Mitochondrial ROS microdomains and neuronal ischemia

线粒体 ROS 微区和神经元缺血

• 批准号: 10198294
• 负责人: Andrew Phillip Wojtovich
• 金额: $ 40.66万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10198294
• 关键词: Address; Affect; Animal Model; Apoptosis; Automobile Driving; Behavior; Binding Sites; Biochemistry; Bioenergetics; Biological; Biological Assay; Biology; Biosensor; Blood flow; Brain; CRISPR/Cas technology; Caenorhabditis elegans; Calcium Signaling; Cells; Chimeric Proteins; Complex; Data; Disease; Electron Transport; Event; Exhibits; Generations; Genetic; Genetic Models; Goals; Hypoxia; Ischemia; Ischemic Stroke; Light; Link; Literature; Location; Measures; Mediating; Metabolic; Metabolism; Mitochondria; Mitochondrial Electron Transport Complex I; Mitochondrial Matrix; Modeling; Molecular; NADH dehydrogenase (ubiquinone); Nature; Nerve Degeneration; Neurons; Neuropathy; Organism; Outcome; Output; Oxidation-Reduction; Pathologic; Pathology; Pathway interactions; Phenotype; Physiological; Physiology; Play; Predisposition; Process; Production; Proteins; Protons; Publishing; Reactive Oxygen Species; Recovery; Reperfusion Injury; Research Personnel; Resistance; Respiration; Respiratory Chain; Role; Second Messenger Systems; Seminal; Severities; Signal Pathway; Signal Transduction; Site; Stress; Stroke; System; Techniques; Testing; Tissues; Titrations; Translating; Translations; Work; biological adaptation to stress; career; enzyme activity; in vivo; insight; light effects; neural circuit; neuronal circuitry; novel; optogenetics; oxidation; oxidative damage; pleiotropism; programs; protein activation; respiratory; response; sensor; spatiotemporal; stroke model; therapeutic target; tool

Project Summary Reactive oxygen species (ROS) contribute to pathology, but conversely, in limited measure they can also act as second messengers, whereby they contribute to beneficial cellular signaling. Similar to calcium signaling or other second messengers, the precise location, timing, and duration of ROS production likely determine divergent signaling outputs. The mechanism underlying this functional dichotomy in redox biology is currently under studied. An intriguing example of an apparent paradoxical impact of ROS occurs at complex I of the mitochondrial electron transport chain. In the case of detrimental effects of oxidation, mitochondrial complex I ROS production is mechanistically linked to oxidative damage in ischemia reperfusion (IR) injury, the pathology of stroke. In the case of beneficial signaling, complex I ROS production is implicated in protective hypoxic signaling. Indeed, the fact that some ROS production is a normal consequence of mitochondrial respiration supports the idea that ROS contribute to normal physiology. Therefore, describing the nuances of complex I ROS production and its context- dependent metabolic effects is necessary to fully determine the mechanisms of mitochondrial redox signaling, both damaging and physiologic. To achieve that goal, precise experimental control of ROS production is required. Until recently, controlling ROS production as an independent variable has been difficult. This renewal leverages an optogenetic approach championed by our lab to overcome this barrier, and isolates ROS production at complex I in the genetic model organism C. elegans. Previously, we have shown that ROS production at the complex II microdomain differentially affects redox-sensitive outcomes in models of IR injury, depending on whether the ROS were produced inside the mitochondrial matrix or in the intermembrane space. Using our published novel CRISPR/Cas9 technology optimized for rapid use in C. elegans, we will target well-characterized light-activated ROS generating proteins (RGPs) to endogenous complex I in order to precisely control the location, timing, and duration of complex I ROS production with light. This will provide a model of either complex I redox signaling, or oxidative damage, depending on the light-titration of RGP activation, where more light will produce more ROS. Combined with tissue-specific expression, we will determine the effects of each of these spatiotemporal parameters on normal mitochondrial function, neuronal function, and stress-resistance signaling programs in response to simulated IR injury. We will focus on the neuronal outcomes of complex I ROS production, both in response to strong literature support for the importance of neurons in mediating hypoxic stress signaling, and to determine neuronal circuits that could be targeted for translation to mammalian models of stroke. This approach is perfectly suited to the powerful C. elegans genetic system. We expect that completion of our aims will provide novel, fundamental insights with clear answers to questions about how the mitochondrial complex I ROS microdomain controls diverse outcomes in both disease and physiology.

项目摘要 活性氧（ROS）有助于病理学，但相反，在有限的措施，他们也可以作为 第二信使，从而它们有助于有益的细胞信号传导。类似于钙信号或其他 第二信使，ROS产生的精确位置，时间和持续时间可能决定了不同的 信号输出。氧化还原生物学中这种功能二分法的机制目前正在研究中。 研究了一个有趣的例子，一个明显的矛盾影响的活性氧发生在复合物I的线粒体 电子传递链在氧化的不利影响的情况下，线粒体复合物I ROS产生 在机械上与缺血再灌注（IR）损伤中的氧化损伤（中风的病理学）相关。在 在有益信号传导的情况下，复合物I ROS产生涉及保护性缺氧信号传导。持续增 一些ROS产生是线粒体呼吸的正常结果，这一事实支持ROS 有助于正常的生理机能。因此，描述复合物I ROS产生的细微差别及其背景- 依赖性代谢效应对于完全确定线粒体氧化还原信号传导的机制是必要的， 伤害性和生理性的。为了实现这一目标，精确的实验控制活性氧的生产是 必需的.直到最近，控制活性氧的生产作为一个独立的变量一直很困难。此续订 利用我们实验室倡导的光遗传学方法来克服这一障碍， 在遗传模型生物C中的复合体I上。优美的以前，我们已经表明，ROS的生产在 复合物II微结构域在IR损伤模型中不同程度地影响氧化还原敏感性结果，这取决于 ROS是在线粒体基质内还是在膜间隙中产生。使用我们 已发表的新型CRISPR/Cas9技术优化用于C. elegans，我们将针对特征良好的 光激活的ROS产生蛋白（RGPs）与内源性复合物I的结合，以精确控制 位置，时间和持续时间的复合物I ROS生产与光。这将提供一个模型， I氧化还原信号传导，或氧化损伤，取决于RGP激活的光滴定，其中更多的光将 产生更多的ROS。结合组织特异性表达，我们将确定每一个的影响， 正常线粒体功能、神经元功能和抗应激信号传导的时空参数 模拟红外线损伤的程序。我们将重点关注复合物I ROS的神经结果 生产，无论是在响应强有力的文献支持的重要性，神经元介导缺氧 应激信号传导，并确定可用于哺乳动物模型翻译的神经元回路 中风这种方法非常适合强大的C。elegans遗传系统我们希望完成 我们的目标将提供新颖的、基本的见解，并明确回答有关线粒体如何作用的问题 复合物I ROS微结构域控制疾病和生理学的多种结果。