates were sonicated for 45 seconds, placed on ice for 20 minutes with vortex- mixing every 5 minutes and kept at −80°C until analysis. 2.6 | ELISA Aβ peptides were analyzed by sandwich ELISA with specific 2.4 | Preparation of Aβ peptide solution mouse monoclonal antibodies, as described previously.41 For human Aβ40 measurement, antibody MM27-33.1.1 For preparation of the 10 µM peptide stock, lyophilized (raised against human Aβ 1-16) was used for capture and Aβ40 peptide (2 µg) was dissolved and equilibrated in dime- MM32-13.1.1-HRP (raised against Aβ35-40) for detec- thyl sulfoxide (Sigma-Aldrich) for 15 minutes at room tem- tion. For Aβ42, anti-Aβ42 antibody MM26-2.1.3 (Aβ35-42) perature with vortex-mixing every 3 minutes. Subsequently, was used for capture and HRP-conjugated 4G8 antibody | 15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License PÉREZ-GONZÁLEZ et al     12925 (Aβ17-24) was used for detection. Aβ antibodies except for a 24-hour incubation, generating both α- and β-APP-CTFs 4G8 (Covance, Princeton, NJ, USA) were from Mayo Clinic (Figure 2A,B) at levels higher than in EVs isolated from the (Rochester, MN, USA). brain of wild-type mice (Figure 2A). Incubation in the pres- ence of the γ-secretase inhibitor, L685,458 had no significant effect on the levels of the APP-CTFs (Figure 2A). An indi- 2.7 | Data analysis and graphing cation for the γ-secretase cleavage of the APP-CTFs in the incubated EVs is the increase in the level of AICD following The statistical and graphing software Prism 5.01 (GraphPad 24-hour incubation and the absence of the increase in AICD Software, La Jolla, CA, USA) was used for data analysis and level when the EVs were treated with the γ-secretase inhibi- graphical representation. For column graphs, column bars tor, L685,458 (Figure 2A,C). represent means ± SEM. To compare the relative changes in In order to investigate whether Aβ is generated in situ in the levels of α- and β-APP-CTFs, Aβ monomers, Aβ dimers EVs isolated from the brain of Tg2576 mice, we performed and AICD, analysis of variance (one-way ANOVA) followed Western blotting experiments. We observed a decrease in the by Tukey's post hoc multiple comparison was performed level of monomeric Aβ (Figure 3A,B) and an increase in the (Figures 2 and 3). level of Aβ dimers (Figure 3A,C) following 24 hours of EV incubation. These findings were not affected by the treat- ment with the γ-secretase inhibitor, L685,458 (Figure 3A- 3 | R E S U LTS C). While the SDS-PAGE band of ~8-9 kDa could derive from N-terminal extended (NTE) Aβ forms,45,46 the absence 3.1 | APP present in EVs is enzymatically of an effect of the γ-secretase inhibitor on the levels of the processed to generate APP-CTFs ~8-9 kDa band (Figure 3A), excludes the possibility of NTE Aβ generation during the incubation. These results suggest In order to study whether flAPP is cleaved in situ in EVs, that EV-associated monomeric Aβ was oligomerized into Aβ we investigated the presence in EVs of the α-secretase dimers during the incubation. The level of Aβ was also an- ADAM10,42 the β-secretase BACE1,43 and all the compo- alyzed by sensitive ELISA measurements of Aβ peptides in nents of the γ-secretase, a membrane protein complex com- EVs incubated for 24 hours in DMEM. We found a robust de- prised of Presenilin-1 (PS1) or Presenilin-2 (PS2), APH-1a or crease in the level of monomeric Aβ40 (Figure 3D), consis- APH-1b, Nicastrin, and PEN-2.44 In addition to the confirma- tent with the Western blotting results. The level of Aβ42 was tion of the presence of previously identified secretases, we below the assay detection level (data not shown). The reduc- found all the γ-secretase complex subunits in EVs isolated tion in Aβ level could be due to the activity of EV-associated from wild-type mouse brain (Figure 1A). Aβ-degrading enzymes such as neprilysin,47 insulin-degrad- We then tested whether these secretases are functional in ing enzyme (IDE),48 or endothelin-converting enzymes-1 and situ, generating APP-CTFs. EVs