Dissertations / Theses on the topic 'MGlu Receptors'

Les récepteurs métabotropiques du glutamate (mGlus) sont connus pour moduler la transmission excitatrice dans le système nerveux central. Parmi ces récepteurs, ceux localisés au niveau de la pré-synapse, exercent un rôle d’autorécepteur, entrainant une diminution de la libération de glutamate à la suite de leur activation. L’étude du rôle fonctionnel de tel ou tel sous-type de mGlus est complexe compte-tenu du manque d’outils pharmacologiques sélectifs permettant de cibler spécifiquement un sous-type mGlus donné. Dans un premier temps, nous avons, par des techniques d’électrophysiologie et de fluorométrie calcique, validé de nouveaux outils pharmacologiques spécifiques de mGlu2 (un « nanobody » modulateur allostérique positif, PAM) et mGlu4 (OptoGluNAM4.1, un modulateur allostérique négatif, NAM), respectivement sur tranches d’hippocampe et de cervelet de rongeur. Nous avons ensuite, au sein du cortex cérébelleux, utilisé l’OptoGluNAM4.1 pour démontrer pour la première fois, l’implication de mGlu4 dans un contexte physio-pathologique: l’ischémie cérébelleuse. A l’aide d’outils pharmacologiques plus classiques nous avons également pu mettre en évidence, au sein des synapses qu’établissent les fibres parallèles avec les cellules de Purkinje l’existence d’un “dialogue” entre les récepteurs mGlu4 et les récepteurs A1 (récepteurs à l’adénosine de type 1), conséquence d’intéractions fonctionnelles entres les voies de signalisation de ces récepteurs présynaptiques dimériques et/ou conséquence de l’association physique de ces récepteurs au sein d’hétérodimères, fonctionnels
Metabotropic glutamate receptors (mGlus) are known to modulate excitatory transmission in the Central Nervous System. Among them, those situated at the pre-synaptic level behave like autoreceptors, their activation leading to a decrease in glutamate release. Functional studies of the different mGlus have been hampered by the lack of selective pharmacological tools specifically targeting a given subgroup of these receptors. As a first step, using electrophysiological and calcium fluorometry techniques, we validated new specific pharmacological tools acting on mGlu2 (a nanobody, positive allosteric modulator, PAM) and mGlu4 (OptoGluNAM4.1, a negative allosteric modulator, NAM) on rodent hippocampal and cerebellar slices, respectively. We then used the OptoGluNAM4.1 in the cerebellar cortex to demonstrate, for the first time, the involvement of mGlu4 in a physiopathological condition: cerebellar ischemia. Using more conventional pharmacological tools, we were also able to show the existence of a dialog between mGlu4 and A1 (Adenosine type 1) receptors at the level of the synapse between parallel fibers and Purkinje cells. Whether this dialog results from functional interactions between the signaling pathways of these pre-synaptic dimeric receptors and/or is a consequence of their physical association in heterodimers is presently under study

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 I_TRPC, is the focus of this thesis.

A specific objective of this thesis is to study the role of I_TRPC 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, I_TRPC 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 I_TRPC 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 I_TRPC, 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 I_TRPC in temporal summation, a specific aim was to study how its decay time constant (τ_decay) 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 τ_decay 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 τ_decay, and we then used a pair-pulse paradigm to get an indirect estimate of τ_decay.

I am not aware of any computational model work taking into account the synaptically evoked I_TRPC current, prior to the current study, and believe that it is the first of its kind. We suggest that I_TRPC 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

Compelling evidence has emerged to indicate that astrocytes play crucial roles in neurotransmitter signalling, in addition to CNS homeostatic functions. Astrocytes can communicate with each other and with neurons, giving rise to the concept of the ‘tripartite synapse’. Expression of type 5 metabotropic glutamate (mGlu5) receptors in astrocytes is now well-established. Agonist stimulation of mGlu5 receptors initiates robust oscillatory changes in cytosolic Ca2+ concentration in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving PKC and unidentified protein phosphatase activities in recombinant and native mGlu5 receptor-expressing systems. Each of the mGlu5 receptor positive allosteric modulators (PAMs) studied (DFB, CPPHA, CDPPB and ADX47273), failed to stimulate a Ca2+ response when applied alone, but increased the frequency of Ca2+ oscillations induced by glutamate or other orthosteric agonists. PAMs and negative allosteric modulators (NAMs) caused respectively graded increases and decreases in the Ca2+ oscillation frequency stimulated by orthosteric agonist. These data demonstrate that allosteric modulators can “tune” the Ca2+ oscillation frequency initiated by mGlu5 receptor activation and this might allow pharmacological modification of the downstream processes (e.g. transcriptional regulation) not achievable through orthosteric ligand interactions. Further investigation into the mechanism of action of PAMs revealed marked differences with respect to their modulation of orthosteric agonist affinity and efficacy. Thus, while DFB and CDPPB primarily exert their allosteric modulatory effects through modifying orthosteric agonist affinity, effects of ADX47273 are primarily mediated through an efficacy-driven modulation. Investigating the mechanisms underlying mGlu5 receptor-mediated Ca2+ oscillations in astrocytes has provided further evidence for the involvement of PKC(s) and protein phosphatase(s) in the rapid phosphorylation/dephosphorylation cycles occurring at the C-terminal of mGlu5 receptors, a process termed ‘dynamic uncoupling’. This work has been extended using specific siRNA knockdown to elucidate the potential PKC isoenzyme(s) that determine mGlu5 receptor regulation of Ca2+ oscillation frequency in astrocytes.

Le glutamate est le principal neurotransmetteur excitateur du système nerveux central. Il agit notamment sur huit récepteurs métabotropiques (mGluR) qui sont des récepteurs couplés aux protéines G (RCPG) responsables de la modulation de la transmission synaptique. Les mGluR constituent des cibles de choix pour le traitement de maladies neurologiques, psychiatriques et neurodégénératives comme la schizophrénie, la dépression, ou la maladie de Parkinson. Aucun médicament agissant sur les mGluR n'existe à l'heure actuelle, mais plusieurs molécules sont en phase clinique pour différentes pathologies. L'objectif principal de mon travail de thèse a été d'étudier la dynamique structurale des mGluR, pour lesquels le mécanisme moléculaire d'activation reste mal connu. Ces récepteurs forment des homodimères constitutifs, dont chaque sous-unité possède un grand domaine extracellulaire qui lie le glutamate et un domaine transmembranaire responsable de l'activation des protéines G et où se fixent des modulateurs allostériques synthétiques. Une des étapes clé de l'activation serait la réorientation relative des deux domaines extracellulaires induite par le glutamate au sein du dimère. En développant tout d'abord une stratégie de marquage orthogonale des sous-unités de mGlu par fusion avec des enzymes suicide (SNAP-/CLIP-tag) combinée à une technique de transfert d'énergie par résonance de type Förster en temps résolu (TR-FRET), nous avons montré qu'en système hétérologue, les mGluR peuvent s'associer sous forme d'hétérodimères fonctionnels. De plus, nos expériences ont révélé une spécificité d'association au sein de la famille des mGluR : les sous-unités mGlu du group I, mGlu1 et mGlu5, peuvent former des hétérodimères entre elles, mais pas avec celles du groupe II et III, qui elles peuvent toutes s'associer entre elles. Puis nous avons fait évoluer la technologie précédente pour développer le premier biosenseur conformationnel de l'activation des mGluR. Nous avons ainsi identifié sur cellules vivantes les changements conformationnels nécessaires à l'activation du récepteur, et démontré que la variation de signal de FRET entre les deux sous-unités au sein du dimère correspondant au réarrangement relatif des domaines externes est corrélée avec l'état d'activation du récepteur. Nous avons ainsi confirmé le modèle d'activation des mGluR initialement proposé à partir des premières structures cristallines des domaines extracellulaires isolés. D'autre part, ce senseur permet de discriminer facilement les agonistes partiels des agonistes complets, et permet de mieux comprendre les mécanismes allostériques régulant l'activité au sein des mGluR (notamment le mode d'action des modulateurs allostériques positifs et négatifs qui se lient dans le domaine membranaire). Cette stratégie de senseurs conformationnels a également pu être appliquée à l'étude d'autres récepteurs membranaires (RCPG et récepteurs tyrosines kinases), et au développement de tests de criblage à haut débit. Enfin, nous nous sommes attachés à développer de nouveaux types de molécules ciblant les mGluR, en utilisant des anticorps simple domaine provenant de lamas. Ces ligands qui agissent sur de nouveaux sites d'activation à la surface des mGluR, représentent de nouvelles pistes pour développer de meilleures solutions thérapeutiques
Glutamate is the main excitatory neurotransmitter in the central nervous system. It notably acts on eight metabotropic glutamate receptors (mGluR), which are G protein coupled receptor responsible for the modulation of synaptic transmission. mGluRs are promising pharmacological targets to treat neurological, psychiatric or neurodegenerative diseases such as depression, schizophrenia or Parkinson's disease. Unfortunately, so far, no drug acting at mGluR is accessible to patients, but several molecules are in clinical trials. The main objective of my thesis has been the study of the structural dynamics of mGluR, for which the molecular mechanism allowing activation are still poorly understood. These receptors are known to form constitutive dimers, with each subunit composed of a large extracellular domain which bind glutamate and a transmembrane domain responsible for G protein activation and where synthetic allosteric modulators bind. A key step in the activation process could be the relative reorientation of the two extracellular domains in the dimer upon glutamate binding. We first developed an orthogonal labeling method of each mGlu subunits by fusion with a suicide enzyme (SNAP-/CLIP-tag) that we combined with time-resolved Förster resonance energy transfer measurements to show that in a heterologous system, mGlu subunits can associate as strict and functional heterodimers. Our experiments also revealed a specific association pattern: mGlu subunits from group I, mGlu1 and mGlu5, can associate with each other, but not with those from group II and III, which can also associate with each other. Then we improved the technology to develop the first conformational sensor to monitor mGluR activation. We were able to monitor in real time in live cells the conformational changes occurring in the mGlu receptor upon activation, and we proved that the variation in FRET signal is correlated with the activation state of the receptor. This allowed us to confirm the activation model proposed based on the crystal structures of the isolated extracellular domains, which consist of a relative movement of the dimer extracellulair domains upon activation. Moreover, this sensor makes it possible to easily discriminate between full and partial agonists, and to better understand the allosteric mechanisms occurring in the mGluR (especially the action mode of positive and negative allosteric modulators binding in the transmembrane domain). This conformational sensor strategy was further applied to study the activation of other receptors (GPCR or tyrosine kinase receptors), and to develop screening assays compatible with high-throughput formats. Finally, we developed innovative ligands acting on mGluRs using single-domain antibodies from llamas. These activating ligands seem to bind to a new site on the surface of the receptor, offering new possibilities to develop better treatment acting at mGluRs

