Fidgetin/NDEL1介导轴突微管骨架重构的机制与轴突再生研究

微管切割蛋白（MSP）是一类切断微管、增加微管动态性的AAA蛋白家族成员，能介导胞内各种微管形式转换，是轴突微管重构和轴突再生的正向调节因子。课题组发现，敲除MSP成员Fidgetin（FIGN）反而增加了轴突长度和分支，与切割稳定微管的MSP的效应相反。进一步研究表明FIGN切割酪氨酸修饰的不稳定微管，因此我们提出FIGN与切割稳定微管的MSP共同组成微管重构的调节系统，影响轴突再生。本研究拟通过检测不同修饰微管比例、体外微管蛋白聚合切割、活细胞拍摄等方法，研究FIGN调节Spastin和Katanin介导轴突生长和分支的机制；免疫共沉淀寻找FIGN互作蛋白，蛋白竞争结合、磷酸化位点突变等方法，论证FIGN通过与其他MSP竞争一个神经发育相关蛋白NDEL1的结合、介导轴突微管重构的机制；神经元特异敲除FIGN，研究其促脊髓损伤修复作用。本研究为FIGN调整微管重构和轴突再生提供了新机制。

Microtubule severing proteins (MSP), members of the AAA (ATPases associated with a variety of cellular activities) protein family, play key roles in intracellular microtubules remodeling and promoting axon regeneration. We previously found that, the depletion of Fidgetin (FIGN), a microtubule severing protein, could significantly increase the axonal length and branches, which is contrary to the effects of Spastin or Katanin severing stable microtubules. Further study found that FIGN removes the unstable Tyr-tubulin at the plus end of microtubules. Therefore, we hypothesize that FIGN could combine with other MSP members to regulate systematically axonal microtubule remodeling, and affect the microtubule dynamics and axonal growth. In this project, we plan to investigate that: FIGN mediates the processes of axon growth and branching with Spastin and Katanin by the methods of detecting alteration of different modified microtubules ratio, in vitro assays of microtubule polymerization and severing, and live imaging; FIGN and Katanin/Spastin co-regulate axonal microtubule remodeling by competitively interacting with NDEL1, an important neurodevelopment related protein by the methods of immunoprecipitation- identifying the FIGN interacting protein, protein synthesis, interaction site mutation, and phosphorylation. And finally, the rat spinal cord injury model of neuron-specific knocking down is established by aav9-syn-shFign injection, to investigate the possibility and procedures of promoting axonal regeneration and spinal cord injury repair. This study provides a novel molecular mechanism for FIGN to reorganize axonal microtubule skeleton, and a new strategy to repair the spinal cord injury.