isolated from the brain of −2 (ECE-1 and ECE-2),49 suggested to be active in the extra- wild-type mice were incubated in DMEM in the absence of cellular space.50 Thus, we pretreated the brain EVs with the cells at 37°C for different periods of time. Following incuba- metalloprotease inhibitors phosphoramidon and thiorphan, tion, the EVs with the medium in which they were incubated in order to inhibit the activities of these enzymes. Treatment were lysed in RIPA buffer and analyzed by Western blotting. with these inhibitors did not affect the monomeric Aβ levels The results showed that flAPP is enzymatically processed to (Figure 3E), suggesting that the decrease in Aβ levels was generate α- and β-APP-CTFs in a time-dependent manner not due to degradation by the metalloproteases against which (Figure 1B), confirming the de novo generation of APP-CTFs inhibitors were used. We also found no effect of the inhib- in brain EVs. itors on the level of Aβ dimers (Figure 3F). An increase in the ratio of Aβ dimers to Aβ monomers during EV incuba- tion was observed in the absence and presence of the inhibi- 3.2 | APP-CTFs are further processed into tors (Figure 3G), suggesting a sustained Aβ oligomerization AICD in EVs and Aβ peptides oligomerize during EVs incubation. We next determined whether the γ-secretase complex in EVs is enzymatically active to generate Aβ and AICD. Given the 4 | DISCUSSION low Aβ production in the brain of wild-type mice, we iso- lated EVs from the brain of APP overexpressing transgenic We have previously reported that EVs are enriched with mice, Tg2576,38 at an age prior to the initiation of amyloid APP-CTFs.8,9 Given that an early event that characterizes deposition. We first corroborated that APP processing oc- AD is a dysfunctional endosomal-lysosomal system (re- curs in EVs isolated from the brain of Tg2576 mice during viewed in 51), we proposed that the release of endosomal |    15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12926 PÉREZ-GONZÁLEZ et al F I G U R E 1 flAPP is enzymatically processed in brain EVs to generate α- and β-APP-CTFs in a time dependent manner. A, Western blot analyses revealing the presence of the APP processing enzymes ADAM10 (α-secretase), BACE1 (β-secretase), and of all the components of the γ-secretase complex: PS1, PS2 APH1a, APH1b, Nicastrin, and PEN-2. Wild-type mouse brain homogenate (BH) was used as a positive control and arrow-heads indicate the specific protein band for each panel. B, Western blot analysis using an antibody against the carboxyl-terminus of APP (C1/6.1) showing an increase in the levels of α- and β-APP-CTFs in wild-type brain EVs during incubation at 37°C for 24, 48, and 72 hours. Antibodies against the exosomal markers HSC70 and CD63 were used in the lower panels F I G U R E 2 APP-CTFs in brain EVs are enzymatically cleaved by the γ-secretase complex, leading to the generation of AICD. A, Western blot analysis with the C1/6.1 antibody of EVs isolated from the brain of 4-month-old Tg2576 and wild-type (WT) mice prior to (0 hour) and after incubation at 37°C in DMEM without (24 hours) or with 10 µM of the γ-secretase inhibitor L-685,458 (24 hours+). B, Quantification of α- and β-APP-CTFs levels in EVs isolated from the brain of Tg2576 mice revealed an increase in the levels of both metabolites. C, The levels of AICD also increased and the increase was blocked by inhibition of the γ-secretase complex. PVDF membranes were used for APP and APP-CTFs detection, whereas nitrocellulose was used for detection of AICD. One-way ANOVA followed by Tukey's post hoc multiple comparison, n = 7 independent experiments (B), n = 3 independent experiments (C). Data are shown as mean ± SEM (**P < .01; ****P < .0001) | 15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License PÉREZ-GONZÁLEZ et al     12927 materials into the extracellular space via EVs is a benefi- and release can exacerbate7 and perhaps lead to endoso- cial mechanism by which neurons remove accumulated mal-lysosomal pathway