| 1. |
- Carlström, Karl
(author)
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Demyelinating conditions regulated by ROS pathways outside and inside the brain
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Multiple sclerosis (MS) is a chronic inflammatory condition of the central nervous system with immune-mediated damage on myelinated nerve tracts. Accumulating evidence support a role for autoreactive T-lymphocytes orchestrating immuneattacks on myelinating oligodendrocytes (OLs). While oligodendrocyte precursor cells (OPCs) may initially compensate for loss of myelin by differentiating into OLs, later stages of MS are characterized by arrest of OPC differentiation and clinical disease progression. Reactive oxygen species (ROS) are important in many contexts, but their role in regulation of adaptive immune processes and remyelination in MS is still uncertain. We here first characterized the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)- activating MS-therapy dimethyl fumarate (DMF/ Tecfidera™) in cell cultures and an experimental model for neuroinflammation. In MS patients we assessed the effect of DMF on transcription and epigenetic modification in immune cells, as well as ROS production by monocytes. Our findings indicate that monocytederived ROS is linked to the clinical efficacy of DMF by modulating the adaptive immune response, in turn highlighting modulation of redox processes as a therapeutic target in autoimmune disease. In order to study the effect of DMF and other redox active agents, we developed a cell based reporter system (pTRAF) that enabled the visualization of transcription factor activity in real-time. Using pTRAF and other in vitro and in vivo models we further characterized the downstream effects of DMF and a set of more selective Nrf2-activating compounds. Finally, we performed extensive in vitro and in vivo characterization of Glutathione S-transferase 4α (Gsta4), which is regulated by Nrf2 and serves an important role for scavenging of the lipid peroxidation product 4-hydroxynonenal (4-HNE). Interestingly, higher levels of Gsta4 was associated with faster and more complete remyelination by facilitating the differentiation of OPC into mature myelinating OLs. This was suggested to be mediated through modulation of the Fas/Casp8/Bid-axis, leading to increased survival of differentiating OPCs. Collectively, our findings support a role for redox processes in regulating adaptive immune responses in MS. Furthermore, a specific redox-related process involving 4-HNE and Gsta4 was found to regulate OPC survival during remyelination. Both these mechanisms may represent interesting novel therapeutic targets in MS, as well as in other autoimmune and demyelinating conditions.
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| 2. |
- Carlström, Karl E., et al.
(author)
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Characterization of More Selective Central Nervous System Nrf2-Activating Novel Vinyl Sulfoximine Compounds Compared to Dimethyl Fumarate
- 2020
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In: NEUROTHERAPEUTICS. - : SPRINGER. - 1933-7213 .- 1878-7479. ; 17, s. 1142-1152
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Journal article (peer-reviewed)abstract
- The Nrf2 transcription factor is a key regulator of redox reactions and considered the main target for the multiple sclerosis (MS) drug dimethyl fumarate (DMF). However, exploration of additional Nrf2-activating compounds is motivated, since DMF displays significant off-target effects and has a relatively poor penetrance to the central nervous system (CNS). We de novo synthesized eight vinyl sulfone and sulfoximine compounds (CH-1-CH-8) and evaluated their capacity to activate the transcription factors Nrf2, NF kappa B, and HIF1 in comparison with DMF using the pTRAF platform. The novel sulfoximine CH-3 was the most promising candidate and selected for further comparison in vivo and later an experimental model for traumatic brain injury (TBI). CH-3 and DMF displayed comparable capacity to activate Nrf2 and downstream transcripts in vitro, but with less off-target effects on HIF1 from CH-3. This was verified in cultured microglia and oligodendrocytes (OLs) and subsequently in vivo in rats. Following TBI, DMF lowered the number of leukocytes in blood and also decreased axonal degeneration. CH-3 preserved or increased the number of pre-myelinating OL. While both CH-3 and DMF activated Nrf2, CH-3 showed less off-target effects and displayed more selective OL associated effects. Further studies with Nrf2-acting compounds are promising candidates to explore potential myelin protective or regenerative effects in demyelinating disorders.
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| 3. |
- Carlström, Karl E., et al.
