We hypothesize that a major cause of aging is damage to mitochondrial, cytosolic and nuclear functions resulting from the toxicity of mitochondrially-generated reactive oxygen species (ROSs). The mitochondria generate O2.- as a toxic by-product of oxidative phosphorylation (OXPHOS). Mitochondrial 02.- is converted to H2O2 by mitochondrial manganese superoxide dismutase (MnSOD) and mitochondrial H2O2 is converted to water by glutathione peroxidase and catalase. To test our hypothesis, we propose to increase the mitochondrial anti-oxidant defenses in mice by inserting inducible anti-oxidant enzymes into either the constitutive ROSA26 locus or into a human GAPDH BAC. The anti-oxidant transgenes will include a mitochondrially-targeted human catalase cDNA (mCAT), a cytosol-specific human catalase cDNA (cCAT) or a combination of mCAT plus the mouse MnSOD cDNAs, separated by a internal ribosome entry site (IRES) (mCAT + MnSOD). Initially, transgenic constructs will be kept off by separating the ROSA26 or GAPDH promoters from the anti,oxidant cDNAs by a transcriptional stop signal, flanked by LoxP sites. Mice with the anti-oxidant cassettes will be combined with transgenes encoding chemically inducible Cre recombinase. The double transgenic animals will then be induced to recombine out the transcriptional stop signal at three months of age causing life long expression of the anti-oxidant transgenes. The tissue distribution, subcellular distribution and level of expression of the transgenes will be confirmed biochemically, and then the induced and uniduced animals will be monited for changes in phenotype, mitochondrial OXPHOS, mitochondrial ROS production, sensitization of the mitochondrial permeability transition pore (mtPTP) and apoptosis, accumulation of mitochondrial DNA (mtDNA) mutations, and changes in MITOCHIP mitochondrial gene expression profiles at 5 months, 18 months and 28 months of age. These mitochondrial parameters will then be correlated with differences in longevity between the induced and uninduced animals. If increased mitochondrial anti-oxidant defenses (mCAT and mCAT + MnSOD) preserve mitochondrial functions and extends mouse life span to a greater extent than increased cytosolic anti-oxidant defenses (cCAT), then this will provide strong evidence that mitochondrial ROS toxicity is an important factor in aging.
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