Each year in the US, severe TBI in children results in <7400 deaths and 60 000 hospitalizations. Fifty percent of surviving children with severe TBI have poor neurological outcome at six months. Severe TBI in children is thus a critical problem in desperate need of impactful therapies. Free radicals and oxidative stress have been accepted as universal pathogenic mechanisms of TBI prompting the therapeutic use of antioxidants. Invariably, the clinical trials of non-specific free radical scavengers/antioxidants failed. Unless the true sources and mechanisms of TBI redox imbalance are identified, the use of sacrificial antioxidants and free radical scavengers will fail. Thus, a paradigm-shifting approach is required. During prior funding period, we discovered that selective peroxidation of a mitochondria-specific phospholipid, cardiolipin (CL), occurs in severe pediatric TBI and represents a required mitochondrial stage of neuronal apoptosis. We further identified cytochrome c (cyt c) as a catalyst of CL peroxidation occurring via the formation of cyt c/CL complexes with peroxidase activity triggered by H2O2 . Thus cyt c/CL redox interactions and CL peroxidation represent a missing causal link between known reactive oxygen species production and mitochondrial pro-apoptotic responses. Importantly, our preliminary data show that a mitochondria-targeted small molecule inhibitor of CL peroxidation suppressed TBI-induced apoptosis in vivo and preserved cognitive function in postnatal day (PND) 17 rats. In normal mitochondria, CL and cyt c are physically separated: CL is confined almost exclusively to the inner mitochondrial membrane. Binding of cyt c with CL depends on the collapse of CL asymmetry and translocation of CL to the outer mitochondrial membrane. While the mechanisms of collapse of CL asymmetry are poorly understood, preliminary data show that two candidate proteins - nucleoside diphosphate kinase (NDPK-D) and phospholipid scramblase-3 (PLSCR3) -facilitate CL translocation. Our hypothesis is that collapse of CL asymmetry and formation of cyt c/CL peroxidase complexes trigger - in the presence of H2O2 - CL peroxidation and release of proapoptotic factors from mitochondria in immature brain after TBI. Thus, maintaining CL asymmetry and suppression of peroxidase activity will improve neuronal survival and outcome after TBI. To test our hypothesis and its successful translation, we will employ multi-disciplinary approach combining in vitro (mechanical stretch injury of neurons) and in vivo (controlled cortical impact in PND17 rats) TBI models, biochemical (oxidative lipidomics) and biophysical (new protocols for assessment of asymmetry and externalization of CL in mitochondria) methodology, genetic and pharmacological approaches as well as computational modeling and organic chemical synthesis. These studies will provide important mechanistic information on the role of CL asymmetry and oxidation in neuronal apoptosis after TBI. The ability to selectively modulate CL oxidation, a critical early event in apoptosis, could lead to targeted therapies for TBI and ultimately improve outcome for children after brain injury.

Public Health Relevance

Trauma is the leading cause of death in children. Severe traumatic brain injury (TBI) is the most important component of this mortality and associated morbidity. A specific therapy for TBI does not yet exist and standard treatment remains supportive in nature. This project will identify role of oxidized phospholipids in neuronal death after pediatric TBI using state of the art oxidative lipidomics approach. We will test novel therapeutic compounds in experiments to prevent neuronal death, improve outcome after pediatric TBI. The ability to selectively modulate this pathway could lead to targeted therapies for TBI and ultimately improve outcome for children.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS061817-06A1
Application #
8771719
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Bellgowan, Patrick S F
Project Start
2008-07-01
Project End
2019-04-30
Budget Start
2014-07-01
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Type
University-Wide
DUNS #
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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