ENHANCED FUNCTION OF PLCG2 VARIANT P522R IN MACROPHAGES AND MICROGLIA IS ASSOCIATED WITH PROTECTION FROM ALZHEIMER’S DISEASE

Abstract

Evidence from GWAS suggests the P522R variant in the enzyme PLCg2 is protective against Alzheimer's disease (AD). This variant lies in the auto-inhibitory component of the PLCg2 gene, and such mutations could possibly alter the activation of PLCg2. Expression of PLCg2 in the brain is predominantly found in microglia and microglia have been implicated in the risk of AD. PLCg2 catalyses the hydrolysis of PIP2 to IP3 and DAG, with IP3 causing intracellular Ca2+ release. Intracellular Ca2+ release causes multiple cellular responses, such as phagocytosis. This suggests that the P522R variant may alter the responsiveness of microglia in conditions such as AD. Several techniques were utilized to investigate function of PLCg2 within the variant. This included molecular dynamic stimulation (GROMACS), run in the NPT ensemble and resulting structures were analysed using RMSD and visualized for structural differences using VMD. In addition, we investigated cytoplasmic Ca2+ increase (Fura2 and Fluo-8), indicative of a signalling response, following activation of PLCg2 in human (using anti-CD32) and murine (using anti-CD16/32 and Ab) models. Novel human P522R KOLF2 iPSC lines and P522R knockin mice were generated using CRISPR-assisted gene-targeting. We used iPSC-derived human microglia and primary murine cortical and hippocampal microglia alongside macrophages derived from conditionally-immortalized mouse macrophage precursor cell lines. The requirement for PLCg2 in the intracellular Ca2+ changes were investigated using specific PLCg inhibitors (edelfosine, U73122) and a specific GapmeR knockdown system. Computational models suggest that structural changes in the P522R mutant would reduce binding of inhibitors. In addition, Ca2+ signalling assays in all the cellular models consistently demonstrated increased cytosolic Ca2+ release in P522R variants compared to controls following activation of PLCg2. Our results are the first to show that the protective P522R variant exhibits increased activity in microglia and macrophages in response to physiological stimuli. This protection most likely results from alteration of microglia response after exposure to the environmental cues present in AD. PLCg2 represents a strong pharmacological target suggesting the possibility of a new intervention site for AD.