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Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Increasing evidence suggests that propagation of the motor neuron disease amyotrophic lateral sclerosis (ALS) involves the pathogenic aggregation of disease-associated proteins that spread in a prion-like manner. We have identified two aggregate strains of human superoxide dismutase 1 (hSOD1) that arise in the CNS of transgenic mouse models of SOD1-mediated ALS. Both strains transmit template-directed aggregation and premature fatal paralysis when inoculated into the spinal cord of adult hSOD1 transgenic mice. This spread of pathogenic aggregation could be a potential target for immunotherapeutic intervention. Here we generated mouse monoclonal antibodies (mAbs) directed to exposed epitopes in hSOD1 aggregate strains and identified an aggregate selective mAb that targets the aa 143–153 C-terminal extremity of hSOD1 (αSOD1143–153). Both pre-incubation of seeds with αSOD1143–153 prior to inoculation, and weekly intraperitoneal (i.p.) administration attenuated transmission of pathogenic aggregation and prolonged the survival of seed-inoculated hSOD1G85R Tg mice. In contrast, administration of a mAb targeting aa 65–72 (αSOD165–72), which exhibits high affinity towards monomeric disordered hSOD1, had an adverse effect and aggravated seed induced premature ALS-like disease. Although the mAbs reached similar concentrations in CSF, only αSOD1143–153 was found in association with aggregated hSOD1 in spinal cord homogenates. Our results suggest that an aggregate-selective immunotherapeutic approach may suppress seeded transmission of pathogenic aggregation in ALS. However, long-term administration of αSOD1143–153 was unable to prolong the lifespan of non-inoculated hSOD1G85R Tg mice. Thus, spontaneously initiated hSOD1 aggregation in spinal motor neurons may be poorly accessible to therapeutic antibodies. Originally published in thesis in manuscript form with title: "An aggregate-selective monoclonal antibody attenuates seeded but not spontaneously evolving SOD1 aggregation in ALS model mice" and authors: "Manuela Lehmann, Matthew Marklund, Anna-Lena Bolender, Elaheh E. Bidhendi, Anders Olofsson, Peter M. Andersen, Thomas Brännström, Stefan L. Marklund, Jonathan D. Gilthorpe, Ulrika Nordström"

Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50 bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50 bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50 bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50 bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population.

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low O2 tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression. Electronic supplementary material The online version of this article (10.1007/s00401-019-01986-1) contains supplementary material, which is available to authorized users.

Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

Aggregation of mutant superoxide dismutase 1 (SOD1) is a pathological hallmark of a subset of familial ALS patients. However, the possible role of misfolded wild type SOD1 in human ALS is highly debated. To ascertain whether or not misfolded SOD1 is a common pathological feature in non-SOD1 ALS, we performed a blinded histological and biochemical analysis of post mortem brain and spinal cord tissues from 19 sporadic ALS, compared with a SOD1 A4V patient as well as Alzheimer’s disease (AD) and non-neurological controls. Multiple conformation- or misfolded-specific antibodies for human SOD1 were compared. These were generated independently by different research groups and were compared using standardized conditions. Five different misSOD1 staining patterns were found consistently in tissue sections from SALS cases and the SOD1 A4V patient, but were essentially absent in AD and non-neurological controls. We have established clear experimental protocols and provide specific guidelines for working, with conformational/misfolded SOD1-specific antibodies. Adherence to these guidelines will aid in the comparison of the results of future studies and better interpretation of staining patterns. This blinded, standardized and unbiased approach provides further support for a possible pathological role of misSOD1 in SALS.

Objective: To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Methods: Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Results: Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72HRE. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Conclusion: Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

Motor neurons containing aggregates of superoxide dismutase 1 (SOD1) are hallmarks of amyotrophic lateral sclerosis (ALS) caused by mutations in the gene encoding SOD1. We have previously reported that two strains of mutant human (h) SOD1 aggregates (denoted A and B) can arise in hSOD1-transgenic models for ALS and that inoculation of such aggregates into the lumbar spinal cord of mice results in rostrally spreading, templated hSOD1 aggregation and premature fatal ALS-like disease. Here, we explored whether mutant hSOD1 aggregates with prion-like properties also exist in human ALS. Aggregate seeds were prepared from spinal cords from an ALS patient carrying the hSOD1G127Gfs*7 truncation mutation and from mice transgenic for the same mutation. To separate from mono-, di- or any oligomeric hSOD1 species, the seed preparation protocol included ultracentrifugation through a density cushion. The core structure of hSOD1G127Gfs*7 aggregates present in mice was strain A-like. Inoculation of the patient- or mouse-derived seeds into lumbar spinal cord of adult hSOD1-expressing mice induced strain A aggregation propagating along the neuraxis and premature fatal ALS-like disease (p

Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.

