Bridging genetic variation with behavior: Molecular and functional mechanisms of quantitative trait gene regulation of the stimulant and addictive properties of methamphetamine in mice

将遗传变异与行为联系起来：数量性状基因调节小鼠甲基苯丙胺的兴奋和成瘾特性的分子和功能机制

• 批准号: 9926356
• 负责人: CAMRON D BRYANT
• 金额: $ 0.85万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926356
• 关键词: ADRA1B gene; AGFG1 gene; Addictive Behavior; Adrenergic Agents; Alleles; Alternative Splicing; Animal Model; Behavior; Biological; Chromosomes, Human, Pair 11; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Congenic Mice; Corpus striatum structure; DARPP; Data; Disease; Down-Regulation; Early Endosome; Environmental Risk Factor; Etiology; Exhibits; FDA approved; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genetic Variation; Genetic study; Genomics; Genotype; Glutamates; Heroin Dependence; Human; Human Genetics; Immunoblotting; Immunohistochemistry; Inherited; Intercistronic Region; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Link; Measures; Mediating; Mental Health; Methamphetamine; Methamphetamine dependence; Microdialysis; Midbrain structure; Molecular; Motor Activity; Mus; Neurobiology; Neurons; Nucleotides; Oral; Pathway interactions; Patients; Phenotype; Predisposition; Prevention; Property; Proteins; Psychostimulant dependence; Public Health; Quantitative Trait Loci; RNA-Binding Proteins; Regulatory Element; Relapse; Research; Self Administration; Series; Signal Transduction; Signaling Molecule; Synaptic Transmission; System; Testing; Therapeutic; Transcription Coactivator; Translating; United States; Variant; Wild Type Mouse; addiction; congenic; differential expression; dopaminergic neuron; gene discovery; genetic variant; genome wide association study; glutamatergic signaling; in vivo; insight; mRNA sequencing; monoamine; mu opioid receptors; mutant; nerve supply; neurobiological mechanism; neurochemistry; neuron development; neurotransmission; neurotransmitter release; noradrenergic; novel; novel therapeutics; nuclease; null mutation; psychostimulant; public health relevance; receptor; resilience; response; targeted nucleases; tool; trait; transcription activator-like effector nucleases; transcriptome; transcriptome sequencing; transcriptomics; transmission process; violent crime

 DESCRIPTION (provided by applicant): Methamphetamine (MA) addiction is a pervasive public health concern in the U.S. and is associated with violent crime, mental health decline, and a high rate of relapse in recovering patients. Despite decades of research, there are still no FDA-approved treatments for MA addiction. Both genetic and environmental factors contribute to the etiology of MA addiction; however, genome-wide association studies in humans are limited in their power to identify common variants associated with this devastating disease. Mouse genetics offers a powerful, complementary tool for accelerating novel gene discovery and biological mechanisms. We recently identified a 210 kb region on mouse chromosome 11 containing two protein coding genes (Hnrnph1 and Rufy1) that contributes to the locomotor stimulant response to MA. The primary objective of this proposal is to identify the novel quantitative trait gene (QTG) and the neurobiological mechanism by which this QTG regulates the stimulant and addictive properties of MA. In Aim 1, we will identify the QTG(s) that regulates MA sensitivity. We generated a 112 kb congenic mouse containing only Hnrnph1 and Rufy1, providing the unique opportunity to determine whether this region is both necessary and sufficient for MA sensitivity. Furthermore, we recently used transcriptional activator-like effecto nucleases (TALENs) to introduce null mutations in Hnrnph1 and Rufy1. Preliminary studies provide strong, replicable evidence that Hnrnph1 is a QTG for reduced MA sensitivity. To fully tackle all of the polymorphisms within the 210 kb region that could potentially contribute to behavior, we will also delete a 150 kb intergenic region using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to introduce double stranded breaks that flank the intergenic region. Finally, we will use CRISPR/Cas9 to generate knockin mice carrying SNPs from the candidate QTG. In Aim 2, we will use transcriptome analysis of the striatum and ventral midbrain via mRNA sequencing (RNA- seq) in mice carrying the congenic allele, the knockout allele of the QTG, or both alleles relative to wild-type mice to hone the molecular pathways and neurobiological mechanisms that link genetic variation with behavior. Preliminary analysis of a congenic line capturing the QTL for reduced MA sensitivity suggests perturbations in midbrain dopaminergic neuron development, glutamate signaling, and adrenergic signaling as potentially responsible for a reduction in MA-induced behavior. In Aim 3, we will use in vivo microdialysis, immunoblotting, immunohistochemistry, and operant oral MA self-administration in congenics and knockouts to functionally assess the neurochemical mechanism by which the QTG influences the stimulant and addictive properties of MA. The proposed studies employ a unique combination of genetics, genomics, and functional analyses to uncover the neurobiological mechanism by which a novel genetic factor contributes to addiction-relevant behaviors. These findings will likely have translational relevance in the genetic and molecular mechanisms that confer susceptibility/resilience to MA addiction and could inform new therapeutic avenues.

