Functional relationships linking C99/APP-βCTF dimerization, proteostasis disruption, and organelle dysfunction.

backgroundThe amyloid β (Aβ) precursor C99 (or APP-βCTF) accumulates in Alzheimer's disease and has been proposed to display Aβ-independent toxicity, notably by affecting the endosomal-lysosomal-autophagic (ELA) network. Our previous findings suggested that some ELA-associated C99 could correspond to dimeric and oligomeric species, but the intracellular sites of C99 dimerization, as well as the toxicity linked to it, remains unknown.

methodsWe here developed a bimolecular fluorescence complementation (BiFC) probe to visualize de novo C99 dimerization and dimer trafficking, as well as to identify possible cellular responses specifically linked to C99 dimerization. Moreover, to confirm dimer localizations and toxicities, the localization and cellular effects of the dimerization mutant C99

resultsBiFC-C99 dimers were first detected within the TGN, in which monomers initially accumulate. The proteasomal inhibitor MG-132 led to increased dimer formation, indicating that the proteasomal activity status is a key determinant of C99 dimerization. Conversely, TGN-associated C99 dimerization had a negative impact on both the ubiquitin-proteasome system (UPS) and the TGN, as highlighted by the appearance of p62/SQSTM1-positive aggresomes and fragmented Golgi, then suggesting a two-way relationship between UPS function and C99 dimerization. Dimerization also led to lysosome repositioning and to the accumulation of LC3B-positive autophagy vesicles, agreeing with the well-known interplay between autophagy and proteasome in protein turnover. P62/SQSTM1 and LC3B accumulation could similarly be observed in cells expressing C99

conclusionsTaken together, our findings indicate that the cellular status of UPS, autophagy and γ-secretase activities are all determinant for C99 expression levels, and are thus crucial for both the level of C99 dimerization and for the fate of the dimers. Moreover, our data show that C99 dimerization itself negatively affects these activities thereby indicating a two-way relationship between C99 dimerization, proteostasis disruption and organelle dysfunction.