海马CA1中TET介导的DNA去甲基化调控吗啡成瘾相关记忆形成的机制

Drug-related memory is hypothesized to underlie the high relapse rate in addiction. How addiction memory forms is one of the most important question in this research field. The long-term regulation of gene transcription plays a critical role in long-term memory, but the underlying mechanisms remain poorly understood. The DNA methylation, a stable epigenetic modification, may persistently influence gene expression. Our previously study also suggested that the DNA methylation in hippocampal CA1 is required for the formation of drug related memory. However, recent studies showed that ten-eleven translocation (TET) enzyme modulated the DNA demethylation, which may influence the maintenance of DNA methylation and negatively regulate the formation of addiction memory. Therefore, focus on hippocampal CA1, one of the most important brain region for the formation of drug-related memories, the present proposal seeks to test this idea. We will use morphine induced self-administration model to examine the effects of different subtypes of TET in the CA1 on the formation of opiate-related memories. In addition, patch clamp will be used to reveal the role of synaptic plasticity in this process. Moreover, we will test the 5hmC and 5mC statuses within whole genome wide by using HMST-Seq (hydroxymethylation and methylation sensitive tag sequencing). The transcription and translation of ten target genes would be detected to confirm the role of TET in the regulation of target genes and the roles of target genes in the formation of morphine induced self-administration. The present investigation will advance our understanding of molecular mechanisms of the formation of long-term drug related memory.

复吸是治疗成瘾的难点，成瘾记忆持续存在是内在原因，但长时程成瘾记忆形成的分子机制还不清楚。DNA甲基化可以终身调控基因转录，可能是长时程记忆的核心分子基础之一。我们在前期工作中发现成瘾记忆的形成须要海马中发生DNA甲基化。新近研究表明，10-11易位酶TET介导DNA主动去甲基化，因此可能在甲基化的保持及成瘾记忆的形成中发挥关键的负调控作用。本研究拟从这一假设出发，以吗啡大鼠自身给药为行为模型，选择记忆加工的关键脑区海马CA1，比较研究不同亚型TET调控的DNA去甲基化在成瘾相关记忆形成中的作用，并利用膜片钳技术，研究TET影响形成的突触可塑性机制，此外还将检测CA1中全基因组DNA羟甲基化和甲基化位点，分析TET调控的靶基因，并结合分子生物学、生物化学和行为学方法，对其中关键靶基因的转录和翻译水平、及其在成瘾相关记忆形成中的作用进行验证。本工作可望为认识成瘾长期记忆的分子机制提供新证据。

药物成瘾相关记忆的长期存在是成瘾高复吸率的内在原因之一。但是成瘾相关记忆如何形成，其机制还不清楚。本课题以大鼠吗啡自给药为行为模型，结合行为药理、分子生物学、病毒转染过表达等方法，比较研究伏隔核壳区与海马CA1中的DNA甲基化修饰在调控吗啡成瘾相关记忆形成中功能的异同，并探索催化DNA甲基化和去甲基化的关键酶。研究结果发现：（1）吗啡自给药的形成过程伴随海马CA1（而非伏隔核壳区）中DNA甲基转移酶DNMT3a（而非DNMT3b）表达的上调；（2）抑制CA1中的DNA甲基化、或者敲减CA1中DNMT3a的表达，都可以破坏吗啡自给药的形成；抑制伏隔核中DNA甲基化对吗啡自给药的形成没有显著影响；（3）吗啡自给药形成的早期，伴随CA1（而非伏隔核壳区）中DNA去甲基化酶TET3表达的上调；形成的后期伴随CA1（而非伏隔核壳区）中TET1表达的下调；（4）敲减CA1中TET3的表达，可以促进吗啡自给药的形成；过表达CA1中TET1，可以抑制吗啡自给药的形成。以上结果共同提示，海马CA1中，DNA甲基化的关键酶DNMT3a与DNA去甲基化的关键酶TET1和TET3可能共同调控吗啡成瘾相关记忆的形成过程。DNA甲基化和去甲基化之间的失衡，可能是阿片类药物成瘾记忆形成的关键机制。