The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function – Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation. The hypermorphic PLCγ2 S707Y variant dysregulates microglial cell function – Insight into PLCγ2 activation in brain health and disease, and opportunities for therapeutic modulation

Phospholipase C-gamma 2 (PLCγ2) is highly expressed in hematopoietic and immune cells, where it is a key signalling node enabling diverse cellular functions. Within the periphery, gain-of-function (GOF) PLCγ2 variants, such as the strongly hypermorphic S707Y, cause severe immune dysregulation. The milder hypermorphic mutation PLCγ2 P522R increases longevity and confers protection in central nervous system (CNS) neurodegenerative disorders, implicating PLCγ2 as a novel therapeutic target for treating these CNS indications. Currently, nothing is known about what consequences strong PLCγ2 GOF has on CNS functionality, and more precisely on the specific biological functions of microglia. Using the PLCγ2 S707Y variant as a model of chronic activation we investigated the functional consequences of strong PLCγ2 GOF on human microglia. PLCγ2 S707Y expressing human inducible pluripotent stem cells (hiPSC)-derived microglia exhibited hypermorphic enzymatic activity under both basal and stimulated conditions, compared to PLCγ2 wild type. Despite the increase in PLCγ2 enzymatic activity, the PLCγ2 S707Y hiPSC-derived microglia display diminished functionality for key microglial processes including phagocytosis and cytokine secretion upon inflammatory challenge. RNA sequencing revealed a downregulation of genes related to innate immunity and response, providing molecular support for the phenotype observed. Our data suggests that chronic activation of PLCγ2 elicits a detrimental phenotype that is contributing to unfavourable CNS functions, and informs on the therapeutic window for targeting PLCγ2 in the CNS. Drug candidates targeting PLCγ2 will need to precisely mimic the effects of the PLCγ2 P522R variant on microglial function, but not those of the PLCγ2 S707Y variant. Overall design: RNA was extracted from PLCγ2 WT and S707Y hiPSC-derived microglia from three separately generated hematopoietic inductions, at different myeloid precursor differentiation ages (early, middle and late). Libraries were prepared using the KAPA RNA HyperPrep Kit and sequenced on an Illumina HiSeq 4000 sequencer at a minimum of 25 million paired-end reads (75 bp) per sample performed by UCL Genomics (London, England).

磷脂酶C-γ2（Phospholipase C-gamma 2, PLCγ2）在造血与免疫细胞中呈高表达，是介导多种细胞功能的关键信号节点。在外周组织中，功能获得性（gain-of-function, GOF）PLCγ2变异体（如强超形态突变体S707Y）可引发严重的免疫失调。而温和的超形态突变PLCγ2 P522R则能够延长生物体寿命，并对中枢神经系统（central nervous system, CNS）神经退行性疾病起到保护作用，这提示PLCγ2可作为治疗此类中枢神经系统适应症的新型治疗靶点。

目前学界尚未明确强效PLCγ2功能获得性突变对中枢神经系统功能，尤其是小胶质细胞的特定生物学功能所产生的影响。本研究以PLCγ2 S707Y变异体作为慢性激活模型，探究了强效PLCγ2功能获得性突变对人类小胶质细胞的功能效应。实验结果显示，相较于PLCγ2野生型细胞，表达PLCγ2 S707Y的人类诱导多能干细胞（human inducible pluripotent stem cells, hiPSC）衍生小胶质细胞，在基础状态与刺激条件下均表现出增强的酶活性。尽管PLCγ2的酶活性有所升高，但经炎症刺激后，PLCγ2 S707Y hiPSC衍生小胶质细胞的关键小胶质细胞功能（包括吞噬作用与细胞因子分泌）却出现显著减弱。RNA测序结果表明，与先天免疫及免疫应答相关的基因表达出现下调，为所观察到的表型提供了分子层面的实验佐证。

本研究数据表明，PLCγ2的慢性激活会诱发有害表型，损害中枢神经系统正常功能，并为中枢神经系统靶向PLCγ2的治疗窗口提供了理论依据。靶向PLCγ2的候选药物需要精准模拟PLCγ2 P522R变异体对小胶质细胞功能的调控效应，而非复刻PLCγ2 S707Y变异体的作用效果。

整体实验设计：从3批独立诱导生成的造血前体细胞中，分别提取处于不同髓系前体分化阶段（早期、中期、晚期）的PLCγ2野生型与S707Y hiPSC衍生小胶质细胞的总RNA。采用KAPA RNA HyperPrep试剂盒构建测序文库，并由英国伦敦大学学院基因组学中心（UCL Genomics, London, England）在Illumina HiSeq 4000测序仪上完成测序，每个样本的双端测序读长均不低于2500万条（读长75 bp）。