Dans certaines aires cérébrales, l’action synergique du glutamate et de la dopamine est nécessaire pour induire et maintenir la plasticité synaptique. Un dialogue fonctionnel entre le récepteur métabotropique du glutamate mGlu5 et le récepteur de la dopamine D1 a été mise en évidence. Par ailleurs, de nombreuses études ont démontré que les récepteurs couplés aux protéines G ont la capacité de former des hétéromères créant ainsi de nouvelles entités fonctionnelles. En s’appuyant sur l’hypothèse d’une hétéromérisation des récepteurs, l’objectif de ce projet de thèse était d’étudier les mécanismes moléculaires qui sous-tendent une synergie fonctionnelle entre les récepteurs mGlu5 et D1. Dans la première partie de ce travail, j’ai caractérisé les bénéfices de la Nanoluciférase, une luciférase très lumineuse, pour améliorer la technique de BRET en imagerie (Bioluminescence Resonance Energy Transfer imaging) qui permet d’étudier la dynamique des interactions entre protéines dans les cellules vivantes. Les bénéfices mis en évidence en termes de résolution spatio-temporelle, de stabilité et de sensibilité du signal ont été exploités pour la suite de ce projet. Dans la seconde partie de ce travail, les améliorations techniques mentionnées ci-dessus ont permis de mettre en évidence pour la première fois des hétéromères mGlu5/D1 dans des neurones en culture. En outre, nous avons montré que la co-expression des récepteurs mGlu5 et D1 en système hétérologue favorise la signalisation calcique, d’une part en augmentant l'activité constitutive de mGlu5 et, d’autre part, en créant une voie de libération du calcium intracellulaire atypique induite par l'agoniste D1.Ces résultats apportent de nouveaux éléments de compréhension des bases moléculaires du dialogue fonctionnel glutamate/dopamine dans le contrôle de la communication neuronale en conditions physiologiques et ouvrent la voie à de nouvelles stratégies thérapeutiques capables de moduler sélectivement la fonction des hétéromères
In some specific brain areas, synergism between glutamate and dopamine transmission is required to induce synaptic plasticity. Metabotropic glutamate receptor mGlu5 and dopamine receptor D1 are both known to control synaptic plasticity. Moreover, multiple lines of evidence converge toward the ability of G-protein coupled receptors to form dynamic heteromers thereby creating new entities with unique properties. Focusing on the hypothesis of receptor heteromerization, my PhD project aimed at investigating the molecular mechanisms underlying a functional interplay between mGlu5 and D1 receptors.To address this issue, a first part of this work consisted in improving single-cell Bioluminescent Resonance Energy Transfer (BRET) imaging, a technology enabling to study real time protein-protein interaction dynamics in living cells. Using the Nanoluciferase, an extremely bright luciferase, we characterized a faster and higher resolution single-cell BRET imaging technique with unprecedented performance in terms of temporal and spatial resolution, duration of signal stability and signal sensitivity. In the second part of this project, we showed that mGlu5 and D1 can form heteromers in heterologous expression system. The above-mentioned improvements of single-cell BRET imaging technique allowed to evidence the occurrence and the dynamics of mGlu5/D1 heteromers in cultured primary neurons. Furthermore, our results showed that the co-expression of mGlu5 and D1 receptors modifies single receptor properties to favor calcium signaling by increasing mGlu5 constitutive activity and creating a D1 agonist-induced activation of Ca2+ release from intracellular stores.These findings advance our knowledge about the molecular basis of the glutamate/dopamine functional dialogue to control neuronal communication in physiological conditions. Further investigation will help the dissection of the mGlu5/D1 heteromer specific signaling pathway with the hope of defining new therapeutics that may selectively modulate heteromer function and thus bypass undesirable side effects

La schizophrénie est un trouble mental débilitant multifactoriel affectant 1 % de la population mondiale qui est caractérisé par trois classes de symptômes : les symptômes positifs (hallucinations), les symptômes négatifs (isolement social) et les déficits cognitifs (altération de la mémoire de travail). Les antipsychotiques actuels tels que les antipsychotiques typiques et atypiques, ciblant respectivement le récepteur D2 de la dopamine et le récepteur 5-HT2A de la sérotonine, sont capables de traiter efficacement les symptômes positifs et peu efficaces contre les symptômes négatifs. Le récepteur métabotropique du glutamate mGlu2 (mGluR2) continue d'attirer une attention particulière compte tenu de son implication dans la schizophrénie. En effet, ce récepteur couplé aux protéines G (RCPG) est la cible principale d'une nouvelle génération d'antipsychotiques actuellement en essai clinique qui traite efficacement les trois classes de symptômes de la schizophrénie sans pour autant entraîner d’effets secondaires notables. Néanmoins, sa signalisation dans le cerveau et ses perturbations pathologiques restent mal caractérisées. En effet, la caractérisation spécifique de la signalisation native de mGluR2 est encore difficile en raison d'une forte homologie de séquences avec mGluR3, complexifiant la production de ligands ou d'anticorps spécifiques. Nous avons tiré parti d'un anticorps monocaténaire de lama (nanocorps) ciblant spécifiquement mGluR2 afin de purifier le récepteur endogène et ses interacteurs du cortex préfrontal de souris, une région cérébrale connue pour exprimer fortement mGluR2 mais également fortement perturbée dans la schizophrénie. Cet interactome a été caractérisé par spectrométrie de masse à haute résolution (AP-MS), et les annotations bioinformatiques des ontologies de gènes de ces partenaires protéiques ont révélé une grande pertinence pour les fonctions de mGluR2. L'un de ces interacteurs s'est révélé plus qu'attractif : le récepteur aux neurotrophines TrkB. Très significatif dans notre analyse AP-MS, il a été démontré que l'ARNm de TrkB est diminué dans le tissu cérébral des patients atteints de schizophrénie par rapport aux tissus témoins. Nous avons pu déterminer que : 1) mGluR2 et TrkB interagissent spécifiquement à une faible proximité, 2) cette interaction est modulée en fonction de leur état d'activation, 3) la stimulation de mGluR2 transactive TrkB dans les neurones corticaux dans le cortex préfrontal, 4) l'activation de TrkB conduit à la modulation de l'activité canonique de mGluR2 et 5) les effets antipsychotique des agonistes de mGluR2 dans un modèle préclinique de schizophrénie sont médiés par TrkB pour le sauvetage des déficits négatifs et cognitifs. En bref, cette transactivation réciproque de mGluR2 et TrkB est prometteuse puisque TrkB s’avère avoir un rôle central dans la médiation des effets antipsychotiques des ligands de mGluR2 dans les symptômes résistants jusqu'alors
Schizophrenia is a multifactorial debilitating mental disorder affecting 1 % of the world population characterized by three classes of symptoms: positive symptoms (e.g. hallucinations), negative symptoms (e.g. social isolation) and cognitive deficits (e.g. impaired working memory). Current antipsychotics such as typical and atypical antipsychotics, targeting dopamine D2 receptor and serotonin 5-HT2A receptor respectively, are able to treat efficiently the positive symptoms and partially the negative symptoms. However, antipsychotics treating efficiently the three classes of symptoms is still an unmet need. The metabotropic glutamate mGlu2 receptor (mGluR2) keeps on attracting particular attention given its implication in schizophrenia. This G protein-coupled receptor (GPCR) is the main target of a new generation of antipsychotics currently under clinical trial, treating efficiently the three class of symptoms without displaying any side effects. However, mGluR2 signaling in the brain and its pathological disturbances remain poorly characterized. Specific characterization of mGluR2 signaling in the brain was previously challenging due to a high homology of sequences with the structurally close metabotropic glutamate mGluR3 receptor, which complicated the production of specific ligands or antibodies. We have taken advantage of a single lama chain antibody (nanobody) specifically targeting mGluR2 in order to purify the endogenous receptor and its interacting proteins from mouse prefrontal cortex, a brain region known to strongly express mGluR2 but also highly disturbed in schizophrenia. This interactome was characterized by high resolution mass spectrometry and bioinformatics annotations of the gene ontologies of the candidate protein partners revealed high relevance to mGluR2 functions. One of these interactors revealed to be very attractive: the receptor tyrosine kinase TrkB. Highly significant in our AP-MS analysis, the mRNA of this rec eptor tyrosine kinase has been shown to be decreased in the brain tissue of patients with schizophrenia in comparison to control tissues. We were able to decipher that: 1) mGluR2 and TrkB interact specifically, 2) this interaction is modulated by the conformational state of both receptors, 3) mGluR2 stimulation by its agonist transactivates TrkB in cortical neurons in the prefrontal cortex, 4) the activation of TrkB leads to the modulation of the canonical activity of mGluR2 and 5) the antipsychotic-like effects of mGluR2 agonists in preclinical model of schizophrenia are mediated by TrkB for negative and cognitive deficits rescue. In summary, this reciprocal transactivation of mGluR2 and TrkB is highly promising and might have a pathophysiological influence in psychosis such as schizophrenia, but most importantly, TrkB has a pivotal role in mediating the antipsychotic-like effect of mGluR2 ligand for symptoms resisting so far to current antipsychotics

