CRMPs结合spastin调控轴突侧枝形成机制的研究

轴突形成新生侧枝重建与靶细胞的联系是其再生的有效方式。我们前期的研究证明CRMPs和spastin均具有促进轴突形成侧枝的作用；进一步用pull down 和免疫共沉淀证实CRMPs与spastin在体内和体外均存在直接的相互作用，共转染发现spastin促进轴突形成侧枝时不能缺少CRMPs参与。那么二者的结合部位在哪里？磷酸化修饰是否导致二者解离？二者结合又是如何参与侧枝形成并重建与靶细胞的联系？我们首次提出CRMPs与spastin结合调控轴突侧枝形成的假说。本项目拟采用pull down、免疫共沉淀、基因截短、共聚焦和示踪等技术，旨在获得CRMPs与spastin结合的确切部位，分析结合部位磷酸化修饰后CRMPs和spastin结合状态，评价二者结合在侧枝形成中的作用及能否通过新生侧枝重建与靶细胞的联系。本项目有望揭示侧枝形成的新机制，为确立神经损伤后修复的新靶点提供充分的科学依据。

It is the main approach that the collateral branches sprout from the injured axon proximal to the lesion to form synaptic connection with the denervated target neuron along appropriate pathway. Collateral branches are initiated by actin filament based axonal protrusions that subsequently become invaded by microtubules thereby allowing the branch to mature and continue extending. Our previous studies have shown that the collapsin response mediator proteins (CRMPs) family of proteins and microtubule-severing proteins seem to be critical in axon outgrowth induction. CRMPs regulate axonal growth and branching as a partner of the tubulin heterodimer, and microtubule-severing proteins regulate axon morphology by creating short microtubules for transport in the axon to promote selectively the formation of collateral branches. Our additional studies have shown that the interaction between CRMPs and spastin played the critical role in the formation of collateral branches. We have identified that CRMPs and spastin could interact each other in neurons by pull down and immunoprecipitation. Moreover, the axon branches could not be initiated when knockdown of CRMP5 expression by small interfering RNA in neurons overexpressed spastin gene. When overexpression of CRMPs, the axon was initiated more elaborated branches in the distal. The resultes suggested that the interaction between CRMPs and spastin should be the critical step during formation of axon branches. Hence, where is the domain they interact each other? Can phosphorylation results in separating each other? How do both of them regulate axon branching to rebuild the neural circuites? Based on all above, we propose a simple interaction-coulped collateral formation model for CRMPs and spastin regulating axon branching. In this study, the following techniques such as pull down, immunoprecipitation, truncated genes, gene transfect and confocal microscopy will be used. We will identify the exacted interacted domain between CRMPs and spastin, and analyze the phosphorylated domain how to influence their interaction. We will elucidate the mechanisms that interaction of CRMPs and spastin regulates axon branching and regeneration. The results promise to yield many advances in our understanding of the mechanisms of axon branching evoked by the interaction of CRMPs and spastin. It is likely to bridge the present gap in knowledge of how targeting CRMPs and spastin could yield directed axon branching after axotomy.