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Glutamatergic synaptic transmission, targeting and distribution of metabotropic glutamate receptor in neuronal cells
Techlovská, Šárka ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Fusek, Martin (referee)
Stránka 7│146 ABSTRACT An evolutionary developed eukaryotic proteins for transformation of extracellular signal into intracellular targets are receptors. There are known receptors integrated to intracellular organelle membranes e.g. inositol-3-phosphate receptor (Inositol-3-phoshpate receptor; IP3R) specific for endoplasmic reticulum or nuclear receptors e.g. receptors for gonad hormones transforming the intracellular signals, however the vast majority of receptors are present on plasmatic membrane and recognize an extracellular signals. High percentage of these receptors are included in family of proteins called G-protein coupled receptors, (G-protein coupled receptors, GPCRs) due to their signal mediators heterotrimeric G-proteins. Each receptor of this family has the preference to specific type of G-protein influencing the intracellular concentrations of Ca2+ , cyclic adenosinmonophosphate or inositol-3- phosphate. Some of them are able to signalise by dual manner or through G-protein independent pathways. Independently on cellular target membrane, GPCRs integration into membranes is enables by the content of GPRCs-specific 7-transmembrane domain (7-transmembrane domain, 7TM) which is consist of hydrophobic amino acids. The relation between the structure and functionality is very tightly related. Many...
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Role of NMDA NR1 subunit in pathophysiology of schizophrenia
Vrajová, Monika ; Horáček, Jiří (advisor) ; Blahoš, Jaroslav (referee) ; Vyklický, Ladislav (referee)
Our work is focused on the role of NR1 subunit of N-methyl-D-aspartate receptor in pathophysiology of schizophrenia. In animal model using separately or in combination, antisense oligodeoxynucleotide (aODN) for NR1, NR2A and NR2B subunit of NMDAR, we affected expression of these proteins in rat hippocampus. We assessed prepulse inhibition of acoustic startle reaction (PPI) in rats and protein expression of NMDAR subunits and expression of PSD proteins. There were significant differences in expression of PSD-95 and NR1 between groups. Application of aODN (NR2A, NR2B) was associated with a significant decrease of PSD-95. PPI and expression of NR2A, NR2B and PSD-93 were not changed after aODN application.The next part of the work concentrates on a human post mortem study. To assess actual changes in the expression of the NR1 subunit and its isoforms, we measured absolute differences in the levels of mRNA/protein for panNR1, as well as the individual mRNA/protein isoforms in the post mortem left/right hippocampus of patients with schizophrenia in comparison with non-psychiatric subjects. There were no significant differences in the panNR1 subunit mRNA expression, but the absolute left/right differences were much more pronounced in the patients with schizophrenia. The expression of splice variants in the...
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Neuropharmacology of spatial navigation, cognitive coordination and flexibility tests in animal models
Prokopová, Iva ; Stuchlík, Aleš (advisor) ; Vyklický, Ladislav (referee) ; Blahoš, Jaroslav (referee)
Spatial navigation, cognitive coordination and behavioral flexibility belong amongst cognitive functions, which play a role in many neuropsychiatric disorders. Behavioral tasks have proved to be useful paradigms to test these functions in pharmacological or genetic animal models. First aim was to determine a potential interaction between β-adrenergic and α1-adrenergic or D2-dopaminergic systems. Spatial navigation and coordination were impaired in both studies during co-aplication of subthreshold doses of drugs. Used substances belong to group of widely prescribed drugs, thus our results could be implicated in clinical practice. Another study examined an acute effect of MK-801 (animal model of schizophrenia) on behavioral flexibility in Carousel maze and the Morris water maze (MWM). Carousel maze showed higher sensitivity with impairments from 0.08 mg.kg-1 compared to 0.10 mg.kg- 1 in MWM. The final experiment aimed at testing the effect of reduced expression of Nogo-A protein on spatial navigation and behavioral flexibility of rats. A battery of tests in the Carousel maze revealed impairment in cognitive functions, MWM showed unaffected working memory of rats. Our results support the hypothesis linking Nogo-A knock-down rats with neuropsychiatric symptoms and cognitive disorders. Key words:...
