AbstractBackgroundAlzheimer’s disease (AD) is the most common form of dementia of which the incidence increases with age. No cures are available yet, urging to explore new strategies for the identification of pathways that can be targeted therapeutically. Research in AD has been majorly driven by studying familial AD (FAD)‐associated mutations in the APP and PSEN genes, albeit they represent only a minor fraction of all cases. GWAS studies have identified ≈30 late‐onset AD (LOAD) susceptibility loci that, when functionally clustered, are directly or indirectly linked to cellular trafficking routes and functioning. These include genes linked to cholesterol metabolism (e.g. APOE4, ABCA7, INPP5D), endocytic transport regulation (e.g. BIN1, CD2AP, PICALM, SORL1) and lysosomal processes (e.g. GRN, PLD3). The genetic findings concur with the appearance of endolysosomal abnormalities at preclinical stages, before the appearance of senile plaques and tangles.MethodTo investigate the role of LOAD risk factors involved endosomal transport regulation, we generated knock‐out (KO), and their corresponding rescued cell lines for several proteins including PSENs, PICALM and PLD3 and this in fibroblasts and neuronal SHS5Y5Y cells. These are now subjected to a comparative analysis of the endolysosomal pathway and lysosomal function.ResultPSENdKO cells present with enlarged late endosomes/lysosomes that (i) display an increased co‐localization with early endosomal markers, (ii) accumulate various lipids as shown by lipidome profiling of isolated organelles, and (iii) accumulate various cargoes and adaptors such as the retromer subunit VPS35. These aberrant accumulations could be corrected by the re‐expression of catalytic active and inactive human PSEN1, indicating that a γ‐secretase‐independent role of PSEN1 is partially connected to recycling. Interestingly, activation of recycling could significantly alleviate endolysosomal defects observed in PSEN KO cells. Ongoing analysis of LOAD risk genes shows both similar as well as distinct endolysosomal defects underscoring that a possible common endosomal deregulations may originate from different upstream mechanisms.ConclusionTogether, these observations are shedding light on the potential of modulating/restoring endosomal transport regulation as a strategy to alleviate observed early endolysosomal dysfunctions at preclinical stages of AD pathogenesis.