USING TRANSGENIC MICE TO EXAMINE THE ROLE OF CNIH3 VARIANTS IN OPIOID DEPENDENCE

 描述：本申请是应RFA-DA-16-004的要求提交的，建议从功能上验证NIDA资助的R01(DA17305；Pi E.Nelson)中确定的遗传变异，在该R01中，对仅限于阿片类药物滥用者的样本进行的GWAS分析测试了遗传多态影响从暴露到严重阿片依赖进展的假设。最强的关联是Cornichon家族AMPA受体辅助蛋白3(CNIH3)的SNPs。在两个非依赖阿片类药物滥用的样本中进行的确证分析产生了rs10799590全基因组显著的荟萃分析p；4.3E-9，rs10799590是一种与降低风险相关的内含子CNIH3 SNP[OR 0.64(0.54-0.74)]。大效应可解释1.2%至5.8%的表型差异。此外，CNIH3的相关性与动物模型中阿片类药物的研究一致，强调了谷氨酸信号的重要性。这项应用的R21阶段建议使用CRISPR技术的创新适应来产生已引入人类CNIH3(风险和保护性单倍型)的转基因小鼠。R33阶段建议使用这些小鼠来测试我们的总体假设，即CNIH3多态改变基因表达，影响AMPA信号和对阿片类药物的行为反应。我们将使用与阿片类药物依赖相关的行为分析(条件性位置偏爱、消失和恢复；上下文依赖的敏化)，它以前将AMPA信号与阿片类药物联系起来。我们将确定吗啡反应的基因表达，重点关注已报道AMPA受体亚单位组成变化的成瘾相关脑区，以确定保护性单倍型和风险相关单倍型小鼠之间是否存在显著差异。我们还将研究可能介导单倍型相关效应的潜在途径。该项目的R21部分的具体目标是：目的1产生携带已识别的保护性和风险相关单倍型的人源化Cnih3小鼠。该项目R33部分的具体目标是：1目的1检查具有保护性和风险相关单倍型的转基因小鼠组(以野生型和Cnih3基因敲除小鼠为对照)在吗啡CPP表达、其消亡、和恢复和上下文依赖敏化(CDS)目的2确定保护性单倍型是否改变这些行为范例中Cnih3的表达(阿片前)或对吗啡的反应。目的3检查这些单倍型是否改变与吗啡诱导的行为反应相关的脑区突触后密度中的Cnih3和相关蛋白质含量。AIM 4检查表观遗传学调节与吗啡CPP相关的这些SNP相关基因表达改变的证据，包括识别改变增强子活性的SNP

 DESCRIPTION: This application, submitted in response to RFA-DA-16-004, proposes to functionally validate genetic variants identified in a NIDA-funded R01 (DA17305; PI E. Nelson) in which GWAS analyses in a sample limited to opioid misusers tested the hypothesis that genetic polymorphisms influence progression from exposure to severe opioid dependence. The strongest association was for SNPs in cornichon family AMPA receptor auxiliary protein 3 (CNIH3). Confirmation analyses performed in two samples with non-dependent opioid misusers yielded a genome-wide significant meta-analytic p of <4.3E-9 for rs10799590, an intronic CNIH3 SNP associated with reduced risk [OR 0.64 (0.54-0.74)].The large effect accounts for between 1.2 and 5.8% of the phenotypic variance. Furthermore, the association of CNIH3 is consistent with studies of opioids in animal models highlighting the importance of glutamate signaling. This application's R21 phase proposes to generate transgenic mice into which human CNIH3 has been introduced (both the risk and protective haplotypes) using innovative adaptations of CRISPR technology. The R33 phase proposes to use these mice to test our overarching hypothesis that CNIH3 polymorphisms alter gene expression impacting AMPA signaling and behavioral response to opioids. We will use behavioral assays related to opioid dependence (conditioned place preference, extinction, and reinstatement; context-dependent sensitization), which previously connected AMPA signaling to opioids. We will determine gene expression in response to morphine, focusing on addiction-related brain regions in which AMPA receptor subunit composition changes have been reported, to determine whether significant differences are found between mice with the protective and risk-associated haplotypes. We will also examine potential routes by which haplotype-associated effects may be mediated. The R21 component of the project's specific aim is: AIM 1 To generate "humanized" Cnih3 mice carrying the identified protective and risk-associated haplotypes The specific aims of the R33 component of the project are: AIM 1 To examine phenotypic differences between groups of transgenic mice with the protective and risk- associated haplotypes (with wild type and Cnih3 knockout mice as controls) in the expression of morphine CPP, its extinction, and reinstatement and context-dependent sensitization (CDS) AIM 2 To determine whether the protective haplotype alters Cnih3 expression at baseline (pre-opioids) or in response to morphine in these behavioral paradigms AIM 3 To examine whether these haplotypes alter Cnih3 and related protein content in the post-synaptic density of brain areas associated with morphine-induced behavioral responses AIM 4 To examine evidence for epigenetic mediation of altered gene expression associated with these SNPs in response to morphine CPP, including identifying SNPs that alter the activity of enhancers