Reactive oxygen species (ROS) contribute to a diverse range of neurodegenerative diseases and the pathology of acute injuries such as stroke in mammals. However, not all ROS are equal, and the disease output depends strongly on the timing and location of ROS production. Furthermore, ROS in limited quantities have been shown to trigger adaptive signaling pathways and may contribute to mitohormesis. These facts lead to our central hypothesis that the biological impact of ROS, like other second messengers, depends on their timing, quantity and site of generation. However, due to their reactive nature, there is currently no means to control the site or timing of ROS production. This proposal addresses this gap by combining novel photo- inducible genetically-encoded ROS generating proteins with the power of C. elegans genetics to determine the evolutionarily-conserved role of ROS signaling in the context of neuronal ischemic sensitivity and stress resistance. In brief, new optogenetic tools for making ROS will be expressed as protein fusions using CRISPR/Cas9 technology with nuclear genes that encode mitochondrial respiratory chain subunits. The proposal focuses on the complex II subunit SDHC and utilizes expertise in biosensors, the model organism C. elegans, and optics to study ROS signaling with an unprecedented degree of precision. Parallel approaches utilizing cortical neurons in culture will address the evolutionarily conserved role of mitochondrial ROS in regulating neuronal sensitivity to ischemia. This approach could potentially yield new therapeutic strategies for diseases in which redox homeostasis has been disrupted. Overall, this novel approach will advance our understanding of mitochondrial redox signaling, allowing us to ask questions that have previously been unanswerable using conventional methodologies.
Oxidative damage is a major contributor to many diseases. However, in some situations reactive oxygen species (ROS) can help to protect the cell. Here, we will utilize novel proteins that can generate ROS in response to light to determine what factors make some ROS beneficial and other ROS toxic. Using the genetic model organism C. elegans will further allow us to integrate our results with conserved stress response pathways, facilitating translation into a mammalian model system.
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