A common cause of amyotrophic lateral sclerosis is mutations in superoxide dismutase-1, which provoke the disease by an unknown mechanism. We have previously found that soluble hydrophobic misfolded mutant human superoxide dismutase-1 species are enriched in the vulnerable spinal cords of transgenic model mice. The levels were broadly inversely correlated with life spans, suggesting involvement in the pathogenesis. Here, we used methods based on antihuman superoxide dismutase-1 peptide antibodies specific for misfolded species to explore the composition and amounts of soluble misfolded human superoxide dismutase-1 in tissue extracts. Mice expressing 5 different human superoxide dismutase-1 variants with widely variable structural characteristics were examined. The levels were generally higher in spinal cords than in other tissues. The major portion of misfolded superoxide dismutase-1 was shown to be monomers lacking the C57-C146 disulfide bond with large hydrodynamic volume, indicating a severely disordered structure. The remainder of the misfolded protein appeared to be non-covalently associated in 130- and 250-kDa complexes. The malleable monomers should be prone to aggregate and associate with other cellular components, and should be easily translocated between compartments. They may be the primary cause of toxicity in superoxide dismutase-1-induced amyotrophic lateral sclerosis.

Background: As the lens is constantly exposed to light and oxygen that generate harmful reactive oxygen species, the importance of the intracellular antioxidant enzyme copper-zinc superoxide dismut ...

Objectives: Our objectives were to study the relationship between iron stores, HFE genotypes and the risk for first-ever myocardial infarction. Methods: First-ever myocardial infarction cases (n=61 ...

Several of the superoxide dismutase-1 (SOD1) mutations linked to amyotrophic lateral sclerosis (ALS) lead to synthesis of structurally defective molecules, suggesting that any cytotoxic conformatio ...

We demonstrated that, in vivo, at resting membrane potential, spinal motor neurones of the adult G127X mice do not show an increased excitability. However, when depolarized they show evidence of an ...

Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.

The most common of the amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase-1 (SOD1) mutations, D90A, differs from others in its high structural stability and by the existence of both recessive and dominant inheritance. Here SOD1 in CNS and peripheral organs from five ALS patients homozygous for D90A were compared to controls. In most areas, including ventral horns, there were no significant differences in SOD1 activities and Western blotting patterns between controls and D90A cases. The SOD1 activities in areas vulnerable to mutant SOD1s, ventral horns and precentral gyrus were intermediate among CNS areas and much lower than in kidney and liver. Thus, the vulnerability of motor areas is not explained by high SOD1 content. The findings argue against the idea of expression-reducing protective factors being present near the D90A locus in recessive pedigrees. The similarity to wild-type SOD1 prompts speculations on the involvement of the latter in sporadic ALS.

Antioxidant levels were determined in five patients with the Lesch-Nyhan syndrome. The erythrocyte glutathione peroxidase activity was in average 1.8 times higher in Lesch-Nyhan patients than in controls (1.68 +/- 0.36 versus 0.92 +/- 0.17 mu kat/g hemoglobin, p less than 0.001). Plasma CuZn-superoxide dismutase activity was two times higher (p less than 0.001) and Mn-superoxide dismutase activity was 1.5 times higher (p less than 0.05) than in controls, whilst erythrocyte CuZn-superoxide dismutase, plasma extracellular-superoxide dismutase and lymphocyte superoxide dismutase did not differ between Lesch-Nyhan patients and healthy controls. These data might indicate that Lesch-Nyhan patients are exposed to a higher oxidative load than healthy control persons.

Introduction The motor system is selectively vulnerable to mutations in the ubiquitously expressed aggregation-prone enzyme superoxide dismutase-1 (SOD1). Results Autophagy clears aggregates, and factors involved in the process were analyzed in multiple areas of the CNS from human control subjects (n = 10) and amyotrophic lateral sclerosis (ALS) patients (n = 18) with or without SOD1 mutations. In control subjects, the key regulatory protein Beclin 1 and downstream factors were remarkably scarce in spinal motor areas. In ALS patients, there was evidence of moderate autophagy activation and also dysregulation. These changes were largest in SOD1 mutation carriers. To explore consequences of low autophagy capacity, effects of a heterozygous deletion of Beclin 1 were examined in ALS mouse models expressing mutant SOD1s. This caused earlier SOD1 aggregation, onset of symptoms, motor neuron loss, and a markedly shortened survival. In contrast, the levels of soluble misfolded SOD1 species were reduced. Conclusions The findings suggest that an inherent low autophagy capacity might cause the vulnerability of the motor system, and that SOD1 aggregation plays a crucial role in the pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0274-y) contains supplementary material, which is available to authorized users.

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.

