| 1. |
- Achour, Cyrinne, 1991-
(författare)
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Canonical and non-canonical functions of METTL3 in breast cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gene expression is spatially and temporally regulated at multiple levels. N6-methyladenosine (m6A) is the most prevalent internal modification in messenger RNA (mRNA) and long noncoding RNA (lncRNAs). m6A plays important roles in multiple cellular processes including stem cell pluripotency, adipogenesis, spermatogenesis, neurogenesis, circadian rhythm and development by modulating RNA splicing, export, stability, degradation and translation. Although aberrant m6A methylation has been reported in various types of cancer, the underlying molecular functions of METTL3, the solely catalytic subunit of the m6A-methylase complex, has yet to be defined.m6A has been recently identified in nascent pre-mRNA, and more specifically intronic m6A has been linked to exon skipping events. The occurrence of impaired alternative splicing (AS) is frequently found during the development of cancer. We performed transcriptome wide analysis in breast cancer cell lines and explored AS events. Our results define an AS signature for breast tumorigenesis. We found that METTL3 modulates AS directly through m6A deposition at the intron-exon junctions or indirectly by the m6A deposition in transcripts encoding for splicing factors and transcription factors. In particular, we show that MYC mRNA harbours the m6A mark, suggesting that METTL3 regulates AS indirectly via the regulation of MYC expression. Indeed, the targets of MYC overlapped with METTL3-associated AS events. Importantly, five of the AS events identified and validated in vitro, are linked to a worse prognosis in breast cancer patients. Additionally, we show that METTL3 enhances the breast cancer phenotype through a dual mechanism depending on its sub-cellular localization. We find that the canonical nuclear function of METTL3 decorates transcripts that are involved in cell proliferation and migration. We observe that METTL3 is highly expressed in the cytoplasmic compartment of breast cancer cells from patients. Remarkably, we uncover that the cytoplasmic METTL3 interacts with subunits of the exocyst, whose subunit EXOC7 has been linked to cell adhesion, migration and invasion. Notably, we show that breast cancer cell lines depleted of METTL3 display less gelatinase activity and invadopodia formation, supporting the role of METTL3 in cell invasion via exocytosis.m6A is a reversible modification, which can be demethylated by the erasers FTO and ALKBH5. Depletion of FTO has been shown to increase the level of m6A in mRNA, however recent studies have reported that FTO could demethylate N6,2´-O-dimethyladenosine (m6Am), adjacent to the 7-methylguanosine cap on mRNA. In the cellular model of colorectal cancer CRC1, depletion of FTO leads to a cancer stem cell phenotype and confers chemotherapy resistance. By performing m6A-RNA immunoprecipitation followed by sequencing (MeRIP), we show that knockdown of FTO in CRC1 cells does not affect the global level of m6A in mRNA but of m6Am level.
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| 2. |
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| 3. |
- Alhouayek, Mireille, et al.
(författare)
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Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panxl substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [H-3]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.
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| 4. |
- Andreae, Laura C, et al.
(författare)
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Analysis of Lrrn1 expression and its relationship to neuromeric boundaries during chick neural development
- 2007
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Ingår i: Neural Development. - : Springer Science and Business Media LLC. - 1749-8104. ; 2:22, s. 1-16
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The Drosophila leucine-rich repeat proteins Tartan (TRN) and Capricious (CAPS) mediate cell affinity differences during compartition of the wing imaginal disc. This study aims to identify and characterize the expression of a chick orthologue of TRN/CAPS and examine its potential function in relation to compartment boundaries in the vertebrate central nervous system.RESULTS: We identified a complementary DNA clone encoding Leucine-rich repeat neuronal 1 (Lrrn1), a single-pass transmembrane protein with 12 extracellular leucine-rich repeats most closely related to TRN/CAPS. Lrrn1 is dynamically expressed during chick development, being initially localized to the neural plate and tube, where it is restricted to the ventricular layer. It becomes downregulated in boundaries following their formation. In the mid-diencephalon, Lrrn1 expression prefigures the position of the anterior boundary of the zona limitans intrathalamica (ZLI). It becomes progressively downregulated from the presumptive ZLI just before the onset of expression of the signalling molecule Sonic hedgehog (Shh) within the ZLI. In the hindbrain, downregulation at rhombomere boundaries correlates with the emergence of specialized boundary cell populations, in which it is subsequently reactivated. Immunocolocalization studies confirm that Lrrn1 protein is endocytosed from the plasma membrane and is a component of the endosomal system, being concentrated within the early endosomal compartment.CONCLUSION: Chick Lrrn1 is expressed in ventricular layer neuroepithelial cells and is downregulated at boundary regions, where neurogenesis is known to be delayed, or inhibited. The timing of Lrrn1 downregulation correlates closely with the activation of signaling molecule expression at these boundaries. This expression is consistent with the emergence of secondary organizer properties at boundaries and its endosomal localisation suggests that Lrrn1 may regulate the subcellular localisation of specific components of signalling or cell-cell recognition pathways in neuroepithelial cells.
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| 5. |
- Andreae, Laura C., et al.