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Steroid - NMDA receptor interaction: Structure-activity study and effect on mutant forms of human NMDA receptors
Krausová, Barbora ; Vyklický, Ladislav (advisor) ; Anděrová, Miroslava (referee) ; Tureček, Rostislav (referee)
N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium permeable ion channels that play a key role in excitatory synaptic transmission and plasticity, and their dysfunction underlies several neuropsychiatric disorders. The overactivation of NMDA receptors by tonically increased ambient glutamate can lead to excitotoxicity, associated with various acute and chronic neurological disorders, such as ischemia, Alzheimer and Parkinson's disease, epilepsy or depression. On the opposite, NMDA receptor hypofunction is thought to be implicated in autism, schizophrenia, or intellectual disability. Recent DNA screening for neurological and psychiatric patients revealed numerous mutations in genes encoding for NMDA receptor subunits. The activity of NMDA receptors is influenced by a wide variety of allosteric modulators, including neurosteroids that could both inhibit and potentiate the activity of NMDA receptors, which makes them promising therapeutic targets. In this thesis, we describe new classes of neurosteroid analogues which possess structural modifications at carbons C3 and C17 of the steroidal core, and analogues without D-ring region (perhydrophenanthrenes). We evaluated the structure-activity relationship (SAR) for their modulatory effect on recombinant GluN1/GluN2B receptors. Our results...
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Function of the GABA and glycine receptors in the mammalian MNTB
Hrušková, Bohdana ; Tureček, Rostislav (advisor) ; Vyklický, Ladislav (referee) ; Horák, Martin (referee)
GABA and glycine are major inhibitory neurotransmitters in the central nervous system. They act on ionotropic and metabotropic receptors that form oligomeric complexes in plasma membrane of neuronal cells. Pharmacological properties, subcellular distribution and function of GABA and glycine receptors depend on their subunit composition. The thesis was aimed to find subunit composition and physiological role of ionotropic glycine and metabotropic GABAB receptors in neurons of medial nucleus of trapezoid body, MNTB. The nucleus resides in the mammalian auditory brainstem and it is characterized by well defined excitatory and inhibitory inputs. Excitatory fibers form giant glutamatergic nerve terminals, calyces of Held, whereas inhibitory fibers form small GABA/glycinergic boutons. Both types of afferents innervate somatodenritic parts of MNTB principal neurons. The nucleus thus represents a suitable model for studying molecular and cellular mechanisms of interactions between excitation and inhibition. Our experiments were performed using electrophysiology and immunohistochemistry methods. Patch clamp technique was used to record membrane currents and voltages from neurons in live MNTB slices isolated from rats or mice. Spontaneous and sound-evoked activity of murine MNTB neurons was recorded by in...
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Characterization of molecular components in cannabinoid signaling pathways.
Hájková, Alena ; Blahoš, Jaroslav (advisor) ; Vyklický, Ladislav (referee) ; Maletínská, Lenka (referee)
The cannabinoid receptor 1 (CB1R), a member of the G-protein coupled receptors superfamily, is a key player in endocannabinoid signalling. The CB1R is found presynaptically in neurones where it modulates synaptic plasticity. Precise description of the molecular mechanisms of synaptic neurotransmission is crucial for understanding of brain diseases and development of new therapeutic aproaches. Possible pharmacological targets of CB1R signalling include the treatment of various ailments such as energy imbalance disorders (anorexia, obesity), drug addiction, pain, insomnia, and some psychiatric conditions. This study reveals the "Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1" (SGIP1) as a novel interacting partner of the CB1R. The SGIP1 is an intracellular neuronal protein localized predominantly in axon terminals and is involved in clathrin mediated endocytosis. The overexpression of SGIP1 imbalance energy homeostasis and leads to obesity. We show that SGIP1 affects CB1R signalling via ERK1/2 whereas G-protein signallization remains unaltered. The SGIP1 also hinders CB1R internalization from the cell surface and supports its interaction with β-arrestin2. Also, we demonstrated heterodimerization of the main splice variants of metabotropic glutamate...
