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Microglia, the resident immune cells of the CNS, play pivotal roles in the pathogenesis of Alzheimer’s disease (AD), such as mediating neuroinflammation and excessive synapse loss. Genome-wide association studies have identified several genes associated with AD risk that are expressed exclusively in microglia in the brain, including INPP5D. INPP5D encodes the phosphatase SHIP1, whose levels are reduced in human AD brains. Lower SHIP1 levels have been associated with improved plaque pathology and cognitive outcomes in mouse studies of AD, making INPP5D an emerging therapeutic target. However, changes in human microglia function upon reduced SHIP1 levels and precise mechanisms underlying these changes remain unknown. We have previously shown that acute SHIP1 inhibition with 3AC or knockdown of INPP5D via CRISPR/Cas9 in human iPSC-derived microglia (iMGs) activates the NLRP3 inflammasome, an inflammatory program that results in the release of pro-inflammatory cytokines IL-18 and IL-1B. Here, we investigated the cell-autonomous and non-autonomous changes induced by heterozygous INPP5D loss-of-function in iMGs to better understand the biological roles of SHIP1. First, we found that SHIP1 is important for maintaining the membrane stability and proper functioning of lysosomes in microglia. INPP5D knockdown induced lysosomal membrane permeabilization, assessed by immunostaining for galectin, and resulted in the leakage of lysosomal cathepsin enzymes into the cytosol. Moreover, BODIPY staining revealed increased lipid droplet accumulation in iMGs heterozygous for INPP5D, potentially due to decreased lysosome-mediated degradation of lipid droplets. Second, we found that SHIP1 regulates transcriptional immune response, and that reduction of SHIP1 levels primes microglia for inflammasome activation regardless of inflammatory signal exposure. While nuclear factor kappa B (NF-kB)-dependent transcription of NLRP3 inflammasome-related genes, including genes encoding proinflammatory cytokines, comprise the priming step of inflammasome activation, we found that iMGs heterozygous for INPP5D do not display a higher baseline expression of these genes. Intriguingly, these microglia also failed to upregulate the expression of these genes upon bacterial LPS exposure, a known activator of NF-kB-dependent transcription. These findings point to an unusual endotoxin tolerance in microglia with INPP5D knockdown and suggest that these cells may be primed for inflammasome activation via a mechanism other than NF-kB-dependent transcription. Third, we found that SHIP1 regulates phagocytosis selective for synapses. Using flow cytometry, we showed that INPP5D knockdown in iMGs leads to selective upregulation of synaptosome phagocytosis, while it does not affect fibrillar amyloid beta phagocytosis. Using immunostaining, we also identified a reduction of synapse numbers in iPSC-derived neurons co-cultured with iMGs with INPP5D knockdown, which indicates increased synapse pruning by these microglia. Taken together, these results suggest that SHIP1 regulates microglia functioning and inflammatory response, and that reduced microglial SHIP1 levels in AD promotes neuroinflammation and synapse loss.
