This is a continuation of a long term study of the nerve growth factor protein, NFG its binding to its receptor, NGFR, and NGF effects relevant to regeneration. NGF plays a role in the development, maintenance and regeneration of some PNS and CNS neurons. NGF has ameliorative effects on certain lesioned neural structures where it plays a role in axon sprouting and regeneration. The primary event associated with NGF action is binding to NGFR. Subsequently, there is a broad spectrum of NGF effects on target tissues. Two hypotheses are to be tested. One is that NGFR from different species or tissues is made up of similar proteins but different carbohydrate compositions that affect NGF binding. The other hypothesis is that NGF has a regulatory role on oxidant-antioxidant metabolism that is relevant to regenerative outcome. The initial binding of NGF to NGFR in CNS tissues is to be compared to that already established for PNS and the NGF-responsive neuronal cell lines, PC12, SY5Y and LAN-1. NGF binding will be characterized using a soluble receptor assay that uses iodinated beta NGF as ligand. Additional ligands to be used as controls will be cytochrome C, insulin and EGF as well as synthetic peptides homologous to part of the NGF sequence. NGFR will be immunoprecipitated using two monoclonal antibodies, the MC192 to rodent NGFR, and a well established monoclonal antibody to human NGFR. Immunoprecipitated NGFR will be characterized by SDS-PAGE, preparative electrofocusing and HPLC. NGFR apoprotein and sugar moieties will be perturbed with retinoic acid, tunicamycin and lectins to test effects on receptor activity and structure. NGF induces the antioxidant enzymes glutathione peroxidase and catalase in vitro. Thus, the mechanism of NGF sparing of lesioned cells, due to regulatory effects on oxidant-antioxidant metabolism, will continue to be studied in the NGF sensitive cell lines PC12 and SY5Y. ln rats, spinal hemisection, or a regimen of exposure to anti-NGF has irreversible developmental consequences on sensory neurons. There is a decrease in the number of neurons and an increase in sensory axons. Here, antibodies to and assays for key antioxidants will be applied to both the in vitro and in vivo paradigms. Because of its effects on CNS cholinergic neurons involved in Alzheimers, it has been suggested that expression of NGF activity may be one factor in the development of this disease. It has also been suggested that an understanding of NGF action may be relevant to aspects of neurofibromatosis, ALS and CNS trauma.
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