Astroglial Glutamate Transporters, Energetics, and Mitochondria

描述（由申请人提供）：谷氨酸是哺乳动物中枢神经系统中的主要兴奋性神经递质。谷氨酸的细胞外积累通过兴奋性毒性机制引起谷氨酸受体的过度激活和细胞死亡。与其他直接循环到突触前神经末梢的经典神经递质不同，大多数谷氨酸被两种称为GLAST和GLT-1（或EAAT 1和EAAT 2）的星形胶质细胞谷氨酸转运蛋白清除。这些转运蛋白在含有毫摩尔浓度谷氨酸的环境中维持非常低的谷氨酸突触浓度，估计约为25 nM。这些转运蛋白在包裹突触的星形胶质细胞的精细突起上富集。我们最近开发的物理证据（共免疫沉淀，质谱，反向免疫沉淀）和解剖学证据（共定位在单个星形胶质细胞的器官型切片培养），这些转运蛋白存在于一个复杂的Na+/K+ ATP酶，大多数的糖酵解酶，和线粒体。该复合物在星形胶质细胞的精细过程中观察到。在第一个目标中，我们将鉴定支持相互作用/共区室化的GLT-1和GLAST的特定结构域。我们希望确定潜在的支架蛋白，可能会形成一个连接之间的转运和线粒体。正如已经观察到的线粒体在突触或在节点的兰维尔，我们建议，神经活动招募线粒体的转运蛋白富集的地区。在第二个目标中，我们将研究神经元活动对这种共区室化的影响，并确定所涉及的机制。最后，我们将测试的假设，这些复合物的形成所需的谷氨酸依赖的糖酵解的变化和谷氨酸代谢的转变（从转换为谷氨酰胺谷氨酸氧化）。星形胶质细胞谷氨酸转运蛋白与这些蛋白质和线粒体的区室化提供了在空间上匹配能量产生和缓冲能力的机会。这也暗示了谷氨酸的分布。因此，我们提出的研究将影响我们对谷氨酸处理和代谢基本方面的理解。他们还将定义一种新的分子机制，将星形胶质细胞的能量需求与神经元活动的变化相匹配。

DESCRIPTION (provided by applicant): Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. An extracellular accumulation of glutamate causes excessive activation of glutamate receptors and cell death through excitotoxic mechanisms. Unlike other classical neurotransmitters, that are recycled directly into the presynaptic nerve terminal, most glutamate is cleared by two astroglial glutamate transporters, called GLAST and GLT-1 (or EAAT1 and EAAT2). These transporters maintain very low synaptic concentrations of glutamate, estimated at ~25 nM, in an environment that contains millimolar concentrations of glutamate. These transporters are enriched on the fine processes of astrocytes that sheath synapses. We recently developed physical evidence (co-immunoprecipitation, mass spectrometry, reverse immunoprecipitations) and anatomic evidence (co-localization in individual astrocytes in organotypic slice cultures) that these transporters exst in a complex with the Na+/K+ ATPase, most of the enzymes in glycolysis, and mitochondria. This complex is observed in fine processes of astroglia. In the first aim, we will identify specifi domains of GLT-1 and GLAST that support the interactions/co- compartmentalization. We wish to identify potential scaffolding proteins that may form a linkage between the transporters and mitochondria. As has been observed with mitochondria at synapses or at nodes of Ranvier, we propose that neural activity recruits mitochondria to regions where transporters are enriched. In the second aim, we will study the effects of neuronal activity on this co-compartmentalization and define the mechanisms involved. Finally, we will test the hypothesis that formation of these complexes is required for glutamate-dependent changes in glycolysis and a shift in glutamate metabolism (from conversion to glutamine to glutamate oxidation). Compartmentalization of the astroglial glutamate transporters with these proteins and mitochondria provides an opportunity to spatially match energy production and buffering capacity. It also has implications for disposition of the glutamate. Therefore, our proposed research will impact our understanding of fundamental aspects of glutamate handling and metabolism. They will also define a novel molecular mechanism that matches astroglial energetic demands to changes in neuronal activity.