Copper and Zinc Metallation Status of Copper-Zinc Superoxide Dismutase from Amyotrophic Lateral Sclerosis Transgenic Mice

Abstract

Mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1) cause one form of familial amyotrophic lateral sclerosis (ALS), and metals are suspected to play a pivotal role in ALS pathology. To learn more about metals in ALS, we determined the metallation states of human wild-type or mutant (G37R, G93A, and H46R/H48Q) SOD1 proteins from SOD1-ALS transgenic mice spinal cords. SOD1 was gently extracted from spinal cord and separated into insoluble (aggregated) and soluble (supernatant) fractions, and then metallation states were determined by HPLC inductively coupled plasma MS. Insoluble SOD1-rich fractions were not enriched in copper and zinc. However, the soluble mutant and WT SOD1s were highly metallated except for the metal-bindingregion mutant H46R/H48Q, which did not bind any copper. Due to the stability conferred by high metallation of G37R and G93A, it is unlikely that these soluble SOD1s are prone to aggregation in vivo, supporting the hypothesis that immature nascent SOD1 is the substrate for aggregation. We also investigated the effect of SOD1 overexpression and disease on metal homeostasis in spinal cord cross-sections of SOD1-ALS mice using synchrotron-based x-ray fluorescence microscopy. In each mouse genotype, except for the H46R/H48Q mouse, we found a redistribution of copper between gray and white matters correlated to areas of high SOD1. Interestingly, a diseasespecific increase of zinc was observed in the white matter for all mutant SOD1 mice. Together these data provide a picture of copper and zinc in the cell as well as highlight the importance of these metals in understanding SOD1-ALS pathology.First described in 1869 by French neurologist Jean-Martin Charcot (1), amyotrophic lateral sclerosis (ALS) 4 is defined by the progressive demise of lower and upper motor neurons leading to the degeneration of muscle tissue and eventual paralysis (2). Familial ALS was linked in 1993 to mutations in the gene that encodes the metalloenzyme copper-zinc, superoxide dismutase (SOD1) (3), and since that time more than 140 different disease-causing mutations have been identified (4). Before the discovery linking SOD1 and ALS, epidemiological studies on ALS suggested that exposure to metallic trace elements was a significant factor among many other environmental factors such as viruses, strenuous exercise, and excitotoxic chemicals that may have a role in ALS etiology (4 -6). Studies on exposure to and tissue accumulation of metallic elements in ALS patients show a correlative yet unspecified role for many metals including copper and zinc in SOD1-related ALS (7-19). Over the years, a number of hypotheses relating metal toxicity to ALS have been proposed. For example, abnormal accumulations of redox-active metal ions such as iron or copper in ALS patients were generally believed to be detrimental due to their ability to mediate oxidative damage through participation in reactions that lead to formation of reactive oxygen species. A more specific hypothesis linking metallation levels of mutant SOD1 to...

Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

“…These findings suggest that SOD1 palmitoylation occurs prior to the intramolecular disulfide-bonding step in SOD1 maturation and that palmitoylation is increased under conditions when there is more immature disulfide-reduced SOD1. Importantly, the immature form of SOD1 is known to be unstable (30) and is proposed to be a critical component in the misfolding and toxicity of mtSOD1 (31)(32)(33)(34). Our findings that palmitoylation occurs on disulfidereduced SOD1, is increased for mtSOD1, and correlates with SOD1 membrane association suggest this modification may play a role in the pathogenesis of mtSOD1-induced FALS.…”

Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110

“…The abundant misfolded states are evidently prone to aggregation in vivo, considering the large fluorescent aggregates observed in motor neurons of G85R SOD1YFP mice as early as weaning age, and, as such, provide a substrate for the Hsc70/DnaJ/Hsp110 disaggregation machinery. In contrast, G93A SOD1 substantially populates the native active state, but it also populates nonnative, aggregationprone forms (15,18). This latter mutant form was also affected by the presence of the Hsp110 transgene, judging from the increased survival data, albeit survival was not extended as much as for G85R SOD1YFP.…”

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

“…Therefore, an impaired maturation process would lead to the accumulation of immature SOD1 species, which are prone to oligomerization. Recent studies reported a higher propensity for deficient protein maturation in vivo or in-cell culture models for some fALS mutants 39,40 . However, most of the studies addressing the properties of fALS SOD1 mutants and their effects on the maturation process have been performed in vitro, therefore, far from physiological conditions [41][42][43] .…”