Mutants of superoxide dismutase-1 (SOD1) cause ALS by an unidentified cytotoxic mechanism. We have previously shown that the stable SOD1 mutants D90A and G93A are abundant and show the highest levels in liver and kidney in transgenic murine ALS models, whereas the unstable G85R and G127X mutants are scarce but enriched in the CNS. These data indicated that minute amounts of misfolded SOD1 enriched in the motor areas might exert the ALS-causing cytotoxicity. A hydrophobic interaction chromatography (HIC) protocol was developed with the aim to determine the abundance of soluble misfolded SOD1 in tissues in vivo . Most G85R and G127X mutant SOD1s bound in the assay, but only minute subfractions of the D90A and G93A mutants. The absolute levels of HIC-binding SOD1 were, however, similar and broadly inversely related to lifespans in the models. They were generally enriched in the susceptible spinal cord. The HIC-binding SOD1 was composed of disulfide-reduced subunits lacking metal ions and also subunits that apparently carried nonnative intrasubunit disulfide bonds. The levels were high from birth until death and were comparable to the amounts of SOD1 that become sequestered in aggregates in the terminal stage. The HIC-binding SOD1 species ranged from monomeric to trimeric in size. These species form a least common denominator amongst SOD1 mutants with widely different molecular characteristics and might be involved in the cytotoxicity that causes ALS.

We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in copper/zinc superoxide dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins' net repulsive charge. For each SOD1 monomer this charge is normally -6. Because biomolecules as a rule maintain net negative charge to assure solubility in the cellular interior, the result lends support to the hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of the preferential reduction of repulsive charge is higher in SOD1-associated ALS than in other inherited protein disorders.

Lenses from mice lacking the antioxidant enzyme copper-zinc superoxide dismutase (SOD1) show elevated levels of superoxide radicals and are prone to developing cataract when exposed to high levels of glucose in vitro. As superoxide may react further with nitric oxide, generating cytotoxic reactive nitrogen species, we attempted to evaluate the involvement of nitric oxide in glucose-induced cataract. Lenses from SOD1-null and wild-type mice were incubated with high or normal levels of glucose (55.6 and 5.56 mM). A nitric oxide synthase inhibitor (L-NAME) or a nitric oxide donor (DETA/NO) was added to the culture medium. Cataract development was assessed using digital image analysis of lens photographs and cell damage by analyzing the leakage of lactate dehydrogenase. The levels of superoxide radicals in the lenses were also measured. L-NAME was found to reduce cataract development and cell damage in the SOD1-null lenses exposed to high glucose. On the other hand, DETA/NO accelerated cataract development, especially in the SOD1-null lenses. These lenses also showed a higher leakage of lactate dehydrogenase than wild-type controls. We conclude that a combination of high glucose and absence of SOD1 increases the formation of cataract and that nitric oxide probably contributes to this process.

Background: High iron levels can increase the formation of noxious oxygen radicals, which are thought to contribute to cerebrovascular disease. The aim of this prospective study was to determine if iron status and HFE genotypes constitute risk factors for stroke. Methods: First-ever stroke cases (231 ischemic and 42 hemorrhagic) and matched double referents from the population-based Northern Sweden cohorts were studied in a nested case-referent setting. Results: For total iron binding capacity, an increased risk of ischemic stroke was seen in the highest quartile (OR 1.80; 95% CI 1.14–2.83; p for trend 0.012). The highest quartile of transferrin iron saturation showed a decreased risk of ischemic stroke in men (OR 0.44; 95% CI 0.22–0.87; p for trend 0.028), but not in women. There was an increased risk of hemorrhagic stroke in the second (OR 4.07; 95% CI 1.09–15.20) and third quartile (OR 4.22; 95% CI 1.08–16.42) of ferritin. Neither quartiles of plasma iron concentrations nor the HFE C282Y and H63D genotypes were associated with ischemic or hemorrhagic stroke. Conclusions: Iron stores were not positively related to increased risk of ischemic stroke. Furthermore, HFE genotypes did not influence the risk of ischemic or hemorrhagic stroke.

Oxidative stress has been suggested to contribute to the development of diabetic nephropathy. Manganese superoxide dismutase (MnSOD) protects the cells from oxidative damage by scavenging free radicals. The demand for antioxidants is increased by smoking, which could disturb the balance between antioxidants and radicals. The present study aimed to determine whether a valine/alanine polymorphism in MnSOD (V16A, rs4880), alone or in combination with smoking, can contribute to development of diabetic nephropathy in 1,510 Finnish and Swedish patients with type 1 diabetes. Overt diabetic nephropathy (n = 619) was defined as having an albumin excretion rate (AER) >200 μg/min or renal replacement therapy; incipient diabetic nephropathy was defined as having an AER of 20–200 μg/min (n = 336). The control subjects had diabetes duration of ≥20 years, without albuminuria (AER

Mutant human CuZn-superoxide dismutases (hSOD1s) cause amyotrophic lateral sclerosis (ALS). The most common mutation is the wild type-like D90A and to explore its properties, transgenic mice were generated and compared with mice expressing wild-type hSOD1. D90A hSOD1 was both in vivo in mice and in vitro under denaturing conditions nearly as stable as the wild-type human enzyme. It appeared less toxic than other tested mutants, but mice homozygous for the transgene insertion developed a fatal motor neuron disease. In these mice, the disease progression was slow and there were bladder disturbances similar to what is found in human ALS cases homozygous for the D90A mutation. The homozygous D90A mice accumulated detergent-resistant hSOD1 aggregates in spinal cords, and abundant hSOD1 inclusions and vacuoles were seen in the ventral horns. Mice expressing wild-type hSOD1 at a comparable rate showed similar pathologic changes but less and later. Hemizygous D90A mice showed even milder alterations. At 600 days, the wild-type hSOD1 transgenic mice had lost more ventral horn neurons than hemizygous D90A mice (38% vs 31% p < 0.01). Thus, wild-type hSOD1 shows a significant neurotoxicity in the spinal cord, that is less than equal but more than half as large as that of D90A mutant enzyme.