(författare)
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Chick Lrrn2, a novel downstream effector of Hoxb1 and Shh, functions in the selective targeting of rhombomere 4 motor neurons
- 2009
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Ingår i: Neural development. - : Springer Science and Business Media LLC. - 1749-8104. ; 4, s. 27-
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Tidskriftsartikel (refereegranskat)abstract
- Background; Capricious is a Drosophila adhesion molecule that regulates specific targeting of a subset of motor neurons to their muscle target. We set out to identify whether one of its vertebrate homologues, Lrrn2, might play an analogous role in the chick.Results; We have shown that Lrrn2 is expressed from early development in the prospective rhombomere 4 (r4) of the chick hindbrain. Subsequently, its expression in the hindbrain becomes restricted to a specific group of motor neurons, the branchiomotor neurons of r4, and their pre-muscle target, the second branchial arch (BA2), along with other sites outside the hindbrain. Misexpression of the signalling molecule Sonic hedgehog (Shh) via in ovo electroporation results in upregulation of Lrrn2 exclusively in r4, while the combined expression of Hoxb1 and Shh is sufficient to induce ectopic Lrrn2 in r1/2. Misexpression of Lrrn2 in r2/3 results in axonal rerouting from the r2 exit point to the r4 exit point and BA2, suggesting a direct role in motor axon guidance.Conclusion; Lrrn2 acts downstream of Hoxb1 and plays a role in the selective targeting of r4 motor neurons to BA2.
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| 6. |
- Aparicio, S, et al.
(författare)
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Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes.
- 1995
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 92:5, s. 1684-1688
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Tidskriftsartikel (refereegranskat)abstract
- Comparative vertebrate genome sequencing offers a powerful method for detecting conserved regulatory sequences. We propose that the compact genome of the teleost Fugu rubripes is well suited for this purpose. The evolutionary distance of teleosts from other vertebrates offers the maximum stringency for such evolutionary comparisons. To illustrate the comparative genome approach for F. rubripes, we use sequence comparisons between mouse and Fugu Hoxb-4 noncoding regions to identify conserved sequence blocks. We have used two approaches to test the function of these conserved blocks. In the first, homologous sequences were deleted from a mouse enhancer, resulting in a tissue-specific loss of activity when assayed in transgenic mice. In the second approach, Fugu DNA sequences showing homology to mouse sequences were tested for enhancer activity in transgenic mice. This strategy identified a neural element that mediates a subset of Hoxb-4 expression that is conserved between mammals and teleosts. The comparison of noncoding vertebrate sequences with those of Fugu, coupled to a transgenic bioassay, represents a general approach suitable for many genome projects.
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| 7. |
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| 8. |
- Bergman, Joakim, et al.
(författare)
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Intrathecal treatment trial of rituximab in progressive MS: An open-label phase 1b study
- 2018
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Ingår i: Neurology. - : Wolters Kluwer. - 1526-632X .- 0028-3878. ; 91:20
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To perform a phase 1b assessment of the safety and feasibility of intrathecally delivered rituximab as a treatment for progressive multiple sclerosis (PMS) and to evaluate the effect of treatment on disability and CSF biomarkers during a 1-year follow-up period. METHODS: Three doses of rituximab (25 mg with a 1-week interval) were administered in 23 patients with PMS via a ventricular catheter inserted into the right frontal horn and connected to a subcutaneous Ommaya reservoir. Follow-ups were performed at 1, 3, 6, 9, and 12 months. RESULTS: Mild to moderate vertigo and nausea were common but temporary adverse events associated with intrathecal rituximab infusion, which was otherwise well tolerated. The only severe adverse event was a case of low-virulent bacterial meningitis that was treated effectively. Of 7 clinical assessments, only 1 showed statistically significant improvement 1 year after treatment. No treatment effect was observed during the follow-up period among 6 CSF biomarkers. CONCLUSIONS: Intrathecal administration of rituximab was well tolerated. However, it may involve a risk for injection-related infections. The lack of a control group precludes conclusions being drawn regarding treatment efficacy. CLINICALTRIALSGOV IDENTIFIER: NCT01719159. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that intrathecal rituximab treatment is well tolerated and feasible in PMS but involves a risk of severe infections. © 2018 American Academy of Neurology.
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| 9. |
- Bergman, Joakim, et al.
(författare)
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Neurofilament light in CSF and serum is a sensitive marker for axonal white matter injury in MS.
- 2016
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Ingår i: Neurology® neuroimmunology & neuroinflammation. - 2332-7812. ; 3:5
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Tidskriftsartikel (refereegranskat)abstract
- In an ongoing, open-label, phase 1b study on the intrathecal administration of rituximab for progressive multiple sclerosis, an intraventricular catheter was inserted for drug delivery. The objective of this study was to characterize the limited white matter axonal injury evoked by catheter insertion by analyzing a panel of markers for tissue damage in CSF and serum.
