In dieser Arbeit wird der Transport von Glutamatrezeptoren an der neuromuskulären Synapse von Drosophila-Larven untersucht. Dies geschieht anhand der Analyse von Mikroskopaufnahmen von mit Fluorophoren markierten Glutamatrezeptoren. Hierfür wurden statistische Parameter entwickelt oder angewendet, um die Verteilung der Glutamatrezeptoren quantitativ zu beschreiben. Die Ergebnisse zeigen, dass die Leitfähigkeit der Rezeptoren eine wichtige Rolle bei der Rezeptorlokalisation spielt. Glutamatrezeptoren mit verminderter Leitfähigkeit haben eine erheblich veränderte Lokalisation und akkumulieren verstärkt an jungen Synapsen. Außerdem ist die Anzahl der Rezeptoren nicht mehr an den Aufbau der prä-synaptischen Seite gekoppelt. Diese Arbeit zeigt auch, das Glutamatrezeptoren um synaptische Ankerplätze miteinander konkurrieren. Deshalb ist die Lokalisation eines Rezeptors nicht nur von seinem strukturellen Aufbau abhängig, sondern auch von den Eigenschaften anderer Rezeptoren. / In this thesis the neuromuscular junction of the Drosophila larva is used as a model system to study basic mechanisms of glutamate receptor trafficking. In particular confocal images of glutamate receptors, tagged to fluorophores, are analysed. For this, statistical parameters are developed and used to quantitatively describe the distributions of glutamate receptors. The results show that gating dynamics are an important variable in glutamate receptor trafficking. Glutamate receptors with reduced charge transfer have strong altered localisation and accumulate prematurely at young synapses. Furthermore, their abundance are uncoupled from presynaptic assembly. The thesis also shows that glutamate receptors compete for synaptic anchoring sites in postsynaptic densities. Therefore, the trafficking of one receptor is not only determined by its own structure but is also dependent on the properties of other receptors.

Coffin-Lowry Syndrome (CLS), an X-linked form of intellectual disability, is caused by mutations of the RPS6KA3 gene encoding the growth factor regulated kinase RSK2. To understand the consequences of RSK2 deficiency in the hippocampus we performed a comparison of the hippocampal gene expression profiles from Rsk2-KO and WT mice. It revealed differentialexpression of 100 genes, encoding proteins acting in various biological pathways. We further analyzed the consequences of deregulation of one of these genes, Gria2 encoding GluR2, a subunit of the glutamate AMPAR. An abnormal two-fold increased expression of GluR2 was found in the hippocampus of Rsk2-KO mice. Electrophysiology studies showed a reduction of basal AMPAR and NMDAR mediated transmission, in the hippocampus of Rsk2-KO mice. Activity of ERK1/2 was also abnormally increased in the adult hippocampus of Rsk2-KO mice. P-Sp1 level was also significantly higher in RSK2 deficient cells. Together, my results suggested that over expression of GluR2 in RSK2 deficient cells, is caused by increased Sp1 transcriptional activity on the Gria2 gene, which, itself, is the result of ERK1/2 increased signaling.

No description available.

Le syndrome de Coffin-Lowry (CLS), une déficience intellectuelle liée à l'X, est causée par des mutations du gène RPS6KA3 codant pour la kinase RSK2 régulée par les facteurs de croissance.Pour comprendre les conséquences du déficit en RSK2 dans l'hippocampe nous avons effectué une comparaison des profils d'expression génique d'hippocampes de souris Rsk2-KO et WT. Elle a révélé l'expression différentielle de 100 gènes, codant pour des protéines agissant dans divers processus biologiques. Nous avons analysé les conséquences de la dérégulation de l'un de ces gènes Gria2 codant pour GluR2, une sous-unité du récepteur glutamate AMPA. Un niveau d'expression doublé de GluR2 a été relevé dans l'hippocampe des souris Rsk2-KO et les études électrophysiologiques y ont révélé une réduction des transmissions AMPAR et NMDAR. L’activité de ERK1/2 était aussi anormalement augmentée dans l'hippocampe des souris Rsk2-KO, ainsi que le niveau de P-Sp1. Ensemble, mes résultats ont suggéré que la surexpression de GluR2 dans les neurones déficients en RSK2, était causée par une augmentation de l'activité transcriptionnelle de Sp1 sur le gène Gria2, qui, elle-même, est le résultat de l’augmentation anormale de l’activité de ERK1 / 2. / Coffin–Lowry Syndrome (CLS), an X-linked form of intellectual disability, is caused by mutations of the RPS6KA3 gene encoding the growth factor regulated kinase RSK2. To understand the consequences of RSK2 deficiency in the hippocampus we performed a comparison of the hippocampal gene expression profiles from Rsk2-KO and WT mice. It revealed differential expression of 100 genes, encoding proteins acting in various biological pathways. We further analyzed the consequences of deregulation of one of these genes, Gria2 encoding GluR2, a subunit of the glutamate AMPAR. An abnormal two-fold increased expression of GluR2 was found in the hippocampus of Rsk2-KO mice. Electrophysiology studies showed a reduction of basal AMPAR and NMDAR mediated transmission, in the hippocampus of Rsk2-KO mice. Activity of ERK1/2 was also abnormally increased in the adult hippocampus of Rsk2-KO mice. P-Sp1 level was also significantly higher in RSK2 deficient cells. Together, my results suggested that over expression of GluR2 in RSK2 deficient cells, is caused by increased Sp1 transcriptional activity on the Gria2 gene, which, itself, is the result of ERK1/2 increased signaling.

