Proteolytic Fragments and Mitochondrial Dysfunction in TBI

TBI 中的蛋白水解片段和线粒体功能障碍

• 批准号: 7631880
• 负责人: BRIAN M POLSTER
• 金额: $ 26.25万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7631880
• 关键词: Acute Brain Injuries; Address; Adopted; Adult; Amyloid; Antibodies; Apoptosis; Apoptotic; Biochemical; Bioenergetics; Brain; Brain Injuries; Calcium; Calpain; Calpain I; Cell Culture Techniques; Cell Death; Cells; Cessation of life; Child; Childhood; Classification; Cleaved cell; Clinical; Clinical Management; Cognitive; Data; Digestion; Dyes; Emotional; Enzymes; Evaluation; Event; Excision; Family; Fluorescence Microscopy; Fluorogenic Substrate; Future; Genetic; Genus Hippocampus; Glucose; Glutamates; Goals; Grant; Guidelines; Hour; Impairment; In Situ; In Vitro; Individual; Injury; Knowledge; Label; Liquid Chromatography; Measures; Mediating; Membrane; Mitochondria; Mitochondrial Proteins; Modeling; Monitor; Mus; Mutant Strains Mice; Necrosis; Nerve Degeneration; Neuronal Injury; Neurons; Neuroprotective Agents; Nuclear; Organelles; Outcome; Outer Mitochondrial Membrane; Oxidative Phosphorylation; Oxygen; Oxygen Consumption; Pathology; Pathway interactions; Peptide Hydrolases; Peptides; Platelet Factor 4; Play; Prevalence; Process; Propidium Diiodide; Protein Fragment; Proteins; Proteolysis; Proteomics; Protons; Quality Control; Relative (related person); Reporting; Research; Role; Serine Protease; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Supportive care; Survivors; System; Technology; Testing; Time; Toxic effect; Traumatic Brain Injury; Up-Regulation; Work; apoptosis inducing factor; brain research; brain tissue; controlled cortical impact; cytochrome c; deprivation; disability; drug candidate; improved; in vivo; inhibitor/antagonist; injured; member; mitochondrial dysfunction; mouse model; neuron apoptosis; neuroprotection; neurotoxic; novel; overexpression; oxidative damage; polypeptide; prevent; programs; protein function; psychologic; public health relevance; research study; respiratory; tandem mass spectrometry; therapy development

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a leading cause of disability and death among children in the U.S. Pediatric TBI victims suffer from neurodegeneration that continues for hours to many days after the injury. Evidence indicates that mitochondrial dysfunction and proteases contribute to the progressive pathology, but the relationship between the two has not been reported. While apoptotic, programmed mechanisms of cell death are more prevalent in the developing compared to the adult brain, almost nothing is known regarding actual mechanisms of mitochondrial dysfunction in the immature brain following TBI. Limiting damage to mitochondria, the primary energy-generating organelles of the cell, is crucial for neuroprotection. This study will test the central hypothesis that bioactive polypeptides generated by pathological protease activation and/or impaired removal contribute to apoptotic mechanisms of mitochondrial dysfunction in models of pediatric TBI. The experiments proposed in aim 1 will apply the recently developed iTRAQ system of isotopic labeling (Applied Biosystems) in a novel way to identify mitochondrial substrates of the calcium-dependent protease calpain and the quality control protease Htra2/Omi. The pathological relevance of these proteolytic activities will be established in a mouse model of TBI using mnd2 mutant mice that are deficient in Htra2/Omi activity. The ability to multiplex several different treatment groups and conduct proteomic analyses in a parallel and quantitative manner using the iTRAQ technology will provide the most comprehensive proteomics study of mitochondrial changes in TBI to date. Studies proposed in aims 1 and 2 will address the specific hypotheses that: 1) protein fragments generated by mitochondrial calpain activity can be degraded by Htra2/Omi; 2) an imbalance between calpain and Htra2/Omi activity occurs following pathological rises in intracellular calcium that leads to the build-up of protein fragments in mitochondria; 3) these fragments contribute to mitochondrial dysfunction and apoptosis by inhibiting oxidative phosphorylation and promoting the release of apoptotic factors. New XF24 technology (Seahorse Biosciences) for measuring the oxygen consumption of cells in semi-high throughput fashion will for the first time allow an efficient assessment of changes in mitochondrial function using in vitro neuronal injury paradigms. The experiments proposed in aim 3 will provide the ultimate test of the central hypothesis by determining whether an upregulation of mitochondrial quality control protease activity prevents the build-up of neurotoxic mitochondrial protein fragments and inhibits apoptotic or excitotoxic cell death pathways in cell culture models related to TBI. This study will broadly advance knowledge on the mechanisms of mitochondrial injury that contribute to neurodegeneration following pediatric TBI. In addition, it will take the vital first steps toward the long-term goal of identifying novel neuroprotective drug candidates by 1) accomplishing the first proteomic screen for mitochondrial protease substrates relevant to acute brain injury and 2) conducting the first evaluation of how protease activities impact mitochondrial function in intact neurons. PUBLIC HEALTH RELEVANCE: Survivors of pediatric traumatic brain injury (TBI) suffer from many long-term physical, cognitive, psychological, and emotional impairments. Current therapy is limited to supportive care, and the majority of clinical management guidelines are extrapolated from studies on adult TBI. By focusing specifically on understanding injury mechanisms in the developing brain, the research proposed in this grant will promote the development of treatments with the ability to improve the long-term clinical outcome for pediatric victims of TBI.

