ETOH Teratogenesis: Apoptotic Death of Cortical Neurons

将发育中的大脑暴露在乙醇(E)中会增加神经元的死亡。不可避免的结论是，神经元死亡的增加，高于正常值，是导致E对正在发育的大脑产生破坏性影响的关键因素。我们和其他人之前的研究已经证明，发育中的大脑中存在与E相关的氧化应激(OS)。此外，我们最近确定E相关神经元死亡的增加是凋亡的，这是在 线粒体(M)水平，可能继发于OS的快速发作。 假设。我们推测，暴露于E的发育中大脑皮质神经元的死亡增强是由OS引起的线粒体介导的细胞凋亡。这项建议的目的是确定E刺激的凋亡途径，建立这一现象的特定机制，确定受E影响的特定神经元群体，并确定可以增强的关键M防御系统，以减轻E诱导的细胞死亡。 方法：研究方法。原代培养的大鼠或小鼠胚胎皮质神经元将暴露于临床相关水平的E，并将通过生化方法、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成分。 具体目的1.确定乙醇诱导的线粒体介导的细胞凋亡途径和激活这些途径的线粒体损伤。这一目的的实验将直接检验假设的组成部分，即乙醇在线粒体水平上启动神经元凋亡，它们将确定特定的凋亡事件，最终导致凋亡死亡，它们将确定E相关M损伤的时间起点。具体目标2.确定酒精相关的氧化应激和受损的GSH稳态是否是 胎儿神经元凋亡性死亡。AIM 2中的实验将确定刺激AIM 1中定义的凋亡途径的潜在机制。我们实验室的研究表明，在培养的胎鼠神经元中，存在与GSH稳态破坏和脂质过氧化损伤产物产生有关的E相关氧化应激的快速启动。这项提议的新内容将是双标记实验，它将确定对E有反应的“代表性”群体，并明确将细胞对E的反应联系起来。具体目标3.线粒体抗氧化防御和神经细胞凋亡。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，可能保护细胞免受E的这种毒性反应，这可能会产生干预。M中的GSH将被正常化，生育酚将增加，以剖析凋亡途径的特定事件。实验将利用培养的胎鼠皮质神经元，这些神经元要么低表达M谷胱甘肽过氧化物酶(对脂质过氧化氢活性)，要么过度或低表达MnSOD，以检验OS介导细胞凋亡的假设，并确定产生E介导的细胞凋亡的特定事件。

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.