Targeting γ-secretase triggers the selective enrichment of oligomeric APP-CTFs in brain extracellular vesicles from Alzheimer cell and mouse models

Background: We recently demonstrated an endolysosomal accumulation of the beta-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Moreover, we showed that the treatment with the gamma-secretase inhibitor (D6) led to further increased endolysosomal APP-CTF levels, but also revealed extracellular APP-CTF-associated immunostaining. We here hypothesized that this latter staining could reflect extracellular vesicle (EV)-associated APP-CTFs and aimed to characterize these gamma-secretase inhibitor-induced APP-CTFs. Methods: EVs were purified from cell media or mouse brains from vehicle- or D6-treated C99 or APP(swedish) expressing cells/mice and analyzed for APP-CTFs by immunoblot. Combined pharmacological, immunological and genetic approaches (presenilin invalidation and C99 dimerization mutants (GXXXG)) were used to characterize vesicle-containing APP-CTFs. Subcellular APP-CTF localization was determined by immunocytochemistry. Results: Purified EVs from both AD cell or mouse models were enriched in APP-CTFs as compared to EVs from control cells/brains. Surprisingly, EVs from D6-treated cells not only displayed increased C99 and C99-derived C83 levels but also higher molecular weight (HMW) APP-CTF-immunoreactivities that were hardly detectable in whole cell extracts. Accordingly, the intracellular levels of HMW APP-CTFs were amplified by the exosomal inhibitor GW4869. By combined pharmacological, immunological and genetic approaches, we established that these HMW APP-CTFs correspond to oligomeric APP-CTFs composed of C99 and/or C83. Immunocytochemical analysis showed that monomers were localized mainly to the trans-Golgi network, whereas oligomers were confined to endosomes and lysosomes, thus providing an anatomical support for the selective recovery of HMW APP-CTFs in EVs. The D6-induced APP-CTF oligomerization and subcellular mislocalization was indeed due to.-secretase blockade, since it similarly occurred in presenilin-deficient fibroblasts. Further, our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis, gamma-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN. Conclusions: This is the first study to demonstrate the presence of oligomeric APP-CTFs in AD mouse models, the levels of which are selectively enriched in endolysosomal compartments including exosomes and amplified by gamma-secretase inhibition. Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset, progression and spreading.