Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice

Mutations of cytosolic Cu/Zn superoxide dismutase 1 (SOD1) in humans and overexpression of mutant human SOD1 genes in transgenic mice are associated with the motor neuron degenerative condition known as amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease). Gain-of-function toxicity from the mutant protein expressed in motor neurons, associated with its misfolding and aggregation, leads to dysfunction and cell death, associated with paralyzing disease. Here, using hydrogen-deuterium exchange in intact mice in vivo, we have addressed whether an ALS-associated mutant protein, G85R SOD1-YFP, is subject to the same rate of turnover in spinal cord both early in the course of the disease and later. We find that the mutant protein turns over about 10-fold faster than a similarly expressed wild-type fusion and that there is no significant change in the rate of turnover as animals age and disease progresses.     

Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to disulfide reduction

We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the beta-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity.     

Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis

More than 100 different mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) cause preferential motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS). Although the cellular target(s) of mutant SOD1 toxicity have not been precisely specified, evidence to date supports the hypothesis that ALS-related mutations may increase the burden of partially unfolded SOD1 species. Influences that may destabilize SOD1 in vivo include impaired metal ion binding, reduction of the intrasubunit disulfide bond, or oxidative modification. In this study, we observed that metal-deficient as-isolated SOD1 mutants (H46R, G85R, D124V, D125H, and S134N) with disordered electrostatic and zinc-binding loops exhibited aberrant binding to hydrophobic beads in the absence of other destabilizing agents. Other purified ALS-related mutants that can biologically incorporate nearly normal amounts of stabilizing zinc ions (A4V, L38V, G41S, D90A, and G93A) exhibited maximal hydrophobic behavior after exposure to both a disulfide reducing agent and a metal chelator, while normal SOD1 was more resistant to these agents. Moreover, we detected hydrophobic SOD1 species in lysates from affected tissues in G85R and G93A mutant but not wildtype SOD1 transgenic mice. These findings suggest that a susceptibility to the cellular disulfide reducing environment and zinc loss may convert otherwise stable SOD1 mutants into metal-deficient forms with locally destabilized electrostatic and zinc-binding loops. These abnormally hydrophobic SOD1 species may promote aberrant interactions of the enzyme with itself or with other cellular constituents to produce toxicity in familial ALS.     

Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause approximately 2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALS-related SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133 Delta) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.     

Abnormal exocytotic release of glutamate in a mouse model of amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.     

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H₂O₂, mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation.     

Altered levels and distribution of microtubule-associated proteins before disease onset in a mouse model of amyotrophic lateral sclerosis

Alterations of the axonal transport and microtubule network are potential causes of motor neurodegeneration in mice expressing a mutant form of the superoxide dismutase 1 (SOD1G37R) linked to amyotrophic lateral sclerosis (ALS). In the present study, we investigated the biology of microtubule-associated proteins (MAPs), responsible for the formation and stabilization of microtubules, in SOD1G37R mice. Our results show that the protein levels of MAP2, MAP1A, tau 100 kDa and tau 68 kDa species decrease significantly as early as 5 months before onset of symptoms in the spinal cord of SOD1G37R mice, whereas decrease in levels of tau 52-55 kDa species is most often noted with the manifestation of the clinical symptoms. Interestingly, there was no change in the protein levels of MAPs in the brain of SOD1G37R mice, a CNS organ spared by the mutant SOD1 toxicity. Remarkably, as early as 5 months before disease onset, the binding affinities of MAP1A, MAP2 and tau isoforms to the cytoskeleton decreased in spinal cord of SOD1G37R mice. This change correlated with a hyperphosphorylation of the soluble tau 52-55 kDa species at epitopes recognized by the antibodies AT8 and PHF-1. Finally, a shift in the distribution of MAP2 from the cytosol to the membrane is detected in SOD1G37R mice at the same stage. Thus, alterations in the integrity of microtubules are early events of the neurodegenerative processes in SOD1G37R mice.     

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.     

Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Delta) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by approximately 1-6 degrees C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at approximately 4-12 degrees C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.     

Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative damage and antioxidant defences: relevance to Down's syndrome and familial amyotrophic lateral sclerosis

Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.     

Stabilization of hyperdynamic microtubules is neuroprotective in amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

Features of wild-type human SOD1 limit interactions with misfolded aggregates of mouse G86R Sod1

Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best-studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.     

Recruitment of mitochondria into apoptotic signaling correlates with the presence of inclusions formed by amyotrophic lateral sclerosis-associated SOD1 mutations

Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.     

Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice

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-binding-region 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 disease-specific 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.     

Mutant SOD1-induced neuronal toxicity is mediated by increased mitochondrial superoxide levels

Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease in adults, is characterized by the selective degeneration and death of motor neurons leading to progressive paralysis and eventually death. Approximately 20% of familial ALS cases are associated with mutations in SOD1, the gene encoding Cu/Zn-superoxide dismutase (CuZnSOD). Previously, we reported that overexpression of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD or SOD2) attenuates cytotoxicity induced by expression of the G37R-SOD1 mutant in a human neuroblastoma cell culture model of ALS. In the present study, we extended these earlier findings using several different SOD1 mutants (G93C, G85R, and I113T). Additionally, we tested the hypothesis that mutant SOD1 increases mitochondrial-produced superoxide (O(2) (*)) levels and that SOD2 overexpression protects neurons from mutant SOD1-induced toxicity by reducing O(2) (*) levels in mitochondria. In the present study, we demonstrate that SOD2 overexpression markedly attenuates the neuronal toxicity induced by adenovirus-mediated expression of all four SOD1 mutants (G37R, G93C, G85R, or I113T) tested. Utilizing the mitochondrial-targeted O(2) (*)-sensitive fluorogenic probe MitoSOX Red, we observed a significant increase in mitochondrial O(2) (*) levels in neural cells expressing mutant SOD1. These elevated O(2) (*) levels in mitochondria were significantly diminished by the overexpression of SOD2. These data suggest that mitochondrial-produced O(2) (*) radicals play a critical role in mutant SOD1-mediated neuronal toxicity and implicate mitochondrial-produced free radicals as potential therapeutic targets in ALS.     

Structural properties and neuronal toxicity of amyotrophic lateral sclerosis-associated Cu/Zn superoxide dismutase 1 aggregates

The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death.     

A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo

Mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis. 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 three disease-causing and four 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.