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Glutamate receptors in NG2-glial cells: gene profiling and functional changes after ischemic brain injury
Waloschková, Eliška ; Anděrová, Miroslava (advisor) ; Růžička, Jiří (referee)
Glutamate is the main excitatory neurotransmitter in the mammalian brain and its transmission is responsible for higher brain functions, such as learning, memory and cognition. Glutamate action is mediated by a variety of glutamate receptors, though their properties were until now studied predominantly in neurons. Glutamate receptors are expressed also in NG2-glia, however their role under physiological conditions as well as in pathological states of the central nervous system is not fully understood. The aim of this work is to elucidate the presence, composition and function of these receptors in NG2-glia under physiological conditions and following focal cerebral ischemia. For this purpose we used transgenic mice, in which NG2-glia are labeled by a fluorescent protein for their precise identification. To analyze the expression pattern of glutamate receptors in NG2-glia we employed single-cell RT-qPCR. Furthermore, we used calcium imaging to characterize their functional properties.
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Calcium signalling in astrocytes under physiological and pathological conditions
Svatoňová, Petra ; Anděrová, Miroslava (advisor) ; Kolář, David (referee)
Calcium signalling in astrocytes represents an important component, which enables proper neuronal functioning under physiological conditions. Alterations in Ca2+ signalling, accompanied by an increase in intracellular calcium levels is a hallmark for numerous pathological states of central nervous system, such as traumatic and ischemic brain/spinal cord injuries, epilepsy as well as neurodegenerative diseases, such as Alzheimer's disease and psychiatric disorders, such as schizophrenia. The research analyzing the molecular components of astrocytic Ca2+ signalling can help us understand the control mechanisms used in calcium signalling and thus be greatly beneficial for further therapeutic research. Powered by TCPDF (www.tcpdf.org)
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Proliferation and differentiation of NG2-glia following ischemic brain injuries
Kirdajová, Denisa ; Anděrová, Miroslava (advisor) ; Machová Urdzíková, Lucia (referee)
NG2-glia, a fourth major glial cell population, were shown to posses wide proliferation and differentiation potential in vitro and in vivo, therefore the aim of this study was to compare the rate of proliferation and differentiation potential of NG2-glia after different types of brain injuries, such as global and focal cerebral ischemia (GCI, FCI) or stab wound (SW), as well as during aging. Moreover, we aimed to determine the role of Sonic hedgehog (Shh) in NG2-glia proliferation/differentiation after FCI. We used transgenic mice, in which tamoxifen triggers the expression of red fluorescent protein (tdTomato) in NG2-glia and cells derived therefrom. Proliferation and differentiation potential of tdTomato+ cells in sham operated animals (controls) and those after injury were determined by immunohistochemistry employing antibodies against proliferating cell nuclear antigen and glial fibrillary acidic protein. FCI was induced by middle cerebral artery occlusion, GCI by carotid occlusion with hypotension and SW by sagittal cortical cut. Shh signaling in vivo was activated or inhibited by Smoothened agonist or Cyclopamine, respectively. Compared to controls, the proliferation rate of tdTomato+ cells was increased after all types of injuries, while it declined in aged mice (15-18- months-old) after...
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The role of the Wnt signalling pathway in proliferation and differentiation of neural stem cells in the neonatal and adult mouse brain
Koleničová, Denisa ; Anděrová, Miroslava (advisor) ; Janečková, Lucie (referee)
The canonical Wnt/β-catenin signalling pathway plays an important role in proliferation and differentiation of neural progenitors during embryogenesis as well as postnatally. In the present study, the effect of the Wnt signalling pathway on the differentiation potential of neonatal and adult neural stem cells (NS/PCs) isolated from subventricular zone (SVZ) of lateral ventricles and their membrane properties were studied eight days after the onset of in vitro differentiation. To manipulate Wnt signalling at different cellular levels, three transgenic mouse strains were used, which enabled inhibition or activation of the pathway using the Cre- loxP system. We showed that the activation of the Wnt signalling pathway leads to higher expression of β-catenin in both postnatal as well as adult NS/PCs, while Wnt signalling inhibition results in the opposite effect. To follow the fate of NS/PCs, the patch-clamp technique, immunocytochemistry, and Western blot were employed. After eight days of NS/PCs differentiation we identified three electrophysiologically and immunocytochemically distinct cell types of which incidence was significantly affected by the canonical Wnt signalling pathway, only in differentiated neonatal NS/PCs. Activation of this pathway suppressed gliogenesis, and promoted neurogenesis,...
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Membrane properties of NG2 glia in CNS
Knotek, Tomáš ; Anděrová, Miroslava (advisor) ; Hrčka Krausová, Barbora (referee)
NG2 glia represent a new type of glial cells in central nervous system, which does not belong to astrocytes, oligodendrocyte or microglia. and their most frequent marker is chondroitine sulphate proteoglycan NG2. These cells keep their proliferation ability in adult brain and it is generally accepted that they can differentiate into oligodendrocytes. This thesis summarize the current knowledge about membrane properties of NG2 glia, namely expression of numerous types of ion channels and ionotropic and metabotropic receptor on their membrane. NG2 glia express outwardly and inwardly rectifying K+ channels, Ca2+ activated K+ channels and two-pore domain K+ channels. Interestingly, they also express voltage gated Na+ channels, L, T, P/Q and N type Ca2+ channels and voltage gated Cl- channels. Furthermore, nonspecific cationic channels, such as HCN and TRP, were identified in NG2 glia and they express Na+ /Ca2+ exchanger at high level. There are also ionotropic and metabotropic glutamate and GABA receptors on NG2 glia membrane, together with nicotinic and muscarinic receptors, adrenergic and glycine receptors, metabotropic and ionotropic purinergic receptors, receptors for serotonine, dopamine and histamine. Ion channels and receptors in NG2 glia play an important role in their proliferation,...