disturbances.52 Here, we demon- materials, and that failure of efficient exosome production strate that APP-CTFs are generated by an active secretase |    15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12928 PÉREZ-GONZÁLEZ et al F I G U R E 3 Aβ present at time zero in EVs oligomerizes into dimers. A, Western blot analysis of EVs isolated from the brain of 4-month- old Tg2576 and wild-type (WT) mice prior to (0 hour) and after incubation without (24 hours) or with 10 µM of L-685,458 (24 hours+) shows the bands for Aβ monomers (lower panel) and dimers. Quantification of the corresponding bands shows a decrease in the level of monomeric Aβ (B) and an increase in the level of the dimers (C) following incubation. DMEM with 10 nM of Aβ40 peptide was used as a positive control of Aβ monomeric and oligomeric forms. D, Aβ40 measurements of brain EVs by ELISA prior to (0 hour) or after a 24-hour incubation without (24 hours) or with L-685,458 (24 hours+). E and F, Western blot analysis of brain EVs after incubation for 1, 4, and 24 hours without (−) or with the metalloprotease inhibitors Phosphoramidon (Pho) or Thiorphan (Thi). Aβ monomers (E) and dimers (F) are indicated by arrowheads. G, The protein bands were quantified in all conditions and presented as the ratio between Aβ dimers and Aβ monomers. Nitrocellulose membranes were used for detection of Aβ monomers (E), whereas PVDF was used for detection of Aβ dimers (F). One-way ANOVA followed by Tukey's post hoc multiple comparison, n = 3 independent experiments (B), n = 7 independent experiments (C), n = 5 independent experiments (D), and n = 4 independent experiments (G). Data are shown as mean ± SEM (*P < .05; **P < .01; ***P < .001; ****P < .0001) machinery and accumulate in EVs over time. Therefore, demonstrated that EVs promote Aβ aggregation on their EVs may contribute to the accumulation in the brain of surface.33,34,65 Since APP processing occurs in endosomes, β-APP-CTFs, suggested to be involved in the initiation of EV-associated Aβ may have an intracellular origin through neurodegenerative processes associated with endosomal- its incorporation into intraluminal vesicles that will be re- lysosomal21,27,53 and autophagy dysfunction,54,55 neu- leased as exosomes upon fusion of the late endosome with roinflammation,28,53 and synaptic alterations leading to the plasma membrane. While we detected an increase in the cognition and memory deficits in AD.26,28,56 Consistently, levels of APP-CTFs in EVs during ex vivo incubation, the it was shown that the treatment with γ-secretase inhibitors levels of EV-associated monomeric Aβ decreased dramat- leading to APP-CTFs accumulation impairs cognitive func- ically. Although EVs have been implicated in the extracel- tion in transgenic mice57 and worsens cognitive deficits in lular enzymatic degradation of Aβ,48,68 the reduction in Aβ AD patients.58 Interestingly, inhibition of γ-secretase in- levels that we observed was not due to active Aβ-degrading creased the levels of APP-CTFs and oligomeric APP-CTFs metalloproteases present in EVs. However, we cannot rule in EVs in an AD mouse model.59 Here, we demonstrate out the possibility that other proteases present in EVs are that AICD, previously identified in EVs,11,17 is also gener- mildly contributing to the degradation of Aβ. The decrease ated in brain EVs. Given the orientation of flAPP in the in the levels of the monomeric Aβ during the incubation membrane, it is likely that AICD is released into and accu- was mostly associated with rapid assembly of the exist- mulates within EV lumen. It was demonstrated that in ad- ing monomeric Aβ into dimers, which was the most en- dition to AICD being a regulator of gene expression60 and riched Aβ oligomer species in association with the EVs. cell-signaling pathways,61 it has deleterious effects such as Altogether, the data suggest that only negligible de novo causing hyperphosphorylation and age-dependent aggrega- generation of Aβ occurs in EVs and that Aβ associated with tion of