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| 10. |
- Bergman, Joakim, 1989-
(författare)
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Studies of the Biology of Intrathecal Treatment in Progressive MS
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Multiple Sclerosis (MS) is a chronic, inflammatory, autoimmune disease, affecting the central nervous system (CNS). About 85% of afflicted present with a relapsing-remitting form of the disease (RRMS), for which a breakthrough in treatment was made in 2008 with rituximab, an antibody directed towards CD20, a surface antigen on B-cells. These findings also contributed to cementing the importance of the B-cell’s role in MS pathophysiology. However, MS also exist as a progressive phenotype, affecting most MS patients either from onset or after a transition from RRMS, and for progressive MS the same treatment effect of anti-CD20 has not been observed. Still, studies have found follicle-like structures containing B-cells in meninges and subarachnoid space of the cortex in progressive MS brains, supporting the involvement of B-cells. Evidence also support the existence of a chronic, low-grade inflammatory process compartmentalised within the CNS that correlates with the progressive phase of MS, which may present a treatment barrier towards anti-CD20. Peripherally administrated therapeutic antibodies cross the intact blood-brain barrier with low efficiency with only 0.1-0.5% of the plasma concentration occurring in the cerebrospinal fluid (CSF). Intrathecal (IT) administration circumvents the blood-brain barrier, presenting an opportunity to better target the CNS B-cells.Aims: To evaluate the safety and feasibility of intrathecal anti-CD20 therapy with rituximab in progressive MS, its effect on disease progression through clinical parameters, and impact on biomarkers in CSF. Furthermore, this thesis aimed to evaluate the effect on biomarkers representative of cell injury related to insertion of a ventricular catheter for drug administration and to examine the interstitial milieu in the brain through microdialysis (MD).Methods: The thesis is based on the open-label, phase IIb, multicentre clinical trial Intrathecal Treatment Trial in Progressive Multiple Sclerosis (ITT-PMS; EudraCT 2008-002626-11), in which 23 participants received IT treatment with rituximab, and the extended follow-up study, ITT-PMS extension (EudraCT 2012-000721-53). All participants received a ventricular catheter and an Ommaya reservoir for drug administration through a neurosurgical procedure, and 10 participants received a MD catheter in parallel to the ventricular catheter for 10 days. The treatment effect was evaluated by regular clinical evaluations and analyses of CSF. The clinical outcome was evaluated through walking and upper-limb function tests, and by clinical evaluation scales. Levels of selected CSF biomarkers were analysed from the same time-points as the clinical evaluations.Results: After the completion of the extension trial, one clinical parameter (cognitive performance) showed improvement but could most likely be explained by a learning effect. Worsening of walking speed was observed, while the remaining clinical parameters showed no change. Two severe adverse events occurred in the form of low-virulent bacterial meningitis caused by Propionibacterium, but both were treated effectively with antibiotics without residual symptoms. A ‘spike’ was noticed in the level of lumbar CSF neurofilament light (NFL) following surgery but returned to pre-surgery baseline within 6-12 months. No change was observed for any of the other lumbar CSF biomarkers. No meaningful correlation of protein levels was observed when comparing MD samples, ventricular CSF, and lumbar CSF.Conclusions: Intrathecal treatment through intraventricular administration was well tolerated but not without risks. A continued progression was observed in gait impairment. The insertion of the ventricular catheter caused white matter injury, measured through an increase in NFL in lumbar CSF, in direct association with the surgical procedure. No impact was observed on other CSF biomarkers. There was a poor correlation between different CNS compartments regarding protein levels, arguing for caution in drawing conclusions about brain pathophysiology from lumbar CSF samples.
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| 11. |
- Brend, Tim, et al.
(författare)
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Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression
- 2003
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 130:12, s. 2717-2728
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Tidskriftsartikel (refereegranskat)abstract
- Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3' untranslated region (region B) are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4 expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3'-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4 transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.
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| 12. |
- Broom, Emma R, et al.
(författare)
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The roof plate boundary is a bi-directional organiser of dorsal neural tube and choroid plexus development.
- 2012
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 139:22, s. 4261-4270
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Tidskriftsartikel (refereegranskat)abstract
- The roof plate is a signalling centre positioned at the dorsal midline of the central nervous system and generates dorsalising morphogenic signals along the length of the neuraxis. Within cranial ventricles, the roof plate gives rise to choroid plexus, which regulates the internal environment of the developing and adult brain and spinal cord via the secretion of cerebrospinal fluid. Using the fourth ventricle as our model, we show that the organiser properties of the roof plate are determined by its boundaries with the adjacent neuroepithelium. Through a combination of in ovo transplantation, co-culture and electroporation techniques in chick embryos between embryonic days 3 and 6, we demonstrate that organiser properties are maintained by interactions between the non-neural roof plate and the neural rhombic lip. At the molecular level, this interaction is mediated by Delta-Notch signalling and upregulation of the chick homologue of Hes1: chairy2. Gain- and loss-of-function approaches reveal that cdelta1 is both necessary and sufficient for organiser function. Our results also demonstrate that while chairy2 is specifically required for the maintenance of the organiser, its ectopic expression is not sufficient to recapitulate organiser properties. Expression of atonal1 in the rhombic lip adjacent at the roof plate boundary is acutely dependent on both boundary cell interactions and Delta-Notch signalling. Correspondingly, the roof plate boundary organiser also signals to the roof plate itself to specify the expression of early choroid plexus markers. Thus, the roof plate boundary organiser signals bi-directionally to acutely coordinate the development of adjacent neural and non-neural tissues.
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| 13. |
- Coutinho, Ana P, et al.
(författare)
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Induction of a parafacial rhythm generator by rhombomere 3 in the chick embryo.
- 2004
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Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 24:42, s. 9383-9390
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Tidskriftsartikel (refereegranskat)abstract
- Observations of knock-out mice suggest that breathing at birth requires correct development of a specific hindbrain territory corresponding to rhombomeres (r) 3 and 4. Focusing on this territory, we examined the development of a neuronal rhythm generator in the chick embryo. We show that rhythmic activity in r4 is inducible after developmental stage 10 through interaction with r3. Although the nature of this interaction remains obscure, we find that the expression of Krox20, a segmentation gene responsible for specifying r3 and r5, is sufficient to endow other rhombomeres with the capacity to induce rhythmic activity in r4. Induction is robust, because it can be reproduced with r2 and r6 instead of r4 and with any hindbrain territory that normally expresses Krox20 (r3, r5) or can be forced to do so (r1, r4). Interestingly, the interaction between r4 and r3/r5 that results in rhythm production can only take place through the anterior border of r4, revealing a heretofore unsuspected polarity in individual rhombomeres. The r4 rhythm generator appears to be homologous to a murine respiratory parafacial neuronal system developing in r4 under the control of Krox20 and Hoxa1. These results identify a late role for Krox20 at the onset of neurogenesis.
