The electrical stimulation of neurons during the development and maintenance of the nervous system has both immediate and longer term effects on cellular physiology. The long term neuronal responses include alterations in neuronal sprotting, and changes in neurotransmitter, transmitter receptor and ion channel protein production. These cellular changes appear to require the activation of new gene expression, and may be fundamental to processes such as neural development, and information storage. The long-range objectives of the proposed research are to understand the mechanisms by which electrical stimulation controls the expression of genes in neurons, and the function of these electrical stimulation controls the expression of genes in neurons, and the function of these electrically-regulated genes during the development, and maintenance of the nervous system. Recent studies have identified a class of """"""""immediate early genes"""""""" that encode mRNAs whose transcription is activated rapidly as a response to electrical stimulation. Many of these genes encode transcription factors that have been hypothesized to control the neuronal cell response to trans-synaptic stimulation. Experiments are proposed that will elucidate the biochemical pathway by which membrane depolarizing agents induce the transcription of two members of the immediate early gene family c-fos and nur/77. Activation of these genes in the pheochromocytoma cell line PC12 by electrical stimulation requires an influx of calcium from the extracellular medium. Depolarization-activation of c-fos is controlled by a calcium response element (CaRE) within the c- fos promoter that binds the transcription factor CREB. Membrane depolarization activates the phosphorylation of CREB raising the possibility that electrical stimulation of gene expression in neurons is mediated by the phosphorylation-activation of a specific transcription factor.
The specific aims of the proposed research are: (1) to determine whether the sites of phosphorylation on CREB are critical for transcriptional activation; (2) to characterize the protein kinases that mediate the depolarization response; (3) to identify novel CaREs within the immediate early gene promoters, and CaRE binding proteins, that control alternative pathways for electrical stimulation of gene expression; (4) to test the hypothesis that varying the duration, strength, or repetitive nature of the electrical stimulation alters the pattern of activation of immediate early genes. Given the function of these genes in the process of normal cell growth and differentiation it is likely that alterations in the developmental process that lead to cancer, neurodegenerative diseases, or seizure disorders will involve changes in the regulation of function of the immediate early genes.
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