| 1. |
- Paré, Bastien, et al.
(författare)
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Misfolded SOD1 pathology in sporadic Amyotrophic Lateral Sclerosis
- 2018
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation of mutant superoxide dismutase 1 (SOD1) is a pathological hallmark of a subset of familial ALS patients. However, the possible role of misfolded wild type SOD1 in human ALS is highly debated. To ascertain whether or not misfolded SOD1 is a common pathological feature in non-SOD1 ALS, we performed a blinded histological and biochemical analysis of post mortem brain and spinal cord tissues from 19 sporadic ALS, compared with a SOD1 A4V patient as well as Alzheimer's disease (AD) and non-neurological controls. Multiple conformation-or misfolded-specific antibodies for human SOD1 were compared. These were generated independently by different research groups and were compared using standardized conditions. Five different misSOD1 staining patterns were found consistently in tissue sections from SALS cases and the SOD1 A4V patient, but were essentially absent in AD and non-neurological controls. We have established clear experimental protocols and provide specific guidelines for working, with conformational/misfolded SOD1-specific antibodies. Adherence to these guidelines will aid in the comparison of the results of future studies and better interpretation of staining patterns. This blinded, standardized and unbiased approach provides further support for a possible pathological role of misSOD1 in SALS.
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| 2. |
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| 3. |
- Bergh, Johan, 1983-
(författare)
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Structural investigation of SOD1 aggregates in ALS : identification of prion strains using anti-peptide antibodies
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative syndrome characterized by progressive degeneration of motor neurons that result in muscle wasting. The symptoms advance gradually to paralysis and eventually death. Most patients suffer from sporadic ALS (sALS) but 10% report a familial predisposition. Mutations in the gene encoding superoxide dismutase-1 (SOD1) were the first identified cause of ALS. The disease mechanism is debated but there is a consensus that mutations in this protein confer a cytotoxic gain of function. SOD1 aggregates in motor neurons are hallmarks of ALS both in patients and in transgenic mouse models expressing a mutated form of human SOD1 (hSOD1). Recently, our group showed that SOD1 aggregates are present also in sALS patients, thus indicating a broader involvement of this protein in ALS. Misfolding and aggregation of SOD1 are difficult to study in vivo since aggregate concentration in the central nervous system (CNS) is exceedingly low. The aim of this thesis was to find a method circumventing this problem to investigate the hSOD1 aggregate structure, distribution and spread in ALS disease.Many studies provide circumstantial evidence that the wild-type hSOD1 protein can be neurotoxic. We developed the first homozygous mouse model that highly overexpresses the wild-type enzyme. These mice developed an ALS-like syndrome and become terminally ill after around 370 days. Motor neuron loss and SOD1 aggregate accumulation in the CNS were observed. This lends further support to the hypothesis of a more general involvement of SOD1 in human disease.A panel of polyclonal antibodies covering 90% of the SOD1 protein was developed by our laboratory. These antibodies were shown to be highly specific for misfolded SOD1. Aggregated hSOD1 was purified from the CNS of terminally ill hSOD1 mice. Disordered segments in aggregated hSOD1 could be identified with these antibodies. Two aggregate strains with different structural architectures, molecular properties, and growth kinetics, were found using this novel method. The strains, denoted A and B, were also associated with different disease progression. Aggregates formed in vitro were structurally different from these strains. The results gave rise to questions about aggregate development and possible prion-like spread. To investigate this, inoculations of purified strain A and B hSOD1 seeds was performed in lumbar spinal cords of 100-day old mice carrying a hSOD1G85R mutation. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill 200 days earlier than mice inoculated with control preparation. Interestingly, a templated spread of aggregates along the neuraxis was concomitantly observed, with strain A and B provoking the buildup of their respective hSOD1 aggregate structure. The phenotypes initiated by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. To further establish the importance of hSOD1 aggregates in human disease, purification and inoculation of aggregate seeds from spinal cords of ALS patients and mice carrying the hSOD1G127X mutation were performed. Inoculation of both human and mouse seeds as described above, induced strain A aggregation and premature fatal ALS-like disease.In conclusion, the data presented in this thesis provide a new, straightforward method for characterization of aggregate strains in ALS, and plausibly also in other neurodegenerative diseases. Two different prion strains of hSOD1 aggregates were identified in mice that resulted in ALS-like disease. Emerging data suggest that prion-like growth and spread of hSOD1 aggregation could be the primary pathogenic mechanism not only in hSOD1 transgenic models, but also in human ALS.
