Two of the most striking effects of ethanol exposure during development are the disruption of neuronal migration and the deletion of neurons in certain areas of the central nervous system (CMS). The cellular/ molecular mechanisms underlying these damages remain unclear. Glycogen synthase kinase-3/beta (GSK3beta) is predominantly expressed in neurons in the developing CMS, and emerges as a critical signaling component regulating diverse events of neuronal development, such as neuronal migration and proliferation/survival. It has also been implicated in abnormalities in the adult brain, such as neurodegenerative diseases. The activity of GSK3beta is regulated by phoshorylation and subcellular localization. GSK3beta is activated by various cellular stresses including endoplasmic reticulum (ER) stress. We have demonstrated that ethanol exposure induces ER stress and significantly alters the phosphorylation state of a GSK3beta in vitro and in vivo event at modest concentrations (50-100 mg/dl); blocking GSK3beta activation mitigates ethanol-induced neuronal death. Although ethanol alone at relatively low concentrations does not cause neuronal death, it greatly potentiates apoptosis caused by various exogenous insults which also activate GSK3beta. In contrast, ethanol does not affect cell death induced by pro-apoptotic signals which are independent of GSK3beta. We hypothesize that GSK3beta is a critical mediator of ethanol neurotoxicity. In this proposal, we will characterize the effect of ethanol on phosphorylation/activity, subcellular localization and expression of GSK3beta in the developing neurons using various model systems (cultured neurons, organotypic slice culture of rat cerebellum and the developing cerebellum). Subsequently, we will determine the role of GSK3beta in ethanol-induced cell cycle arrest, apoptosis, inhibition of neurite outgrowth and aberrant migration patterns; we will determine whether blocking GSK3beta activation by selective inhibitors or over- expression of dominant negative GSK3beta (DN-GSK3beta) or GSK3beta binding protein (GBP) provides neuroprotection against ethanol-induced damage. In addition, we will investigate the role of Chop (GADD153), another ER stress responsive factor, in ethanol-induced neuronal damages. Futhermore, we will elucidate the mechanisms underlying GSK3beta activation; we will systematically investigate up-stream signaling of GSK3beta and the contribution of ER stress-responsive components, such as protein phosphatase 2A, to ethanol-induced GSK3beta activation. Finally, we will investigate whether ethanol-induced sensitization or potentiation to apoptosis caused by other insults is dependent on GSK3beta we will determine whether over- expression of DN- GSK3beta and GBP abolishes ethanol-induced potentiation to neuronal death triggered by other pro-apoptotic stimuli. As a unit, these experiments will explore a novel mechanism underlying ethanol- induced damage to the CMS using in vitro and in vivo model systems. The systematical study may establish GSK3beta as a potential therapeutic target for the treatment of alcohol neurotoxicity
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