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Differentiation potential of polydendrocytes in pathological states of central nervous system
Pavlištová, Tereza ; Anděrová, Miroslava (advisor) ; Smejkalová, Terézia (referee)
NG2 cells also called polydendrocytes or oligodendrocyte progenitors comprise fourth type of glial cells in the brain. Ng2 glia express distinct markers on their cell surface, which can be used for their identification. Particularly, NG2 proteoglycan chondroitin sulphate and receptors for platelet-derived growth factor belong to the main markers. Polydendrocytes arise in two canals of spinal cord and also in the ventral part of frontal brain, telencephalon and diencephalon. Population of NG2 cells is heterogeneous because they differ morphologically, by their electrophysiological properties and distinct differentiation potential depending on localization in brain. In conditions in vitro, polydendrocytes can differentiate into oligodendrocytes, protoplasmic astrocytes or neurons. In early postnatal brain, NG2 glia give rise to astrocytes, but most of these cells remain in the mature state or change themselves to oligodendrocytes. Pathological states of the central nervous system cause an activation of polydendrocytes, they start to be hypertrophied and increase expression of NG2 proteoglycan. Reaction of these cells is influenced by environment and chemical factors, for example growth factors, morphogens and cytokines. Depending on the type of CNS disorder the differentiation potential of NG2 glia...
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Polydendrocytes and their role in CNS
Suchá, Petra ; Anděrová, Miroslava (advisor) ; Tvrdoňová, Vendula (referee)
Polydendrocytes (NG2+ cells) are recently discovered glial cells in central nervous system (CNS) distinct from neurons, oligodendrocytes, astrocytes and microglia. Polydendrocytes could be identified mainly by the expression of the proteoglycan NG2 and platelet derived growth factor receptor alpha. They could be found in grey and white matter and represent the largest proliferating cell population in adult CNS. It is accepted that a subpopulation of polydendrocytes gives rise to oligodendrocytes not only in development, but also in adult CNS and after demyelination. A subpopulation gives rise also to protoplasmic astrocytes in embryonic development. In in vitro studies was observed that neurons and astrocytes may arise from polydendrocytes. Electrophysiological studies revealed that polydendrocytes form synapses with neurons and that their rate of proliferation could be controlled this way. Polydendrocytes are very important in study of remyelination after ischemia and demyelinating diseases, as they might serve as source of new oligodendrocytes or possibly of another glial cells. This thesis summaries general knowledge about polydendrocytes. Initially, I focus on their immunohistochemical markers and morphology. Next, I summarise findings about their development and fate in both embryonic and adult CNS. A...
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Physiological and pathological potential of astroglial NMDA receptors
Džamba, Dávid ; Anděrová, Miroslava (advisor) ; Vyklický, Ladislav (referee) ; Paleček, Jiří (referee)
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 during ageing as well as before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in cortical GFAP/EGFP- positive (GFAP/EGFP+ ) glial cells. 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...
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Early morphogenesis of lower cheek teeth in mice with gene defects.
Lagronová, Svatava ; Peterková, Renata (advisor) ; Anděrová, Miroslava (referee) ; Buchtová, Marcela (referee)
Tooth number is reduced in humans and mice when compared to the presumed basic tooth formula in mammals. In the regions, where teeth had been suppressed during evolution, a supernumerary tooth can appear as a result of abnormal development. However development of a supernumerary tooth, as well as origin of other anomalies, cannot be directly investigated in human embryos. That is the development of a supernumerary tooth was studied in a mouse model of this anomaly. The aims of the thesis were focused to verifying the hypothesis: Development of the supernumerary tooth in mutant mice is based on the revitalization of the rudimentary primordia of the teeth suppressed during evolution. We compared the morphological and quantitative aspects of the developing epithelium of the largest rudimentary (premolar) tooth primordia, called MS and R2, in the mandibles of WT, Spry2-/- , Spry4-/- , Spry2-/- ;Spry4-/- and Tabby mutant mice. Similarly, the upper incisor in WT mice was analysed and compared to the development of the duplicated incisor in Spry2+/- ;Spry4-/- mutant mice. In comparison to controls, decreased cell apoptosis and increased cell proliferation together with an enlarged volume of the dental epithelium were found during rudimentary tooth development in Spry mutant mice. These changes showed the...
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Neurogeneze a gliogeneze v dospělém mozku po ischemickém poškození
Honsa, Pavel ; Anděrová, Miroslava (advisor) ; Vyklický, Ladislav (referee) ; Mazurová, Yvona (referee)
Ischemic brain injury belongs to the most common cause of death in the developed countries. High complexity of this disorder significantly slows and limits the possible treatment. Currently, there is only one treatment available - the application of the thrombolytic, tissue plasminogen activator, while thousands of other drugs failed during clinical testing. Great expectations were seen in the stroke treatment employing neural stem cells obtained from several external sources; nevertheless, low survival rate, limited favorable outcome and enormous ethical issues complicate the application of such therapy. On the other hand, in the adult mammalian brain exist two endogenous processes - neurogenesis and gliogenesis. These processes need to be fully described and understood, in order to employ them as a source of new cells after injury. Therefore, this thesis focuses on the processes of adult neurogenesis and gliogenesis predominantly after ischemia. Adult neurogenesis and gliogenesis are processes, by which neurons or glial cells are generated from stem/progenitor cells. Both these processes are strongly influenced by brain injury; nevertheless, their contribution to regeneration after ischemia in the human brain is negligible. Here, we aimed to describe the role of polydendrocytes in the...
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