CRMPs结合spastin调控轴突侧枝形成机制的研究

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.

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。

项目旨在解析神经元突起生长和分支的基本生物学问题。我们用分子生物学、细胞学以及神经电生理技术证明了微管相关蛋白CRMPs和spastin在突起生长和分支形成过程中的作用模式。主要的结论包括以下5个方面：①CRMP5与spastin通过释放微管动力促进神经元突起生长：阐明了CRMP5与sapstin之间存在相互作用，具体的结合部位在CRMP5的C末端和spastin的MTBD结构域；二者失去结合抑制微管的重排，二者结合可以通过促进微管的聚合促进突起的生长，包括轴突和树突。②CRMP2与spastin结合的磷酸化修饰抑制与微管结合调控神经元突起生长：CRMP2和spastin之间的结合与CRMP5相似，具体的结合部位也是在CRMP2的C末端和spastin的MTBD结构域；磷酸化修饰并不影响二者结合，但却抑制二者与微管之间的结合，二者促进轴突和树突的生长离不开二者的结合，失去二者结合突起的生长受到抑制，微管的动力性释放是突起生长的主要原因。③CRMP4通过微丝聚集促进树突棘形成：CRMP4促进树突棘的形成，其机制是通过与微丝结合改变微丝的聚合状态，失去与微丝的结合树突棘的形成受到抑制。④CRMP2的磷酸化和SUMO修饰调控树突棘的可塑性：CRMP2存在磷酸化和SUMO化修饰位点，CRMP2磷酸化和SUMO化修饰抑制树突棘形成和功能，去磷酸化和SUMO化则促进树突棘的成熟，两种修饰方式是独立发挥作用的。⑤CRMP2和sapstin促进神经元树突野的形成：CRMP2和spastin能够促进树突的形成，增加树突野的复杂性，而这些新形成的树突具有接受信息的功能，属于功能性的树突。这为后续研究CRMP2与spastin如何调控树突棘的功能提供了确切的实验证据。项目明确了突起生长的CRMPs和spastin调控模式，为以此为靶点进行神经纤维损伤和修复提供了理论基础。