描述（由申请人提供）：创伤性脑损伤 (TBI) 是美国儿童残疾和死亡的主要原因。小儿 TBI 受害者患有神经退行性病变，这种退行性病变在受伤后持续数小时至数天。有证据表明线粒体功能障碍和蛋白酶导致进行性病理学，但两者之间的关系尚未报道。虽然与成人大脑相比，细胞凋亡、程序性死亡机制在发育中更为普遍，但对于 TBI 后未成熟大脑中线粒体功能障碍的实际机制几乎一无所知。限制线粒体（细胞的主要能量产生细胞器）的损伤对于神经保护至关重要。这项研究将检验一个中心假设，即病理性蛋白酶激活和/或去除受损产生的生物活性多肽有助于儿科 TBI 模型中线粒体功能障碍的凋亡机制。目标 1 中提出的实验将以一种新的方式应用最近开发的同位素标记 iTRAQ 系统（Applied Biosystems）来识别钙依赖性蛋白酶钙蛋白酶和质量控制蛋白酶 Htra2/Omi 的线粒体底物。这些蛋白水解活性的病理相关性将使用缺乏 Htra2/Omi 活性的 mnd2 突变小鼠在 TBI 小鼠模型中建立。使用 iTRAQ 技术对多个不同的治疗组进行多重处理并以并行和定量的方式进行蛋白质组学分析的能力将提供迄今为止最全面的 TBI 线粒体变化蛋白质组学研究。目标 1 和 2 中提出的研究将解决以下具体假设：1）线粒体钙蛋白酶活性产生的蛋白质片段可以被 Htra2/Omi 降解； 2) 细胞内钙病理性升高后，钙蛋白酶和 Htra2/Omi 活性之间发生不平衡，导致线粒体中蛋白质片段的堆积； 3）这些片段通过抑制氧化磷酸化和促进凋亡因子的释放而导致线粒体功能障碍和凋亡。用于以半高通量方式测量细胞耗氧量的新 XF24 技术（Seahorse Biosciences）将首次允许使用体外神经元损伤范例有效评估线粒体功能的变化。目标 3 中提出的实验将通过确定线粒体质量控制蛋白酶活性的上调是否阻止神经毒性线粒体蛋白片段的积累并抑制与以下相关的细胞培养模型中的凋亡或兴奋毒性细胞死亡途径来提供中心假设的最终检验。创伤性脑损伤。这项研究将广泛增进对线粒体损伤机制的了解，线粒体损伤导致儿科创伤性脑损伤后的神经退行性变。此外，它将朝着确定新型神经保护候选药物的长期目标迈出重要的第一步，方法是：1）完成与急性脑损伤相关的线粒体蛋白酶底物的首次蛋白质组学筛选，2）首次评估蛋白酶活性如何影响完整神经元的线粒体功能。公众健康相关性：儿科创伤性脑损伤 (TBI) 的幸存者遭受许多长期的身体、认知、心理和情感障碍。目前的治疗仅限于支持性护理，大多数临床管理指南都是从成人 TBI 研究中推断出来的。通过专门关注发育中大脑的损伤机制，本次资助中提出的研究将促进治疗方法的开发，能够改善 TBI 儿童受害者的长期临床结果。