Mitochondrial dynamics in astrocytic processes after transient ischemia

描述(申请人提供)：星形胶质细胞是大脑中最丰富的细胞类型。它们负责从突触中清除细胞外谷氨酸，这是主要的兴奋性神经递质，以维持清晰的信号并防止兴奋性毒性。在前脑，星形细胞谷氨酸转运体GLT1负责绝大多数谷氨酸的摄取。线粒体被包裹在星形细胞的细小突起中，在那里它们与GLT1共存。我们最近发现了GLT1、多种糖酵解酶和线粒体之间的物理和功能相互作用。在完成我最初的拨款申请的目标1时，我们得出结论，星形细胞突起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，这种分布受到神经元谷氨酸释放、星形细胞谷氨酸摄取和Na+/Ca~(2+)交换逆转的调节。线粒体可以通过提供ATP和缓冲离子来支持谷氨酸的摄取，越来越多的证据表明，部分运输的谷氨酸在线粒体中被氧化，在这些隔室中产生能量。线粒体功能障碍和星形胶质细胞谷氨酸摄取失败引起的兴奋性毒性是缺血性损伤后持续存在的延迟细胞死亡的核心。除了导致体温过低外，这种病理目前还无法治愈。我观察到星形细胞突起中线粒体密度的丧失是对氧糖剥夺(OGD)的反应，这种反应发生在这种体外模型和体内中风常见的延迟性神经细胞死亡之前。在第一个目标中，我将描述OGD诱导的星形细胞突起线粒体丢失的特征，并确定在此之前是否有线粒体膜电位和活性氧物种的变化。我将通过药物阻断或激活NMDA受体来研究兴奋性毒性与星形细胞突起线粒体占有率减少之间的关系。在初步研究中，我发现环孢素A减少了细胞死亡，并减轻了OGD后星形细胞突起中线粒体密度的丧失。环孢素A可增加线粒体的钙缓冲能力。环孢菌素A治疗将被评估为减轻突起线粒体丢失的干预机制。我还观察到OGD后星形细胞突起中有丝分裂的增加(功能失调的线粒体降解的一种机制)。在第二个目标中，我将描述OGD后有丝分裂吞噬的变化。我还将评估药物和遗传抑制有丝分裂对OGD后星形细胞突起线粒体占有率和迟发性神经元死亡的影响。通过首次研究线粒体动力学在缺血损伤期间星形细胞过程中的作用，该项目的实施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.