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The genetic defect in FXS is a CGG trinucleotide repeat expansion (>200) in the promoter region of the FMR1 (fragile X mental retardation 1) gene; this amplification causes the absence of the encoded protein FMRP (Fragile X Mental Retardation Protein). FMRP is an RNA-binding protein involved in mRNA transport and translation. Despite numerous studies, the available treatments are only symptomatic. There is no cure to replace FMRP expression, yet. FMRP can interact with RNA-binding proteins such as FXR1P, FXR2P, NUFIP and 82-FIP, and with proteins that do not bind RNA, like CYFIP1 and CYFIP2. The interaction with these different proteins may modulate FMRP functions and its RNA affinity. A new role of FMRP in mRNA metabolism as component of stress granules (SGs) has been identified. FMRP seems to lead mRNAs in SGs upon cellular stress, during which protein synthesis is blocked. SGs are ribonucleoproteic aggregates containing translation initiation components and RNA binding proteins, like eIF2a and FMRP. Several data also indicate that some of the FXS symptoms are a consequence of a defect in group-I metabotropic glutamate receptor, namely mGlu5; pharmacological blockade of mGlu5 receptors provide a therapeutic target in FXS. mGlu5 receptor, like FMRP, regulates protein synthesis but in a functionally opponent manner: mGlu5 receptor activates protein synthesis, FMRP suppresses it. In the absence of FMRP, mGlu5-dependent protein synthesis is unchecked, with consequent excessive translation. Activation of mGlu5 receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but its role in SGs formation is unknown.The aim of this PhD thesis was to better investigate FMRP function studying the relationship of FMRP with its interacting proteins and the role of FMRP in stress response under activation of mGlu5 receptor. In Paper I, we analyzed the expression pattern of FMRP and its interacting proteins in different brain areas, at different ages in wild type (WT) mice to better define the interplay between FMRP and its interacting proteins during development. FMRP was strongly expressed at P3, peaked at P7-P14 and gradually decreases thereafter. The analysis of expression pattern of several proteins carried out, indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental stages. We found that the RNA binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development. In Paper II we studied FMRP under stress condition using WT and Fmr1 knockout (KO) astrocytes. We have demonstrated that the lack of FMRP impairs SGs formation and furthermore that activation of mGlu5 receptor affects SGs formation through a FMRP-mediated mechanism in WT. Interestingly, the mGlu5 receptor blockade restores SGs formation in Fmr1 KO. Also, mGlu5 receptor activation before stress reduced FMRP recruitment in SGs and phosphorylation of eIF2a and FMRP. In contrast, mGlu5 receptor activation did not affect SGs formation in Fmr1 KO astrocytes. Since phosphorylation of eIF2a and FMRP are two crucial key events in SGs formation and modulation of protein synthesis, mGlu5 receptors may act by shifting the balance from inhibition to activation of protein synthesis during stress. These findings suggest a potential novel role for mGlu receptors in SGs formation. We suggest that FMRP may have a positive role in stress response, facilitating and enhancing SGs formation to prevent stress damages. This process is useful to understand what happens in FXS, in which can occur abnormal modulation of different proteins during development with consequent abnormal response during adversal conditions, like oxidative stress that represent a frequent component in FXS and neurodegenerative disorders.

Les douleurs chroniques et notamment les douleurs neuropathiques sont particulièrement difficiles à traiter par les solutions thérapeutiques actuellement disponibles. Par conséquent, il existe un besoin crucial de découvrir et d'exploiter de nouveaux concepts d'antalgiques afin de traiter ce type de douleurs. Parmi les différentes pistes possibles, le système glutamatergique semble particulièrement intéressant puisque le glutamate est le principal neurotransmetteur propageant l'information douloureuse. Notre hypothèse est que l'activation du récepteur mGlu4 spinal inhiberait la neurotransmission glutamatergique et réduirait donc l'excès de douleur observé lors de douleurs chroniques. Grâce notamment au développement du premier agoniste orthostérique sélectif de mGlu4, nous avons mis en évidence que le récepteur mGlu4 n'altère pas la perception de la douleur aiguë mais qu'il influe, au contraire, sur l'aspect pathologique de la douleur en inhibant l'allodynie et l'hyperalgie mécanique ou thermique présentes lors de douleurs chroniques. Nous montrons que la modulation de l'hypersensibilité par mGlu4 semble provenir de sa capacité à inhiber la transmission glutamatergique via un couplage négatif avec les canaux calciques de type N au niveau de la couche II de la moelle épinière et plus particulièrement au niveau des fibres exprimant le transporteur vésiculaire VGLUT3. L'ensemble de nos résultats permettent de valider le récepteur mGlu4 spinal comme une cible thérapeutique potentielle pour le traitement des douleurs chroniques. En effet, les agonistes de mGlu4 pourraient être des antalgiques puissants et sélectifs des douleurs pathologiques
Chronic pain and in particular neuropathic pain are particularly difficult to treat by therapeutic options currently available. Therefore, it is a crucial to develop new concepts of analgesics to treat this type of pain. Among the various possibilities, targeting the glutamatergic system seems to be particularly interesting since glutamate is the main neurotransmitter propagating the pain information. Our hypothesis is that the activation of spinal mGlu4 receptor would inhibit the spinal glutamatergic neurotransmission and would thus reduce the excess of pain observed in chronic pain.Thanks to the development of the first orthosteric agonist selective for mGlu4 and the use of transgenic animals, we demonstrated that mGlu4 receptor does not alter the perception of acute pain but that it does affect the pathological aspect of pain by inhibiting the allodynia and the hyperalgesia (mechanical and thermal) usually observed in chronic pain. We show that the mGlu4 modulation of the hypersensitivity seems to result from the ability of the receptor to inhibit the glutamatergic transmission through a negative coupling with N-type calcium channels in the lamina II of the spinal cord and especially at the level of fibers expressing the vesicular transporter VGLUT3. Taken together, our results validate spinal mGlu4 as a potential therapeutic target for the treatment of chronic pain. Indeed, mGlu4 agonists could be potent and selective painkillers of pathological pain

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

Ces cinquante dernières années ont été marquées par la percée des maladies neurodégénératives comme la maladie d’Alzheimer. À ce jour, il existe uniquement des traitements symptomatiques. De plus, face à cette maladie multifactorielle, il apparaît donc nécessaire d’identifier et d’étudier de nouvelles cibles thérapeutiques. Parmi elles, le récepteur à l’adénosine A2A (A2AR) a fait l’objet de nombreuses études ces dernières années. En effet, ses antagonistes tels que la caféine ont montré de nombreux effets bénéfiques en permettant d’améliorer les performances cognitives par une diminution des dépôts amyloïdes et de la phosphorylation de la protéine Tau. Bien que plusieurs antagonistes d’A2AR aient atteint les phases cliniques, les efforts actuels sont maintenant dirigés vers le développement de nouveaux antagonistes avec de meilleures propriétés ADME. D’autre part, le récepteur métabotropique au glutamate 5 (mGluR5) et ses modulateurs allostériques négatifs jouent un rôle important dans les conditions physiopathologiques associées à la maladie d’Alzheimer. En effet, il a été montré que bloquer l’activité d’mGluR5 entraîne une diminution de la neurotoxicité et de la synaptoxicité du peptide amyloïde in vitro mais aussi in vivo. Basées sur des études de modélisation moléculaire, nous avons développé deux nouvelles familles d’antagonistes présentant un hétérocycle central quinazolinique et benzofuranique. Par la suite, nous avons mis en place une stratégie de ligands duaux ciblant de manière conjointe les récepteurs A2A et mGlu5. Les relations structure-affinité autour de ces deux hétérocycles nous ont permis d’identifier des composés d’affinité nanomolaire pour le récepteur A2A. Les travaux ont également conduit à l’obtention d’une nouvelle structure co-cristallisée. Parmi les composés développés, certains présentent une affinité micromolaire pour le récepteur mGlu5
The past fifty years have been marked by the breakthrough of neurodegenerative diseases such as Alzheimer’s. Unfortunately, only symptomatic treatments are available. Furthermore, facing this multifactorial disease, the search for new and innovative therapeutic targets becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AR) has been the subject of much research in recent years. Indeed, it has been shown that A2AR antagonists such as caffeine improve memory performance as it reduces β-amyloid deposits and Tau-phosphorylation. Though several A2AR antagonists have reached clinical trials, current research efforts are focused on developing new antagonists with relevant ADME properties. On the other hand, negative allosteric modulators of the metabotropic glutamate receptor 5 (mGluR5) also play an important role in the pathological conditions associated with Alzheimer’s disease. It has been found that blocking the activity of mGlu5 reduces the neurotoxicity and synaptoxicity of the amyloid peptide in vitro but also in vivo. Based on a molecular modeling-guided design, we developed new A2AR antagonists with quinazoline and benzofuran as central scaffold and a dual ligands strategy targeting both A2A and mGlu5 receptors. Hit-to-lead optimization has led to nanomolar affinity compounds for A2AR and a new co-crystallized structure. Among them, some hit compounds have been identified with micromolar affinities towards mGluR5

