Chronic ethanol exposure can damage the nervous system, in part, by altering the growth of neural processes (neurites). In some regions of the brain, ethanol increases neurite growth. This could harm the nervous system by slowing conduction down dendrites to the cell body, and by disturbing the balanced development and organization of synapses. PC12 cells, which form neurites in response to nerve growth factor (NGF), have been used to study mechanisms involved in neurite outgrowth. Ethanol enhances NGF-induced neurite outgrowth by a process that is dependent on protein kinase C (PKC). Chronic ethanol exposure increases levels of two PKC isozymes, delta and epsilon, and increases PKC activity in PC12 cells. Studies outlined in this proposal will examine whether these PKC isozymes mediate enhancement of neurite outgrowth by ethanol in PC12 cells and primary cultures of cerebellar Purkinje cells which are known to respond to NGF and ethanol with increased neurite growth. In addition to increasing NGF-induced neurite outgrowth, ethanol also enhances NGF- induced stimulation of mitogen-activated protein kinases, suggesting that ethanol regulates NGF signal transduction. Studies are planned to investigate whether ethanol enhances NGF signaling by promoting the activity of the GTP-binding protein Ras or the kinase Raf-1, which are critical for NGF-induced neural differentiation. Methods to be used include PKC assay in cell homogenates and permeabilized cells, MAP kinase assays, assays for Ras-GTP formation, Ras GTPase activity, and Raf-1 activity, Western analyses, and stable transfection of PC12 cells to over- express PKC isozymes and to express antisense RNA and dominant-negative mutants. In addition, oligodeoxynucleotides and new PKC antagonists will be studied for isozyme-specific activity in PC12 cells. These will then be used to inhibit specific PKC isozymes in Purkinje cell cultures. These studies will provide new information about a biochemical mechanism that may be important in the pathogenesis of brain damage in alcoholics and of neurologic abnormalities in children born to alcoholic mothers. In addition, these studies will identify and test PKC isozyme-selective agents that may eventually prove useful as drugs in the treatment of ethanol neurotoxicity.
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