The involvement of metal ions in copper-zinc superoxide dismutase related amyotrophic lateral sclerosis
Bourassa, Megan Whitney
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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the motor neurons of the spinal cord. While most ALS cases are sporadic with no known cause, some are linked to mutations in the antioxidant protein copper-zinc superoxide dismutase (SOD1), which is associated with the formation of small SOD1 aggregates in motor neurons. While the aggregation of SOD1 has been studied extensively in vitro, much less is known about aggregate-formation and its effects on metal homeostasis and neurodegeneration in intact cells and tissues. Therefore, the goal of this thesis was to understand how SOD1 aggregation plays a role in metal homeostasis and oxidative damage in SOD1-ALS by combining high-resolution X-ray and infrared imaging methodologies with both mouse and cell culture models of the disease. Results showed that the SOD1 aggregates were unmetallated within the cells, suggesting that aggregate formation occurs with the nascent protein and prior to delivery of copper and zinc to the enzyme's active site. It has been suggested that aggregation protects the cells by preventing cytotoxic reactions arising from improperly bound metal in soluble SOD1 mutants. Cells containing SOD1 aggregates also showed a reduced intracellular copper concentration, which is consistent with the inability of the misfolded protein to be metallated by copper chaperone proteins. In contrast, the copper content in the spinal cords of a SOD1-ALS mouse model was dependent upon the ability of the SOD1 mutant to bind metal, i.e. only mutations that permitted copper-binding showed elevated copper in the spinal cord. This suggests that there could be more soluble metallated SOD1 in the tissues than in the cells, which quickly form dense unmetallated aggregates, and would account for the difference between the two models. Interestingly, the copper levels in the spinal cord tissue were not correlated with increased oxidative stress, as most spinal cords showed evidence of oxidative damage. One exception was G37R, which is a mutation with a protected copper active site and increased aggregation propensity, both of which reduce aberrant forms of SOD1 that can contribute to oxidative damage. Thus, this research identifies complex relationships in the disease pathology, where soluble forms of mutant SOD1 can lead to damaging redox reactions due to improper copper binding whereas SOD1 aggregation prevents the binding of copper, essentially neutralizing the redox toxicity of the mutant SOD1. In both cells and tissues, SOD1-ALS is associated with altered copper homeostasis, which can affect other antioxidant mechanisms beyond SOD1. This new understanding of aggregate formation, metal homeostasis, and oxidative damage in ALS could lead to improved treatment options and ultimately a cure.