Les récepteurs métabotropes du glutamate (mGluR) sont des récepteurs couplés aux protéines G qui régulent la transmission synaptique. Ce sont des cibles de choix pour le traitement de maladies neurologiques et psychiatriques telles que la maladie de Parkinson et la schizophrénie.J'ai développé une stratégie d'étude de l'assemblage multimérique des protéines membranaires dans des cellules vivantes, à l'aide de techniques de marquage orthogonal et de FRET en temps-résolu. De façon inattendue, j'ai montré que certaines sous-unités de mGluR, en plus de former des récepteurs homodimériques, peuvent former des récepteurs hétérodimériques fonctionnels. D'autre part, j'ai appliqué ces techniques à l'étude du mécanisme d'activation des mGluR et de leur régulation allostérique. J'ai démontré qu'un mouvement relatif des domaines extracellulaires au sein du dimère était responsable de l'action du glutamate.Ce travail a permis de mieux comprendre le fonctionnement des mGluR, et permet la conception de nouveaux tests de criblage
Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate synaptic transmission. They are relevant therapeutic targets for neurological and psychiatric disorders, such as Parkinson disease and schizophrenia.I developed a strategy to study the multimeric assembly of membrane proteins in living cells, through a combination of orthogonal labeling and time-resolved FRET. Unexpectedly, some subunits of mGluRs, in addition to forming homodimeric receptors, were found capable of forming functional heterodimeric receptors. Then, I applied these techniques to study the activation mechanism of mGluRs and their allosteric regulation. I demonstrated that a conformational change of the dimeric extracellular domain is responsible for the action of glutamate.In addition to increase our understandings of how mGluRs function, this work opens new avenues for the design of drug screening tests

Le cervelet est un petit cerveau dans le cerveau. Il contient plus de la moitié du nombre total de neurones du cerveau. Sa structure très régulière est bien connue, toutefois son rôle demeure mystérieux. Le développement essentiellement postnatal du cervelet chez les rongeurs permet d’y étudier la formation activité-dépendante du réseau de neurones. C’est aussi le siège où s’opèrent diverses formes de plasticité synaptique, ce qui en fait un modèle d’étude idéal pour la plasticité synaptique développementale et adulte. Au cours de cette thèse, à l’aide d’enregistrements électophysiologiques en patch-clamp et en extracellulaire sur des tranches aigües de cervelet de souris et grâce aux techniques immunohistochimiques, j’ai étudié trois acteurs importants de la plasticité synaptique et du développement des cellules de Purkinje, les neurones centraux du cortex cérébelleux. Nous avons démontré que l’activation du récepteur métabotropique glutamatergique de type 1 (mGlu1) déclenche l’activation et l’ouverture de GluD2, un récepteur nécessaire au développement et à la plasticité des synapse des cellules de Purkinje (CPs). Nous avons également mis en évidence que les Pannexines 1, des canaux potentiellement impliqué dans la synchronisation neuronale récemment découverts et encore mal caractérisés, sont exprimées par les cellules de Purkinje Zebrine II –immunopositives, suivant les bandes parasagittales que délimitent les microdomaines du cervelet. Enfin, nous avons étudié la physiologie du cortex cérébelleux des souris néonatales, cherchant à caractériser les différents acteurs essentiels à l’activité neuronale de ce cortex en développement très particulier et peu étudié. L’activation du récepteur GluD2 médiée par mGlu1 dans la synapse entre Fibre Parallèle et cellule de Purkinje (synapse PF-PC). GluD2 est classifié parmi les récepteurs ionotropiques glutamatergique, pourtant aucun ligand n’est capable d’induire l’ouverture de son canal. Nous avons identifié pour la première fois un mécanisme physiologique d’ouverture du canal de GluD2 en démontrant que l’activation de mGlu1 déclenche l’ouverture du canal de GluD2 pour une voie intracellulaire, aussi bien dans un système d’expression en culture que dans les tranches aigues de cervelet murin. Cela nous permettra d’étudier la contribution du courant médié à travers GluD2 dans la plasticité à long terme, avec des perspectives totalement nouvelles. L’expression de Pannexine 1 par les CPs se superpose aux stries Zebrine II- immunopositives du cervelet. Les CPs adultes constituent une population hétérogènes, les différents sous-types étant organisés sur le plan parasagittal. Nous avons montré que l’expression des protéines Pannexine 1 (Panx1) We have shown that Pannexin1 (Panx1) déssine un gradient rostrocaudal discontinu dans les lobules de tranches parasagittales. Sur les coupes coronales, leur distribution forme une série de bandes parasagittales. Les canaux Panx1 médient la libération d’ATP en réponse à divers stimuli et pourrais de cette façon contribuer à une activité neuronale orientée sur le plan parasagittal en réponse aux signaux des fibres parallèles. Caractérisation de l’activité GABAergique des CPs immatures dans les souris néonatales. Le cortex cérébelleux entre les jours postnataux P0 et P4 consistent principalement en une multicouche dense de CPs fortement interconnectées. A cet âge, les CPs sont remplies de GABA extrasynapstique qui est libéré dans le milieu extracellulaire par un mécanisme qui n’est pas clairement identifié. Nos recherches préliminaires sur la première semaine de développment postnatal, nous montrons que l’activation de récepteur au GABA de type A induit une réponse excitatrice chez les CPs. Avec notre préparation, cet effet est indépendant de la présence de corps cétoniques ou de lactate comme substrats énergétiques dans le milieu extracellulaire. (...)
The cerebellum is a little brain in the brain. It houses more than half the total number of neurons in the brain. Its crystalline structure is very well known but, still, its function remain unclear to date. Its mainly postnatal development in rodents allows the study of the physiology of activity-dependent neuronal wiring. It is also the place of many types of neuronal plasticity, making it an ideal model to study both developmental and adult synaptic plasticity. In this thesis, using mainly patch-clamp and extracellular recordings in cerebellar slices as well as immunohistochemistry in mice, I have studied three important actors of synaptic plasticity and development in the Purkinje cells, the principal neurons of the cerebellar cortex. We have established that the type 1metabotropic glutamate receptor (mGlu1) triggers the gating of GluD2, a receptor necessary for Purkinje cells (PCs) synapses development and synaptic plasticity. We have also shown that the Pannexins 1, some channels likely involved in neuronal synchronization that have been recently discovered but yet remain poorly characterized, are expressed by Zebrin II immmunopositive Purkinje cells in the classical Zebra stripes formed by microdomains of the cerebellum. Last, we have studied the physiology of the primitive cerebellar cortex in neonatal mice, establishing the first elements of the neuronal activity of this very particular developing cortex at a stage still very poorly characterized. The mGlu1-mediated gating of Glu2D receptors at Parallel Fiber to PC (PF-PC) synapse. GluD2 are classified among ionotropic glutamate receptors, but no ligand has proved capable of gating their channel. We have identified for the first time a physiological mechanism of gating GluD2 channels by demonstrating that the activation of mGlu1 triggers the opening of GluD2 channels through intracellular pathways, both in expression systems and in acute murine cerebellar slices. This will allow us to study the contribution of GluD2-mediated current in long-term plasticity in a totally new way. Expression of Pannexin1 by PCs matches with adult Zebrin II immunopositive cerebellar stripes. Adult PCs constitute an heterogeneous population, the different subtypes being parasagittaly organized. We have shown that Pannexin1 (Panx1) proteins expression by PCs, draw a rostrocaudal discontinuous gradient in lobules of parasagittal slices. In transverse slices, their distribution forms an array of parasagittal stripes. Panx1 channels mediate ATP release in response to various stimuli and may in this way contribute to parasagittally oriented response to PF inputs. Characterization of GABAergic activity of immature Purkinje cells of newborn mice. The cerebellar cortex during postnatal days P0 to P4 essentially consists in a dense multilayer and highly interconnected network of PCs. At this age, PCs are filled with extrasynaptic GABA which is released in the extracellular space by a mechanism that is not clear. In our preliminary investigation of first week postnatal development, we show that activation of GABA-A receptors leads to excitatory responses in PCs. In our preparation, this effect is independent of the presence of keton bodies or lactate as energetic substrates in the extracellular medium. The complete inhibition of spontaneous discharge of PCs by Panx1 channel blockers, suggests that they mediate ion fluxes or release of neuromediators, such as ATP or GABA

