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

Zhx2 在 CYP2D 调节、羟考酮代谢和阿片类药物成瘾模型行为中的作用。

• 批准号: 10466122
• 负责人: William Lynch
• 金额: $ 4.77万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-08 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10466122
• 关键词: Accounting; Analgesics; Behavior; Behavioral; Biological Assay; Brain; CRISPR/Cas technology; CYP2D6 gene; Candidate Disease Gene; Cessation of life; Chromosome 15; Chromosome Mapping; Clinical; Code; Complex; Corpus striatum structure; Cytochrome P450; DNA Insertion Elements; Data; Elements; Environment; Enzymes; Epidemic; Excision; Female; Genes; Genetic; Genetic Transcription; Genotype; Goals; Harvest; Hepatic; Heritability; High Pressure Liquid Chromatography; Hippocampus; Homeobox; Human; Inbred BALB C Mice; Inbred Mouse; Learning; Link; Liver; Liver Extract; Locomotion; Maps; Mass Spectrum Analysis; Messenger RNA; Metabolism; Modeling; Molecular; Monitor; Mus; Neurobiology; Opiate Addiction; Opioid; Oral; Orthologous Gene; Oxycodone; Oxymorphone; Phenotype; Plasma; Predisposition; Privatization; Production; Proteins; Proteome; Proteomics; Public Health; Quantitative Trait Loci; Regulation; Rewards; Rodent; Rodent Model; Role; Sample Size; Self Administration; Site; Substance Use Disorder; System; Testing; Therapeutic; Therapeutic Uses; Tissues; Training; Transcription Repressor; United States; Validation; Variant; Zinc Fingers; Zinc deficiency; addiction; adeno-associated viral vector; behavioral phenotyping; brain tissue; candidate validation; causal variant; conditioned place preference; drug metabolism; genetic manipulation; genetic variant; genome wide association study; genome-wide; in vivo; individualized medicine; insight; interdisciplinary approach; liver metabolism; model organism; mouse genetics; novel; opioid mortality; opioid use disorder; overexpression; personalized medicine; pharmacologic; prescription opioid; protein expression; rodent genome; small hairpin RNA; therapeutic opioid; trait; transcriptome; transcriptome sequencing; transcriptomics

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.

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 有记录的 OUD 相关死亡人数。处方阿片类药物占 OUD 相关疾病的很大一部分 死亡，其中阿片类药物羟考酮 (OXY) 是最常用的阿片类药物之一，并且具有显着的潜在风险 OUD 的百分比。 OUD 相关特征，包括使用和误用、行为特征和新陈代谢，对 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- 具有数量性状基因座（QTL）作图的广泛关联研究（GWAS）可以有效地识别数量性状 通过强大的样本量、受控的环境条件和 进行直接遗传操作以验证候选基因的能力；所有明显的优势 人类系统遗传学。我们之前发现 OXY 状态依赖奖励学习的增加和 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 亚种之间仅存在约 8,000 个遗传变异。这些行为变化对应于 女性 Js 大脑内的高效 OXY 代谢物羟吗啡酮 (OMOR)。降低复杂性的交叉 Js 和 ByJs 之间在 15 号染色体上发现了一个稳健的 QTL，占潜在方差的 29% J 女性的全脑 [OMOR] 增加。使用纹状体和海马进一步表达 QTL 分析 脑组织将转录抑制基因 Zhx2 确定为我们的首要候选基因。 js包含鼠标 Zhx2 内的内源性逆转录病毒元件 (MERV)，减少转录和随后的蛋白质 表达。 Zhx2 调节肝脏（主要部位）中多种细胞色素 P450 (CYP) 酶的表达 药物代谢的影响，有趣的是我们观察到大脑 CYP2D11 的增加，CYP2D11 是人类的小鼠直系同源物。 CYP2D6 将 OXY 代谢为 OMOR，与女性 J 中 ZHX2 的减少相关。我们的研究结果表明 ZHX2 表达减少会增加 CYP2D 表达，从而增加大脑 [OMOR] 和 OXY 成瘾模型行为。该提案的主要目标是评估 Zhx2 对 OXY通过调节CYP2D表达和随后的OMOR产生来模拟成瘾行为 大脑与肝脏。在目标 1 中，我们将确定肝脏转录组和蛋白质组与 [OMOR] 的关联 和 OXY 通过 RNA 测序和蛋白质质谱分析成瘾模型行为。在目标 2 中，我们将删除 Zhx2 MERV通过CRISPR/Cas9基因编辑来确定这种遗传变异的必要性 增加 OMOR 的产生和 OXY 成瘾模型行为。在目标 3 中，我们将确定以下方面的相对作用： 大脑与肝脏 Zhx2 在调节 CYP2D 表达、OMOR 产生和 OXY 成瘾模型行为中的比较 通过组织特异性 AAV 基因操作。我们的结果将为遗传、 OXY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 治疗中的个性化医疗产生影响。