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| 14. |
- Cunningham, Thomas J., et al.
(författare)
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DNA Editing for Amyotrophic Lateral Sclerosis : Leading Off First Base
- 2020
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Ingår i: The CRISPR Journal. - : Mary Ann Liebert. - 2573-1599 .- 2573-1602. ; 3:2, s. 75-77
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Gene therapy in a mouse model for amyotrophic lateral sclerosis (ALS) illustrates the rapid deployment of base editing in therapeutic modeling of neurodegenerative disease.
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| 15. |
- Devoy, Anny, et al.
(författare)
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Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models
- 2021
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 24:12
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Tidskriftsartikel (refereegranskat)abstract
- Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.
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| 16. |
- Falker-Gieske, Clemens, et al.
(författare)
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Resistance to chicken amyloid arthropathy is associated with a dysfunctional mutation in serum amyloid A
- 2023
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Ingår i: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 37:1
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Tidskriftsartikel (refereegranskat)abstract
- Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.
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| 17. |
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| 18. |
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| 19. |
- 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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| 20. |
- Gilthorpe, Jonathan, et al.
(författare)
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Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia.
- 2013
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Ingår i: F1000Research. - : F1000 Research Ltd. - 2046-1402. ; 2:148
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Tidskriftsartikel (refereegranskat)abstract
- In neurodegenerative conditions and following brain trauma it is not understood why neurons die while astrocytes and microglia survive and adopt pro-inflammatory phenotypes. We show here that the damaged adult brain releases diffusible factors that can kill cortical neurons and we have identified histone H1 as a major extracellular candidate that causes neurotoxicity and activation of the innate immune system. Extracellular core histones H2A, H2B H3 and H4 were not neurotoxic. Innate immunity in the central nervous system is mediated through microglial cells and we show here for the first time that histone H1 promotes their survival, up-regulates MHC class II antigen expression and is a powerful microglial chemoattractant. We propose that when the central nervous system is degenerating, histone H1 drives a positive feedback loop that drives further degeneration and activation of immune defences which can themselves be damaging. We suggest that histone H1 acts as an antimicrobial peptide and kills neurons through mitochondrial damage and apoptosis.
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| 21. |
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| 22. |
- Gilthorpe, Jonathan, et al.
(författare)
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Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY.
- 2002
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Ingår i: Development. - 0950-1991 .- 1477-9129. ; 129:16, s. 3887-99
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Tidskriftsartikel (refereegranskat)abstract
- Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.
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| 23. |
- Gilthorpe, Jonathan, et al.
(författare)
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The migration of cerebellar rhombic lip derivatives.
- 2002
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Ingår i: Development. - : F1000 Research Ltd. - 0950-1991 .- 1477-9129. ; 129:20, s. 4719-4728
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Tidskriftsartikel (refereegranskat)abstract
- We have used cell labelling, co-culture and time-lapse confocal microscopy to investigate tangential neuronal migration from the rhombic lip. Cerebellar rhombic lip derivatives demonstrate a temporal organisation with respect to their morphology and response to migration cues. Early born cells, which migrate into ventral rhombomere 1, have a single long leading process that turns at the midline and becomes an axon. Later born granule cell precursors also migrate ventrally but halt at the lateral edge of the cerebellum, correlating with a loss of sensitivity to netrin 1 and expression of Robo2. The rhombic lip and ventral midline express Slit2 and both early and late migrants are repelled by sources of Slit2 in co-culture. These studies reveal an intimate relationship between birthdate, response to migration cues and neuronal fate in an identified population of migratory cells. The use of axons in navigating cell movement suggests that tangential migration is an elaboration of the normal process of axon extension.
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| 24. |
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| 25. |
- Günther, René, et al.
(författare)
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Alteration of Mitochondrial Integrity as Upstream Event in the Pathophysiology of SOD1-ALS
- 2022
-
Ingår i: Cells. - : MDPI. - 2073-4409. ; 11:7
-
Tidskriftsartikel (refereegranskat)abstract
- Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.
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| 26. |
- Gutman, Alejandro, et al.
(författare)
-
Multiple positive and negative regulatory elements in the promoter of the mouse homeobox gene Hoxb-4.
- 1994
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Ingår i: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 14:12, s. 8143-8154
-
Tidskriftsartikel (refereegranskat)abstract
- Mouse Hoxb-4 (Hox-2.6) is a homeobox gene that belongs to a family which also includes Hoxa-4, Hoxc-4, and Hoxd-4 and that is related to the Deformed gene in Drosophila melanogaster. We have determined the sequence of 1.2 kb of 5' flanking DNA of mouse Hoxb-4 and by nuclease S1 and primer extension experiments identified two transcription start sites, P1 and P2, 285 and 207 nucleotides upstream of the ATG initiator codon, respectively. We have shown that this region harbors two independent promoters which drive CAT expression in several different cell lines with various efficiencies, suggesting that they are subject to cell-type-specific regulation. Through detailed mutational analysis, we have identified several cis-regulatory elements, located upstream and downstream of the transcription start sites. They include two cell-type-specific negative regulatory elements, which are more active in F9 embryonal carcinoma cells than in neuroblastoma cells (regions a and d at -226 to -186 and +169 to +205, respectively). An additional negative regulatory element has been delimited (region b between +22 and +113). Positive regulation is achieved by binding of HoxTF, a previously unknown factor, to the sequence GCCATTGG (+148 to +155) that is essential for efficient Hoxb-4 expression. We have also defined the minimal promoter sequences and found that they include two 12-bp initiator elements centered around each transcription start site. The complex architecture of the Hoxb-4 promoter provides the framework for fine-tuned transcriptional regulation during embryonic development.
