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

摘要 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谷氨酸盐的增加 纹状体中的信号传导，其中皮质神经元轴突主要发送其信号以控制运动 功能、习惯和动机。我们发现EAAT 2表达在ENT 1基因敲除小鼠中被降低， 蛋白质组学，并通过Western印迹分析证实了这一点，这表明， ENT 1敲除小鼠可能部分是由于星形胶质细胞中EAAT 2的突触谷氨酸摄取减少。 尽管有证据表明EAAT 2（G603 A）的遗传变异与酗酒有关， 对星形胶质细胞EATT 2表达和功能在乙醇反应中的调节知之甚少， 分子、细胞和行为水平。考虑到ENT 1和EAAT 2主要是 我们的主要假设是，抑制ENT 1会导致EAAT 2 在星形胶质细胞中通过细胞内机制表达。该假设将通过以下方式进行检验： 培养的星形胶质细胞、ENT 1缺失小鼠和表达绿色的新型双转基因小鼠系 星形胶质细胞中的荧光蛋白。首先，我们将确定乙醇是否会改变ENT 1 和EAAT 2在星形胶质细胞中的表达和功能。我们还将研究星形胶质细胞特异性信号 使用蛋白质组学技术分析参与ENT 1和EAAT 2表达和功能的途径。使用 串联质谱和新开发的电生理学方法，我们将确定是否 乙醇改变了星形胶质细胞的腺苷和谷氨酸的释放。第二，调查 ENT 1和EAAT 2在星形胶质细胞中的生理作用，我们将在ENT 1缺失的星形胶质细胞中检测星形胶质细胞的功能。 使用双转基因小鼠、GFAP-EGFP/ENT 1缺失小鼠或GFAP-EGFP/ENT 1野生型小鼠。我们 将使用FACS（荧光辅助细胞分选）分离星形胶质细胞，然后将检测蛋白质 使用质量标签标记蛋白质组学技术iTRAQ进行基因型比较， 生理盐水或不同剂量的乙醇。在此之后，我们将使用 功能蛋白质组学，例如蛋白质印迹和免疫荧光实验。最后我们将 确定EAAT 2的药理学或遗传调节是否改变酒精偏好和奖励 对小鼠该项目的总体目标是鉴定一种新的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，这些相互作用可能包含 开发治疗人类酒精使用障碍的新疗法。

Abstract 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.