Ceramide and Oligodendrocyte Protection in Stroke

描述(由申请人提供)： 中风目前在退伍军人中是一种流行和高风险的疾病。少突胶质细胞(OLs)富含白质和灰质，是大脑中唯一的髓鞘形成细胞。酒精损伤对白质的功能有深远的影响，白质参与了人类几乎所有的缺血性中风病例。此外，OL损伤可能引发对神经元的继发性损伤。此前，更多的研究集中在神经保护方面，许多治疗中风的药物都是基于它们在动物中风模型中减少神经元损伤的能力而开发的。然而，神经保护性药物的临床试验结果一直令人失望，部分原因是这些药物未能改善对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，以设计有效的治疗方法来保护细胞和最终治疗中风。中风时脑组织损伤的一个特征是线粒体功能障碍和线粒体蛋白的释放，线粒体蛋白是细胞凋亡的起始者。尽管线粒体完整性丧失的机制尚不清楚，但神经酰胺，一种膜鞘磷脂和细胞应激反应的重要中介，可能在脑IR诱导的OLS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，是细胞凋亡的普遍因素。我们已经鉴定了神经酰胺合成酶的几种亚型，包括CerS1、CerS2和CerS6，它们位于纯化的脑线粒体中。我们的数据提示了一种新的机制，即IR后由于CerS6的选择性激活导致神经酰胺在脑线粒体过度堆积。我们还表明，人工合成的天然神经酰胺类似物对线粒体造成的损伤类似于IR后脑线粒体的损伤。这一建议验证了IR触发神经酰胺合成酶CerS6的激活和神经酰胺在脑线粒体中堆积导致线粒体功能障碍和凋亡性OL死亡的假说。第一个特异性目的是确定CerS6在IR诱导的OL损伤和脑损伤中的作用。第二个目的是探讨CerS6/神经酰胺介导的脑缺血再灌注后线粒体功能障碍的机制。我们将采用一种综合的方法，将转基因和基因敲除小鼠的体外和体内研究结合起来，以实现这些目标。一种强大的新方法，串联质谱仪，将被用于准确检测天然神经酰胺种类和神经酰胺合成酶活性。本方案设计的研究将建立线粒体鞘脂神经酰胺作为参与OL损伤的关键分子，并可为开发突破性的中风治疗方法提供基础。它将改善退伍军人的治疗和生活质量，最终将改善护理负担对家庭和美国公众的影响。 公共卫生相关性： 叙述性中风是导致退伍军人死亡的3D主要原因，也是导致长期残疾的主要原因。我们的研究应该建立一种新的细胞损伤机制和神经鞘脂神经酰胺作为参与卒中后细胞死亡的关键分子。这些研究将促进我们对实验性卒中脑损伤机制的了解，并揭示特定的靶点，为开发新的治疗干预方法提供基础。它将改善退伍军人的治疗和生活质量，最终将改善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. PUBLIC HEALTH RELEVANCE: NARRATIVE Stroke is the 3d leading cause of death and the leading cause of long-term disability in Veterans. Our studies should establish a novel mechanism of cell injury and sphingolipid ceramide as a key molecule involved in cell death after stroke. The proposed studies will advance our understanding of the mechanisms of brain injury in experimental stroke and reveal specific targets that can provide the basis for developing new approaches for therapeutic intervention. 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.