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Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in erythrocytes in ALS patients and their relatives
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
Umeå University, Faculty of Medicine, Department of Medical Biosciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Our objective was to in blood samples from 3723 individuals including ALS patients without a coding SOD1 mutation and 372 control individuals characterize stabilities of mutant SOD1s, 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. Erythrocyte SOD1 enzymatic activities normalized to hemoglobin content were determined. Coding SOD1 sequences were analyzed by Sanger sequencing, copy number variations by fragment length analysis and by TaqMan Assay. Hemoglobin disorders were searched for. Of the 46 SOD1 mutations found, ¾ caused severe destabilization of the mutant protein but in ¼ SOD1 was essentially physically stable. Mutations producing structural changes all caused halved SOD activities. There were no differences in SOD1 activities between controls and patients without any detected SOD1 mutations or patients with C9ORF72HRE or TBK1 mutations. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Also, no uncommon variants in exon-flanking sequences were detected. Thalassemias and iron deficiency anemia were associated with increased SOD1 activity/hemoglobin ratios. In conclusion, adjunct erythrocyte SOD activity analysis is of value to signal the presence of exon and splice-site-intron mutations that influence the SOD1 structure.

National Category
Biochemistry and Molecular Biology Medical and Health Sciences
Identifiers
URN: urn:nbn:se:umu:diva-124955OAI: oai:DiVA.org:umu-124955DiVA: diva2:956771
Available from: 2016-08-31 Created: 2016-08-31 Last updated: 2016-09-01Bibliographically approved
In thesis
1. SOD, ORF and ALS: On the role of SOD1 and C9ORF72 in the pathogenesis of ALS
Open this publication in new window or tab >>SOD, ORF and ALS: On the role of SOD1 and C9ORF72 in the pathogenesis of ALS
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset degeneration of upper and lower motor neurons. Symptoms begin focally in one muscle and then spread contiguously, resulting in progressive paralysis and death from respiratory failure. Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause, however, mutations in SOD1 were the first identified and are found in 1-9% of patients. Misfolded SOD1 aggregates in the CNS are hallmarks of ALS associated with SOD1 mutations. However, accumulation of misfolded or aggregated SOD1 protein has also been reported in sporadic and familial ALS without SOD1 mutations, suggesting that wild-type SOD1 could play a role in ALS pathology in general.

The aims of this thesis are: 1) To describe the resulting disease phenotype and specific characteristics of the SOD1 protein carrying the stable disease- associated mutation L117V. 2) To set up cell-based in vitro models to study the mechanisms of SOD1 misfolding and aggregation under physiologically relevant expression levels. 3) To compare SOD1 activity in patient-derived samples and screen for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations.

1) We identified a novel L117V SOD1 mutant in two families of Syrian origin that co-segregated with the disease. This mutation was associated with slow disease progression, reduced penetrance and a uniform phenotype. The L117V mutant protein was indistinguishable from wild-type SOD1 in terms of stability, dismutation activity and misfolding in patient-derived cell lines.

2) We established patient-derived fibroblast and iPSC-MN lines expressing mutant SOD1 at physiological levels as in vitro models to study misfolding and aggregation of SOD1. We investigated the effects of several cellular pathway disturbances on SOD1 misfolding. Misfolded SOD1 was increased by inhibition of the ubiquitin-proteasome pathway in fibroblasts derived from both patients and controls. An age-related decline in proteasome activity could contribute to the late onset of ALS.

Next, we studied the effects of low oxygen tension on misfolding and aggregation of SOD1 in patient-derived cells. Low O2 tensions were found to markedly increase C57-C146 disulphide reduction, misfolding and aggregation of SOD1. Importantly, the largest effects were detected in iPSC-MNs. This suggests that motor neurons are specifically vulnerable to misfolding and aggregation of SOD1 under low O2 tension.

3) We compared the enzymatic activity of SOD1 in blood samples from a large number of ALS patients and controls. We screened for potential underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. No aberrations in copy number, other large structural changes in introns and exons or intronic mutations in the 30-50 bp flanking the exons were found in the 142 outliers, with either very low or very high SOD1 dismutation activities. However, hemoglobinopathies, including thalassemias and iron deficiency anemia, were associated with high SOD1/mg Hb ratios. Erythrocytes from patients with destabilizing SOD1 mutations showed half the normal activity. There were no significant differences in SOD1 activity between control individuals and ALS patients without a coding SOD1 mutation, or carriers of TBK1 mutations or the hexanucleotide repeat expansion in C9ORF72. Our result suggests that SOD1 enzymatic activity is not associated with the disease in non-SOD1 mutation ALS.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 89 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1829
Keyword
ALS, SOD1, Misfolded species
National Category
Biochemistry and Molecular Biology Neurology
Identifiers
urn:nbn:se:umu:diva-124917 (URN)978-91-7601-531-5 (ISBN)
External cooperation:
Public defence
2016-09-23, Föreläsningssal A, Unod T 9, Norrlands universitetssjukhus, Umeå, 13:00 (English)
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Available from: 2016-09-02 Created: 2016-08-30 Last updated: 2016-09-01Bibliographically approved

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Keskin, IsilBirve, AnnaBerdynski, MariuszHjertkvist, KarinRofougaran, RezaNilsson, Torbjörn K.Marklund, Stefan L.Andersen, Peter M.
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