Mechanisms of In Vivo Ethanol-Induced Mitochondrial Depolarization

摘要：该项目研究酒精如何导致线粒体功能障碍和肝病。它将填补了解酒精性肝病的一个关键空白。 理由：该项目解决了一个对生物医学研究和公共卫生很重要的问题。酒精性肝病(ALD)在美国影响着超过250万人，每年的治疗和生产力损失超过15亿美元。乙醇损害肝脏的机制还远不清楚，在开发有效的治疗方法之前，必须填补我们知识中的显著空白。这项研究的重点是利用最先进的活体多光子/共聚焦显微镜技术了解乙醇导致活体动物线粒体功能障碍的机制，并有望填补了解ALD机制的关键空白。活体共聚焦/多光子显微镜是一项新的技术，将在拟议的研究中开发和优化，作为从细胞器和分子水平研究活体动物乙醇毒性的强有力的新工具。由于酒精生物反应的一些重要特征不能在体外系统中复制，因此这种技术是必不可少的。拟议项目将立即设立3个新的全职职位，其中包括2名博士后研究员和1名技术员，以及另外两个职位的部分支助。通过这笔ARRA资金，总共将创建或保留3.3个FTE。预计这些招聘将很快完成，而且从Pi已经拥有的候选人名单中毫无困难地进行。已经提交了一份详细的预算，并将其包括在这项请求中。在ARRA支持的两年结束时，调查人员将完成前两个具体目标。首先，他们将描述乙醇处理后体内线粒体去极化与SIAM和肝脏脂肪变性的剂量依赖性、时间进程和恢复的关系。其次，他们将评估乙醇脱氢(ADH)、微粒体醇氧化系统(MEOS)和乙醛脱氢酶(ALDH)在乙醇诱导的线粒体去极化中的作用。在ARRA支持的2年中，这项工作将开发和应用新的活体多光子显微镜技术，并为进一步研究线粒体功能障碍在ALD病理生理中的作用奠定坚实的基础。在两年的ARRA资助期内，将向国家和国际会议提交4-6篇摘要，向同行评审期刊提交2-4篇论文。这是对RFA-AA-08-002(线粒体在酒精诱导的组织损伤中的作用)的响应应用。在那些非资助的RFA申请中，这项申请的优先级分数(179)是最好的。R01的RFA支付线为171，R21的RFA支付线为183。PI可以解决评审员的所有主要问题。然而，由于这是一份RFA申请，PI不允许对批评做出回应、修改申请和重新提交。

Summary: The project studies how alcohol causes mitochondrial dysfunction and liver disease. It will fill a critical gap in understanding alcoholic liver disease. Justification: The project addresses a problem important for biomedical research and public health. Alcoholic liver disease (ALD) affects more than 2.5 million people in the U.S. and costs over $1.5 billion/year for treatment and lost productivity. Mechanisms by which ethanol damages the liver are far from clear, and striking gaps in our knowledge must be filled before effective therapies can be developed. The study focuses on understanding the mechanisms by which ethanol causes mitochondrial dysfunction in living animals using state-of-the-art intravital multiphoton/confocal microscopy techniques and is expected to fill a critical gap in understanding the mechanisms of ALD. Intravital confocal/multiphoton microscopy is a novel technology that will be developed and optimized in the proposed study as a powerful new tool to investigate ethanol toxicity at organelle and molecular levels in living animals. Since ,some important features of the biological response to alcohol cannot be reproduced in in vitro systems, such technology is essential. The proposed project will create 3 new full-time positions immediately, including 2 postdoctoral fellows and one technician, in addition to partial support of two other positions. Total 3.3 FTEs will be created or retained through this ARRA funding. These hires are expected to be made quickly and without difficulty from a candidate list that PI already has. A detailed budget has been submitted and included with this request. At the end of two years of ARRA support, the investigators will complete the first 2 specific aims. First, they will characterize the dose-dependency, time course and recovery of mitochondrial depolarization in vivo after ethanol treatment in relation to SIAM and hepatic steatosis. Second, they will evaluate the role of alcohol dehydrogense (ADH), the microsomal ethanoloxidizing system (MEOS) and acetaldehyde dehydrogenase (ALDH) in ethanol-induced mitochondrial depolarization. The work during 2 years of ARRA support will develop and apply new technologies of intravital multiphoton microscopy and lay a solid foundation for further research to elucidate the mechanisms by which mitochondrial dysfunction contributes to the pathophysiology of ALD. During the 2-Year ARRA funding period, 4-6 abstracts will be submitted to national and international meetings and 2-4 papers will be submitted to peer-reviewed journals. This is an application in response to RFA-AA-08-002 (The Role Of Mitochondria In Alcohol-Induced Tissue Injury). The priority score of this application (179) is the best among those non-funded RFA applications. The RFA payline for R01 was 171 The RFA payline for R21 was 183. The PI can address all major concerns of the reviewers. However, because this is a RFA application, the PI is not allowed to respond to the critiques, revise the application, and resubmit.