Validating PLCG2 as a target for the treatment of Alzheimer's Disease through cell biology and pharmacological tools

Indiana University Indianapolis, Indianapolis IN

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Genetic studies have identified novel Alzheimer’s Disease (AD) protective and risk associated genes and genetic variants. PLCG2 is one such gene with protective (P522R) and risk (M28L) variants making it a useful protein to target for the treatment of AD. However, the particular phenotype to be targeted through small molecule modulation of PLCG2 is unknown. Without this knowledge, the validation of PLCG2 as a target for the treatment of AD will be greatly impeded. Therefore, we hypothesize that activation of PLCG2 will mimic the protective effect of PLCG2P522R by inducing molecular signaling changes to PLCG2WT in a manner similar to the protective variant. We will address this hypothesis through the following Specific Aims: Aim 1: Determine how PLCG2 variants associated with AD risk and protection affect PLCG2 related signaling, in vitro. We hypothesize that these variants cause their respective effects (protection and risk) by modulating the preference of PLCG2 to be recruited to the cell membrane, its phosphorylation state, its preferred interaction complexes, and its turnover rate. We will test this hypothesis by stimulating human induced pluripotent stem cell (iPSC) derived microglia like cells (MGLs) expressing either wildtype (WT), protective, or risk variants of PLCG2, and then use both mass spectrometry and traditional biochemical methods to measure these molecular effects. Aim 2: Determine how modulation of PLCG2 activity affects PLCG2WT microglia, in vitro, compared to protective and risk PLCG2 variants. We hypothesize that activation of PLCG2 will produce an activation profile that overlaps with PLCG2P522R microglia. We will test this hypothesis by comparing pAKT levels and Aβ uptake between WT MGLs treated with already developed, novel, small molecule modulators of PLCG2, and vehicle treated MGLs bearing the protective and risk variants. With respect to outcomes as a consequence of the work proposed, we expect to identify changes in the phosphorylation state, subcellular localization, interactome, and turnover rate of PLCG2 caused by the protective and risk variants. We also expect to identify the effect of small molecule modulation of PLCG2 activity on pAKT levels and Aβ uptake with reference to the protective and risk variants. These results will advance our understanding of how PLCG2P522R protects from AD and provide insight into the pharmacological requirements of a commercial PLCG2 modulator as a potential AD therapeutic. Additionally, we expect that this study will guide the design of therapeutic interventions that mimic the protective functions of the PLCG2P522R variant. Finally, this study will contribute to the continued understanding of the role of microglia in response to AD pathology. Overall, this study will have a positive impact because it will provide a biological basis for understanding the pharmacology of novel therapeutics targeting PLCG2 for the treatment of AD.