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主要的兴奋性神经递质。细胞外谷氨酸的积累导致谷氨酸受体的过度激活和细胞通过兴奋毒性机制死亡。与其他直接循环进入突触前神经终端的经典神经递质不同，大多数谷氨酸被两种星形胶质谷氨酸转运体清除，称为GLAST和GLT-1（或EAAT1和EAAT2）。这些转运体在含有毫摩尔浓度谷氨酸的环境中维持非常低的突触谷氨酸浓度，估计约为25 nM。这些转运蛋白富集于星形胶质细胞鞘突触的精细突起上。我们最近发现了物理证据（共免疫沉淀，质谱，反向免疫沉淀）和解剖学证据（在器官型切片培养的单个星形细胞中共定位），这些转运体存在于Na+/K+ atp酶，糖酵解中的大多数酶和线粒体的复合体中。这种复合体在星形胶质细胞的精细突起中可见。在第一个目标中，我们将确定支持相互作用/共区隔的GLT-1和GLAST的特定结构域。我们希望鉴定可能在转运体和线粒体之间形成连接的潜在支架蛋白。正如在突触或Ranvier节点上观察到的线粒体一样，我们提出神经活动将线粒体招募到转运蛋白富集的区域。在第二个目标中，我们将研究神经元活动对这种共区隔化的影响，并确定所涉及的机制。最后，我们将验证这些复合物的形成是糖酵解中谷氨酸依赖变化和谷氨酸代谢转变（从转化为谷氨酰胺到谷氨酸氧化）所必需的假设。星形胶质谷氨酸转运体与这些蛋白质和线粒体的区隔化提供了空间匹配能量产生和缓冲能力的机会。这对谷氨酸的处理也有影响。因此，我们提出的研究将影响我们对谷氨酸处理和代谢的基本方面的理解。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. PUBLIC HEALTH RELEVANCE: For approximately two decades, it has been clear that failure to clear glutamate contributes to the brain damage observed in stroke, neurodevelopmental disorders, and neurodegenerative diseases. This failure to clear glutamate occurs, at least in part, because of impaired energy mobilization. Our long-term goal is to understand how extracellular glutamate is controlled so that it will be possible to intervene and prevent the debilitating effects of these disorders.