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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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Functional and pharmacological properties of GluN1/GluN2 and GluN1/GluN3 subtypes of NMDA receptors
Kolcheva, Marharyta ; Horák, Martin (advisor) ; Bohačiaková, Dáša (referee) ; Balaštík, Martin (referee)
(EN) N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors and they play a critical role in excitatory synaptic transmission in the mammalian central nervous system (CNS). Hyperactivity or hypoactivity of NMDARs can lead to a wide spectrum of pathological conditions and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy, schizophrenia. NMDARs form a heterotetrameric complex made up of GluN1, GluN2(A-D) and/or GluN3(A, B) subunits. Different subtypes of NMDARs could have various effects on disease pathogenesis and therefore it is crucial to investigate the specific role of each subunit in the regulation of normal NMDAR functioning. The regulation of NMDARs occurs at different levels, from early processing, including synthesis, assembly, quality control in the endoplasmic reticulum (ER), trafficking to the cell surface, to internalization, recycling, and degradation. In this dissertation, we mainly focused on determining the roles of extracellular and transmembrane regions of different subtypes of NMDARs in the regulation of their function. In particular, using electrophysiology and microscopy methods on HEK293 cells and cultured hippocampal neurons, we investigated: (i) the impact of N-glycosylation and different lectins on...
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Neurosteroid effects on intracellular calcium and excitotoxicity
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Adámek, Pavel (referee)
NMDA receptors belong to the family of ionotropic glutamate receptors, and are involved in synaptic plasticity, learning and memory. However, overactivation by the agonist glutamate can lead to neuronal death - excitotoxicity. Exitotoxicity is a result of excessive calcium influx into the cell through NMDA receptors, and is associated with many cental nervous system (CNS) diseases. Neurosteroids are endogenous compounds capable of NMDA receptor modulation, thus they may have pharmacological potential in the treatment of CNS disorders. The aim of this work was to investigate how pregnanolone sulfate (PA-S) and pregnanolone hemipimelate (PA-hPim) influence somatic calcium and excitotoxicity. We used fluorescence microscopy for recording changes in somatic calcium concentration. We observed that PA-S had no influence on relative somatic calcium concentration. Synthetic analog PA-hPim increased somatic calcium levels slightly. Next, we used oxygen-glucose deprivation (OGD) in vitro to study the influence of neurosteroids on excitotoxicity. Both PA-S and PA-hPim were neuroprotective in the model of acute OGD in vitro. Moreover, PA-S or PA-hPim pretreatment induced ischemic tolerance to a subsequent OGD episode. Our results suggest that neurosteroids PA-S and PA-hPim are potential candidates for the development...
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Computational Studies of Interactions of Small Molecules with their Biological Targets
Nekardová, Michaela ; Hobza, Pavel (advisor) ; Berka, Karel (referee) ; Kabeláč, Martin (referee)
The thesis specializes in the computational description of pharmaceutically important compounds. A substantial number of pharmaceutical drugs are small molecules that are bound to an active site of an enzyme by the "lock (binding site) and key (drug)" model through non-covalent interactions. The association of enzymes with drugs cause an increase or decrease in the activity of enzymes. The main topic is focused on the computational elucidation of the structural basis for the interactions of the purine-like compounds with the enzyme cyclin- dependent kinase 2 that belongs to the protein-kinase enzyme family. These enzymes play an important role in the cell cycle regulation; their increased activity significantly contributes to the loss of control over cell proliferation, which is one of the primary causes of cancer cell formation. The study describes the binding motifs of roscovitine, which shows an inhibitory effect on the function of cyclin-dependent kinases, and its analogues containing bioisosteric central heterocycles in the complex with cyclin-dependent kinase 2. The binding affinity between the cyclin-dependent kinase 2 enzyme and the inhibitors was quantified as calculated binding scores and evaluated in relation to the conformation of the optimized structures. The hybrid model combining the...
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Possible influencing the motor performance of developing rats by repeated administration of the NMDA receptor antagonist specific for NR2 subunit
Kozlová, Lucie ; Mareš, Pavel (advisor) ; Valeš, Karel (referee)
Nonspecific NMDA receptor antagonists induce hyperlocomotion in rats. The aim of this work is to determine whether the NMDA receptor antagonist specific for NR2 subunit exhibit similar negative effect as nonspecific antagonists. This subunit is predominant in the brain in the early postnatal period. The introduction summarizes the data on NMDA receptors and the development of rat. The experimental part deals with the action of a specific NMDA receptor antagonist Ro 25-6981 on motor performance of developing rats. Substance was repeatedly administered to rats at postnatal days 7 to 11. Spontaneous locomotion and motor performance of the animals were repeatedly tested up to adulthood by battery of tests appropriate for individual ages. Our research demonstrated that this substance does not have significant effect on motor system of laboratory rat and that it might be further tested as a possible age-bound antiepileptic drug.
