Role and Regulation of SIRT3 in White Matter

描述（由申请人提供）：白色物质损伤是卒中、创伤性脑损伤和围产期缺氧/缺血的基本特征。少突胶质细胞（Oligodendrocytes，OL）是脑中形成髓鞘的神经胶质细胞，因此OL损伤对白色物质的功能具有深远的影响。我们的长期目标是深入揭示OL损伤的机制，以设计新的和更有效的治疗策略。该项目的重点是脑缺血/再灌注（IR）后线粒体功能障碍导致凋亡性OL死亡的新机制。OL代谢的一个标志是产生大量鞘脂，鞘脂是髓鞘的关键脂质成分。除了它们的结构功能之外，生物活性鞘脂在增殖和凋亡的调节中发挥重要作用。神经酰胺是一种促凋亡鞘脂，在细胞中受到严格调控，其参与细胞死亡信号传导途径受神经酰胺快速转化为毒性较小的鞘脂（包括鞘氨醇-1-磷酸，一种促存活鞘脂）控制。Sirtuins是一个蛋白质脱乙酰基酶家族，是代谢和长寿的重要调节因子。Sirtuin 3（SIRT 3）协调线粒体中几种代谢途径的适应性反应。我们的初步数据表明，SIRT 3参与了生物活性鞘脂代谢产物的相互调节，最终导致OL凋亡。我们的目标 是为了确定SIRT 3如何参与脑IR后OL的凋亡程序，并阐明如何操纵SIRT 3用于预防和治疗目的。我们的中心假设是，脑IR触发SIRT 3介导的神经酰胺依赖性OL凋亡，鞘氨醇-1-磷酸是OL中SIRT 3活性的关键调节因子。我们的具体目标是：（1）确定SIRT 3介导的OL凋亡的机制;（2）确定调节OL中SIRT 3活性的机制。我们将使用多学科和综合的方法，将转基因和基因敲除小鼠中风模型的体外和体内研究与现代药理学，生物化学和生物能量学方法相结合。为了监测活OL中的线粒体功能，将使用配备飞秒激光和Meta光谱检测的激光扫描共聚焦/多光子显微镜。多种天然鞘脂种类的准确定量将通过最先进的基于质谱的方法实现。在这项研究计划的结论，我们应该建立一个机制之间的联系SIRT 3，线粒体代谢途径的关键调节器，线粒体功能障碍导致OL凋亡。这些研究将揭示中风中细胞死亡的新决定因素，这些决定因素极有可能成为合理设计治疗方案的新靶点。 更有效的治疗方法。所提出的研究将产生SIRT 3在OL的线粒体病理生物学中的作用的一致和统一的观点，并且这种知识将有益于中风、创伤性脑损伤、围产期缺氧/缺血和以OL损失为特征的慢性神经系统疾病的研究。

DESCRIPTION (provided by applicant): White matter damage is an essential feature of stroke, traumatic brain injury and perinatal hypoxia/ischemia. Oligodendrocytes (OLs) are the myelin-forming glial cells of the brain, thus OL damage has profound consequences for the function of white matter. Our long-term goal is to reveal, in-depth, the mechanisms of OL damage for the design of novel and more effective therapeutic strategies. The focus of this project is on a novel mechanism of mitochondrial dysfunction leading to apoptotic OL death after brain ischemia/reperfusion (IR). A hallmark of OL metabolism is the generation of large quantities of sphingolipids which are key lipid components of myelin. In addition to their structural function, bioactive sphingolipids play fundamental roles in the regulation of proliferation and apoptosis. Ceramide, a pro-apoptotic sphingolipid, is tightly regulated in cells and its participation in cell death signaling pathways is controlled by a rapid conversion of ceramide into less deleterious sphingolipids, including sphingosine-1-phosphate, a pro-survival sphingolipid. Sirtuins, a family of protein deacetylases, are important regulators of metabolism and longevity. Sirtuin 3 (SIRT3) coordinates the adaptive responses of several metabolic pathways in mitochondria. Our preliminary data indicate that SIRT3 is involved in the reciprocal regulation of bioactive sphingolipid metabolites that ultimately results in OL apoptosis. Our goals are to define how SIRT3 participates in the apoptotic program of OLs after cerebral IR and to unravel how SIRT3 could be manipulated for preventive and therapeutic purposes. Our central hypothesis is that cerebral IR triggers SIRT3- mediated, ceramide-dependent OL apoptosis and that sphingosine-1-phosphate is a key regulator of SIRT3 activity in OLs. Our Specific Aims are: (1) Identify the mechanisms of SIRT3-mediated OL apoptosis; (2) Determine the mechanisms regulating SIRT3 activity in OLs. We will use a multi-disciplinary and integrative approach, combining in vitro and in vivo studies in transgenic and knockout mouse stroke models with modern pharmacological, biochemical and bioenergetics methodologies. To monitor mitochondrial function in live OLs, a laser scanning confocal/multi-photon microscope equipped with femtosecond laser and META spectral detection will be used. Accurate quantification of multiple natural sphingolipid species will be achieved by a state-of-the-art mass spectrometry-based methodology. At the conclusion of this research program, we should establish a mechanistic link between SIRT3, the key regulator of mitochondrial metabolic pathways, and mitochondrial dysfunction leading to OL apoptosis. These studies will reveal novel determinants of cell demise in stroke which are highly likely to serve as new targets for the rational design of more effective therapies. The proposed studies will generate a coherent and unified view of SIRT3's role in the mitochondrial pathobiology of OLs and this knowledge will benefit the research in stroke, traumatic brain injury, perinatal hypoxia/ischemia and chronic neurological disorders characterized by OL loss.