Important results in 2018
1. A single-cell analysis reveals multiple roles of oligodendroglial lineage cells during post-ischemic regeneration
Here we identified several yet unknown differences between the gene expression profiles of NG2 cells and oligodendrocytes, and characterized specific genes contributing to oligodendrocyte maturation and phenotypical changes of NG2 cells after focal cerebral ischemia. Moreover, our results suggest that unlike in Alzheimer’s-like pathology, NG2 cells acquire a multipotent phenotype following ischemic injury.
Important roles of oligodendroglial lineage cells during post-ischemic regeneration. NG2 cells acquire a multipotent phenotype after cerebral ischemia; they first participate in the glial scar formation by differentiating into reactive astrocytes, then they give rise to oligodendrocytes – thus taking part in remyelination. Arrows indicate upregulation (green arrows) or downregulation (red arrows) of genes.
Institute of Biotechnology AS CR, v.v.i.; Department of Neuroscience, 2nd Faculty of Medicine, Charles University
Valný, M; Honsa, P; Waloschková, E; Matušková, H; Kriška, J; Kirdajová, D; Androvič, P; Valihrach, L; Kubista, M; Anděrová, M. A single-cell analysis reveals multiple roles of oligodendroglial lineage cells during post-ischemic regeneration. Glia. 2018 May;66(5):1068-1081. doi: 10.1002/glia.23301.
Important results in 2017
1. A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice
Deficiency in a link protein Bral2, which stabilizes the extracellular matrix complexes, leads to a reduction of perineuronal nets and axonal coats in the thalamic ventral posteromedial nucleus (VPM) of both young and aged mice However, a decrease in the extracellular volume was observed only in aged animals. Our data suggest that the effect of Bral2 deficiency on the VPM diffusion is probably indirect and mediated through the enhanced age-related damage of neurons lacking protective shields.
Age-related changes in thalamic diffusivity and ECM composition in Bral2 deficient mice. Deficiency for a link protein Bral2, associated with a disruption of brevican-based axonal coats (arrow), significantly affect tissue diffusivity in the thalamic ventroposteromedial nucleus in the aged but not in the young mice.
Cicanič, M; Edamatsu, M; Bekku, Y; Voříšek, I; Oohashi, T; Vargová, L. A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice. J Neurosci Res. 2018 Feb;96(2):313-327. doi: 10.1002/jnr.24136.
2. Multipotency and therapeutic potential of NG2 cells
Here, we summarize the current knowledge about NG2 cell proliferation, their fate plasticity during embryogenesis and in postnatal CNS under physiological and pathological conditions, with the emphasis on the role of signaling molecules, growth factors, hormones or even neurotransmitters on the fate potential of NG2 cells. They are well-known for their ability to generate new oligodendrocytes, however following CNS injuries, such as demyelination, trauma or ischemia, the proliferative capacity of NG2 cells rapidly increases and their differentiation potential broadens.
During embryogenesis of CNS and postnatally NG2 cells give rise to astrocytes and oligodendrocytes, while following injury they give rise to oligodendrocytes, reactive astrocytes and rarely, to immature neurones.
Publication: Valný, M; Honsa, P; Kriška, J; Anděrová, M. Multipotency and therapeutic potential of NG2 cells, Biochemical Pharmacology, 2017, Volume: 141 Pages: 42-55 Special Issue DOI: 10.1016/j.bcp.2017.05.008
Important results in 2016
1. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog
NG2 cells produce oligodendrocytes in the healthy nervous tissue. Under pathological conditions, they display a wide differentiation potential and could give rise to reactive astrocytes. In this study we have shown that morphogene sonic hedgehog (Shh) is an important factor that influences differentiation of NG2 cells into astrocytes during focal cerebral ischemia. We suggest that Shh has a direct influence on the formation and composition of a glial scar, which consequently affects the degree of the brain damage.
Honsa, P; Valný, M; Kriška, J; Matušková, H; Harantová, L; Kirdajová, D; Valihrach, L; Androvič, P; Kubista, M; Anděrová, M. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog. Glia. 2016 Sep;64(9):1518-31. doi: 10.1002/glia.23019.
