Alcohol and Adenosine-Mediated Glutamate Signaling in Neuro-Glial Interaction

描述(由申请人提供)：对ENT1缺失小鼠的研究表明，ENT1调节乙醇中毒和偏好，以及乙醇的激励效应。这些行为似乎归因于纹状体谷氨酸信号的增加，纹状体皮质谷氨酸能轴突主要发送信号来控制运动功能、习惯和动机。我们通过蛋白质组学方法发现ENT1缺失小鼠的EAAT2表达降低，并通过Western印迹分析证实，ENT1缺失小鼠谷氨酸水平的升高可能部分是由于EAAT2减少了星形胶质细胞对突触谷氨酸的摄取。尽管有证据表明EAAT2(G603A)的一个基因变异与酒精中毒有关，但在分子、细胞和行为水平上对酒精对星形胶质细胞EATT2表达和功能的调控知之甚少。鉴于ENT1和EAAT2主要在星形胶质细胞中表达，我们的主要假设是抑制ENT1可能通过细胞内机制降低EAAT2的表达。这一假设将通过培养的星形胶质细胞、ENT1缺失小鼠和一种新型的双转基因小鼠品系进行验证，该品系在ENT1缺失背景下的星形胶质细胞中表达绿色荧光蛋白。首先，我们将确定乙醇是否会改变星形胶质细胞中ENT1和EAAT2的表达和功能。我们还将使用蛋白质组学技术研究星形胶质细胞特有的信号通路，涉及ENT1和EAAT2的表达和功能。利用串联质谱仪和一种新开发的电生理学方法，我们将确定乙醇是否会改变星形胶质细胞的腺苷和谷氨酸释放。其次，为了研究ENT1和EAAT2在星形胶质细胞中的生理作用，我们利用双转基因小鼠、GFAP-EGFP/ENT1缺失小鼠和GFAP-EGFP/ENT1野生型小鼠检测了ENT1缺失小鼠的星形胶质细胞功能。我们将使用FACS(荧光辅助细胞分类)分离星形胶质细胞，然后将使用质量标签标记蛋白质组技术iTRAQ来检测蛋白质谱，以比较生理盐水和不同剂量乙醇的基因型。随后，我们将使用蛋白质印迹和免疫荧光实验等功能蛋白质组学来验证改变后的蛋白质表达。最后，我们将确定EAAT2的药理学或遗传调控是否会改变小鼠的酒精偏好和奖赏。这个项目的总体目标是确定一个新的ENT1和EAAT2信号通路，它调节星形胶质细胞中的乙醇反应和神经胶质细胞的相互作用，这可能包含开发治疗人类酒精使用障碍的新疗法的靶点。 公共卫生相关性：我们将研究乙醇敏感的腺苷转运体ENT1和谷氨酸转运体EAAT2在纹状体星形胶质细胞中的作用。我们将利用培养的星形胶质细胞、ENT1缺失小鼠和星形胶质细胞特异性标志物表达小鼠进行细胞、分子、生化、药理和行为研究。我们的研究结果有望定义一个新的ENT1和EAAT2信号通路，调节星形胶质细胞中的乙醇反应和神经胶质细胞的相互作用，这可能会引导我们开发治疗酒精使用障碍的新疗法。

DESCRIPTION (provided by applicant): Studies with ENT1 null mice indicate that ENT1 regulates ethanol intoxication and preference, as well as motivational effects of ethanol. These behaviors appear to be attributed to the increased glutamate signaling in the striatum where cortical glutamatergic axons mainly send their signaling to control motor functions, habits and motivations. We found that EAAT2 expression was reduced in ENT1 null mice by proteomics, and confirmed it by Western blot analysis, indicating that the increased glutamate levels in ENT1 null mice may be partly due to the reduced synaptic glutamate uptake by EAAT2 in astrocytes. Despite evidence demonstrating that a genetic variant of EAAT2 (G603A) is implicated in alcoholism, regulation of astrocytic EATT2 expression and function in response to ethanol is poorly understood in molecular, cellular and behavioral levels. Considering that ENT1 and EAAT2 are predominantly expressed in the astrocytes, our main hypothesis is that inhibition of ENT1 causally reduces EAAT2 expression through intracellular mechanisms in the astrocytes. This hypothesis will be tested using cultured astrocytes, ENT1 null mice, and a novel bi-transgenic mouse line that expresses green fluorescent protein in astrocytes in ENT1 null background. First, we will determine if ethanol alters ENT1 and EAAT2 expression and function in the astrocytes. We will also investigate astrocyte-specific signaling pathways involved in ENT1 and EAAT2 expression and function using a proteomic technique. Using tandem mass spectrometry and a newly developed electrophysiological method, we will determine if ethanol alters adenosine and glutamate releases from the astrocytes. Secondly, to investigate the physiological role of ENT1 and EAAT2 in the astrocytes, we will examine astrocytes function in ENT1 null mice using bi-transgenic mice, GFAP-EGFP/ENT1 null mice, or GFAP-EGFP/ENT1 wild-type mice. We will isolate the astrocytes using FACS (fluorescent-assisted cell sorting), and then will examine protein profiles using a mass-tag labeling proteomic technique, iTRAQ, to compare between genotypes with saline or different ethanol doses. Following this, we will validate the altered protein expressions using a functional proteomics such as Western blot and immunofluorescence experiments. Finally, we will determine whether pharmacological or genetic regulations of EAAT2 alter alcohol preference and reward in mice. The overall goal of this project is to identify a novel ENT1 and EAAT2 signaling pathway that regulates ethanol responses in the astrocytes and neuro-glial interactions which may contain targets for the development of new therapeutics to treat alcohol use disorders in humans. PUBLIC HEALTH RELEVANCE: We will investigate the role of ethanol-sensitive adenosine transporter, ENT1 and a glutamate transporter, EAAT2, in the striatal astrocytes. We will use cultured astrocytes, ENT1 null mice, and astrocyte-specific marker expressing mice to carry out cellular, molecular, biochemical, pharmacological, and behavioral studies. The results of our studies are expected to define a novel ENT1 and EAAT2 signaling pathway that regulates ethanol response in the astrocytes and neuro-glial interactions, which may lead us to develop new therapeutics to treat alcohol use disorders.