Project IV will focus on two important gene products in the nervous system. S100-beta is a glial-specific protein that may be a neurotrophic factor. Choline Acetyltransferase (ChAT) is the key enzyme in acetylcholine synthesis and is expressed exclusively in cholinergic neurons. ChAT and S100-beta have been implicated in a number of neurologic disorders including Down's Syndrome and Alzheimer's Disease. Project IVa will investigate the regulation of Sl00-beta expression. S100- beta is a Ca2+ binding protein synthesized and secreted by glial cells in the nervous system. S100-beta may function as an important neurotrophic factor in the nervous system. It has been shown to induce neurite outgrowth from chick embryonic cortical neurons in tissue culture. It is a member of the """"""""S100 Protein Family"""""""", which has been proposed to exert various biochemical effects by inhibiting the phosphorylation of specific """"""""Effector Proteins"""""""" including Tau, GAP43, 87k, and Calpactin, which are Protein Kinase C and tyrosine kinase substrates. The S100 proteins are, therefore, likely to modulate the activities of important cellular phosphoproteins and kinases. S100 proteins are involved in cell cycle progression, differentiation and cellular transformation, Recently it has been proposed that S100-beta is involved in the pathogenesis of Down's Syndrome. The S100 gene is located on a segment of chromosome 21, which when triplicated, produces Down's Syndrome. We have obtained cDNA and genomic clones of S100-beta and have described the complex regulation of S100-beta mRNA and protein expression by cAMP and glucocorticoids --- two important intracellular signal transduction systems. We propose to study the CAMP and glucocorticoid transcriptional regulation of S100-beta gene expression by use of nuclear """"""""run-on"""""""" experiments, promoter/enhancer fusions, DNA sequence analyses, and DNase footprinting. These experiments will significantly increase our knowledge of the mechanism of transcriptional regulation of this important gene. S100-beta is exclusively expressed by glia but not neurons in the nervous system. Furthermore, we have demonstrated that the gene is expressed only in the glia cell types in a group of rat nervous system tumor cell lines. These experiments, coupled with previous observations, demonstrate that S100-beta is a glia-specific protein in the nervous system. We therefore hypothesize that the S100-beta gene contains a specific DNA sequence which functions as a glia-specific enhancer. Such a motif would promote the tissue specific expression of S100-beta in glial but not neuronal cells. S100-beta gene structure will be examined to locate and characterize such a sequence. Project lVb will focus on the regulation of the expression of chorine acetyltransferase (ChAT), the key enzyme in acetylcholine synthesis. ChAT is expressed exclusively by cholinergic neurons and arises developmentally with differentiation and the onset of synaptic transmission in such neurons. A recent series of studies in this laboratory suggests that a number of different types of effectors regulate ChAT expression. These include thymid hormone, vanadate, and cyclic AMP. Our data indicate further that these effectors may act at different mechanistic levels, from transcription to post-translational modifications; however, the mechanisms are at present poorly understood. Specifically, we propose to examine the molecular mechanisms underlying regulation of CHAT expression by thyroid hormone, vanadate, and cyclic AMP in cholinergic neurons in culture, and to investigate the role of these effectors of CHAT expression in neurite proliferation. Understanding the molecular actions of these regulatory influences is expected to provide important knowledge concerning control of expression of this important enzyme as well as the pathophysiology of cholinergic dysfunction in a number of disorders of the nervous system that involve mental impairment, including Down's Syndrome and Alzheimer's Disease. In summary, these experiments will shed new fight on the molecular mechanisms of neuronal and glial differentiation and cell type specific gene expression in the nervous system.
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