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The study of functional and molecular properties of NMDA receptors
Vyklický, Vojtěch ; Vyklický, Ladislav (advisor) ; Chvátal, Alexandr (referee) ; Tureček, Rostislav (referee)
N-methyl-D-aspartate (NMDA) receptors are a subtype of ionotropic glutamate receptors, which mediate fast synaptic transmission in the vertebrate central nervous system and play a key role in synaptic plasticity. NMDA receptor overexcitation leads to cell death that underlies many serious neurological and psychiatric disorders. The aim of our research was to characterize novel drugs that modulate NMDAR activity. We have tested a series of newly synthesized steroidal compounds for their activity on recombinant receptors. We have demonstrated a relationship between the structure of the analogues of a naturally occurring neurosteroid pregnanolone sulfate and their ability to modulate NMDA receptors. The results of our experiments characterize the role of substituents at the steroid A and D ring. We have found specific substituents on carbons C3 and C17 of the steroidal skeleton that lead to a substantial increase of steroid efficacy and the ability to positively affect the ratio between the inhibition of stationary and non-stationary receptor activation. These two states represent receptors long-term activated (tonically) and synaptically activated (phasically), respectively. Using electrophysiological and optical methods in combination with mathematical modeling we have shown that the plasma membrane plays an...
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Věkové a pohlavní rozdíly v GABAergní transmisi v substantia nigra pars reticulata u krys.
Chudomel, Ondřej ; Bojar, Martin (advisor) ; Vyklický, Ladislav (referee) ; Mareš, Pavel (referee)
Many experimental as well as clinical studies have shown that subcortical neuronal circuitries including the anterior part of the substantia nigra pars reticulata (SNR) are closely involved in the control of seizures propagation and termination. In vivo studies in rats demonstrated that inhibition of the GABAergic SNRA electrical output increases the seizure threshold in the flurothyl model of generalized clonic seizures. The anticonvulsant properties of the SNRA are largely age- and sex-depended. In the current experiments were used 3 age groups of Sprague Dawley rats (PN5-9, PN11-16 and PN25-32 days, PN = postnatal) to study GABAergic inhibition. Two types of GABAergic inhibition were studied: a) a synaptic inhibition, which is generated by α1 and α3-containing GABAARs and b) a tonic inhibition mediated by extrasynaptic δ subunit-containing GABAARs. Immunohistochemistry showed that the α1 subunit expression was generally more abundant in adult rats and females while the α3 subunit dominated in the early development and gradually decreased by the age of PN30. The more α1 subunit was expressed the faster were the kinetics, higher the mean amplitudes and frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs). The α subunit subtype underlies sensitivity to zolpidem, which preferentially...
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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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Multiple regulatory roles of the transmembrane adaptor protein NTAL in gene transcription and mast cell physiology
Polakovičová, Iva ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
(EN) This thesis focuses mainly on understanding of the regulatory roles of the transmembrane adaptor proteins, non-T cell activation linker (NTAL) and phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), in murine mast cell signaling. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) activation pathways in mast cells. Studies carried out on mast cells prepared from NTAL knock-out mice have indicated that NTAL is a negative regulator of FcεRI signaling, whereas experiments performed on human mast cells and rat basophilic leukemia cells with silenced NTAL expression have suggested its positive regulatory role. To thoroughly examine the involvement of NTAL in FcεRI-mediated signaling events in mouse mast cells and to determine whether different methodologies of NTAL ablation have different physiological consequences, we utilized a broad range of assays. Using bone marrow-derived mast cells (BMMCs) as a model, we obtained cells from NTAL wild type and knock-out cells and using lentiviral delivery approach we transduced part of the wild type cells, with vector bearing NTAL shRNA or empty vector to generate NTAL knock-down cells and control cells, respectively. Comparison of all four groups of generated cells in our assays...
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