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 (??m), our central hypothesis is that neuronal ischemic outcomes depend on the timing and degree of ??m (de)polarization. Currently, the field lacks tools to assess the contribution of ??m to ischemic outcomes independent of other variables. Our proposal addresses this gap by applying a novel optogenetic approach to regulate the ??m, 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 ??m 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 ??m 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 ??m, an elusive, dependent variable, to stress outcomes in order to better guide future therapeutic strategies.

Public Health Relevance

Mitochondria are important for stroke survival and can contribute to either adaptation or energetic failure through mechanisms that remain incompletely understood. We propose a novel approach that employs light to directly regulate mitochondrial function to determine when, where, and how it alters the balance between damage and protection during ischemic stroke. We anticipate that this approach will allow us to better understand how mitochondrial function regulates stroke outcomes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS115906-01
Application #
9939131
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Bosetti, Francesca
Project Start
2020-02-01
Project End
2024-11-30
Budget Start
2020-02-01
Budget End
2020-11-30
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Rochester
Department
Anesthesiology
Type
School of Medicine & Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627