Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament-L

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO⁻), which may be the damaging gain-of-function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD-catalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

Rapid Communication: Cu/Zn Superoxide Dismutase Activity in Familial and Sporadic Amyotrophic Lateral Sclerosis

Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that is inherited as an autosomal dominant trait in ~ 10% of cases. Recently we and others identified several single-base mutations in the Cu/Zn superoxide dismutase (SOD1) gene in patients with familial ALS (FALS). Using single-strand conformational polymorphism, we studied the C to G mutation in exon 2 of the SOD1 gene (resulting in a leucine to valine substitution in position 38) in affected and unaffected members of a large Belgian family with FALS. We measured the SOD1 activity in red blood cell lysates in 14 members of this family, including the only surviving clinically affected patient. SOD1 activity of the family members carrying the mutation was less than half that of members without the mutation. In addition, in 11 patients with sporadic ALS and 11 age- and sex-matched controls, red blood cell SOD1 activity was normal. These studies indicate that SOD1 activity is reduced in these FALS patients but not in sporadic ALS patients. Moreover, this SOD1 enzyme abnormality is detectable years before onset of clinical ALS in carriers of this FALS mutation.     

Normal Binding and Reactivity of Copper in Mutant Superoxide Dismutase Isolated from Amyotrophic Lateral Sclerosis Patients

Abstract: In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn-superoxide dismutase. The mutant CuZn-superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active-site Cu ions. Asp⁹⁰Ala mutant CuZn-superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp⁹⁰Ala mutant CuZn-superoxide dismutase was 45 Da lower than that of the wild-type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn-superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild-type enzyme in enzymic activity, in content and affinity for active-site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn-superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active-site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.     

Superoxide Dismutase Activity, Oxidative Damage, and Mitochondrial Energy Metabolism in Familial and Sporadic Amyotrophic Lateral Sclerosis

The cause of neuronal death in amyotrophic lateral sclerosis (ALS) is unknown. Recently, it was found that some patients with autosomal-dominant familial ALS (FALS) have point mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1). In this study of postmortem brain tissue, we examined SOD activity and quantified protein carbonyl groups, a marker of oxidative damage, in samples of frontal cortex (Brodmann area 6) from 10 control patients, three FALS patients with known SOD1 mutations (FALS-1), one autosomal-dominant FALS patient with no identifiable SOD1 mutations (FALS-0), and 11 sporadic ALS (SALS) patients. Also, we determined the activities of components of the electron transport chain (complexes I, II-III, and IV) in these samples. The cytosolic SOD activity, which is primarily SOD1 activity, was reduced by 38.8% (p < 0.05) in the FALS-1 patients and not significantly altered in the SALS patients or the FALS-0 patient relative to the control patients. The mitochondrial SOD activity, which is primarily SOD2 activity, was not significantly altered in the FALS-1, FALS-0, or SALS patients. The protein carbonyl content was elevated by 84.8% (p < 0.01) in the SALS patients relative to the control patients. Finally, the complex I activity was increased by 55.3% (p < 0.001) in the FALS-1 patients relative to the control patients. These results from cortical tissue demonstrate that SOD1 activity is reduced and complex I activity is increased in FALS-1 patients and that oxidative damage to proteins is increased in SALS patients.     

Increased Superoxide Dismutase Activity Improves Survival of Cultured Postnatal Midbrain Neurons

Abstract: Copper/zinc superoxide dismutase (Cu/Zn-SOD) is a major free radical scavenging enzyme. Increased Cu/Zn-SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn-SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn-SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn-SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn-SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn-SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn-SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn-SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn-SOD activity does not seem to play a critical role in determining the mode of cell death in this culture system.     

Evidence of Increased Oxidative Damage in Both Sporadic and Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal dominant familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1), suggesting that oxidative damage may play a role in ALS pathogenesis. To further investigate the biochemical features of FALS and sporadic ALS (SALS), we examined markers of oxidative damage to protein, lipids, and DNA in motor cortex (Brodmann area 4), parietal cortex (Brodmann area 40), and cerebellum from control subjects, FALS patients with and without known SOD mutations, SALS patients, and disease controls (Pick's disease, progressive supranuclear palsy, diffuse Lewy body disease). Protein carbonyl and nuclear DNA 8-hydroxy-2'-deoxyguanosine (OH⁸dG) levels were increased in SALS motor cortex but not in FALS patients. Malondialdehyde levels showed no significant changes. Immunohistochemical studies showed increased neuronal staining for hemeoxygenase-1, malondialdehyde-modified protein, and OH⁸dG in both SALS and FALS spinal cord. These studies therefore provide further evidence that oxidative damage may play a role in the pathogenesis of neuronal degeneration in both SALS and FALS.     