A formação de memórias de medo contextuais, como as estudadas no presente trabalho, requer a indução da plasticidade sináptica iniciada pela ativação de receptores transmembrana localizados nos neurônios de estruturas encefálicas como o hipocampo. O fluxo iônico mediado pelos receptores N-metil-D-aspartato (NMDARs) é essencial para ativar vias de sinalização intracelular que darão suporte à formação da memória. No entanto, esses receptores parecem não ser necessários em situações onde os animais passaram por uma experiência prévia similar a que está sendo aprendida. Dessa forma, um aprendizado anterior pode modificar os mecanismos de plasticidade que serão utilizados para codificar uma nova informação, caracterizando um fenômeno de metaplasticidade. Esse fenômeno ocorre quando os animais são pré-expostos ao local onde posteriormente serão submetidos a um aprendizado associativo ou quando são re-submetidos a mesma tarefa comportamental com dicas contextuais/espaciais diferentes. No presente trabalho, investigamos (i) os mecanismos de plasticidade sináptica (receptores) e de plasticidade não-sináptica (excitabilidade neuronal) recrutados para a formação do segundo aprendizado e (ii) se a independência dos NMDARs é mantida quando a memória anterior foi adquirida remotamente. Os animais utilizados nesse trabalho (camundongos ou ratos) foram expostos a dois aprendizados sequenciais realizados na tarefa de condicionamento aversivo ao contexto (CAC). O intervalo entre os condicionamentos foi de dois dias nos experimentos do Capítulo I e de três ou quarenta dias nos experimentos do Capítulo II. Cada aprendizado ocorreu em uma caixa de condicionamento com características próprias de formato, odor e iluminação (contexto A ou contexto B), sendo que o primeiro aprendizado ocorreu no contexto A e o segundo no contexto B. Nos experimentos do Capítulo I foram avaliadas no hipocampo dorsal as modificações na excitabilidade neuronal hipocampal induzidas pelo primeiro condicionamento, bem como os receptores envolvidos com a aquisição da memória subsequente e a sobreposição neuronal entre os dois aprendizados. Com a utilização do camundongo transgênico Teg-Tag foi possível identificar os neurônios recrutados para o primeiro aprendizado. Esse animal tem a expressão da proteína fluorescente verde (GFP, do inglês, green fluorescent protein) controlada pela ativação do gene c-fos, que é fisiologicamente transcrito após a atividade neuronal. Dessa forma, os neurônios ativados pelo aprendizado são marcados com GFP. Através da técnica de patch clamp foi observado que os neurônios GFP+ mantiveram a excitabilidade elevada por até dois dias após o treinamento no CAC. Além disso, a identificação dos neurônios recrutados 8 para o aprendizado subsequente foi realizada através da marcação imunofluorescente da proteína Fos, no seu pico de expressão endógena, noventa minutos após o re-treino. Foi observada uma maior sobreposição neuronal (GFP+, Fos+) quando os animais foram retreinados no mesmo contexto dois dias após o primeiro treino. Uma sobreposição intermediária (GFP+, Fos+) foi vista quando os animais tiveram o segundo condicionamento no contexto B, sendo ela significativamente maior do que a sobreposição nos animais não re-treinados. Adicionalmente, foi demonstrado que a aquisição do aprendizado subsequente é mediada por receptores metabotrópicos glutamatérgicos (mGluRs) ao invés de NMDARs. No Capítulo II foi investigado se uma memória remota, adquirida há quarenta dias, ainda seria capaz de influenciar nos mecanismos de plasticidade recrutados para aquisição do aprendizado subsequente. A dinâmica da consolidação sistêmica foi considerada nesses experimentos já que a evocação da memória remota passa a depender de estruturas encefálicas neocorticais, sem recrutar a atividade hipocampal. Apesar da evocação da memória remota não requerer a atividade hipocampal, foi observado que a aquisição do aprendizado subsequente a uma memória remota necessita a atividade de pelo menos uma sub-região do hipocampo (dorsal ou ventral). Complementarmente, os resultados indicaram que, quando o intervalo entre os aprendizados é aumentado (de três para quarenta dias), a formação do aprendizado subsequente, que era independente de NMDARs, volta a depender da plasticidade sináptica mediada por esses receptores no hipocampo (dorsal e ventral). Juntos, nossos resultados sugerem que o primeiro aprendizado causa um aumento da excitabilidade neuronal e modifica a plasticidade sináptica recrutada para o aprendizado subsequente, sendo este último mediado por mGluRs ao invés de NMDARs. Além disso, a metaplasticidade induzida pelo primeiro condicionamento é transiente; quando o intervalo entre as exposições é aumentado, o segundo aprendizado passa a depender novamente da ativação dos NMDARs.
Contextual fear memory formation, like the ones explored in the current work, requires the induction of the synaptic plasticity mediated by the activation of transmembrane receptors that are present in the brain structures as the hippocampus. The ionic flux through the N-methylaspartate- D-aspartate is crucial for activation of the intracellular signaling pathways that will support memory formation. However, these receptors are not necessary when animals had a prior similar learning. In this way, a previous learning can modify the plasticity mechanism that will be recruited to encode a new information, featuring a metaplasticity phenomenon. This phenomenon occurs when animals are pre-exposed to an environment where they will learn an associative learning later or when animals are re-exposed to the same behavioral task with distinct contextual/spatial cues. In the present study, we investigated (i) the synaptic plasticity mechanisms (receptors) and the non-synaptic plasticity mechanisms (neuronal excitability) required for the acquisition of the second learning and (ii) whether a subsequent learning that occurs in a remote time-point is still NMDAR-independent. The animals used in this study (mice or rats) were exposed to two sequential learnings that were performed in the contextual fear conditioning (CFC). The interval between conditionings were two days in the experiments of Chapter I and three or forty days in the experiments of the Chapter II. Each learning was performed in a box with differences on shape, odor and illumination (context A or context B). The first learning occurred in the context A followed by learning on context B. In the experiments of Chapter I it was evaluated the changes in the hippocampal neuronal excitability induced by the first conditioning, the receptors involved with the acquisition of the subsequent memory and the neuronal overlapping between the two sequential learnings. The Teg-Tag transgenic mouse allowed to identify the neurons activated for the first learning experience. This animal has the GFP expression under control of c-fos promoter that is activated by neuronal activity. It was shown by patch clamp that GFP+ neurons are still more excitable two days after learning. Also, the identification of neurons recruited for the subsequent learning was made through immunofluorescent staining of the Fos protein in its peak of endogenous expression, ninety minutes after learning. A greater overlapping (GFP+, Fos+) was observed when animals were retrained in the same context two days after first training. An intermediate overlapping was observed when animals were conditioned in the context B and this expression was significantly higher when compared to animals that were not 10 retrained in either context. Additionally, it was shown that acquisition of the subsequent learning is mediated by metabotropic glutamate receptors (mGluRs) instead of NMDARs In the Chapter II it was investigated whether a remote memory, acquired forty days earlier, is still able to influence in the synaptic plasticity mechanisms recruited for the acquisition of the subsequent learning. Systems consolidation dynamics was considered in these experiments because memory retrieval of a remote memory depends on neocortical brain regions, it not requires hippocampal activity. It was confirmed that hippocampus is not necessary for remote memory retrieval, however at least one longitudinal division of the hippocampus (dorsal or ventral) is essential for learning following a prior remote memory. Moreover, the results indicate that acquisition of the second learning is once again mediated by NMDARs in the hippocampus when the interval between learnings is extended from three to forty days. Altogether, our results suggest that the first learning lead to an increase in the neuronal excitability and modify the synaptic plasticity mechanism recruited for following learning, mGluR are required instead of NMDAR. Furthermore, the metaplasticity induced by first conditioning is transient; the second learning once again requires NMDARs activation when the interval between learnings is longer.

