Phosphorylation-dependent neurofilament epitopes are reduced at the node of Ranvier.
Neurofilaments in axons are highly phosphorylated at multiple sites on the 200 kDa neurofilament (neurofilament-H) and 160 kDa (neurofilament-M) subunit peptides. We used a panel of monoclonal and polyclonal antibodies against distinct neurofilament epitopes to study the distribution of these epitopes along the axons of large myelinated fibres in rat sciatic nerve using quantitative electron microscopic immunocytochemistry with colloidal gold. Antibodies specific for phosphorylated epitopes on neurofilament-H showed a 60% reduction in density of immunoreactivity at the node of Ranvier, compared to the internodal axon. Antibodies directed against neurofilament-M, which recognized phosphorylated epitopes preferentially, showed a 40% reduction in density of immunoreactivity at the node. Following dephosphorylation of the neurofilaments in tissue sections by alkaline phosphatase treatment, antibodies which recognized dephosphorylated forms of neurofilament-H showed no reduction in density of immunoreactivity at the node. Similarly, an antibody directed against the 70 kDa subunit (neurofilament-L), showed no reduction in density of immunoreactivity at the node. An antibody against tubulin also showed no decrease in the density of immunoreactivity at the node of Ranvier. Tubulin immunoreactivity was similar in myelinated and unmyelinated fibres. In contrast to phosphorylated neurofilament epitopes, immunoreactivity was much greater in myelinated than unmyelinated fibres. These results suggest that the degree of phosphorylation of neurofilament-H and neurofilament-M subunits is reduced at the node of Ranvier, in comparison to internodal neurofilaments, and imply that a post-translational modification of neurofilaments must occur along the length of the axon at the node.
Department of Neurology, University of Michigan, Ann Arbor.
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D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D016900:Neurofilament Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011901:Ranvier's Nodes / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012584:Sciatic Nerve / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000032:analysis
D000939:Epitopes; D016900:Neurofilament Proteins; D014404:Tubulin; D000469:Alkaline Phosphatase