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，这种高于正常值的神经元死亡的增加是E对生长中的大脑造成破坏性影响的关键因素。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）。此外，我们最近已经确定，E相关神经元死亡的增加是凋亡性的，这是在细胞凋亡中介导的。 线粒体（M）水平，可能继发于OS的快速发作。 假说.我们假设，在发展中的大脑暴露于E的皮层神经元的增强死亡是由OS引起的神经细胞介导的凋亡。这个建议的目的是确定由E刺激的凋亡途径，建立这种现象的具体机制，确定特定的神经元群体受E影响，并定义关键M防御系统，可以增强，以减轻E诱导的凋亡细胞死亡。 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，并通过生物化学方法、流式细胞术以及活神经元的共聚焦和多光子显微镜来确定凋亡死亡途径的组分。 具体目标1。确定乙醇引起的线粒体介导的凋亡途径和可能激活这些途径的线粒体损伤。本目标中的实验将直接测试乙醇在线粒体水平上启动凋亡性神经元死亡的假设的组成部分，并且它们将确定最终产生凋亡性死亡的特定的非致死性事件。它们将确定E相关的M损伤的时间发作。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中的实验将定义目标1中定义的细胞凋亡途径刺激的潜在机制。我们实验室的研究表明，在培养的胎鼠神经元中，存在与E相关的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的产生有关。新的建议将是双标签实验，将确定“代表性”的人口响应E，这将明确联系细胞反应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。第一个将建立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，该系统可以保护细胞免受E的毒性反应，这可能会产生干预。M中的GSH将被标准化，生育酚增加以剖析凋亡途径的特定事件。实验将利用来自胎鼠的培养的皮质神经元，其要么低表达M谷胱甘肽过氧化物酶（对脂质氢过氧化物有活性），要么过表达或低表达MnSOD，以测试OS介导凋亡性死亡的假设，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.