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.     

Effects of Wild-Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l-DOPA-Induced Toxicity in Postnatal Midbrain Culture

Abstract: Mutations in the free radical-scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn-SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild-type (WT SOD) and mutant (Gly⁹³→ Ala; G93A) Cu/Zn-SOD enzyme on the fate of postnatal midbrain neurons. One-week-old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non-transgenic cultures. In contrast, 1-week-old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l -DOPA. l -DOPA at 200 µ M caused ∼50% loss of tyrosine hydroxylase (TH)-positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µ M l -DOPA did not cause any significant reductions in TH-positive or GABA neuron numbers in transgenic WT SOD cultures. l -DOPA at 50 µ M had opposite effects, in that it significantly increased TH-positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l -DOPA-induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn-SOD enzyme have inverse effects. As the spontaneous loss and l -DOPA-induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn-SOD enzyme kills cells by oxidative stress.     

Elevated "Hydroxyl Radical" Generation In Vivo in an Animal Model of Amyotrophic Lateral Sclerosis

Abstract: Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

Aberrant Copper Chemistry as a Major Mediator of Oxidative Stress in a Human Cellular Model of Amyotrophic Lateral Sclerosis

We have investigated the response to oxidative stress in a model system obtained by stable transfection of the human neuroblastoma cell line SH-SY5Y with plasmids directing constitutive expression of either wild-type human Cu,Zn superoxide dismutase or a mutant of this enzyme (H46R) associated with familial amyotrophic lateral sclerosis. We report that expression of mutant H46R Cu,Zn superoxide dismutase induces a selective increase in paraquat sensitivity that is reverted by addition of D-penicillamine. Furthermore, expression of this mutant enzyme affects the activity of the endogenous wild-type enzyme both in basal conditions and in copper overloading experiments. Our data indicate that aberrant metal chemistry of this mutant enzyme is the actual mediator of oxidative stress and that concurrent impairment of the activity of wild-type endogenous enzyme compromises the cell's ability to respond to oxidative stress.     

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

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.     

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

Molecular Analyses of the Cu/Zn Superoxide Dismutase Gene in Patients with Familial Amyotrophic Lateral Sclerosis (ALS) in Japan

1. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by selective damage to the neural system that mediates voluntary movement. Although the pathophysiologic process of ALS remains unknown, about 5 to 10% of cases are familial. According to genetic linkage studies, the familial ALS (FALS) gene has been mapped on chromosome 21 in some families and recent work identified some different missense mutations in the Cu/Zn superoxide dismutase gene in FALS families.2. We recently identified five mutations in six FALS families. The mutations identified in our FALS families are H46R, L84V, I104F, S134N, and V148I. The H46R mutation that locates in the active site of Cu/Zn SOD gene is associated with two Japanese families with very slow progression of ALS. On the other hand, the L84V mutation associated with a rapidly progressive loss of motor function with predominant lower motor neuron manifestations.3. In the family with the V148I, the phenotype of the patient varied very much among the affected members. One case had weakness of the lower extremities at first and died without bulbar paresis. The second case first noticed wasting of the upper limbs with bulbar symptoms, but the third had weakness of upper extremities without developing dysarthria nor dysphagia until death. These mutations account for 50% of all FALS families screened, although Cu/Zn SOD gene mutations are responsible for less than about 13–21% in the Western population.4. Our results indicate that the progression of disease with mutations of Cu/Zn SOD is well correlated with each mutation. The exact mechanism by which the abnormal Cu/Zn SOD molecules selectively affect the function of motor neurons is still unknown.     

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.     

Motor neuronal protection by l-arginine prolongs survival of mutant SOD1 (G93A) ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to motor neuron degeneration. Despite the fact that many different therapeutic strategies have been applied to prevent disease progression, no cure or effective therapy is currently available for ALS. We found that l-arginine protects cultured motor neurons from excitotoxic injury. We also found that l-arginine supplementation both prior to and after the onset of motor neuron degeneration in mtSOD1 (G93A) transgenic ALS mice significantly slowed the progression of neuropathology in lumbar spinal cord, delayed onset of motor dysfunction, and prolonged life span. Moreover, l-arginine treatment was associated with preservation of arginase I activity and neuroprotective polyamines in spinal cord motor neurons. Our findings show that l-arginine has potent in vitro and in vivo neuroprotective properties and may be a candidate for therapeutic trials in ALS.