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Mutant superoxide dismutase-1-caused pathogenesis in amyotrophic lateral sclerosis
Umeå University, Faculty of Medicine, Medical Biosciences, Clinical chemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their late mid-life, with fatal outcome usually within a few years. The progressive degeneration of neurons responsible for muscle movement (motor neurons) throughout the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually affects respiratory function. Most cases have no familial background (sporadic) whereas about 10% of cases have relatives affected by the disease. A substantial number of familial cases are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). Since the initial discovery of this relationship about 17 years ago, numerous workers have tried to identify the pathogenicity of mutant SOD1 but without any final agreement or consensus regarding mechanism. The experiments in this thesis have been aimed at finding common pathogenic mechanisms by analyzing transgenic mouse models expressing mutant SOD1s with widely different properties.

    Mitochondrial pathology and dysfunction have been reported in both ALS patients and murine models. We used density gradient ultracentrifugation for comparison of mitochondrial partitioning of SOD1 in our transgenic models. It was found that models with high levels of mutant protein, overloaded mitochondria with high levels of SOD1-protein whereas models with wild type-like levels of mutant protein did not. No significant association of the truncation mutant G127X with mitochondria was found. Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis this is unlikely to be caused by physical association of mutant SOD1 with mitochondria.

    Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the ventral horns of the spinal cord but also in the liver and kidney, although at lower levels. This showed that such signs of the disease can also be found outside the CNS.

    This method was used further to characterize SOD1 inclusions with regard to the properties of mutant SOD1 and the presence of other proteins. The inclusions were found to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57-C146 being the major inclusion protein, constituting at least 50% of the protein content. Ten co-aggregating proteins were isolated, some of which were already known to be present in cellular inclusions. Of great interest was the presence of several proteins that normally reside in the endoplasmic reticulum (ER), which is in accordance with recent data suggesting that the unfolded protein response (UPR) has a role in ALS.

    To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to the difference between transgenic and control mice. From 53 proteins finally identified, we found pathways such as mitochondrial function, oxidative stress, and protein degradation to be affected by the disease. We also identified a previously uncharacterized covalent SOD1 dimer.

   In conclusion, the work described in this thesis suggests that mutant SOD1 affects the function of mitochondria, but not mainly through direct accumulation of SOD1 protein. It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues, mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be deleterious as motor neurons, unable to renew themselves, are dependent on proper protein folding and degradation.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 89 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1285
Keyword [en]
ALS, SOD1, mitochondria, proteome, transgenic mice, inclusion
National Category
Other Clinical Medicine
Research subject
Biochemistry; Neurology; Clinical Chemistry; Pathology
Identifiers
URN: urn:nbn:se:umu:diva-31116ISBN: 978-91-7264-838-8 (print)OAI: oai:DiVA.org:umu-31116DiVA: diva2:291039
Public defence
2010-02-26, Betula, NUS, 90185, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-02-05 Created: 2010-01-29 Last updated: 2010-02-05Bibliographically approved
List of papers
1. Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models
Open this publication in new window or tab >>Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models
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2006 (English)In: Journal of Neuroscience, ISSN 0270-6474, Vol. 26, no 16, 4147-4154 p.Article in journal (Refereed) Published
Keyword
Amyotrophic Lateral Sclerosis/*enzymology/genetics, Animals, Disease Models; Animal, Enzyme Stability/genetics, Humans, Mice, Mice; Transgenic, Mitochondria/*enzymology/genetics, Mutation, Spinal Cord/enzymology, Superoxide Dismutase/*biosynthesis/genetics, Variation (Genetics)
Identifiers
urn:nbn:se:umu:diva-15270 (URN)10.1523/JNEUROSCI.5461-05.2006 (DOI)16624935 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2010-02-05Bibliographically approved
2. Inclusions of amyotrophic lateral sclerosis-linked superoxide dismutase in ventral horns, liver, and kidney
Open this publication in new window or tab >>Inclusions of amyotrophic lateral sclerosis-linked superoxide dismutase in ventral horns, liver, and kidney
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2008 (English)In: Annals of Neurology, ISSN 0364-5134, Vol. 63, no 5, 671-675 p.Article in journal (Refereed) Published
Keyword
Adult, Amyotrophic Lateral Sclerosis/*enzymology/*genetics/pathology, Anterior Horn Cells/*metabolism/pathology, Humans, Kidney/*enzymology/pathology, Liver/*enzymology/pathology, Mutation, Organ Specificity, Superoxide Dismutase/*genetics/*metabolism, Tissue Distribution
Identifiers
urn:nbn:se:umu:diva-11525 (URN)10.1002/ana.21356 (DOI)18409196 (PubMedID)
Available from: 2009-01-13 Created: 2009-01-13 Last updated: 2010-02-05Bibliographically approved
3. Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice
Open this publication in new window or tab >>Superoxide dismutase-1 and other proteins in inclusions from transgenic amyotrophic lateral sclerosis model mice
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from 4 different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intrasubunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, 4 were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.

Keyword
Amyotrophic lateral sclerosis, Superoxide dismutase-1, transgenic mice, inclusion, proteomics, endoplasmic reticulum
National Category
Other Clinical Medicine
Research subject
Medicine; Neurology; Biochemistry
Identifiers
urn:nbn:se:umu:diva-31092 (URN)
Projects
Mutant Superoxide Dismutase-1-caused pathogenesis in Amyotrophic Lateral Sclerosis
Available from: 2010-01-29 Created: 2010-01-28 Last updated: 2010-02-05Bibliographically approved
4. Changes in the spinal cord proteome of an amyotrophic lateral sclerosis murine model determined by differential in-gel electrophoresis
Open this publication in new window or tab >>Changes in the spinal cord proteome of an amyotrophic lateral sclerosis murine model determined by differential in-gel electrophoresis
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2009 (English)In: Molecular and cellular proteomics, ISSN 1535-9484, Vol. 8, no 6, 1306-1317 p.Article in journal (Refereed) Published
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis. To date, more than 140 different mutations in the gene encoding CuZn-superoxide dismutase (SOD1) have been associated with ALS. Several transgenic murine models exist in which various mutant SOD1s are expressed. We have used differential in-gel electrophoresis (DIGE) to analyze the changes in the spinal cord proteome induced by expression of the unstable SOD1 truncation mutant G127insTGGG (G127X) in mice. Unlike mutants used in most other models, G127X lacks SOD activity and is present at low levels, thus reducing the risk of overexpression artifacts. The mice were analyzed at their peak body weights, just before onset of symptoms. Variable importance plot (VIP) analysis showed that 420 of 1,800 detected protein spots contributed significantly to the differences between the groups. By MALDI-TOF MS analysis, 54 proteins were identified. One spot was found to be a covalently linked mutant SOD1 dimer, apparently analogous to SOD1 immunoreactive bands migrating at double the molecular weight of SOD1 monomers previously detected in humans and mice carrying mutant SOD1s and in sporadic ALS cases. Analyses of affected functional pathways, and the subcellular representation of alterations suggest that the toxicity exerted by mutant SODs induces oxidative stress and affects mitochondria, cellular assembly/organization, and protein degradation.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology,Inc, 2009
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-22132 (URN)10.1074/mcp.M900046-MCP200 (DOI)19357085 (PubMedID)
Available from: 2009-04-24 Created: 2009-04-24 Last updated: 2012-04-19Bibliographically approved

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