We have recently demonstrated in high density, virtually pure sympathetic neuronal cultures that perikaryal aggregation, with attendant cell membrane contact, regulates neurotransmitter phenotypic expression. Preliminary observations suggest that the membrane component (s) mediating transmitter regulation may be extracted in soluble form and purified.
The specific aims of this proposal are to determine the molecular characteristics of the membrane component(s) that selectively induces neuronal substance P and choline acetyltransferase; to purify, isolate and identify this component; to determine the scope of activity of the membrane factor(s); to characterize its distribution, species specificity and ontogeny; to examine the critical role of this factor(s) during development and maturity; and to define the molecular mechanisms underlying neuronal responses to the membrane factor(s). Cultures of pure sympathetic neurons derived from the neonatal superior cervical ganglion will serve as a model system. Using various chromatographic and electrophoretic techniques, the membrane-associated factor(s) will be isolated and purified to homogeneity. In its purified form, the factor(s) will be added to cultures to determine its transmitter phenotypic, tissue and age specificity. Polyclonal antisera to the factor will be generated in rabbits and used to characterize distribution, species specificity and ontogeny by immunohistochemistry and radioimmunoassay. Molecular mechanisms underlying neuronal responses to the factor will be defined by examining transcription-related processes, using pre-protachykinin, and transduction-related processes, using second messenger agonists and antagonists. Knowledge of the molecular basis of cell contact-mediated transmitter regulation may foster the development of new therapeutic approaches to such congenital neurologic diseases as Familial Dysautonomia and dystonia musculorum deformans. Purification and characterization of factors mediating cell aggregation effects may provide an entirely new class of therapeutic agents to treat birth defects.
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