Hypoglycemic signaling targets in astrocytes

描述（由申请人提供）： 低血糖是糖尿病管理中常见的并发症。严重的低血糖可导致癫痫发作、意识丧失和不可逆的认知障碍，可能是由于神经元损伤和死亡。星形胶质细胞是遍布大脑的星形胶质细胞，在支持和保护神经元方面发挥重要作用。最近的工作表明，在低血糖症，星形胶质细胞能够迅速产生底物，以饲料能量耗尽的神经元，可能是通过细胞内糖原的分解。星形胶质细胞感知葡萄糖水平下降的机制，以及控制糖原代谢的下游信号通路，目前还知之甚少。我们提出了一个发现为基础的，但集中的方法来确定星形胶质细胞降糖信号通路的组成部分，利用两个强大的和互补的方法：蛋白质组学和一种新的表达克隆的方法。我们将测试一个普遍的假设：星形胶质细胞具有专门的葡萄糖信号分子和机制，使快速调节底物的神经元代谢低血糖症。该项目的具体目标是： 1)使用两种独立和互补的方法鉴定星形胶质细胞中急性、亚急性和发作性低血糖的分子靶点：a）蛋白质组学（通过质谱法进行蛋白质鉴定的二维凝胶电泳）;和B）FIMS，我们实验室开发的一种新型表达克隆方法。 2)使用细胞培养和低血糖症体内模型进行靶点筛选。整套已鉴定的低血糖调节蛋白将作为研究界的数据库提供。确定的目标的一个子集将是我们实验室未来假设驱动的研究项目的主题。长期目标是开发药理学或分子方法来增强或调节星形胶质细胞对低血糖反应的神经保护作用。

DESCRIPTION (provided by applicant): Hypoglycemia is a common complication in the management of diabetes. Severe hypoglycemia can cause seizures, unconsciousness, and irreversible cognitive impairment, presumably because of neuronal injury and death. Astrocytes, the star-shaped glial cells found throughout the brain, perform important roles to support and protect neurons. Recent work indicates that in hypoglycemia, astrocytes are capable of rapidly generating substrates to feed energy-depleted neurons, probably by breakdown of intracellular glycogen. The mechanisms by which astrocytes sense dropping glucose levels, and the downstream signaling pathways controlling glycogen metabolism, are poorly understood. We propose a discovery-based, but focused approach to identify components of astrocytic hypoglycemic signaling pathways, utilizing two powerful and complementary methodologies: proteomics and a novel method of expression cloning. We will test a general hypothesis: Astrocytes possess specialized glucose signaling molecules and mechanisms enabling rapid regulation of substrates for neuronal metabolism in hypoglycemia. The specific aims of the project are: 1) Identify molecular targets of acute, subacute and episodic hypoglycemia in astrocytes using two independent and complementary approaches: a) proteomics (two-dimensional gel electrophoresis with protein identification by mass spectrometry); and b) FIMS, a novel expression cloning method developed in our laboratory. 2) Validate targets using cell culture and in vivo models of hypoglycemia. The entire set of identified hypoglycemia-regulated proteins will be made available as a database for the research community. A subset of the targets identified will be the subject of future hypothesis-driven research projects in our laboratory. The longterm goal is to develop pharmacologic or molecular methods to augment or modulate neuroprotective aspects of the astrocytic response to hypoglycemia.