Les récepteurs ionotropiques AMPA (AMPAR) activés par le glutamate sont les principaux acteurs de la transmission synaptique excitatrice rapide du cerveau. Ils jouent également un rôle crucial dans les processus de plasticité synaptique, reconnus pour être à la base des fonctions cognitives. Les récepteurs canaux P2X sont activés par l'adénosine-5'-triphosphate (ATP) extracellulaire libéré par les neurones ou les cellules gliales. Ils sont exprimés dans le cerveau en périphérie des synapses glutamatergiques, où ils participent à l’excitabilité neuronale et modulent la transmission synaptique ainsi que la plasticité synaptique. Bien que la signalisation purinergique ait de multiples effets sur la transmission et la plasticité synaptique, la fonction des récepteurs P2X au niveau des synapses du cerveau reste à établir. Ici, nous montrons dans les neurones d'hippocampe en culture que l'activation des récepteurs P2X postsynaptiques par l'ATP exogène ou via la libération d'ATP endogène par les cellules gliales diminue l'amplitude des courants miniatures et évoqués des AMPAR postsynaptiques. En utilisant des approches d’électrophysiologie, de biochimie et d'imagerie en temps réel, nous démontrons que l'afflux de calcium passant par les canaux P2X déclenche l’internalisation des AMPAR par un mécanisme d’endocytose clathrine et dynamine dépendante. Cette diminution de surface altère par conséquent la transmission synaptique médiée par les AMPAR. Nous avons aussi démontré par des approches moléculaires et pharmacologiques la cascade de signalisation engagée dans l’altération du trafic des AMPAR de surface après activation des récepteurs P2X. Cette inhibition par les récepteurs P2X, serait dépendante de l’activation de kinases et des phosphatases qui régulent le niveau de phosphorylation des AMPAR. Nos travaux de recherche suggèrent ainsi, que les récepteurs postsynaptiques P2X jouent un rôle essentiel dans la régulation de l'expression de surface des AMPAR et régulent ainsi la force et la plasticité synaptique. / Ionotropic AMPA receptors (AMPAR) activated by glutamate are the main actors of the fast excitatory synaptic transmission in the brain. They also play a crucial role in the process of synaptic plasticity that are widely recognized to be the basis cognitive functions. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular adenosine-5’-triphosphate (ATP) released by neurons or glia. P2X receptors are located et the periphery of glutamatergic synapses and although purinergic signaling has multiple effects on synaptic transmission and plasticity, the function of P2X receptors at brain synapses remains to be established.Here, we show in cultured hippocampal neurons that activation of postsynaptic P2X receptors by exogenous ATP or glial release of endogenous ATP decreases the amplitude of miniature excitatory postsynaptic currents and AMPA-evoked currents. Using a combination of electrophysiology, surface or internalization assays and real time imaging, we demonstrate that the calcium influx through the ATP-gated channels triggers AMPA receptor internalization through clathrin-mediated dynamin-dependent endocytosis leading to reduced surface AMPA receptors and therefore, altered AMPA-mediated current. We also identified by molecular and pharmacological approaches the signaling cascade involved in the P2X-mediated alteration of surface AMPAR trafficking. P2X-mediated AMPAR internalization is dependent on the activation of kinases CamKII and phosphatases which regulate the phosphorylation level of AMPARs. Our finding indicates that postsynaptic P2X receptors play a critical role in regulating the surface expression of AMPAR and thereby regulate the synaptic strength.

