Spy1/CRMP1及其介导的Sema3A信号通路在周围神经损伤及修复中的功能及意义

Whether the injuried nerves could regenerate or not mainly dependens on the regenerate microenvironment they are living in. Sema3A is a member of neurotrophic factor family existing in regeneration microenvironment. Sema3A could induce the effect of axon guidance and the development of dendritic ridge, which play an important role in the formation of the connections between the neurons.CRMP1 is main downstream effector molecule in Sema3A signaling pathways. Spy1 is considered to be one of the important function genes and plays a role in the process of cell cycle regulation, apoptosis, differentiation and other envents. Our early research found that Spy1 could interact with CRMP1 and regulate the phosphorylation of CRMP1, which is involved in the regulation of repaired and developed nervous system. So our study will would concentrate on the mechanism of Spy1 regulating the phosphorylation of CRMP1, searching for the kinase participating in it and further exploring its function in the repair of peripheral nerve injury. Our study aims at exploring the molecular mechanism of regeneration of peripheral nerve injury from the view of signal pathway transduction in regenerate microenvironment and further could provid new theoretical basis for the looking for specific drug therapeutic targets.

周围神经损伤后能否实现成功再生很大程度上取决于是否具备适合轴突生长的再生微环境，Sema3A是再生微环境中的神经营养因子家族成员，通过影响下游效应分子CRMP1的磷酸化调节轴突生长及导向。本课题组发现一个调节细胞周期进程、凋亡、分化等重要功能的分子Spy1与CRMP1相互作用。进一步研究发现，Spy1可以调节CRMP1的磷酸化，并影响神经元突起生长。鉴于此，提出Spy1通过影响CRMP1的磷酸化修饰，从而参与Sema3A信号通路对周围神经轴突再生调节的假说。本课题拟首先研究Spy1和CRMP1与周围神经损伤再生的关系，接着在体外探讨Spy1对CRMP1的调节机制，以及该调节通路在神经元突起生长、生长锥塌陷中的作用，最后回到整体模型，分析Spy1/CRMP1调控通路在周围神经再生中的意义。本课题从再生微环境信号通路调节的角度，探讨周围神经损伤再生的分子机制，为寻找新药物治疗靶点提供理论依据。

周围神经损伤后，是否存在适合轴突生长的再生微环境是神经损伤后能不能实现成功再生的关键。课题组在项目进行过程中发现：（1）在PC12未分化细胞中Spyl和 CRMP1存在相互作用；免疫荧光显示Spy1和CRMP1在未分化的PC12中都存在于胞浆；NGF（100ng／ml）刺激PC12未分化细胞分化和原代培养DRG神经元过程中都观察到Spy1和 CRMPI在突起末端和边缘的聚集。（2）HEK293T工具细胞中发现Spy1域与 CRMP1的530~572结构域结合；Spy1可有效的增强CDK5的激酶活性，从而介导对于 CRMP1的磷酸化调节作用。（3）发现CRMP1与actin存在相互作用；Spy1对CRMP1的磷酸化的增强效应会干扰CRMP1与actin的相互作用，从而影响细胞骨架的动态性，并介导Sema3A的生长锥塌陷效应；体外培养的DRG神经元用Sema3A刺激后发现明显的生长锥塌陷现象：体外 siSpy1病毒感染原代DRG神经元并进行Sema3A刺激，发现生长锥的塌陷受到明显的抑制；而 siSpy1与CRMP1的拟磷酸化点突变质粒 CRMP1T509D或 CRMP1S522D共同感染并在Sema3A刺激下，发现生长锥的塌陷现象得到部分回复。（4）CRMP1表达在坐骨神经损伤模型中升高并在第3天达到顶峰。免疫荧光显示CRMP1和NF200（轴突标记物）共定位在坐骨神经损伤组相对于正常组明显增强。CRMP1 /actin结合在第三天受到干扰，而Spy1 / CRMP1结合增强。siSpy1注射组坐骨神经损伤后在第16天和24天功能得到了恢复。课题组从再生微环境信号通路调节的角度，探讨了周围神经损伤再生的分子机制，为寻找新药物治疗靶点提供了理论依据。