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Misfolded superoxide dismutase-1 in sporadic and familial Amyotrophic Lateral Sclerosis
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Felveckat superoxid dismutas-1 i sporadisk och familiär amyotrofisk lateralskleros (Swedish)
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features.

The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder.

Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates.

In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2011. , 78 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1438
Keyword [en]
ALS, SOD-1 motor neuron, protein misfolding, intranuclear, antibodies, CNS, brain
National Category
Neurosciences
Research subject
Pathology
Identifiers
URN: urn:nbn:se:umu:diva-47550ISBN: 978-91-7459-256-6 (print)OAI: oai:DiVA.org:umu-47550DiVA: diva2:442928
Public defence
2011-10-14, Betula, Umeå University, Norrlands Universitetssjukhus, byggnad 6M, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-09-23 Created: 2011-09-22 Last updated: 2011-09-26Bibliographically approved
List of papers
1. Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients
Open this publication in new window or tab >>Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients
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2010 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 7, e11552- p.Article in journal (Refereed) Published
Abstract [en]

Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

Place, publisher, year, edition, pages
Public library of science, 2010
Keyword
amyotrophic-lateral-sclerosis; cu/zn superoxide-dismutase; motor-neuron degeneration; molecular pathology; gene mutation; linked SOD1; mutant SOD1; mice; disease; immunoreactivity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medicine
Identifiers
urn:nbn:se:umu:diva-40739 (URN)10.1371/journal.pone.0011552 (DOI)000279884900009 ()20644736 (PubMedID)
Available from: 2011-03-08 Created: 2011-03-08 Last updated: 2017-12-11Bibliographically approved
2. Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis
Open this publication in new window or tab >>Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis
2011 (English)In: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 121, no 5, 623-634 p.Article in journal (Refereed) Published
Abstract [en]

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from 9 ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia and oligodendrocytes in ALS patients carrying and as well as lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.

Place, publisher, year, edition, pages
SpringerLink, 2011
Keyword
Amyotrophic lateral sclerosis, superoxide dismutase, motoneurons, intranuclear, glia
National Category
Neurosciences
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-47510 (URN)10.1007/s00401-011-0805-3 (DOI)
Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2017-12-08Bibliographically approved
3. High expression of wild-type human superoxide dismutase-1 gives a model of sporadic ALS
Open this publication in new window or tab >>High expression of wild-type human superoxide dismutase-1 gives a model of sporadic ALS
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(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-47520 (URN)
Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2011-09-23Bibliographically approved
4. ALS patients with the SOD1 D90A mutation show both spinal cord and frontal cortical pathology
Open this publication in new window or tab >>ALS patients with the SOD1 D90A mutation show both spinal cord and frontal cortical pathology
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(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Research subject
Pathology
Identifiers
urn:nbn:se:umu:diva-47528 (URN)
Available from: 2011-09-22 Created: 2011-09-22 Last updated: 2011-09-23Bibliographically approved

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