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| 4. |
- Canosa, Antonio, et al.
(författare)
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A novel p.Ser108LeufsTer15 SOD1 mutation leading to the formation of a premature stop codon in an apparently sporadic ALS patient : insights into the underlying pathomechanisms
- 2018
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Ingår i: Neurobiology of Aging. - : Elsevier. - 0197-4580 .- 1558-1497. ; 72
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Tidskriftsartikel (refereegranskat)abstract
- We report an apparently sporadic amyotrophic lateral sclerosis patient carrying a heterozygous novel frameshift SOD1 mutation (p.Ser108LeufsTer15), predicted to cause a premature protein truncation. RTPCR analysis of SOD1 mRNA and SDS-PAGE/Western blot analysis of PBMC demonstrated that mRNA from the mutant allele is expressed at levels similar to those of the wild-type allele, but the truncated protein is undetectable also in the insoluble fraction and after proteasome inhibition. Accordingly, the dismutation activity in erythrocytes is halved. Thus, the pathogenic mechanism associated with this mutation might be based on an insufficient activity of SOD1 that would make motor neurons more vulnerable to oxidative injury. However, it cannot be excluded that p.Ser108LeufsTer15 SOD1 is present in the nervous tissue and, being less charged and hence having less repulsive forces than the wild-type protein, may trigger toxic mechanisms as a consequence of its propensity to aggregate.
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| 5. |
- Ekhtiari Bidhendi, Elaheh, et al.
(författare)
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Mutant superoxide dismutase aggregates from human spinal cord transmit amyotrophic lateral sclerosis
- 2018
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Ingår i: Acta Neuropathologica. - : Springer. - 0001-6322 .- 1432-0533. ; 136:6, s. 939-953
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Tidskriftsartikel (refereegranskat)abstract
- Motor neurons containing aggregates of superoxide dismutase 1 (SOD1) are hallmarks of amyotrophic lateral sclerosis (ALS) caused by mutations in the gene encoding SOD1. We have previously reported that two strains of mutant human (h) SOD1 aggregates (denoted A and B) can arise in hSOD1-transgenic models for ALS and that inoculation of such aggregates into the lumbar spinal cord of mice results in rostrally spreading, templated hSOD1 aggregation and premature fatal ALS-like disease. Here, we explored whether mutant hSOD1 aggregates with prion-like properties also exist in human ALS. Aggregate seeds were prepared from spinal cords from an ALS patient carrying the hSOD1G127Gfs*7 truncation mutation and from mice transgenic for the same mutation. To separate from mono-, di- or any oligomeric hSOD1 species, the seed preparation protocol included ultracentrifugation through a density cushion. The core structure of hSOD1G127Gfs*7 aggregates present in mice was strain A-like. Inoculation of the patient- or mouse-derived seeds into lumbar spinal cord of adult hSOD1-expressing mice induced strain A aggregation propagating along the neuraxis and premature fatal ALS-like disease (p < 0.0001). Inoculation of human or murine control seeds had no effect. The potencies of the ALS patient-derived seed preparations were high and disease was initiated in the transgenic mice by levels of hSOD1G127Gfs*7 aggregates much lower than those found in the motor system of patients carrying the mutation. The results suggest that prion-like growth and spread of hSOD1 aggregation could be the primary pathogenic mechanism, not only in hSOD1 transgenic rodent models, but also in human ALS.