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Effects of sulfated neurosteroids on proteins involved in excitatory synaptic transmission
Naimová, Žaneta ; Smejkalová, Terézia (advisor) ; Mrózková, Petra (referee)
The discovery of steroid compounds capable of synthesize or accumulation in CNS and PNS led to a question about their function. Neurosteroid compounds are capable of modulating synaptic transmission. Effect is direct and fast mediated through nongenomic mechanisms. They are known to affect wide array of channels and receptors - both excitatory and inhibitory. This thesis summarizes findings about effect of sulfated neurosteroid on proteins involved in excitatory synaptic transmission. Thesis covers findings about ionotropic glutamate receptors, TRP channels, metabotropic receptors, sodium and potassium channels. Excessive or insufficient activity of these proteins involved in synaptic transmission can lead to a pathological condition. The purpose of this thesis is to summarize findings about effect of these compounds, point out structural and function characteristic probably responsible for their action and to outline possible pharmacological usage.
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The influence of the neuroactive steroids inhibiting NMDA receptors on behaviour
Chvojková, Markéta ; Valeš, Karel (advisor) ; Mareš, Pavel (referee)
The neuroactive steroid pregnanolone glutamate (Pg glu), a synthetic analogue of the naturally occurring pregnanolone sulfate (3alpha5betaS), has neuroprotective properties and a minimum of adverse effects. The subject of my thesis is the influence of selected structural modifications of the molecule Pg glu on biological effects. The first modification involves an increase of lipophilicity, the second involves the attachment of a positively charged group to C3. All these neuroactive steroids are use-dependent inhibitors of NMDA receptors. The first aim of this thesis was to determine the neuroprotective effectiveness of the neuroactive steroids chosen. The second aim was to explore the influence of selected neuroactive steroids on motor coordination, reflexes, anxiety and locomotor activity, as well as the effect of their high doses. The third aim was to create a battery of behavioural tests for screening the biological effects of analogues of Pg glu in laboratory rodents. The neuroprotective effects were evaluated in a model of excitotoxic damage of hippocampus in the rat on the basis of its behavioural consequences. The neuroprotective efficacy of androstane glutamate (And glu) and Pg glu was demonstrated. In the case of positively charged molecules, neuroprotective efficacy was not demonstrated....
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Synthesis and Properties of Neuroactive Steroids
Kapras, Vojtěch
Herein is reported the synthesis of molecular probes for action of neuroactive steroids in vitro and in living organisms. In the first part, preparation of enantiomeric pregnane steroids is investigated, ultimately resulting into the total synthesis of ent-progesterone. The chirality of the target molecule is introduced by a highly effective organocatalytic asymmetric Robinson annulation. A new method for the sequential construction of five-membered carbocyclic ring is introduced as the key step. This is composed of substrate-controlled copper-catalyzed conjugate addition followed by radical oxygenation and subsequent thermal cyclization employing the persistent radical effect. The synthesis of truncated neurosteroid analogs is described and their biological activity at the NMDA receptor is compared with the native hormone. In the second part, methodology for specific deuterium labeling of both angular methyls of the 5β-pregnane steroid core is explored. Special attention was paid to the Barton-McCombie deoxygenation as the tool for introduction of the last deuterium atom into the methyl group. Both positions were labelled with total of three deuterium atoms in high isotopic purity.
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Molecular mechanisms of regulation of trafficking and function of different subtypes of NMDA receptors in hippocampal neurons
Skřenková, Kristýna ; Horák, Martin (advisor) ; Balík, Aleš (referee) ; Bendová, Zdeňka (referee)
of Ph.D. thesis Molecular mechanisms of regulation of trafficking and function of different subtypes of NMDA receptors in hippocampal neurons Mgr. Kristýna Skřenková N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that play a key role in the mammalian central nervous system. Under physiological conditions, these receptors are important for excitatory synaptic transmission and memory formation. However, under pathological conditions, their abnormal regulation or activation may lead to many neurological and psychiatric disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, epilepsy or schizophrenia. Previous studies have shown that the number and type of NMDA receptors on the cell surface are regulated at multiple levels, including their synthesis, folding, internalization or degradation. During the trafficking of NMDA receptors to the cell surface membrane, both the agonist binding and receptor activation are examined. Moreover, NMDA receptors undergo many posttranslational modifications such as palmitoylation, phosphorylation or N-glycosylation. In this thesis, we studied the molecular mechanisms that may affect the trafficking and functional properties of NMDA receptors in mammalian cells and rat hippocampal neurons. Specifically, we studied i)...
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Computational Studies of Interactions of Small Molecules with their Biological Targets
Nekardová, Michaela ; Hobza, Pavel (advisor) ; Berka, Karel (referee) ; Kabeláč, Martin (referee)
The thesis specializes in the computational description of pharmaceutically important compounds. A substantial number of pharmaceutical drugs are small molecules that are bound to an active site of an enzyme by the "lock (binding site) and key (drug)" model through non-covalent interactions. The association of enzymes with drugs cause an increase or decrease in the activity of enzymes. The main topic is focused on the computational elucidation of the structural basis for the interactions of the purine-like compounds with the enzyme cyclin- dependent kinase 2 that belongs to the protein-kinase enzyme family. These enzymes play an important role in the cell cycle regulation; their increased activity significantly contributes to the loss of control over cell proliferation, which is one of the primary causes of cancer cell formation. The study describes the binding motifs of roscovitine, which shows an inhibitory effect on the function of cyclin-dependent kinases, and its analogues containing bioisosteric central heterocycles in the complex with cyclin-dependent kinase 2. The binding affinity between the cyclin-dependent kinase 2 enzyme and the inhibitors was quantified as calculated binding scores and evaluated in relation to the conformation of the optimized structures. The hybrid model combining the...
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