Role of the transcriptional regulator Lmo4 in alcohol consumption and reward

转录调节因子 Lmo4 在饮酒和奖励中的作用

• 批准号: 10322460
• 负责人: Rajani Maiya
• 金额: $ 32.61万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10322460
• 关键词: Address; Affinity; Affinity Chromatography; Alcohol consumption; Alcohol dependence; Alcohols; Amygdaloid structure; Binding; Brain; Candidate Disease Gene; Cues; Dependence; Desire for food; Development; Disintegrins; Dopamine; Down-Regulation; Ethanol; Event; Extracellular Matrix; Fright; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Goals; Heavy Drinking; Heparitin Sulfate; Human; Knowledge; Lead; Learning; Lentivirus; Mediating; Messenger RNA; Metalloproteases; Molecular; Motivation; Mus; Neurons; Ontology; Pathway Analysis; Pathway interactions; Phenocopy; Procedures; Process; RNA Interference; Regulation; Rewards; Rodent; Role; Structure; Sulfatases; System; Testing; Therapeutic; Thrombospondins; Transcript; Viral; Work; alcohol exposure; alcohol reward; alcohol use disorder; base; cell type; chromatin immunoprecipitation; conditioned place preference; density; differential expression; drinking; drug reward; experimental study; gene network; hippocampal pyramidal neuron; kappa opioid receptors; knock-down; new therapeutic target; novel; preference; problem drinker; promoter; recruit; relating to nervous system; response; small hairpin RNA; transcription factor; transcriptome; transcriptome sequencing

Project Summary Repeated exposure to alcohol leads to neuroadapative changes that underlie the transition from moderate to excessive alcohol intake. Gene expression profiling studies in human alcoholics and rodents have led to the identification of a multitude of ethanol-responsive gene networks and pathways. However, there is a gap in knowledge in our understanding of how these networks are coordinated into a neuroadaptive response. One potential mechanism could involve the recruitment of transcription factors and transcriptional co-regulators that could modulate the expression of several downstream genes. However, very few studies have examined the role of transcriptional co-regulators in alcohol use disorders. Our preliminary results implicate a novel role for the transcriptional co-regulator Lim-Only 4 (LMO4) in regulating alcohol intake. LMO4 knockdown in the basolateral amygdala (BLA) led to a significant decrease in alcohol consumption in the intermittent access procedure and a significant deficit in conditioned place preference to alcohol suggesting a role for LMO4 in the BLA in regulating both alcohol consumption and reward. Unbiased transcriptome analysis of the BLA from WT and Lmo4gt/+ mice, which make 50% less Lmo4 than WT mice, using RNASeq revealed several genes that were differentially expressed including the kappa opioid receptor (Oprk1). Weighted gene co-expression network analysis (WGCNA) revealed extracellular matrix (ECM)-related genes as being dysregulated upon LMO4 knockdown. These results led us to hypothesize that a LMO4-regulated transcriptional network in the BLA regulates alcohol consumption. We propose the following specific aims to test this hypothesis. In Aim 1, we will determine whether Oprk1 functions downstream of LMO4 in the BLA to regulate alcohol consumption. We will use a combination of approaches including cell-type specific shRNA-mediated knockdown of Lmo4 and Oprk1 in BLA pyramidal neurons and chromatin immunoprecipitation to address this question. In Aim 2, we will determine whether LMO4 downregulation in the BLA causes abnormalities in the density of perineuronal nets (PNNs), a highly specialized form of ECM in the brain. We will next determine whether enzymatic dissolution of PNNs will reduce ethanol consumption. We will also determine whether a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) 2 and sulfatase 2 (Sulf2) function downstream of LMO4 to regulate alcohol consumption. Finally, in Aim 3, we will use a viral-based translational affinity purification strategy to determine how the LMO4-regulated transcriptome changes with alcohol exposure. The proposed experiments are significant because identification of transcriptional targets functioning downstream of LMO4 to regulate alcohol consumption could lead to the identification of novel therapeutic targets to treat alcohol use disorders.

项目概要 反复接触酒精会导致神经适应性变化，这是从中度到中度过渡的基础 过量饮酒。对人类酗酒者和啮齿动物的基因表达谱研究导致 鉴定大量乙醇反应基因网络和途径。然而，存在差距 我们理解这些网络如何协调成神经适应性反应的知识。一 潜在的机制可能涉及转录因子和转录共调节因子的招募 可以调节多个下游基因的表达。然而，很少有研究考察 转录共调节因子在酒精使用障碍中的作用。我们的初步结果表明， 转录辅助调节因子 Lim-Only 4 (LMO4) 调节酒精摄入量。 LMO4 敲低 基底外侧杏仁核（BLA）导致间歇性访问中酒精消耗量显着减少 程序和对酒精的条件性位置偏好的显着缺陷表明 LMO4 在 BLA 监管酒精消费和奖励。 WT BLA 的无偏转录组分析 和 Lmo4gt/+ 小鼠，其 Lmo4 比 WT 小鼠少 50%，使用 RNASeq 揭示了几个基因 差异表达包括 kappa 阿片受体 (Oprk1)。加权基因共表达 网络分析（WGCNA）揭示了细胞外基质（ECM）相关基因的失调 LMO4 击倒。这些结果使我们推测 LMO4 调节的转录网络 BLA 监管酒精消费。我们提出以下具体目标来检验这一假设。在目标 1 中， 我们将确定 Oprk1 是否在 BLA 中 LMO4 下游发挥作用来调节酒精消耗。 我们将使用多种方法的组合，包括细胞类型特异性 shRNA 介导的 Lmo4 敲低和 BLA 锥体神经元中的 Oprk1 和染色质免疫沉淀可以解决这个问题。在目标 2 中，我们将 确定 BLA 中 LMO4 下调是否会导致神经周围网络密度异常 （PNN），大脑中高度专业化的 ECM 形式。接下来我们将确定是否酶促溶解 PNN 将减少乙醇消耗。我们还将确定解整合素和金属蛋白酶是否 血小板反应蛋白基序 (ADAMTS) 2 和硫酸酯酶 2 (Sulf2) 在 LMO4 下游发挥调节作用 饮酒量。最后，在目标 3 中，我们将使用基于病毒的翻译亲和纯化策略 确定 LMO4 调节的转录组如何随酒精暴露而变化。拟议的实验 意义重大，因为识别在 LMO4 下游发挥作用的转录靶点以调节 饮酒可能导致确定治疗酒精使用障碍的新治疗靶点。