Preliminary data have shown that among 135 monoclonal antibodies to rat brain, 37 were neuron-specific, out of which 10 were specific for the 200 and 150 kDa neurofilament (Nf) subunits. Immunocytochemically, 5 of these antibodies stained long fiber tracts and terminal axons but not cell bodies, dentrites or proximal axons (group II), 4 stained cell bodies, dendrites and proximal axons but not terminal axons (group III), and 1 stained both sets of structures. Each antibody within the groups described microheterogeneity of neurofilaments, while intergroup differences expressed macroheterogneity. Further work has shown that macroheterogeneity is posttranslational and is due to phosphorylation of Nf. Group II antibodies reacted with phosphorylated, and group III antibodies with nonphosphorylated epitopes in Nf preparations. In tissue sections, dephosphorylation by phosphatase required trypsin pretreatment. Trypsin alone had no effect on staining with group II antibodies, but abolished that of group III antibodies. Phosphatase after trypsin treatment diminished staining with group II antibodies, but caused reappearance of staining with group III antibodies. However, the reappearing staining with group III antibodies had been coverted from group III to group II patterns. These preliminary data suggested that immunocytochemistry with monoclonal antibodies could be used to trace posttranslational changes in situ and that phosphorylated forms of Nf may be more compact than nonphosphorylated forms. Since analysis for such changes may have significance in neuropathology, it appeared indicated to explain, with the use of monoclonal antibodies, the relationship of individual phosphorylation sites to normal Nf structure and compactness in different regions of the neuron and its projections. Dephosphorylation and rephosphorylation will be carried out in Nf suspension and in fixed tissue sections. Phosphorylation sites and sites contributing to Nf compactness will be dissected by degradative treatments with cyanogen bromide, trypsin, Alpha-chymotrypsin, V8 protease, and Nf-associated calcium-dependent protease. Analysis of treated Nf suspension will be by electrophoresis-electroblot, followed by immunocytochemistry and autoradiography, and by HPLC, followed by radioimmuno-precipitation. Analysis of treated tissue sections will be by immunocytochemistry. Data will be obtained on distribution and heterogeneity of individual phosphorylation sites and epitopes within Nf dromains and on regional variations of posttranslational changes (phosphorylation, limited proteolysis, and compactness) in normal, developing, and regenerating nervous tissue and in Alzheimer lesions.
