ETOH Teratogenesis: Apoptotic Death of Cortical Neurons

将发育中的大脑暴露于乙醇（E）会增强神经元的死亡。不可避免的结论是， 高于正常值的神经元死亡的增加是E对生长中的大脑的破坏作用的关键因素。 我们和其他人之前的研究已经记录了发育中大脑中与E相关的氧化应激（OS）。另外我们 最近已经确定，E相关神经元死亡的增加是凋亡，这是介导的， 线粒体（M）水平，可能继发于OS的快速发作。 假说.我们假设，在暴露于E的发育中的大脑中，皮层神经元的死亡增加， 这一建议的目的是确定凋亡途径 通过E刺激，建立这种现象背后的特定机制，确定特定的神经元群体 受E的影响，并定义了关键的M防御系统，可以增强以减轻E诱导的凋亡性细胞死亡。 方法.将大鼠或小鼠胎儿皮质神经元的原代培养物暴露于临床相关水平的E和 细胞凋亡死亡途径的组分将通过生物化学方法、流式细胞术和 活神经元的共聚焦和多光子显微镜。 具体目标1。确定乙醇和乙醇诱导的线粒体介导的细胞凋亡途径 线粒体损伤可能激活这些途径。本目标中的实验将直接测试 假设乙醇在线粒体水平启动凋亡神经元死亡，他们将确定 最终导致凋亡性死亡的特异性凋亡事件将决定E相关M 损害具体目标2。确定乙醇相关的氧化应激和受损的GSH稳态是否是 胎儿神经元的凋亡。目标2中的实验将定义刺激的潜在机制， Aim 1中定义的凋亡途径。我们实验室的研究表明，在培养的胎鼠神经元中， E-相关氧化应激的快速发作，其与GSH稳态的破坏和 脂质过氧化的有毒产物。新的建议将是双标签实验，将确定 “代表性的”群体响应E，并将明确联系细胞响应E。 具体目标3。线粒体抗氧化防御与神经元凋亡。这一目标的意图是双重的。 第一部分将建立E介导的氧化应激和M相关的神经元凋亡之间的因果关系， 第二个将定义细胞防御系统，它可以保护细胞免受E的毒性反应， 进行干预。将M中的GSH标准化，并增加生育酚以分析 凋亡途径实验将利用来自胎鼠的培养皮层神经元，这些神经元要么表达M 谷胱甘肽过氧化物酶（对脂质过氧化氢有活性）或过或过表达MnSOD以测试 假设OS介导凋亡性死亡，并定义产生E介导的凋亡的特定事件。

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.