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The detection of superoxide and implications for amyotrophic lateral sclerosis Public Deposited

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  • Mutations to superoxide dismutase were the first proven cause of Lou Gehrig’s disease (amyotrophic lateral sclerosis; ALS) implicating superoxide in the selective death of motor neurons that characterizes ALS. Nitric oxide competes effectively with superoxide dismutase for superoxide to form the powerful oxidant peroxynitrite. Endogenous formation of peroxynitrite can kill motor neurons in vitro and has been implicated in the pathogenesis of ALS in vivo. To further investigate the role of superoxide and peroxynitrite in the pathogenesis of ALS, several new approaches were developed. First, the synthesis of peroxynitrite from nitrite and hydrogen peroxide was simplified to provide a stable source for in vitro experimentation. Second, the products from the peroxynitrite-mediated oxidation of the antioxidant, urate, were determined. Urate is an efficient inhibitor of radicals derived from peroxynitrite without scavenging peroxynitrite directly. Radicals derived from peroxynitrite were found to oxidize urate to the ring-opened product, triuret, which helps explain the previously reported formation of aminocarbonyl radicals from urate. Urate is protective in cell culture models of ALS and in vivo in the treatment of stroke and muscular dystrophy. Unfortunately, oral administration of urate failed to inhibit disease progression in a mouse ALS model. The third new approach was the development of a method to measure superoxide in vivo, overcoming current limitations of specificity, sensitivity and intracellular access. The assay is based upon the newly discovered reaction of superoxide with the hydroethidine radical to form a transient peroxide that spontaneously decomposes to leave a hydroxyl group. The hydroxyl product can be selectively detected by fluorescence using 396 nm excitation. This novel excitation wavelength is advantageous to the current practice of 500 nm excitation which detects non-specific oxidation. Comparison of fluorescence using both excitations can provide a useful ratio of superoxide-dependent versus non-specific oxidation. Furthermore, covalent modification with the triphenylphosphonium cation targets the dye to mitochondria, providing direct measurement of superoxide in mitochondria. The method we developed revealed that mitochondrially-generated superoxide was increased in astrocytes expressing the ALS-associated mutation, SODG93A. These new tools allow superoxide generation to be measured in ALS models and provide evidence that free radicals killed Lou Gehrig.
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