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| 6. |
- Ekhtiari Bidhendi, Elaheh, 1986-
(författare)
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SOD1 prions transmit templated aggregation and fatal ALS-like disease
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease characterized by a progressive degeneration of the upper and lower motor neurons. The resulting paresis begins focally, usually in one muscle, and spreads contiguously, leading to muscle wasting, progressive paralysis and eventually death. 90% of all ALS cases are sporadic, with no genetic background (sALS), while 10% are hereditary or familial (fALS). The first identified cause of ALS was mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1), which are found in 3-6% of the ALS patients. Mutations in SOD1 confer a cytotoxic gain of function on the enzyme. Cytosolic inclusions containing aggregated SOD1 in motor neurons are a hallmark of ALS, both in patients and transgenic (Tg) mice carrying mutant human SOD1s (hSOD1). These inclusions have also been reported in sporadic and familial ALS cases without SOD1 mutations, suggesting a broader role of this protein in the ALS pathology. However, the mechanism of SOD1 misfolding and aggregation, and their contribution to the disease pathogenesis, is unclear.Our research group has recently identified two structurally different strains of hSOD1 aggregates (denoted A and B) in the central nervous system of Tg murine models expressing full-length hSOD1 variants.The aim of this thesis is to investigate if the SOD1 aggregation is a collateral byproduct in the process of the disease, or if it drives ALS pathogenesis. In addition, this work investigates the spreading characteristic of the disease in vivo.Human SOD1 A and B seeds were prepared from spinal cords of terminally ill hSOD1 Tg mice by ultracentrifugation through a density gradient. Minute amounts of the aggregate seeds were micro-inoculated into the lumbar spinal cord of asymptomatic recipient Tg mice, overexpressing G85R mutant hSOD1 (hSOD1G85R). Mice inoculated with A or B aggregates developed early-onset fatal ALS-like disease, becoming terminally ill around 100 days after inoculation. This is nearly 200 days earlier than hSOD1G85R Tg mice inoculated with a control preparation or non-inoculated mice. Concomitantly, exponentially growing templated hSOD1 aggregation developed in the recipient mice, spreading all along the neuraxis. The pathology provoked by the A and B strains differed in aggregation growth rates, disease progression rates, aggregate distribution along the neuraxis, rates of weight loss, end-stage amounts of aggregates, and histopathology.Next, we explored the existence of mutant hSOD1 aggregates with prion-like properties in the spinal cord of ALS patients. To this end, aggregate seeds were prepared from the spinal cord of the autopsy material of an ALS patient carrying the hSOD1G127X truncation mutation, as well as from mice transgenic for the same mutation. The aggregates showed a strain A-like core structure. Inoculation of both the murine and human derived seeds into the lumbar spinal cord of hSOD1 expressing mice efficiently transmitted strain A aggregation, propagating rostrally throughout the neuraxis and causing premature fatal ALS-like disease. The inoculation of human or murine control seeds had no effect. The potency of the ALS patient-derived seed was exceedingly high, and the disease was initiated under conditions plausible to exist also in the human motor system. These results demonstrate for the first time, the presence of hSOD1 aggregates with prion-like properties in human ALS.We extended the exploration of hSOD1 prion mechanisms by inoculating another recipient mouse line, with wild-type-like stability and essentially normal SOD activity. Mice that are hemizygous for the hSOD1D90A transgene insertion do not develop ALS pathology and have normal murine lifespans (>700 days). Homozygous mice develop ALS-like disease around 400 days-of-age. Interestingly, inoculations of both strain A and B seeds into the lumbar spinal cord of hemizygous hSOD1D90A mice induced progressive hSOD1 aggregations and premature fatal ALS-like disease after around 250 and 350 days, respectively. In contrast, hemizygous hSOD1D90A mice inoculated with a mouse control seed died from senescence-related causes at ages beyond 700 days.Altogether, data in this thesis shows that the hSOD1 aggregate strains are ALS transmitting prions, suggesting that prion-like growth and spread of hSOD1 aggregation is the core pathogenic mechanism of SOD1-induced ALS.
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| 7. |
- Ekhtiari Bidhendi, Elaheh, et al.
(författare)
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Two superoxide dismutase prion strains transmit amyotrophic lateral sclerosis-like disease
- 2016
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 126:6, s. 2249-2253
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Tidskriftsartikel (refereegranskat)abstract
- Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.
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| 8. |
- Ingre, Caroline, et al.
(författare)
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A 50bp deletion in the SOD1 promoter lowers enzyme expression but is not associated with ALS in Sweden
- 2016
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Ingår i: Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. - : Informa UK Limited. - 2167-8421 .- 2167-9223. ; 17:5-6, s. 452-457
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population.
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| 9. |
- Keskin, Isil, et al.
(författare)
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Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in ALS patients and their relatives
- 2017
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Ingår i: Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. - : TAYLOR & FRANCIS LTD. - 2167-8421 .- 2167-9223. ; 18:5-6, s. 457-463
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations.Methods: Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay.Results: Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72(HRE). In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios.Conclusion: Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.
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| 10. |
- Keskin, Isil, et al.
(författare)
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Effects of Cellular Pathway Disturbances on Misfolded Superoxide Dismutase-1 in Fibroblasts Derived from ALS Patients
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.
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