Ceramide and Oligodendrocyte Protection in Stroke

描述（由申请人提供）： 中风目前是退伍军人中普遍存在的高风险疾病。少突胶质细胞（Oligodendrocytes，OL）是脑内唯一的髓鞘形成细胞，富含白色和灰质。OL损伤对白色物质的功能具有深远的影响，这涉及人类几乎所有的缺血性卒中病例。同时，OL损伤可引起神经元的继发性损伤。以前，更多的研究集中在神经保护上，并且已经开发了许多用于中风治疗的药物，这些药物基于它们在动物中风模型中减少神经元损伤的能力。然而，神经保护药物的临床试验结果令人失望，部分原因是药物未能改善OL的缺血/再灌注（IR）损伤。因此，我们建议研究IR诱导的OL损伤的新机制，以设计用于细胞保护和最终中风治疗的有效疗法。 中风中脑组织损伤的标志是线粒体功能障碍和线粒体蛋白质的释放，线粒体蛋白质是细胞凋亡的起始物。虽然线粒体完整性丧失背后的机制尚不清楚，但神经酰胺（一种膜鞘脂和细胞应激反应的重要介质）可能在脑IR诱导的OL线粒体损伤中起关键作用。此外，强有力的证据表明神经酰胺的产生是对细胞应激刺激的反应，是细胞凋亡的一个普遍因素。我们已经确定了几种异构体的神经酰胺合酶，包括CerS 1，CerS 2和CerS 6，定位于纯化的脑线粒体。我们的数据表明，由于IR后CerS 6的选择性激活，脑线粒体中神经酰胺过量积聚的新机制。我们还表明，天然神经酰胺的合成类似物会造成线粒体损伤，类似于IR后脑线粒体中发生的损伤。该提议测试了IR触发神经酰胺合成酶CerS 6激活和脑线粒体中神经酰胺积聚的假设，导致线粒体功能障碍和凋亡性OL死亡。第一个具体的目的是确定CerS 6在IR诱导的OL损伤和脑损伤中的作用。第二个目的是确定CerS 6/神经酰胺介导的IR后脑线粒体功能障碍的机制。我们将使用一种整合的方法，通过结合转基因和基因敲除小鼠的体外和体内研究来实现这一目标。一个强大的新方法，串联质谱，将用于准确检测天然神经酰胺物种和神经酰胺合酶活性。本提案中设计的研究将确立线粒体鞘脂神经酰胺作为参与OL损伤的关键分子，并可为开发突破性卒中治疗方法提供基础。它将改善退伍军人的待遇和生活质量，最终减轻护理负担对家庭和美国公众的影响。

DESCRIPTION (provided by applicant): Stroke is currently a prevalent and high-risk condition among Veterans. Oligodendrocytes (OLs), abundant in white and grey matter, are the only myelin-forming cells in the brain. OL injury has profound consequences for the function of white matter, which is involved in almost all instances of ischemic stroke in humans. Also, OL damage could trigger secondary injury to neurons. Previously, more research was focused on neuroprotection, and many drugs for stroke treatment have been developed based on their ability to reduce neuronal damage in animal stroke models. However, clinical trial outcomes for neuroprotective drugs have been disappointing partly due to the drugs' failure to ameliorate ischemia/reperfusion (IR) damage to OLs. Therefore, we propose to investigate a novel mechanism of IR-induced OL injury for the design of effective therapeutics for cell protection and eventual stroke treatment. A hallmark of brain tissue injury in stroke is mitochondrial dysfunction and release of mitochondrial proteins, initiators of apoptosis. Although the mechanism behind the loss of mitochondrial integrity is obscure, ceramide, a membrane sphingolipid and an essential mediator of cell-stress responses, could be critical in brain IR-induced mitochondrial damage in OLs. Moreover, strong evidence implicates ceramide generation in response to cell- stress stimuli as a universal element of apoptosis. We have identified several isoforms of ceramide synthase, including CerS1, CerS2, and CerS6, localized in purified brain mitochondria. Our data suggest a novel mechanism of excessive ceramide accumulation in brain mitochondria due to selective activation of CerS6 after IR. We have also shown that synthetic analogs of natural ceramide inflict mitochondrial injury similar to that occurring in brain mitochondria after IR. This proposal tests the hypothesis that IR triggers activation of ceramide synthase CerS6 and ceramide accumulation in brain mitochondria leading to mitochondrial dysfunction and apoptotic OL death. The first specific aim is to determine the role of CerS6 in the IR-induced OL injury and brain damage. The second aim is to determine the mechanisms of CerS6/ceramide-mediated mitochondrial dysfunction in brain after IR. We will use an integrative approach by combining in vitro and in vivo studies in transgenic and knockout mice to accomplish the objectives. A powerful new methodology, tandem mass spectrometry, will be utilized for accurate detection of natural ceramide species and ceramide synthase activity. The studies designed in this proposal will establish the mitochondrial sphingolipid ceramide as a key molecule involved in OL injury and can provide the basis for development of groundbreaking therapeutics for stroke. It will improve the treatment and the quality of life of Veterans that will ultimately ameliorate the impact of the burden of care on the families and the American public.