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Superoxide dismutase 1 and amyotrophic lateral sclerosis
Umeå University, Faculty of Medicine, Medical Biosciences, Pathology. Umeå University, Faculty of Medicine, Medical Biosciences, Clinical chemistry. Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Neurology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Superoxid dismutas 1 och amyotrofisk lateralskleros (Swedish)
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the spinal cord, brain stem and motor cortex, leading to paralysis, respiratory failure and death. In about 5% of ALS cases, the disease is associated with mutations in the CuZn-superoxide dismutase (hSOD1) gene. As a rule, ALS caused by hSOD1 mutations is inherited dominantly and the mutant hSOD1s cause ALS by the gain of a noxious property.

The present study focused on two hSOD1 mutations with widely differing characters. In Scandinavia, ALS caused by the D90A mutation is inherited in a recessive pattern. Elsewhere, families with dominant inheritance have been found. The properties of D90A mutant hSOD1 are very similar to those of the wild-type protein. The G127insTGGG (G127X) mutation causes a 21 amino acid C-terminal truncation which probably results in an unstable protein.

The aim of this thesis was to generate transgenic mice expressing D90A and G127X mutant hSOD1s and to compare these mice with each other and with mice expressing other mutant hSOD1s, in search of a common noxious property. The findings were also compared with the results from studies of human CNS tissue.

The cause of the different inheritance patterns associated with D90A mutant hSOD1 was investigated by analyzing erythrocytes from heterozygous individuals from dominant and recessive pedigrees. There was no evidence that a putative protective factor in recessive pedigrees acts by down-regulating the synthesis of D90A mutant hSOD1.

In cerebrospinal fluid, there was no difference in hSOD1 content between homozygous D90A patients, ALS patients without hSOD1 mutations and controls. hSOD1 cleaved at the N-terminal end was found in both controls and D90A patients, but the proportion was significantly larger in the latter group. This indicates a difference in degradation routes between mutant and wild-type hSOD1.

Both D90A and G127X transgenic mice develop an ALS-like phenotype. Similar to humans, the levels of D90A protein were high. The levels of G127X hSOD1 were very low in the tissues but enriched in the CNS. Similarly, in an ALS patient heterozygous for G127X hSOD1, the levels of the mutant protein were overall very low, but highest in affected CNS areas. Despite the very different levels of mutant hSOD1, both D90A and G127X transgenic mice developed similar levels of detergent-resistant aggregates in the spinal cord when terminally ill. Surprisingly, mice overexpressing wild-type hSOD1 also developed detergent-resistant aggregates, although less and later. Most of the hSOD1 in the CNS of transgenic mice was inactive due to deficient copper charging or because of reduced affinity for the metal. The stabilizing intrasubunit disulfide bond of hSOD1 was partially or completely absent in the different hSOD1s. Both these alterations could increase the propensity of mutant hSOD1s to misfold and form aggregates.

The results presented here suggest that the motor neuron degeneration caused by mutant hSOD1s may be attributable to long-term exposure to misfolded, aggregation-prone, disulfide-reduced hSOD1s and that the capacity to degrade such hSOD1s is lower in susceptible CNS areas compared with other tissues. The data also suggest that wild-type hSOD1 has the potential to participate in the pathogenesis of sporadic ALS.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap , 2005. , 119 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 981
Keyword [en]
Neurosciences, aggregates, ALS, amyotrophic lateral sclerosis, cerebrospinal fluid, disulfide-reduced, inclusions, misfolded, protective factor, SOD1, transgenic
Keyword [sv]
Neurovetenskap
National Category
Neurology
Research subject
Clinical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-611ISBN: 91-7305-936-6 (print)OAI: oai:DiVA.org:umu-611DiVA: diva2:143981
Public defence
2005-11-04, Sal B, 9 tr, 1D, Tandläkarhögskolan, 90185 NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2005-10-13 Created: 2005-10-13 Last updated: 2009-11-18Bibliographically approved
List of papers
1. CuZn-superoxide dismutase in D90A heterozygotes from recessive and dominant ALS pedigrees.
Open this publication in new window or tab >>CuZn-superoxide dismutase in D90A heterozygotes from recessive and dominant ALS pedigrees.
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2002 (English)In: Neurobiology of Disease, ISSN 0969-9961, Vol. 10, no 3, 327-333 p.Article in journal (Refereed) Published
Abstract [en]