2. Brain diffusivity and structural changes in the R6/2 mouse model of Huntington‘s disease
To clarify the nature of brain diffusivity changes in Huntington‘s disease (HD), we correlated results of two diffusion methods with histological tissue analysis. Magnetic resonance changes were mostly related to changes in the extracellular volume associated with a neuronal loss. Different degree of astrogliosis and/or extracellular matrix expression affect the brain diffusion as well and partially compensates the cellular loss, which may result in inconsistent results of numerous HD studies.
Changes in the brain diffusivity and tissue structure in the globus pallidus of R6/2 mouse model of Huntington‘s disease The extracellular volume increase and ADCw decrease in globus pallidus result from the alterations in the tissue structure which may have opposing effect on the brain diffusivity: profound neuronal loss, reduction of extracellular matrix and remodeling of astrocytes due to astrogliosis.
Voříšek, I; Syka, M; Vargová, L. Brain Diffusivity and Structural Changes in the R6/2 Mouse Model of Huntington Disease. J Neurosci Res. 2016 Oct 11. doi: 10.1002/jnr.23965.
Important result in 2015
Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors
Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP+) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP+ glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP+ glial cells and that its expression is increased after ischemia. In situ and in vitro Ca2+ imaging revealed that Ca2+ elevations evoked by the application of NMDA were diminished in GFAP/EGFP+ glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP+ glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.
Focal ischemia increases expression of most of the glutamate receptors (GluRs) in GFAP/EGFP glia. As for the NMDA receptor subunits, immunohistochemical analysis confirmed their presence in GFAP/EGFP glia, and their detection was even increased after ischemic insult. The Ca2+ imaging results indicate diminished NMDA receptor Ca2+ permeability after focal ischemia, which is probabl due to the involvement of GluN3A subunit.
Immunohistochemical analysis of the GluN1, GluN2A-D and GluN3A-B subunits of the NMDA receptors in the cortex of adult GFAP/EGFP mice under control conditions (CTRL) and 14 days after MCAo (D14). Coronal brain sections from CTRL (A) and D14 (B) animals stained with triphenyltetrazolium chloride. The white color in B indicates the volume of ischemic tissue at D14. The boxed areas indicate the regions in which the immunohistochemical analysis was performed. The arrowheads in C - P indicate the overlay of GFAP/EGFP+ cells and NMDA subunit staining – see figure insets for detailed images of cells in white rectangles. Note the overlap of the EGFP signal with GluN3A staining in CTRL tissue and GluN1, GluN2B-D and GluN3A staining at D14. The same scale bar applies to all non-inset images.
Important Results in 2014
1. Increased expression of hyperpolarization-activated cationic channels in reactive astrocytes following ischemia
Following cerebral ischemia we have identified hyperpolarization-activated (HCN) cationic channels in astrocytes. Until now, these channels were described only in neurons. Since HCN channels are mainly permeable for sodium and potassium ions, their increased expression in reactive astrocytes indicates that they may markedly influence the basic astrocytic functions in central nervous system, and consequently, an extent of nervous tissue damage following ischemia. Astrocytic HCN channels could therefore be an important therapeutic target in post-stroke therapy.
The expression of Hcn genes is strikingly increased in cortical astrocytes from GFAP/EGFP mice following focal cerebral ischemia – single-cell RT-qPCR profiling. (A) Scheme depicting the brain regions, which were used for EGFP+ cells isolations. These brain slices were stained with tetrazolium chloride to visualize the ischemic regions. (B) Percentage of EGFP+ cells in the postischemic mouse cortex (7 and 14 days after focal cerebral ischemia; D7, 2W) expressing Hcn1, 2, 3 and 4. (C)The relative expression of Hcn1–4 genes in EGFP+ cells in the control mouse cortex and in the post-ischemic cortex revealed the strong upregulation of Hcn1–4 expression 2W after FCI. HCN1 staining in the CA1 region of the rat hippocampus in controls and five weeks after global cerebral ischemia. Arrowheads indicate the HCN-positive astrocytes after ischemia (s.p., stratum pyramidale; s.r., stratum radiatum). Scale bars, 50 μm.
