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

基本信息

批准号：
10268581
负责人：
Rajani Maiya
金额：
$ 29.35万
依托单位：
LSU HEALTH SCIENCES CENTER
依托单位国家：
美国
项目类别：
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/10268581
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 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

项目摘要

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，转录共调节因子 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 中的 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。拟议的实验是重要的，因为识别在 LMO4 下游发挥作用的转录靶标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。