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Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic ALS model mice
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Neurology.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 23, 20130-20136 p.Article in journal (Refereed) Published
Abstract [en]

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011. Vol. 286, no 23, 20130-20136 p.
Keyword [en]
Amyotropic Lateral Sclerosis (Lou Gehrig's Disease), Heat Shock Protein, Neurodegeneration, Protein-Protein Interactions, Superoxide Dismutase (SOD), Transgenic Mice
National Category
Other Clinical Medicine
Research subject
Clinical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-43895DOI: 10.1074/jbc.M111.218842PubMedID: 21493711ISBN: 1083-351X (Electronic) 0021-9258 (Linking) (print)OAI: oai:DiVA.org:umu-43895DiVA: diva2:417136
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2017-12-11Bibliographically 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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1421
Keyword
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
Identifiers
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)
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Available from: 2011-05-20 Created: 2011-05-16 Last updated: 2011-05-20Bibliographically approved

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