Mitochondrial signaling dynamics in cocaine use disorder

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PROJECT SUMMARY Cocaine use disorder continues to be a significant health burden, with psychostimulant overdose deaths rising significantly over the past few years. Cocaine exposure can alter aspects of mitochondrial function, but the tools to access and manipulate mitochondria in vivo are limited. Mitochondria contribute a significant amount of the energy required for events in the synapse in the form of adenosine triphosphate (ATP). Production of ATP in mitochondria near synapses is tightly regulated by the influx of calcium into mitochondria and leads to the generation of reactive oxygen species within the mitochondrial matrix. The studies in this proposal aim to measure and manipulate mitochondrial function, specifically calcium and hydrogen peroxide signaling in vivo, to determine how mitochondrial activity in dopamine neurons is altered in response to cocaine exposure. We will target three mitochondria-specific protein-encoding genes using a novel CRISPR viral strategy to knock out genes from dopamine neurons in DATCre mice in vivo. The target genes are involved in regulating calcium and hydrogen peroxide concentrations in mitochondria: Micu1, which is part of the mitochondrial calcium uniporter complex and allows mitochondrial calcium influx; Crls1, which encodes an enzyme necessary for the synthesis of cardiolipin, a mitochondrial phospholipid involved in integrating calcium and reactive oxygen species signaling; and Sod2, a mitochondria-specific superoxide dismutase that converts superoxide into hydrogen peroxide and enables removal of ROS from the mitochondrial matrix. Notably, Micu1 and Crls1 encode mitochondrial proteins that have been identified as targets of lead compounds, allowing for a relatively rapid determination of whether these systems can be modulated in vivo for substance use disorder treatments. To begin developing drugs for Sod2 and to refine drugs targeting Micu1 and Crls1, these studies will use mitochondrially-targeted fluorescent sensors for calcium and hydrogen peroxide in combination with fiber photometry in vivo. My preliminary evidence shows that HyPerRed, a hydrogen peroxide sensor, can be used in vivo in cortical neurons to record responses to drugs of abuse. The studies in this proposal will expand on these characterizations and further determine how cocaine may alter reactive oxygen species activity in presynaptic terminals of dopamine neurons in the nucleus accumbens core. I will also test mitochondrially- targeted GCaMP to develop a comprehensive understanding of how calcium and reactive oxygen species activity is coordinated within mitochondria. These sensors will be combined with the CRISPR viral knockout strategies and pharmacological tools targeting mitochondria to further characterize the specificity of these signals and the possible impact of these pharmacological approaches on neural function and behavior. Together, these studies will enable the exploration of a novel avenue of research by monitoring and manipulating mitochondrial function in vivo to develop treatments for cocaine use disorders.