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）是美国儿科TBI受害者患有神经变性的儿童残疾和死亡的主要原因，受伤后持续了数小时至多天。证据表明，线粒体功能障碍和蛋白酶有助于进行性病理学，但尚未报道两者之间的关系。与成人大脑相比，在发育中，细胞死亡的凋亡，编程的机制更为普遍，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。将细胞的主要产生细胞器的线粒体损害限制，对于神经保护至关重要。这项研究将检验中心假设，即病理蛋白酶激活和/或去除受损产生的生物活性多肽在儿科TBI模型中有助于线粒体功能障碍的凋亡机制。 AIM 1中提出的实验将采用最近开发的同位素标记的ITRAQ系统（应用生物系统），以新颖的方式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的小鼠模型中建立。使用ITRAQ技术以平行和定量方式进行多个不同治疗组并以平行和定量方式进行蛋白质组学分析的能力将为迄今为止TBI的线粒体变化提供最全面的蛋白质组学研究。目标1和2中提出的研究将解决以下特定的假设：1）HTRA2/OMI可以降解线粒体钙蛋白酶活性产生的蛋白质片段； 2）钙蛋白酶和htra2/omi活性之间的失衡发生在细胞内钙中的病理上升，从而导致线粒体中蛋白质碎片的积累； 3）这些片段通过抑制氧化磷酸化并促进凋亡因子的释放而导致线粒体功能障碍和凋亡。新的XF24技术（Seahorse Biosciences）用于以半高吞吐量的方式测量细胞的氧气消耗，这将首次使用体外神经元损伤范式有效评估线粒体功能的变化。 AIM 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可以防止神经毒性线粒体蛋白片段的积累，并抑制与TBI相关的细胞培养模型中神经毒性的线粒体蛋白片段的上调，从而提供中枢假设的最终检验。这项研究将广泛地提高有关线粒体损伤机制的知识，这些线粒体损伤有助于小儿TBI后神经退行性。此外，它将采取至关重要的第一步，朝着长期目标迈出新的神经保护药物候选物，通过1）完成与急性脑损伤有关的线粒体蛋白酶底物的第一个蛋白质组学筛选，以及与急性脑损伤相关的第一个蛋白酶底物，2）对蛋白酶在完整神经元中的影响如何影响线粒体功能。公共卫生相关性：小儿创伤性脑损伤（TBI）的幸存者患有许多长期的身体，认知，心理和情绪障碍。当前的疗法仅限于支持性护理，大多数临床管理指南都从成人TBI的研究中推断出来。通过专门研究发展中大脑中的损伤机制，该赠款中提出的研究将促进治疗的发展，以改善TBI儿科受害者的长期临床结果的能力。