中枢神经损伤talin调控胞内张力依赖轴突生长再生机制研究

Axon regeneration and then neuron network reconstruction make great contributions to the recovery of brain function after central nerve injury. The elongation of axons depends strictly on the effect of intracellular tension. Based on fluorescence resonance energy transfer (FRET), an innovative force probe that can convert microscopic mechanics into optical signals, was designed to real-timely detect the intracellular force in the progression of axons growth and regeneration. Tension sensor talin was presumed to modulate intracellular mechanics in nerve cells through integrin pathway. The scar tissue-associated regeneration inhibitors might downregulate the talin/integrin pathway and intracellular tension through upregulating competitive binding proteins of talin. By means of constructing talin mutants, the different roles of distinct domains of talin were determined in integrin signals to modulate intracellular tension of neuron and F-actin pull effect on non-branch growth of axon. Besides, the mechanism underlying how talin was activated after phosphorylation at specific sites and its effect on intracellular mechanic was examined, with the help of site mutation technology. The aim of the studies is to propose a novel therapeutic approach to promote axon regeneration and find new pharmacal targets to enhance intracellular mechanic, and provide new research methods for the recovery of central nervous function.

神经元轴突再生及神经网络重建是中枢神经损伤后脑功能恢复的关键，而轴突的有效延伸严格依赖于胞内张力持续增强。通过开创性的构建以荧光共振能量转移（FRET）为基础的力学检测探针，将微观力学变化转换为光学信号，能实时检测胞内张力改变对神经轴突生长及再生的影响。评价张力感受器talin调控神经胞内力学变化的分子机制。结合生物信息学筛查胶质瘢痕抑制因子通过上调talin竞争性结合蛋白，抑制talin/整合素信号调控的力学效应。通过构建talin突变和截短体，识别talin不同结构域在激活整合素信号和诱导F-actin牵拉效应调控胞内张力及轴突定向生长的功能差异；另用点突变技术，鉴定特异性位点磷酸化调控talin活性变化的信号机制及其对胞内张力的影响。旨在阐明能识别以“细胞张力”为药物靶点，促进轴突生长再生的治疗新途径，并为中枢神经功能恢复提供新的检测手段及新药研发方向。

胞内力学活动是维持细胞生命活动的基本形式，受多种张力协同调节。依据荧光共振能量转移原理，构建了微丝、微管、中间纤维和牵拉蛋白等多种荧光张力检测探针，将细胞内微观力学变化转换为光学信号，用于识别细胞内张力的组成、数值和方向等力学活动，揭示多种高发疾病的力学调控机制及治疗新举措。研究发现：1、Talin1调控的化学和力学活动是神经元极化必不可缺的。Talin向内的牵拉力及其相关的渗透压向外膨胀力参与了神经元生长调节，瘢痕抑制因子能减弱这些力学效应抑制神经元再生，整合素活化参与了talin张力及其相关渗透压力调控，而DOK1能减弱整合素诱导的talin力学效应。2、神经生长因子能上调胞内微丝张力，瘢痕抑制因子能减弱这种张力。Talin能通过上调微丝张力拮抗瘢痕抑制因子的作用，且talin能通过NF-κB通路参与E-cadherin的功能调节，拮抗其介导的微丝张力下调和神经生长抑制。3、在谷氨酸诱导的脑水肿模型中，微丝和微管骨架解聚能诱导蛋白纳米颗粒生成及其相关渗透压和总渗透压的升高以及胶质细胞水肿的发生。SSH/cofilin和PP2B/stathmin-1的激活是胶质细胞肿胀发生的重要分子机制。临床五种药物组合能有效抑制其活性、阻止微丝和微管的解聚、降低渗透压、恢复细胞体积和改善脑水肿损伤。4、在细胞焦亡模型中，微管向外张力调控了细胞膨胀和出泡，而微丝向内张力调节的血影蛋白牵拉力，能对抗细胞焦亡形态改变。钙通道抑制、膜孔堵塞、自由基清除、caspase-1活性下调能显著降低血影蛋白、微丝和微管张力，参与细胞焦亡形态改变调节。5、在肿瘤诱发炎性微环境中，iNOS激活能诱导ezrin硝基化，促进非小细胞肺癌依赖其牵拉力的侵袭性。Cys117被识别是其硝基化的唯一活性位点。微丝而不是微管张力被发现调节了ezrin向内的张力，尤其是其硝基化后。靶向ezrin硝基化及其向质膜的力学效应传递，可能是抑制非小细胞肺癌发生的潜在治疗靶点。该项目研究审视了细胞力学活动的调控机制，从力学矢量组合的角度阐明了多种疾病发生的物理机制，并探寻了新的治疗药物发展方向。