MODELS FOR EXPLORING FREE RADICAL DAMAGE

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Superoxide and hydrogen peroxide are produced during normal metabolism. However, when produced in excess, the oxidant species can be converted by reaction with free iron to reactive hydroxyl radicals which can degrade DNA, proteins, and membrane lipids resulting in cellular degeneration. The brain is particularly susceptible to free-radical damage due to its unique cellular membrane composition, and its high levels of oxygen metabolism and free iron. Brain cells have developed several defense against these oxidant species including production of antioxidant enzymes and storage of iron in forms that will not catalyze formation of reactive radicals. Specifically, the antioxidant enzyme superoxide dismutase reduces superoxide to hydrogen peroxide, which is in turn reduced by either glutathione peroxidase or catalase to H20. Iron, in turn, is normally bound to ferritin, the major iron storage molecule in the brain. However, when balance in the brain is disturbed and free-radicals are allowed to accumulate, this can lead to iron- catalyzed production of hydroxyl radical and subsequent cell damage. It has been postulated that oxidative stress may play a role in the pathogenesis of neurodegenerative diseases like Parkinson Disease (PD) and cellular degeneration during normal aging. To examine the role of antiodixants in preventing neuronal cell damage, transgenic mice will be create which over-express the iron-binding protein ferritin, or the anti- oxidant enzymes glutathione peroxidase or catalase in neurons using neuron-specific promoters. Constructs will be evaluated for their ability to confer transgene activity on cultured cells by Northern analysis, immunocytochemistry, enzyme assays, and for sensitivity to iron, MPTP, 6-OHDA, and H202. Following injection into mouse embryos, genomic insertion of constructs will be confirmed by PCR and Southern blot analysis. Transgenic mice will be bred to create new lines and progeny examined for transgene expression in brain by Northern analysis, in situ hybridization, immunocytochemistry, and enzyme assays. Transgenic lines will be evaluated during normal aging for changes in lipid peroxidation by MDA accumulation, protein oxidation by glutamine synthetase activity, oxidative stress as assessed by GSH/GSSG ratios, and for cell loss or atrophy as measured by cell number and sizes in the substantia nigra using tyrosine hydroxylase immunocytochemistry compared with normal controls. Levels of striatal dopamine and its metabolite, DOPAC, tyrosine hydroxylase activity, 3H-dopamine binding, and dopaminergic cell numbers and sizes will be assessed in transgenic compared to controls following MPTP injection. Changes in brain morphology during development, and in protecting against the effects of oxidative stress in the brain, exploring the hypothesis that a genetic increase in one of these molecules may be involved in predisposition to or protection against PD or neuronal degeneration during aging.