Mutants of human superoxide dismutase-1 (hSOD1) cause amyotrophic lateral sclerosis (ALS), and mitochondria are thought to be primary targets of the cytotoxic action. The high expression rates of hSOD1s in transgenic ALS models give high levels of the stable mutants G93A and D90A as well as the wild-type human enzyme, significant proportions of which lack Cu and the intrasubunit disulfide bond. The endogenous murine SOD1 (mSOD1) also lacks Cu and is disulfide reduced but is active and oxidized in mice expressing the low-level unstable mutants G85R and G127insTGGG. The possibility that the molecular alterations may cause artificial loading of the stable hSOD1s into mitochondria was explored. Approximately 10% of these hSOD1s were localized to mitochondria, reaching levels 100-fold higher than those of mSOD1 in control mice. There was no difference between brain and spinal cord and between stable mutants and the wild-type hSOD1. mSOD1 was increased fourfold in mitochondria from high-level hSOD1 mice but was normal in those with low levels, suggesting that the Cu deficiency and disulfide reduction cause mitochondrial overloading. The levels of G85R and G127insTGGG mutant hSOD1s in mitochondria were 100- and 1000-fold lower than those of stable mutants. Spinal cords from symptomatic mice contained hSOD1 aggregates covering the entire density gradient, which could contaminate isolated organelle fractions. Thus, high hSOD1 expression rates can cause artificial loading of mitochondria. Unstable low-level hSOD1s are excluded from mitochondria, indicating other primary locations of injury. Such models may be preferable for studies of ALS pathogenesis.

Mice lacking the secreted extracellular superoxide dismutase (EC-SOD) or the cytosolic copper- and zinc-containing SOD (CuZn-SOD) show relatively mild phenotypes. To explore the possibility that the isoenzymes have partly overlapping functions, single and double knockout mice were examined. The absence of EC-SOD was found to be without effect on the lifespan of mice, and the reduced lifespan of CuZn-SOD knockouts was not further shortened by EC-SOD deficiency. The urinary excretion of isoprostanes was increased in CuZn-SOD knockout mice, and plasma thiobarbituric acid-reactive substances levels were elevated in EC-SOD knockout mice. These oxidant stress markers showed potentiated increases in the absence of both isoenzymes. Other alterations were mainly found in CuZn-SOD knockout mice, such as halved glutathione peroxidase activity in the tissues examined and increased glutathione and iron in the liver. There were no changes in tissue content of the alternative superoxide scavenger ascorbate, but there was a 25% reduction in ascorbate in blood plasma in mice lacking CuZn-SOD. No increase was found in the urinary excretion of the terminal metabolites of NO, nitrite, and nitrate in any of the genotypes. In conclusion, apart from the increases in the global urinary and plasma oxidant stress markers, our phenotype studies revealed no other evidence that the copper- and zinc-containing SOD isoenzymes have overlapping roles.

The purpose of this study was to evaluate the involvement of the superoxide radical in glucose-induced cataract using lenses from mice lacking the cytosolic copper-zinc superoxide dismutase (SOD1). Lenses from wild-type mice and SOD1 null mice were kept in organ culture with either 5.6 or 55.6 mM glucose for 6 days. The cataract formation was followed with digital image analysis and ocular staging. The lens damage was further quantified by analysis of the leakage of lactate dehydrogenase into the medium by the uptake of 86Rb and by determining the water content of the lenses. The formation of superoxide radicals in the lenses was assessed with lucigenin-derived chemiluminescence. Immunohistochemical staining for SOD1 was also performed on murine lenses. The SOD1 null lenses exposed to high glucose developed more cataract showed an increased leakage of lactate dehydrogenase and developed more oedema compared to the control lenses. At 5.6 mM glucose there was no difference between the SOD1 null and wild-type lenses. Staining for SOD1 was seen primarily in the cortex of the wild-type lens. This in vitro model suggests an involvement of the superoxide radical and a protective effect of SOD1 in glucose-induced cataract formation.

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.

Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors. Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation ( n -acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO -releasing compounds. In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.

