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The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and A\u03b2 generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for \u03b2-secretase (BACE1)-mediated \u03b2-secretase cleavage of \u03b2-amyloid precursor protein (APP) to generate Amyloid-\u03b2 (A\u03b2), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in \u03b2-secretase mediated cleavage of APP and A\u03b2 generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on \u03b2-secretase-mediated cleavage of APP and A\u03b2 generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1\/BIN1V9 on \u03b2-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure A\u03b2 generation using A\u03b2 ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates \u03b2-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated A\u03b2 generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced A\u03b2 generation\u2014all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated \u03b2-secretase processing of APP and A\u03b2 generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern A\u03b2 production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.<\/jats:p>","DOI":"10.1038\/s41598-022-07372-4","type":"journal-article","created":{"date-parts":[[2022,3,3]],"date-time":"2022-03-03T11:05:35Z","timestamp":1646305535000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["The neuronal-specific isoform of BIN1 regulates \u03b2-secretase cleavage of APP and A\u03b2 generation in a RIN3-dependent manner"],"prefix":"10.1038","volume":"12","author":[{"given":"Raja","family":"Bhattacharyya","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catarina Amelia Fidalgo","family":"Teves","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra","family":"Long","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madison","family":"Hofert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rudolph E.","family":"Tanzi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,3,3]]},"reference":[{"key":"7372_CR1","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1186\/1750-1326-9-31","volume":"9","author":"X Wang","year":"2014","unstructured":"Wang, X. et al. 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