The role of DNA breaks and repairs in opioid addiction

摘要 阿片类药物使用障碍是一种使人衰弱的疾病，尽管目前正在努力治疗，但复发率很高。临床 临床前研究发现，阿片类药物诱导的基因表达的慢性变化是神经可塑性的基础， 大脑奖励回路的改变，包括前额叶皮层（PFC），这导致了终身的 复发倾向最近的研究强调，表观遗传变化协调了阿片类药物滥用诱导的 转录可塑性然而，表观基因组变化和随之而来的转录组的原因 变更尚不清楚。 DNA损伤和修复过程在染色质完整性中起重要作用， 表观遗传标记和细胞中的基因表达。有丝分裂后的神经元特别容易受到DNA 因为它们的高代谢和转录输出而受到损害。DNA断裂引发DNA损伤修复 反应，这涉及广泛的染色质重塑纳入DNA修复机制。因此，DNA 断裂和修复可以逐渐改变染色质结构以改变神经元中的基因表达模式， 最终影响行为。 与DNA损伤和阿片类药物成瘾相关的研究一致，我们的初步数据表明，DNA 在阿片类药物使用障碍患者和小鼠的死后PFC组织中， 自我注射海洛因（阿片类药物成瘾的有效动物模型）。此外，DNA断裂的增加 伴随着与DNA损伤修复有关的基因表达的改变。更重要的是， 在PFC中实验性地引入DNA断裂增强了小鼠的海洛因寻求行为。综合起来看， 这些初步数据提供了DNA断裂和修复过程与阿片成瘾有关的证据。我们 该提案的目标是确定和描述DNA断裂和修复在阿片类药物成瘾中的确切作用。 使用基因组学、表观基因组学和翻译组学方法的独特组合， 纯化，我们将充分表征海洛因诱导的神经元特异性（PFC小白蛋白中间神经元和 锥体神经元）DNA断裂和修复位点的改变（即，以及相关的变化 在染色质可接近性（即，表观基因组）和基因表达（即，翻译）。此外，使用 通过CRISPR/Cas9基因组编辑方法，我们将确定海洛因诱导的DNA断裂变化是否会导致DNA断裂， 修复位点与海洛因诱导的行为可塑性有直接的因果关系。 该提案将为未来广泛的研究工作提供坚实的基础， 阿片类药物诱导的长期转录适应不良的遗传和表观遗传机制， 最终导致阿片类药物成瘾。

Abstract Opioid use disorder is a debilitating disease with a high rate of relapse despite current treatment efforts. Clinical and preclinical studies have found that opioid-induced chronic changes in gene expression underlie neuroplastic alterations within the brain reward circuitry, including in the prefrontal cortex (PFC), which lead to the life-long propensity for relapse. Recent studies highlighted that epigenetic changes orchestrate the opioid abuse-induced transcriptional plasticity. However, the causes for the epigenome changes and consequent transcriptome alterations remain unclear. DNA damage and repair processes play important roles in chromatin integrity relating to proper epigenetic signature and gene expression in cells. Postmitotic neurons are particularly susceptible to DNA damage because of their high metabolic and transcriptional output. DNA breaks trigger the DNA damage repair response, which involves extensive chromatin remodeling to incorporate DNA repair machinery. Therefore, DNA breaks and repairs can progressively alter chromatin structure to alter gene expression patterns in neurons and, ultimately, affect behaviors. Consistent with studies linking DNA damage and opioid addiction, our preliminary data suggest that DNA breaks are increased in the postmortem PFC tissues from individuals with opioid use disorder and from mice that self-administered heroin (a valid animal model for opioid addiction). Additionally, the increase in DNA breaks was accompanied by altered expression of genes that are involved in DNA damage repair. More importantly, experimentally introducing DNA breaks in the PFC potentiated heroin-seeking behavior in mice. Taken together, these preliminary data provide evidence that DNA break and repair processes are linked to opioid addiction. Our goal in this proposal is to identify and characterize the precise role of DNA breaks and repairs in opioid addiction. Using a unique combination of genomic, epigenomic, and translatomic approaches coupled with affinity purification, we will fully characterize heroin-induced neuron-specific (PFC parvalbumin interneurons and pyramidal neurons) alterations in DNA break and repair sites (i.e., the breakome) and the associated changes in chromatin accessibility (i.e., the epigenome) and gene expression (i.e., the translatome). Additionally, using CRISPR/Cas9 genome editing approaches, we will identify whether heroin-induced changes in DNA break and repair sites are directly and causally linked to heroin-induced behavioral plasticity. This proposal will provide a solid foundation for a wide range of future research endeavors addressing the genetic and epigenetic mechanisms that underlie opioid-induced long-term transcriptional maladaptation and ultimately contribute to opioid addiction.