Over 60 different dominant missense mutations to the Cu, Zn superoxide dismutase gene are associated with motor neuron death in amyotrophic lateral sclerosis (ALS). The apparent gain-of-function conferred by these SOD mutations remains elusive. Four broad theories have been proposed to account for the gain-of-function: an amyloid effect due to aberrant protein folding unrelated to free radicals; toxicity due to reactions of SOD with hydrogen peroxide; the loss of zinc leading to altered redox reactions by SOD; and increased tyrosine nitration. Our preliminary data suggests that zinc-deficient SOD causes increased tyrosine nitration and apoptosis in motor neurons. In the present application, we propose to test these four general theories utilizing new lines of ALS- SOD transgenic mice where mutant ALS SOD expression is controlled by a tetracycline- inducible promoter. The inducible expression will allow us to determine how long expression of mutant SOD is necessary to induce motor neuron death and whether down regulating expression of ALS SOD allows motor neurons to be rescued. Other transgenic lines expressing ALS-SODs with additional mutations to eliminate zinc and copper binding will be made to determine the roles of these metals in the development of motor neuron disease. We have developed novel assays to measure formation of hydrogen peroxide, accumulation of zinc-deficient SOD and tyrosine nitration in vivo, which will be used to determine whether expression of these mutant SODs affects these factors as mice develop disease. From in vitro expression experiments, we have discovered that one cysteine residue renders the ALS-SOD mutant proteins vulnerable to aggregation. We will determine whether mutation of this cysteine residue increases or decreases the toxicity of ALS-SODs in transgenic mice and how it affects intracellular aggregation. These experiments will critically test the contributions of protein aggregation, metal ions and oxidative stress in SOD-induced degeneration of motor neurons in vivo.
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