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...

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide.Mutations in the gene encoding superoxide dismutase 1 (SOD1) 7 induce familial amyotrophic lateral sclerosis (ALS), and aggregation of the mutant SOD1 protein is hypothesized to cause pathogenesis (1). Support for the aggregation hypothesis includes pathological, cell-culture, and biophysical data such as: 1) the appearance of fibrillar, spherical or irregularly shaped SOD1-containing aggregates in murine and human ALS spinal cord (2-8); 2) the formation of detergent-insoluble SOD1 aggregates with the onset of motor neuron degeneration in ALS mice (2, 9 -12); 3) the observation that cultured neural cells that form SOD1 aggregates die faster than cells that do not (13); and 4) various biophysical and structural perturbations to ALS variant SOD1 proteins (14 -18) that accelerate aggregation in vitro (1).Although the aggregation of SOD1 is a conspicuous signature of SOD1-linked ALS, the protein composition of SOD1 aggregates remains unclear. Identifying proteins that might be co-aggregated with SOD1 could help explain how the aggregates are formed and why they are apparently toxic. For example, the expression of ALS-SOD1 variants in mice and cell cultures can induce: the slowing of axonal transport (19), glutamate excitotoxicity (20,21), and proteasomal inhibition (22,23). Data also suggest that variant SOD1 proteins aggregate with other proteins and lower the concentration of these proteins below a threshold for normal viability (24 -27). Therefore, the identification of binding interactions or co-aggregation between SOD1 and specific proteins involved in any of these processes could provide clues about the cellular processes involv...

Mutations in SOD1 cause FALS. The Cu binding capacity of SOD1 has spawned hypotheses that implicate metal-mediated production of reactive species as a potential mechanism of toxicity. In past experiments, we have tested such hypotheses by mutating residues in SOD1 that normally coordinate the binding of Cu, finding that such mutants retain the capacity to induce motor neuron disease. We now describe the lack of disease in mice that express a variant of human SOD1 in which residues that coordinate the binding of Cu and Zn have been mutated (SODMD). SODMD encodes 3 disease-causing and 4 experimental mutations that ultimately eliminate all histidines involved in the binding of metals; and includes one disease-causing and one experimental mutation that eliminate secondary metal binding at C6 and C111. We show that the combined effect of these mutations produces a protein that is unstable but does not aggregate on its own, is not toxic, and does not induce disease when co-expressed with high levels of wild-type SOD1. In cell culture models, we determine that the combined mutation of C6 and C111 to G and S, respectively, dramatically reduces the aggregation propensity of SODMD and may account for the lack of toxicity for this mutant.

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce misfolding and aggregation of the protein with the inherent propensity of mutant SOD1 to aggregate generally correlating, with a few exceptions, to the duration of illness in patients with the same mutation. One notable exception was the D101N variant, which has been described as wild-type-like. The D101N mutation is associated with rapidly progressing motor neuron degeneration but shows a low propensity to aggregate. By assaying the kinetics of aggregation in a well characterized cultured cell model, we show that the D101N mutant is slower to initiate aggregation than the D101G mutant. In this cell system of protein over-expression, both mutants were equally less able to acquire Zn than WT SOD1. Additionally, both of these mutants were equivalently less able to fold into the trypsin-resistant conformation that characterizes WT SOD1. A second major difference between the two mutants was that the D101N variant more efficiently formed a normal intramolecular disulfide bond. Overall, our findings demonstrate that the D101N and D101G variants exhibit clearly distinctive features, including a different rate of aggregation, and yet both are associated with rapidly progressing disease.

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis (ALS), is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with ALS mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In the present study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time-stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly-made protein, are prone to misfolding and aggregation.