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，在三种主要轴突细胞骨架中，稳定聚合型所占比例最高。在本研究项目中，我计划分析再生大鼠坐骨神经后神经丝蛋白的溶解性和轴突运输及其磷酸化状态的变化，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，无论与细胞体的距离如何，通过含有1%Triton X-100的缓冲液在磷酸酶抑制剂的存在下处理，15%-25%的高分子量神经丝亚单位(NF-H)被恢复为可溶部分。而在损伤神经中，损伤部位远端神经的神经丝蛋白在损伤后1周内完全消失。在损伤后2周或以后的再生芽中，超过一半的NF-H(&gt；50%)可溶于C-末端区域，与对照神经相比，其磷酸化水平较低。L-[35&gt；S]-蛋氨酸标记的核因子-H在损伤时正处于近侧轴突的中间，其溶解度的增加也是明显的。在正常神经和再生神经中，低分子质量的亚基都保留在不溶性部分，这表明在再生过程中，神经丝发生了选择性的释放，而不是神经细丝的完全解体。

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.