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

 产品说明：本申请是根据RFA-DA-16-004提交的，旨在对NIDA资助的R 01（DA 17305; PI E）中鉴定的遗传变异进行功能验证。纳尔逊），其中在仅限于阿片类药物滥用者的样本中进行的GWAS分析检验了遗传多态性影响从暴露到严重阿片类药物依赖的进展的假设。最强的关联是cornichon家族AMPA受体辅助蛋白3（CNIH 3）中的SNP。在两个非依赖性阿片类药物滥用者样本中进行的确认分析得出rs 10799590的全基因组显著性荟萃分析p <4.3E-9，rs 10799590是一种与风险降低相关的内含子CNIH 3 SNP [OR 0.64（0.54-0.74）]。此外，CNIH 3的相关性与动物模型中阿片类药物的研究一致，突出了谷氨酸信号传导的重要性。 该申请的R21阶段提出使用CRISPR技术的创新适应性来产生已引入人CNIH 3（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CNIH 3多态性改变影响AMPA信号传导和对阿片类药物的行为反应的基因表达。我们将使用与阿片类药物依赖相关的行为测定（条件性位置偏好，消退和恢复;上下文依赖性敏化），这些行为测定先前将AMPA信号传导与阿片类药物联系起来。我们将确定响应吗啡的基因表达，重点关注已报道AMPA受体亚基组成变化的成瘾相关脑区，以确定具有保护性和风险相关单倍型的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为了产生“人源化的”Cnih 3小鼠携带确定的保护性和风险相关的单倍型该项目的R33部分的具体目标是：目的1检测具有保护性和风险相关单倍型的转基因小鼠之间的表型差异（以野生型和Cnih 3敲除小鼠作为对照）在吗啡CPP的表达，其消退，目的2确定保护性单倍型是否改变基线时Cnih 3的表达（前阿片样物质）或在这些行为范例中对吗啡的反应AIM 3为了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域的突触后密度中的Cnih 3和相关蛋白质含量AIM 4为了检查在对吗啡CPP的反应中与这些SNP相关的改变的基因表达的表观遗传介导的证据，包括鉴定改变增强子活性的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