PARP-mediated gene regulation in alcohol drinking behavior

PARP介导的饮酒行为基因调控

• 批准号: 9898316
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898316
• 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; Military Personnel; 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; Veterans; 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/antagonist; innovation; knock-down; mRNA Expression; male; men; 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.

在美国退伍军人中，酒精使用障碍（AUD）非常普遍。过度饮酒， 定义为获取血液酒精浓度（BAC）≥0.08g/dl（binging）或≥15饮料/周 男性是许多严重的医学和精神病患者（包括AUD）的重要危险因素。 媒体前额叶皮层（MPFC）对于整合各种内部和外部状态很重要 确定方法/回避行为以奖励或厌恶刺激。先前的研究表明多聚 ADP核糖聚合酶（PARP）通过调节合成可塑性基因表达的能力促进 可卡因添加剂行为。 PARP酶活性是否在添加特性中起着相似的作用 乙醇（ETOH）尚未研究。该赠款提案的假设是EtoH增加了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降低的活性 最终增加饮酒行为。 ETOH增加了细胞培养中的PARP酶活性，成人 在胎儿发育过程中，动物的动物。 PAR可以通过催化反应沉默基因表达 将PAR组（Parylation）添加到下游基因调节蛋白，包括转录因子 过氧化物组增殖物激活的受体-γ（PPARγ）和KDM4D，转录的脱甲基酶 Hisstone 3（H3K9me2）的抑制性二甲基化赖氨酸9。在AUD中实施的基因示例 由H3K9ME2调节的是脑衍生的神经营养因子（BDNF）和PPARγ。 bdnf和 PPARγ由皮质中的兴奋性神经元表达，在皮质中，两者在突触可塑性中都有作用，并且 神经元生存。因此，酒精诱导的BDNF和PPARγ表达的抑制作用将是 预计将改变MPFC兴奋性产量，从而促进持续的饮酒行为。这是由 报告表明较高的BDNF表达和PPARγ激动剂减少了酒精消耗。我们报告了这一点 PARP活性部分负责ETOH诱导的BDNF IV和IXA mRNA表达的下降 原发性皮质神经元培养物。我们发现了几条证据，表明PPARγ启动子减少 结合可以作为增加PARP活性和降低BDNF表达之间的中间步骤。 现在，我们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 自愿消耗的eTOH在黑暗中饮酒（DID）范式中增加了PARP mRNA PFC中的表达和酶活性。消耗了ETOH降低了BDNF和PPARγ的表达， PARP抑制剂治疗逆转的影响。 EtOH消耗降低了PPARγDNA 一般，特别是在BDNF IXA启动子上的结合能力。另外，消耗了ETOH的全球增加 H3K9ME2和PARP抑制水平降低了PPARγ和BDNF IXA基因启动子的H3K9me2。最后， 我们发现，PARP抑制减少了DID范式的ETOH消耗。在第一个目标中，我们提出了 剖析将PARP连接到AUD中涉及基因表达的变化的分子机制 MPFC以下是DID和2瓶自由选择的饮用范式。在第二个目标中，我们将研究 EtOH诱导的PARP在皮质兴奋神经元中的基因调节作用。这种特定的细胞方法将 利用翻译核糖体亲和力纯化（TRAP）方法进行RNA隔离和荧光 KDM4D，H3K9ME2和PPARγ结合的激活细胞分选（FACS）分离兴奋性神经元核的分离 实验。我们还将使用从兴奋性神经元中分离的RNA进行数据驱动的实验 整个转录组微阵列分析。在第三个目标中，我们将研究PARP在ETOH饮用中的作用 行为，例如ID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。拟议研究的结果有望阐明PARP在 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的有用的药物工具。