Ca2+/Cofilin信号通路在电刺激促进神经元突起再生中的作用及其机制研究

Electrical stimulation (ES) can accelerate axon growth and nerve regeneration, but the mechanism is still unclear, which limits the application in clinical treatment. Studies have shown that the dynamic formation of neuron growth cone microfilament cytoskeleton is the molecular basis of axon growth. Our early studies have found that ES could raise intracellular calcium concentration ([Ca2+]i) in neurons, dynamically regulate Ca2+-dependent level of Cofilin phosphorylation (P-cofilin) and promote Ca2+-dependent neurite regrowth. Based on these previous reaserches, we presume that ES could activate Ca2+-dependent signaling pathways by adjusting [Ca2+]i in neurons, then dually and dynamically regulate the level of P-cofilin with the influence of cytoskeletal dynamics to realize the promotion of neurite regrowth. In this project, we intend to study the effects and mechanisms of Ca2+-activating double signaling pathways (CaN-SSH1 and CaMKs-LIMK1) and dynamically regulating P-cofilin levels under ES. Based on these studies, we intend to investigate the growth cone cytoskeleton formation and neurite regrowth by interventing the upstream molecules in the signaling pathways, and to clarify the mechanisms of Ca2+-dependent signaling pathways mediating ES-induced neurite regrowth. Practically, our study is of great importance as to possibly provide experimental data and theoretical basis for clinical application of ES therapy on nerve injury and for bringing about new strategies to promotion of nerve regeneration, which has important medical and social significances.

电刺激能促进轴突生长、加速神经再生，但机制不明，限制了其在临床中的广泛应用。研究表明生长锥微丝细胞骨架动态形成是轴突生长的分子基础。我们前期研究发现电刺激能上调神经元内Ca2+浓度，动态调控钙依赖性Cofilin磷酸化（P-cofilin）水平，促进钙依赖性神经元突起再生。据此推测，电刺激可能通过调节神经元内Ca2+浓度，激活钙依赖性信号通路，对P-cofilin水平进行双重调控和动态调节，影响细胞骨架动力学，促进神经元突起再生。本课题拟探究电刺激作用下，Ca2+通过激活双信号通路（CaN-SSH1及CaMKs-LIMK1）、动态调节P-cofilin水平的作用机制。在此基础上观察干预上述通路，对生长锥细胞骨架形成和突起再生的影响，明确通路介导电刺激促进神经元突起再生的机制。本研究有望为临床推广电刺激治疗神经损伤、发现新的促进神经再生策略提供实验数据和理论依据，具有重要的医学和社会意义。

电刺激能促进轴突生长、加速神经再生，但机制不明，限制了其在临床中的广泛应用。研究表明生长锥微丝细胞骨架动态形成是轴突生长的分子基础。我们前期研究发现电刺激能上调神经元内Ca2+浓度，动态调控钙依赖性Cofilin磷酸化（P-cofilin）水平，促进钙依赖性神经元突起再生。据此推测，电刺激可能通过调节神经元内Ca2+浓度，激活钙依赖性信号通路，对P-cofilin水平进行双重调控和动态调节，影响细胞骨架动力学，促进神经元突起再生。本课题研究了电刺激作用下，Ca2+通过激活双信号通路（CaN-SSH1及CaMKs-LIMK1）、动态调节P-cofilin水平的作用机制。在此基础上观察了干预上述通路，对生长锥细胞骨架形成和突起再生的影响，明确了通路介导电刺激促进神经元突起再生的机制。通过本课题的实施，我们观察到在电刺激的作用下，神经元胞内Ca2+浓度上升，一方面CaN被激活，SSH1活性增强，加强Cofilin去磷酸化作用，增强F-actin解聚；同时，另一方面，胞内Ca2+浓度上升也会激活CaMKIV，活化LIMK1，加强Cofilin磷酸化修饰，增强F-actin重组。此过程中Cofilin去磷酸化和磷酸化作用同时存在，可能以SSH1对Cofilin的去磷酸化作用为主，出现电刺激期间P-cofilin水平降低的现象。这两种调节加速生长锥Cofilin去磷酸化/磷酸化的动态转换，促进F-actin解聚/重组的循环转化，提高神经元生长锥细胞骨架的动态形成能力，实现电刺激对神经元突起再生的促进作用。本研究有望为临床推广电刺激治疗神经损伤、发现新的促进神经再生策略提供实验数据和理论依据，具有重要的医学和社会意义。