Mucosal tissue damage and dysfunction in chronic inflammatory bowel disease (IBD) are partly caused by an enduring exposure to excessive amounts of reactive oxygen metabolites (ROMs). Although the three human isoforms of superoxide dismutase (SOD), copper/zinc (Cu/Zn)-SOD, manganese (Mn)-SOD, and extracellular (EC)-SOD, form the primary endogenous defence against ROMs, their expression levels and cellular localization in IBD mucosa are largely unknown. The present study used enzyme-linked immunosorbent assays (ELISAs), spectrophotometric activity assays, and immunohistochemistry to evaluate the protein concentration, enzymatic activity, and distribution of Cu/Zn-, Mn-, and EC-SOD in paired inflamed and non-inflamed mucosal resection specimens of patients with Crohn's disease (CD) or ulcerative colitis (UC) and compared these with the levels obtained in normal control mucosa. Gut mucosal SOD isoform expression was found to be differentially affected in IBD patients, without major differences between CD and UC. A marked step-wise increase in Mn-SOD protein levels was observed in non-inflamed and inflamed IBD mucosae, whereas the Cu/Zn-SOD content decreased with inflammation. EC-SOD was only found in low amounts, which tended to be decreased in IBD patients. Immunohistochemical evaluation confirmed these observations. Mn-SOD and Cu/Zn-SOD were both predominantly expressed in intestinal epithelial cells and the percentage of epithelial cells positive for Mn-SOD was considerably increased in IBD, whereas epithelial Cu/Zn-SOD expression was much less affected. Within the lamina propria, SOD expression was much lower. Cu/Zn-SOD and Mn-SOD were prominently present in neutrophils and macrophages, and EC-SOD was mainly localized in small vessels, stromal cells, and neutrophils. The percentage of lamina propria cells positive for Cu/Zn-, Mn-, or EC-SOD was not affected by inflammation. Enzyme activity measurements showed consistent results for Cu/Zn-SOD and EC-SOD, but the activity of Mn-SOD did not concordantly increase with the immunological assessments, which may indicate that a proportion of the Mn-SOD in IBD is present in an enzymatically inactive form. This study reveals remarkable changes in the expression levels of the three SOD isoforms in IBD, particularly in the epithelium. Disturbances in the carefully orchestrated mucosal antioxidant cascade may contribute to the induction and perpetuation of intestinal inflammation in IBD, and may have important implications for the development of antioxidant treatment of IBD patients.

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.

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to ALS. In most cases ALS is inherited as a dominant trait and there is marked reduction in CuZn-SOD activity in samples from the patients. The D90A mutation, however, mostly causes ALS as a recessive trait and shows near normal CuZn-SOD activity. A few familial and sporadic ALS cases heterozygous for the D90A mutation have also been found. Haplotype analysis of both types of D90A families has suggested that all recessive cases share a common founder and may carry a protective factor located close to the D90A mutant CuZn-SOD locus. To search for effects of a putative protective factor we analysed erythrocytes from D90A heterozygous individuals for SOD activity by a direct assay, subunit composition by immunoblotting, and zymogram pattern formed by isoelectric focusing and SOD staining. Included were heterozygotes from 17 recessive families, and from 2 dominant families and 4 apparently sporadic cases. The CuZn-SOD activity in the recessive and dominant groups was found to be equal, and 95% of controls. The ratio between mutant and wildtype subunits was likewise equal and 0.8:1 in both groups. The zymograms revealed multiple bands representing homo- and heterodimers. There were, however, no differences between the groups in patterns or in ratios between the molecular forms. In conclusion we find no evidence from analyses in erythrocytes that the putative protective factor in recessive families acts by simply downregulating the synthesis or altering the molecular structure or turnover of the mutant enzyme.

Extracellular-superoxide dismutase (EC-SOD) exists primarily in the tissue interstitium and the lung contains particularly large amounts of the enzyme. To determine the roles of EC-SOD and extracellularly formed superoxide radicals in the pulmonary response to the common air pollutant ozone, wild-type mice and mice lacking EC-SOD were exposed to 1·5 ppm ozone for 48 h. The exposure resulted in a marked neutrophilic inflammatory reaction observed both in the bronchoalveolar lavage fluid (BALF) and by histopathology of the lungs, which was much stronger in the mice lacking EC-SOD. Unlike the wild-type mice, the null mutants also showed increased levels of interleukin-6 in the BALF. The ozone exposure also resulted in increased airway mucosal permeability and cell damage as indicated by increased protein and lactate dehydrogenase in the BALF. There was, however, no difference between the two groups of mice. The results suggest that extracellular superoxide radicals are important inflammatory mediators in the pulmonary response to ozone, but in the present model, the radical and the infiltrating neutrophils contributed little to the pulmonary injury. The data, together with previous findings, support a role for EC-SOD as a modulator of inflammatory reactions.

