Mitochondrial Energy Sensing and Neuronal Ischemia

线粒体能量感应和神经元缺血

基本信息

批准号：
10318080
负责人：
Andrew Phillip Wojtovich
金额：
$ 38.36万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318080
关键词：
Address Adult Affect Animal Behavior Animal Model Axon Behavioral Bioenergetics Biological Models Biosensor CRISPR/Cas technology Caenorhabditis elegans Calcium Cations Cell Culture Techniques Cell Death Cell membrane Cell physiology Cells Development Distant Energy Supply Equilibrium Etiology Event Exhibits Failure Future Gene Expression Genes Genetic Genetic Epistasis Hypoxia In Vitro Inner mitochondrial membrane Intervention Ischemia Ischemic Stroke Learning Light Location Measures Mediating Membrane Potentials Metabolic Metabolism Methods Mitochondria Molecular Molecular Probes Morphology Neurons Outcome Output Oxygen Oxygen Consumption Pathologic Pathology Pathway interactions Pharmacology Production Proteins Proton Pump Protons Reactive Oxygen Species Recovery Reperfusion Injury Reperfusion Therapy Reporter Role Severities Signal Pathway Signal Transduction Specificity Stress Stroke Surveys System Techniques Testing Therapeutic Time Tissues Whole Organism biological adaptation to stress cell type genetic approach in vivo insight mitochondrial membrane mutant neuron loss neuronal cell body neuronal circuitry neuronal survival novel novel strategies optogenetics promoter response stroke outcome tool

项目摘要

Neuronal survival or death depends strongly on mitochondrial function and metabolic signaling following stroke. Ischemic stroke causes changes in mitochondrial function that, depending on severity or persistence, can result in either damage or protection. However, the mechanisms that regulate the balance between these outcomes are not fully understood, but likely depend on how, when and where mitochondrial function changes. As mitochondrial function and signaling are dependent on the mitochondrial membrane potential (Δψm), our central hypothesis is that neuronal ischemic outcomes depend on the timing and degree of Δψm (de)polarization. Currently, the field lacks tools to assess the contribution of Δψm to ischemic outcomes independent of other variables. Our proposal addresses this gap by applying a novel optogenetic approach to regulate the Δψm, thereby dissociating it from upstream metabolism. Conventional optogenetic approaches use light-sensitive proteins to control neuronal plasma membrane potentials. Here we will apply this approach in mitochondria to either increase or decrease the Δψm in response to different wavelengths of light. We will use a novel CRISPR/Cas9 method to express our light-activated proteins at single-copy levels to take unprecedented spatial and temporal control of bioenergetics in vivo. This system can mimic or reverse the Δψm changes that occur during stroke. Combined with the power of C. elegans genetics and epistasis, this approach will probe the molecular mechanisms that mediate mitochondrial signaling in hypoxic pathology. Using neuron-selective gene expression, we will test how mitochondrial function affects ischemic outcomes in different types of neurons. In addition, we will characterize how bioenergetics influences the neuronal circuits that signal during ischemic pathology. Overall, the results of our approach will allow us to integrate the contribution of Δψm, an elusive, dependent variable, to stress outcomes in order to better guide future therapeutic strategies.

神经元的存活或死亡在很大程度上取决于线粒体功能和代谢信号缺血性中风会导致线粒体功能发生变化，具体取决于严重程度或持续时间，可能会导致损害或保护，但是调节这些之间平衡的机制。结果尚不完全清楚，但可能取决于线粒体功能如何、何时何地发生变化。由于线粒体功能和信号传导取决于线粒体膜电位 (Δψm)，我们的中心假设是神经缺血的结果取决于 Δψm 的时间和程度目前，该领域缺乏评估 Δψm 对缺血结果影响的工具。我们的建议通过应用一种新颖的光遗传学方法来解决这一差距。调节 Δψm，使其与上游代谢分离，从而使用传统的光遗传学方法。光敏蛋白来控制神经元质膜电位。在这里，我们将应用这种方法。我们将使用线粒体来增加或减少 Δψm 以响应不同波长的光。新颖的 CRISPR/Cas9 方法以单拷贝水平表达我们的光激活蛋白，取得了前所未有的成果该系统可以模拟或逆转体内生物能量的空间和时间控制。结合线虫遗传学和上位性的力量，这种方法将探测中风期间发生的情况。利用神经元选择性介导缺氧病理学中线粒体信号传导的分子机制。基因表达，我们将测试线粒体功能如何影响不同类型的缺血结果此外，我们还将描述生物能量学如何影响在过程中发出信号的神经元回路。总的来说，我们的方法的结果将使我们能够整合 Δψm 的贡献，难以捉摸的因变量，强调结果，以便更好地指导未来的治疗策略。