Evaluation of Mitochondrial Cardiolipin Modification in Neonatal Hypoxia/Ischemia Encephalopathy

项目摘要 新生儿缺氧/缺血可导致婴儿大脑严重损伤。重建血流， 氧气输送（再灌注）对存活至关重要。然而，再灌注诱导了反应性的积聚， 由线粒体产生的氧物种（ROS），最终导致氧化应激和不可逆组织 损害目前的研究表明，心磷脂（CL）及其重塑通过氧化损伤， 单溶血心磷脂（MLCL）直接参与线粒体自噬和线粒体孔形成的激活， 细胞色素C释放并促进诱导程序性细胞死亡。本研究计划的目的 探讨CL及其修饰在缺氧性脑损伤中的分子机制。目的 1将专注于建立CL修饰的临床相关性并研究CL修饰的作用 缺氧缺血性脑病后的脑损伤利用循环离子迁移谱质谱的尖端技术 将在缺氧/缺血后定量分析CL及其亚型， 新生仔猪HIE模型。与传统的质谱法不同，cIMS-MS基于质谱分析分离分子。 结构以及质荷比（m/z），允许详细分析CL物质并直接研究 I/R损伤中的重塑几种新的转基因小鼠系也将用于操纵CL生物合成 和体内重塑。将小鼠幼仔暴露于缺氧/缺血，然后分析脑损伤 评价CL调整对HIE预后的影响。目标2将进一步探讨 缺氧/缺血后CL修饰调节的机制，包括对线粒体的破坏 网络通过线粒体自噬和线粒体动力学。在原代培养中将表征线粒体吞噬通量 来自具有线粒体质量控制（mitoQC）报告基因和线粒体DNA断裂的小鼠， 形态学将使用我们基于机器学习的尖端量化方法进行评估。最后， 线粒体特异性抗氧化剂mitoTEMPO和磷脂酶抑制剂将与 我们的遗传小鼠模型，以确定氧化修饰和脂解对CL修饰的贡献 缺氧/缺血后。总之，这些实验将建立缺氧后CL的机制 修改，询问CL和MLCL在脑损伤进展中的偶然作用，并定义潜在的 治疗目标凭借卓越的科学指导和严格的学术研究，这项研究将 深入了解脂质组学在HIE中的作用。该项目将提供广泛的培训， 使我能够获得各种动物模型和大脑机制分子分析的专业知识 损害拟议的项目创造了一个独特的环境，以促进发展和批判性思维的一个 年轻的科学家

Project Summary Neonatal hypoxia/ischemia can result in severe damage to the infant brain. Reestablishing blood flow and oxygen delivery (reperfusion) is crucial for survival. However, reperfusion induces an accumulation of reactive oxygen species (ROS) produced by the mitochondria, culminating in oxidative stress and irreversible tissue damage. Current studies suggest that cardiolipin (CL) and its remodeling via oxidative injury to monolysocardiolipin (MLCL) directly participate in activation of mitophagy and lipidic pore formation to regulate cytochrome c release and facilitate induction of programmed cell death. The objectives of this research proposal are to investigate of the roles of CL and CL modification as molecular mechanisms in hypoxic brain injury. Aim 1 will focus on establishing the clinical relevance of CL modification and investigate the role of CL modification in brain injury following HIE. Utilizing the cutting-edge technology of cyclic ion mobility spectrometry mass spectrometry (cIMS-MS) CL and its isoforms will be quantitatively analyzed following hypoxia/ischemia in a neonatal piglet model of HIE. Unlike traditional mass spectrometry, cIMS-MS separates molecules based on structure as well as mass to charge ratio (m/z) allowing detailed analysis of CL species and to directly investigate its remodeling in I/R injury. Several novel transgenic mouse-lines will also be used to manipulate CL biosynthesis and remodeling in vivo. Mouse pups will be exposed to hypoxia/ischemia, then brains will be analyzed for injury to evaluate the contribution of CL modification on outcomes following HIE. Aim 2 will further explore the mechanisms regulated by CL modification following hypoxia/ischemia, including disruption to the mitochondrial network through mitophagy and mitochondrial dynamics. Mitophagic flux will be characterized in primary culture from mice possessing the mitochondrial quality control (mitoQC) reporter and disruptions to mitochondrial morphology will be assessed with our cutting-edge machine learning based quantification methodology. Finally, the mitochondrial specific antioxidant mitoTEMPO and phospholipase inhibitors will be used in combination with our genetic mouse models to define the contribution of oxidative modification and lipolysis to CL modification following hypoxia/ischemia. Together, these experiments will establish the mechanism of post-hypoxic CL modification, interrogated the casual role of CL and MLCL in the progression of brain injury , and define potential therapeutic targets. With exceptional scientific mentorship and rigorous academic study, this research will develop a deeper understanding of the role lipidomics play in HIE. This project will allow extensive training and allow me to gain expertise in a diverse range of animal models and molecular analysis of mechanisms of brain damage. The proposed project creates a unique environment to foster the development and critical thinking of a young scientist.