The role of Zhx2 in CYP2D regulation, oxycodone metabolism, and opioid addiction model behaviors.

项目摘要

PROJECT ABSTRACT Opioid Use Disorder (OUD) remains a major public health issue, with 2020 containing the highest number of OUD-related deaths on record. Prescription opioids contribute towards a substantial portion of OUD-related deaths, with the opioid oxycodone (OXY) among the most prescribed opioids and underlying a significant percentage of OUDs. OUD-related traits, including use and misuse, behavioral traits, and metabolism have a heritable component, yet genetic variants underlying OUD traits remain largely undiscovered. Rodent genome- wide association studies (GWAS) with quantitative trait locus (QTL) mapping can efficiently identify quantitative trait genes and mechanisms through robustly powered sample-sizes, controlled environmental conditions, and the ability to perform direct genetic manipulations to validate candidate genes; all distinct advantages over human system genetics. We previously identified increases in OXY state-dependent reward learning and locomotion in female BALB/cJ (J) mice compared to the closely-related BALB/cByJ (ByJ) mice, despite these substrains differing by only ~8,000 genetic variants. These behavioral changes corresponded to increases in the highly potent OXY metabolite oxymorphone (OMOR) within the brain in female Js. A reduced complexity cross between Js and ByJs identified a robust QTL on chromosome 15, accounting for 29% of variance underlying increased whole brain [OMOR] in J females. Further expression QTL analysis using striatal and hippocampal brain tissue identified the transcriptional repressor gene Zhx2 as our top candidate gene. Js contain a mouse endogenous retroviral element (MERV) within Zhx2, decreasing transcription and subsequent protein expression. Zhx2 regulates expression of multiple cytochrome P450 (CYP) enzymes in the liver, the primary site of drug metabolism, and interestingly we observed increases of brain CYP2D11, a mouse ortholog of human CYP2D6 that metabolizes OXY to OMOR, correlated to decreased ZHX2 in female Js. Our findings suggest that decreased ZHX2 expression increases CYP2D expression, consequentially increasing both brain [OMOR] and OXY addiction model behaviors. The primary objective of this proposal is to assess the contribution of Zhx2 to OXY addiction model behaviors through regulation of CYP2D expression and subsequent OMOR production in the brain vs. the liver. In Aim 1, we will identify hepatic transcriptomic and proteomic associations with [OMOR] and OXY addiction model behaviors through RNA-seq and protein mass-spectrometry. In Aim 2, we will delete the Zhx2 MERV through CRISPR/Cas9 gene editing to determine the necessity of this genetic variant in increasing OMOR production and OXY addiction model behaviors. In Aim 3, we will identify the relative role of brain vs. liver Zhx2 in regulating CYP2D expression, OMOR production, and OXY addiction model behaviors through tissue-specific AAV genetic manipulations. Our results will provide critical insight into the genetic, molecular, and tissue-specific mechanisms underlying OXY metabolism and addiction model behaviors that could have implications for personalized medicine in opioid therapeutics and OUD treatments.