Depletion of the AD risk gene SORL1 causes endo‐lysosomal dysfunction in human microglia

Abstract

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder which is currently incurable. Prior to well‐established neuropathological changes in AD, enlarged early endosomes are cytopathological hallmark of AD and strong genetic evidence links genes involved in intra‐cellular trafficking, mediated by endo‐lysosomal pathway, to increased AD risk, suggesting that this pathway is integral to the pathogenesis of AD. We recently identified SORL1, an intracellular sorting receptor as a potential regulator of this pathway. Using CRISPR/Cas9 to deplete SORL1 in human induced pluripotent stem cells (hiPSCs), we showed that loss of SORL1 results in enlarged early endosomes in (hiPSCs)‐derived neurons, but not microglia. This model replicates the earliest AD cytopathology and our data underscores important cell‐type specific differences in response to loss of SORL1. Building on this study, we further examined alterations in lysosomal function, with altered expression of SORL1 in hiPSC‐derived microglia.MethodIsogenic hiPSC lines that are deficient in SORL1 were generated and differentiated into microglia using previous published protocols. Lysosome function was assayed by examining lysosome morphology, lysosomal trafficking of fibrillar Aβ, lysosomal degradation, lysosome enzyme activity and lysosomal exocytosis. Lysosome morphology and number were measured using immunocytochemistry, confocal microscopy and Cell Profiler and Image J software. To assess Aβ transport into lysosomes, pH sensitive fluorescent dye, Phrodo, conjugated to Aβ, was used. Lysosomal degradation was measured using a proteolysis sensitive fluorogenic substrate, DQ‐BSA. Fluorescence intensity of both Phrodo and DQ‐BSA was measured using flow cytometry. Lysosome enzyme activity and exocytosis assays were performed using previously published protocols.ResultLoss of SORL1 results in increased lysosome size and decreased lysosome number in SORL1 KO microglia indicating altered lysosome function. In support of this idea, we observed decreased lysosomal degradation, trafficking of Aβ to lysosomes and lysosomal enzyme activity and also decreased lysosomal exocytosis in SORL1 KO microglia.ConclusionOverall, our results have unraveled a previously unexplored function of the AD risk gene SORL1 in human microglia and our work indicates that maintaining homeostatic balance in the endo‐lysosomal network impacts diverse CNS cell types. Our study will define the endo‐lysosomal network in general, and SORL1 in particular, as a novel therapeutic target for AD.