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| 27. |
- 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
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:2
-
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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| 28. |
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| 29. |
- Keskin, Isil, 1987-, et al.
(författare)
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The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension
- 2019
-
Ingår i: Acta Neuropathologica. - New York : Springer. - 0001-6322 .- 1432-0533. ; 138:1, s. 85-101
-
Tidskriftsartikel (refereegranskat)abstract
- Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low oxygen tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression.
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| 30. |
- Khademi, Mohsen, et al.
(författare)
-
Intense Inflammation and Nerve Damage in Early Multiple Sclerosis Subsides at Older Age : A Reflection by Cerebrospinal Fluid Biomarkers
- 2013
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:5, s. e63172-
-
Tidskriftsartikel (refereegranskat)abstract
- Inflammatory mediators have crucial roles in leukocyte recruitment and subsequent central nervous system (CNS) neuroinflammation. The extent of neuronal injury and axonal loss are associated with the degree of CNS inflammation and determine physical disability in multiple sclerosis (MS). The aim of this study was to explore possible associations between a panel of selected cerebrospinal fluid biomarkers and robust clinical and demographic parameters in a large cohort of patients with MS and controls (n = 1066) using data-driven multivariate analysis. Levels of matrix metalloproteinase 9 (MMP9), chemokine (C-X-C motif) ligand 13 (CXCL13), osteopontin (OPN) and neurofilament-light chain (NFL) were measured by ELISA in 548 subjects comprising different MS subtypes (relapsing-remitting, secondary progressive and primary progressive), clinically isolated syndrome and persons with other neurological diseases with or without signs of inflammation/infection. Principal component analyses and orthogonal partial least squares methods were used for unsupervised and supervised interrogation of the data. Models were validated using data from a further 518 subjects in which one or more of the four selected markers were measured. There was a significant association between increased patient age and lower levels of CXCL13, MMP9 and NFL. CXCL13 levels correlated well with MMP9 in the younger age groups, but less so in older patients, and after approximately 54 years of age the levels of CXCL13 and MMP9 were consistently low. CXCL13 and MMP9 levels also correlated well with both NFL and OPN in younger patients. We demonstrate a strong effect of age on both inflammatory and neurodegenerative biomarkers in a large cohort of MS patients. The findings support an early use of adequate immunomodulatory disease modifying drugs, especially in younger patients, and may provide a biological explanation for the relative inefficacy of such treatments in older patients at later disease stages.
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| 31. |
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| 32. |
- Knyazeva, Anastasia, 1995-, et al.
(författare)
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A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation
- 2024
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 121:17
-
Tidskriftsartikel (refereegranskat)abstract
- The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.
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| 33. |
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| 34. |
- Lehmann, Manuela, et al.
(författare)
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Aggregate-selective antibody attenuates seeded aggregation but not spontaneously evolving disease in SOD1 ALS model mice
- 2020
-
Ingår i: Acta neuropathologica communications. - : BMC. - 2051-5960. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Increasing evidence suggests that propagation of the motor neuron disease amyotrophic lateral sclerosis (ALS) involves the pathogenic aggregation of disease-associated proteins that spread in a prion-like manner. We have identified two aggregate strains of human superoxide dismutase 1 (hSOD1) that arise in the CNS of transgenic mouse models of SOD1-mediated ALS. Both strains transmit template-directed aggregation and premature fatal paralysis when inoculated into the spinal cord of adult hSOD1 transgenic mice. This spread of pathogenic aggregation could be a potential target for immunotherapeutic intervention. Here we generated mouse monoclonal antibodies (mAbs) directed to exposed epitopes in hSOD1 aggregate strains and identified an aggregate selective mAb that targets the aa 143–153 C-terminal extremity of hSOD1 (αSOD1143–153). Both pre-incubation of seeds with αSOD1143–153 prior to inoculation, and weekly intraperitoneal (i.p.) administration attenuated transmission of pathogenic aggregation and prolonged the survival of seed-inoculated hSOD1G85R Tg mice. In contrast, administration of a mAb targeting aa 65–72 (αSOD165–72), which exhibits high affinity towards monomeric disordered hSOD1, had an adverse effect and aggravated seed induced premature ALS-like disease. Although the mAbs reached similar concentrations in CSF, only αSOD1143–153 was found in association with aggregated hSOD1 in spinal cord homogenates. Our results suggest that an aggregate-selective immunotherapeutic approach may suppress seeded transmission of pathogenic aggregation in ALS. However, long-term administration of αSOD1143–153 was unable to prolong the lifespan of non-inoculated hSOD1G85R Tg mice. Thus, spontaneously initiated hSOD1 aggregation in spinal motor neurons may be poorly accessible to therapeutic antibodies.
