DNA Methylation in Ethanol Drinking Behavior

描述(由申请人提供)： 酒精滥用障碍非常普遍，特别是在退伍军人中，分子生物学基础尚未完全阐明。人类和动物研究表明，防止酒精强化效应的基因稳定下调，如脑源性神经营养因子(BDNF)，可能会导致酒精摄入量增加。BDNF是一种已知的突触可塑性调节器，可能在乙醇的Addictiv和神经退行性变特性中发挥重要作用。DNA甲基化是表观遗传基因调控的一个组成部分。它能够承受动物的寿命，甚至跨越几代人，这表明它作为一种编码信息的手段具有潜在的实用价值。BDNF启动子的高甲基化已被证明可降低其表达。研究表明，神经元中活跃的DNA去甲基化是由于GADD45蛋白(GADD45a，b，g)招募胞苷脱氨酶(CDs)和胸苷糖基酶(TGS)到基因启动子。最近，我们报道了精神病患者死后脑标本中BDNF IXabcd mRNA的表达降低，与GADD45b BDNF IXabcd启动子结合减少以及5-甲基胞嘧啶(5MC)和5-羟甲基胞嘧啶(5HmC)增加有关。我们的主要目标是利用两个不同的动物模型来确定DNA去甲基化在饮酒行为中的作用。在第一种方法中，我们将利用酒精研究中最常用的小鼠模型之一，C57BL/6(C57)和DBA/2J(DBA)菌株。平均而言，C57小鼠自愿消耗的酒精是DBA小鼠的10倍。我们的初步数据表明，与DBA小鼠相比，C57小鼠在伏核外壳(NAC)中的BDNF表达较低，而在整个NAC中BDNF IXa mRNA的表达也较低。此外，先前的研究表明，C57和DBA小鼠对急性乙醇的反应在BDNF表达上存在差异。综上所述，这些数据表明，BDNF奖赏回路中的失调可能在一定程度上导致了C57小鼠的高酒精消耗。我们将通过确定将BDNF蛋白注入C57小鼠的NAC壳中是否减少了自愿的乙醇消耗来验证BDNF是否解释了这种差异。我们还将确定C57小鼠NAC中BDNF表达不足是否可以通过DNA甲基化(DNMT1、DNMT3a和Dnmt3b)或DNA去甲基化基因(如CDS、TGS或GADD45s)的表达变化来解释。我们将检测这些转录本的mRNA表达，以及NAC和腹侧被盖区(VTA)中GADD45b和BDNF蛋白的表达。我们将重点研究GADD45蛋白，因为它们似乎是去甲基化过程的关键。我们将确定在C57小鼠的这些脑区中，是否存在GADD45b结合减少和BDNF启动子5MC和5hmC增加。此外，我们将测试C57菌株无法增加与急性乙醇有关的BDNF是否是由于去甲基化机制功能障碍所致。我们将尝试通过siRNA策略在体内敲除NAC壳中的GADD45b来诱导DBA品系C57小鼠的饮酒表型。在我们的第二种方法中，我们将使用GADD45b基因敲除(KO)小鼠，因为GADD45b负责BDNF的活性依赖性去甲基化。我们将评估急性乙醇是否增加缺乏GADD45b的小鼠NAC和VTA中BDNF的表达和启动子去甲基化。最后，我们将测试BDNF缺陷的GADD45b KO小鼠是否增加了酒精偏好。最终，这些研究将确定酒精和酒精饮酒行为作用的新的分子靶点，并促进酒精中毒药物治疗的发展。

DESCRIPTION (provided by applicant): Alcohol abuse disorders are highly prevalent particularly in military combat veterans, with yet to be fully elucidated molecular biological underpinnings. Human and animal studies indicate that the stable downregulation of genes protective against the reinforcing effects of ethanol, such as brain derived neurotrophic factor (BDNF), may result in increased alcohol consumption. BDNF is a known regulator of synaptic plasticity that may play a significant role in ethanol's addictiv and neurodegenerative properties. DNA methylation is one component of epigenetic gene regulation. Its ability to endure an animal's lifespan, and even across generations, indicates its potential utility as a means for encoding information. Hypermethylation of the BDNF promoter has been shown to decrease its expression. Studies suggest active DNA demethylation in neurons results from GADD45 proteins (GADD45a, b, g) recruiting cytidine deaminases (CDs) and thymidine glycosylases (TGs) to gene promoters. Recently, we reported decreased BDNF IXabcd mRNA expression in psychotic subjects' postmortem brain samples associated with decreased GADD45b BDNF IXabcd promoter binding and increased 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC). Our principal objective is to determine the role of DNA demethylation in alcohol-drinking behavior utilizing two separate animal models. In the first approach, we will utilize one of the most frequently used murine models in alcohol studies, the C57BL/6 (C57) and DBA/2J (DBA) strains. On average C57 mice voluntarily consume 10- fold more ethanol than DBA mice. Our preliminary data indicate, by comparison to DBA mice, C57 mice have lower BDNF expression in the shell of the nucleus accumbens (NAc), as well as less BDNF IXa mRNA expression in the NAc as a whole. Additionally, prior studies show divergent responses in BDNF expression between C57 and DBA mice in reaction to acute ethanol. Taken together, these data suggest that BDNF dysregulation in the reward circuitry may, in part, contribute to high ethanol consumption in C57 mice. We will validate that BDNF accounts for this difference by determining if the infusion of BDNF protein into the NAc shell of C57 mice reduces voluntary ethanol consumption. We will also determine if deficient BDNF expression in the NAc in C57 mice may be explained by altered expression of DNA methylating (DNMT1, DNMT3a, and DNMT3b) or DNA demethylating genes, such as CDs, TGs, or the GADD45s. We will measure mRNA expression of these transcripts, as well as GADD45b and BDNF protein in the NAc and ventral tegmental area (VTA). We will focus on GADD45 proteins because they appear to be the lynchpins of the demethylation process. We will determine whether there is reduced GADD45b binding and increased 5MC and 5HMC at BDNF promoters in these brain regions in C57 mice. In addition, we will test if the C57 strain's inability to increase BDNF in relation to acute ethanol is due to dysfunctional demethylating machinery. We will attempt to induce the alcohol-drinking phenotype of C57 mice in the DBA strain by in vivo knockdown of GADD45b in the NAc shell via siRNA strategy. In our second approach, we will use a GADD45b knockout (KO) mouse because GADD45b is responsible for activity-dependent demethylation of BDNF. We will evaluate whether acute ethanol increases BDNF expression and promoter demethylation in the NAc and VTA in mice lacking GADD45b. Finally, we will test whether the BDNF-deficient GADD45b KO mice have increased alcohol preference. Ultimately, these studies will identify novel molecular targets in the actions of ethanol and ethanol drinking behavior, and promote the development of pharmacotherapy for alcoholism.