Mitochondrial Energy Sensing and Neuronal Ischemia

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， 中风缺血性中风导致线粒体功能的变化，根据严重程度或持续时间， 可能会造成损害或保护。然而，调节这些之间平衡的机制 结果尚未完全了解，但可能取决于线粒体功能如何、何时以及在何处发生变化。 由于线粒体功能和信号传导依赖于线粒体膜电位（Δ μ m），因此我们的 中心假设是神经元缺血的结果取决于Δ Tcm的时间和程度， （去）极化。目前，该领域缺乏工具来评估Δ m对缺血性结局的贡献 独立于其他变量。我们的建议通过应用一种新的光遗传学方法来解决这一差距， 调节Δ λ m，从而使其与上游代谢分离。传统的光遗传学方法使用 控制神经元质膜电位的光敏蛋白。在这里，我们将应用这种方法， 线粒体响应不同波长的光而增加或减少Δ λ m。我们将使用一个 新的CRISPR/Cas9方法以单拷贝水平表达我们的光激活蛋白， 在体内生物能量学的空间和时间控制。该系统可以模拟或逆转Δ μ m的变化， 发生在中风期间。结合C. elegans遗传学和上位性，这种方法将探索 缺氧病理学中介导线粒体信号传导的分子机制。使用神经元选择性 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性结局。 神经元此外，我们将描述生物能量学如何影响神经元回路，在 缺血病理学总的来说，我们的方法的结果将使我们能够整合Δ λ m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。

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.