Mitochondrial ROS microdomains and neuronal ischemia

项目摘要 活性氧物种(ROS)有助于病理，但反过来，它们在有限的程度上也可以起到作用 第二信使，从而为有益的细胞信号做出贡献。类似于钙信号或其他 第二信使，ROS产生的准确位置、时间和持续时间可能决定分歧 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尚不清楚 学习。一个有趣的例子表明，ROS的一个明显的矛盾影响发生在线粒体的复合体I上 电子传输链。在氧化的有害影响的情况下，线粒体复合体i ROS的产生 在机制上与缺血再灌注(IR)损伤中的氧化损伤有关，这是中风的病理。在 在有益信号的情况下，复杂的i ROS的产生与保护性低氧信号有关。事实上， 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 对正常的生理有贡献。因此，描述复杂的iROS生产及其背景的细微差别- 依赖的代谢效应是充分确定线粒体氧化还原信号机制所必需的， 无论是破坏性的还是生理性的。为了实现这一目标，对ROS生产的精确实验控制是 必填项。直到最近，将ROS生产作为一个自变量来控制一直是困难的。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，并分离ROS产生 在遗传模式生物秀丽线虫中的复合体I。之前，我们已经证明了ROS的生产在 在IR损伤模型中，复杂II微域不同地影响氧化还原敏感的结果，这取决于 ROS是在线粒体基质内还是在膜间隙产生。使用我们的 发布了针对线虫快速使用而优化的新型CRISPR/Cas9技术，我们将针对具有良好特性的 光激活的ROS生成蛋白(RGPS)到内源复合体I，以便精确地控制 用光产生复杂的iROS的位置、时间和持续时间。这将提供任一复合体的模型 I氧化还原信号，或氧化损伤，取决于RGP激活的光滴定，那里会有更多的光 产生更多的RO。结合组织特异性表达，我们将确定每一种方法的效果 时空参数对正常线粒体功能、神经元功能和抗逆信号的影响 程序以响应模拟的IR损伤。我们将关注复合体i ROS的神经元结果。 产生，都是为了回应强有力的文献支持，神经元在调节低氧中的重要性 应激信号，并确定可以靶向翻译成哺乳动物模型的神经元回路 中风的可能性。这种方法非常适合于强大的线虫遗传系统。我们期待着这一完成 我们的目标将提供新的，基本的见解，清楚地回答有关线粒体如何 复杂的i ROS微域控制着疾病和生理上的不同结果。

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.