The nervous system, among all tissues, is uniquely composed of non-renewable elements, the neurons. Consequently, the initial ontogenetic production of neurons is critical for mature brain organization. Although extensive evidence documents the complex, integrated process of neuron generation during development, there is virtually no information on regulation of neuronal proliferation. We have devised a new model culture system in which neurons undergo mitosis. Further, we have found that insulin plays a regulatory role. We are now in a position to define molecular mechanisms governing neuron proliferation.
Our specific aims are to define the relationship of insulin-like growth factors to insulin in neuronal mitosis, to characterize modulation of insulin mitogenesis by molecular factors localized to the brain, to define intracellular mechanisms underlying mitosis and transducing the insulin effect and to characterize mitosis in vivo, guided by insights derived in vitro. Our strategy employs a pure population of dissociated rat, embryonic sympathetic neurons, specifically identified by immunocytochemistry for neurotransmitter enzyme, tyrosine hydroxylase. Serum-free culture medium avoids confounding effects of undefined components. We plan to assay the effects of insulin-like growth factors as well as other growth factors, peptide transmitters and hormones localized to brain, on 3H- thymidine incorporation, employing autoradiography and scintillation spectroscopy. Further, we will define the role of ions and calmodulin, phospholipid and cAMP metabolism in mitosis and the insulin effect. Finally, we will apply insights from culture to study of development, performing neuronal counts and autoradiography after manipulating in vivo neurogenesis. By characterizing extracellular signals and intracellular transduction processes in neuronal mitosis, we may elucidate multiple mechanisms regulating neuronal proliferation. In turn, we may identify heretofore unrecognized loci where disease processes intervene to produce deranged brain formation. Hopefully, new therapeutic approaches may be designed to alleviate symptoms of perinatal asphyxia and stroke, congenital spinal muscular atrophy and neural tube dysgenesis.
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