Protective variant in PLC?2 mitigates Alzheimer's disease associated pathology via enhancing beneficial microglia functions

PLC?2-P522R (phospholipase C gamma 2, proline 522 to arginine) is a protective variant that reduces the risk for late onset Alzheimer's disease (LOAD). Recently, it was shown to decrease ß-amyloid pathology in 5XFAD mouse model of AD. In this study, our goal was to investigate the protective functions of PLC?2-P522R variant in less aggressive mouse model of AD as well as to assess underlying mechanisms at the molecular and cellular level using mouse and human microglia models. The effects of the protective PLC?2-P522R variant on microglia activation, AD-related ß-amyloid and neuronal pathologies, as well as behavioral changes were investigated in PLC?2-P522R knock-in mice crossbred with an APP/PS1 mouse model of AD. Transcriptomic, proteomic, and functional studies were carried out in cultured and acutely isolated adult PLC?2-P522R mouse microglia to study molecular mechanisms. Finally, microglia-like cell models generated from blood and skin biopsy samples of the PLC?2-P522R variant carriers were employed to translate the key findings in human cells. Our results demonstrate that the PLC?2-P522R variant reduces brain ß-amyloid plaque burden of APP/PS1 mice. Simultaneously, PLC?2-P522R variant increased non-proinflammatory microglia activation and microglia clustering around ß-amyloid plaques, leading to reduced ß-amyloid plaque-associated neuronal dystrophy. In cultured mouse primary microglia, PLC?2-P522R variant decreased accumulation of large lipid droplets, reduced cell stress, and increased acute response to strong inflammatory stimuli. Transcriptomic and proteomic analyses in acutely isolated adult mouse microglia as well as in human monocyte-derived microglial cells showed that PLC?2-P522R upregulates mitochondrial fatty acid oxidation and downregulates inflammatory/interferon signaling pathways. Accordingly, PLC?2-P522R increased mitochondrial respiration in iPSC -derived microglial cells. Together, these findings suggest that PLC?2-P522R variant exerts protection against AD-associated ß-amyloid and neuronal pathologies via enhancing microglial barrier formation around ß-amyloid plaques, but suppressing pro-inflammatory activation. Observed changes in fatty acid metabolism and mitochondrial flexibility as well as the downregulation of genes involved in inflammatory signaling pathways suggest that these protective effects of the PLC?2-P522R variant are mediated through an anti-ageing mechanism. Overall design: APP/PS1xPLCG2-P522R knock in mice (n=4) and respective APP/PS1 control animals (n=4) were sacrificed and transcardially perfused with saline at the age 13-months. Half of the brain was dissected into regional blocks and snap frozed in liquid nitrogen. Temporo-occipital cortex was then homogenized on DPBS and an aliquot was subjected to RNA sequencing (see protocol section). The whole brain of subset of above-described mice was used for acute CD11b+ microglia isolation utilizing magnetic bead-based isolation and subjected to RNA sequencing. Wildtype animals without the AD background were included as controls in the above mentioned analyses. Similarly, CD11b+ microglia isolated from 13-month-old PLCG2-P522R knock in (n=4) and wildtype (n=3) mice without the AD background were used for RNA sequencing studies.

PLCγ2-P522R（磷脂酶Cγ2，即phospholipase C gamma 2，脯氨酸522突变为精氨酸）是一种保护性变异体，可降低晚发型阿尔茨海默病（late onset Alzheimer's disease, LOAD）的发病风险。既往研究表明，该变异体可减轻AD的5XFAD小鼠模型中的β-淀粉样蛋白（β-amyloid）病理负荷。本研究旨在探究PLCγ2-P522R变异体在低侵袭性AD小鼠模型中的保护功能，并利用小鼠及人类小胶质细胞（microglia）模型，从分子与细胞层面解析其潜在作用机制。

我们通过将PLCγ2-P522R敲入小鼠（knock-in mice）与AD的APP/PS1小鼠模型杂交，探究了保护性PLCγ2-P522R变异体对小胶质细胞活化、AD相关β-淀粉样蛋白与神经元病理，以及行为学改变的影响。为解析分子机制，我们对培养的及急性分离的成年PLCγ2-P522R小鼠小胶质细胞开展了转录组学（transcriptomic）、蛋白质组学（proteomic）及功能学研究。最后，我们利用来自PLCγ2-P522R变异携带者的血液与皮肤活检样本构建的小胶质细胞样细胞模型，将关键研究发现转化至人类细胞体系中进行验证。

本研究结果显示，PLCγ2-P522R变异体可降低APP/PS1小鼠的脑内β-淀粉样蛋白斑块负荷。与此同时，该变异体可促进非促炎型小胶质细胞活化，并增强小胶质细胞向β-淀粉样蛋白斑块周围的募集，进而减少β-淀粉样蛋白斑块相关的神经元营养不良。在培养的小鼠原代小胶质细胞中，PLCγ2-P522R变异体可减少大脂滴的蓄积、减轻细胞应激，并增强对强炎症刺激的急性应答反应。对急性分离的成年小鼠小胶质细胞及人类单核细胞来源的小胶质细胞（monocyte-derived microglial cells）进行的转录组学与蛋白质组学分析显示，PLCγ2-P522R可上调线粒体脂肪酸氧化过程，并下调炎症/干扰素信号通路。相应地，PLCγ2-P522R可增强诱导多能干细胞（iPSC, induced pluripotent stem cell）来源的小胶质细胞的线粒体呼吸功能。

综上，上述研究结果表明，PLCγ2-P522R变异体通过增强β-淀粉样蛋白斑块周围的小胶质细胞屏障形成，同时抑制促炎型活化，从而对AD相关的β-淀粉样蛋白与神经元病理发挥保护作用。观察到的脂肪酸代谢与线粒体灵活性的改变，以及炎症信号通路相关基因的下调，提示PLCγ2-P522R变异体的这些保护效应可能通过抗衰老机制介导。

总体实验设计：将APP/PS1xPLCG2-P522R敲入小鼠（n=4）及其对应的APP/PS1对照小鼠（n=4）在13月龄时处死，经心脏灌注生理盐水。将一半脑组织切成区域组织块，于液氮中快速冷冻。随后将颞枕皮层在Dulbecco's磷酸缓冲盐溶液（DPBS）中匀浆，取部分样本进行RNA测序（具体流程见实验方案章节）。将上述部分小鼠的全脑用于基于磁珠分选的急性CD11b+小胶质细胞分离，并进行RNA测序。无AD背景的野生型动物作为上述分析的对照。同样，我们从13月龄、无AD背景的PLCG2-P522R敲入小鼠（n=4）及野生型小鼠（n=3）中分离CD11b+小胶质细胞，用于RNA测序研究。