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Misfolded superoxide dismutase-1 in amyotrophic lateral sclerosis
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.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Felveckat superoxiddismutas-1 i amyotrofisk lateralskelros (Swedish)
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 [en]
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: urn:nbn:se:umu:diva-43898ISBN: 978-91-7459-215-3OAI: oai:DiVA.org:umu-43898DiVA: diva2:417745
Public defence
2011-09-09, Hörsal Betula, Building 6M, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-05-20 Created: 2011-05-16 Last updated: 2011-05-20Bibliographically approved
List of papers
1. Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models
Open this publication in new window or tab >>Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models
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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.

Keyword
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)
Identifiers
urn:nbn:se:umu:diva-7589 (URN)10.1073/pnas.0700477104 (DOI)17715066 (PubMedID)
Available from: 2008-10-16 Created: 2008-10-16 Last updated: 2011-05-20Bibliographically approved
2. Structure of soluble misfolded superoxide dismutase-1 in murine ALS models
Open this publication in new window or tab >>Structure of soluble misfolded superoxide dismutase-1 in murine ALS models
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:umu:diva-43897 (URN)
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2011-05-20Bibliographically approved
3. Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic ALS model mice
Open this publication in new window or tab >>Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic ALS model mice
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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
Keyword
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:nbn:se:umu:diva-43895 (URN)10.1074/jbc.M111.218842 (DOI)21493711 (PubMedID)1083-351X (Electronic) 0021-9258 (Linking) (ISBN)
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2012-08-16Bibliographically approved
4. Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients
Open this publication in new window or tab >>Misfolded superoxide dismutase-1 in CSF from amyotrophic lateral sclerosis patients
2011 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 117, no 1, 91-99 p.Article in journal (Refereed) Published
Abstract [en]

Several of the superoxide dismutase-1 (SOD1) mutations linked to amyotrophic lateral sclerosis (ALS) lead to synthesis of structurally defective molecules, suggesting that any cytotoxic conformational species common for all mutations should be misfolded. SOD1 can be secreted and evidence from ALS model systems suggests that extracellular SOD1 may be involved in cytotoxicity. Three ELISAs specifically reacting with different sequence segments in misfolded SOD1 species were used for analysis of CSF from 38 neurological controls and from 96 ALS patients, 57 of whom were sporadic cases and 39 familial, including 22 patients carrying SOD1 mutations. Misfolded SOD1 was found in all samples. There were, however, no significant differences between patients with and without mutations, and between all the ALS patients and the controls. The estimated concentration of misfolded SOD1 in the interstitium of the CNS is a 1000 times lower than that required for appreciable cytotoxicity in model systems. The results argue against a direct cytotoxic role of extracellular misfolded SOD1 in ALS. Misfolded SOD1 in CSF cannot be used as a biomarker of ALS in patients with and without mutations in the enzyme.

Keyword
amyotrophic lateral sclerosis;biomarker;CSF;ELISA;misfolding;superoxide dismutase-1
National Category
Other Clinical Medicine
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:umu:diva-43892 (URN)10.1111/j.1471-4159.2011.07177.x (DOI)21226712 (PubMedID)
Available from: 2011-05-16 Created: 2011-05-16 Last updated: 2011-05-20Bibliographically approved

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