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to ALS. In most cases ALS is inherited as a dominant trait and there is marked reduction in CuZn-SOD activity in samples from the patients. The D90A mutation, however, mostly causes ALS as a recessive trait and shows near normal CuZn-SOD activity. A few familial and sporadic ALS cases heterozygous for the D90A mutation have also been found. Haplotype analysis of both types of D90A families has suggested that all recessive cases share a common founder and may carry a protective factor located close to the D90A mutant CuZn-SOD locus. To search for effects of a putative protective factor we analysed erythrocytes from D90A heterozygous individuals for SOD activity by a direct assay, subunit composition by immunoblotting, and zymogram pattern formed by isoelectric focusing and SOD staining. Included were heterozygotes from 17 recessive families, and from 2 dominant families and 4 apparently sporadic cases. The CuZn-SOD activity in the recessive and dominant groups was found to be equal, and 95% of controls. The ratio between mutant and wildtype subunits was likewise equal and 0.8:1 in both groups. The zymograms revealed multiple bands representing homo- and heterodimers. There were, however, no differences between the groups in patterns or in ratios between the molecular forms. In conclusion we find no evidence from analyses in erythrocytes that the putative protective factor in recessive families acts by simply downregulating the synthesis or altering the molecular structure or turnover of the mutant enzyme.

Identifiers
urn:nbn:se:umu:diva-27728 (URN)10.1006/nbdi.2002.0508 (DOI)12270693 (PubMedID)
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2009-11-18
2. Superoxide dismutase in CSF from amyotrophic lateral sclerosis patients with and without CuZn-superoxide dismutase mutations
Open this publication in new window or tab >>Superoxide dismutase in CSF from amyotrophic lateral sclerosis patients with and without CuZn-superoxide dismutase mutations
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2001 (English)In: Brain, ISSN 0006-8950, Vol. 124, no 7, 1461-1466 p.Article in journal (Refereed) Published
Abstract [en]

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to familial amyotrophic lateral sclerosis (ALS), and motor neurone death is caused by the gain of a toxic property of the mutant protein. Here we determined amounts, activity and molecular forms of CuZn-SOD in CSF from ALS patients carrying the D90A and other CuZn-SOD mutations and patients without such mutations. There were no differences in amount of protein and enzymic activities of CuZn-SOD between 37 neurological controls, 54 sporadic and 12 familial ALS cases, and 10 cases homozygous for the D90A mutation. Three cases heterozygous for the A89V, S105L and G114A CuZn-SOD mutations showed low amounts of CuZn-SOD. There was no evidence for accumulation of inactive protein in any of the groups. Immunoblots showed no evidence for the presence of any precipitates or other molecular forms of CuZn-SOD with higher molecular weight in the groups. About 25% of the CuZn-SOD subunits in CSF from controls shows an N-terminal truncation. This truncated portion does not differ between controls and ALS groups not carrying CuZn-SOD mutations, but is 70% larger in samples from D90A homozygous ALS patients. The findings suggest an essentially normal amount and activity of D90A mutant CuZn-SOD in CNS tissues of ALS cases. The increased occurrence of N-terminally truncated mutant subunits may indicate a difference in degradation routes compared with the wild-type enzyme, resistance against subsequent proteolytic steps and/or a compromised downstream proteolytic machinery. Molecular fragments accumulated to a greater extent from the D90A mutant enzyme might contribute to the motor neurone degeneration. We also determined the other SOD isoenzymes: in the controls, CuZn-SOD contributed 75%, extracellular SOD 25% and Mn-SOD <5% of the total SOD activity. There was no difference in the amount of extracellular SOD between any of the groups.

