Mitochondrial Effects on Sensitivity to Anesthetics

描述(申请人提供)：我们实验室利用一个非常简单的动物模型，线虫线虫，来研究挥发性麻醉剂作用的分子机制。我们已经确定，改变线虫中麻醉敏感性的基因和生理途径也改变了哺乳动物的敏感性。相同的分子途径影响不同物种的敏感性，这一事实的重要性怎么强调都不为过。我们已经确定了一组改变线粒体功能和控制线虫对挥发性麻醉剂(VAS)敏感性的基因。GAS-1编码线粒体电子传递链复合体I的49 kDa亚基，它的一个点突变导致动物对所有VAS都非常敏感。此外，我们还发现，一组患者，即代谢缺陷儿童，最明显地与复杂的I功能有关，对七氟醚高度敏感。这一发现为非常简单的动物模型的基础研究提供了临床相关性。总而言之，我们的工作清楚地表明，线粒体功能是一种有助于控制麻醉反应的新机制。在这项提案中，我们将扩展我们对线虫的研究，以检验我们的假设，即复合体I功能的变化通过直接影响新陈代谢和间接影响特定下游蛋白来改变麻醉敏感性。验证这些假说的具体目的是：1.使用RNAi筛选，确定我的复合体的哪些亚基改变线虫的麻醉敏感性。我们的数据表明，这个复合体的功能是调节线虫VAS行为的关键，可能在线粒体疾病患者中也有类似的作用。2.利用基因芯片分析，我们将确定基因和基因集的表达变化作为复杂的I功能障碍的下游效应。我们将通过一种专注于神经元特异性变化的减法策略，将我们的分析限制在改变麻醉敏感性的表达谱上。微阵列分析将通过使用RNAi来具体降低在这一目标中发现的基因的表达水平来验证，并测试由此对VAS行为的影响。这一卓越的动物模型允许通过评估整个动物的行为来确认或驳斥VAS的假定分子靶点。一个易于处理的动物模型对于揭示这类非常重要的麻醉剂的作用机制至关重要。

DESCRIPTION (provided by applicant): Our laboratory exploits a very simple animal model, the nematode C. elegans, to investigate the molecular mechanism of volatile anesthetic action. We have established that genes and physiologic pathways that alter anesthetic sensitivity in the nematode also alter sensitivity in mammals. The importance of the fact that the same molecular pathways affect sensitivity across different species is hard to overstate. We have identified a group of genes that alter mitochondrial function and control the sensitivity of C. elegans to volatile anesthetics (VAs). A point mutation in gas-1, which encodes the 49 kDa subunit of complex I of the mitochondrial electron transport chain, causes the animal to be very hypersensitive to all VAs. In addition, we have found that a subset of patients, children with metabolic defects most clearly related to complex I function, are profoundly hypersensitive to sevoflurane. This finding provides a clinical correlate to the basic study of very simple animal model. The sum of our work clearly implicates mitochondrial function as a novel mechanism that contributes to the control of anesthetic response. In this proposal we will extend our studies in the nematode to test our hypothesis that alterations in complex I function change anesthetic sensitivity via direct effects on metabolism, and via indirect effects on specific downstream proteins. The specific aims to test these hypotheses are: 1. Using an RNAi screen, determine which subunits of complex I alter anesthetic sensitivity in C. elegans. Our data indicate that the function of this complex is key to mediating behavior in VAs in nematodes, and may have a parallel role in patients with mitochondrial disease. 2. Using gene array analysis, we will determine genes and gene sets whose expression changes as a downstream effect of complex I dysfunction. We will restrict our analysis to expression profiles that alter anesthetic sensitivity by a subtraction strategy that focuses on changes specific to neurons. The microarray analysis will be validated by using RNAi to specifically reduce levels of expression of genes discovered in this aim, and test the resultant effects on behavior in VAs. This superb animal model allows putative molecular targets of VAs to be confirmed or refuted by assessing whole animal behavior. A tractable animal model is crucial to unraveling the mechanism of action of this very important class of anesthetic agents.