Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition involving the upper and lower motor neurons, leading to progressive muscle atrophy, paresis, and death, usually by paralysis of the respiratory muscles. The majority of patients are considered sporadic with no reported hereditary background. However, about 5-10% of patients have a genetic predisposition. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are one of the most common causes of familial ALS. Over 240 mutations spread over the entire SOD1 gene have been described in ALS. SOD1 plays a significant role in the cellular defense machinery against damage caused by the superoxide anion radical. A large body of evidence advocates for a gain of novel toxic function to be the major cause of SOD1-linked ALS by forming pathological aggregates. However, children homozygous for mutations resulting in total loss of SOD1 function develop a motor neuron disease phenotype within the first 6 months of life. This indicates a potential threshold below which the loss of SOD1 activity is deleterious. The approval of the antisense oligonucleotide tofersen designed to reduce SOD1 mRNA levels and hence prevent protein aggregation, marked the beginning of a new era where a subgroup of patients has access to disease-modifying therapy with a real clinical effect. Not all patients respond to treatment, and most continue to clinically progress, albeit at a slower pace, and the reason for this is not fully understood. This thesis aims to investigate the role of SOD1 enzymatic activity in ALS. We used specific SOD antibodies and immunocapture, combined with biochemical and quantitative methods, to investigate and better understand the effects of altered SOD1 levels and activity in CSF and answer questions emerging from these findings. We quantified SOD1 content in different tissues, including blood, thereby mapping SOD1 abundance in the CNS and peripheral tissues as a baseline for SOD1-reducing therapies. Even though SOD1 is an abundant protein, it accounts for only 0.16% of total protein levels, which is 10-fold lower than stated in the literature. ALS mainly affects the CNS, and CSF reflects the status of the CNS better than erythrocytes. We therefore developed a method that allows the specific measurement of SOD1 activity in CSF. We then analyzed SOD1 activity in 171 CSF samples collected from ALS patients with and without SOD1 mutations and compared them with controls. The SOD1 activity varies greatly in CSF. Consequently, we asked how SOD1 activity is influenced by SOD1-reducing drugs and studied SOD1 activity in longitudinal CSF samples of ALS patients treated with tofersen up to 3 years. The activity was reduced to different degrees in all patients. The treatment response time varied from 4 to 12 months and is clinically relevant for deciding whether the drug has a beneficial effect or not in individual ALS patients, and might allow for individual dosing. In CSF samples, a band on immunoblots 3 kDa below monomeric SOD1, indicated a N-truncated variant of SOD1 in CSF. Further investigation revealed the truncation site to be between amino acids 26 and 27 and that the cleaved peptide stays connected to the truncated SOD1 monomer in a folded state. With mass spectrometry, the cleaved peptide was identified to be identical to the N-terminal sequence found in native SOD1. The truncation event does not seem to have an effect on SOD1 misfolding. We could not explain what causes the truncation of SOD1 in CSF, and our data indicate that N-truncation does not contribute to ALS pathogenesis.
Laura Leykam (Thu,) studied this question.