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的功能和随后的代谢信号 卒中。缺血性中风会导致线粒体功能的改变，这取决于严重程度或持续性， 既可能造成损害，也可能导致保护。然而，调节这两者之间平衡的机制 结果还不完全清楚，但很可能取决于线粒体功能如何、何时和在哪里改变。 由于线粒体的功能和信号依赖于线粒体膜电位(Δψm)，我们的 中心假说是神经元缺血的结果取决于Δψm的时间和程度。 (去)极化。目前，该领域缺乏评估Δψm对缺血预后的贡献的工具。 独立于其他变量的。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Δψm，从而使其与上游代谢分离。传统的光遗传方法使用 控制神经细胞质膜电位的光敏蛋白。这里，我们将把这种方法应用于 线粒体对不同波长的光作出反应，增加或减少Δψm。我们将使用 新的CRISPR/Cas9方法以单拷贝水平表达我们的光激活蛋白史无前例 活体生物能量学的时空调控。该系统可以模拟或反转Δψm的变化， 发生在中风期间。结合线虫遗传学和上位性的力量，这种方法将探索 缺氧病理中线粒体信号转导的分子机制。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脑缺血预后 神经元。此外，我们还将描述生物能量学是如何影响神经回路的 缺血病理学。总体而言，我们方法的结果将允许我们集成Δψm、 难以捉摸的依赖变量，以强调结果，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.