(author)
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Therapeutic efficacy of dimethyl fumarate in relapsing-remitting multiple sclerosis associates with ROS pathway in monocytes
- 2019
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10:1, s. 1-13
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Journal article (peer-reviewed)abstract
- Dimethyl fumarate (DMF) is a first-line-treatment for relapsing-remitting multiple sclerosis (RRMS). The redox master regulator Nrf2, essential for redox balance, is a target of DMF, but its precise therapeutic mechanisms of action remain elusive. Here we show impact of DMF on circulating monocytes and T cells in a prospective longitudinal RRMS patient cohort. DMF increases the level of oxidized isoprostanes in peripheral blood. Other observed changes, including methylome and transcriptome profiles, occur in monocytes prior to T cells. Importantly, monocyte counts and monocytic ROS increase following DMF and distinguish patients with beneficial treatment-response from non-responders. A single nucleotide polymorphism in the ROS-generating NOX3 gene is associated with beneficial DMF treatment-response. Our data implicate monocyte-derived oxidative processes in autoimmune diseases and their treatment, and identify NOX3 genetic variant, monocyte counts and redox state as parameters potentially useful to inform clinical decisions on DMF therapy of RRMS.
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| 4. |
- Dominguez, Cecilia A, et al.
(author)
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Variability in C-type lectin receptors regulates neuropathic pain-like behavior after peripheral nerve injury
- 2014
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In: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 10
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Journal article (peer-reviewed)abstract
- INTRODUCTION: Neuropathic pain is believed to be influenced in part by inflammatory processes. In this study we examined the effect of variability in the C-type lectin gene cluster (Aplec) on the development of neuropathic pain-like behavior after ligation of the L5 spinal nerve in the inbred DA and the congenic Aplec strains, which carries seven C-type lectin genes originating from the PVG strain.RESULTS: While both strains displayed neuropathic pain behavior early after injury, the Aplec strain remained sensitive throughout the whole study period. Analyses of several mRNA transcripts revealed that the expression of Interleukin-1β, Substance P and Cathepsin S were more up-regulated in the dorsal part of the spinal cord of Aplec rats compared to DA, indicating a stronger inflammatory response. This notion was supported by flow cytometric analysis revealing increased infiltration of activated macrophages into the spinal cord. In addition, macrophages from the Aplec strain stimulated in vitro displayed higher expression of inflammatory cytokines compared to DA cells. Finally, we bred a recombinant congenic strain (R11R6) comprising only four of the seven Aplec genes, which displayed similar clinical and immune phenotypes as the Aplec strain.CONCLUSION: We here for the first time demonstrate that C-type lectins, a family of innate immune receptors with largely unknown functions in the nervous system, are involved in regulation of inflammation and development of neuropathic pain behavior after nerve injury. Further experimental and clinical studies are needed to dissect the underlying mechanisms more in detail as well as any possible relevance for human conditions.
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| 5. |
- Jackson, Johanna, et al.
(author)
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Altered synaptic properties during integration of adult-born hippocampal neurons following a seizure insult.
- 2012
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4
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Journal article (peer-reviewed)abstract
- Pathological conditions affect several stages of neurogenesis in the adult brain, including proliferation, survival, cell fate, migration, and functional integration. Here we explored how a pathological environment modulates the heterogeneous afferent synaptic input that shapes the functional properties of newly formed neurons. We analyzed the expression of adhesion molecules and other synaptic proteins on adult-born hippocampal neurons formed after electrically-induced partial status epilepticus (pSE). New cells were labeled with a GFP-retroviral vector one week after pSE. One and three weeks thereafter, synaptic proteins were present on dendritic spines and shafts, but without differences between pSE and control group. In contrast, at six weeks, we found fewer dendritic spines and decreased expression of the scaffolding protein PSD-95 on spines, without changes in expression of the adhesion molecules N-cadherin or neuroligin-1, primarily located at excitatory synapses. Moreover, we detected an increased expression of the inhibitory scaffolding protein gephyrin in newborn but not mature neurons after SE. However, this increase was not accompanied by a difference in GABA expression, and there was even a region-specific decrease in the adhesion molecule neuroligin-2 expression, both in newborn and mature neurons. Neuroligin-2 clusters co-localized with presynaptic cholecystokinin terminals, which were also reduced. The expression of neuroligin-4 and glycine receptor was unchanged. Increased postsynaptic clustering of gephyrin, without an accompanying increase in GABAergic input or neuroligin-2 and -4 expression, the latter important for clustering of GABA(A) and glycine receptors, respectively, could imply an increased but altered inhibitory connectivity specific for newborn neurons. The changes were transient and expression of both gephyrin and NL-2 was normalized 3 months post-SE. Our findings indicate that seizure-induced brain pathology alters the sub-cellular expression of synaptic adhesion molecules and scaffolding proteins related to particularly inhibitory but also excitatory synapses, which may yield functional consequences for the integration of adult-born neurons.
