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年的数字最高 有记录的与OUD相关的死亡人数。处方阿片类药物在很大程度上导致了与OUD相关的 死亡，阿片类药物羟考酮(Oxy)是处方最多的阿片类药物之一，其潜在的显著 OUD百分比。与OUD相关的特征，包括使用和误用、行为特征和新陈代谢 这是一种可遗传的成分，然而，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)作图的广谱关联研究可以有效地识别数量性状 通过强有力的样本大小、受控的环境条件和 执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状态依赖的奖赏学习和 雌性BALB/CJ(J)小鼠与亲缘关系密切的BALB/cByJ(ByJ)小鼠相比，尽管有这些 亚株之间只有大约8000个遗传变异。这些行为变化对应于 女性Js脑内高效氧代谢产物羟吗啡酮(Omor)。降低复杂性的交叉 在Js和ByJs之间，在15号染色体上发现了一个强大的QTL，可以解释29%的变异 J女性的全脑[Omor]增加。利用纹状体和海马区进一步分析基因表达的QTL 脑组织发现转录抑制基因ZX2是我们的首选候选基因。JS包含一个鼠标 ZX2中的内源性逆转录病毒元件(MERV)减少转录和随后的蛋白 表情。Zhx2调节肝脏多种细胞色素P450(CYP)酶的表达 对药物代谢的影响，有趣的是，我们观察到大脑中人类的同源基因--脑细胞色素P450_2D11的增加 将Oxy代谢为OMOR的CYP2D6与女性Js中ZHX2的降低有关。我们的发现表明 ZHX2表达降低会增加CYP2D的表达，从而增加大脑[Omor]和 氧成瘾模型行为。这项建议的主要目标是评估zhx2对 氧成瘾模型动物行为通过调节细胞色素P450-2D的表达和随后的Omor的产生 大脑对抗肝脏。在目标1中，我们将确定肝脏转录和蛋白质组学与[OMOR]的关联 并通过RNA-SeQ和蛋白质质谱仪建立氧瘾行为模型。在目标2中，我们将删除 通过CRISPR/Cas9基因编辑来确定该基因变异在中国的必要性 增加Omor的产生和Oxy成瘾模型的行为。在目标3中，我们将确定 脑与肝ZX2在调节CYP2D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。我们的结果将提供对基因的关键洞察， 氧代谢和成瘾模型行为背后的分子和组织特异性机制 可能会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.