Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.

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.disulfide bond ͉ motor neuron ͉ neurodegeneration A myotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration, resulting in progressive paralysis, and death from respiratory failure. Approximately 10% of ALS cases are familial (1) and in some of these the disease is linked to mutations in the CuZn-superoxide dismutase (SOD1) gene (2). Overall, Ϸ6% of all cases with ALS show SOD1 mutations, and more than 100 different such mutations have been identified (3). The mutations confer a cytotoxic gain of function to the enzyme (4, 5). SOD1 is ubiquitously expressed, and in several organs at higher levels than in the motor areas in the CNS (6, 7). Both the nature of the cytotoxicity, and the reasons for the particular susceptibility of some parts of the CNS remain unexplained.It is likely that the different mutant SOD1s cause ALS by essentially the same mechanism. Still, the levels of different mutant SOD1s in the human CNS differ by more than 200-fold (7). Similar large differences are observed in the murine ALS models. In spinal cords from mice expressing the D90A, G93A, G85R and G127insTGGG (G127X) mutant human SOD1s (hSOD1s), the levels are 20, 14, 0.9, and 0.45 times as large, respectively, as the levels of the endogenous murine SOD1 (mSOD1) (8). In the high-level models, D90A and G93A, most hSOD1 lacks enzymatic activity owing to Cu-deficiency, and significant proportions lack the stabilizing intrasubunit disulfide bond. The G85R and G127X hSOD1s lack both enzymatic activity and the disulfide bond. Whereas the liver and kidney contain the highest lev...

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.

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.