Les récepteurs métabotropiques au glutamate (mGluR), qui appartiennent à la classe C des récepteurs couplés aux protéines G (RCPG), sont bien connus pour leurs rôles importants dans les troubles neurologiques et psychiatriques. La compréhension de leur mécanisme d’activation est essentielle pour la mise au point de nouveaux agents thérapeutiques. Récemment, le nombre de structures de RCPG cristallisées a augmenté de façon exponentielle grâce à l'application des méthodes de stabilisation de la protéine. Cependant, certaines ambiguïtés et incohérences ont été révélées au cours des études cristallographiques. En outre, des études en molécules uniques, y compris par transfert d'énergie d’excitation électronique de Förster (smFRET), ont montré la nature très dynamique des RCPG en général, et du domaine d’activation de mGluR en particulier. Ici, nous nous sommes intéressés au mécanisme d'activation des mGluR entiers en utilisant des techniques de FRET d’ensemble et sur molécules uniques. Les techniques de HTRF ont permis l’optimisation de la préparation des échantillons. Un protocole a été mis au point, permettant d'extraire les mGlu2 entiers dans du détergent, à partir de cellules HEK293T, sans affecter de manière importante la pharmacologie et de la stabilité des récepteurs. Les expériences de FRET en molécules uniques ont été effectuées avec la technique MFD-PIE. Une analyse poussée de ces données, par mesure de l'efficacité de FRET ratiométrique, de durée de vie des fluorophores dans l’état excité, et d’analyse en corrélation (FCS), ont permis de montrer un changement conformationel rapide (sub-milliseconde) des récepteurs mGlu2 entiers. Par ailleurs, le rôle de stabilisation du domaine transmembranaire en faveur de l’état actif a été prouvé. / Metabotropic glutamate receptors (mGluR), which belong to class C of G protein-coupled receptors (GPCR), are well-known for their important roles in neurological and psychiatric disorders. Understanding of receptor activation is essential to decipher the receptor functioning, and thus orientate drugs design for targeted therapeutics. Recently, the number of GPCR crystal structures has increased exponentially thanks to the application of protein stabilization methods. However, these crystallography studies have revealed certain ambiguities and discrepancies, and these approaches do not take into account the dynamic nature of GPCR activation. Indeed, single-molecule studies, including single-molecule FRET (smFRET), have revealed the highly dynamic nature of GPCR in general, and fast conformational changes of mGluR domains in particular. Here, we study the activation mechanism of the full-length mGluR by FRET techniques at ensemble and single-molecule level. Homogenous time-resolved fluorescence (HTRF) was applied for optimizing the sample preparation. An appropriate protocol was established, allowing to extract mGlu2 full-length in detergent from the HEK293T cells without significantly affecting its pharmacology and stability. smFRET experiments were performed using the combination of multiparameter fluorescence detection (MFD) with pulsed interleaved excitation (PIE). Advanced data analysis such as ratiometric FRET efficiency, lifetime-based FRET measurement, and fluorescence correlation spectroscopy (FCS) revealed that the fast dynamic oscillation in sub-millisecond timescale of the full-length mGlu2, and prove the stabilization role of the transmembrane domain of the full-length receptor in favor of the active state.