En dehors de son rôle de stockage de l’énergie cellulaire, l’adénosine-triphosphate (ATP) est également une molécule de signalisation extracellulaire qui agit sur deux familles de récepteurs, les récepteurs métabotropiques P2Y et les récepteurs ionotropiques P2X. Dans le système nerveux central, les récepteurs P2X et la signalisation purinergique sont impliqués dans de nombreuses fonctions physiologiques comme la modulation de la transmission synaptique et la communication neurone-glie ainsi que dans diverses pathologies telles que les douleurs chroniques, l’épilepsie ou les maladies neurodégénératives. Enregistrer l’activité des récepteurs P2X in situ reste aujourd’hui encore difficile de par la paucité des outils pharmacologiques et de par les propriétés biophysiques particulières de ces récepteurs canaux. De surcroit, les mécanismes de libération de l’ATP sont encore mal caractérisés et la détection de cette molécule dans l’espace extracellulaire est limitée par la faible résolution spatio-temporelle des techniques disponibles. A ce jour, il n’existe aucune méthode satisfaisante permettant de suivre l’activité des récepteurs P2X ou détecter la libération d’ATP en temps réel. Pour pallier à ces manques, nous avons développé de nouveaux outils fluorescents basés sur la fusion du rapporteur calcique GCaMP6s aux récepteurs P2X.Notre étude montre d’abord que ces biosenseurs P2X-GCaMP6s sont capables de rapporter spécifiquement et dynamiquement, en fluorescence, l’activité des récepteurs P2X dans différentes lignées cellulaires (HEK, astrocytes, macrophages), ainsi que dans des neurones hippocampiques en culture. Dans un deuxième temps, l’outil P2X2-GCaMP6s a été modifié afin de créer un biosenseur de haute affinité pour l’ATP extracellulaire. Deux mutants, dont l’affinité apparente est de l’ordre de la centaine de nanomolaire, ont permis de détecter et de quantifier une libération d’ATP endogène. En combinant l’utilisation de ces biosenseurs avec des approches pharmacologiques et génétiques, nous avons montré que lors d’un choc hypotonique l’activation du canal VRAC LRRC8 contribue à la libération d’ATP par les cellules HEK et par des monocytes humains différenciés en macrophages. Enfin, nous avons montré que la libération d’ATP lors du gonflement des cellules déclenchait le phénomène de régulation du volume cellulaire, permettant aux cellules de retrouver leur volume initial.Ces biosenseurs fluorescents permettent donc de visualiser de façon dynamique l’activité des récepteurs P2X et la libération d’ATP. Ces outils étant compatibles avec des approches in vivo, ils devraient permettre une meilleure caractérisation des mécanismes moléculaires de la communication purinergique. / Adenosine 5'-triphosphate (ATP) is an extracellular signaling molecule acting on two major classes of membrane receptors, metabotropic P2Y receptors and ionotropic P2X receptors. In the central nervous system, P2X receptors are involved in diverse functions such as modulation of synaptic transmission or neuron-glia communication and are implicated in different pathologies including chronic pain, epilepsy or neurodegenerative diseases. Recording P2X receptor activity is difficult because of the paucity of pharmacological tools and because P2X receptors are prone to desensitization. In addition, measuring extracellular ATP concentration is challenging since the mechanisms and the source of ATP are still poorly characterized. In addition, classical ATP detecting approaches have clear spatial and temporal limitations. As a consequence, following P2X activity and visualizing or quantifying ATP release in real time remains challenging. To overcome these issues, we developed new fluorescent biosensors based on the fusion of the fluorescent calcium reporter GCaMP6s to P2X receptors.We first determined that fluorescence specifically reports on the activity of the P2X2 channel in different cell line (HEK, astrocytes, macrophages) and in primary culture of hippocampal neurons. We next engineered P2X2 receptor to create high affinity ATP biosensors. We identified two mutants with EC50s for ATP in the 100 nanomolar range that allow for the detection and the quantification of endogenous ATP release evoked by cell swelling. Using pharmacological approaches and knock-out cells, we demonstrated the implication in ATP release of the recently identify volume-regulated anion channel, LRRC8A in HEK cells and differentiated human macrophages. Finally, we provided evidence that the LRRC8-dependant ATP release is necessary for the cellular regulation of volume decrease after swelling.Our results show that these fluorescent ATP biosensors can be used to dynamically track P2X channel activity and can be used in vivo to decipher the molecular mechanisms involved in purinergic signaling.

