is a critical early event in apoptotic cell death. Cyt c is bound to the inner mitochondrial membrane by its association with cardiolipin (CL), an anionic phospholipid found exclusively in the inner mitochondrial membrane of eukarydtic cells. Recently we have shown novel redox catalytic properties of cyt c realized though its interactions with CL and PS resulting in their selective oxidation. The resulting products, CL and PS hydroperoxides act as important signals in two apoptotic pathways - regulation of release of apoptotic factors from mitochondria into cytosol, and externalization of PS marking apoptotic cells for phagocytosis. Our hypothesis is that TBI initiates excessive production of ROS and oxidation of CL catalyzed by CL/cyt c complex, which is required for the release of pro-apoptotic factors from mitochondria. CL oxidation is catalyzed by a pool of cyt c that is tightly bound to inner mitochondrial membrane by its complex with CL in neurons. As a consequence, we predict that TBI induced CL oxidation and apoptosis can be prevented by antioxidant strategies and treatments decreasing susceptibility of CL to oxidation by dietary manipulation of its fatty acid residues. In this proposal we will use controlled cortical impact (CCI) model of TBI in post-natal day (PND) 17 rats coupled with in vitro studies to test these specific hypotheses and address the following specific aims: 1) Determine the degree, spatial and temporal pattern of ROS production, antioxidant depletion and CL oxidation in immature brain after TBI and in neurons after glutamate exposure. 2) Determine the potential of antioxidants and dietary manipulation to inhibit mitochondrial CL oxidation and protect against apoptosis in immature brain after TBI and in neurons after glutamate exposure. These studies will employ the newly developing technology of oxidative lipidomics to provide important mechanistic information on the role of cyt c -CL interactions in neuronal apoptosis after pediatric TBI in an experimental model. Relevant to the specific aims of this proposal in vivo studies linking overall CL oxidation with cyt c release and apoptosis have been lacking. The ability to selectively modulate Cyt c release could lead to targeted therapies for TBI and ultimately improve outcome for children. Project Description Page 6
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