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| 35. |
- Lehmann, Manuela, 1986-
(författare)
-
SOD1 misfolding and aggregation in ALS : in the light of conformation-specific antibodies
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mutations in the superoxide dismutase 1 (SOD1) gene are linked to the progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS). ALS-associated mutations affect the stability of the SOD1 protein and promote its unfolding. As a consequence, disordered SOD1 species can misfold and accumulate into insoluble aggregates. Cytoplasmic inclusions containing misfolded SOD1 are a hallmark of ALS pathology in patients as well as transgenic mouse models. However, it remains unclear, which SOD1 species are pathogenic and how they arise and contribute to the disease.The aim of this thesis was to use antibodies as tools to study the role of disordered and aggregated SOD1 species in ALS. These antibodies recognize epitopes exposed in disordered SOD1 species and hence, discriminate between natively folded SOD1 and the disordered or misfolded protein.SOD1 is expressed in all cell types, but aggregates of misfolded SOD1 are predominantly found in motor neurons and associated glial cells in the spinal cord of ALS patients. To understand why misfolded SOD1 targets the motor system, we used ELISA and immunocapture methods to quantify soluble SOD1 species in patient-derived cell models of ALS. The highest levels of soluble disordered SOD1 were detected in induced pluripotent stem cell (iPSC)-derived motor neuron and astrocytes cultures (MNACs) compared to fibroblasts, iPSCs and sensory neuron cultures. These results suggest that the selective vulnerability of motor areas to SOD1-ALS could derive from an enhanced burden of disordered SOD1.To understand factors that might promote SOD1 unfolding, we focussed on the disulfide bond that is required for the stability of natively folded SOD1. Formation of the bond is oxygen-dependent and reduction of the bond promotes SOD1 unfolding. We studied the stability of SOD1 in patient-derived cells exposed to lowered oxygen tensions. This induced increases in disulfide-reduced, disordered mutant and wild-type SOD1. The response was time- and concentration-dependent and more pronounced in MNACs, where even increased aggregation of mutant SOD1 was observed. These results are consistent with the enhanced vulnerability of the motor system in ALS and suggest that conditions causing impaired oxygen perfusion could contribute to the initiation and progression of the disease.Inclusions containing aggregated misfolded wild-type SOD1 have been found in sporadic ALS (sALS) patients without SOD1 mutations and those carrying mutations in genes other than SOD1. However, other groups have reported contrasting results and the contribution of misfolded wild-type SOD1 to ALS pathology is controversial. Guidelines for preservation, storage, and analysis of tissues under standardized conditions would facilitate the comparison of results between different laboratories. We established an optimized immunohistochemistry protocol to detect misfolded wild-type SOD1 in paraffin-embedded spinal cord samples from sALS patients. We also developed a method to immunocapture disordered SOD1 from frozen post-mortem tissue. High, but variable, levels of disordered SOD1 were detected in spinal cords from sALS patients. Our data support a possible pathological role of misfolded wild-type SOD1 in sALS.Recent evidence suggests that SOD1 aggregates can induce templated aggregation of disordered SOD1 and spread from cell-to-cell via a prion-like mechanism. To test if antibodies could block this process in vivo, we conducted an immunotherapy study in a model of prion-like spread, where SOD1 aggregate seeds are inoculated into the lumbar spinal cord of SOD1G85R transgenic mice and lead to accelerated disease onset and progression. Novel monoclonal antibodies (mAb) against disordered domains of SOD1 aggregates were developed and validated for their reactivity to disordered and aggregated SOD1 species in vitro and in vivo. Immunotherapy using a mAb against the C-terminal end of SOD1 attenuated the onset and progression of prion-like SOD1 spread. However, no effect was seen on onset, duration or progression of the underlying disease. This suggests that, under the conditions tested, immunotherapy against disordered domains of SOD1 does not affect intracellular aggregation and additional strategies might be needed to reduce intracellular accumulation of misfolded SOD1 aggregation.In conclusion, we show that conformation-specific antibodies are powerful tools to investigate disordered and potentially pathogenic species of SOD1 in various biochemical, cellular and in vivo contexts. The development of the novel immunocapture strategy could facilitate future research on characterizing SOD1 aggregates from mouse tissues, patient-derived cells or post-mortem tissues with the goal of determining their role in ALS disease pathogenesis.
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| 36. |
- Leppert, Axel, et al.
(författare)
-
ATP-independent molecular chaperone activity generated under reducing conditions
- 2022
-
Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:8
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra- to intermolecular disulfide bond relay mechanism. Chaperone-active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol-containing compounds and proteins, and appear in parallel with reduction-induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space.Significance: Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction-induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.
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| 37. |
- Lindqvist, Richard, et al.
(författare)
-
Cell-type- and region-specific restriction of neurotropic flavivirus infection by viperin
- 2018
-
Ingår i: Journal of Neuroinflammation. - : BioMed Central. - 1742-2094. ; 15
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Flaviviruses are a group of diverse and emerging arboviruses and an immense global health problem. A number of flaviviruses are neurotropic, causing severe encephalitis and even death. Type I interferons (IFNs) are the first line of defense of the innate immune system against flavivirus infection. IFNs elicit the concerted action of numerous interferon-stimulated genes (ISGs) to restrict both virus infection and replication. Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an ISG with broad-spectrum antiviral activity against multiple flaviviruses in vitro. Its activity in vivo restricts neurotropic infections to specific regions of the central nervous system (CNS). However, the cell types in which viperin activity is required are unknown. Here we have examined both the regional and cell-type specificity of viperin in the defense against infection by several model neurotropic flaviviruses.Methods: Viral burden and IFN induction were analyzed in vivo in wild-type and viperin(-/-) mice infected with Langat virus (LGTV). The effects of IFN pretreatment were tested in vitro in primary neural cultures from different brain regions in response to infection with tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV).Results: Viperin activity restricted nonlethal LGTV infection in the spleen and the olfactory bulb following infection via a peripheral route. Viperin activity was also necessary to restrict LGTV replication in the olfactory bulb and the cerebrum following CNS infection, but not in the cerebellum. In vitro, viperin could restrict TBEV replication in primary cortical neurons, but not in the cerebellar granule cell neurons. Interferon-induced viperin was also very important in primary cortical neurons to control TBEV, WNV, and ZIKV.Conclusions: Our findings show that viperin restricts replication of neurotropic flaviviruses in the CNS in a region- and cell-type-specific manner. The most important sites of activity are the olfactory bulb and cerebrum. Activity within the cerebrum is required in the cortical neurons in order to restrict spread. This study exemplifies cell type and regional diversity of the IFN response within the CNS and shows the importance of a potent broad-spectrum antiviral ISG.