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to familial amyotrophic lateral sclerosis (ALS), and motor neurone death is caused by the gain of a toxic property of the mutant protein. Here we determined amounts, activity and molecular forms of CuZn-SOD in CSF from ALS patients carrying the D90A and other CuZn-SOD mutations and patients without such mutations. There were no differences in amount of protein and enzymic activities of CuZn-SOD between 37 neurological controls, 54 sporadic and 12 familial ALS cases, and 10 cases homozygous for the D90A mutation. Three cases heterozygous for the A89V, S105L and G114A CuZn-SOD mutations showed low amounts of CuZn-SOD. There was no evidence for accumulation of inactive protein in any of the groups. Immunoblots showed no evidence for the presence of any precipitates or other molecular forms of CuZn-SOD with higher molecular weight in the groups. About 25% of the CuZn-SOD subunits in CSF from controls shows an N-terminal truncation. This truncated portion does not differ between controls and ALS groups not carrying CuZn-SOD mutations, but is 70% larger in samples from D90A homozygous ALS patients. The findings suggest an essentially normal amount and activity of D90A mutant CuZn-SOD in CNS tissues of ALS cases. The increased occurrence of N-terminally truncated mutant subunits may indicate a difference in degradation routes compared with the wild-type enzyme, resistance against subsequent proteolytic steps and/or a compromised downstream proteolytic machinery. Molecular fragments accumulated to a greater extent from the D90A mutant enzyme might contribute to the motor neurone degeneration. We also determined the other SOD isoenzymes: in the controls, CuZn-SOD contributed 75%, extracellular SOD 25% and Mn-SOD

Chronic cobalt exposure is characterized by severe cardiac insufficiency. Since the mechanisms of cobalt toxicity are not yet clear, we analysed the effects of chronic cobalt exposure on antioxidant enzyme activities and myocardial mitochondrial ATP production rate in a rat model. One group of rats was fed a conventional diet and another a cobalt supplemented diet for 24 weeks. The manganese-superoxide dismutase activity was markedly reduced in the cobalt rats (18+/-4.7 U/mg protein) compared to the control rats (100+/-22 U/mg protein; p

Endogenous superoxide anion[Formula: see text] interferes with the bioactivity of nitric oxide (NO) in endothelium-dependent arterial relaxation (EDR). Using the lucigenin chemiluminescence assay, we measured[Formula: see text] in the thoracic and abdominal aortas and the carotid artery of rabbits to determine whether ambient[Formula: see text] varies among the three arteries and differentially diminishes the effect of NO. Basal levels of[Formula: see text] were significantly higher in carotid arteries than in the thoracic aorta [23 ± 6.1 vs. 3.9 ± 1.4 chemiluminescence units (CU); P < 0.05], whereas EDR in response to ACh (10−8–10−5M) was not significantly different on ANOVA. After treatment with the superoxide dismutase (SOD) inhibitor diethyldithiocarbamate (DDC; 10 mM), [Formula: see text] levels were significantly elevated, becoming greater in the carotid artery and abdominal aorta than in the thoracic aorta (185 ± 31.2 and 202 ± 40.3 vs. 89 ± 18 CU; P < 0.05). DDC significantly reversed EDR in the thoracic aorta but not in the carotid artery; at 10−6M ACh, the decrease seen with DDC was 48 ± 6.2 vs. 6.8 ± 8.0% of maximal relaxation in the thoracic aorta and carotid artery, respectively. In the thoracic aorta, exogenous SOD reversed the inhibition of EDR caused by DDC. Moreover, DDC/[Formula: see text]-resistant EDR in the carotid artery was ablated by the addition of nitro-l-arginine methyl ester (300 μM; P < 0.05), an NO synthase inhibitor, consistent with peroxynitrite or an[Formula: see text]-resistant NO donor being involved in carotid relaxation. Indeed, exogenous peroxynitrite caused similar relaxation of the carotid artery and thoracic aorta, which was unaffected by DDC. Our studies show a greater production of nitrite and[Formula: see text] per unit area by the carotid artery, suggesting a greater amount of their product peroxynitrite. These findings support the hypothesis that peroxynitrite is the relaxing agent that resists high [Formula: see text] in the carotid artery.

Concentrations of up to 1.5 milliunits/ml xanthine oxidase (XO) (1.1 micrograms/ml) are found circulating in plasma during diverse inflammatory events. The saturable, high affinity binding of extracellular XO to vascular endothelium and the effects of cell binding on both XO catalytic activity and differentiated vascular cell function are reported herein. Xanthine oxidase purified from bovine cream bound specifically and with high affinity (Kd = 6 nM) at 4 degreesC to bovine aortic endothelial cells, increasing cell XO specific activity up to 10-fold. Xanthine oxidase-cell binding was not inhibited by serum or albumin and was partially inhibited by the addition of heparin. Pretreatment of endothelial cells with chondroitinase, but not heparinase or heparitinase, diminished endothelial binding by approximately 50%, suggesting association with chondroitin sulfate proteoglycans. Analysis of rates of superoxide production by soluble and cell-bound XO revealed that endothelial binding did not alter the percentage of univalent reduction of oxygen to superoxide. Comparison of the extent of CuZn-SOD inhibition of native and succinoylated cytochrome c reduction by cell-bound XO indicated that XO-dependent superoxide production was occurring in a cell compartment inaccessible to CuZn-SOD. This was further supported by the observation of a shift of exogenously added XO from extracellular binding sites to intracellular compartments, as indicated by both protease-reversible cell binding and immunocytochemical localization studies. Endothelium-bound XO also inhibited nitric oxide-dependent cGMP production by smooth muscle cell co-cultures in an SOD-resistant manner. This data supports the concept that circulating XO can bind to vascular cells, impairing cell function via oxidative mechanisms, and explains how vascular XO activity diminishes vasodilatory responses to acetylcholine in hypercholesterolemic rabbits and atherosclerotic humans. The ubiquity of cell-XO binding and endocytosis as a fundamental mechanism of oxidative tissue injury is also affirmed by the significant extent of XO binding to human vascular endothelial cells, rat lung type 2 alveolar epthelial cells, and fibroblasts.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TTR and Arg213G1y EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.

