胶质细胞活化的Wnt信号途径与中脑多巴胺稳态调控研究

Accumulating evidences have shown that brain glial cell activation might act as a critical factor in the pathological progression of Parkinson's disease (PD). Currently,however,the critical controlling mechanism underling glial activation and proliferation remains unclear. Our preliminary observations have shown that the activated astrocytes appeared early,maintained shorter time and exhibited neuroprotective role, while the reactive microglial cells occurred later, maintained longer peroid and showed neurotoxic effect. While Wnt signaling pathway actively functions in cell division and differentiation, cellular localization and expression has been detected in the activated glial cells under PD condition. We thus hypothesize that Wnt/β-catenin signaling pathway may play an important role in dopaminergic system balance and disease progression by regualtion of the glial activation and cell proliferation. In this project, we shall perform these following experiments: 1)Dynamic relationship of Wnt/β-catenin signaling activation with astrocytic and microglial cell reaction in PD model prepared from TOPgal (Wnt reporter) mice; 2)Promotion effect of Wnt signaling to trigger astrocytic activation , neurotrophic factor production and neuroprotection, and effect of Wnt signaling blocker to inhibit microglial reaction, inflammatory cytokine generation and neurotoxicity by in vitro co-culture system; 3)Neuroprotective and therapeutic effects of Wnt signaling and glial activation intervention by in vivo animal model study. It is expected that Wnt signaling mechanism underling glial cell activation and potential application for therapeutic intervention target in PD will be elucidated in this study.

资料表明脑内胶质细胞活化是影响帕金森病（PD）进程的重要因素，但目前对于其活化增殖的关键调控机制尚不清楚。我们前期研究发现：星形胶质活化早而短暂、具有神经营养特性，小胶质活化晚而持久、产生神经毒性效应；Wnt信号作为调节细胞增殖分化的主要因子、定位表达于活化胶质细胞。我们推测：Wnt/β-catenin信号可能具有调控胶质细胞活化增殖、多巴胺稳态和PD疾病进程的重要作用。本项目拟通过：1）采用Wnt报告基因鼠PD模型，分析Wnt/β-catenin激活与星形胶质、小胶质细胞活化的关系；2）应用细胞共培养，明确激活Wnt信号促进星形胶质活化、营养因子合成和神经保护作用，阻断Wnt信号对小胶质活化、炎症因子分泌和神经毒性的抑制作用；3）运用体内干预实验，探讨早期激活/晚期抑制胶质细胞Wnt信号的神经保护、多巴胺稳态和疾病治疗效果，阐明胶质细胞活化的Wnt信号调控机制及PD干预治疗新靶点价值。

项目主要目标是研究中脑胶质细胞功能活化的Wnt信号调节机制及其多巴胺神经元保护意义，课题通过TOPgal（Wnt-reporter小鼠）、MPTP诱导小鼠PD模型、细胞培养体系、RT-PCR、Western blot、MTT、ELISA、siRNA干涉、流式细胞术、免疫细胞化学、激光共聚焦显微镜观察等方法，研究Wnt/β-catenin信号参与星形胶质细胞和小胶质细胞功能活化、选择性干预的生物效应问题，结果发现：1）中脑黑质星形胶质细胞具有Wnt信号通路分子Wnt1、Wnt3a、Wnt5a、β-catenin等阳性定位，表明Wnt/β-catenin信号是星形胶质细胞的重要调控因子；2）MPTP诱导PD状态下，黑质星形胶质细胞和小胶质细胞明显活化、Wnt信号分子表达水平变化（上调为主）和多巴胺神经元丢失，表明Wnt/β-catenin信号激活可能与胶质细胞功能活化和多巴胺神经元生存活力的调节有关。3）体外培养状态下，Wnt信号激动剂LiCI或Wnt信号抑制剂IWR-1处理对于星形胶质细胞增殖活力无显著影响，但Wnt-inducible Gas1表达水平上调能够诱导LPS炎症刺激星形胶质细胞自身的凋亡发生；4）MPTP模型体内状态下，Wnt信号激动剂LiCI能够减轻MPTP诱导的TH表达下降或中脑多巴胺神经元丢失。另外，星形胶质细胞针对不同炎症刺激呈现M1和M2样极化现象，其可塑性变化和Wnt相关调节因子是一个引人关注的新问题。本研究结果提示Wnt/β-catenin信号通路参与星形胶质细胞的功能活化调节，适度激活Wnt信号促进星形胶质细胞的神经保护作用，但选择性干预Wnt信号通路方法和PD疾病治疗价值有待进一步研究阐明。