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.