伏隔核组蛋白乙酰化及其介导的基因转录对大鼠可卡因成瘾觅药动机作用的研究

【作者】 王雷；

【导师】 马兰；

【作者基本信息】 复旦大学 ， 药理学， 2010， 博士

【摘要】 药物成瘾是一种慢性复发性脑病,它以强迫性的觅药行为和不计后果的药物滥用为特征。个体一旦成瘾几乎没有有效的临床治疗方案,绝大部分患者短期就会复吸。越来越多的研究表明,成瘾性药物长期滥用导致的关键奖赏脑区基因表达异常,是成瘾最重要的发病机制之一表观遗传学机制介导染色体结构的重构是调控基因表达的重要方式,它通过改变相应基因启动子区域染色体结构调控基因的转录,其只影响基因表达而不影响基因序列。组蛋白乙酰化是表观遗传学机制介导染色体重构的重要调节方式,通常认为基因启动子区组蛋白H3和H4的乙酰化水平升高与基因表达活性增强相关,而它们的去乙酰化则与基因的表达活性减弱或沉默相关。近年来,很多研究探讨了染色体重构和可卡因诱导的神经及行为可塑性之间的关系。目前,组蛋白乙酰化如何参与主动成瘾行为的发生、发展和复发的机制还尚未完全阐明。自身给药模型是目前国际上广泛应用的研究药物成瘾的动物模型,它是一种主动成瘾模型,能更好的模拟吸毒患者的整个成瘾过程特别是检测成瘾状态(如对药物的渴求程度即觅药动机)的改变等；同时它也是研究成瘾药物正性强化的重要工具模型。本研究采用大鼠可卡因自身给药模型中的固定比率(FR)给药及递增比率(PR)两种给药方案,研究可卡因成瘾长期发展阶段大鼠伏隔核组蛋白乙酰化水平及其介导的基因转录活性改变对大鼠可卡因自身给药中成瘾行为的影响。我们的研究结果显示：1.伏隔核壳部微注射去乙酰化酶(HDAC)抑制剂增强大鼠在可卡因自身给药中的觅药动机。每天给予伏隔核壳部核团微注射HDAC抑制剂TSA或SAHA不仅可以上移实验动物在固定比率(FR)给药方案中的剂量反应和剂量摄入曲线,而且可以增加递增比率(PR)给药方案下的破发点(Break Point),动物的上述行为学表现揭示了成瘾动物觅药动机的增强,而在伏隔核中央部或中央前额叶皮层核团微注射TSA处理后,均未见上述变化。上述行为学表现提示HDAC抑制剂对成瘾动物觅药动机增强可能归因于持续HDAC抑制而导致的组蛋白乙酰化的增加。2HDAC抑制剂上调慢性可卡因经历诱导的伏隔核壳部乙酰化水平的升高。我们采用免疫印迹及免疫组化的方法检测了急性可卡因给药及慢性可卡因给药经历诱导的大鼠伏隔核内组蛋白H3和H4乙酰化水平的变化。我们发现,慢性可卡因经历能够引起大鼠伏隔核壳部组蛋白H3和H4乙酰化水平增高,而这种乙酰化水平的增高在伏隔核中央部和中央前额叶皮层均不明显；进一步的研究发现,HDAC抑制剂能够上调慢性可卡因经历诱导的伏隔核壳部乙酰化水平的升高。