HCN1 staining in the CA1 region of the rat hippocampus in controls and five weeks after global cerebral ischemia. Brain slices were stained with anti-HCN1 antibodies and an antibody directed against glial fibrillary acidic protein (GFAP) in controls and fi ve weeks (5W) after global cerebral ischemia (GCI). Arrowheads indicate the HCN-positive astrocytes after ischemia (s.p., stratum pyramidale; s.r., stratum radiatum). Scale bars, 50 μm.
Institute of Biotechnology CAS, v.v.i. – Prof. Mikael Kubista
Honsa, P; Pivoňková, H; Harantová, L; Butenko, O; Kriška, J; Džamba, D; Rusnaková, V; Valihrach, L; Kubista, M; Anděrová, M. Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia. Glia 62 (12), 2004–2021. IF 5,466
2. Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice
We have showed that knockout of α-syntrophin, which is a protein responsible for aquaporin-4 anchoring on the astrocytic membrane, aff ects cell swelling/volume regulation in individual astrocytes in situ when exposed to pathological stimuli. Astrocyte volume quantifi cation revealed that α-syntrophin deletion results in signifi cantly smaller/slower astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K+.
Volume changes in the astrocytic soma and processes during hypotonic stress, increased extracellular K+ concentration and oxygen-glucose deprivation. (A–C) Time-dependent changes in the volume of the astrocytic soma (top) and processes (bottom) in GFAP/EGFP (red) and GFAP/EGFP/α-Syn-/- mice (green) during a 30-minute application of aCSFH-100 (A), a 20-minute application of aCSFK+50 (B) or 20-minute OGD (C), followed by a 60- or 40-minute washout. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn-/- mice.
The contribution of processes and cell soma to total astrocyte swelling is altered in astrocytes lacking α-syntrophin. A scheme depicting differences in swelling of astrocytic processes and cell soma in response to severe hypoosmotic stress, 50 mM K+ and OGD (oxygen-glucose deprivation), highlighting smaller swelling of astrocytic processes in GFAP/EGFP/α-Syn-/- mice compared to those in GFAP/EGFP mice due to altered distribution of aquaporin4 and Kir4.1 channels.
Department of Neuroscience, 2nd Faculty of Medicine, Charles University in Prague Institute of Biotechnology AS CR, v.v.i. – Prof. Mikael Kubista
Anděrová, M; Benešová, J; Mikešová, M; Džamba, D; Honsa, P; Kriška, J; Butenko, O; Novosadová, V; Valihrach, L; Kubista, M; Dmytrenko, L; Cicanič, M; Vargová, L. Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice, PLoS One. 9(11):e113444. IF 3.534
3. Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes
Using patch clamp technique we have demonstrated that in primary cultured rat cortical astrocytes, elevations of [Na+]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity-evoked current mediated by volume-regulated anion channel (VRAC). These results provide the first evidence that intracellular Na+ dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na+ accumulation might be a favorable strategy to counteract the development of brain edema.
High elevation of intracellular sodium (Na+) concentration in cultured rat astrocytes decreases the activity of volume-regulated anion channels (VRAC) measured by patch clamp technique. We speculate that in ischemic conditions intracellular Na+ elevation either through the augmented activity of the Na+-dependent glutamate transporter (GLT-1) or by Na+-permeable channels could modulate the astrocytic volume and glutamate release (Glu-) regulated by VRAC.
University of Bologna, Italy, Prof. Stefano Ferroni
Minieri, L; Pivoňková, H; Harantová, L; Anděrová, M; Ferroni, S. Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes, J Neurochemistry, doi: 10.1111/jnc.12962., IF 3.973
, Suchá, P., Bochin, M., Voříšek, I., Pivoňková, H., Heřmanová, Z., Anděrová, M., Vargová, L.: (2018) The role of aquaporin-4 and transient receptor potential vaniloid isoform 4 channels in the development of cytotoxic edema and associated extracellular diffusion parameter changes. Eur J Neurosci. 2019 Jan 11. doi: 10.1111/ejn.14338.
