Molecular anatomical analysis of neurofilament

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Four main new results concerning neurofilament (NF)organizations were obtained in the present study. 1. When NFs reassembeld in various combinations of NF subunits after purified from bovine spinal cord were examined by quick-freeze deep-etch mica electron microscopy, NF-H, the largest NF subunit, was found to be the most essential component for forming cross-bridges between core filaments. 2. NF-H, the most phosphorylated subunit, is heavily phosphorylated in the axon but poorly phosphorylated in the dendrite or perikaryon. Further, axonal NFs are considerably different in structural organization from the dendritic or perikaryal NFs. From these facts and the result 1, we examined with various electron microscopic techniques how NF-H phosphorylation state influences the NF structure in vitro, and found that crossbridge structures were significantly altered by dephosphorylation. Thus, we provide the evidence of phosphorylation-de-pendent structural change in the NF-H carboxy-terminal tail domain. 3. To clarify why NF-H is heavily phosphorylated in the axon but not in the other compartments, we examined rat central neurons with immunogold labeling. We found in dendrites that when NFs appeared singly their NF-H was not phosphorylated while when NFs were bundled NF-H became phosphorylated. The result suggests that phosphorylation of NF-H is necessary for forming crossbridges and unphosphorylation is necessary for NF interaction with other proteins. 4. From all above results, crossbridges are considered to be structurally changeable according to the NF-H phosphorylation degree. To confirm this idea in vivo, we compared NFs accumulated in perikaryon or dendrite following aluminum intoxication with axonal NFs. Our examination showed that core filaments have less regular alignment, fewer crossbridges and lower degree of NF-H phosphorylation in accumulated NFs than in axonal NFs. In conclusion, our data indicate that NF-H phosphorylation is necessary for the formation of de