N-methyl-D-aspartate (NMDA) receptors are a class of ionotropic glutamate receptors, involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They form heterotetrameric complexes composed of GluN1, GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Previous reports showed that different subunits of NMDA receptors, especially the GluN2 subunits, confer different functional and pharmacological properties on the receptor complexes. However, the subunit-dependent differences in the regulation of intracellular processing and transport of NMDA receptor subtypes has not been clearly elucidated. The aim of this work was to clarify the mechanisms of regulation of the NMDA receptor transport. In our experiments we performed immunocytochemistry of receptors on heterologous COS-7 cells and cultured cerebellar granule cells (CGC), both expressing recombinant NMDA receptors. The results of my work show that the transport of NMDA receptors is regulated by presence of GluN2A and GluN2B subunits. Our results further showed that transport of the GluN1/GluN2C receptors is regulated by three specific areas of the GluN2C subunit: i) the A2 segment within the amino- terminal domain, ii.) the M3 domain, and iii.) the proximal part of the C-terminus containing the...

In order to understand how the brain functions, under normal as well as pathological conditions, it is important to study the mechanisms underlying information integration. Depending on the nature of an input arriving at a synapse, different strategies may be used by the neuron to integrate and respond to the input. Naturally, if a short train of high-frequency synaptic input arrives, it may be beneficial for the neuron to be equipped with a fast mechanism that is highly sensitive to inputs on a short time scale. If, on the contrary, inputs arriving with low frequency are to be processed, it may be necessary for the neuron to possess slow mechanisms of integration. For example, in certain working memory tasks (e. g. delay-match-to-sample), sensory inputs may arrive separated by silent intervals in the range of seconds, and the subject should respond if the current input is identical to the preceeding input. It has been suggested that single neurons, due to intrinsic mechanisms outlasting the duration of input, may be able to perform such calculations. In this work, I have studied a mechanism thought to be particularly important in supporting the integration of low-frequency synaptic inputs. It is mediated by a cascade of events that starts with activation of group I metabotropic glutamate receptors (mGlu1/5), and ends with a membrane depolarization caused by a current that is mediated by canonical transient receptor potential (TRPC) ion channels. This current, denoted ITRPC, is the focus of this thesis. A specific objective of this thesis is to study the role of ITRPC in the integration of synaptic inputs arriving at a low frequency, < 10 Hz. Our hypothesis is that, in contrast to the well-studied, rapidly decaying AMPA and NMDA currents, ITRPC is well-suited for supporting temporal summation of such synaptic input. The reason for choosing this range of frequencies is that neurons often communicate with signals (spikes) around 8 Hz, as shown by single-unit recordings in behaving animals. This is true for several regions of the brain, including the entorhinal cortex (EC) which is known to play a key role in producing working memory function and enabling long-term memory formation in the hippocampus. Although there is strong evidence suggesting that ITRPC is important for neuronal communication, I have not encountered a systematic study of how this current contributes to synaptic integration. Since it is difficult to directly measure the electrical activity in dendritic branches using experimental techniques, I use computational modeling for this purpose. I implemented the components necessary for studying ITRPC, including a detailed model of extrasynaptic glutamate concentration, mGlu1/5 dynamics and the TRPC channel itself. I tuned the model to replicate electrophysiological in vitro data from pyramidal neurons of the rodent EC, provided by our experimental collaborator. Since we were interested in the role of ITRPC in temporal summation, a specific aim was to study how its decay time constant (τdecay) is affected by synaptic stimulus parameters. The hypothesis described above is supported by our simulation results, as we show that synaptic inputs arriving at frequencies as low as 3 - 4 Hz can be effectively summed. We also show that τdecay increases with increasing stimulus duration and frequency, and that it is linearly dependent on the maximal glutamate concentration. Under some circumstances it was problematic to directly measure τdecay, and we then used a pair-pulse paradigm to get an indirect estimate of τdecay. I am not aware of any computational model work taking into account the synaptically evoked ITRPC current, prior to the current study, and believe that it is the first of its kind. We suggest that ITRPC is important for slow synaptic integration, not only in the EC, but in several cortical and subcortical regions that contain mGlu1/5 and TRPC subunits, such as the prefrontal cortex. I will argue that this is further supported by studies using pharmacological blockers as well as studies on genetically modified animals. / QC 20101005

Yes / Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. / BBSRC, MRC and the European Research Council

Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.

