| 1. |
- Bergemalm, Daniel, 1977-
(författare)
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Mutant superoxide dismutase-1-caused pathogenesis in amyotrophic lateral sclerosis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Wuolikainen, Anna, 1980-
(författare)
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Metabolomics studies of ALS : a multivariate search for clues about a devastating disease
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyotrophic lateral sclerosis (ALS), also known as Charcot’s disease, motor neuron disease (MND) and Lou Gehrig’s disease, is a deadly, adult-onset neurodegenerative disorder characterized by progressive loss of upper and lower motor neurons, resulting in evolving paresis of the linked muscles. ALS is defined by classical features of the disease, but may present as a wide spectrum of phenotypes. About 10% of all ALS cases have been reported as familial, of which about 20% have been associated with mutations in the gene encoding for CuZn superoxide dismutase (SOD1). The remaining cases are regarded as sporadic. Research has advanced our understanding of the disease, but the cause is still unknown, no reliable diagnostic test exists, no cure has been found and the current therapies are unsatisfactory. Riluzole (Rilutek®) is the only registered drug for the treatment of ALS. The drug has shown only a modest effect in prolonging life and the mechanism of action of riluzole is not yet fully understood. ALS is diagnosed by excluding diseases with similar symptoms. At an early stage, there are numerous possible diseases that may present with similar symptoms, thereby making the diagnostic procedure cumbersome, extensive and time consuming with a significant risk of misdiagnosis. Biomarkers that can be developed into diagnostic test of ALS are therefore needed. The high number of unsuccessful attempts at finding a single diseasespecific marker, in combination with the complexity of the disease, indicates that a pattern of several markers is perhaps more likely to provide a diagnostic signature for ALS. Metabolomics, in combination with chemometrics, can be a useful tool with which to study human disease. Metabolomics can screen for small molecules in biofluids such as cerebrospinal fluid (CSF) and chemometrics can provide structure and tools in order to handle the types of data generated from metabolomics. In this thesis, ALS has been studied using a combination of metabolomics and chemometrics. Collection and storage of CSF in relation to metabolite stability have been extensively evaluated. Protocols for metabolomics on CSF samples have been proposed, used and evaluated. In addition, a new feature of data processing allowing new samples to be predicted into existing models has been tested, evaluated and used for metabolomics on blood and CSF. A panel of potential biomarkers has been generated for ALS and subtypes of ALS. An overall decrease in metabolite concentration was found for subjects with ALS compared to their matched controls. Glutamic acid was one of the metabolites found to be decreased in patients with ALS. A larger metabolic heterogeneity was detected among SALS cases compared to FALS. This was also reflected in models of SALS and FALS against their respective matched controls, where no significant difference from control was found for SALS while the FALS samples significantly differed from their matched controls. Significant deviating metabolic patterns were also found between ALS subjects carrying different mutations in the gene encoding SOD1.
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| 3. |
- Ahmed, Niaz, et al.
(författare)
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Consensus statements and recommendations from the ESO-Karolinska Stroke Update Conference, Stockholm 11-13 November 2018.
- 2019
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Ingår i: European Stroke Journal. - : SAGE Publications. - 2396-9873 .- 2396-9881. ; 4:4, s. 307-317
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of the European Stroke Organisation-Karolinska Stroke Update Conference is to provide updates on recent stroke therapy research and to give an opportunity for the participants to discuss how these results may be implemented into clinical routine. The meeting started 22 years ago as Karolinska Stroke Update, but since 2014 it is a joint conference with European Stroke Organisation. Importantly, it provides a platform for discussion on the European Stroke Organisation guidelines process and on recommendations to the European Stroke Organisation guidelines committee on specific topics. By this, it adds a direct influence from stroke professionals otherwise not involved in committees and work groups on the guideline procedure. The discussions at the conference may also inspire new guidelines when motivated. The topics raised at the meeting are selected by the scientific programme committee mainly based on recent important scientific publications. This year's European Stroke Organisation-Karolinska Stroke Update Meeting was held in Stockholm on 11-13 November 2018. There were 11 scientific sessions discussed in the meeting including two short sessions. Each session except the short sessions produced a consensus statement (Full version with background, issues, conclusions and references are published as web-material and at www.eso-karolinska.org and http://eso-stroke.org) and recommendations which were prepared by a writing committee consisting of session chair(s), scientific secretary and speakers. These statements were presented to the 250 participants of the meeting. In the open meeting, general participants commented on the consensus statement and recommendations and the final document were adjusted based on the discussion from the general participants Recommendations (grade of evidence) were graded according to the 1998 Karolinska Stroke Update meeting with regard to the strength of evidence. Grade A Evidence: Strong support from randomised controlled trials and statistical reviews (at least one randomised controlled trial plus one statistical review). Grade B Evidence: Support from randomised controlled trials and statistical reviews (one randomised controlled trial or one statistical review). Grade C Evidence: No reasonable support from randomised controlled trials, recommendations based on small randomised and/or non-randomised controlled trials evidence.