Identifiers
urn:nbn:se:umu:diva-4749 (URN)10.1093/brain/124.7.1461 (DOI)11408340 (PubMedID)
Available from: 2005-10-13 Created: 2005-10-13Bibliographically approved
3. Motor neruon disase in mice expressing the wild-type like D90A mutant superoxide dismutase-1
Open this publication in new window or tab >>Motor neruon disase in mice expressing the wild-type like D90A mutant superoxide dismutase-1
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4750 (URN)
Available from: 2005-10-13 Created: 2005-10-13 Last updated: 2010-01-13Bibliographically approved
4. Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis.
Open this publication in new window or tab >>Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis.
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2004 (English)In: Brain, ISSN 0006-8950, Vol. 127, no Pt 1, 73-88 p.Article in journal (Refereed) Published
Abstract [en]

Mutant forms of superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS) by an unknown noxious mechanism. Using an antibody against a novel epitope in the G127insTGGG mutation, mutant SOD1 was studied for the first time in spinal cord and brain of an ALS patient. The level was below 0.5% of the SOD1 level in controls. In corresponding transgenic mice the content of mutant SOD1 was also low, although it was enriched in spinal cord and brain compared with other tissues. In the mice the misfolded mutant SOD1 aggregated rapidly and 20% occurred in steady state as detergent-soluble protoaggregates. The misfolded SOD1 and the protoaggregates form, from birth until death, a potentially noxious burden that may induce the motor neuron injury. Detergent-resistant aggregates, as well as inclusions of mutant SOD1 in motor neurons and astrocytes, accumulated in spinal cord ventral horns of the patient and mice with terminal disease. The inclusions and aggregates may serve as terminal markers of long-term assault by misfolded SOD1 and protoaggregates.

Keyword
Amyotrophic Lateral Sclerosis/enzymology/*genetics, Animals, Anterior Horn Cells/enzymology, Brain/enzymology, Detergents/pharmacology, Disease Models; Animal, Female, Humans, Mice, Mice; Transgenic, Middle Aged, Mutation, Phenotype, Solubility, Spinal Cord/enzymology, Superoxide Dismutase/drug effects/*genetics/immunology/metabolism, Survival Analysis
Identifiers
urn:nbn:se:umu:diva-15192 (URN)10.1093/brain/awh005 (DOI)14534160 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2009-11-18Bibliographically approved
5. Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models.
Open this publication in new window or tab >>Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models.
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2006 (English)In: Brain, ISSN 0006-8950, Vol. 129, no Pt 2, 451-644 p.Article in journal (Refereed) Published
Abstract [en]

Mutant forms of superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS) by an unknown noxious mechanism. Using an antibody against a novel epitope in the G127insTGGG mutation, mutant SOD1 was studied for the first time in spinal cord and brain of an ALS patient. The level was below 0.5% of the SOD1 level in controls. In corresponding transgenic mice the content of mutant SOD1 was also low, although it was enriched in spinal cord and brain compared with other tissues. In the mice the misfolded mutant SOD1 aggregated rapidly and 20% occurred in steady state as detergent-soluble protoaggregates. The misfolded SOD1 and the protoaggregates form, from birth until death, a potentially noxious burden that may induce the motor neuron injury. Detergent-resistant aggregates, as well as inclusions of mutant SOD1 in motor neurons and astrocytes, accumulated in spinal cord ventral horns of the patient and mice with terminal disease. The inclusions and aggregates may serve as terminal markers of long-term assault by misfolded SOD1 and protoaggregates.

Keyword
Amyotrophic Lateral Sclerosis/*enzymology, Animals, Central Nervous System/*enzymology, Copper/metabolism, Disulfides/metabolism, Humans, Mice, Mice; Transgenic, Models; Animal, Molecular Chaperones, Motor Neurons/*enzymology, Protein Conformation, Superoxide Dismutase/*genetics/metabolism
Identifiers
urn:nbn:se:umu:diva-15241 (URN)10.1093/brain/awh704 (DOI)16330499 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2009-11-18Bibliographically approved

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