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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.

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.

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.