Subcellular-targeted CYP2E1 and alcohol in the brain

项目总结 酒精(即乙醇)在不同脑区和不同亚细胞内的乙醛代谢 隔室，以及这种新陈代谢如何随着长期使用乙醇的使用而发生变化，目前还不是很清楚。长的- 学期目标是确定慢性酒精滥用中发生的适应性变化，并确定治疗方法 预防或逆转酒精所致神经损害的策略。这项建议的目的是 测定线粒体和内质网乙醇代谢的亚细胞调控 在急性和慢性乙醇使用期间，并确定不同靶向的后果。 中心假说是，在长期饮酒过程中，细胞色素P4502对线粒体的靶向性增加 在大脑中对酒精引起的损伤敏感的一些区域，以及高线粒体 靶向将推动线粒体乙醛的高产生，并导致线粒体功能障碍和 氧化应激。这一假说背后的基本原理是，CYP2E1的总体表达在 对酒精敏感的大脑区域，包括前额叶皮质、海马体和小脑，以及 在酒精使用过程中，这些区域还会出现线粒体功能障碍和氧化应激。中环 假设将通过追求三个特定的目标来检验：1)评估诱导的亚细胞特异性 通过急性和慢性乙醇在大脑中的作用；2)确定线粒体和ER靶向的作用 慢性酒精使用对线粒体的影响；以及3)测量线粒体和内质网的贡献。 乙醇诱导的氧化应激中细胞色素P450酶2的定位。我们将以创新的策略追求这些目标。 三个互补的系统：小鼠、线虫和培养细胞。在每个系统中，我们都有一个空 背景缺乏针对线粒体和内质网的野生型CYP2E1基因，内质网靶向基因 和线粒体靶向的细胞色素P4502。拟议的研究具有重要意义，因为它将阐明 在长期使用乙醇的范例中，细胞色素P450 2 1如何改变乙醇代谢，并可能揭示 线粒体CYP2E1与乙醇中毒的易感性它也很重要，因为它产生了有用的 研究亚细胞定位依赖的细胞色素P450-2E1效应的平台。这项工作将会得到发展 将被其他研究人员使用的基础资源。这项工作的最接近预期结果是 了解慢性和急性期脑内乙醇对细胞色素P450-2的影响 酗酒。结果将立即产生重要的积极影响，因为它们将确立 更好地理解脑内乙醇代谢与毒性的关系，以及在 长期的，因为它们为确定治疗机会奠定了基础。

PROJECT SUMMARY Metabolism of alcohol (i.e., ethanol) to acetaldehyde within different brain regions and in different subcellular compartments, and how that metabolism changes with chronic ethanol use, is not well-understood. The long- term goal is to identify adaptive changes that occur in chronic ethanol abuse and to identify therapeutic strategies to prevent or reverse neurological damage from ethanol. The objective of this proposal is to determine subcellular regulation of ethanol metabolism in mitochondria and endoplasmic reticulum (ER) by CYP2E1 during acute and chronic ethanol use, and to determine the consequences of the differential targeting. The central hypothesis is that targeting of CYP2E1 to mitochondria will be increased during chronic alcohol use in some regions of the brain that are sensitive to ethanol-induced damage, and that high mitochondrial targeting will drive high mitochondrial acetaldehyde production and resulting mitochondrial dysfunction and oxidative stress. The rationale underlying this hypothesis is that CYP2E1 expression overall is increased in brain regions that are sensitive to ethanol, including the prefrontal cortex, hippocampus, and cerebellum, and these regions also develop mitochondrial dysfunction and oxidative stress during ethanol use. The central hypothesis will be tested by pursuing three specific aims: 1) Evaluate subcellular specificity of induction of CYP2E1 by acute and chronic ethanol in the brain; 2) Determine the role of mitochondria- and ER-targeted CYP2E1 in mitochondrial effects of chronic ethanol use; and 3) Measure contribution of mitochondria- and ER- localized CYP2E1 to ethanol-induced oxidative stress. We will pursue these aims using an innovative strategy of three complementary systems: mice, C. elegans, and cultured cells. In each system we have a null background lacking CYP2E1, a wild-type CYP2E1 gene targeted to both mitochondria and ER, an ER-targeted CYP2E1, and a mitochondrial-targeted CYP2E1. The proposed research is significant because it will elucidate how ethanol metabolism by CYP2E1 changes over a chronic ethanol use paradigm, and could reveal mitochondrial CYP2E1 as a liability for ethanol toxicity. It is also significant because it generates useful platforms for studying subcellular localization-dependent effects of CYP2E1. The work will develop foundational resources that will be used by other researchers. The proximate expected outcome of this work is an understanding of how CYP2E1 contributes to the effects of ethanol in the brain during chronic and acute binge drinking. The results will have an important positive impact immediately because they will establish better understanding of the relationship between ethanol metabolism in brain regions with toxicity, and in the long-term because they lay the groundwork for identifying therapeutic opportunities.