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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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The use of chemogenetic tools in the research of brain diseases
Neřoldová, Markéta ; Stuchlík, Aleš (advisor) ; Tabáková, Petra (referee)
Chemogenetics is a newly developed set of tools that allow for selective manipulation of cell activity. They consist of a receptor mutated irresponsive to endogenous ligands and a synthetic ligand that does not interact with the wild-type receptors. Many different types of these receptors and their respective ligands for inhibition or excitation of neuronal subpopulations were designed in the past few decades. It has been mainly the G-protein coupled receptors (GPCRs) selectively responding to clozapine-N-oxide (CNO), namely Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), that have been employed in research. Chemogenetics offer great possibilities since the activity of the receptors is reversible, inducible on demand by the ligand, and non-invasive. Also, specific groups or types of neurons can be selectively manipulated thanks to the delivery by viral vectors. Furthermore, the effect of the chemogenetic receptors on neurons lasts longer, and even chronic activation can be achieved. That can be useful for behavioural testing. The great advantage of chemogenetic tools is especially apparent in the case of the research of brain diseases since they can manipulate whole neuronal circuits and connections between different brain areas. Many psychiatric or other brain diseases...
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Maternal separation and its impact on neurotransmitter systems of the brain
Přítulová, Eliška ; Novotný, Jiří (advisor) ; Černá, Barbora (referee)
Adverse early life stress conditions during the postnatal period can alter normal brain and neuroendocrine system development and increase the susceptibility of the individual to various disorders. Material separation is a model used to investigate the effects of early life stress on organism. The principle of this model is to provoke the stress of the infant by disrupting its contact with the mother, which is important for its proper development. The aim of this paper is to unify the existing knowledge about the impact of maternal separation on signaling pathways in the brain and to outline the directions of future research. Despite all the differences between the individual studies, there is a basic finding that maternal separation leads to deficits in the functions of important neurotransmitter systems, can induce long-term changes in different brain signaling pathways and can be accompanied by changes at the neuroendocrine and behavioral levels.
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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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