, Heřmanová, Z., Kirdajová, D., Awadová, T., Malínský, J., Valihrach, L., Žucha, D., Kubista, M., Gálisová, A., Jirák, D., Anděrová, M.: (2018) The Contribution of TRPV4 Channels to Astrocyte Volume Regulation and Brain Edema Formation. Neuroscience. 2018 Dec 1;394:127-143. doi: 10.1016/j.neuroscience.2018.10.028. Epub 2018 Oct 24.
Awadová, T., Pivoňková, H., Heřmanová, Z., Kirdajová, D., Anděrová, M., Malínský, J.: (2018) Cell volume changes as revealed by fluorescence microscopy: Global vs local approaches. J Neurosci Methods. 2018 Aug 1;306:38-44. doi: 10.1016/j.jneumeth.2018.05.026. Epub 2018 Jun 7.
Edamatsu, M., Bekku, Y., Voříšek, I., Oohashi, T., Vargová, L.: (2018) A deficiency of the link protein Bral2 affects the size of the extracellular space in the thalamus of aged mice. J Neurosci Res. 96(2):313-327.
Honsa, P., Waloschková, E., Matušková, H., Kriška, J., Kirdajová, D., Androvič, P., Valihrach, L., Kubista, M., Anděrová, M.: (2018) A single-cell analysis reveals multiple roles of oligodendroglial2 lineage cells during post-ischemic regeneration. Glia. 2018 May;66(5):1068-1081. doi: 10.1002/glia.23301. Epub 2018 Feb 2.
Anděrová M.: (2017) Altered Homeostatic Functions in Reactive Astrocytes and Their Potential as a Therapeutic Target After Brain Ischemic Injury. Curr Pharm Des. 23(33):5056-5074.
Rychmach, P., Drahorádová, I., Konrádová, Š., Růžičková, K., Voříšek, I., Forostyak, S., Homola, A., Bojar, M.: (2017) Transplantation of Mesenchymal Stromal Cells in Patients With Amyotrophic Lateral Sclerosis: Results of Phase I/IIa Clinical Trial. Cell Transplantation. 26(4): 647-658.
Honsa P., Kriška J., Anděrová M.: (2017) Multipotency and therapeutic potential of NG2 cells. Biochem Pharmacol. 141:42-55.
Syka, M., Vargová, L.: (2017) Brain Diffusivity and Structural Changes in the R6/2 Mouse Model of Huntington Disease. Journal of Neuroscience Research. 95(7): 1474-1484.
Harantová L., Butenko O., Anděrová, M.: (2015) Glial cells – the key elements of Alzheimer´s disease. Curr. Alzheimer Res., 13(8): 894-911.
Valihrach, L., Kubista, M., Anděrová, M.: (2016) The correlation between expression profiles measured in single cells and in traditional bulk samples. Scientific Reports, 16(6): 37022.
Forostyak, O., Butenko, O., Anděrová, M., Forostyak, S., Syková, E., Verkhratsky, A., Dayanithi, G.: (2016) Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells. Stem Cell Res., 16(3):622-634.
Valný, M., Kriška, J., Matušková, H., Harantová, L., Kirdajová, D., Valihrach, L., Androvič, P., Kubista, M., Anděrová, M.: (2016) Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog. Glia, 64(9): 1518-1531.
Fafílek, B., Krausová, M., Horázná, M., Vojtěchová, M., Alberich-Jorda, M., Sloncová, E., Galušková, K., Sedláček, R., Anděrová, M., Kořínek, V.: (2016) Wnt Signaling Inhibition Deprives Small Intestinal Stem Cells of Clonogenic Capacity. Genesis, 54(3): 101-114.
Honsa, P., Džamba, D., Butenko, O., Koleničová, D., Janečková, L., Nahacká, Z., Anděra, L., Kozmík, Z., Taketo, M.M., Kořínek, V., Anděrová, M.: (2016) Manipulating Wnt signaling at different subcellular levels affects the fate of neonatal neural stem/progenitor cells. Brain Res., 1651: 73-87.
Lee, C.Y., Dallérac, G., Ezan, P., Anděrová, M., Rouach, N.: (2016) Glucose Tightly Controls Morphological and Functional Properties of Astrocytes. Front. Aging Neurosci., 8(82): 1-12.
