Mitochondrial ROS microdomains and neuronal ischemia

 描述（由申请人提供）：活性氧（ROS）导致多种神经退行性疾病和急性损伤的病理学，如哺乳动物中的中风。然而，并不是所有的ROS都是相等的，并且疾病输出强烈依赖于ROS产生的时间和位置。此外，有限数量的ROS已被证明可以触发适应性信号通路，并可能有助于有丝分裂。这些事实导致了我们的中心假设，即ROS的生物学影响，就像其他第二信使一样，取决于它们产生的时间，数量和地点。然而，由于它们的反应性质，目前没有方法控制ROS产生的位点或时间。该提议通过将新的光诱导遗传编码的ROS生成蛋白与C. elegans遗传学来确定ROS信号传导在神经元缺血敏感性和应激抗性背景下的进化保守作用。简而言之，用于制造ROS的新的光遗传学工具将使用CRISPR/Cas9技术与编码线粒体呼吸链亚基的核基因表达为蛋白质融合物。该提案的重点是复合物II亚基SDHC，并利用生物传感器的专业知识，模式生物C。elegans和光学以前所未有的精确度研究ROS信号传导。平行的方法，利用皮层神经元的文化将解决进化保守的作用，线粒体活性氧在调节神经元的敏感性缺血。这种方法可能会产生新的治疗策略，其中氧化还原稳态已被破坏的疾病。总的来说，这种新方法将促进我们对线粒体氧化还原信号的理解，使我们能够提出以前使用传统方法无法回答的问题。

 DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) contribute to a diverse range of neurodegenerative diseases and the pathology of acute injuries such as stroke in mammals. However, not all ROS are equal, and the disease output depends strongly on the timing and location of ROS production. Furthermore, ROS in limited quantities have been shown to trigger adaptive signaling pathways and may contribute to mitohormesis. These facts lead to our central hypothesis that the biological impact of ROS, like other second messengers, depends on their timing, quantity and site of generation. However, due to their reactive nature, there is currently no means to control the site or timing of ROS production. This proposal addresses this gap by combining novel photo- inducible genetically-encoded ROS generating proteins with the power of C. elegans genetics to determine the evolutionarily-conserved role of ROS signaling in the context of neuronal ischemic sensitivity and stress resistance. In brief, new optogenetic tools for making ROS will be expressed as protein fusions using CRISPR/Cas9 technology with nuclear genes that encode mitochondrial respiratory chain subunits. The proposal focuses on the complex II subunit SDHC and utilizes expertise in biosensors, the model organism C. elegans, and optics to study ROS signaling with an unprecedented degree of precision. Parallel approaches utilizing cortical neurons in culture will address the evolutionarily conserved role of mitochondrial ROS in regulating neuronal sensitivity to ischemia. This approach could potentially yield new therapeutic strategies for diseases in which redox homeostasis has been disrupted. Overall, this novel approach will advance our understanding of mitochondrial redox signaling, allowing us to ask questions that have previously been unanswerable using conventional methodologies.