Mitochondrial ROS microdomains and neuronal ischemia

线粒体 ROS 微区和神经元缺血

基本信息

批准号：
10552598
负责人：
Andrew Phillip Wojtovich
金额：
$ 39.81万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
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/10552598
关键词：
Address Affect 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 Proliferating 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 model organism neural circuit neuronal circuitry novel optogenetics oxidation oxidative damage pleiotropism programs protein activation respiratory response sensor spatiotemporal stroke model therapeutic target tool

项目摘要

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.

项目概要活性氧 (ROS) 会导致病理，但相反，在有限的范围内，它们也可以充当第二信使，它们有助于有益的细胞信号传导。与钙信号传导或其他类似作为第二信使，ROS 产生的精确位置、时间和持续时间可能决定了不同的结果信号输出。氧化还原生物学中这种功能二分法背后的机制目前正在研究中研究过。 ROS 明显矛盾影响的一个有趣例子发生在线粒体复合物 I 处。电子传输链。在氧化产生有害影响的情况下，线粒体复合物 I ROS 产生在机制上与缺血再灌注（IR）损伤（中风病理学）中的氧化损伤有关。在在有益信号传导的情况下，复合物 I ROS 的产生与保护性缺氧信号传导有关。确实，一些 ROS 的产生是线粒体呼吸的正常结果，这一事实支持了 ROS 的观点有助于正常的生理机能。因此，描述复杂的 I ROS 产生及其背景的细微差别- 依赖性代谢效应对于充分确定线粒体氧化还原信号传导机制是必要的，既有损害性的，也有生理性的。为了实现这一目标，需要对 ROS 产生进行精确的实验控制必需的。直到最近，将 ROS 产生作为自变量进行控制一直很困难。此次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，并隔离 ROS 的产生位于遗传模型生物秀丽隐杆线虫中的复合体 I。之前，我们已经证明了 ROS 的产生复合体 II 微区对 IR 损伤模型中氧化还原敏感的结果有不同的影响，具体取决于 ROS 是在线粒体基质内还是在膜间隙中产生。使用我们的发表了针对在线虫中快速使用而优化的新型 CRISPR/Cas9 技术，我们将瞄准具有良好特征的光激活ROS生成蛋白（RGP）内源性复合物I，以精确控制光产生复合物 I ROS 的位置、时间和持续时间。这将提供一个复杂的模型 I 氧化还原信号或氧化损伤，取决于 RGP 激活的光滴定，其中更多的光会产生更多的ROS。结合组织特异性表达，我们将确定其中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响应模拟红外损伤的程序。我们将重点关注复杂 I ROS 的神经元结果生产，都是对神经元在介导缺氧中的重要性的强有力的文献支持的回应应激信号传导，并确定可用于转化为哺乳动物模型的神经元回路中风。这种方法非常适合强大的线虫遗传系统。我们预计完成我们的目标将为有关线粒体如何复杂的 I ROS 微域控制着疾病和生理学的不同结果。