Role of Striatal Molecular Rhythms in Sleep and Opioid Use Disorder - PROJECT SUMMARY Opioid use and deaths from overdoses have skyrocketed in the United States over recent years. Most people with opioid use disorder (OUD) relapse within weeks to months despite treatment. Relapse vulnerability is strongly associated with severe and persistent sleep and circadian rhythm disruptions, suggesting therapeutics that mitigate these alterations during withdrawal and abstinence may reduce craving and risk for relapse. However, our understanding of the mechanisms across cellular and molecular levels in the brains of people with OUD is extremely limited. We recently demonstrated alterations in several pathways related to dopamine, opioid, and glutamate signaling in human brain associated with OUD. We also reported significant disruptions in molecular rhythms associated with OUD in human striatum, encompassing major neural substrates for reward and motivation, as well as arousal, sleep, and circadian rhythms. We and others developed a novel and innovative approach for using time-of-death (TOD) of the subject to measure molecular rhythms in human postmortem brain associated with various psychiatric disorders, gaining insights into disease-specific neurobiological mechanisms and pathways in human brain. In this proposal, we will use TOD computational tools combined with single nuclei RNA-sequencing (snRNA-seq) to investigate the relationship between molecular rhythm disruption in specific cell types of the striatum (nucleus accumbens (NAc), caudate, putamen) in OUD using postmortem brains from unaffected subjects and subjects with OUD, followed by mechanistic studies using brain region- and cell type-specific ablation of molecular rhythms in mouse models of opioid self- administration. In Aim 1, we will use snRNA-seq and TOD analyses to create a cell type specific map of molecular rhythms in subregions of human striatum. In Aim 2, we will compare molecular rhythms in specific cell types of the striatum from unaffected subjects to molecular rhythms in cell types from subjects with OUD to determine the extent of molecular rhythm disruptions associated with OUD at the cellular level. Results will be integrated with human GWAS sleep and opioid traits. snRNA-seq findings will be validated using in situ RNAscope hybridization of the top rhythmic transcripts and cell type specific markers. We then directly investigate the functional relevance of molecular rhythm ablation in specific striatal cell types during opioid self- administration and sleep physiology and assess the impact of opioids on cell type-specific molecular rhythms in mice in Aim 3. The findings from our proposal will resolve molecular rhythm alterations at the cell type-specific level in the brains of subjects with OUD to begin to identify the mechanisms underlying the relationships between circadian rhythms and OUD in the hopes of progressing towards strategies that target the circadian system in the treatment of OUD.