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| 6. |
- Köhler, Christian, et al.
(author)
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Backbone 1H, 13C, and 15N resonance assignments of the ligand binding domain of the human wildtype glucocorticoid receptor and the F602S mutant variant
- 2018
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In: Biomolecular NMR Assignments. - : Springer Science and Business Media LLC. - 1874-2718 .- 1874-270X. ; 12:2, s. 263-268
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Journal article (peer-reviewed)abstract
- The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the 1H–15N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.
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| 7. |
- Lindholm Carlström, Eva, et al.
(author)
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Linkage and exome analysis implicate multiple genes in non-syndromic intellectual disability in a large Swedish family
- 2019
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In: BMC Medical Genomics. - : Springer Science and Business Media LLC. - 1755-8794. ; 12:1
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Journal article (peer-reviewed)abstract
- BackgroundNon-syndromic intellectual disability is genetically heterogeneous with dominant, recessive and complex forms of inheritance. We have performed detailed genetic studies in a large multi-generational Swedish family, including several members diagnosed with non-syndromic intellectual disability. Linkage analysis was performed on 22 family members, nine affected with mild to moderate intellectual disability and 13 unaffected family members.Methods Family members were analyzed with Affymetrix Genome-Wide Human SNP Array 6.0 and the genetic data was used to detect copy number variation and to perform genome wide linkage analysis with the SNP High Throughput Linkage analysis system and the Merlin software. For the exome sequencing, the samples were prepared using the Sure Select Human All Exon Kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced using the Ion Proton (TM) System. Validation of identified variants was performed with Sanger sequencing.ResultsThe linkage analysis results indicate that intellectual disability in this family is genetically heterogeneous, with suggestive linkage found on chromosomes 1q31-q41, 4q32-q35, 6p25 and 14q24-q31 (LOD scores of 2.4, simulated p-value of 0.000003 and a simulated genome-wide p-value of 0.06). Exome sequencing was then performed in 14 family members and 7 unrelated individuals from the same region. The analysis of coding variation revealed a pathogenic and candidate variants in different branches of the family. In three patients we find a known homozygous pathogenic mutation in the Homo sapiens solute carrier family 17 member 5 (SLC17A5), causing Salla disease. We also identify a deletion overlapping KDM3B and a duplication overlapping MAP3K4 and AGPAT4, both overlapping variants previously reported in developmental disorders.Conclusions DNA samples from the large family analyzed in this study were initially collected based on a hypothesis that affected members shared a major genetic risk factor. Our results show that a complex phenotype such as mild intellectual disability in large families from genetically isolated populations may show considerable genetic heterogeneity.
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| 8. |
- Perez-Alcazar, Marta, et al.
(author)
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Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3.
- 2014
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In: Experimental neurology. - : Elsevier BV. - 1090-2430 .- 0014-4886. ; 253C, s. 154-164
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Journal article (peer-reviewed)abstract
- Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.
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| 9. |
- Xie, Meng, et al.
(author)
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Secondary ossification center induces and protects growth plate structure
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In: eLIFE. - 2050-084X.
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Journal article (peer-reviewed)abstract
- Growth plate and articular cartilage constitute a single anatomical entity, but later separate into two distinct structures by the formation of secondary ossification center (SOC). The reason for such spatial separation remains unknown. Here, we demonstrate that evolutionarily SOC first appears in amniotes. Mathematical modelling reveals that SOC reduces mechanical stress within the growth plate. Analysis of mammals with specialized extremities reveals that SOC size correlates with the extent of mechanical demands. Ex and in vivo experiments demonstrate that SOC allows epiphyseal chondrocytes to withstand a several-fold higher load before activation of the YAP-p73 signalling pathway and caspase-dependent apoptosis, with hypertrophic chondrocytes being the most load-sensitive cells. Atomic force microscopy shows that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC is evolved to protect epiphyseal chondrocytes, especially the hypertrophic chondrocytes, from the high mechanical stress encountered in the terrestrial environment.
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