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DISP3/PTCHD2 function in neural cells
Konířová, Jana ; Bartůněk, Petr (advisor) ; Anděrová, Miroslava (referee) ; Pacherník, Jiří (referee)
DISP3 protein, also known as PTCHD2, belongs to the PTCHD family of proteins, which contain a sterol-sensing domain in their structure. The expression of the Disp3 gene is high in neural tissues and is regulated by thyroid hormone. The DISP3 gene is associated with development and progression of certain types of tumors, as well as with development of some neural pathologies. Neural stem cells also display high expression of the Disp3 gene. Neural stem cells are defined by their capability to self-renewal and capacity to differentiate into the basic types of neural cells - neurons, astrocytes, and oligodendrocytes. Precise regulation of the balance between proliferation and differentiation of neural stem cells is crucial for development of the central nervous system and its subsequent proper functioning, and disruption of this balance may lead to development of various pathologies. In this work we mainly focused on describing the function of the DISP3 protein in neural cells and tissues. We have shown that during differentiation of neural stem cells, the expression of the Disp3 gene is significant decreased. Furthermore, we have found that in neural stem and progenitor cells, the increased expression of the Disp3 gene promotes their proliferation. Moreover, when Disp3 expression was disrupted, the...
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Astrocyte volume regulation during aging
Eliášová, Barbora ; Anděrová, Miroslava (advisor) ; Vargová, Lýdia (referee)
Astrocytes, as one of the glial cell types, have many important functions in healthy functioning of the central nervous system (CNS) but also in its pathology. Since they play a key role in maintenance of ionic, neurotransmitter and water homeostasis in CNS, they possess the ability to regulate their volume. Hypo- or hyperosmotic stress can trigger regulatory volume decrease or increase in astrocytes in order to stabilize their volume. During aging, astrocytes undergo many changes together with the rest of the brain. In order to determine whether these alterations involve also regulatory volume mechanisms, we employed three dimensional morphometry, which comprises confocal microscope scanning of fluorescently labelled astrocytes in brain slices of EGFP/GFAP mice and quantification of astrocyte volume during different pathological stimuli. Time-dependent volume changes of hippocampal astrocytes were recorded while applying either hypoosmotic solution or solution with high extracellular potassium concentration. In the four different age groups studied in the experiment, several differences in volume changes were discovered together with some sex-dependent alterations in astrocyte volume. Additionally, in accordance with previous studies, two subpopulation of astrocytes were identified using...
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Calcium signaling of oligodendroglial lineage cells in the animal model of schizophrenia
Kročianová, Daniela ; Anděrová, Miroslava (advisor) ; Stuchlík, Aleš (referee)
Schizophrenia is a neurological disorder with a complex psychopathology, which is far from fully elucidated. In the patients with this disorder, changes on anatomical, cellular, and neurotransmitter level have been found. The aim of this work is to elucidate the function of specific ionotropic glutamate receptors in NG2 glia in the hippocampus of a mouse model of schizophrenia. For this purpose, a mouse model of schizophrenia was generated and validated using immunohistochemistry and behavioural testing. Mice with NG2 glia labelled by a fluorescent protein with a calcium indicator also in NG2 glia were used to observe the activity of glutamate channels and the properties of the extracellular space in these mice. Changes were found in the schizophrenic animals when compared to control animals in the numbers of hippocampal oligodendrocyte lineage cells, in prepulse inhibition and in both volume fraction and tortuosity of the extracellular space in hippocampus. Moreover, the percentage of cells responding to glutamate receptor agonists in NG2 glia in hippocampus also differed significantly between the schizophrenic and the control animals. In conclusion, it can be said that we were able to observe significant changes in the mouse model of schizophrenia that we generated in comparison to control...
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The role of the Wnt signaling pathway in the regeneration following ischemic brain injury
Kriška, Ján ; Anděrová, Miroslava (advisor) ; Stuchlík, Aleš (referee) ; Zíková, Martina (referee)
The role of the Wnt signaling pathway in the regeneration following ischemic brain injury Focal cerebral ischemia results in the loss of neural cells, which may cause permanent disability. At the same time, there are precursor cells (neural stem/progenitor cells and NG2 glia) that naturally reside in the postnatal brain and may proliferate and give rise to other cell types. Their fate is to a large extent influenced by morphogens of the Wnt and Shh family. However, the role of these cellular pathways in differentiation of precursor cells is still enigmatic. For this reason, we employed transgenic mice that enabled us to inhibit or hyper-activate the canonical Wnt signaling pathway, or to map the fate of NG2 cells. The induction of ischemia was achieved by the occlusion of the middle cerebral artery. The changes in the differentiation potential were characterized at the mRNA, protein, and functional levels. First, we evaluated neural stem/progenitor cells isolated from neonatal mice under physiological conditions and found out that Wnt signaling promotes neurogenesis and suppresses gliogenesis. Next, we focused on adult mice and detected a smaller impact of Wnt signaling on their differentiation potential. Nonetheless, its effect was more profound after the induction of ischemia, as we identified...
