Role of Dopamine Neuron-Specific Gene Enhancers in Cocaine Relapse

多巴胺神经元特异性基因增强剂在可卡因复发中的作用

• 批准号: 10157603
• 负责人: Luis Miguel Tuesta
• 金额: $ 5.37万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10157603
• 关键词: Abstinence; Address; Affect; Behavior; Behavioral; Bioinformatics; Brain; Brain Diseases; CRISPR/Cas technology; Candidate Disease Gene; Cell Nucleus; Cells; Cessation of life; Chromatin; Chronic; Cocaine; Cocaine Dependence; Complex; Cues; DNase I hypersensitive sites sequencing; Data Analyses; Disease; Distal; Dopamine; Drug Addiction; Drug usage; Enhancers; Epigenetic Process; Exhibits; Fluorescence; Gene Expression; Genes; Genetic Enhancer Element; Genetic Transcription; Health; Heterogeneity; Human; Hypersensitivity; Intravenous; Label; Measures; Mediating; Mentored Research Scientist Development Award; Molecular; Mus; National Institute of Drug Abuse; Neurons; Nuclear; Nucleic Acid Regulatory Sequences; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Population; Relapse; Research; Rewards; Role; Self Administration; Site; Sorting - Cell Movement; Specificity; System; Techniques; Testing; Training; Transcriptional Regulation; United States; addiction; base; cell type; cocaine relapse; comparative; cost; craving; dopaminergic neuron; drug relapse; epigenetic profiling; epigenetic regulation; epigenomics; genome editing; high risk; in vivo; inducible gene expression; insight; mouse model; new therapeutic target; novel; relapse risk; social; therapeutic development; tool; transcriptome; transcriptome sequencing; web site

PROJECT SUMMARY Drug addiction is a complex and devastating disease characterized by compulsive drug seeking and use, despite harmful consequences. This urgent social and health problem contributes to 90,000 deaths per year and an annual cost of over $700 billion in the United States alone (see NIDA website). Despite its complexity, it is generally believed that long-term maladaptive changes in the mesolimbic dopamine (DA) reward system play a central role in the progression from casual to compulsive drug use and to contribute to the high rates of relapse in human addicts. Indeed, while extensive efforts have aimed to achieve abstinence, understanding the molecular mechanisms underlying drug relapse is critical for maintaining a drug-free state. Accordingly, accumulating evidence suggests that drug-induced epigenetic and chromatin changes play an important role in mediating addiction-related behavior, but due to technical hurdles in dealing with cellular heterogeneity of the mammalian brain, most of the molecular studies so far have been performed using mixed cell populations, thus confounding data interpretation. To address this issue, I have led the effort to develop a robust in vivo nuclear tagging system with neuron subtype specificity that facilitates transcriptome and chromatin accessibility profiling. Accessible chromatin profiles are cell-type specific and can be used to identify regulatory regions such as enhancers of active genes. This proposal therefore seeks to understand the role of dopaminergic enhancers during cocaine craving in order to regulate relapse to cocaine-seeking. I hypothesize that enhancer elements play an important role in regulating gene expression changes induced by cocaine craving, and that these changes can affect the likelihood and magnitude of relapse to cocaine-seeking. To test this hypothesis, I propose to profile chromatin accessibility and gene expression changes associated with cocaine craving in DA neurons, using a mouse model of intravenous cocaine self-administration (IVSA). The epigenomics and bioinformatics training I will receive during this K01 Award will allow me to reveal candidate genes and molecular pathways specific to this phase of addiction. Further, using CRISPR/Cas9 genome editing techniques, I will edit top craving- associated enhancers in DA neurons to validate their ability to regulate transcriptional activity in vivo. Finally, I will test the functional role of these enhancers in regulating cocaine relapse behavior by IVSA. Completion of the proposed studies will not only advance our understanding of molecular mechanisms underlying cocaine addiction and identify novel therapeutic targets, but will also provide a novel, broadly applicable strategy for manipulating and understanding epigenetic regulation in brain with cell-type specificity.

项目摘要 药物成瘾是一种复杂而毁灭性的疾病，其特征是强迫性的药物寻求和使用， 尽管会有有害的后果。这一紧迫的社会和健康问题每年造成9万人死亡， 仅在美国，每年的成本就超过7000亿美元（见NIDA网站）。尽管它的复杂性， 一般认为，中脑边缘多巴胺（DA）奖赏系统的长期适应不良变化， 在从偶然吸毒到强迫吸毒的发展过程中起着核心作用，并导致高复吸率 在人类成瘾者身上事实上，尽管人们为实现禁欲做出了广泛的努力， 药物复发的分子机制对于维持无药物状态至关重要。因此，委员会认为， 越来越多的证据表明，药物诱导的表观遗传和染色质变化在 介导成瘾相关行为，但由于在处理细胞异质性的技术障碍， 哺乳动物大脑，迄今为止，大多数分子研究都是使用混合细胞群进行的，因此， 混淆数据解释。为了解决这个问题，我领导了一个强大的体内核 标记系统，其具有神经元亚型特异性，有助于转录组和染色质可及性分析。 可分辨的染色质谱是细胞类型特异性的，并且可用于鉴定调控区域，例如 活性基因的增强子。因此，这项建议旨在了解多巴胺能增强剂的作用， 以调节可卡因成瘾的复发。我假设增强子元件 在调节可卡因渴求诱导的基因表达变化中起重要作用， 这些变化可能影响可卡因成瘾复发的可能性和程度。为了验证这个假设，我建议 为了分析DA神经元中与可卡因渴求相关的染色质可及性和基因表达变化， 使用静脉内可卡因自我给药（IVSA）的小鼠模型。表观基因组学与生物信息学 在K 01奖期间，我将接受的培训将使我能够揭示候选基因和分子途径 特别是在这个阶段的成瘾。此外，使用CRISPR/Cas9基因组编辑技术，我将编辑顶级渴望- 相关的增强子，以验证它们在体内调节转录活性的能力。最后我 将通过IVSA测试这些增强剂在调节可卡因复吸行为中的功能作用。完成 拟议中的研究不仅将促进我们对可卡因成瘾分子机制的理解， 并确定新的治疗靶点，但也将提供一种新的，广泛适用的策略， 以及理解具有细胞类型特异性的脑中的表观遗传调节。