This proposal is submitted to pursue our investigation of the pathogenesis of amyotrophic lateral sclerosis (ALS) using transgenic mice expressing the glycine-93 yields arginine mutant copper/zinc superoxide dismutase (SOD1G93A). Pertinent to this goal, first, we have shown that inducible nitric oxide synthase (iNOS) is upregulated in glial cells in the spinal cords of affected transgenic SOD1G93A mice. To elucidate the role of iNOS in this model of ALS, Specific Aim (SA)-I will determine the effect of iNOS inhibition or ablation on SOD1G93A-mediated neurodegeneration. Second, we have evidence that the production of the highly-reactive tissue damaging species hypochlorous acid is increased in the spinal cords of affected transgenic SOD1G93A mice. To acquire a better understanding the cytotoxic role of hypochlorous acid in this model of ALS, SA-II will quantify spinal cord levels of chlorotyrosine and nitrotyrosine, the two main fingerprints of hypochlorous acid-induced protein oxidative attack, at different disease stages, in different lines of transgenic mice that express either mutant or wild-type SOD1 and, in transgenic SOD1G93A mice after ablation of neuronal NOS (nNOS), iNOS, or myeloperoxidase (MOP), which is the only mammalian enzyme which produced hypochlorous acid. To explore further the role of MPO. SA-III will (1) define spinal cord expression of MPO mRNA and protein, as in SA-II, at different disease stages and transgenic lines; and (2) assess the effect of MPO ablation on SOD1G93A-mediated neurodegeneration. Third, we have observed that cyclooxygenase-2 (Cox-2), a key enzyme in the synthesis of the pro-inflammatory prostaglandin PGE2, is also markedly increased in the spinal cord of affected transgenic SOD1G93A mice. To demonstrate whether Cox-2 upregulation plays a role in SOD1G93A-mediated neurodegeneration, SA-IV will (1) characterize spinal cord Cox-2 mRNA and protein expression, and PGE2 content, as in SA-II, at different disease stages, transgenic lines, and in transgenic SOD1G93A mice after ablation of iNOS; and (2) assess the effects of Cox-2 inhibition or ablation on SOD1G93A-mediated neurodegeneration. This proposal contains a comprehensive set of experiments, which should provide insights into the role of iNOS, hypochlorous acid and its synthesizing enzyme MPO, as well as into Cox-2 in transgenic SOD1G93A mice. It should also shed light onto the mechanisms of neurodegeneration in ALS.
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