Dynamic Analysis of Axonal Cytoskeletons by Most Advanced Visualization Technique.

神经丝是轴突细胞骨架中最重要和最丰富的成员之一，其稳定聚合形式在轴突细胞骨架的三大类中所占比例最高。在这个研究项目中，我计划利用再生大鼠坐骨神经，分析神经丝蛋白的溶解度和轴突转运的变化及其磷酸化状态，以了解神经损伤后轴突细胞骨架的重组机制。在对照神经中，在磷酸酶抑制剂存在的情况下，用含有1% Triton X-100缓冲液处理15-25%的神经丝高分子质量亚基（NF-H）在可溶性部分中被回收，而与细胞体的距离无关。而在受损神经中，损伤部位远端神经的神经丝蛋白在沃勒氏变性损伤后1周内完全消失。在损伤后2周或更晚的再生芽中，检测到半数以上的NF-H （bbb50 %）可溶，与对照神经相比，其c端结构域磷酸化水平较低。损伤时在近端轴突运输的L-[^<35>S]-蛋氨酸标记的NF-H的溶解度也明显增加。低分子质量亚基在正常神经和再生神经中都保持在不溶性部分，这表明NF-H在再生过程中是选择性释放的，而不是神经丝的完全分解。

Neurofilament is one of the most important and abundant members of axonal cytoskeleton, and the proportion of stably polymerized form is the highest among three main classes of axonal cytoskeleton. In this research project, I planned to analyze the change in solubility of neurofilement protein and in axonal transport as well as in their phosphosrylation state using regenerating rat sciatic nerve, in order to know the reorganization mechanism of axonal cytoskeletons after nerve injury.In the control nerve, 15-25% of high-molecular-mass subunit of neurofilament (NF-H) was recovered in soluble fraction by treating with 1% Triton X-100 containing buffer in the presence of phosphatase inhibitors, irrespective of the distance from the cell body. In injured nerve, however, neurofilement protein in the nerve distal to injured site completely disappeared within 1 week after injury by Wallerian degeneration. In the regenerating sprouts at 2 weeks or later after injury, more than half of NF-H (>50%) was detected soluble with lower phosphorylation level in C-terminal domain compared with the control nerve. Increase in solubility was also apparent with L-[^<35>S]-methionine-labeled NF-H that was intransit in the proximal axon at the time of injury. The low-molecular-mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective release of NF-H rather than total disassembly of neurofilament occurs during regeneration.