上述现象提示慢性可卡因给药经历诱导的伏隔核壳部组蛋白乙酰化的升高与自身给药中的可卡因觅药动机相关。3.伏隔核壳部过表达组蛋白去乙酰化酶4(HDAC4)下调慢性给药导致的组蛋白乙酰化水平的升高并降低实验动物的觅药动机。为了更为深入的研究组蛋白乙酰化改变与可卡因觅药动机之间的关系,我们通过伏隔核壳部过表达HDAC4的方法来下调慢性可卡因诱导的组蛋白乙酰化水平的增加,进一步观察其对自身给药中成瘾行为的影响。研究表明,伏隔核壳部过表达全长HDAC4,而非HDAC催化活性缺失的突变体显著抑制了可卡因诱导的组蛋白乙酰化水平的增加,同时行为学上表现为固定比率(FR)给药方案中的剂量反应和剂量摄入曲线的下移以及递增比率(PR)给药方案中的Break Point降低。上述现象说明,伏隔核壳部HDAC4过表达降低自身给药中的觅药动机,而这种作用是HDAC催化活性依赖的。然而,伏隔核中央部或中央前额叶皮层过表达HDAC4未见上述行为学表现。该结果进一步确立了慢性可卡因诱导的伏隔核壳部的组蛋白乙酰化水平的升高在调控可卡因觅药动机中发挥重要作用。4.可卡因觅药动机与伏隔核壳部组蛋白H3乙酰化正相关。采用染色质免疫沉淀(CHIP)的方法研究了慢性可卡因自身给药中伏隔核成瘾相关基因转录激活水平的变化情况,我们发现慢性可卡因诱导的成瘾相关基因转录激活主要存在于伏隔核壳部,并且大多数成瘾相关基因的转录激活的由组蛋白H3乙酰化介导。与此同时,觅药动机的改变与组蛋白H3乙酰化介导的成瘾相关基因转录激活水平的变化方向一致。该现象提示组蛋白乙酰化对可卡因觅药动机调控作用是通过调控基因转录活性来实现的,而H3乙酰化介导的成瘾相关基因转录激活可能在可卡因觅药动机调控中扮演更为重要的角色。5.伏隔核壳部下调CaMKⅡα的表达减弱自身给药中的觅药动机。在伏隔核壳部由H3乙酰化介导的转录激活的成瘾相关基因中,钙调激酶Ⅱ(CaMKⅡ)的亚型CaMKⅡα,而非CaMKⅡβ,其基因表达与可卡因的觅药动机成正相关。由慢病毒载体介导的基因敲低后减弱自身给药中的觅药动机,提示大鼠伏隔核壳部CaMKⅡα的基因表达对维持可卡因自身给药中的觅药动机是必需的。也揭示了自身给药中组蛋白H3乙酰化介导的基因转录激活,如CaMKⅡα等基因的表达,是调控可卡因觅药动机的潜在的重要机制。本论文的主要创新点在于,首次揭示了(1)慢性可卡因经历诱导的伏隔核的壳部组蛋白H3乙酰化水平增加以及(2)组蛋白H3乙酰化介导的CaMKⅡα的转录激活在可卡因自身给药觅药动机中发挥的重要作用。更多还原

