LRRK2调控小胶质细胞突触修剪影响5xFAD小鼠认知行为异常的机制

Abnormal synaptic pruning by microglia leads to synaptic plasticity dysfunction in Alzheimer's disease (AD). LRRK2 is highly expressed in microglia, involves in neuroimmune response and regulates the synaptogenesis in central nervous system. However, it remain unclear that the role and its mechanism of LRRK2 on the AD. Interestingly, our preliminary studies firstly found that the expressions of LRRK2, complement production C1qa, C3 and the complement receptor CR3 mRNA in hippocampus and striatum were significantly higher in 5XFAD mice than that of age-match wild-type mice. In vitro, the LRRK2 protein and inflammatory transcription factor NFAT2 were increased in Aβ oligomers-stimulated microglia. Notably, LRRK2 knockout alleviated the loss of TH positive synaptic fiber density in striatum and improved the moter coordinative function in the early AD model mice (3 month-old 5xFAD mice). However, the immunologic mechanism of synaptic pruning regulated by microglial LRRK2 is not yet clear. Studies have shown that LRRK2 or complement C1q modulated PKA activity. We hypothesize that LRRK2 modulates synaptic pruning and synaptic plasticity by microglia through complement system, eventually could affect the neuropathologic and immunologic damages and the abnormality of cognition and behaviors in AD. Therefore, this project will set up a mouse model of 5xFAD;LRRK2-cKO;CX3CR1-CreER that time-induced by tamoxifen-conditional knockouting microglial LRRK2 gene in 5xFAD mice. Using the electrophysiologic, confocal microscope and molecular biological technologies, we will explore how LRRK2 regulate complement activation, lysosomal pathaway and synaptic pruning in microglia whereby fine-tuned by PKA signals in the AD model.The present study will support an important conceptual advance that complement system and microglia-mediated synaptic pruning are major triggers of AD caused by LRRK2, which provide a new strategy for targeting microglial LRRK2 against AD.

小胶质细胞异常突触修剪引起阿尔茨海默病(AD)突触可塑障碍。LRRK2在小胶质细胞高表达、参与免疫反应、调控突触发生，但在AD中作用及机制不清。我们前期研究发现AD鼠海马和纹状体LRRK2和补体C1qa、C3mRNA表达显著增高;体外Aβ寡聚体能刺激小胶质细胞LRRK2蛋白升高;敲除LRRK2减轻了早期AD鼠纹状体TH突触丢失、改善运动行为能力。研究已显示LRRK2、补体可调节PKA活力。我们推测:抑制小胶质细胞LRRK2可调节小胶质细胞补体/PKA信号影响突触修剪及重塑、减轻AD免疫损害和认知行为异常。本项目构建他莫昔芬诱导条件敲除小胶质细胞LRRK2基因5xFAD痴呆鼠模型(5xFAD;LRRK2-cKO;CX3CR1-CreER),应用电生理/共聚焦/分子技术,从整体-细胞-分子水平验证上述假说,揭示LRRK2调控小胶质细胞活化及突触修剪的分子机制,为LRRK2靶点防治AD提供新思路

研究显示LRRK2突变与AD病理改变明显关联，然而LRRK2在AD中的具体作用及其机制还不清楚。我们前期研究发现AD转基因鼠海马和纹状体LRRK2和补体C1qa、C3及CR3受体mRNA表达显著增高；Aβ寡聚体刺激小胶质细胞LRRK2及炎症转录因子（NFAT2）蛋白明显增高；早期敲除LRRK2基因的5xFAD小鼠海马或纹状体C1qa、C3及CR3 mRNA表达减低；敲除LRRK2减轻早期5xFAD小鼠纹状体TH+突触纤维丢失、减轻了小胶质细胞激活、提高AD小鼠的运动协调能力，但LRRK2如何调节补体和PKA信号激活影响小胶质细胞异常活化和突触修剪？以及LRRK2调节补体活化后吞噬、修剪突触的具体途径是什么还不清楚？因此，在前期基础上，我们大胆地提出“在痴呆早期敲除小胶质细胞LRRK2基因可能减轻中老年期AD痴呆鼠的行为异常和神经病理免疫损害，其机制可能是通过小胶质细胞补体系统和内吞溶酶体信号影响突触修剪发挥作用”的科学假说。本项目应用5xFAD敲减LRRK2基因小鼠模型，分别从动物、细胞、分子层次证明我们的假说。结果发现：LRRK2基因敲减可以减轻AD转基因小鼠小胶质细胞产生的免疫炎症反应，抑制小胶质细胞和星型胶质细胞的激活、促进Aβ的清除，减轻5xFAD转基因小鼠的突触损伤和丢失，改善AD小鼠的运动及认知能力，其机制可能通过降低5xFAD小鼠中LRRK2基因剂量从而改变小胶质细胞反应性重编程来实现。同时LRRK2基因剂量的降低对5xFAD转基因小鼠表型的多方面的益处与其LRRK2激酶活性的缺失有特异相关性。这些为LRRK2为靶标防治AD脑免疫紊乱、改善AD病理病程提供了新思路和策略。同时，以本研究为基础的临床前研究所开发的LRRK2分子探针和LRRK2的生物学标记物的样本检测试剂盒，将在未来应用于LRRK2相关的认知障碍神经变性病的临床免疫检测和临床治疗，具有很好的科学意义和临床应用前景。