The Disulfide Bond,but Not Zinc or Dimerization,Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of post-translational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57–Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.     

Species-dependent neuropathology in transgenic SOD1 pigs

Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.     

Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.     

Human SOD1 before harboring the catalytic metal: solution structure of copper-depleted, disulfide-reduced form

SOD1 has to undergo several post-translational modifications before reaching its mature form. The protein requires insertion of zinc and copper atoms, followed by the formation of a conserved S-S bond between Cys-57 and Cys-146 (human numbering), which makes the protein fully active. In this report an NMR structural investigation of the reduced SH-SH form of thermostable E,Zn-as-SOD1 (E is empty; as is C6A, C111S) is reported, characterizing the protein just before the last step leading to the mature form. The structure is compared with that of the oxidized S-S form as well as with that of the yeast SOD1 complexed with its copper chaperone, CCS. Local conformational rearrangements upon disulfide bridge reduction are localized in the region near Cys-57 that is completely exposed to the solvent in the present structure, at variance with the oxidized forms. There is a local disorder around Cys-57 that may serve for protein-protein recognition and may possibly be involved in intermolecular S-S bonds in familial amyotrophic lateral sclerosis-related SOD1 mutants. The structure allows us to further discuss the copper loading mechanism in SOD1.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys(6)- and Cys(111)-disulfide cross-linked form and targeted it for proteasomal degradation.     

Cysteine 111 affects aggregation and cytotoxicity of mutant Cu,Zn-superoxide dismutase associated with familial amyotrophic lateral sclerosis

Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.     

Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

Modulation of mutant superoxide dismutase 1 aggregation by co-expression of wild-type enzyme

Mutations in superoxide dismutase 1 (SOD1, EC 1.15.1.1) cause familial amyotrophic lateral sclerosis; with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop amyotrophic lateral sclerosis-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared with mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, hSOD1 or mouse SOD1, delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mouse SOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates the aggregation of mutant SOD1.     

Using yeast models to probe the molecular basis of amyotrophic lateral sclerosis

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative disease attributable to the death of motor neurons. Associated with ALS are mutations in the genes encoding SOD1 (superoxide dismutase 1), FUS (fused in Sarcoma) protein and TDP-43 (TAR DNA-binding protein-43) each of which leads to aggregation of the respective protein. For example, the ALS-associated mutations in the hSOD1 (human SOD1) gene typically destabilize the native SOD homodimer, leading to misfolding, aggregation and degradation of SOD1. The ALS-associated pathology is not a consequence of the functional inactivation of SOD1 itself, but is rather due to a toxic gain-of-function triggered by mutant SOD1. Recently, the molecular basis of a number of human neurodegenerative diseases resulting from protein misfolding and aggregation, including fALS (familial ALS), was probed by using the baker's yeast, Saccharomyces cerevisiae, as a highly tractable model. Such studies have, for example, identified novel mutant SOD1-specific interactions and demonstrated that mutant SOD1 disrupts mitochondrial homoeostasis. Features of ALS associated with TDP-43 aggregation have also been recapitulated in S. cerevisiae including the identification of modulators of the toxicity of TDP-43. In this paper, we review recent studies of ALS pathogenesis using S. cerevisiae as a model organism and summarize the potential mechanisms involved in ALS progression.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

Fully metallated S134N Cu,Zn-superoxide dismutase displays abnormal mobility and intermolecular contacts in solution

S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metal-deficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.     

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status

The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis.     

Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Absence of Nrf2 or Its Selective Overexpression in Neurons and Muscle Does Not Affect Survival in ALS-Linked Mutant hSOD1 Mouse Models

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1^G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.     

Cysteine to Serine Conversion at 111th Position Renders the Disaggregation and Retains the Stabilization of Detrimental SOD1 A4V Mutant Against Amyotrophic Lateral Sclerosis in Human—A Discrete Molecular Dynamics Study

Protein aggregation is a hallmark of various neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) in humans. Mutations in Cu/Zn superoxide dismutase (SOD1) protein were found to be a prominent cause behind the majority of the familial ALS cases with abnormal protein aggregates. Herein, we report the biophysical characterization of the beneficial mutation C111S that stabilizes the SOD1 harboring A4V mutation, one of the most lethal diseases causing mutant that leads to protein destabilization and aggregation. In this study, we utilized discrete molecular dynamics (DMD) simulations, which stipulated an outlook over the systematic action of C111S mutation in the A4V mutant that stabilizes the protein and impedes the formation of protein aggregation. Herewith, the findings from our study manifested that the mutation of C111S in SOD1 could aid in regaining the protein structural conformations that protect against the formation of toxic aggregates, thereby hindering the disease pathogenicity subtly. Hence, our study provides a feasible pharmaceutical strategy in developing the treatment for incurable ALS affecting the mankind.