The role of DNA breaks and repairs in opioid addiction

摘要 阿片类药物使用障碍是一种衰弱的疾病，尽管目前的治疗努力很高，但复发率很高。临床 临床前研究发现，阿片类药物诱导的慢性基因表达变化是神经整形的基础 大脑奖励回路内的变化，包括前额叶皮质(PFC)，这将导致终生 故态复萌。最近的研究强调，表观遗传变化协调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后的转录组的原因 目前尚不清楚是否有变化。 DNA损伤和修复过程在染色质完整性中起着重要作用 表观遗传特征和细胞中的基因表达。有丝分裂后神经元对DNA特别敏感 因为它们的高代谢和转录输出而造成损害。DNA断裂触发DNA损伤修复 反应，这涉及广泛的染色质重塑，以纳入DNA修复机制。因此，DNA 断裂和修复可以逐渐改变染色质结构，从而改变神经元中的基因表达模式， 最终，它会影响人们的行为。 与DNA损伤和阿片成瘾有关的研究一致，我们的初步数据表明，DNA 阿片类药物使用障碍患者和小鼠死后PFC组织中的断裂增加 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 伴随着参与DNA损伤修复的基因表达的变化。更重要的是， 在PFC中引入DNA断裂的实验增强了小鼠的海洛因寻找行为。加在一起， 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。我们的 这项提案的目标是确定和表征DNA断裂和修复在阿片成瘾中的确切作用。 使用基因组学、表观基因组学和翻译基因组学方法的独特组合，并结合亲和力 纯化后，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断裂和修复部位(即断骨)的变化及其相关变化 染色质的可及性(即表观基因组)和基因表达(即翻译组)。此外，使用 CRISPR/Cas9基因组编辑方法，我们将确定海洛因诱导的DNA断裂和 修复部位与海洛因诱导的行为可塑性有直接和因果关系。 这项建议将为今后广泛的研究工作提供坚实的基础，解决 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.