The present study was designed to establish whether or not an increased production of oxygen-derived free radicals is involved in the causation of myringosclerosis. Sclerotic lesions in the tympanic membrane were experimentally elicited by keeping rats with perforated tympanic membranes in an atmosphere containing roughly 40% oxygen. The animals were treated daily with a solution containing either copper zinc-supcroxide dismutase plus catalase, deferoxamine, or copper sulfate plus iron chloride, applied to the traumatized area. After 1 week the extension of myringosclerotic plaques was determined otomicroscopically. The pars tensa and pars flaccida were then dissected free and prepared for light microscopic studies. The results showed that treatment with copper zinc-superoxide dismutase plus catalase and deferoxamine inhibited or reduced the development of myringosclerosis, whereas die ears treated with copper sulfate plus iron chloride appeared unaffected. Consequently, the findings support the hypothesis that the formation of oxygen free radicals contributes significantly to the development of myringosclerosis.

The importance of early microcirculatory changes in the rat colon after exposure to acetic acid was investigated. Administration of 4% acetic acid for 15 sec into an exteriorized colonic segment induced a marked, transient (starting 2 min after the challenge with acetic acid and persisting for 15 min) decrease in the colonic blood flow as estimated by a laser–Doppler flowmeter. Four days after acetic acid administration, a uniform colitis had developed in the exteriorized colonic segment with a total morphological score (TMS) of 15.1 ± 0.8, myeloperoxidase activity (MPO) increased more than threefold, and plasma exudation into the colonic lumen increased sevenfold. Administration of hydrochloric acid (HCl) with the same pH as the acetic acid or sodium acetate (pH 7.0) did not affect colonic blood flow or produce colitis. Mechanical colonic ischemia, induced by a controlled increase in the intraluminal pressure, resulted in several pathological features of colitis with a TMS of 7.3 ± 0.2, combined with a significant increase in MPO activity. The TMS and MPO were further increased when mechanical colonic ischemia was combined with HCl or sodium acetate. Pretreatment with SOD and catalase 5 or 15 min before acetic acid administration did not affect the transient ischemia immediately following acetic acid administration. However, it partially prevented the development of colitis. It is concluded that immediate transient ischemia accompanied by the generation of oxygen free radicals might be of importance in the pathogenesis of acetic acid-induced colitis in the rat.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

Extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a secreted Cu- and Zn-containing tetrameric glycoprotein, the bulk of which is bound to heparan sulfate proteoglycans in the interstitium of tissues. To test the function of EC-SOD in vivo, mice carrying a targeted disruption of the EC-SOD gene were generated. The EC-SOD null mutant mice develop normally and remain healthy until at least 14 months of age. No compensatory induction of other SOD isoenzymes or other antioxidant enzymes was observed. When stressed by exposure to > 99% oxygen, the EC-SOD null mutant mice display a considerable reduction in survival time compared to wild-type mice and an earlier onset of severe lung edema. These findings suggest that while under normal physiological conditions other antioxidant systems may substitute for the loss of EC-SOD; when the animal is stressed these systems are unable to provide adequate protection.

A common cause of amyotrophic lateral sclerosis (ALS) is mutations in the gene encoding superoxide dismutase-1. There is evolving circumstantial evidence that the wild-type protein can also be neurotoxic and that it may more generally be involved in the pathogenesis of ALS. To test this proposition more directly, we generated mice that express wild-type human superoxide dismutase-1 at a rate close to that of mutant superoxide dismutase-1 in the commonly studied G93A transgenic model. These mice developed an ALS-like syndrome and became terminally ill after around 370 days. The loss of spinal ventral neurons was similar to that in the G93A and other mutant superoxide dismutase-1 models, and large amounts of aggregated superoxide dismutase-1 were found in spinal cords, but also in the brain. The findings show that wild-type human superoxide dismutase-1 has the ability to cause ALS in mice, and they support the hypothesis of a more general involvement of the protein in the disease in humans.

