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，这些变化是从中度到中度过度的基础。 过量饮酒。在人类酗酒者和啮齿动物中的基因表达谱研究已经导致了 鉴定大量乙醇响应基因网络和途径。然而，在以下方面存在差距： 我们对这些网络如何协调成神经适应性反应的理解。一 潜在的机制可能涉及转录因子和转录辅助调节因子的募集， 可以调节下游几个基因的表达。然而，很少有研究检查 转录辅助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作用， 转录辅助调节因子Lim-Only 4（LMO 4）在调节酒精摄入方面的作用。LMO 4敲除在 基底外侧杏仁核（BLA）导致酒精消耗量的显着减少，在间歇性访问 程序和一个显着的赤字，条件性位置偏好酒精表明LMO 4的作用， BLA在调节酒精消费和奖励方面的作用。来自WT的BLA的无偏倚转录组分析 和Lmo 4gt/+小鼠，其Lmo 4比WT小鼠少50%，使用RNASeq揭示了几个基因， 包括κ阿片受体（Oprk 1）。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 LMO 4击倒。这些结果使我们假设，LMO 4调控的转录网络在细胞中的表达可能是一个重要的机制。 BLA调节酒精消费。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Oprk 1是否在BLA中的LMO 4下游起作用以调节酒精消耗。 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Lmo 4敲低， Oprk 1在BLA锥体神经元和染色质免疫沉淀来解决这个问题。在目标2中，我们 确定BLA中的LMO 4下调是否引起神经元周围网密度的异常 （PNNs）是大脑中高度专业化的ECM形式。我们接下来将确定是否酶促溶解 PNN将减少乙醇的消耗。我们还将确定是否有解整合素和金属蛋白酶 血小板反应蛋白基序（ADAMTS）2和硫酸酯酶2（Sulf 2）在LMO 4下游发挥作用， 酒精消费。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LMO 4调节的转录组如何随着酒精暴露而变化。拟议的实验 是重要的，因为识别LMO 4下游的转录靶点， 酒精消费可能导致识别新的治疗目标，以治疗酒精使用障碍。

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.