Mitochondrial dynamics in astrocytic processes after transient ischemia

描述（由申请人提供）：星形胶质细胞是脑中最丰富的细胞类型。它们负责清除突触中的细胞外谷氨酸（主要的兴奋性神经递质），以维持清晰的信号传导并防止兴奋性毒性。在前脑中，星形胶质细胞谷氨酸转运体GLT 1负责绝大多数谷氨酸摄取。线粒体被投资在整个精细的星形胶质细胞的过程中，他们与GLT 1共定位。我们最近发现了GLT 1，多种糖酵解酶和线粒体之间的物理和功能相互作用。在完成我最初的资助申请的目标1时，我们得出结论，星形胶质细胞过程中的线粒体保留在谷氨酸转运体和突触附近。我们的数据表明，这种分布是由神经元谷氨酸的释放，星形胶质细胞谷氨酸的摄取，和逆转的Na+/Ca 2+交换。线粒体可以通过提供ATP和缓冲离子来支持谷氨酸摄取，并且越来越多的证据表明，一部分转运的谷氨酸在线粒体中被氧化以在这些隔室中产生能量。线粒体功能障碍和兴奋性毒性从失败的星形胶质细胞摄取谷氨酸是在延迟细胞死亡的核心，持续缺血性损伤后。除了引起体温过低，这种病理目前是无法治疗的。 我已经观察到在星形胶质细胞过程中响应于氧葡萄糖剥夺（OGD）的线粒体密度的损失，所述氧葡萄糖剥夺（OGD）先于迟发性神经元细胞死亡，所述迟发性神经元细胞死亡对于该离体模型和体内中风是常见的。在第一个目标中，我将描述OGD诱导的星形胶质细胞过程中线粒体的损失，并确定它是否是由线粒体膜电位和活性氧的变化引起的。我将通过阻断或激活NMDA受体来研究兴奋性毒性和星形胶质细胞过程中线粒体占有率降低之间的关系。在一项初步研究中，我发现环孢菌素A减少了细胞死亡，并减弱了OGD后星形胶质细胞过程中线粒体密度的损失。环孢菌素A增加线粒体的钙缓冲能力。将评价环孢菌素A治疗作为减轻线粒体从过程中损失的干预机制。我还观察到OGD后星形胶质细胞过程中线粒体自噬（功能障碍线粒体的降解机制）增加。在第二个目标中，我将描述OGD后线粒体自噬的变化。我还将评估药物和遗传抑制线粒体自噬对星形胶质细胞过程的线粒体占有率和OGD后迟发性神经元死亡的影响。通过提供有史以来第一次检查线粒体动力学在缺血性损伤期间星形胶质细胞过程中的作用，该项目的执行可能有助于为迫切需要它们的医学领域带来新的治疗靶点。

DESCRIPTION (provided by applicant): Astrocytes are the most abundant cell type in brain. They are responsible for clearing extracellular glutamate, the predominant excitatory neurotransmitter, from the synapse to maintain crisp signaling and prevent excitotoxicity. In forebrain, the astrocytic glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake. Mitochondria are invested throughout fine astrocytic processes where they colocalize with GLT1. We recently discovered physical and functional interactions between GLT1, multiple glycolytic enzymes and mitochondria. In completing Aim 1 of my original grant submission, we concluded that mitochondria in astrocytic processes are retained near glutamate transporters and synapses. Our data suggest that this distribution is regulated by neuronal glutamate release, astrocytic glutamate uptake, and reversal of the Na+/Ca2+ exchanger. Mitochondria can support glutamate uptake by providing ATP and buffering ions, and there is growing evidence suggesting that a portion of transported glutamate is oxidized in mitochondria to generate energy in these compartments. Mitochondrial dysfunction and excitotoxicity from failure of astrocytic glutamate uptake are at the core of the delayed cell deat that persists after an ischemic insult. Aside from inducing hypothermia, this pathology is currently untreatable. I have observed a loss of mitochondrial density in astrocytic processes in response to oxygen glucose deprivation (OGD) that precedes the delayed neuronal cell death that is common to this ex vivo model and to stroke in vivo. In the first aim I will characterize th OGD-induced loss of mitochondria from astrocytic processes, and determine if it is preceded by changes in mitochondrial membrane potential and reactive oxygen species. I will pharmacologically block or activate NMDA receptors to investigate the relationship between excitotoxicity and reduced mitochondrial occupancy of astrocytic processes. In a preliminary study, I found that cyclosporin A reduced cell death and attenuated the loss of mitochondrial density in astrocytic processes after OGD. Cyclosporin A increases mitochondrial capacity for calcium buffering. Treatment with cyclosporin A will be evaluated as mechanism of intervention for attenuating the loss of mitochondria from processes. I have also observed increased mitophagy (a mechanism for degradation of dysfunctional mitochondria) in astrocytic processes after OGD. In aim two I will characterize changes in mitophagy after OGD. I will also evaluate the effects of pharmacological and genetic inhibition of mitophagy on mitochondrial occupancy of astrocytic processes and delayed neuronal death after OGD. By providing the first ever examination of the role of mitochondrial dynamics in astrocytic processes during ischemic injury, execution of this project could help lead to new therapeutic targets for a field of medicine that desperately needs them.