Mitochondrial Ceramide in Traumatic Brain Injury

这项提议的长期目标是按顺序理解脑损伤的分子机制。 设计创伤性脑损伤(TBI)治疗的新治疗方法。TBI是两者共同作用的结果 脑组织的即刻机械性破坏(原发损伤)和迟发性损伤(继发损伤) 机械装置。颅脑损伤继发性脑组织损伤的一个特征是线粒体功能障碍和释放 线粒体促凋亡蛋白导致神经细胞死亡。尽管这种分子的性质 导致线粒体完整性丧失和功能不明，膜鞘磷脂神经酰胺，a 细胞应激反应的关键调节因子可能起到一定作用。强有力的证据表明， 内源性神经酰胺对细胞凋亡刺激的反应，作为细胞凋亡的普遍成分。我们的预赛 数据显示实验性脑损伤后神经酰胺过度蓄积是由于神经酰胺的激活 生物合成。重要的是，我们已经鉴定了几种神经酰胺合成酶的异构体，定位于高纯度。 大脑线粒体。我们还显示了天然神经酰胺对线粒体造成类似损伤的能力。 到脑损伤后线粒体的表达。 我们推测，脑损伤触发了神经酰胺生物合成的激活，从而导致过量 神经酰胺在线粒体中的积累导致线粒体促凋亡蛋白和神经的释放 细胞死亡。我们计划通过追求两个具体目标来验证我们的假设：1)确定神经酰胺的激活 颅脑损伤后继发性脑损伤需要生物合成，并确定相关机制；2) 确定神经酰胺介导的脑损伤后线粒体损伤的机制。我们将使用 计算机控制的颅脑损伤皮质撞击小鼠模型。在拟议的研究中，我们将使用 将蛋白质表达和活性分析与磁共振成像相结合的跨学科方法 (MRI)、转基因和基因敲除小鼠的行为和治疗研究。我们将使用一个强大的新 用于准确检测天然神经酰胺种类和神经酰胺的串联质谱法 合酶活性。建议的研究将促进我们对脑外伤机制的理解，并揭示 可以为开发新的治疗干预方法提供基础的具体目标。 此外，拟议的研究将把创伤后细胞功能障碍的新细胞机制与 对死亡病例进行临床相关损伤的MRI评估，以制定最有意义的 颅脑损伤患者成功康复的结果措施。

The long-term goal of this proposal is to understand the molecular mechanisms of brain injury in order to devise novel therapeutic approaches to traumatic brain injury (TBI) treatment. TBI is a result of both immediate mechanical disruption of brain tissue (the primary injury) and delayed (secondary) injury mechanisms. A hallmark of secondary brain tissue injury in TBI is mitochondrial dysfunction and release of mitochondrial pro-apoptotic proteins leading to neural cell death. Although the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure, the membrane sphingolipid ceramide, a key regulator of cell-stress responses could play a role. Strong evidence implicates the production of endogenous ceramide in response to apoptotic stimuli as a universal element of apoptosis. Our preliminary data indicate excessive ceramide accumulation in brain after experimental TBI due to activation of ceramide biosynthesis. Importantly, we have identified several isoforms of ceramide synthase localized in highly purified cerebral mitochondria. We have also shown the ability of natural ceramide to inflict mitochondrial injury similar to that occurring in mitochondria after TBI. We hypothesize that TBI triggers activation of ceramide biosynthesis that results in excessive accumulation of ceramide in mitochondria leading to release of mitochondrial pro-apoptotic proteins and neural cell death. We plan to test our hypothesis by pursuing 2 specific aims: 1) Determine if activation of ceramide biosynthesis is required for the secondary brain damage after TBI and identify the mechanisms involved; 2) Determine the mechanisms underlying the ceramide-mediated mitochondrial injury in TBI. We will use a computer-controlled cortical impact mouse model of TBI. In the proposed studies, we will use an interdisciplinary approach combining protein expression and activity assays with magnetic resonance imaging (MRI), behavioral and treatment studies in transgenic and knockout mice. We will use a powerful new methodology, tandem mass spectroscopy, for accurate detection of natural ceramide species and ceramide synthase activity. The proposed studies will advance our understanding of the mechanisms of TBI and reveal specific targets that can provide the basis for developing new approaches for therapeutic intervention. Furthermore, the proposed studies will link a novel cellular mechanism of post-traumatic cell dysfunction and death to the clinically relevant assessment of the injury by MRI in order to develop the most meaningful outcome measures for successful rehabilitation of the head-injured patient.