Role of the transcriptional regulator Lmo4 in alcohol consumption and reward

项目摘要 反复接触酒精会导致神经适应性变化，这是从中度到 过量饮酒。人类酗酒者和啮齿动物的基因表达谱研究导致了 大量乙醇反应基因网络和通路的鉴定。然而，有一个缺口是在 了解这些网络是如何协调成神经适应性反应的。一 潜在的机制可能涉及招募转录因子和转录共调节因子， 可以调节几个下游基因的表达。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。我们的初步结果暗示了一个新的角色 转录共调控因子LIM-Only 4(LMO4)在调节酒精摄入中的作用。LMO4击倒在 杏仁基底外侧核(BLA)导致间歇性通路中的酒精消耗量显著减少 程序和对酒精的条件性位置偏爱的显著缺陷表明LMO4在 BLA在调节酒精消费和奖励方面的作用。WT中BLA的无偏转录组分析 使用RNAseq的Lmo4gt/+小鼠比WT小鼠产生的Lmo4少50%，揭示了几个基因 差异表达的基因包括kappa阿片受体(OPRK1)。加权基因共表达 网络分析(WGCNA)显示细胞外基质(ECM)相关基因在 LMO4击倒。这些结果导致我们假设LMO4调控的转录网络在 BLA调节酒精摄入量。我们提出了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下游发挥调节酒精消耗的功能。 我们将使用多种方法，包括细胞类型特异性shRNA介导的LMO4和LMO4的敲除 OPRK1在BLA锥体神经元和染色质免疫沉淀中解决了这个问题。在目标2中，我们将 确定BLA中LMO4的下调是否导致神经周网络密度的异常 (PNNS)，大脑中一种高度专业化的ECM形式。我们接下来将确定酶促溶解是否 PNNS将减少乙醇消耗。我们还将确定一种去整合素和金属蛋白酶 利用凝血酶反应蛋白基序(ADAMTS)2和硫酸酯酶2(Sulf2)功能调节LMO4下游 饮酒。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来 确定LMO4调节的转录组如何随酒精暴露而变化。拟议中的实验 之所以意义重大，是因为识别了LMO4下游发挥调节作用的转录靶标 酒精消费可能导致确定治疗酒精使用障碍的新治疗靶点。

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.