Amyotrophic Lateral Sclerosis (ALS or MND) is a lethal neurodegenerative disease characterized by the progressive death of motor neurons. Mutations to Cu, Zn Superoxide Dismutase (SOD1) remain the only known cause in humans and transgenic animals, inducing neurotoxicity by an unknown mechanism. The only clues as to how and why the enzyme is selectively toxic to motor neurons come from work with recombinant enzymes. The properties which give rise to the "gain of function" toxicity of SOD1 mutants has yet to be identified, however, mounting evidence suggests that Zn binding and labile cysteine residues play key roles. The goal of this project was to characterize a mutant SOD1 (hS0D1G93A) purified from transgenic rat spinal cords as a function of disease onset and progression, and test these proposed properties in vivo . This required developing new methodology to purify the enzyme in such a way to preserve its native properties. This was accomplished using a novel HPLC purification method that yielded > 99% pure enzyme. In addition, new experimental protocols were designed and optimized to determine the SOD1 specific activity, the content of Cu, Zn, and reduced thiols, all as a function of disease severity. Along with the first report of specific activity, status of the Cu, Zn and thiol content for in vivo expressed SOD1, these results revealed that a substantial fraction of SOD1 was Zn-deficient in vivo which increases with disease progression. This finding has potential implications in ALS as Zn-deficient SOD1 was demonstrated to be both pro-oxidant and pro-aggregatory in vitro. Thus, these results provide both proof of concept for a novel method of measuring the in vivo metallation state of Zn-binding proteins, and support the fundamental hypothesis that Zn-deficient SOD1 correlates with the severity of motor neuron disease. To summarize, these results identified Zn-deficient SOD1 as a primary toxic species or toxic intermediate in causing ALS and thus, could be a novel target for therapeutic intervention.
|Advisor:||Crow, John P.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 70/04, Dissertation Abstracts International|
|Subjects:||Neurosciences, Pharmacology, Biochemistry|
|Keywords:||Amyotrophic lateral sclerosis, G93A mutant SOD1, HPLC, Neurotoxic, Superoxide dismutase, Wild-type SOD1, Zinc|
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