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Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
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2007 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 35, 14157-14162 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2007. Vol. 104, no 35, 14157-14162 p.
Keyword [en]
Animals, Disease Models; Animal, Humans, Mice, Mice; Transgenic, Motor Neuron Disease/enzymology/*genetics, Mutation, Oxidation-Reduction, Protein Denaturation, Protein Folding, Protein Subunits, Sequence Deletion, Spinal Cord/growth & development/*physiopathology, Superoxide Dismutase/*genetics/metabolism, Variation (Genetics)
URN: urn:nbn:se:umu:diva-7589DOI: 10.1073/pnas.0700477104PubMedID: 17715066OAI: diva2:147260
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2011-05-20Bibliographically approved
In thesis
1. Misfolded superoxide dismutase-1 in amyotrophic lateral sclerosis
Open this publication in new window or tab >>Misfolded superoxide dismutase-1 in amyotrophic lateral sclerosis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Felveckat superoxiddismutas-1 i amyotrofisk lateralskelros
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a disease in which the motor neurons die in a progressive manner, leading to paralysis and muscle wasting. ALS is always fatal, usually through respiratory failure when the disease reaches muscles needed for breathing. Most cases are sporadic, but approximately 5–10% are familial. The first gene to be linked to familial ALS encodes the antioxidant enzyme superoxide dismutase-1 (SOD1). Today, more than 160 different mutations in SOD1 have been found in ALS patients.  The mutant SOD1 proteins cause ALS by gain of a toxic property that should be common to all. Aggregates of SOD1 in motor neurons are hallmarks of ALS patients and transgenic models carrying mutant SOD1s, suggesting that misfolding, oligomerization, and aggregation of the protein may be involved in the pathogenesis. SOD1 is normally a very stable enzyme, but the structure has several components that make SOD1 sensitive to misfolding. The aim of the work in this thesis was to study misfolded SOD1 in vivo.

Small amounts of soluble misfolded SOD1 were identified as a common denominator in transgenic ALS models expressing widely different forms of mutant SOD1, as well as wild-type SOD1. The highest levels of misfolded SOD1 were found in the vulnerable spinal cord. The amounts of misfolded SOD1 were similar in all the different models and showed a broad correlation with the lifespan of the different mouse strains. The misfolded SOD1 lacked the C57-C146 intrasubunit disulfide bond and the stabilizing zinc and copper ions, and was prinsipally monomeric. Forms with higher apparent molecular weights were also found, some of which might be oligomers. Misfolding-prone monomeric SOD1 appeared to be the principal source of misfolded SOD1 in the CNS. Misfolded SOD1 in the spinal cord was found to interact mainly with chaperones, with Hsc70 being the most important. Only a minor proportion of the Hsc70 was sequestered by SOD1, however, suggesting that chaperone depletion is not involved in ALS.

 SOD1 is normally found in the cytoplasm but can be secreted. Extracellular mutant SOD1 has been found to be toxic to motor neurons and glial cells. Misfolded SOD1 in the extracellular space could be involved in the spread of the disease between different areas of the CNS and activate glial cells known to be important in ALS. The best way to study the interstitium of the CNS is through the cerebrospinal fluid (CSF), 30% of which is derived from the interstitial fluid. Antibodies specific for misfolded SOD1 were used to probe CSF from ALS patients and controls for misfolded SOD1. We did find misfolded SOD1 in CSF, but at very low levels, and there was no difference between ALS patients and controls. This argues against there being a direct toxic effect of extracellular SOD1 in ALS pathogenesis.

In conclusion, soluble misfolded SOD1 is a common denominator for transgenic ALS model mice expressing widely different mutant SOD1 proteins. The misfolded SOD1 is mainly monomeric, but also bound to chaperones, and possibly exists in oligomeric forms also. Misfolded SOD1 in the interstitium might promote spread of aggregation and activate glial cells, but it is too scarce to directly cause cytotoxicity.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2011. 126 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1421
ALS, SOD1, protein misfolding, SOD1 conformation, disulfide-reduced, transgenic mice, cerebrospinal fluid, protein-protein interaction, antibodies
National Category
Other Clinical Medicine
Research subject
Clinical Chemistry
urn:nbn:se:umu:diva-43898 (URN)978-91-7459-215-3 (ISBN)
Public defence
2011-09-09, Hörsal Betula, Building 6M, Umeå University, Umeå, 09:00 (English)
Available from: 2011-05-20 Created: 2011-05-16 Last updated: 2011-05-20Bibliographically approved

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Zetterström, PerBergemalm, DanielJonsson, P AndreasGraffmo, Karin SAndersen, Peter MBrännström, ThomasMarklund, Stefan L
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