Single-cell molecular rhythm alterations in human nucleus accumbens associated with 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 nucleus accumbens (NAc), a major neural substrate for reward and motivation, and a key region involved in 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 postmortem brain associated with various psychiatric disorders to gain 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 NAc in OUD using postmortem brains from unaffected subjects and subjects with OUD. In Aim 1, we will use snRNA-seq and TOD analyses to create a cell type specific map of molecular rhythms in the human NAc using control, ‘neurotypical’ subjects. In Aim 2, we will compare molecular rhythms in specific cell types of the NAc from unaffected subjects to molecular rhythms in NAc cell types from subjects with OUD to determine the extent of molecular rhythm disruptions associated with OUD at the cellular level. Our studies will define the loss and gain of molecular rhythms in NAc cell types associated with OUD, in addition to providing a framework for uncovering the temporal relationships (i.e., circadian) in transcript expression between different cell types in human brain. snRNA-seq findings will be validated using spatial transcriptomics (MERSCOPE). The findings from our proposal will resolve molecular rhythm alterations at the cellular 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.