Exposure of the developing brain to ethanol (E) enhances death of neurons. The inescapable conclusion is that this increase in neuron death, above normal values, is a key factor underlying the damaging effects E on the growing brain. Prior studies by us and by others have documented E-related oxidative stress (OS) in the developing brain. Also, we have recently determined that the increase in E-related neuron death is apoptotic, which is mediated at the mitochondrial (M) level, likely secondary to a rapid-onset of OS. Hypothesis. We hypothesize that the enhanced death of cortical neurons in developing brains exposed to E is mitochondrially-mediated apoptosis caused by OS. The intent of this proposal is to determine the apoptotic pathways stimulated by E, establish the specific mechanisms underlying this phenomenon, determine specific neuron populations affected by E, and define key M defense systems which can be augmented to mitigate E-induced apoptotic cell death. Methods. Primary cultures of rat or mouse fetal cortical neurons will be exposed to clinically relevant levels of E and components of the apoptotic death pathways will be determined by biochemical approaches, flow cytometry, and by confocal and multiphoton microscopy of live neurons.
Specific Aim 1. Determine the Mitochondrially-Mediated Pathways of Apoptosis Elicited by Ethanol and Mitochondrial Damage that May Activate These Pathways. Experiments in this Aim will directly test the component of the hypothesis that ethanol initiates apoptotic neuron death at the mitochondrial level and they will determine the specific apoptofic events ultimately producing apoptotic death They will determine the temporal onset of E-related M damage.
Specific Aim 2. Determine if Ethanol-Related Oxidative Stress and Damaged GSH Homeostasis Underlie the Apoptotie Death of the Fetal Neurons. The experiments in Aim 2 will define mechanisms underlying the stimulation of the apoptotic pathways defined in Aim 1. Studies in our laboratory illustrated that, in cultured fetal rat neurons, there is a rapid onset of E-related oxidative stress which is associated with a disruption of GSH homeostasis and a production of toxic products of lipid peroxidafion. New to this proposal will be dual-tag experiments that will determine """"""""representative"""""""" populations responding to E and which will definitively link cellular responses to E.
Specific Aim 3. Mitochondrial Antioxidant Defenses and Apoptotic Neuronal Death. Intents of this Aim are twofold. The First will establish causality between E-mediated oxidative stress and M-related apoptotic death of neurons and the Second will define cellular defense systems which may protect the cell from this toxic response to E, that could generate interventions. GSH in M will be normalized and a tocopherol increased to dissect specific events of the apoptotic pathways. Experiments will utilize cultured cortical neurons from fetal mice that either underrexpress M glutathione peroxidase (active towards lipid hydroperoxides) or which over or underrexpress MnSOD to test the hypothesis that OS mediates the apoptotic death and to define specific events which generate the E-mediated apoptosis.
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