Proteolytic Fragments and Mitochondrial Dysfunction in TBI

描述(申请人提供)：创伤性脑损伤(TBI)是导致美国儿童残疾和死亡的主要原因。儿童脑损伤患者在受伤后会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水解酶参与了进行性病理过程，但两者之间的关系尚未见报道。虽然与成人脑相比，细胞凋亡和程序性死亡机制在发育中更为普遍，但对于脑外伤后未成熟脑线粒体功能障碍的实际机制几乎一无所知。限制对线粒体的损伤，线粒体是细胞的主要能量产生细胞器，对神经保护至关重要。本研究将验证这一中心假设，即病理性蛋白水解酶激活和/或清除受损所产生的生物活性多肽参与了儿童脑外伤模型线粒体功能障碍的凋亡机制。在Aim 1中提出的实验将应用最近开发的iTRAQ同位素标记系统(应用生物系统)以一种新的方式来鉴定钙依赖的蛋白水解酶Calain和质量控制蛋白Htra2/Omi的线粒体底物。这些蛋白分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技术对几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分析的能力，将提供迄今为止关于脑损伤线粒体变化的最全面的蛋白质组学研究。AIMS 1和2中提出的研究将解决这样的特定假设：1)线粒体Calain活性产生的蛋白质片段可以被Htra2/Omi降解；2)细胞内钙的病理性升高导致线粒体中蛋白质片段的积聚，导致Calain和Htra2/Omi活性之间的失衡；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细胞凋亡。用于以半高通量方式测量细胞耗氧量的新XF24技术(海马生物科学)将首次允许使用体外神经元损伤范例有效地评估线粒体功能的变化。目标3中提出的实验将通过确定在与脑损伤相关的细胞培养模型中，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，并抑制细胞凋亡或兴奋性毒性细胞死亡途径，来提供对中心假设的终极检验。这项研究将广泛促进对线粒体损伤机制的了解，线粒体损伤是导致儿童脑外伤后神经退行性变的原因。此外，它还将朝着确定新的神经保护药物候选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筛选；2)首次评估蛋白酶活性如何影响完整神经元中的线粒体功能。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治疗，大多数临床治疗指南都是从成人脑外伤的研究中推断出来的。通过专门关注了解脑发育中的损伤机制，这项拨款中提议的研究将促进有能力改善脑外伤儿童受害者的长期临床结果的治疗方法的开发。

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.