L’ensemble des cellules, qu’elles soient procaryotes ou eucaryotes, est doté d’une couche de glycosylation externe riche et diversifiée, composant la face dominante immédiate en relation à leur environnement. Elles résultent de processus enzymatiques complexes liant les sucres entre eux et sur des protéines ou lipides. Des variations du « glycome » peuvent apparaître dans certaines pathologies. Les cancers sont les pathologies les plus fréquentes présentant des anomalies de ces glycosylations. Ces altérations sont quasi systématiques à la surface des cellules cancéreuses. Parmi celles-ci, l’antigène Thomsen-nouveau (Tn), un N-acétylgalactosamine (GalNAc) sur une sérine ou une thréonine, est fortement exprimé dans 90% des carcinomes mammaires ainsi que dans les cancers de la vessie, du col de l’utérus, de l’ovaire, du colon, de l’estomac et de la prostate. L’omniprésence de l’antigène Tn dans de nombreux cancers, associés à son absence dans les cellules saines, en fait une cible de choix pour la thérapie ciblée ou des vaccins synthétiques antitumoraux. Aucun anticorps ciblant l’antigène Tn n’est à ce jour disponible du fait de la difficulté à développer un anticorps avec une telle spécificité. Ainsi, nous nous sommes intéressés à une autre stratégie de ciblage, basée sur l’utilisation d’une molécule capable de reconnaître l’antigène Tn. Les lectines de type C sont une famille de protéines capables de se lier spécifiquement et de façon réversible à certains glucides, en présence de calcium. La macrophage galactose lectine (MGL) est une lectine de type C ayant une affinité très importante pour le GalNac et ses dérivés comme l’antigène Tn. Ce travail a consisté, dans un premier temps, à l’utilisation d’une forme recombinante soluble de la MGL pour valider le potentiel de cet outil pour le ciblage des cellules cancéreuses. Les différentes expériences, in vitro et in vivo, impliquant la MGL, ont démontré la capacité de cette dernière à cibler spécifiquement les tumeurs humaines via l’antigène Tn. La partie extracellulaire de la MGL est de ce fait un très bon candidat de vecteur pour le diagnostic et l’imagerie de tumeurs humaines et potentiellement pour l’administration de médicaments. Dans un deuxième temps, diverses stratégies de développement d’un outil bifonctionnel exploitant cette lectine ont été exploré. Le but était de créer une plateforme peptidique fonctionnalisable d’une part avec plusieurs domaines lectines, afin de contrôler l’affinité de reconnaissance, et d’autre part des groupements fonctionnels variable selon l’application recherché (diagnostique, thérapeutique, ...). Les différentes stratégies de couplage employées nous ont permis d’accrocher plusieurs CRD de lectine sur un support peptidique, cela en conservant l’état tridimensionnel et fonctionnel des protéines. Les caractérisations effectuées démontrent une importante augmentation de l’affinité directement fonction du nombre de lectine ajouté sur la plateforme. Ce travail ouvre la voie vers de nouveaux systèmes de ciblage des sucres modulable à façon
All cells, whether prokaryotic or eukaryotic, have a rich and diversified external glycosylation layer, forming the immediate dominant face in relation to their environment. They result from complex enzymatic processes linking sugars to each other and to proteins or lipids. Variations of the "glycome" can appear in certain pathologies. Cancers are the most frequent pathologies with abnormalities in these glycosylations. These alterations are almost systematic on the surface of cancer cells. Among them, the Thomsen-new antigen (Tn), an N-acetylgalactosamine (GalNAc) on a serine or threonine, is strongly expressed in 90% of mammary carcinomas as well as in cancers of the bladder, cervix, ovary, colon, stomach and prostate. The ubiquitous presence of the Tn antigen in many cancers, combined with its absence in healthy cells, makes it a target of choice for targeted therapy or synthetic anti-tumor vaccines. No antibody targeting the Tn antigen is currently available because of the difficulty in developing an antibody with such specificity. Thus, we were interested in an alternative targeting strategy, based on the use of a molecule capable of recognizing the Tn antigen. C-Type lectins are a family of proteins capable of specifically and reversibly binding to certain carbohydrates in the presence of calcium. Macrophage galactose lectin (MGL) is a C-type lectin with a high affinity for GalNac and its derivatives such as the Tn antigen. This work consisted, initially, in the use of a soluble recombinant form of MGL to validate the potential of this tool for the targeting of cancer cells. The different experiments, in vitro and in vivo, involving MGL, demonstrated the latter's ability to specifically target human tumors via the Tn antigen. The extracellular portion of MGL is therefore a very good vector candidate for the diagnosis and imaging of human tumors and potentially for drug delivery. In a second step, various strategies for the development of a bifunctional tool exploiting this lectin were explored. The goal was to create a peptide platform that could be functionalized on one hand with several lectin domains, in order to control recognition affinity, and on the other hand with functional groups that could be variable according to the application (diagnostic, therapeutic, ...). The different coupling strategies employed allowed us to attach several lectin CRDs to a peptide support, while preserving the three-dimensional and functional state of the proteins. The characterizations carried out show a significant increase in affinity directly related to the number of lectins added to the platform. This work paves the way to new customizable sugar-targeting systems

Dissertação de mestrado em em Biologia Celular e Molecular, especialização em Neurociências, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
Glutamato é o principal neurotransmissor excitatório no cérebro, sendo bastante importante em várias funções do sistema nervoso central. Alterações no sistema glutamatérgico estão envolvidas em doenças como esquizofrenia e ansiedade. Tem vindo a ser demonstrado que a activação de receptores mGlu2 reduz a transmissão glutamatérgica nas regiões cerebrais associadas a estas doenças. Por esta mesma razão, a activação destes receptores tem sido alvo de investigação para desenvolvimento de novas técnicas terapêuticas, especialmente com o uso de modeladores alostericos positivos (PAMs); estes modeladores ligam-se a uma zona do receptor diferente do local de ligação do glutamato. Dado que a área de estudo dos PAM do receptor mGlu2 se encontra em expansão e o primeiro ensaio clínico está a decorrer é bastante importante obter mais informacão sobre o correcto local de ligacão destes ligandos. Esta informacão também pode suportar e facilitar os esforços da investigação química. Com o objectivo de identificar os aminoácidos responsáveis pela interação entre os PAMs e o receptor mGlu2, foi efectuada modulação molecular e docking de PAMs de receptores mGlu2 em paralelo com mutagénese dirigida. Nos receptores mGlu2 mutantes foi avaliado o impacto das mutações na actividade e afinidade dos PAMs. Este estudo confima a importância de aminoácidos previamente demonstrados como importantes na actividade de PAMs estruturalmente diferentes nestes receptores. É tambem demonstrado que adicionais aminoácidos seleccionados com base na comparação de sequencias entre mGlu2/3 parecem não ser importantes na actividade dos PAMs. A informação obtida neste estudo tambem demonstra que a actividade dos modeladores testados é reduzida devido à diminuição da afinidade de ligação. Toda esta informação oferece um melhor entendimento sobre o ‘binding pocket’ para PAMs do receptor mGlu2.
Glutamate is the major excitatory neurotransmitter in the brain and plays an important role in a wide variety of central nervous system functions. Alterations in the glutamatergic system are involved in disorders like schizophrenia and anxiety. It has been shown that activation of the metabotropic glutamate 2 receptor reduces the glutamatergic transmission in brain regions associated with these disorders. Therefore, activation of mGlu2 receptor is being pursued as a novel therapeutic approach, specially using positive allosteric modulators (PAMs), which bind to a site other than that of the endogenous mGlu2 receptor agonist glutamate. Since the field of mGlu2 PAMs is expanding and the first clinical studies are ongoing with mGlu2 PAMs, it will be important to get more insight into the actual binding site of these ligands. This knowledge may also facilitate and support future chemistry endeavors. In order to identify the amino acids important for the activity of mGlu2 PAMs, homology modeling and docking of mGlu2 receptor PAMs were performed in parallel with site-directed mutagenesis. Mutant mGlu2 receptors were generated and the impact of these mutations on activity and affinity of PAMs was evaluated. This study confirms the importance of several amino acids previously shown as crucial for the activity of structurally diverse mGlu2 receptor PAMs. It furthermore demonstrates that additional amino acids that were selected based on mGlu2/3 comparison did not seem to be important for PAM activity. Our data also suggest that their activity is reduced due to lower binding affinity. All this sheds further light on the mGlu2 PAM binding pocket.

Dissertação de mestrado em Biologia Celular e Molecular apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
O sucesso dos receptores acoplados à proteína G (GPCRs) enquanto alvos terapêuticos para o tratamento de doenças do sistema nervoso central torna estes receptores alvos pertinentes de investigação. Especificamente, a activação de receptores mGlu2 tem mostrado reduzir a transmissão glutamatérgica em áreas do cerebro onde o excesso de sinalização glutamatérgica parece estar implicado na patofisiologia de doenças como a ansiedade e a esquizofrenia. Deste modo, a activação dos receptores mGlu2 está actualmente a ser encarada como uma potencial estratégia para o tratamento destas doenças. Diversos moduladores alostéricos positivos (PAMs), que se ligam a um local do receptor diferente do local de ligação do glutamato, têm revelado modular os receptores mGlu2 de uma forma selectiva. Com o objectivo de identificar aminoácidos potencialmente importantes para a interacção entre o PAM e o receptor mGlu2, foi efectuada a modelação molecular e o docking de PAMs de receptores mGlu2 em paralelo com mutagénese dirigida. Receptores mGlu2 mutantes foram produzidos e o impacto dessas mutações na actividade de diversos PAMs foi avaliado, de forma a confirmar o papel destes aminoácidos na actividade destes compostos. Este estudo identifica aminoácidos importantes para a actividade dos PAMs e sugere um potencial local de ligação destes compostos.
The proven success of G protein-coupled receptors (GPCRs) as drug targets for the treatment of CNS disorders renders them attractive targets of research. Specifically, activation of the G protein-coupled or metabotropic glutamate (mGlu2) receptor has been shown to reduce glutamatergic transmission in brain regions where excess glutamate signaling may be implicated in the pathophysiology of disorders such as anxiety and schizophrenia. Hence, activation of the mGlu2 receptor is being pursued as a novel therapeutic approach for the treatment of these diseases. Multiple positive allosteric modulators (PAMs), which bind to a site other than that of the endogenous mGlu2 receptor agonist glutamate, have been shown to modulate mGlu2 receptors in a selective way. In order to identify amino acids potentially important for the interaction between PAMs and the mGlu2 receptor, homology modelling and docking of mGlu2 receptor PAMs was performed in parallel with experimental site directed mutagenesis. Mutant mGlu2 receptors were produced and the impact of the selected receptor mutations on the activity of several PAM compounds towards the receptor was evaluated, in order to confirm the role of these amino acids in the activity of the compounds. This study identifies crucial amino acids for the activity of mGlu2 receptor PAMs and suggests a potential binding pocket of PAMs.

