CRMP-2介导的微管组装在Cdc42调节神经元轴突发生中的作用

A progressive axonal degeneration, which precedes neuron death, is a major pathological hallmark in Alzheimer’s disease (AD) and other neurodegenerative diseases. Cdc42 in AD brain is upregulated and overlaps with early cytoskeletal abnormalities. Although studies have shown that Cdc42 is involved in regulating axonogenesis, but the underlying mechanisms have not been elucidated. Based on the latest research progress and our previous work, we propose the hypothesis that Cdc42 regulates axonogenesis via collapsin response mediator protein-2 (CRMP-2)-mediated microtubule assembly with the mechanisms involving Par6-aPKC complex and GSK-3beta. The experiment is designed as follows: (1) It will be studied that Cdc42 regulates axonogenesis via CRMP-2-mediated microtubule assembly in cultured primary hippocampal neurons, embryonic rats and Alzheimer-like rat models in vivo; (2) Technologies such as transfection with dominate negative plasmid, siRNA and fluorescence resonance energy transfer (FRET) will be used to investigate the roles of Par6-aPKC complex and GSK-3beta in Cdc42 regulating axonogenesis via CRMP-2-mediated microtubule assembly. The project will provide not only new information for the molecular mechanisms underlying the axonogenesis but also new strategies and targets for preserving the axons, preventing and rescuing the axonal degeneration in AD and other neurodegenerative diseases.

先于神经元死亡的进行性轴突退化是阿尔茨海默病（AD）等神经退行性疾病主要的病理学特征，AD脑中Cdc42的水平升高并且与早期的细胞骨架异常共定位，Cdc42调节神经元轴突发生，但其机制尚未阐明。基于最新研究进展及申请者的前期工作，设想Cdc42通过脑衰蛋白反应调解蛋白-2（CRMP-2）介导的微管组装调节神经元轴突发生，其机制涉及Par6-aPKC和GSK-3beta。本项目拟(1)在原代海马神经元、胚胎大鼠和AD模型大鼠整体水平研究Cdc42通过CRMP-2介导的微管组装调节神经元轴突发生；(2) 采用功能缺失基因转染、siRNA和荧光共振能量转移(FRET)等技术探讨Par6-aPKC和GSK-3beta在Cdc42通过CRMP-2介导的微管组装调节轴突发生中的作用。该研究将为神经元轴突发生的分子机制提供新资料，并为AD等神经退行性疾病中轴突的保护和轴突退化的防治提供新的策略和靶点。

先于神经元死亡的进行性轴突退化是阿尔茨海默病（AD）等神经退行性疾病主要的病理学特征，AD脑中Cdc42的水平升高并且与早期的细胞骨架异常共定位，Cdc42调节神经元轴突发生，但其机制尚未阐明。为探讨Cdc42对神经元轴突发生的调节作用及其机制，在大鼠海马神经元中分别转染空载体、野生型Cdc42、Cdc42活性突变体和Cdc42显性负性突变体，应用生物化学试剂盒测定Cdc42的活性，免疫荧光检测神经元轴突的数目和长度，证实了Cdc42促进大鼠海马神经元轴突发生；免疫荧光检测乙酰化a-微管蛋白（acetylated α-tubulin）（稳定微管的标记）和酪氨酸a-微管蛋白（tyrosinated α-tubulin）（动态微管的标记），证实了Cdc42促进大鼠海马神经元微管稳定；微管稳定剂Taxol处理显著抑制转染Cdc42显性负性突变体引起的轴突发生障碍，而且微管稳定剂Taxol处理显著抑制转染Cdc42显性负性突变体引起的微管稳定性的下降；提示Cdc42通过增强微管稳定促进大鼠海马神经元轴突发生；免疫印迹检测脑衰蛋白反应调节蛋白-2(CRMP-2)苏氨酸514位点的磷酸化，证实Cdc42促进大鼠海马神经元CRMP-2苏氨酸514位点的去磷酸化；转染Cdc42L28增高GSK-3β的Ser9位点的磷酸化和降低GSK-3的活性，过表达GSK-3βwt或GSK-3βS9A均能够抑制Cdc42L28诱导的CRMP-2（Thr514位点）的去磷酸化；GSK-3β与aPKC（PKCζ）和Par6均共沉淀；过表达Par6或aPKC（PKCζ）均能够诱导GSK-3β的Ser9位点的磷酸化；过表达Par6 Nt (aa 2-130 of hPar6C) 或失活形式的aPKC (PKCζ KD) 均能够抑制Cdc42活性突变体Cdc42L28诱导的GSK-3β的Ser9位点的磷酸化，提示Par6–aPKC复合体诱导的GSK-3β的失活（Ser9位点磷酸化）介导了Cdc42促进CRMP-2的去磷酸化。本项目证实了Cdc42通过增强微管稳定促进海马神经元轴突发生，其机制为Cdc42通过Par6-aPKC诱导GSK-3β失活（Ser9位点磷酸化）从而抑制CRMP-2的磷酸化。