Abstract
BackgroundWe recently demonstrated an endolysosomal accumulation of the β-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Moreover, we showed that the treatment with the γ-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 γ-secretase inhibitor-induced APP-CTFs.MethodsEVs were purified from cell media or mouse brains from vehicle- or D6-treated C99 or APPswedish 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.ResultsPurified 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 γ-secretase blockade, since it similarly occurred in presenilin-deficient fibroblasts. Further, our data proposed that besides favoring APP-CTF oligomerization by preventing C99 proteolysis, γ-secretase inhibiton also led to a defective SorLA-mediated retrograde transport of HMW APP-CTFs from endosomal compartments to the TGN.ConclusionsThis 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 γ-secretase inhibition. Future studies should evaluate the putative contribution of these exosome-associated APP-CTFs in AD onset, progression and spreading.
Highlights
We recently demonstrated an endolysosomal accumulation of the β-secretase-derived amyloid precursor protein (APP) C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models
(2019) 8:35 (Continued from previous page). 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 γsecretase inhibition
Purified EVs from C99- or APP- expressing cells are enriched in APP-CTFs and contain high molecular weight APP-CTFs, the levels of which are increased by γ-secretase inhibition Our previous data obtained from immunostaining on brains from 3xTgAD and C99-expressing mice proposed the presence of extracellular vesicle-associated APPCTFs, the levels of which should be increased in γsecretase inhibitor treated mice [7]
Summary
We recently demonstrated an endolysosomal accumulation of the β-secretase-derived APP C-terminal fragment (CTF) C99 in brains of Alzheimer disease (AD) mouse models. Our previous work on the 3xTgAD mouse model demonstrated that endolysosomal-associated C99 was only detected with N-terminal-directed and aggregate-specific antibodies but not with C-terminal-directed APP antibodies. This staining was strongly increased in γ-secretase inhibitor treated-mice, in which C99 and C99-derived C83 (hereafter refered to as APP-CTFs) levels were enhanced and those of Aβ reduced, ruling out the possibility that it could be ascribed to Aβ. We confirmed the strong abundance of APP-CTFs in purified extracellular vesicles (EVs) from various cellular and mouse AD models and found that the treatment with the γ-secretase inhibitor D6 led to further increased levels of C99 and C99-derived C83. Our data showed that the selective recovery of dimeric/oligomeric APP-CTFs in EVs is linked to their intracellular mislocalization to the endolysosomal network due partly to a defective SorLA-mediated retrograde sorting of dimeric APP-CTFs to the TGN
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