Mitochondrial Ceramide in Traumatic Brain Injury

这项提案的长期目标是了解脑损伤的分子机制， 为创伤性脑损伤（TBI）的治疗设计新的治疗方法。TBI是两者的结果 脑组织的即时机械性破坏（原发性损伤）和延迟性（继发性）损伤 机制等TBI中继发性脑组织损伤的标志是线粒体功能障碍和线粒体释放。 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 导致线粒体完整性和功能丧失仍然不清楚，膜鞘脂神经酰胺， 细胞应激反应关键调节因子可能发挥作用。有力的证据表明， 内源性神经酰胺作为细胞凋亡的通用元件响应于细胞凋亡刺激。我们的初步 数据表明在实验性TBI后由于神经酰胺的活化， 生物合成重要的是，我们已经确定了几种神经酰胺合酶的亚型，这些亚型定位于高度纯化的神经酰胺合酶。 脑线粒体我们还显示了天然神经酰胺造成线粒体损伤的能力， 与脑外伤后线粒体中发生的变化相对应。 我们假设TBI触发了神经酰胺生物合成的激活，导致过度的神经酰胺合成。 神经酰胺在线粒体中的积累导致线粒体促凋亡蛋白的释放， 细胞死亡我们计划通过追求2个具体目标来测试我们的假设：1）确定是否激活神经酰胺 生物合成是TBI后继发性脑损伤所必需的，并确定所涉及的机制; 2） 确定脑外伤中神经酰胺介导的线粒体损伤的机制。我们将使用一个 计算机控制的TBI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测定与磁共振成像相结合的跨学科方法 (MRI)在转基因和基因敲除小鼠中的行为和治疗研究。我们将使用强大的新 准确检测天然神经酰胺种类和神经酰胺的串联质谱法 合成酶活性这些研究将促进我们对TBI机制的理解，并揭示 具体的目标，可以为开发新的治疗干预方法提供基础。 此外，拟议中的研究将把创伤后细胞功能障碍的新细胞机制与 通过MRI对损伤进行临床相关评估，以制定最有意义的 脑外伤患者成功康复的结果指标。

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.