PARP-mediated gene regulation in alcohol drinking behavior

酒精使用障碍(AUD)在美国退伍军人中非常普遍。过度饮酒， 定义为每周获得血液酒精浓度(BAC)≥0.08g/dl(狂饮)或≥15饮品 男性，是许多严重的医疗和精神疾病的重要风险因素，包括澳门氏症。这个 内侧前额叶皮质(MPFC)在整合多种内外状态中起重要作用。 确定对奖赏或厌恶刺激的接近/回避行为。先前的研究表明，Poly- ADP核糖聚合酶(PARP)通过其调节突触可塑性基因表达的能力，促进 可卡因成瘾行为。PARP酶活性是否在PARP成瘾特性中起类似作用 乙醇(Etoh)还有待研究。这项拨款提案的假设是，乙醇增加了PARP 兴奋性mPFC神经元中某些神经元突触可塑性基因表达降低的活性 最终增加饮酒行为。乙醇增加细胞培养中的PARP酶活性，成人 捕食动物，以及在胎儿发育期间。PARP通过催化反应抑制基因表达 向下游基因调控蛋白添加PAR基团(PAR化)，包括转录因子 过氧化物酶体增殖物激活受体-γ(PPARγ)和KDM4D，一种转录水平的去甲基酶 组蛋白3的抑制性二甲基化赖氨酸9(H3K9me2)。与AUD有关的基因例子很好- 已确定受H3K9me2调控的是脑源性神经营养因子和PPARγ。BDNF和 PPARγ是由皮层中的兴奋性神经元表达的，在那里两者都在突触可塑性和 神经元存活。因此，酒精诱导的脑源性神经营养因子和PPARγ表达的抑制将是 预计会改变mPFC的兴奋性输出，促进持续饮酒行为。这是由支持的 报告表明，更高的脑源性神经营养因子表达和PPARγ激动剂可以减少酒精消费。我们报道了 PARP活性是乙醇诱导BDNF-IV和IXa mRNA表达下降的部分原因 原代皮质神经元培养。我们发现有几条证据表明，减少的PPARγ启动子 结合可能是PARP活性增加和BDNF表达降低之间的中间步骤。 我们现在建议在活体内检查这些相同的途径。我们的初步数据表明， 在类似狂欢的黑暗饮酒(DID)范式中，自愿摄入乙醇增加了PARP mRNA 在PFC中的表达及酶活性测定。乙醇可降低脑源性神经营养因子和PPARγ的表达， 这种作用可被PARP抑制剂治疗逆转。乙醇消费是否降低了PPARγDNA BDNF IXa启动子的一般结合能力和特异性结合能力。此外，消费Etoh是否增加了全球 在PPARγ和BDNFIXa基因启动子上，H3K9me2水平和PARP抑制降低了H3K9me2。最后， 我们发现，在DID范式中，抑制PARP减少了乙醇的消耗。在第一个目标中，我们建议 PARP与AUD相关基因表达变化的分子机制剖析 MPFC遵循了DID和2瓶自由选择饮酒的范例。在第二个目标中，我们将研究 乙醇诱导的PARP对皮层兴奋性神经元的基因调控作用。这种特定于单元的方法将 利用翻译核糖体亲和纯化(TRAP)方法提取RNA，并进行荧光检测 KDM4D、H3K9me2和PPARγ结合的兴奋性神经元核的活化细胞分选 实验。我们还将使用从兴奋性神经元中分离出来的RNA进行数据驱动实验 转录组范围的微阵列分析。在第三个目标中，我们将研究PARP在乙醇饮用中的作用 行为，如DID和2瓶自由选择。我们将使用PARP测试这些更改是否可逆 抑制剂和PPARγ激动剂。拟议研究的结果预计将澄清PARP在 调控与AUD相关的基因表达。根据这些数据，PARP抑制剂可能是 治疗AUD的有用药理工具。

Alcohol use disorder (AUD) is highly prevalent among U.S. military veterans. Excessive alcohol consumption, defined as the acquisition of a blood alcohol concentration (BAC) ≥ 0.08 g/dl (binging) or ≥ 15 drinks/week for men, is an important risk factor for many serious medical and psychiatric conditions, including AUD. The medial prefrontal cortex (mPFC) is important for integrating various internal and external states in order to determine approach/avoidant behavior to rewarding or aversive stimuli. Prior studies demonstrated that Poly- ADP ribose Polymerase (PARP), through its ability to regulate synaptic plasticity gene expression, promotes cocaine addictive behaviors. Whether PARP enzymatic activity plays a similar role in the addictive properties of ethanol (EtOH) has yet to be studied. The hypothesis of this grant proposal is that EtOH increases PARP activity causing reduced expression of certain neuronal synaptic plasticity genes in excitatory mPFC neurons ultimately increasing alcohol drinking behavior. EtOH increases PARP enzymatic activity in cell culture, adult binging animals, and during fetal development. PARP can silence gene expression by catalyzing reactions adding PAR groups (PARylation) to downstream gene regulatory proteins, including the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) and KDM4D, a demethylase of the transcriptionally repressive dimethylated lysine 9 of histone 3 (H3K9me2). Examples of genes implicated in AUD that are well- established to be regulated by H3K9me2 are Brain-derived Neurotrophic Factor (Bdnf) and Pparγ. BDNF and PPARγ are expressed by excitatory neurons in the cortex, where both have roles in synaptic plasticity and neuronal survival. Therefore, an alcohol induced suppression of BDNF and PPARγ expression would be expected to alter mPFC excitatory outputs, promoting continued alcohol drinking behavior. This is supported by reports indicating higher BDNF expression and PPARγ agonists reduce alcohol consumption. We reported that PARP activity is in part responsible for EtOH-induced decreases in Bdnf IV and IXa mRNA expression in primary cortical neuron cultures. We found several lines of evidence indicating that reduced PPARγ promoter binding may serve as an intermediary step between increased PARP activity and decreased Bdnf expression. We now propose to examine these same pathways in vivo. Our preliminary data indicate that mice that voluntarily consumed EtOH in the binge-like drinking-in-the-dark (DID) paradigm had increased PARP mRNA expression and enzymatic activity in the PFC. DID consumed EtOH reduced BDNF and PPARγ expression, effects that were reversed by PARP inhibitor treatment. DID EtOH consumption decreased PPARγ DNA binding ability generally and specifically at the Bdnf IXa promoter. Also, DID consumed EtOH increased global levels of H3K9me2, and PARP inhibition decreased H3K9me2 at Pparγ and Bdnf IXa gene promoters. Finally, we found that PARP inhibition reduced EtOH consumption in the DID paradigm. In the first aim, we propose dissecting the molecular mechanisms connecting PARP to changes in expression of genes involved in AUD in the mPFC following DID and 2-bottle free-choice drinking paradigms. In the second aim, we will examine the gene regulatory effects of EtOH-induced PARP in cortical excitatory neurons. This cell specific approach will utilize the Translating Ribosome Affinity Purification (TRAP) method for RNA isolation, and Fluorescence activated cell sorting (FACS) isolation of excitatory neuronal nuclei for KDM4D, H3K9me2, and PPARγ binding experiments. We will also perform data-driven experiments using RNA isolated from excitatory neurons in transcriptome-wide microarray analysis. In the third aim, we will study the role of PARP in EtOH drinking behavior, such as DID and 2-bottle free-choice. We will test whether these changes are reversible using PARP inhibitors and PPARγ agonists. Results of the proposed studies are expected to clarify PARP’s role in regulating gene expression in relation to AUD. Based on these data it is possible that PARP inhibitors may be useful pharmacological tools for treating AUD.