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，以及乙醇的激励作用。这些行为似乎归因于纹状体中谷氨酸信号的增加，其中皮层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分析发现，在ENT1缺失小鼠中EAAT2表达降低，并通过Western blot分析证实，表明ENT1缺失小鼠中谷氨酸水平升高的部分原因可能是星形胶质细胞中EAAT2突触谷氨酸摄取减少。尽管有证据表明EAAT2的遗传变异（G603A）与酒精中毒有关，但在分子、细胞和行为水平上，对星形细胞EATT2对乙醇的表达和功能的调节知之甚少。考虑到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通过星形胶质细胞内机制导致EAAT2表达降低。这一假设将通过培养的星形胶质细胞、ENT1缺失小鼠和一种新的双转基因小鼠系进行验证，该小鼠系在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异性信号通路，这些信号通路涉及ENT1和EAAT2的表达和功能。利用串联质谱法和新开发的电生理方法，我们将确定乙醇是否改变了星形胶质细胞中腺苷和谷氨酸的释放。其次，为了研究ENT1和EAAT2在星形胶质细胞中的生理作用，我们将使用双转基因小鼠、GFAP-EGFP/ENT1缺失小鼠或GFAP-EGFP/ENT1野生型小鼠检测ENT1缺失小鼠的星形胶质细胞功能。我们将使用FACS（荧光辅助细胞分选）分离星形胶质细胞，然后使用质量标签标记蛋白质组学技术iTRAQ检查蛋白质谱，以比较生理盐水或不同乙醇剂量的基因型。接下来，我们将使用功能蛋白质组学（如Western blot和免疫荧光实验）验证改变的蛋白质表达。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励。该项目的总体目标是确定一种新的ENT1和EAAT2信号通路，该通路调节星形胶质细胞和神经胶质相互作用中的乙醇反应，这可能包含开发治疗人类酒精使用障碍的新疗法的靶点。

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.