Spastin类泛素化在脊髓损伤修复中的作用及其机制研究

To increase axonal branch is critical for the recovery of spinal cord injury (SCI), while microtubule severing is important for axonal branching. Spastin is a microtubule severing protein, regulating cytoskeleton dynamics to promote axonal growth. Till now, the role of Spastin modification, especially Spastin SUMOylation, in axonal branching and SCI remains obscure. Our results showed that Spastin interacted with SUMO protein and colocalized with SUMO1 in axon branch nodes; bioinformatic analysis revealed three potential conservative SUMOylation motifs in Spastin protein sequences; mutations of the motif decreased the interaction with SUMO protein, the tubulin severing ability of Spastin and also axonal branch numbers. Thus, we propose the hypothesis: during SCI, increasing Spastin SUMOylation could elevate its microtubule severing ability to promote new branch formation and the recovery of SCI. By morphological, molecular biological and physiological approaches, in neuronal cultures in vitro and in SCI rat model in vivo, the project aims to obtain the evidences of Spastin SUMOylation, to elucidate its role in microtubule severing, axonal branching and SCI and to provide new targets for the clinical treatment of SCI.

增加轴突分支数量是脊髓损伤修复的关键，而微管切割是轴突分支的必要条件。Spastin是微管切割蛋白，调控微管促进轴突分支。目前尚不清楚Spastin类泛素化修饰在轴突分支和脊髓损伤修复中的作用。我们的前期研究结果表明，Spastin蛋白能够与SUMO蛋白相互作用，且两者在神经元的分支处共定位；Spastin蛋白上存在SUMO化位点，该位点突变后Spastin与SUMO蛋白解离，微管切割功能丧失，神经元轴突分支数量随之减少。据此，我们提出科学假设：脊髓损伤时，增强Spastin 类泛素化修饰可促进微管切割，从而促进轴突新侧枝形成、促进脊髓损伤修复。本项目拟通过形态学、分子生物学、电生理学等技术，从体外细胞培养和体内动物模型获得Spastin被类泛素化修饰的证据，阐明该修饰在微管切割、神经元分支形成及脊髓损伤修复中的作用，为脊髓损伤的临床治疗提供新思路和新靶点。

增加轴突分支数量是脊髓损伤修复的关键，而微管切割是轴突分支的必要条件。Spastin是微管切割蛋白，调控微管促进轴突分支。本项目的研究结果表明，Spastin蛋白能与SUMO蛋白发生相互作用；Spastin荧光信号能够与SUMO1信号存在共定位，定位于树突及树突棘；我们构建了Spastin单点突变（K305，K427及K530）及多位点（pan）突变的质粒，发现K427是关键的类泛素化修饰位点；去SUMO化后的K427R Spastin会进一步促进微管的稳定，促进蘑菇状树突棘的增加，并影响AMPA受体的膜转运。本项目发现了Spastin的类泛素化修饰现象，通过阐明Spastin被类泛素化修饰的机制，分析其在轴突侧枝形成及脊髓损伤修复中的作用，为临床治疗脊髓损伤提供切实的依据、新思路和新靶点。