tau, which recapitulates the features of AD,62 and the EVs has mainly an intracellular origin. During incuba- impairing hippocampal neurogenesis through the induction tion of the EVs, the Aβ dimer/monomer ratio increased in of neuroinflammation.63 While we did not observe sig- a time-dependent manner. These results are consistent with nificant changes in the levels of APP-CTF when the EVs previous reports showing that Aβ oligomers are rapidly se- were incubated in the presence of a γ-secretase inhibitor, questered on membranes due to their low stability in the the decrease in the levels of AICD in the presence of the aqueous extracellular space.69 The lower molecular weight γ-secretase inhibitor indicates that γ-secretase is indeed ac- Aβ oligomers such as dimers have been suggested to be es- tive in EVs. This is likely due to the continuous cleavage pecially neurotoxic because they can act as seeds for further of flAPP by α- and β-secretases leading to the accumula- aggregation.70,71 In addition, a recent study demonstrated tion of α- and β-APP-CTFs in the EV membrane and a low that Aβ dimers derived from AD brain mediated neuronal γ-secretase activity resulting in only a small part of APP- hyperactivity, which characterizes the early stage of AD, CTFs being further processed by γ-secretase. through block of synaptic glutamate reuptake.72 The Aβ di- Although less abundant than APP-CTFs, the peptide Aβ mers-enriched EVs may thus have a neurotoxic role at the has also been identified in association with EVs.9,16 The early stages of AD by Aβ oligomerization into dimers and association between Aβ and EVs is supported by the find- we suggest that EV-associated Aβ dimers could be used as ings of specific surface molecules in EVs that mediate their a diagnostic marker to detect early AD. interaction with Aβ, such as glycosphingolipids glycans,64 More than a decade has passed since EVs were proposed ceramides,65 and the GPI-anchored protein PrPc.66 The to participate in the spread of Aβ pathology in the AD brain.16 interactions of Aβ with PrP, glycosphingolipids, and cer- Accordingly, it has been recently demonstrated that EVs are amide localized in lipid raft domains may also target intra- vehicles for the intercellular transmission of APP and APP cellular amyloids to EVs.67 Regardless the Aβ origin, it was metabolites,17 including oligomeric Aβ.73 Our results support | 15306860, 2020, 9, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202000823R by Readcube (Labtiva Inc.), Wiley Online Library on [03/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License PÉREZ-GONZÁLEZ et al     12929 that brain EVs are not only carriers, but also a site for the gen- R E F E R E NC E S eration of APP-CTFs and AICD, and for the oligomerization 1. van Niel G, D'Angelo G, Raposo G. Shedding light on the of Aβ, all neurotoxic peptides. Thus, EVs may propagate the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. disease within the brain by delivering toxic metabolites into 2018;19:213-228. 2. Colombo M, Raposo G, Thery C. Biogenesis, secretion, and inter- recipient neurons. Indeed, it was shown that inhibition of EV cellular interactions of exosomes and other extracellular vesicles. secretion is protective by decreasing amyloid plaque depo- Annu Rev Cell Dev Biol. 2014;30:255-289. sition and ameliorating the cognitive impairment in trans- 3. Thery C, Witwer KW, Aikawa E, et al. Minimal information for genic APP mice.65 However, apparently contradictory data studies of extracellular vesicles 2018 (MISEV2018): a position suggest that EVs may act as potent scavengers for Aβ in the statement of the International Society for Extracellular Vesicles brain64,74 and contribute to the clearance of toxic proteins by and update of the MISEV2014 guidelines. J Extracell Vesicles. microglia uptake33,75 or by enzymatic degradation of Aβ.76 2018;7:1535750. Whether EVs drive pathology spread or serve as a pathway 4. Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma for the removal of toxic molecules within the brain, as we membrane activities with released vesicles (exosomes). J Biol have previously suggested,7,8 is dependent on a balance be- Chem. 1987;262:9412-9420. tween the release and efficient removal of EVs from the brain 5. Cataldo AM, Mathews PM, Boiteau AB, et al. Down syndrome extracellular space. Given that the contribution of microglia fibroblast model of Alzheimer-related endosome pathology: accel- to Aβ clearance is reduced in advanced AD,77 this balance erated endocytosis promotes late endocytic defects. Am J Pathol. is likely to tilt toward the pathological side of EVs as the 2008;173:370-384. disease progresses. 6. D'Acunzo P, Hargash T, Pawlik M, Goulbourne CN, Perez- To conclude, our data along with a growing body of re- Gonzalez R, Levy E. Enhanced generation of intraluminal vesi- cles in neuronal late endosomes in the brain of a Down syndrome search suggest a working model where at early stages of AD, mouse model with endosomal dysfunction. Dev Neurobiol. EVs have a protective role for cells in the brain. EVs are 2019;79:656-663. secreted loaded with APP and neurotoxic APP metabolites 7. Gauthier SA, Perez-Gonzalez R, Sharma A, et al. Enhanced exo- (APP-CTFs, AICD, and Aβ) to relieve neurons from accu- some secretion in Down syndrome brain - a protective mechanism mulated toxic material. At that stage, EVs transport the ma- to alleviate neuronal endosomal abnormalities. Acta Neuropathol terial to either microglia for degradation, or out of the brain Commun. 2017;5:65. into the circulation. However, in advanced stages of AD with 8. Perez-Gonzalez R, Gauthier SA, Sharma A, et al. A pleiotropic role for exosomes loaded with the amyloid beta precursor protein car- compromised endosomal-lysosomal and removal systems, boxyl-terminal fragments in the brain of Down syndrome patients. EVs can be sites for the generation and accumulation of APP Neurobiol Aging. 2019;84:26-32. metabolites, which would potentiate AD-associated neurode- 9. Perez-Gonzalez R, Gauthier SA, Kumar A, Levy E. The exosome generation. Therefore, determining the spatiotemporal func- secretory pathway transports amyloid precursor protein carbox- tions of brain EVs in AD is crucial to further understand AD yl-terminal fragments from the cell into the brain extracellular pathogenesis. space. J Biol Chem. 2012;287:43108-43115. 10. Sharples RA, Vella LJ, Nisbet RM, et al. Inhibition of gam- ACKNOWLEDGMENTS ma-secretase causes increased secretion of amyloid precursor pro- tein C-terminal fragments in association with exosomes. FASEB J. The authors thank Dr Monika Pawlik for managing the 2008;22:1469-1478. mouse colonies. This work was supported by the Alzheimer's 11. Vingtdeux V, Hamdane M, Loyens A, et al. Alkalizing drugs Association Grant NIRG-14-316622 (to RP-G.) and the induce accumulation of amyloid precursor protein by-prod- National Institute on Aging (NIA) Grants AG017617 and ucts in luminal vesicles of multivesicular bodies. J Biol Chem. AG056732 (to EL). 2007;282:18197-18205. 12. Glenner GG, Wong CW. Alzheimer's disease: initial report of the CONFLICT OF INTERESTS purification and characterization of a novel cerebrovascular amy- The authors declare that they have no conflict of interests. loid protein. Biochem Biophys Res Commun. 1984;120:885-890. 13. Haass C, Kaether C, Thinakaran G, Sisodia S. Trafficking and proteolytic processing of APP. Cold Spring Harb Perspect Med. AUTHOR'S CONTRIBUTIONS 2012;2:a006270. E. Levy and R. Pérez-González conceived the ideas and wrote 14. Passer B, Pellegrini L, Russo C, et al. Generation of an apop- and revised the manuscript. R. Pérez-González, Y. Kim, and totic intracellular peptide by gamma-secretase cleavage of E. Levy designed research. R. Pérez-González, Y. Kim, C. Alzheimer's amyloid beta protein precursor. J Alzheimers Dis. Miller, and J. Pacheco-Quinto performed experiments and 2000;2:289-301. analyzed data. R. Pérez-González, Y. Kim, J. Pacheco- 15. Nixon RA. 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