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| 38. |
- Lindqvist, Richard, et al.
(författare)
-
Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects
- 2016
-
Ingår i: Journal of Neuroinflammation. - : Springer Science and Business Media LLC. - 1742-2094. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. Methods: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR(-/-)) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. Results: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. Conclusions: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.
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| 39. |
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| 40. |
- Lumsden, A, et al.
(författare)
-
Neurobiology.
- 2001
-
Ingår i: Current Opinion in Neurobiology. - 0959-4388 .- 1873-6882. ; 11:3, s. 259-66
-
Forskningsöversikt (refereegranskat)
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| 41. |
- Lövheim, Hugo, et al.
(författare)
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Herpes simplex infection and the risk of Alzheimer's disease : a nested case-control study
- 2015
-
Ingår i: Alzheimer's & Dementia. - : Elsevier BV. - 1552-5260 .- 1552-5279. ; 11:6, s. 587-592
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Herpes simplex virus (HSV) is thought to play an etiological role in the development of Alzheimer's disease (AD).METHODS: Plasma samples from 360 AD cases (75.3% women, mean age 61.2 years) and 360 age- and sex-matched dementia-free controls, taken on average 9.6 years before AD diagnosis, were analyzed for anti-HSV antibodies (immunoglobulin G, IgG, and immunoglobulin M, IgM) by enzyme-linked immunosorbent assays.RESULTS: In the complete sample group, the presence of anti-HSV IgG and IgM antibodies did not increase the risk of AD significantly (odds ratio (OR) 1.636, P = .069 and OR 1.368, P = .299, respectively). In cases with 6.6 years or more between plasma sampling and AD diagnosis (n = 270), there was a significant association between presence of anti-HSV IgG antibodies and AD (OR 2.250, P = .019).CONCLUSION: Among persons with a follow-up time of 6.6 years or more, HSV infection was significantly associated with AD.
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| 42. |
- Lövheim, Hugo, et al.
(författare)
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Reactivated herpes simplex infection increases the risk of Alzheimer's disease
- 2015
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Ingår i: Alzheimer's & Dementia. - : John Wiley & Sons. - 1552-5260 .- 1552-5279. ; 11:6, s. 593-599
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Previous studies have suggested a link between herpes simplex virus (HSV) type 1 and the development of Alzheimer's disease (AD).METHODS: The present analysis included 3432 persons (53.9% women, mean age at inclusion 62.7 ± 14.4 years) with a mean follow-up time of 11.3 years. The number of incident AD cases was 245. Serum samples were analyzed for anti-HSV antibodies (immunoglobulin (Ig)G and IgM) by enzyme-linked immunosorbent assays.RESULTS: The presence of anti-HSV IgG antibodies was not associated with an increased risk for AD, controlled for age and sex (hazard ratio, HR, 0.993, P = .979). However, the presence of anti-HSV IgM at baseline was associated with an increased risk of developing AD (HR 1.959, P = .012).CONCLUSION: Positivity for anti-HSV IgM, a sign of reactivated infection, was found to almost double the risk for AD, whereas the presence of anti-HSV IgG antibodies did not affect the risk.
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| 43. |
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| 44. |
- Marsili, Luca, et al.
(författare)
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SOD1-related cerebellar ataxia and motor neuron disease : Cp variant as functional modifier?
- 2024
-
Ingår i: Cerebellum. - : Springer Nature. - 1473-4222 .- 1473-4230. ; 23, s. 205-209
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Tidskriftsartikel (refereegranskat)abstract
- We describe a novel superoxide dismutase (SOD1) mutation-associated clinical phenotype of cerebellar ataxia and motor neuron disease with a variant in the ceruloplasmin (Cp) gene, which may have possibly contributed to a multi-factorial phenotype, supported by genetic and protein structure analyses.
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| 45. |
- Meyer, Martin P, et al.
(författare)
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Characterization of zebrafish PSD-95 gene family members.
- 2005
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Ingår i: Journal of Neurobiology. - : Wiley. - 0022-3034 .- 1097-4695. ; 63:2, s. 91-105
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Tidskriftsartikel (refereegranskat)abstract
- The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.
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| 46. |
- Muthu, Magesh, et al.
