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

描述（由申请人提供）：对ENT 1缺失小鼠的研究表明，ENT 1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，其中皮质谷氨酸能轴突主要发送其信号以控制运动功能、习惯和动机。我们通过蛋白质组学发现ENT 1敲除小鼠中EAAT 2表达减少，并通过Western印迹分析证实，表明ENT 1敲除小鼠中谷氨酸水平增加可能部分是由于星形胶质细胞中EAAT 2突触谷氨酸摄取减少。尽管有证据表明EAAT 2（G603 A）的遗传变异与酒精中毒有关，但在分子、细胞和行为水平上对星形胶质细胞EATT 2表达和功能的调节还知之甚少。考虑到ENT 1和EAAT 2主要在星形胶质细胞中表达，我们的主要假设是ENT 1的抑制通过星形胶质细胞中的细胞内机制导致EAAT 2表达降低。将使用培养的星形胶质细胞、ENT 1无效小鼠和在ENT 1无效背景下在星形胶质细胞中表达绿色荧光蛋白的新型双转基因小鼠系来检验该假设。首先，我们将确定乙醇是否改变星形胶质细胞中ENT 1和EAAT 2的表达和功能。我们还将使用蛋白质组学技术研究参与ENT 1和EAAT 2表达和功能的星形胶质细胞特异性信号通路。使用串联质谱和一种新开发的电生理方法，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ENT 1和EAAT 2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/ENT 1敲除小鼠或GFAP-EGFP/ENT 1野生型小鼠检测ENT 1敲除小鼠中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。在此之后，我们将使用功能蛋白质组学如Western印迹和免疫荧光实验来验证改变的蛋白质表达。最后，我们将确定EAAT 2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ENT 1和EAAT 2信号通路，该通路调节星形胶质细胞和神经胶质细胞相互作用中的乙醇反应，其中可能包含用于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蛋白ENT 1和谷氨酸转运蛋白EAAT 2在纹状体星形胶质细胞中的作用。我们将使用培养的星形胶质细胞，ENT 1空小鼠，和星形胶质细胞特异性标志物表达小鼠进行细胞，分子，生物化学，药理学和行为学研究。我们的研究结果有望确定一种新的ENT 1和EAAT 2信号通路，调节星形胶质细胞和神经胶质细胞相互作用中的乙醇反应，这可能会导致我们开发新的治疗药物来治疗酒精使用障碍。

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.