Les patients atteints d'épilepsie du lobe temporal (TLE) ainsi que les rats injectés à l'acide kaïnique (KA) exhibent des patrons pathophysiologiques similaires de crises, de sclérose de l'hippocampe et de perte de certains types neuronaux. Parmi les cellules atteintes dans le modèle KA du TLE on retrouve certains interneurones inhibiteurs du CA1. En effet, certains interneurones des couches oriens et alveus (O/A-IN) meurent suite à une injection de KA chez le rat, contrairement aux interneurones à la bordure des couches radiatum et lacunosum/moleculare (R/LM-IN) de la même région. Bien que cette perte soit empêchée par des antagonistes des récepteurs glutamatergiques métabotropes de groupe I (mGluR1/5), la cause de cette perte sélective des O/A-INs reste à être précisée. Au cours des travaux de cette thèse, nous avons effectué des enregistrements de patch-clamp en configuration cellule-entière en modes courant- et voltage-imposé couplés à l'imagerie calcique pour étudier les causes de la vulnérabilité sélective des O/A-INs dans ce modèle. Dans un premier temps, nous avons évalué les effets d'une application aiguë de KA sur les propriétés membranaires et calciques pour voir s'il y avait des différences entre les O/A-INs et R/LM-INs qui pourraient expliquer la vulnérabilité. Nos résultats montrent que les dépolarisations et variations de résistance d'entrée ainsi que les augmentations de calcium intracellulaire, dépendantes principalement des récepteurs -amino-3-hydroxy-5-methyl-4-isoxasole propionic acid (AMPA), sont similaires entre les deux types d'interneurones suite à des applications aigües de KA. Ceci indique que l'effet aigu du KA sur les interneurones ne serait pas la cause de la vulnérabilité des O/A-INs. Dans un second temps nous avons comparé l'implication des sous-types de récepteurs mGluR1 et 5 dans l'activité épileptiforme des deux types d'interneurones évoquée dans un modèle de tranche désinhibée. Dans ce cas, nos données montrent un rôle important des mGluR1 et 5 activés synaptiquement lors des décharges épileptiformes et ce, de manière spécifique aux O/A-INs. Les courants synaptiques sous-tendant ces décharges impliquent des récepteurs ionotropes et métabotropes du glutamate. En présence d'antagonistes des récepteurs ionotropes glutamatergiques, les courants synaptiques sont biphasiques et formés de composantes rapide et lente. Les récepteurs mGluR1 et 5 sont différemment impliqués dans ces composantes: les mGluR5 étant impliqués dans les composantes rapide et lente, et les mGluR1 que dans la composante lente. Ces résultats indiquent que les mGluR1 et 5 contribuent différemment à l'activité épileptiforme, et spécifiquement dans les O/A-INs, et pourraient donc être impliqués dans la vulnérabilité sélective de ces interneurones dans le modèle KA. / Temporal lobe epilepsy (TLE) patients, as well as kainic acid (KA)-treated rodents, display similar pathophysiological patterns of behavioural seizures, hippocampal sclerosis and loss of certain neuronal types in the hippocampus. Among the cell types selectively vulnerable in the experimental KA model of TLE are certain inhibitory interneurons of the CA1 hippocampal region. Specifically, interneurons located in the oriens and alveus layers (O/A-IN) are lost following KA injections, whereas interneurons found in the radiatum/lacunosum-moleculare layers (R/LM-IN) are resistant. Although it has been shown that the group I metabotropic glutamate receptor (mGluR1/5) inhibitors can block this cell loss seen in the KA model, the precise cause of the selective O/A-IN vulnerability remains to be clarified. In this thesis, we have performed whole-cell patch-clamp recordings with simultaneous calcium imaging in an effort to elucidate the cause of the selective vulnerability of O/A-INs. We first determined the effects of acute KA applications on membrane properties and intracellular calcium rises in hippocampal slices to see if they might be different between O/A-INs and R/LM-INs. Our results reveal similar -amino-3-hydroxy-5-methyl-4-isoxasole propionic acid (AMPA) receptor dependent membrane depolarizations, input resistance variations and calcium reponses in these cells following KA applications, suggesting that acute KA actions may not cause the selective vulnerability of O/A-INs. Furthermore, we evaluated the contribution of mGluR1/5 to epileptiform discharges evoked in a disinhibited slice model, comparing responses between O/A-INs and R/LM-INs. Our data show an important role of synaptically activated mGluR1/5 during epileptiform discharges specifically in O/A-INs. In addition we show that the synaptic currents underlying these discharges involve ionotropic and metabotropic glutamate receptors. In the presence of antagonists of ionotropic glutamate receptors, synaptic currents are biphasic and composed of fast and slow components. mGluR1 and mGluR5 are involved differently in these components with mGluR5 implicated in fast and slow components and mGluR1 in the slow component only. Our findings therefore suggest that mGluR1 and 5 contribute differently to epileptiform discharges, and do so specifically in O/A-INs, suggesting that their activation may contribute to the selective vulnerability of these interneurons in the KA model of TLE.