yes
Brain function depends on co-ordinated transmission of signals from both excitatory and inhibitory neurotransmitters acting upon target neurons. NMDA, AMPA and mGluR receptors are the major subclasses of glutamate receptors that are involved in excitatory transmission at synapses, mechanisms of activity dependent synaptic plasticity, brain development and many neurological diseases. In addition to canonical role of regulating presynaptic release and activating postsynaptic potassium channels, GABAB receptors also regulate glutamate receptors. There is increasing evidence that metabotropic GABAB receptors are now known to play an important role in modulating the excitability of circuits throughout the brain by directly influencing different types of postsynaptic glutamate receptors. Specifically, GABAB receptors affect the expression, activity and signaling of glutamate receptors under physiological and pathological conditions. Conversely, NMDA receptor activity differentially regulates GABAB receptor subunit expression, signaling and function. In this review I will describe how GABAB receptor activity influence glutamate receptor function and vice versa. Such a modulation has widespread implications for the control of neurotransmission, calcium-dependent neuronal function, pain pathways and in various psychiatric and neurodegenerative diseases.

Numerous behavioral studies indicate that dopamine transporter (DAT) inhibitors and metabotropic glutamate receptor 5 (mGlu5) negative allosteric modulators (NAMs) possess promising anti-addiction therapeutic properties. However, the lack of human DAT (hDAT) and mGlu5 X-ray crystal structures makes it difficult to understand how these promising anti-addiction compounds interact with their major drug targets at the molecular level. This knowledge gap represents an important problem towards rationally designing new therapeutics for numerous addiction disorders. The objective of this research was to develop irreversible chemical probes based on select DAT inhibitors and mGlu5 NAMs in order to map the corresponding binding sites and poses of these compounds within their major drug targets. The central hypothesis was that these compounds could be derivatized, without significant loss in pharmacological activity, with a functional group capable of forming a covalent bond to their target protein and, if necessary, a tag functional group. These probes would then allow proteomic experiments to be coupled with computational modeling in order to directly map the ligand-binding sites of these promising anti-addiction compounds within their target proteins. The central hypothesis was tested by pursuing two specific aims: 1) identification of non-tropane DAT inhibitor photoprobes suitable for DAT structure-function studies, and 2) identification of irreversible mGlu5 NAM ligands as chemical probes suitable for mGlu5 structure-function studies. Under the first aim, methylphenidate (MP) and GBR-12909 as non-tropane DAT inhibitors were structurally modified to contain a photoreactive group (e.g., aryl azide, benzophenone) and a tag (e.g., 125I, terminal alkyne, aliphatic azide). These compounds were then subjected to DAT pharmacological evaluation in order to identify suitable candidates for DAT structure-function studies. Under the second aim, thiazole- and pyridine-based mGlu5 NAMs were structurally modified to contain either a photoreactive group (e.g., aryl azide, benzophenone) or an affinity labeling group (e.g., methanethiosulfonate, maleimide) and a tag (e.g., 125I, terminal alkyne, aliphatic azide). These compounds were then subjected to mGlu5 pharmacological evaluation in order to identify suitable chemical probe candidates for mGlu5 structure-function studies. At present, several irreversible chemical probes from these specific aims have advanced to the proteomics stage of the experimental research strategy.
Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences;
Medicinal Chemistry
PhD;
Dissertation;

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
The metabotropic glutamate subtype receptor 7 (mGlu7 receptor) is the most highly conserved, the predominant of the group III mGlu receptor in the presynaptic active zone and the most widely distributed among mGlu receptors, suggesting a critical role in regulating excitatory and inhibitory synaptic transmission in the central nervous system (CNS). Nonetheless the characterization of the functional role of the mGlu7 receptor in the CNS has been hampered by the lack of selective agents and limited to mGluR7 knockout mouse studies until valuable pharmacological tools for studying its function are being developed. The main aim of this work was to investigate its role in the control of hippocampal basal synaptic transmission and short-term plasticity. Hippocampal slices from adult and immature Sprague Dawley rats were used to record electrophysiological events using paired-pulse stimulation and a Short-High stimulation paradigm in Schaffer fiber-CA1 synapses and/or dentate gyrus medial perforant path- granule cells synapses. When tested in the CA1 area L-2- amino-4-phosphonobutyric acid (L-AP4, 3 µM to 1 mM) demonstrated a very different EC50 for adult ad immature rats, 1.13 mM and 10.44 µM, respectively. The diverse allosteric modulators used (AMN802, MMPIP and ADX71743) have demonstrated a promising potential when tested in diverse types of cells expressing the mGlu7 receptor. However when tested in brain slices in CA1 none of them modified in a relevant manner the effect of 600 µM of LAP4 ((0.74±0.1); MMPIP (10 μM) and L-AP4 (600 μM), 0.56±0.09; ADX71743 (10 μM) and L-AP4 (600 μM), 0.80±0.09; AMN082 (1 μM) and L-AP4 (600 μM), 0.66±0.1). In the other hand, in the dentage gyrus medial perforant path, LY341495 reversed the effect of L-AP4 (0.97±0.05) and, controversially, MMPIP potentiated it (MMPIP plus L-AP4, 0.51±0.06). In view of the proposed role of mGlu7 as a “low-pass filter”, its role in spontaneous and NMDA evoked spiking was tested with the used of L-AP4. The compound demonstrated a negative effect over spontaneous events (L-AP4 (600 µM), 94.6%± 12) nonetheless when applied before or after elevated doses of NMDA it led to a further increase of the events evoked (NMDA 100 μM and L-AP4 600 μM, 143.4%± 2.85; L-AP4 600 μM and NMDA 100 μM, 1101.4%± 14). The presence of GABAA receptor antagonist picrotoxin (PTX) partially reverted the effects of LAP4 (600 µM) (0.94±0.04 – with PTX vs. 0.74±0.1 –without PTX) and in the presence of 10 μM PTX L-AP4 did not led to an alteration of the PPR. L-AP4 at a concentration of 600 μM significantly reduces both the values for the peak and area under the curve of NMDAR mediated potentials, indicating that the compound is modulating this receptor response (Peak value: L-AP4 600 μM, 0.53±0.04; Area under the curve: L-AP4 600 μM, 0.55±0.03) The results suggest that L-AP4 may be acting on different targets than mGlu receptors with a possible agonist effect over GABAa receptors and/or antagonism of NMDAR responses. This still allows the presence of a small window for mGlu7 receptors that need to be further explored with relevant models and more selective compounds tested in those same models.
O recetor metabotrópico de glutamato do subtipo 7 (recetor mGlu7) é o recetor mais conservado entre espécies, sendo dos elementos do grupo III dos recetores mGlu o que predominantemente se encontra na zona pré-sináptica, este é também o mais amplamente distribuído entre os receptores metabotrópicos de glutamato. Estas características sugerem um papel crítico na regulação da transmissão sináptica excitatória e inibitória no sistema nervoso central (SNC). No entanto, a caracterização funcional deste recetor no SNC continua a ser limitada à existência de modelos knockout para o mGlu7 e pela falta de agentes seletivos até que novos agentes farmacológicos sejam desenvolvidos. O principal objetivo deste trabalho foi discernir o papel que este recetor tem no controlo da transmissão sináptica basal e na plasticidade de curta duração no hipocampo. Para tal, foram obtidos registos eletrofisiológicos a partir de fatias de hipocampo de ratos Sprague Dawley adultos e jovens. Estes registos foram obtidos após estimulação por pulso pareado tanto nas fibras de Schaffer-CA1 como no caminho perfurante medial – células granulares do giro dentado. Quando testado na área CA1 o agonista ácido L-2- amino-4-fosfonobutirico (L-AP4, 3 µM até 1 mM) demonstrou um EC50 muito diferente entre ratos adultos e imaturos, 1.13 mM e 10.44 µM respetivamente. Os diversos moduladores alostéricos usados (AMN802, MMPIP e ADX71743) demonstram um potencial promissor quando testados em diversos tipos de linhas celulares que expressam o recetor mGLU7. Contudo quando testados em fatias de cérebro na área CA1 do hipocampo nenhum dos compostos modificou de maneira significativa os efeitos da aplicação de 600 µM de L-AP4 (0.74±0.1; 10 μM MMPIP e 600 μM L-AP4: 0.56±0.09; 10 μM ADX71743 e 600 μM L-AP4: 0.80±0.09; 1 μM AMN082 e 600 μM L-AP4: 0.66±0.1). Em contraste no giro dentado, o antagonista LY341495, reverteu os efeitos do L-AP4 (0.97±0.05) e contrariamente ao esperado, o MMPIP potenciou os efeitos do agonista (MMPIP e L-AP4: 0.51±0.06). Tendo em conta o pressuposto papel do recetor como filtro passa-baixo, o seu potencial papel no controlo da atividade neuronal espontânea mediada por NMDA foi testado usando o L-AP4. Este composto diminuiu o número de eventos espontâneos (600 µM L-AP4: 94.6%± 12) no entanto, quando aplicado antes ou depois de doses elevadas de NMDA conduziu a um aumento dos eventos evocados por NMDA (100 uM NMDA e 600 uM L-AP4: 143,4% ± 2,85; 600 uM L-AP4 e 100 uM NMDA: 1101,4% ± 14). Na presença de picrotoxina (PTX), um antagonista dos recetores GABAA, os efeitos de L-AP4 foram parcialmente revertidos(600 uM L-AP4: com PTX: 0.94±0.04 versus sem PTX:0.74±0.1) e na presença de 10 μM PTX este não induziu a alteração de PPR. O L-AP4 a uma concentração de 600 uM reduz significativamente tanto os valores de pico como a área sob a curva dos potenciais mediados por NMDAR, indicando que o composto estará a modular a resposta do recetor (valor de pico: L-AP4 600 uM, 0,53 ± 0,04; área sob a curva: L-AP4 600 mM, 0,55 ± 0,03). Os resultados sugerem que o L-AP4 pode estar a atuar em alvos diferentes dos recetores mGlu com um possível efeito agonista sobre os recetores GABAa e/ou antagonista das respostas do NMDAR. Contudo, os dados parecem também indicar a presença de uma pequena janela de ação para os recetores mGlu7, que precisa de ser mais explorada em modelos relevantes e testada com compostos mais seletivos.

