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

项目摘要 阿片类药物使用障碍（OUD）仍然是一个主要的公共卫生问题，2020年的数量最多 与OUD有关的死亡记录处方阿片类药物有助于大部分OUD相关的 死亡，其中阿片类羟考酮（OXY）是处方最多的阿片类药物， %的OUD。OUD相关的特征，包括使用和误用，行为特征和代谢， 然而，OUD性状背后的遗传变异在很大程度上仍未被发现。啮齿动物基因组- 利用数量性状基因座（QTL）定位的广泛关联研究（GWAS）可以有效地识别数量性状， 性状基因和机制，通过强大的动力样本量，控制环境条件， 进行直接遗传操作以验证候选基因的能力;所有明显优于 人类系统遗传学我们之前发现OXY状态依赖性奖励学习的增加， 与密切相关的BALB/cByJ（ByJ）小鼠相比，雌性BALB/cJ（J）小鼠的运动能力，尽管这些 亚株之间仅存在约8，000种遗传变异。这些行为变化对应于 在雌性J的脑内高效氧代谢物羟吗啡酮（OMOR）。一个简化的十字架 在Js和ByJs之间鉴定了15号染色体上的一个稳健的QTL，占潜在方差的29%， J雌性的全脑[OMOR]增加。使用纹状体和海马的进一步表达QTL分析 脑组织鉴定转录抑制基因Zhx 2为我们的首选候选基因。Js包含鼠标 Zhx 2内的内源性逆转录病毒元件（MERV），减少转录和随后的蛋白质 表情Zhx 2调节肝脏中多种细胞色素P450（CYP）酶的表达， 有趣的是，我们观察到大脑CYP 2D 11（人类的小鼠直系同源物）的增加， 将OXY代谢为OMOR的CYP 2D 6与女性Js中ZHX 2的降低相关。我们的发现表明 ZHX 2表达降低会增加CYP 2D表达，从而增加脑[OMOR]和 OXY成瘾模型行为。本提案的主要目的是评估Zhx 2对 通过调节CYP 2D表达和随后的OMOR产生的OXY成瘾模型行为 大脑和肝脏在目标1中，我们将确定肝脏转录组和蛋白质组与[OMOR]的相关性。 和OXY成瘾模型行为通过RNA-seq和蛋白质质谱。在目标2中，我们将删除 Zhx 2 MERV通过CRISPR/Cas9基因编辑，以确定这种遗传变异的必要性， 增加OMOR产生和OXY成瘾模型行为。在目标3中，我们将确定 脑与肝Zhx 2在调节CYP 2D表达、OMOR产生和OXY成瘾模型行为中的作用 通过组织特异性AAV基因操作。我们的研究结果将为遗传学， 氧代谢和成瘾模型行为的分子和组织特异性机制， 可能对阿片类药物治疗和OUD治疗中的个性化药物产生影响。

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.