Role of Dopamine Neuron-Specific Gene Enhancers in Cocaine Relapse

项目总结 毒瘾是一种复杂而毁灭性的疾病，以强迫寻求和使用毒品为特征， 尽管有有害的后果。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)。尽管它很复杂，但它是 普遍认为，中脑边缘多巴胺(DA)奖赏系统的长期适应不良变化在 在从随意吸毒到强制吸毒的过程中发挥核心作用，并导致高复发率 在人类瘾君子身上。事实上，虽然广泛的努力旨在实现禁欲，但理解 药物复发的分子机制是维持戒毒状态的关键。因此， 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 调节与成瘾相关的行为，但由于处理细胞异质性的技术障碍 哺乳动物的大脑，到目前为止，大多数的分子研究都是使用混合细胞群体进行的，因此 数据解释混乱。为了解决这个问题，我领导了开发一种强大的体内核技术的努力 具有神经元亚型特异性的标记系统，便于转录组和染色质可访问性分析。 可访问的染色质图谱是特定于细胞类型的，可用于识别调节区，例如 活性基因的增强剂。因此，这项提议试图了解多巴胺能增强剂的作用。 在可卡因渴求期间，为了调节复发而寻求可卡因。我假设增强剂元素 在调节可卡因渴求引起的基因表达变化方面发挥重要作用，而这些 变化会影响对寻求可卡因的复发的可能性和程度。为了检验这一假设，我建议 为了描述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的变化， 采用小鼠静脉注射可卡因自我给药(IVSA)模型。表观基因组学与生物信息学 我将在K01大奖期间接受的培训将使我能够揭示候选基因和分子途径 专门针对这一阶段的上瘾。此外，使用CRISPR/Cas9基因组编辑技术，我将编辑顶级渴望- DA神经元中的相关增强子，以验证它们调节体内转录活动的能力。最后，我 将通过IVSA测试这些增强剂在调节可卡因复发行为中的功能作用。已完成的 拟议的研究不仅将促进我们对可卡因成瘾的分子机制的理解 并确定新的治疗靶点，但也将提供一种新的、广泛适用的操纵策略 以及了解大脑中具有细胞类型特异性的表观遗传调节。

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.