PARP-mediated gene regulation in alcohol drinking behavior

PARP介导的饮酒行为基因调控

• 批准号: 10552525
• 负责人: Rajiv Pandit Sharma
• 金额: --
• 依托单位: JESSE BROWN VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10552525
• 关键词: Addictive Behavior; Adult; Affect; Affinity Chromatography; Agonist; Alcohol consumption; Alcohols; Animals; Applications Grants; Aversive Stimulus; Behavior; Binding; Blood alcohol level measurement; Brain region; Brain-Derived Neurotrophic Factor; Cell Culture Techniques; Cell Nucleus; Cells; Chromatin; Chronic; Clinical; Cocaine; Cytoskeleton; DNA Binding; Data; Drug Targeting; Enzymes; Ethanol; FDA approved; Factor IXa; Female; Fetal Development; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Glycoside Hydrolases; Heavy Drinking; Histones; Hour; Human; Lysine; Measurement; Measures; Medial; Mediating; Medical; Mental disorders; Methods; Microarray Analysis; Modeling; Molecular; Mus; Neurons; Output; PPAR gamma; Pathway interactions; Pattern; Pharmacology; Pioglitazone; Play; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Prefrontal Cortex; Property; Proteins; RNA; Reaction; Regulator Genes; Reporting; Rewards; Ribosomal Proteins; Ribosomes; Risk Factors; Role; Stimulus; Synaptic plasticity; Testing; Transgenic Mice; Translating; addiction; alcohol effect; alcohol exposure; alcohol seeking behavior; alcohol use disorder; base; cell type; drinking; drinking behavior; excitatory neuron; executive function; experimental study; genetic regulatory protein; human subject; in vivo; inhibitor; innovation; knock-down; mRNA Expression; male; men; military veteran; neuronal survival; overexpression; poly ADP-ribose glycohydrolase; promoter; protein expression; responsible alcohol use; tool; transcription factor; transcriptome

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.

酒精使用障碍（AUD）在美国退伍军人中非常普遍。 定义为血液酒精浓度 (BAC) ≥ 0.08 g/dl（狂饮）或每周饮酒 ≥ 15 杯 男性是许多严重的医疗和精神疾病（包括 AUD）的重要危险因素。 内侧前额皮质 (mPFC) 对于整合各种内部和外部状态非常重要，以便 确定对奖励或厌恶刺激的接近/回避行为。 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 PARP 酶活性是否在可卡因成瘾行为中发挥类似作用。 乙醇 (EtOH) 尚未得到研究 该拨款提案的假设是 EtOH 会增加 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 会增加成人细胞培养物中的 PARP 酶活性。 暴饮暴食的动物以及胎儿发育过程中的 PARP 可以通过催化反应来沉默基因表达。 将 PAR 基团（PARylation）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 (PPARγ) 和 KDM4D，一种转录去甲基化酶 组蛋白 3 的抑制性二甲基化赖氨酸 9 (H3K9me2)。 确定受 H3K9me2 调节的是脑源性神经营养因子 (Bdnf) 和 Pparγ。 PPARγ 由皮质中的兴奋性神经元表达，两者在突触可塑性和突触可塑性中发挥作用。 因此，酒精会抑制 BDNF 和 PPARγ 的表达。 预计会改变 mPFC 兴奋性输出，促进持续饮酒行为，这一点得到了支持。 报告表明，较高的 BDNF 表达和 PPARγ 激动剂可减少饮酒量。 PARP 活性在一定程度上是 EtOH 诱导的 Bdnf IV 和 IXa mRNA 表达降低的原因 我们发现了几条证据表明 PPARγ 启动子减少。 结合可能作为 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小鼠 在黑暗中饮酒（DID）范式中自愿消耗 EtOH 增加了 PARP mRNA PFC 中的表达和酶活性 DID 消耗 EtOH 降低了 BDNF 和 PPARγ 表达， PARP 抑制剂治疗逆转了 EtOH 的消耗是否降低了 PPARγ DNA。 此外，DID 消耗的 EtOH 整体上增加了 Bdnf IXa 启动子的结合能力。 H3K9me2 水平和 PARP 抑制降低了 Pparγ 和 Bdnf IXa 基因启动子处的 H3K9me2。 我们发现 PARP 抑制减少了 DID 范式中的 EtOH 消耗。在第一个目标中，我们提出。 剖析 PARP 与 AUD 相关基因表达变化之间的分子机制 mPFC 遵循 DID 和 2 瓶自由选择饮用范例 在第二个目标中，我们将研究 EtOH 诱导的 PARP 对皮质兴奋性神经元的基因调节作用。 利用翻译核糖体亲和纯化 (TRAP) 方法进行 RNA 分离和荧光 激活细胞分选 (FACS) 分离兴奋性神经元核以进行 KDM4D、H3K9me2 和 PPARγ 结合 我们还将使用从兴奋性神经元中分离的 RNA 进行数据驱动的实验。 在第三个目标中，我们将研究 PARP 在乙醇饮酒中的作用。 行为，例如 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 激动剂的研究结果有望阐明 PARP 在其中的作用。 根据这些数据，PARP 抑制剂可能是调节与 AUD 相关的基因表达。 治疗 AUD 的有用药理学工具。