The Alzheimer’s disease protective R522 variant of PLCg2, consistently enhances PLCγ2 activation, depleting PI(4,5)P 2 levels and altering cell function in in vitro and in vivo assays

Background Recent GWAS studies have identified the R522 variant in PLCg2 as protective against late onset Alzheimer’s disease (AD). Expression of PLCg2 in the brain is predominantly found in microglia. PLCg2 has been shown to catalyse the hydrolysis of PIP 2 to DAG and IP 3 leading to increased intracellular Ca 2+ . Intracellular Ca 2+ has been linked to NLRP3 inflammasome activation and increased phagocytic activity. This suggests that the R522 variant may alter the response time of microglia in conditions such as AD. Method Using CRISPR‐engineering novel PLCG2‐R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model were generated. From these, primary murine microglia, murine macrophages and hIPSC derived microglia cultures were created. The role of PLCg2 in the intracellular Ca 2+ was investigated by detecting cytoplasmic Ca 2+ increase, following activation of PLCg2 in human (anti‐CD32) and murine (anti‐FcgII/III) models. This was further investigated using specific PLCg inhibitors and a GapmeR knockdown system. The phagocytic and endocytic activity of these cells was investigated using pHrodo‐Red bound zymosan, E.coli and dextran, and fluorescent amyloid oligomers. PIP 2 levels were detected using immunostaining and mass ELISAs. Molecular dynamic stimulation was used to ensemble and analyse the variant. Result Cells with the R522 mutation demonstrate hyperfunctionality manifesting as enhanced cellular Ca 2+ store release in response to physiologically relevant stimuli. This resulted in increased PIP 2 depletion in the cells with the R522 variant after exposure to stimuli and reduced basal detection of PIP 2 levels in vivo . These PLCγ2‐R522 associated abnormalities led to impairments to phagocytosis and enhanced endocytosis. The variant was found to be in the auto‐inhibitory domain. Conclusion These results indicate a gain of function mutation in PLCγ2 which increases calcium release from the ER but depletes substrate levels resulting in a smaller reservoir of PIP 2 available for other functions. This is consistent with an alteration in the auto‐inhibitory domain. Phagocytosis, which has a larger PIP 2 requirement, is decreased while endocytosis which has a lower PIP 2 requirement is increased. PLCγ2 sits downstream of disease relevant pathways and as an enzyme is classically druggable thus raises the prospect of PLCγ2 as a therapeutic target for AD.