Validation studies and multi-omics analysis of Zhx2 as a candidate quantitative trait gene underlying brain oxycodone metabolite (oxymorphone) levels and behavior

Sensitivity to the subjective reinforcing properties of opioids has a genetic component and can predict addiction liability of opioid compounds. We previously identified Zhx2 as a candidate gene underlying increased brain concentration of the oxycodone (OXY) metabolite oxymorphone (OMOR) in BALB/cJ (J) versus BALB/cByJ (By) females that could increase OXY state-dependent reward. A large structural intronic variant is associated with a robust reduction of Zhx2 expression in J mice, which we hypothesized enhances OMOR levels and OXY addiction-like behaviors. We tested this hypothesis by modeling the loss-of-function variant through knocking out the Zhx2 coding exon (E3KO). Integrative transcriptomic and proteomic analysis of E3KO mice identified astrocyte function, cell adhesion, extracellular matrix properties, and endothelial cell functions as pathways influencing brain OXY metabolite concentration and behavior. These results support Zhx2 as a quantitative trait gene underlying brain OMOR concentration that is associated with changes in OXY behavior and implicate potential quantitative trait mechanisms that together inform our overall understanding of Zhx2 in brain function. To investigate potential mechanisms for how Zhx2 influences OXY metabolism and state-dependent reward learning, we established a CRISPR-mouse line where Zhx2 Exon3 (the only coding exon) has been constitutively knocked out in all tissues in BALB/cByJ mice. Brains were harvested from drug-naive CRISPR offspring and the University of Chicago Genomics Facility performed RNA-sequencing on right brain hemisphere for gene expression profiling. Pathway enrichment analysis using Gene-Set Enrichment Analysis was performed on transcriptomic data as well as combined transcriptomic and proteomic data of shared genes.阿片类物质的主观强化效应敏感性具有遗传基础，可用于预测阿片类化合物的成瘾易感性。我们此前在BALB/cJ（简称J）与BALB/cByJ（简称By）雌性小鼠中，鉴定出Zhx2为调控脑内羟考酮（oxycodone, OXY）代谢产物羟吗啡酮（oxymorphone, OMOR）浓度升高的候选基因，该差异可能增强羟考酮的状态依赖性奖赏效应。J小鼠中存在一种大型内含子结构变异，该变异与Zhx2表达的显著下调相关，我们推测这一变异会提升羟吗啡酮水平并诱发类羟考酮成瘾行为。我们通过敲除Zhx2编码外显子构建功能缺失变异模型（E3KO），以此验证上述假说。对E3KO小鼠的整合转录组与蛋白质组分析显示，星形胶质细胞功能、细胞黏附、细胞外基质特性以及内皮细胞功能是影响脑内阿片代谢物浓度与行为的关键通路。本研究结果证实Zhx2是调控脑内羟吗啡酮浓度的数量性状基因，该基因与羟考酮相关行为改变存在关联，并揭示了潜在的数量性状调控机制，有助于我们全面理解Zhx2在脑功能中的作用。为探究Zhx2调控羟考酮代谢与状态依赖性奖赏学习的潜在机制，我们构建了CRISPR工程小鼠品系：在BALB/cByJ小鼠的所有组织中，组成型敲除Zhx2的唯一编码外显子Exon3。我们从未接触过药物的CRISPR子代小鼠中提取脑组织，并由芝加哥大学基因组学中心对右侧大脑半球进行RNA测序以开展基因表达谱分析。我们采用基因集富集分析（Gene-Set Enrichment Analysis），分别对转录组数据以及共享基因的整合转录组-蛋白质组数据进行通路富集分析。