Over 170 mutations in superoxide dismutase-1 (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). The properties of SOD1 mutants differ considerably including copper-binding abilities. Nevertheless, they cause the same disease phenotype, suggesting a common neurotoxic pathway. We have previously reported that copper homeostasis is disturbed in spinal cords of SOD1(G93A) mice. However, it is unknown whether copper dyshomeostasis is induced by other SOD1 mutants. Using the additional mouse strains SOD1(G127insTGGG), SOD1(G85R), and SOD1(D90A), which express SOD1 mutants with different copper-binding abilities, we show that copper dyshomeostasis is common to SOD1 mutants. The SOD1 mutants shifted the copper trafficking systems toward copper accumulation in spinal cords of the mice. Copper contents bound to the SOD1 active site varied considerably between SOD1 mutants. Still, copper bound to other ligands in the spinal cord were markedly increased in all. Zinc was also increased, whereas there were no changes in magnesium, calcium, aluminum, manganese and iron. Further support for a role of copper dyshomeostasis in ALS was gained from results of pharmacological intervention. Ammonium tetrathiomolybdate (TTM), a copper chelating agent, prolonged survival and slowed the disease progression of SOD1(G93A) mice, even when the treatment was started after the disease onset. TTM markedly attenuated pathology, including the loss of motor neurons and axons, and atrophy of skeletal muscles. Additionally, TTM decreased amounts of SOD1 aggregates. We propose that pharmacological agents that are capable of modulating copper dyshomeostasis, such as TTM, might be beneficial for the treatment of ALS caused by SOD1 mutations.

Extracellular superoxide dismutase (EC-SOD) is a secretory SOD isoenzyme. 99% of EC-SOD is anchored to heparan sulfate proteoglycans in the tissue interstitium, and 1% is located in the vasculature in equilibrium between the plasma and the endothelium. Analysis of EC-SOD in plasma samples from 504 random blood donors revealed a common (2.2%) phenotypic variant displaying 10-fold increased plasma EC-SOD content. The EC-SOD in the plasma of these individuals, collected both before and after intravenous injection of heparin, displayed a reduced heparin affinity when compared with samples from normal individuals. The specific enzymatic activity was the same as that of normal enzyme. Nucleotide sequence analyses of two of the affected subjects revealed a nucleotide exchange resulting in a substitution of Arg-213 by Gly. The substitution is located in the center of the carboxyl-terminal cluster of positively charged amino acid residues, which defines the heparin-binding domain. Polymerase chain reaction-single-strand conformational polymorphism and allele-specific polymerase chain reaction showed that all 11 affected individuals are heterozygous, carrying the same single-base mutation. Recombinant EC-SOD containing this mutation had a reduced heparin affinity similar to that of EC-SOD C from variant persons. The high plasma activity can be explained by an accelerated release from the tissue interstitium heparan sulfate to the vasculature and should thus be accompanied by significantly reduced tissue EC-SOD activities.

We have targeted the expression of recombinant human extracellular superoxide dismutase, a glycosylated tetrameric metalloprotein, to the mammary gland of transgenic mice. This was achieved by using regulatory elements from either the murine whey acidic protein gene or the ovine beta-lactoglobulin gene to control expression of human extracellular superoxide dismutase cDNA. Whey acidic protein regulatory sequences directed high level mammary gland-specific expression of the recombinant gene and secretion of biologically active extracellular superoxide dismutase into the milk. The produced recombinant protein was fully active, it was in tetrameric form, it showed heparin affinity, and its mass was similar to that of the native enzyme. In addition, the in vivo plasma clearance in a rabbit model was similar to the previously studied native and recombinant forms. To our knowledge, this is the first example of efficient production of a tetrameric, protease-susceptible metalloprotein in milk of transgenic animals. Production at equivalent levels in transgenic farm animals would yield sufficient extracellular superoxide dismutase for therapeutic purposes.

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.

The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.

Comparison of amino acid sequences have suggested similarities between the active site portions of the tetrameric, Cu- and Zn-containing glycoprotein, extracellular superoxide dismutase (EC-SOD) and the dimeric CuZn-SOD. In the present study spectral and physical properties of EC-SOD were analyzed to further knowledge about the enzyme and to extend the comparison with CuZn-SOD. EC-SOD displays an absorbance peak at 652 nm, blue-shifted some 30 nm compared with CuZn-SOD. The molar extinction coefficient, 167 M-1 cm-1 per copper atom, is similar to those of CuZn-SODs. In contrast to bovine and human CuZn-SOD, EC-SOD has a strong uv absorption originating from the rich content of aromatic amino acids. The extinction coefficient at 280 nm is 1.73 ml mg-1 cm-1. The electron paramagnetic resonance spectrum is also similar to those of bovine and human CuZn-SOD, but shows a slightly higher Az and a smaller difference between gx and gy. These spectral findings support the notion that the active sites of the SOD iso-enzymes are similar, but with a higher tetragonal symmetry of the copper ligands in EC-SOD. The circular dichroism spectrum of EC-SOD in the ultraviolet indicates a high alpha-helix content of the amino-terminal and carboxy-terminal parts flanking the central homologous active site portion. Similar to bovine CuZn-SOD, EC-SOD displays a marked physical resistance toward high temperature, pH extremes, and high urea and guanidinium chloride concentrations. This similarity in physical resistance suggests a central role of the homologous active site portion for the structural integrity of the SOD iso-enzymes.