LRRK2基因调控NFAT-CX3CR1表达影响小胶质细胞活化在帕金森病发病中的机制研究

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of late-onset Parkinson’s disease (PD). Accumulating evidence in PD models suggests that microgliosis plays a pivotal role in both disease initiation and progression. LRRK2 is constitutively expressed in not only in neurons but also in microglia that is associated with α-synuclein (α-syn) deposit and clearance in PD. However, little is known about any potential pathophysiological interplay between LRRK2 and α-syn-mediated microglia dysfunction in PD. Our preliminary data showed that LRRK2 knockout mice acted as resistant to microglial activation in striatum elicited by stereotactic injection of lipopolysaccharide (LPS). And LRRK2 Inhibition also attenuated inflammatory and phagocytosis response in LPS-treated primary cultured microglia. Interestingly, evidence indicates that neurons are not only passive targets of microglia but also control microglial activity. Fractalkine (CX3CL1) is one of the neuronal signal that reduce microglial activation by binding a G-protein coupled receptor (CX3CR1) that present on microglia. Of note, using microarray screen and q-PCR, our preliminary confirmed that LRRK2 knockout increased the level of CX3CR1 mRNA and protein in vitro and vivo. Meanwhile, studies have been found that nuclear factor of activated T cells (NFAT) regulates innate immune responses in macrophages, dendritic cells, but little is known about the function of NFAT in brain-resident immune cell, microglia. These findings prompt us to explore the molecular mechanism by which LRRK2 and NFAT regulates CX3CR1 expression and its signaling pathway in α-syn mediated microglia. In this study, we will clarify the role of LRRK2 on α-syn induced microglial activation. Using the Pitx3/A53T mice that conditional overexpressed the human α-syn A53T mutation in the middle brain DA neurons and the LRRK2 knockout (LRRK2-/-) mice, we determine the changes of microglia phenotype in vivo in the Pitx3/A53T mice with or without LRRK2-genotype background. To address this issue, we generated Pitx3/A53T/LRRK2-/- mice. Also, we will profoundly observe the activity of primary cultured microglia from LRRK2-/- mice when exposed of α-syn in vitro. Moreover, we will further investigate how LRRK2 regulate Cx3CR1 expression whereby fine-tuned by the balance of activity of NFAT isoforms in α-syn-treated microglia. The present study will support the hypothesis that LRRK2-mediated NFAT-CX3CR1 signaling pathway may play a crucial role in α-syn-induced microglial activation in PD.

LRRK2基因在帕金森病(PD)发病中起重要作用,但LRRK2引起PD脑免疫紊乱的机制不详。我们前期研究发现LRRK2 基因敲除明显减轻脂多糖(LPS) 纹状体立体定向注射的脑小胶质细胞的激活和培养的小胶质细胞的炎症反应;更重要的是,我们经分子筛选发现抑制LRRK2明显增加特异表达于小胶质细胞表面的趋化因子受体CX3CR1水平,但具体调控机制不清。LRRK2能够调节外周树突状细胞的T-淋巴细胞核因子(NFAT)转录,但对中枢小胶质细胞的作用不明。我们推测“LRRK2使小胶质细胞NFAT活性变化、调控CX3CR1表达，激活小胶质细胞”是帕金森病脑内免疫紊乱的新机制。本项目通过条件过表达α-突触核蛋白的Pitx3/A53T转基因和LRRK2基因敲除的杂交鼠模型，应用siRNA、基因芯片、免疫共沉淀等技术,从整体-细胞-分子层次证明我们的假说。研究将为PD脑免疫紊乱的防治提供新思路和新靶点。

LRRK2基因在帕金森病(PD)发病中起重要作用,但LRRK2引起PD脑免疫紊乱的机制不详。我们前期研究发现LRRK2 基因敲除明显减轻脂多糖(LPS) 纹状体立体定向注射的脑小胶质细胞的激活和培养的小胶质细胞的炎症反应;更重要的是,我们经分子筛选发现抑制LRRK2明显增加特异表达于小胶质细胞表面的趋化因子受体CX3CR1水平,但具体调控机制不清。LRRK2能够调节外周树突状细胞的T-淋巴细胞核因子(NFAT)转录,但对中枢小胶质细胞的作用不明。我们推测“LRRK2使小胶质细胞NFAT活性变化、调控CX3CR1表达，激活小胶质细胞”是帕金森病脑内免疫紊乱的新机制。本项目通过过表达α-突触核蛋白的A53T立体定位注射LRRK2基因敲除模型、LRRK2基因小胶质细胞特异性敲除鼠(Lrrk2fl/fl:Lyz2tm1Cre+/-)；NFATc1基因小胶质细胞特异性敲除鼠 (Nfatc1fl/fl:Lyz2tm1Cre+/- )和Cx3cr1基因敲除鼠，从整体-细胞-分子层次证明我们的假说。结果发现：在PD的病理发展中，α-突触核蛋白诱导小胶质细胞产生强烈的免疫炎症反应。但LRRK2基因敲除减轻了小胶质细胞产生的免疫炎症反应、促进了对α-Syn的趋化迁移和清除能力；相反，NFATc1的敲除促进小胶质细胞的免疫炎症反应。机制研究发现，LRRK2缺失可促进NFATc1的表达与核转位，NFATc1缺失则减少CX3CR1的表达。研究结果进一步证明了 LRRK2通过调节NFATc1-CX3CR1信号影响PD脑α-突触核蛋白诱导的小胶质细胞活化，揭示了LRRK2- NFATc1-CX3CR1在帕金森病脑内免疫紊乱的作用机制。研究为 PD 脑免疫紊乱的防治提供新的思路和靶点。