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Cellular and molecular mechanisms of activation of thermally sensitive TRP ion channels
Máčiková, Lucie ; Vlachová, Viktorie (advisor) ; Anděrová, Miroslava (referee) ; Jakubík, Jan (referee)
The transient receptor potential (TRP) are cation channels mostly permeable to both monovalent and divalent cations. ThermoTRP is a specific group of directly thermally activated TRP channels. The vanilloid transient receptor potential 3 (TRPV3) is an ion channel widely expressed in keratinocytes, that is implicated in the regulation of skin homeostasis, thermo- sensing, nociception and development of itch sensation. Our results show the importance of the cytoplasmic inter-subunit interface in the heat sensitivity of TRPV3. As there is a structural analogy within the vanilloid receptors, our hypothesis of the identified important region is supposed to be valid also for other thermally activated TRPV receptors (TRPV1, TRPV2 and TRPV4). We have proved that TRPV3 is a substrate for ERK1/2 protein kinase (kinase regulated by extracellular signal 1 and 2) and we have identified TRPV3 phosphorylation sites that may be direct targets for ERK1/2. Of these residues, threonine 264 has been shown to be the main phosphorylation site responsible for TRPV3 sensitization mediated by ERK kinase. In human keratinocytes, the phosphorylation might be physiologically and pathophysiologically important in processes of TRPV3 sensitization mediated by MAPK signaling pathway. The transient receptor potential ankyrin 1...
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The role of AQP4 and TRPV4 channels in the ischemic brain edema: focusing on glial cells
Kročianová, Daniela ; Anděrová, Miroslava (advisor) ; Máčiková, Lucie (referee)
Cerebral ischemia, also known as stroke, is one of the most common causes of death. It is accompanied by the formation of edema, which can be characterized as an influx of water and osmolytes into the brain, causing volume alterations. We recognize two types of cerebral edema - vasogenic, characterized by the disruption of the blood-brain barrier (BBB) and increase of the extracellular volume, and cytotoxic, caused by the increase of the volume of cells, mainly glia. The major contributors to the formation of cytotoxic edema are the astrocytes, which, in physiological conditions, are responsible for the maintenance of the BBB and keeping the homeostasis of the brain and spinal cord or central nervous system. The mechanism responsible for the process of volume and osmotic changes are the transmembrane channels, mainly aquaporin 4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4). AQP4 is the main pathway for water influx as well as efflux when the edema subsides. TRPV4 is likely responsible for the maintenance of the osmotic balance of the organism, although its precise role in the formation of the edema has not yet been fully elucidated. The main aim of this thesis was to categorize the types of cerebral ischemia and edema, and to describe the process of cerebral edema formation and the...
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The role of AQP4 and TRVP4 channels in the ischemic brain edema: focusing on glial cells.
Kročianová, Daniela ; Anděrová, Miroslava (advisor) ; Máčiková, Lucie (referee)
Cerebral ischemia, also known as stroke, is one of the most common causes of death. It is accompanied by the formation of edema, which can be characterized as an influx of water and osmolytes into the brain, causing volume alterations. We recognize two types of cerebral edema - vasogenic, characterized by the disruption of the blood-brain barrier (BBB) and increase of the extracellular volume, and cytotoxic, caused by the increase of the volume of cells, mainly glia. The major contributors to the formation of cytotoxic edema are the astrocytes, which, in physiological conditions, are responsible for the maintenance of the BBB and keeping the homeostasis of the brain and spinal cord or central nervous system. The mechanism responsible for the process of volume and osmotic changes are the transmembrane channels, mainly aquaporin 4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4). AQP4 is the main pathway for water influx as well as efflux when the edema subsides. TRPV4 is likely responsible for the maintenance of the osmotic balance of the organism, although its precise role in the formation of the edema has not yet been fully elucidated. The main aim of this thesis was to categorize the types of cerebral ischemia and edema, and to describe the process of cerebral edema formation and the...
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The Role of Aquaporin 4 channels and Transient Receptor Potential Vanilloid 4 channels in astrocytic swelling
Heřmanová, Zuzana ; Anděrová, Miroslava (advisor) ; Machová Urdzíková, Lucia (referee)
Astrocytes posses a wide range of functions within the brain. In response to ischemic conditions they swell due to increased uptake of osmolytes and they are mainly responsible for cytotoxic edema formation. However, they are also able to regulate their volume by releasing osmolytes together with water via the process of regulatory volume decrease (RVD). The Aquaporin 4 (AQP4) channel and Transient receptor potential vanilloid 4 (TRPV4) channel are suspected to be strongly involved in these processes of astrocytic volume regulation. The goal of the present diploma thesis was to clarify the role of both channels in astrocytic swelling in situ. For our experiments we used a subpopulation of green fluorescent protein-labelled astrocytes from AQP4-deficient (AQP4-/- ), TRPV4-deficient (TRPV4-/- ) and control (Ctrl) mice. Cell volume alterations were induced in acute brain slices by hypoosmotic stress or by oxygen-glucose deprivation (OGD). Data were quantified using fluorescence intensity-based approach in the whole cells and in astrocytic endfeet. Our results indicate, that there is no difference in astrocytic swelling or cell volume recovery between astrocytes from AQP4-/- , TRPV4-/- and control mice when exposed to hypoosmotic stress. On the contrary, volume changes induced by OGD varied...
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