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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS064978-01
Application #
7631880
Study Section
Cell Death in Neurodegeneration Study Section (CDIN)
Program Officer
Hicks, Ramona R
Project Start
2009-05-01
Project End
2013-02-28
Budget Start
2009-05-01
Budget End
2010-02-28
Support Year
1
Fiscal Year
2009
Total Cost
$262,500
Indirect Cost
Name
University of Maryland Baltimore
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Bordt, Evan A; Clerc, Pascaline; Roelofs, Brian A et al. (2017) The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species. Dev Cell 40:583-594.e6
Choe, Moran; Brusgard, Jessica L; Chumsri, Saranya et al. (2015) The RUNX2 Transcription Factor Negatively Regulates SIRT6 Expression to Alter Glucose Metabolism in Breast Cancer Cells. J Cell Biochem 116:2210-26
Roelofs, Brian A; Ge, Shealinna X; Studlack, Paige E et al. (2015) Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV. Free Radic Biol Med 86:250-8
Clerc, P; Ge, S X; Hwang, H et al. (2014) Drp1 is dispensable for apoptotic cytochrome c release in primed MCF10A and fibroblast cells but affects Bcl-2 antagonist-induced respiratory changes. Br J Pharmacol 171:1988-99
Polster, Brian M (2013) AIF, reactive oxygen species, and neurodegeneration: a ""complex"" problem. Neurochem Int 62:695-702
Clerc, Pascaline; Young, Christina A; Bordt, Evan A et al. (2013) Magnesium sulfate protects against the bioenergetic consequences of chronic glutamate receptor stimulation. PLoS One 8:e79982
Laird, Melissa D; Clerc, Pascaline; Polster, Brian M et al. (2013) Augmentation of normal and glutamate-impaired neuronal respiratory capacity by exogenous alternative biofuels. Transl Stroke Res 4:643-51
McCranor, Bryan J; Bozym, Rebecca A; Vitolo, Michele I et al. (2012) Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J Bioenerg Biomembr 44:253-63
Clerc, Pascaline; Carey, Gregory B; Mehrabian, Zara et al. (2012) Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry. PLoS One 7:e42487
Clerc, Pascaline; Polster, Brian M (2012) Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons. PLoS One 7:e34465

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