(författare)
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GLUL Ablation Can Confer Drug Resistance to Cancer Cells via a Malate-Aspartate Shuttle-Mediated Mechanism
- 2019
-
Ingår i: Cancers. - : MDPI. - 2072-6694. ; 11:12
-
Tidskriftsartikel (refereegranskat)abstract
- Glutamate-ammonia ligase (GLUL) is important for acid-base homeostasis, ammonia detoxification, cell signaling, and proliferation. Here, we reported that GLUL ablation conferred resistance to several anticancer drugs in specific cancer cell lines while leaving other cell lines non-resistant to the same drugs. To understand the biochemical mechanics supporting this drug resistance, we compared drug-resistant GLUL knockout (KO) A549 non-small-cell lung carcinoma (NSCLC) cells with non-resistant GLUL KO H1299 NSCLC cells and found that the resistant A549 cells, to a larger extent, depended on exogenous glucose for proliferation. As GLUL activity is linked to the tricarboxylic acid (TCA) cycle via reversed glutaminolysis, we probed carbon flux through both glycolysis and TCA pathways by means of 13C5 glutamine, 13C5 glutamate, and 13C6 glucose tracing. We observed increased labeling of malate and aspartate in A549 GLUL KO cells, whereas the non-resistant GLUL KO H1299 cells displayed decreased 13C-labeling. The malate and aspartate shuttle supported cellular NADH production and was associated with cellular metabolic fitness. Inhibition of the malate-aspartate shuttle with aminooxyacetic acid significantly impacted upon cell viability with an IC50 of 11.5 μM in resistant GLUL KO A549 cells compared to 28 μM in control A549 cells, linking resistance to the malate-aspartate shuttle. Additionally, rescuing GLUL expression in A549 KO cells increased drug sensitivity. We proposed a novel metabolic mechanism in cancer drug resistance where the increased capacity of the malate-aspartate shuttle increased metabolic fitness, thereby facilitating cancer cells to escape drug pressure.
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| 47. |
- Muthukrishnan, Uma, 1984-, et al.
(författare)
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The exosome membrane localization of histones is independent of DNA and upregulated in response to stress
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular histones contribute to many acute and chronic diseases but also populate the secretomes of healthy cells and biofluids. However, a secretory pathway for histones has not been described. Here we report that core and linker histones localize to multivesicular bodies and are secreted via exosomes. Histones are tightly associated with the exosome membrane, with N-terminal domains exposed, in a DNA-independent manner. Furthermore, rapid upregulation of exosomal histones occurs following heat stress, accompanied by enhanced vesicle secretion and a shift towards a population of smaller vesicles. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) complex as a possible mechanism underlying increased histone secretion.We show for the first time that membrane-associated histones are actively secreted from intact cells via the multivesicular body/exosomal pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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| 48. |
- Muthukrishnan, Uma, 1984-
(författare)
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The release of histone proteins from cells via extracellular vesicles
- 2018
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes.In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting.In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway.Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent.The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion.In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients. In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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| 49. |
- Månberg, Anna, 1985-, et al.
(författare)
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Altered perivascular fibroblast activity precedes ALS disease onset
- 2021
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Ingår i: Nature Medicine. - : Nature Publishing Group. - 1078-8956 .- 1546-170X. ; 27:4, s. 640-646
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Tidskriftsartikel (refereegranskat)abstract
- Apart from well-defined factors in neuronal cells1, only a few reports consider that the variability of sporadic amyotrophic lateral sclerosis (ALS) progression can depend on less-defined contributions from glia2,3 and blood vessels4. In this study we use an expression-weighted cell-type enrichment method to infer cell activity in spinal cord samples from patients with sporadic ALS and mouse models of this disease. Here we report that patients with sporadic ALS present cell activity patterns consistent with two mouse models in which enrichments of vascular cell genes preceded microglial response. Notably, during the presymptomatic stage, perivascular fibroblast cells showed the strongest gene enrichments, and their marker proteins SPP1 and COL6A1 accumulated in enlarged perivascular spaces in patients with sporadic ALS. Moreover, in plasma of 574 patients with ALS from four independent cohorts, increased levels of SPP1 at disease diagnosis repeatedly predicted shorter survival with stronger effect than the established risk factors of bulbar onset or neurofilament levels in cerebrospinal fluid. We propose that the activity of the recently discovered perivascular fibroblast can predict survival of patients with ALS and provide a new conceptual framework to re-evaluate definitions of ALS etiology.
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| 50. |
- Najafi, Pardis, et al.
(författare)
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Genomic evidence for the suitability of Göttingen minipigs with a rare seizure phenotype as a model for human epilepsy
- 2024
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Ingår i: Neurogenetics. - : Springer Nature. - 1364-6745 .- 1364-6753. ; 25:2, s. 103-117
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Tidskriftsartikel (refereegranskat)abstract
- Epilepsy is a complex genetic disorder that affects about 2% of the global population. Although the frequency and severity of epileptic seizures can be reduced by a range of pharmacological interventions, there are no disease-modifying treatments for epilepsy. The development of new and more effective drugs is hindered by a lack of suitable animal models. Available rodent models may not recapitulate all key aspects of the disease. Spontaneous epileptic convulsions were observed in few Göttingen Minipigs (GMPs), which may provide a valuable alternative animal model for the characterisation of epilepsy-type diseases and for testing new treatments. We have characterised affected GMPs at the genome level and have taken advantage of primary fibroblast cultures to validate the functional impact of fixed genetic variants on the transcriptome level. We found numerous genes connected to calcium metabolism that have not been associated with epilepsy before, such as ADORA2B, CAMK1D, ITPKB, MCOLN2, MYLK, NFATC3, PDGFD, and PHKB. Our results have identified two transcription factor genes, EGR3 and HOXB6, as potential key regulators of CACNA1H, which was previously linked to epilepsy-type disorders in humans. Our findings provide the first set of conclusive results to support the use of affected subsets of GMPs as an alternative and more reliable model system to study human epilepsy. Further neurological and pharmacological validation of the suitability of GMPs as an epilepsy model is therefore warranted.
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