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。过量饮酒， 定义为血液酒精浓度（BAC）≥ 0.08 g/dl（暴饮暴食）或每周饮酒≥ 15次， 男性，是许多严重的医疗和精神疾病的重要风险因素，包括AUD。的 内侧前额叶皮层（mPFC）对于整合各种内部和外部状态非常重要， 确定奖励或厌恶刺激的接近/回避行为。先前的研究表明，聚- ADP核糖聚合酶（PARP）通过其调节突触可塑性基因表达的能力，促进突触可塑性基因表达。 可卡因成瘾行为PARP酶活性是否在药物的成瘾性中起着类似的作用， 乙醇（EtOH）还有待研究。这项拨款提案的假设是，乙醇增加PARP 导致兴奋性mPFC神经元中某些神经元突触可塑性基因表达减少的活性 最终增加饮酒行为。EtOH增加细胞培养物中的PARP酶活性，成人 暴食动物和胎儿发育期间。PARP可以通过催化反应来沉默基因表达 将PAR基团（PAR化）添加到下游基因调控蛋白，包括转录因子 过氧化物酶体增殖物激活受体-γ（过氧化物酶体增殖物激活受体γ）和KDM 4D，一种转录 组蛋白3的抑制性二甲基化赖氨酸9（H3 K9 me 2）。与AUD有关的基因的例子是- 已确定受H3 K9 me 2调节的是脑源性神经营养因子（Bdnf）和Pparγ。BDNF和 PPARγ由皮质中的兴奋性神经元表达，两者都在突触可塑性中发挥作用， 神经元存活因此，酒精诱导的BDNF和PPARγ表达的抑制可能是 预期改变mPFC兴奋性输出，促进持续饮酒行为。都支持这一看法 有报道表明，较高的BDNF表达和PPARγ激动剂可减少饮酒量。我们报道了 PARP活性部分负责EtOH诱导的Bdnf IV和IXa mRNA表达的降低， 原代皮层神经元培养。我们发现了一些证据表明，减少的PPARγ启动子 结合可以作为PARP活性增加和Bdnf表达减少之间的中间步骤。 我们现在建议在体内检查这些相同的途径。我们的初步数据表明， 在狂欢样黑暗饮酒（DID）模式中自愿消耗EtOH增加了PARP mRNA DID消耗EtOH降低BDNF和PPARγ表达， PARP抑制剂治疗可逆转这些效应。DID EtOH消耗降低了PPARγ DNA 通常和特异性地在Bdnf IXa启动子处的结合能力。此外，全球乙醇消耗量增加 抑制PARP可降低Pparγ和Bdnf IXa基因启动子上的H3 K9 me 2。最后， 我们发现PARP抑制减少了DID范例中的EtOH消耗。在第一个目标中，我们建议 剖析PARP与AUD相关基因表达变化的分子机制， mPFC遵循DID和2瓶自由选择饮用模式。在第二个目标中，我们将研究 皮质兴奋性神经元中EtOH诱导的PARP的基因调节作用。这种细胞特异性方法将 利用翻译核糖体亲和纯化（TRAP）方法进行RNA分离， 活化细胞分选（FACS）分离兴奋性神经元核用于KDM 4D、H3 K9 me 2和PPARγ结合 实验我们还将使用从兴奋性神经元中分离的RNA进行数据驱动实验， 全转录组微阵列分析。在第三个目标中，我们将研究PARP在EtOH饮用中的作用 行为，如DID和2瓶自由选择。我们将使用PARP测试这些变化是否可逆 抑制剂和PPARγ激动剂。预计拟议研究的结果将澄清PARP在以下方面的作用： 调节与AUD相关的基因表达。基于这些数据，PARP抑制剂可能是 用于治疗AUD的有用的药理学工具。

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.