Understanding the protective effect of the PLCG2 P522R variant in human microglia

Abstract

AbstractBackgroundAlthough recent advances in the field suggest significant involvement of the shared pathway between the proteins ApoE and TREM2 in the pathogenesis of late onset Alzheimer’s disease (LOAD), the direction that disease‐modifying interventions targeting this pathway should take are still under debate.Interestingly, a variant of the Phospholipase C Gamma 2 (PLCG2), a downstream effector of the TREM2 receptor, has been associated with a reduced risk of developing dementia. This variant results in a single amino acid change, which was reported to increment enzyme activity. However, mutations in this same enzyme (S707Y, L848P), which also result in hyperactivity, were found to lead to auto‐inflammatory immune diseases or increased AD risk (M28L), suggesting that there may be specific modulatory effects of the P522R variant. Alternatively, the existence of diverging downstream signaling pathways may explain its protective potential.MethodWe have generated a complete set of human isogenic iPSCs derived from the Bioni037A parental line using Crispr/Cas9 edition to introduce the mutations for the different PLCG2 variants. We obtained homozygous and heterozygous clones with the protective P522R mutation, the immune disease related S707Y or L848P. We also acquired ApoE4 isogenic iPSCs. Microglia differentiation was induced from these iPSCs using a protocol involving embryoid bodies. Our approach includes lipidomics and proteomics analyses, with focus on microglial lipid metabolism.ResultsHere, we show the generation of new human isogenic iPSC lines to study PLCG2 mutations. Preliminary results from the characterization of human iPSC‐derived ApoE4 microglia employing this multi‐omics approach shows a reduced expression of TREM2 pathway components including the tyrosine kinases SYK and BTK. Since BTK and SYK are both PLCG2 kinases that result in enzyme activation, we are expanding this analysis to PLCG2 mutants and base our working hypothesis on the possibility that PLCG2 hyperactive variants show a reverse phenotype compared to ApoE4 microglia.ConclusionWe present new tools and a rationale to explore the protective effect of the PLCG2 P522R variant against LOAD in human microglia.