Proteolytic Fragments and Mitochondrial Dysfunction in TBI

描述（由申请人提供）：创伤性脑损伤（TBI）是美国儿童致残和死亡的主要原因，儿童TBI受害者在受伤后持续数小时至数天的神经变性。有证据表明，线粒体功能障碍和蛋白酶有助于病理进展，但两者之间的关系尚未报道。与成人大脑相比，细胞凋亡的程序性机制在发育中的大脑中更为普遍，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。限制对线粒体（细胞的主要能量产生细胞器）的损害对神经保护至关重要。本研究将验证由病理性蛋白酶激活和/或受损去除产生的生物活性多肽参与儿童TBI模型线粒体功能障碍的凋亡机制的中心假设。目标1中提出的实验将应用最近开发的iTRAQ同位素标记系统（Applied Biosystems），以一种新的方式识别钙依赖性蛋白酶calpain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在使用缺乏Htra2/Omi活性的mnd2突变小鼠的TBI小鼠模型中建立。使用iTRAQ技术对多个不同治疗组进行并行和定量分析的能力，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和目标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) calpain和Htra2/Omi活性之间的不平衡发生在细胞内钙的病理性升高后，导致线粒体中蛋白质片段的积累；3)这些片段通过抑制氧化磷酸化和促进凋亡因子的释放，参与线粒体功能障碍和凋亡。新的XF24技术（Seahorse Biosciences）以半高通量方式测量细胞的氧气消耗，将首次允许使用体外神经元损伤范式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的凋亡或兴奋性毒性细胞死亡途径，为中心假设提供最终测试。这项研究将广泛推进对线粒体损伤机制的认识，线粒体损伤有助于儿童脑外伤后神经退行性变。此外，该研究还将为确定新的神经保护候选药物的长期目标迈出重要的第一步：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)对完整神经元中蛋白酶活性如何影响线粒体功能进行首次评估。公共卫生相关性：儿童创伤性脑损伤（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。通过专注于理解发育中的大脑损伤机制，本基金提出的研究将促进治疗方法的发展，从而改善儿童创伤性脑损伤受害者的长期临床结果。

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.