Honsa, P., Kirdajová, D., Kameník, Z., Anděrová, M.: (2016) Tamoxifen in the Mouse Brain: Implications for Fate-Mapping Studies Using the Tamoxifen-Inducible Cre-loxP System. Front. Cell. Neurosci., 10: 243.
Honsa, P., Valný, M., Kriška, J., Valihrach, L., Novosadová, V., Kubista, M., Anděrová, M.: (2015) Quantitative Analysis of Glutamate Receptors in Glial Cells from the Cortex of GFAP/EGFP Mice Following Ischemic Injury: Focus on NMDA Receptors. Cell Mol Neurobiol. 35(8): 1187-1202.
Harantová L., Butenko O., Anděrová, M.: (2015) Glial cells – the key elements of Alzheimer´s disease. Current Alzheimer Research IN PRESS
(2015) Discovering the structure of nrve tissue: part 1: from Marcello Malpighi to Christian Berres. J. Hist. Neurosci., 24(3): 268-291.
(2015) Jiří Procháska (1749-1820): Part 2: "De structura nervorum"--studies on a structure of the nervous system. J. Hist. Neurosci., 24(1): 1-25.
(2015) Jan Křtitel Boháč (1724-1768) a jeho disertace o bolesti z roku 1746. (Jan Křtitel Boháč (1724-1768) and dissertation on pain from 1746). Bolest, 18(1): 8-20.
(2015) Výzkum struktury nervové tkáně III: od Jana Evangelisty Purkyně (1787–1869) k Ludwigovi Mauthnerovi (1840–1894). Československá fyziologie, 2: 52-72.
Minieri, L., Pivoňková, H., Harantová, L., Anděrová, M., Ferroni S.: (2014) Intracellular Na+ inhibits volume regulated anion channel in rat cortical astrocytes. J. Neurochem. 132(3): 286-300.
Benešová, J., Mikešová, M., Džamba, D., Honsa, P., Kriška, J., Butenko, O., Novosadová, V., Valihrach, L., Kubista, M., Dmytrenko, L., Cicanič, M., Vargová, L.: (2014) Altered astrocytic swelling in the cortex of α-syntrophin-negative GFAP/EGFP mice. PloS One. 9(11): e113444.
Pivoňková, H., Harantová, L., Butenko, O., Kriška, J., Džamba, D., Rusňáková, V., Valihrach, L., Kubista, M., Anděrová, M.: (2014) Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia. Glia. 62(12): 2004-2021
(2014) Jiří Procháska (1749-1820): Part 1: A Significant Czech Anatomist, Physiologist and Neuroscientist of the Eighteenth Century. J. Hist. Neurosci. 23(4): 367-376
(2014) Discovering the Structure of Nerve Tissue: Part 1: From Marcello Malpighi to Christian Berres. J. Hist. Neurosci. In press.
Mazurová, Y., Anděrová, M., Němečková, I., Bezrouk, A.: (2014) Transgenic Rat Model of Huntington's Disease: A Histopathological Study and Correlations with Neurodegenerative Process in the Brain of HD Patients. Biomed. Res. Int. 2014: 291531.
Dmytrenko, L., Cicanič, M., Anděrová, M., Voříšek, I., Ottersen, O. P., Syková, E., Vargová, L.: (2013) The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions. PLoS One. 8(7): e68044.
Honsa, P., Anděrová, M.: (2013) NMDA Receptors in Glial Cells: Pending Questions. Curr. Neuropharmacol. 11: 250-262.
Pivoňková, H., Anděrová, M.: (2013) Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles. Neuroscience. 240: 39-53.
Minieri, L., Pivoňková, H., Caprini, M., Harantová, L., Anděrová, M., Ferroni, S.: (2013) The inhibitor of volume regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes. Br. J. Pharmacol. 168(5): 1240-1254.
Rusňáková, V., Honsa, P., Džamba, D., Stählberg, A., Kubista, M., Anděrová, M.: (2013) Heterogeneity of Astrocytes: From Development to Injury – Single Cell Gene Expression. PLoS One 8(8): e69734.
Stählberg, A., Rusňáková, V., Forootan, A., Anděrová, M., Kubista, M.: (2013) RT-qPCR work-flow for single-cell data analysis.Methods. 59(1): 80-88.