| 1. |
- Bartolini, Barbara, et al.
(författare)
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Mouse development is not obviously affected by the absence of dermatan sulfate epimerase 2 in spite of a modified brain dermatan sulfate composition.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:7, s. 1007-1016
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 2 (DS-epi2), together with its homologue DS-epi1, transform glucuronic acid into iduronic acid in dermatan sulfate polysaccharide chains. Iduronic acid gives dermatan sulfate increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in brain. Here we report the generation and initial characterization of DS-epi2 null mice. DS-epi2 deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin/dermatan sulfate (CS/DS) isolated from newborn mutated mouse brains had a 38% reduction in iduronic acid compared to wild type littermates and compositional analysis revealed a decrease of 4-O-sulfate and an increase of 6-O-sulfate containing structures. Despite the reduction in iduronic acid, adult DS-epi2-/- brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2.. These results extend previous findings of functional redundancy of brain extracellular matrix components.
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| 2. |
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| 3. |
- Degerman, Eva, et al.
(författare)
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Expression of insulin signalling components in the sensory epithelium of the human saccule.
- 2013
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Ingår i: Cell and Tissue Research. - : Springer Science and Business Media LLC. - 1432-0878 .- 0302-766X. ; 352:3, s. 469-478
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Tidskriftsartikel (refereegranskat)abstract
- Several studies have demonstrated a link between diabetes and the dysfunction of the inner ear. Few studies, however, have reported the signalling mechanisms involved in metabolic control in human inner ear cells. Knowledge of the expression and role of the insulin receptor and downstream signalling components in the inner ear is sparce. Our immunohistochemistry approach has shown that the insulin receptor, insulin receptor substrate 1 (IRS1), protein kinase B (PKB) and insulin-sensitive glucose transporter (GLUT4) are expressed in the sensory epithelium of the human saccule, which also exhibits expression of a calcium-sensitive cAMP/cGMP phosphodiesterase 1C (PDE1C) and the vasopressin type 2 receptor. IRS1 and PDE1C are selectively expressed in sensory epithelial hair cells, whereas the other components are expressed in sensory epithelial supporting cells or in both cell types, as judged from co-expression or non-co-expression with glial fibrillary acidic protein, a marker for supporting cells. Furthermore, IRS1 appears to be localized in association with sensory nerves, whereas GLUT4 is expressed in the peri-nuclear area of stromal cells, as is the case for aquaporin 2. Thus, the insulin receptor, insulin signalling components and selected cAMP signalling components are expressed in the human saccule. In addition to well-known mechanisms of diabetes complications, such as neuropathy and vascular lesions, the expression of these proteins in the saccule could have a role in the observed link between diabetes and balance/hearing disorders.
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| 4. |
- Degerman, Eva, et al.
(författare)
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Identification of new signaling components in the sensory epithelium of human saccule.
- 2011
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Ingår i: Frontiers in Neurology. - : Frontiers Media SA. - 1664-2295. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To locate components and target proteins of relevance for the cAMP and cGMP signaling networks including cAMP and cGMP phosphodiesterases (PDEs), salt-inducible kinases (SIKs), subunits of Na+, K+-ATPases, and aquaporins (AQPs) in the human saccule. Methods: The human saccule was dissected out during the removal of vestibular schwannoma via the translabyrinthine approach and immediately fixed. Immunohistochemistry was performed using PDE, SIK, Na(+), K(+)-ATPase, and AQP antibodies. Results: PDEs selective for cAMP (PDE4A, PDE4D, and PDE8A) and cGMP (PDE9A) as well a dual specificity PDE (PDE10A) were detected in the sensory epithelium of the saccule. Furthermore, AQP2, 4, and 9, SIK1 and the α-1 subunit of the Na(+), K(+)-ATPase were detected. Conclusion: cAMP and cGMP are important regulators of ion and water homeostasis in the inner ear. The identification of PDEs and SIK1 in the vestibular system offers new treatment targets for endolymphatic hydrops. Exactly how the PDEs are connected to SIK1 and the SIK1 substrate Na(+), K(+)-ATPase and to AQPs 2, 4, 9 remains to be elucidated. The dissection of the signaling networks utilizing these components and evaluating their roles will add new basic knowledge regarding inner ear physiology.
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| 5. |
- Geissler, Maren, et al.
(författare)
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Primary hippocampal neurons, which lack four crucial extracellular matrix molecules, display abnormalities of synaptic structure and function and severe deficits in perineuronal net formation.
- 2013
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Ingår i: The Journal of Neuroscience. - 1529-2401. ; 33:18, s. 7742-7742
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.
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| 6. |
- Pacharra, Sandra, et al.
(författare)
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The Lecticans of Mammalian Brain Perineural Net Are O-Mannosylated
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:4, s. 1764-1771
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Tidskriftsartikel (refereegranskat)abstract
- O-Mannosylation is an important protein modification in brain. During the last years, a few mammalian proteins have been identified as targets of the protein-O-mannosyltransferases 1 and 2. However, these still cannot explain the high content of O-mannosyl glycans in brain and the strong brain involvement of congenital muscular dystrophies caused by POMT mutations (Walker-Warburg syndrome, dystroglycanopathies). By fractionating and analyzing the glycoproteome of mouse and calf brain lysates, we could show that proteins of the perineural net, the lecticans, are O-mannosylated, indicating that major components of neuronal extracellular matrix are O-mannosylated in mammalian brain. This finding corresponds with the high content of O-mannosyl glycans in brain as well as with the brain involvement of dystroglycanopathies. In contrast, the lectican neurocan is not O-mannosylated when recombinantly expressed in EBNA-293 cells, revealing the possibility of different control mechanisms for the initiation of O-mannosylation in different cell types.
