Protective variant in PLC2 mitigates Alzheimers disease associated pathology via enhancing beneficial microglia functions

PLC2-P522R (phospholipase C gamma 2, proline 522 to arginine) is a protective variant that reduces the risk for late onset Alzheimers disease (LOAD). Recently, it was shown to decrease -amyloid pathology in 5XFAD mouse model of AD. In this study, our goal was to investigate the protective functions of PLC2-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 PLC2-P522R variant on microglia activation, AD-related -amyloid and neuronal pathologies, as well as behavioral changes were investigated in PLC2-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 PLC2-P522R mouse microglia to study molecular mechanisms. Finally, microglia-like cell models generated from blood and skin biopsy samples of the PLC2-P522R variant carriers were employed to translate the key findings in human cells. Our results demonstrate that the PLC2-P522R variant reduces brain -amyloid plaque burden of APP/PS1 mice. Simultaneously, PLC2-P522R variant increased non-proinflammatory microglia activation and microglia clustering around -amyloid plaques, leading to reduced -amyloid plaque-associated neuronal dystrophy. In cultured mouse primary microglia, PLC2-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 PLC2-P522R upregulates mitochondrial fatty acid oxidation and downregulates inflammatory/interferon signaling pathways. Accordingly, PLC2-P522R increased mitochondrial respiration in iPSC -derived microglial cells. Together, these findings suggest that PLC2-P522R variant exerts protection against AD-associated -amyloid and neuronal pathologies via enhancing microglial barrier formation around -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 PLC2-P522R variant are mediated through an anti-ageing mechanism. 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.PLC2-P522R（磷脂酶Cγ2，脯氨酸522突变为精氨酸）是一种保护性变异体，可降低晚发性阿尔茨海默病（late onset Alzheimer's disease, LOAD）的发病风险。既往研究证实，该变异可减轻5XFAD阿尔茨海默病小鼠模型中的β-淀粉样蛋白病理损伤。本研究旨在探究PLC2-P522R变异体在低侵袭性阿尔茨海默病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。 本研究通过将PLC2-P522R敲入小鼠与APP/PS1阿尔茨海默病小鼠模型杂交，分析了保护性PLC2-P522R变异体对小胶质细胞活化、阿尔茨海默病相关β-淀粉样蛋白及神经元病理损伤，以及行为学变化的影响。研究人员通过体外培养及急性分离的成年小鼠小胶质细胞，开展转录组学、蛋白质组学及功能学实验以解析分子机制。最后，本研究利用来自PLC2-P522R变异携带者的血液及皮肤活检样本构建的小胶质细胞样细胞模型，将关键研究结果转化至人类细胞体系中验证。 本研究结果显示，PLC2-P522R变异体可降低APP/PS1小鼠的脑内β-淀粉样蛋白斑块负荷。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-淀粉样蛋白斑块周边的募集聚集，从而减轻β-淀粉样蛋白斑块相关的神经元营养不良。在体外培养的小鼠原代小胶质细胞中，PLC2-P522R变异体可减少大脂滴蓄积、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进行转录组学与蛋白质组学分析后发现，PLC2-P522R可上调线粒体脂肪酸氧化通路，并下调炎症/干扰素信号通路。相应地，在诱导多能干细胞（induced pluripotent stem cell, iPSC）诱导的小胶质细胞中，PLC2-P522R可提升线粒体呼吸功能。 综上，PLC2-P522R变异体通过增强β-淀粉样蛋白斑块周边的小胶质细胞屏障形成，同时抑制促炎型小胶质细胞活化，从而发挥对抗阿尔茨海默病相关β-淀粉样蛋白及神经元病理损伤的保护作用。本研究观察到的脂肪酸代谢与线粒体可塑性改变，以及炎症信号通路相关基因的下调，提示PLC2-P522R变异体的保护效应可能通过抗衰老机制介导。 本研究对APP/PS1xPLCG2-P522R敲入小鼠（n=4）及同基因型APP/PS1对照小鼠（n=4）在13月龄时实施安乐死，并经心脏灌注生理盐水进行全身灌流。取半侧脑组织制备组织块，于液氮中快速冷冻。随后将颞枕叶皮层在杜氏磷酸盐缓冲液（DPBS）中匀浆，取部分匀浆样本进行RNA测序（具体实验流程参见方法部分）。取部分上述小鼠的全脑组织，利用磁珠分选法分离CD11b阳性小胶质细胞，并对其进行RNA测序。本研究同时纳入无阿尔茨海默病背景的野生型小鼠作为上述分析的对照。同样地，我们对13月龄无阿尔茨海默病背景的PLCG2-P522R敲入小鼠（n=4）及野生型小鼠（n=3）分离得到的CD11b阳性小胶质细胞进行RNA测序分析。