 描述（由适用提供）：甲基苯丙胺（MA）成瘾是美国普遍存在的公共卫生问题，与暴力犯罪，心理健康下降和康复患者的救济率很高有关。尽管进行了数十年的研究，但仍然没有FDA批准的MA成瘾治疗方法。遗传和环境因素都导致了MA成瘾的病因。但是，人类全基因组的关联研究的限制是识别与这种毁灭性疾病相关的常见变异的能力。小鼠遗传学为加速新型基因发现和生物学机制提供了强大的完整工具。我们最近在11个小鼠染色体上鉴定了一个210 Kb区域，其中包含两个蛋白质编码基因（HNRNPH1和RUFY1），该基因有助于对MA的运动刺激响应。该提案的主要目的是确定新的定量性状基因（QTG）和神经生物学机制，该QTG调节MA的兴奋剂和加性特性。在AIM 1中，我们将确定调节MA灵敏度的QTG。我们产生了一个仅包含HNRNPH1和RUFY1的112 KB先生小鼠，提供了独特的机会来确定该区域是否既是必要又足够的MA灵敏度。此外，我们最近使用了转录激活剂样核酸酶（TALENS）在HNRNPH1和RUFY1中引入无效突变。初步研究提供了强有力的，可复制的证据，表明HNRNPH1是降低MA灵敏度的QTG。为了充分解决210 KB区域内的所有多态性，可能有助于行为，我们还将使用簇状的定期散布的短篇小学重复序列（CRISPR）/CAS9系统删除150 KB的基因间区域，以引入侧基因间区域的双链突破。最后，我们将使用CRISPR/CAS9生成携带候选QTG的SNP的敲门蛋白小鼠。在AIM 2中，我们将通过携带友善等位基因的小鼠，QTG的敲除等位基因或相对于野生型小鼠的固定途径和神经生物学变量的构造量相关的概念分析，在携带友善等位基因的淘汰等位基因的小鼠中，通过mRNA测序（RNA-SEQ）对纹状体和腹中脑进行转录组分析，或两个等位基因分析。敏感性表明中脑多巴胺能神经元发展，谷氨酸信号传导和肾上腺素信号传导的扰动是可能导致MA诱导的行为减少的。在AIM 3中，我们将使用体内微透析，免疫印迹，免疫组织化学以及操作型和敲除的口服MA自我给药，并在功能上评估QTG影响MA的刺激性和添加性能的神经化学机制。拟议的研究员工是遗传学，基因组学和功能分析的独特组合，以发现神经生物学机制，通过这种机制，新型遗传因子有助于与成瘾相关的行为。这些发现可能会转化在遗传和分子机制中的相关性，这些遗传和分子机制会涉及对MA成瘾的敏感性/韧性，并可以为新的疗法途径提供信息。