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| 4. |
- 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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| 5. |
- 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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| 6. |
- Forsgren, Elin, 1987-
(författare)
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Using patient-derived cell models to investigate the role of misfolded SOD1 in ALS
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein misfolding and aggregation underlie several neurodegenerative proteinopathies including amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) was the first gene found to be associated with familial ALS. Overexpression of human mutant or wild type SOD1 in transgenic mouse models induces motor neuron (MN) degeneration and an ALS-like phenotype. SOD1 mutations, leading to the destabilization of the SOD1 protein is associated with ALS pathogenesis. However, how misfolded SOD1 toxicity specifically affects human MNs is not clear. The aim of this thesis was to develop patient-derived, cellular models of ALS to help understand the pathogenic mechanisms underlying SOD1.To understand which cellular pathways impact on the level of misfolded SOD1 in human cells, we established a model using patient-derived fibroblasts and quantified misfolded SOD1 in relation to disturbances in several ALS-related cellular pathways. Misfolded SOD1 levels did not change following reduction in autophagy, inhibition of the mitochondrial respiratory chain, or induction of endoplasmic reticulum (ER)-stress. However, inhibition of the ubiquitin-proteasome system (UPS) lead to a dramatic increase in misfolded SOD1 levels. Hence, an age-related decline in proteasome activity might underlie the late-life onset that is typically seen in SOD1 ALS.To address whether or not SOD1 misfolding is enhanced in human MNs, we used mixed MN/astrocyte cultures (MNCs) generated in vitro from patient-specific induced pluripotent stem cells (iPSCs). Levels of soluble misfolded SOD1 were increased in MNCs as well as in pure iPSC-derived astrocytes compared to other cell types, including sensory neuron cultures. Interestingly, this was the case for both mutant and wild type human SOD1, although the increase was enhanced in SOD1 FALS MNCs. Misfolded SOD1 was also found to exist in the same form as in mouse SOD1 overexpression models and was identified as a substrate for 20S proteasome degradation. Hence, the vulnerability of motor areas to ALS could be explained by increased SOD1 misfolding, specifically in MNs and astrocytes.To investigate factors that might promote SOD1 misfolding, we focussed on the stability of SOD1 mediated by a crucial, stabilizing C57-C146 disulphide bond and its redox status. Formation of disulphide bond is dependent on oxidation by O2 and catalysed by CCS. To investigate whether low O2 tension affects the stability of SOD1 in vitro we cultured fibroblasts and iPSC-derived MNCs under different oxygen tensions. Low oxygen tension promoted disulphide-reduction, SOD1 misfolding and aggregation. This response was much greater in MNCs compared to fibroblasts, suggesting that MNs may be especially sensitive to low oxygen tension and areas with low oxygen supply could serve as foci for ALS initiation.SOD1 truncation mutations often lack C146, and cannot adopt a native fold and are rapidly degraded. We characterized soluble misfolded and aggregated SOD1 in patient-derived cells carrying a novel SOD1 D96Mfs*8 mutation as well as in cells fom an unaffected mutation carrier. The truncated protein has a C-terminal fusion of seven non-native amino acids and was found to be extremely prone to aggregation in vitro. Since not all mutation carriers develop ALS, our results suggested this novel mutation is associated with reduced penetrance.In summary, patient derived cells are useful models to study factors affecting SOD1 misfolded and aggregation. We show for the first time that misfolding of a disordered and disease associated protein is enhanced in disease-related cell types. Showing that misfolded SOD1 exists in human cells in the same form as in transgenic mouse models strengthens the translatability of results obtained in the two species. Our results demonstrate disulphide-reduction and misfolding/aggregation of SOD1 and suggest that 20S proteasome could be an important therapeutic target for early stages of disease. This model provides a great opportunity to study pathogenic mechanisms of both familial and sporadic ALS in patient-derived models of ALS.
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| 7. |
- Oxfeldt, Mikkel, et al.
(författare)
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Low Energy Availability Followed by Optimal Energy Availability Does Not Benefit Performance in Trained Females
- 2024
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Ingår i: Medicine & Science in Sports & Exercise. - : Lippincott Williams & Wilkins. - 0195-9131 .- 1530-0315. ; 56:5, s. 902-916
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Short periods of reduced energy availability are commonly undertaken by athletes to decrease body mass, possibly improve the power-to-mass ratio, and enhance physical performance. Our primary aim was to investigate the impact of 10 d of low energy availability (LEA) followed by 2 d of optimal energy availability (OEA) on physical performance parameters in trained females. Second, physiological markers at the whole-body and molecular level related to performance were evaluated. Methods: Thirty young trained eumenorrheic females were matched in pairs based on training history and randomized to a 10-d intervention period of LEA (25 kcal.fat-free mass (FFM)(-1).d(-1)) or OEA (50 kcal.FFM-1.d(-1)) along with supervised exercise training. Before the intervention, participants underwent a 5-d run-in period with OEA + supervised exercise training. After the LEA intervention, 2 d of recovery with OEA was completed. Participants underwent muscle biopsies, blood sampling, physical performance tests, body composition measurements, and resting metabolic rate measurements. A linear mixed model was used with group and time as fixed effects and subject as random effects. Results: Compared with OEA, LEA resulted in reduced body mass, muscle glycogen content, repeated sprint ability, 4-min time-trial performance, and rate of force development of the knee extensors (absolute values; P < 0.05). Two days of recovery restored 4-min time-trial performance and partly restored repeated sprint ability, but performance remained inferior to the OEA group. When the performance data were expressed relative to body mass, LEA did not enhance performance. Conclusions: Ten days of LEA resulted in impaired performance (absolute values), with concomitant reductions in muscle glycogen. Two days of recovery with OEA partially restored these impairments, although physical performance (absolute values) was still inferior to being in OEA. Our findings do not support the thesis that LEA giving rise to small reductions in body mass improves the power-to-mass ratio and thus increases physical performance.
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