Mitochondrial Energy Sensing and Neuronal Ischemia

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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.