The purpose of this project is to further our understanding of the mechanism by which specific neuronotrophic factors act on the mammalian nervous system during development. To date, this project has dealt exclusively with one neuronotrophic factor, the nerve growth factor (NGF), known to be necessary for the development of vertebrate sympathetic and sensory neurons. NGF is a multimeric protein made up of three dissimilar subunits, only one of which is biologically active. A large number of studies would suggest that there are alterations in ambient levels of NGF in humans suffering from a number of neuropathies including the peripheral and central nervous system. However, there are inconsistencies in some of these reports, difficulty in repeating some of the observations, as well as disagreement as to their significance. This is due to our ignorance of the regulatory mechanisms responsible for NGF synthesis, delivery and mode of action on target cells. These ambiguities also stem from the use of murine-NGF as an antigen and standard in assays on human tissues and the use of a rat non-neuronal adrenal clonal cell line (PC12) or of heterogeneous cell populations in primary cultures in the study of NGF effects. In order to circumvent some of these problems, we will focus our effects on NGF of human origin and NGF-responsiveness neuroblastoma clonal lines of human sympathetic origin. We therefore propose to: (1) To use NGF isolated from human term placenta and monoclonal antibodies developed by us and directed against the beta subunit of human term placental NGF in the conclusive phase of our determination of the quaternary structure of human NGF and its synthesis and intracellular processing. (2) To further characterize the cell surface receptors to NGF using isolation procedures developed by us. (3) To use in vitro differentiating agents, such as retinoic acid and dibutyryl cyclic AMP: and metabolic inhibitors, such as tunicamycin, in order to manipulate expression of NGF receptors in vitro. (4) As part of these studies, to develop monoclonal antibodies directed against receptors to NGF. (5) Lastly, to determine the effects of NGF on neuronal injury in vitro. In particular, we wish to determine the mechanism of the NGF stimulation of neuronal survival following free radical damage.
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