Tracking endocytosed APP in the endo-lysosomal pathway in a model of AD

ANTONINO, MAGDALENA; ALMIRON, ROMINA; PICONÊZ PEREIRA, TIAGO MIGUEL; ALFREDO, LORENZO; GUIMAS ALMEIDA, CLAUDIA; BIGNANTE, ELENA ANAHI

Rosario

Congreso; Congreso Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2023

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Alzheimer´s Disease is a neurodegenerative disorder characterized by brain deposition of amyloid-β (Aβ). We recently found that Aβ assemblies, oligomers and fibrils, increase APP and BACE1 convergence and interaction in recycling endosomes of human neurons derived from iPSCs through Go/Gβγ signalling, which leads to an increase in APP processing by BACE1 and intracellular accumulation of Aβ1-42. Now, we are interested in deepen on Aβ effects on the APP trafficking in the endocytic pathway. We started transfecting N2A cells with whether APP:YFP or BiFC (a Bimolecular Fluorescent System to asses APP and BACE1 interaction) and amyloidogenic compartments markers fused to the RFP. Next, we treated the cultures with gallein, a Gβγ subunit inhibitor, and Aβ assemblies. We found that Aβ induced an increment of APP levels and its interaction with BACE1 in recycling endosomes and Golgi Apparatus and decreased APP levels and its interaction with BACE1 in lysosomes, effects that were abrogated by gallein. These results suggest that Aβ induces a redirection of APP to endosomes, avoiding its default route to degradation in lysosomes. Also, in order to test if this enrichment of APP in endosomes could be due to an increase in the APP endocytosis rate, we performed a pulse-chase assay that consisted of starving cells with serum free medium, incubating with anti-APP (clone 22C11) in complete medium (pulse) and chasing for 0, 30 and 60 min. We found that Aβ notably enhanced APP endocytosis rate by a mechanism dependent on Gβγ signalling. Furthermore, Aβ significatively increased the number of endocytosed 22C11-positive vesicles by a Gβγ signalling pathway. Finally, we evaluated if this accumulation of APP and its fragments in endosomes is related to the endosomal dysfunction largely described in AD patients so we measured the size and number of endosomes and we found that Aβ increased the number and size of both recycling and early endosomes by a Gβγ signalling. In conclusion, Aβ assemblies induce an increase in the APP endocytosis rate and a its redirection to endosomes avoiding lysosomes by a Gβγ signalling, which leads to APP accumulation in endosomes where it interacts with BACE1 favouring its amyloidogenic processing. Also, this accumulation of APP and BACE1 in endosomes leads to the enlargement and increase the number of endosomes, another early pathological sign of AD.