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A Reduced Complexity Cross in BALB/c substrains to identify the genetic basis of oxycodone dependence phenotypes

BALB/c 亚种中复杂性降低的杂交以确定羟考酮依赖性表型的遗传基础

基本信息

批准号：
10437702
负责人：
CAMRON D BRYANT
金额：
$ 71.49万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10437702
关键词：
Adaptive Behaviors Address Affective Alleles Animal Model Behavior Behavioral Biological Body Weight decreased Boston Brain region CRISPR/Cas technology Candidate Disease Gene Cell Nucleus Cells Cessation of life Collaborations Collection Complement Cues Dependence Dose Drug Addiction Emotional Epidemic Future Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Genetic Crosses Genetic Variation Genetic study Genomics Genotype Health Hereditary Disease Heritability Human Human Genome Inbred BALB C Mice Inbred Mouse Learning Maps Memory Mental disorders Modeling Molecular Mus Neurobiology Neurons Nucleotides Opiate Addiction Opioid Opioid agonist Outcome Oxycodone Pathway interactions Pharmaceutical Preparations Phenotype Process Quantitative Trait Loci Resolution Retrieval Rewards Risk Small Nuclear RNA Speed Statistical Study Substance Use Disorder Time Tissue-Specific Gene Expression Tissues Training Treatment Protocols United States Universities Validation Variant Withdrawal Work addiction base behavioral outcome brain tissue causal variant cell type drug reward drug withdrawal gene discovery gene network genetic analysis genetic variant genome wide association study glial activation improved in vivo innovation insight motor learning mouse model mu opioid receptors neuroadaptation novel opioid withdrawal trait transcriptome sequencing whole genome

项目摘要

PROJECT SUMMARY Substance use disorders (SUDs) are heritable psychiatric disorders with a significant genetic component. Opioid dependence, one of the most heritable SUDS, has reached epidemic proportions in the United States. Human genome-wide association studies (GWAS) are statistically underpowered to detect the majority of common genetic variation that contributes to opioid dependence. Discovery-based genetics in mammalian model organisms is a powerful complement to human GWAS and can uncover novel genetic factors, biological pathways, and gene networks underlying addiction traits. Mouse models are advantageous because they enable collection of the relevant brain tissue at the appropriate time points under controlled opioid dosing. Furthermore, gene editing permits the validation of functional variants in vivo within the same species on a controlled, genetic background. Reduced Complexity Crosses (RCCs) are genetic crosses between inbred mouse substrains that are nearly genetically identical and can vastly improve the speed at which causal genetic factors can be identified. Our primary objective is to use an RCC between BALB/c substrains to discover the genetic and molecular basis of opioid addiction-relevant traits at two stages of opioid dependence following repeated administration of the mu opioid receptor agonist oxycodone (OXY; the active ingredient of Oxycontin®). We found robust differences between BALB/c substrains in opioid adaptive behaviors, including state-dependent learning of OXY-induced locomotor stimulation and reward following limited, low-dose administration (1.25 mg/kg, IP) as well as the emotional-affective component of opioid withdrawal and weight loss following repeated high-dose administration (40 mg/kg, IP). In Aim 1, we will map quantitative trait loci (QTLs) underlying these OXY phenotypes in an RCC F2 cross. In Aim 2, we will map QTLs controlling gene expression (eQTLs) in the relevant brain tissues of control F2 mice and in OXY-trained F2 mice. We will then nominate candidate causal genes and nucleotides underlying behavior by integrating eQTL with behavioral QTL analysis. To increase precision in assigning candidate variants with the regulation of gene expression and behavior and to identify biological pathways and opioid-adaptive gene networks in specific cell types, we will use single nucleus RNA- seq (snRNA-seq) of brain tissue following limited, low-dose OXY and repeated high-dose OXY. In Aim 3, we will validate candidate functional variants underlying OXY phenotypes using CRISPR/Cas9 gene editing of each of the two alternate alleles onto each reciprocal substrain background. This approach will allow us to demonstrate both necessity and sufficiency of the quantitative trait nucleotides. The proposed studies will identify the genetic basis of unique opioid phenotypes across two stages of opioid dependence. Independent from gene discovery, these studies have broader application in revealing novel, actionable insight toward cellular adaptations at progressive stages of the opioid addiction process and potentially improving behavioral outcomes.

项目概要物质使用障碍 (SUD) 是一种具有显着遗传成分的遗传性精神疾病。阿片类药物依赖性是最具遗传性的 SUDS 之一，在美国已达到流行病的程度。人类全基因组关联研究（GWAS）在统计上不足以检测大多数常见的导致阿片类药物依赖的遗传变异。哺乳动物模型中基于发现的遗传学生物体是人类 GWAS 的有力补充，可以发现新的遗传因素、生物成瘾特征的途径和基因网络。鼠标模型是有利的，因为它们能够在控制阿片类药物剂量的适当时间点收集相关脑组织。此外，基因编辑允许在受控的遗传条件下验证同一物种体内的功能变异背景。降低复杂性杂交 (RCC) 是近交小鼠亚系之间的遗传杂交，基因几乎相同，可以极大地提高因果遗传因素的速度确定。我们的主要目标是利用 BALB/c 亚种之间的 RCC 来发现遗传和反复阿片类药物依赖两个阶段阿片类药物成瘾相关特征的分子基础给予μ阿片受体激动剂羟考酮（OXY；奥施康定®的活性成分）。我们发现 BALB/c 亚株在阿片类药物适应行为（包括状态依赖性）方面存在显着差异在有限的低剂量给药后学习 OXY 诱导的运动刺激和奖励（1.25 mg/kg，IP）以及阿片类药物戒断和反复服用后体重减轻的情绪情感成分高剂量给药（40 mg/kg，IP）。在目标 1 中，我们将绘制这些基因座背后的数量性状位点 (QTL) RCC F2 杂交中的 OXY 表型。在目标 2 中，我们将绘制控制基因表达的 QTL（eQTL）对照 F2 小鼠和 OXY 训练的 F2 小鼠的相关脑组织。然后我们将提名候选因果通过将 eQTL 与行为 QTL 分析相结合，研究行为背后的基因和核苷酸。增加通过基因表达和行为的调节来精确分配候选变体并识别特定细胞类型中的生物途径和阿片类药物适应性基因网络，我们将使用单核 RNA- 有限、低剂量 OXY 和重复高剂量 OXY 后脑组织的 seq (snRNA-seq)。在目标 3 中，我们将使用每个基因的 CRISPR/Cas9 基因编辑来验证 OXY 表型背后的候选功能变异两个交替等位基因到每个相互亚株背景上。这种方法将使我们能够证明数量性状核苷酸的必要性和充分性。拟议的研究将确定遗传跨阿片类药物依赖的两个阶段的独特阿片类药物表型的基础。独立于基因发现，这些研究在揭示细胞适应的新颖的、可操作的见解方面具有更广泛的应用阿片类药物成瘾过程的渐进阶段并可能改善行为结果。