【Abstract】 Drug addiction is a chronic relapsing encephalopathy that is characterized by compulsive drug seeking behavior and taking despite severe adverse consequences. Once an individual becomes addicted to a drug of abuse, there are few effective clinical treatments, and most addicts relapse within a short period of time. The mechanisms of cocaine addiction are very obscure and complicated. Increasing evidence suggests that abnormality of gene expression in the brain reward regions is one of the most important mechanisms involved in a state of addiction caused by drugs of abuse.Chromatin remodeling epigenetically controls gene transcription, indicating that complex mechanisms regulate the accessibility of genes to the transcriptional machinery. It is regarded that increased histone acetylation is associated with DNA relaxation and elevated transcriptional activity, whereas decreased acetylation, brought about by histone deacetylases (HDACs) and methyl-CpG-binding proteins, results in tighter DNA coiling and gene silencing. Nowadays, some researches indicate that histone modification plays a critical in cocaine-induced neuronal and behavioral plasticity. However, research has not completely clarified the epigenetic mechanism in the different phases of drug self-administration including the initial, reinforcement, incubation and reinstatement stages.Intravenous drug self-administration, which better models the human syndrome especially the addicted-state changes, such as increased motivation for the drug, is a useful tool for exploring the neurobiology of drug positive reinforcement in experimental animals. In this study, rats were subjected to cocaine self-administration under fixed-ratio (FR) or progressive-ratio (PR) schedule. The role of histone acetylation and histone-acetylation-mediated gene transcriptional activation in the NAc during the incentive motivation for cocaine following a chronic phase of drug self-administration was examined. Our results showed that:1. HDAC inhibitor infusions into the NAc shell increase the motivation for cocaine self-administration. Daily histone deacetylase (HDAC) inhibitor (TSA or SAHA) infusions in the nucleus accumbens (NAc) shell, but not the core or medial prefrontal cortex (mPFC), caused an upward shift in the dose-response and dose-intake curve under fixed-ratio schedule, and increased the break point under progressive-ratio schedule, indicating enhanced motivation for self-administered drug. These data indicated intra-NAc-shell infusions of HDAC inhibitor enhance motivation for rat to obtain cocaine, which may be attributable to the potentiating of histone acetylation induced by sustained HDAC inhibition.2. HDAC inhibitor augments histone acetylation elevation induced by chronic cocaine experience in the NAc shell. Histone acetylation levels following the acute and chronic administration of cocaine were evaluated by western blotting and immunochemistry analysis. The acetylation levels of H3 and H4 were elevated specifically in the NAc shell, but not in the NAc core or the mPFC following chronic intravenous cocaine self-administration experience. Furthermore, Chronic-cocaine-stimulated increases in histone acetylation in the NAc shell were notably augmented by HDAC inhibitor pretreatment. These data supports the notion that increased histone acetylation in the NAc shell induced by chronic cocaine self-administration is associated with motivation for drug reinforcement.3. Neutralizing the chronic-cocaine-induced increase in histone modification by the bilateral overexpression of HDAC4 in the NAc shell reduced drug motivation. To gain further insight into the relationship between histone acetylation and the motivation for cocaine self-administration, we investigated the effect of neutralizing cocaine-stimulated increase in histone acetylation by overexpressing of HDAC4. Intra-NAc shell overexpression of full-length HDAC4, but not its truncated form, which lacks deacetylase activity, strongly inhibited the cocaine-induced increase in histone acetylation, and induced a downward shift in dose-response and dose-intake curves under FR schedule, and decreased break point under PR schedule, indicating reduced motivation for drug. The effect of HDAC4 expression on the motivation for cocaine self-administration was dependent on its deacetylation activity. However, this effect was not observed when HDAC4 overexpression in the NAc core or the mPFC. These results confirm that the elevation of histone acetylation in the NAc shell is critical for drug motivation.4. Motivation for cocaine is associated with H3 acetylation in the NAc shell. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the association between the motivation for cocaine and the transcriptional activation of addiction-related genes by H3 acetylation in the NAc shell. As is shown in ChIP assay, chronic-cocaine-induced gene transcriptional activation occured primarily in the NAc shell and was predominantly modulated by the acetylation of H3. Futhermore, motivation for cocaine was associated with transcriptional activation of addiction-related genes modulated by H3 acetylation of NAc shell. These data indicated that drug-induced gene transcriptional activation in the NAc shell, modulated by H3 acetylation, is a potential mechanism underlying the motivation for cocaine.5. Localized CaMKⅡαknockdown in the NAc shell decreases the motivation for cocaine. Among the genes activated by chronic cocaine experiences, the expression of CaMKIIa, but not CaMKⅡβ, correlated positively with motivation for the drug. Lentivirus-mediated shRNA knockdown experiments demonstrated that CaMKⅡα, but not CaMKⅡβ, in the NAc shell is essential for the maintenance of motivation to self-administer cocaine. These findings suggest that chronic-drug-use-induced transcriptional activation of genes such as CaMKIIa, modulated by H3 acetylation in the NAc, is a critical regulatory mechanism underlying motivation for drug reinforcement.Taken together, our results establish an important role for the chronic-cocaine-induced increase in histone H3 acetylation and the transcriptional activation of the gene encoding CaMKⅡαin the shell of the NAc in the motivation for cocaine reinforcement firstly, and suggest a new therapeutic mechanism for anti-addiction treatment.更多还原