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| 7. |
- Rauch, Uwe
(författare)
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Detection of neurocan in cerebrospinal fluid.
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 836, s. 87-95
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Tidskriftsartikel (refereegranskat)abstract
- Cerebrospinal fluid (CFS) is the most easily accessible component of the human central nervous system and has been successfully used for the analysis of disease-associated molecular imbalances, particularly for extracellular matrix components. Alterations in the presence of the nervous system-associated chondroitin sulfate proteoglycan neurocan had been reported from active multiple sclerosis lesions. Neurocan could be detected as a component of human CFS after enrichment of proteoglycans by anion exchange chromatography from pooled liquor as well as individual 300 μL samples by Western blot. However, a general alteration in neurocan levels in CFS sample with high immunoglobulin content could not be demonstrated. To further reduce the sample size, the development of a PG capturing assay based on polybrene-coated 96-well plates was initiated. This approach could be an interesting alternative option for the analysis of PGs in biological fluid and tissue samples.
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| 8. |
- Rauch, Uwe, et al.
(författare)
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Increased Neointimal Thickening in Dystrophin-Deficient mdx Mice.
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation. METHODOLOGY/PRINCIPAL FINDINGS: We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice. CONCLUSIONS/SIGNIFICANCE: These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.
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| 9. |
- Rauch, Uwe, et al.
(författare)
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Laminin isoforms in atherosclerotic arteries from mice and man.
- 2011
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Ingår i: Histology and Histopathology. - 1699-5848. ; 26:6, s. 711-724
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Tidskriftsartikel (refereegranskat)abstract
- The properties of the arterial vasculature depend to a large extent on the activities of smooth muscle cells, which, in turn, are determined by their extracellular environment. During pathological conditions, such as atherosclerosis, this interaction is altered. In close proximity to medial smooth muscle cells are basement membrane components, such as different isoforms of laminin. These proteins can have great impact on cellular function via interaction with cell surface integrins. However, knowledge of laminins in smooth muscle cell basement membranes during normal and pathological conditions is scarce. Therefore, we have analyzed the presence of laminin isoforms in atherosclerotic lesions of apolipoprotein E (ApoE)-deficient mice. Our study revealed that the laminin chain isotype composition within atherosclerotic plaque tissue was different from the chain composition in the media. In addition, obvious differences in laminin chain composition could be observed in areas of the media, which were or were not associated with plaque tissue. Our major findings demonstrate that laminin gamma3 was exclusively present in media associated with plaque tissue. Laminin alpha2 was also enriched in these medial areas. Plaque tissue was predominantly enriched in laminin alpha5 chains. This general distribution applied to lesions both with and without a fibrous cap-like structure. The differential distribution of laminin chains were partially accompanied by changes in the presence of the integrin alpha subunits 7 and V. The distribution of laminin chains in human atherosclerotic arteries, with different size and morphology, grossly resembled their distribution in mouse arteries.
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| 10. |
- Saxena, Amit, et al.
(författare)
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The Vascular Repair Process after Injury of the Carotid Artery is regulated by IL-1RI and MyD88 Signalling.
- 2011
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Ingår i: Cardiovascular Research. - : Oxford University Press (OUP). - 1755-3245 .- 0008-6363. ; 91, s. 350-357
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Tidskriftsartikel (refereegranskat)abstract
- Aim: The aim of this study was to determine if innate immune signalling influences the vascular repair process in response to mechanical injury of arteries in mice. Methods and Results: A non-obstructive collar was introduced around the carotid artery of MyD88-deficient mice and neointima formation was compared to that observed in MyD88-competent mice. MyD88-deficient mice are characterized by impaired signal transduction from interleukin (IL)-1/IL-18 receptors and most Toll-like receptors. The vascular response to injury was severely impaired in MyD88-deficient mice as neointima formation was not different from sham operated mice, whereas MyD88-competent mice displayed robust neointima formation. Furthermore, infiltration of CD68 positive leukocytes was dependent on MyD88. During the early response to injury, 3 days after collar placement, a transient increase in the expression of Toll-like receptor (TLR) 4 on vascular smooth muscle cells was observed. To determine the relative importance of IL-1 receptor and TLR4 activation in the vascular response to injury mice were injected with blocking antibodies to these receptors prior to the collar placement. Neointima formation was reduced by 80% in mice administered IL-1RI blocking antibodies compared to mice given a control antibody, whereas administration of TLR4 blocking antibodies was without effect. Conclusion: These results show that inhibition of MyD88- or IL-1 receptor signalling reduces neointima formation in response to vascular injury and could offer therapeutic options for reducing clinical complications of excessive smooth muscle cell proliferation, such as that observed in in-stent restenosis.
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