Throughout this work, we looked at P2X coupled pathways in macrophages. We worked on three different models of macrophages in order to establish the best model to understand the role of P2X4 receptors in the inflammation. Our work also consisted of further characterizing P2X7 receptor dependent pathways in these models. <p>P2X4 and P2X7 receptors are ionotropic receptors which are expressed by a variety of immune cells including macrophages. Macrophages play a very important host defense function as they are major actors in the innate immune system and they can initiate the activation of the adaptive immune system. The endogenous ligand of P2X receptors is ATP for which they share very different sensitivities. P2X4 receptors are relatively sensitive to this agonist while P2X7 receptors require concentrations > 100 μM ATP to be activated. <p>Our study supports the expression of P2X4 and P2X7 receptors in J774.2 murine macrophages and in human macrophages. Additionally, we worked on murine peritoneal macrophages for which the existence of P2X4 and P2X7 receptor expression had previously been shown in our lab. <p>A wide range of different macrophage phenotypes exist. Two extremes determine an array of phenotypes which are delimited by M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages while Mφ macrophages define the center of the array. Most of the work exposed in this study was carried out on pro-inflammatory macrophages which were obtained either by priming the cells with LPS alone (Mφ + LPS) or by polarizing them with LPS in association with IFNγ (M1). <p>We show in this study that LPS-primed J774.2 murine macrophages are not a good model to study the role of surface P2X4 receptor in pro-inflammatory macrophages. Additionally, we support that murine peritoneal macrophages primed with LPS are a good model to understand the hypothetical role of P2X4 receptors in the inflammation. Finally, we suggest that human M1 macrophages could be as well. Next, we also confirm that J774.2 murine macrophages, murine peritoneal macrophages and human macrophage express functional P2X7 receptors. In this study, we show that P2X7 receptors are coupled to RONS formation in J774.2 murine macrophages and to AA release through PLA2 activation in peritoneal macrophages. We show that activation of J774.2 murine macrophages with high concentrations of ATP (>600 μM) stimulates ROS formation including mitochondrial superoxide anions. In addition, our work shows the importance in using different dyes and suggests that different types of ROS play different functions in P2X7 receptors downstream pathways. <p>Next, we show that P2X7 receptor activation is coupled to an iPLA2 activity and that the release of free fatty acids mediated by 1 mM ATP is p-ERK1/2 dependent in LPS-primed murine peritoneal macrophages. <p>In addition, we have evaluated the effect of hypoxia on pathways which have been reported to be coupled to P2X7 receptor activation in pro-inflammatory human macrophages. Hypoxia does not seem to modulate P2X7 receptor functionality. However, both acute and chronic hypoxia influenced P2X7 receptors downstream pro-inflammatory coupled pathways. Finally, our work has enabled us to suggest for the first time that IFNγ plays an important function in host defense mediated by human P2X7 receptor activation in a hypoxic environment. <p>The effect of extracellular environment and thus different macrophage phenotypes have also been evaluated throughout this work in which we looked at the effect of polarization on P2X4 and P2X7 receptor functionality. Our work shows that LPS-priming does not modulate P2X4 receptor functionality in murine macrophages. Next, through our work, we suggest that polarization of human macrophages affects P2X4 receptor functionality in human macrophages. Additionally, our work shows that LPS affects ATP-mediated RONS formation in J774.2 murine macrophages but not P2X7-mediated AA release in primary murine macrophages. <p><p>Overall, first, our work has enabled us to suggest macrophage models to use in order to study the hypothetical role of P2X4 receptor in the inflammation mediated by macrophages. Second, it has allowed us to further understand how P2X7 receptors can act as important mediators of the immune system mediated by macrophages. In addition, many interesting observations were made in this study which allows us to propose several options for future directions. To finish, our work underlines the importance of the extracellular environment in some pathways mediated by ATP in macrophages.<p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished