Dynamic Analysis of Axonal Cytoskeletons by Most Advanced Visualization Technique.

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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.