Fragile-X syndrome (FXS) is the most common form of inherited intellectual impairment. Caused by the transcriptional repression of the Fmr1 gene on the X chromosome, FXS results in the loss of the Fragile-X Mental Retardation Protein (FMRP). Human female patients with FXS are heterozygous for the Fmr1 mutation whereas males are hemizygous. FXS has been studied far less in females than in males due to a generally less severe clinical phenotype. Previous research has implicated the metabotropic glutamate receptor (mGluR) in synaptic plasticity alterations in the cornu ammonis area 1 (CA1) region of the juvenile male Fmr1 knock-out (KO) hippocampus. In contrast, our investigations into the young adult dentate gyrus (DG) subfield of the hippocampus have revealed N-methyl-D-aspartate receptor (NMDAR)-associated impairments in synaptic plasticity. The current study sought to extend these investigations to the young adult female Fmr1 heterozygous (Het) and Fmr1 KO mouse as well as investigate NMDAR- and mGluR-mediated long-term depression (LTD) in the DG and CA1 of the young adult male Fmr1 KO mouse. Input-output curves and paired pulse measures of short-term plasticity were also evaluated in all genotypes. Field electrophysiology revealed a significant impairment in long-term potentiation (LTP) and LTD in male Fmr1 KO and female Fmr1 Het mice that was associated with NMDAR alteration. A more robust synaptic protocol was not able to rescue LTP in the male Fmr1 KO DG. Paired-pulse low-frequency stimulation and (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced mGluR-LTD was intact in all genotypes and brain regions examined. Although further investigation will be required to expand our understanding of FXS and to fully elucidate the mechanisms behind intact synaptic plasticity in the female Fmr1 KO mouse, our results suggest that NMDARs may be poised as important contributors to hippocampal pathophysiology in FXS.
Graduate

Yes
Group I metabotropic glutamate receptor (mGluR) dependent long-term depression (LTD) is a major form of synaptic plasticity underlying learning and memory. The molecular mechanisms involved in mGluR-LTD have been investigated intensively for the last two decades. In this 60th anniversary special issue article, we review the recent advances in determining the mechanisms that regulate the induction, transduction and expression of mGluR-LTD in the hippocampus, with a focus on the mitogen-activated protein kinase (MAPK) pathways. In particular we discuss the requirement of p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. The recent advances in understanding the signaling cascades regulating mGluR-LTD are then related to the cognitive impairments observed in neurological disorders, such as fragile X syndrome and Alzheimer's disease.

Yes
Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasomedependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.
Open access funded by Biotechnology and Biological Sciences Research Council

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
A perturbação do espectro do autismo (PEA) inclui um grupo heterogéneo de condições de neurodesenvolvimento, caracterizadas por défices na linguagem, alterações na interação social e na comunicação e por comportamentos repetitivos e estereotipados. Estudo epidemiológicos estimam que mais de 1% da população mundial tem uma PEA e que, 70% dos indivíduos com PEA também apresentam défice intelectual (DI).Há duas teorias para explicar a patofisiologia das PEA: uma destas teorias considera que é a perda da homeostasia neuronal, devido à disfunção sináptica e ao desequilíbrio entre a excitação e inibição que desencadeia as comorbidades características das PEAs. A outra teoria admite que são as alterações de circuitos neuronais específicos (conetividade neuronal) que desencadeiam as PEAs. Contudo, o exato mecanismo que desencadeia as PEAs não está totalmente definido. Estudo epidemiológicos sugerem uma forte componente genética nesta desordem. Para além disto, estudos genéticos têm identificado proteínas envolvidas na função sináptica com implciações nas PAE.Os recetores acoplados às proteínas G (GPCRs) desempenham várias funções na regulação da sinalização sináptica. Assim, as proteínas que interagem ou regulam estes recetores, permitindo a sua modulação, poderão emergir como possíveis genes associados a PAE. Um exemplo de uma proteína que poderá interagir e/ou modular os GPCRs é a GPRASP2, tendo já sido associada a desordens neuropsiquiátricas, incluindo PAE. A GPRASP2 regula a sinalização das GPCR, incluindo dos recetores metabotrópicos de glutamato (mGLuRs), que estão igualmente associados a PEA e DI.De forma a perceber o papel da GPRASP2 no sistema nervoso, um modelo de murganhos knockout (KO) para o gene Gprasp2 foi desenvolvido. Este modelo apresenta diferentes fenótipos associados aos PEA, incluindo alterações no comportamento social. A GPRASP2 é expressa em diferentes regiões do cérebro, como no hipocampo, tálamo e hipotálamo. Para além disto, alterações na expressão dos níveis de GPRASP2 afetam a morfologias as espiculas dendríticas, a complexidade neuronal e o tráfico dos mGluRs, em culturas do hipocampo. Contudo, a função sináptica desta proteína e o seu papel na modulação da sinalização dos GPCR continua por concluir.De forma a entender a função da GPRASP2 nos recetores mGluR5s, recorreu-se a culturas organotáticas do hipocampo incubadas na presença e ausência de DHPG de forma a estudar as vias de sinalização ERK, PI3K e mTOR utilizando a técnica de western blot. Foram também averiguadas as alterações em proteínas sinápticas associadas à deleção do gene Gprasp2. Paralelamente, animais Gprasp2-/y e Gprasp2+/y foram sujeitos a tarefas de comportamento dependentes do hipocampo. De forma a estudar possíveis alterações na comunicação neonatal destes animais, as vocalizações foram detetadas em animais nas primeiras duas semanas de vida. Estas tarefas tiveram como objetivos analisar a função da GPRASP2 na sinapse e os efeitos da deleção deste gene no hipocampo.
Autism spectrum disorder (ASD) comprises a group of heterogeneous neurodevelopmental conditions, characterised by deficits in language, impairments in social interaction and communication and restricted interests and repetitive or stereotyped behaviours. Epidemiological studies estimate that more than 1% of the world’s human population has an ASD and that more than 70% of those with autism also have intellectual disability. There are two main theories to explain the pathophysiology of ASD: one involves failure in maintaining neural homoeostasis (synaptic dysfunction and E/I imbalance) and another involves alterations in specific neural circuits (cerebral connectivity). However, the exact mechanism that triggers an ASD is not yet fully understood.Twin and epidemiological studies strongly suggest that genetic factors play an important role in this disease. Moreover, genetic studies have now identified several proteins involved in synaptic function as strongly implicated in ASD.G-protein coupled receptors (GPCRs) have a role in the regulation of synaptic signalling. As such, GPCRs partners that modulate these receptors might be candidate genes for ASD. One example is the family of G-protein coupled receptor associated sorting proteins (GPRASP). GPRASP2, in particular, has been implicated in neuropsychiatric disorders, including ASD. GPRASP2 regulates GPCRs signalling, including metabotropic glutamate receptor (mGluR), themselves associated with ASD and ID pathophysiology. To understand the role of GPRASP2 in the nervous system, a new knockout (KO) mouse model was generated, and several autistic phenotypes, including abnormal social behaviour, were identified in this animal model. GPRASP2 is expressed in several brain regions, such as the hippocampus, thalamus, and hypothalamus. Additionally, changes in the expression levels of GPRASP2 can affect dendritic spine morphology, neuronal complexity and mGluR5 trafficking in rat hippocampal neuronal cultures. However, the synaptic function of GPRASP2 and its role in modulating GPCR signalling remains largely unknown.To elucidate the role of GPRASP2 upon mGluR5, hippocampal organotypic slices were incubated in the presence/absence of DHPG and downstream signalling was analysed using western blot techniques to study ERK, PI3K and mTOR pathways. We also assessed alterations in synaptic protein when GPRASP2 is ablated in vivo. Additionally, we performed hippocampal-dependent tasks and measured ultrasonic vocalisations in GPRASP2 knockout mice to complement the characterisation of this line as a model for ASD. These tasks were performed to understand the role of GPRASP2 in the synapse function and to dissect the downstream effects of its deletion.