Institutes of Brain Science and Key Laboratory of Medical Neurobiology, Neurology Department, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
Brain Res. 2012 Nov 27;1486:1-13. doi: 10.1016/j.brainres.2012.08.054. Epub 2012 Sep 6.
Cofilin is the major actin-depolymerizing factor in the CNS for the regulation of actin dynamics. Neurodegenerative stimuli can induce the formation of cofilin rod, a pathological structure composed of cofilin and actin. The formation of cofilin rod was found to disrupt synapse function and cause neurite loss. The aim of the present study is to study the whole process of cofilin rod formation pattern in cultured hippocampal neurons under excitotoxic stimulation and to explore its underlying pharmacological mechanism. By using live cell imaging of neurons overexpressing EGFP-tagged wild type cofilin, we found a two-phase pattern of rod formation induced by glutamate stimulation. The early phase of rod formation occurred shortly after stimulation (∼0.5h) but quickly dissolved within 2h. The second phase happened within a much longer time window, 8h after stimulation. Immunostaining of endogenous cofilin in neurons also confirmed this glutamate stimulation induced two-phase rod formation pattern. The first phase was co-related with intracellular calcium concentration and pH increase while the second phase was not. These two phases of cofilin rod formation induced by glutamate stimulation was antagonized by both non-NMDA and NMDA receptor antagonist DNQX and AP5, respectively. Our results for the first time demonstrate the dynamic cofilin rod formation pattern under stress stimulation in detail by time lapse imaging. These findings reveal a novel time course of excitotoxicity induced neuronal damage and indicate a potential target of neuropathy treatment of neurodegenerative diseases.
丝切蛋白是中枢神经系统中主要的肌动蛋白解聚因子，可调节肌动蛋白动力学。神经退行性刺激可诱导丝切蛋白形成杆状结构，这是一种由丝切蛋白和肌动蛋白组成的病理性结构。研究发现，丝切蛋白杆的形成会破坏突触功能并导致轴突丢失。本研究旨在研究兴奋性刺激下培养的海马神经元中丝切蛋白杆形成模式的全过程，并探讨其潜在的药理学机制。通过对过表达 EGFP 标记野生型丝切蛋白的神经元进行活细胞成像，我们发现谷氨酸刺激诱导的杆形成呈现两阶段模式。杆的早期形成发生在刺激后很短的时间（约 0.5h），但在 2h 内迅速溶解。第二阶段发生在更长的时间窗口内，即在刺激后 8h。神经元内源性丝切蛋白的免疫染色也证实了这种谷氨酸刺激诱导的双相杆形成模式。第一阶段与细胞内钙浓度和 pH 值增加有关，而第二阶段则没有。谷氨酸刺激诱导的这两个阶段的丝切蛋白杆形成分别被非 NMDA 和 NMDA 受体拮抗剂 DNQX 和 AP5 拮抗。我们的结果首次通过延时成像详细展示了应激刺激下丝切蛋白动态杆形成模式。这些发现揭示了兴奋性毒性诱导的神经元损伤的新的时间过程，并表明神经退行性疾病神经病变治疗的一个潜在靶点。
