Dissertations / Theses on the topic 'GluK1 receptors'

Nrf2 project: The protein nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that provides protection against oxidative stress and the dysfunction of this pathway has been suggested to be implicated in many neurodegenerative diseases. The aim of this thesis was to identify novel Nrf2 activators that disrupt the protein-protein interaction between Nrf2 and Keap1 and thereby induce increased expression of antioxidant enzymes and protective genes. The crystal structure of the Keap1-Nrf2 interface was used to perform a virtual screen and compounds from the screen were assayed using a cellular nuclear complementation assay that measures the nuclear translocation of Nrf2 from the cytosol. Although two novel compounds were found to increase the Nrf2 nuclear translocation, they had low activity and further characterisation did not provide sufficient evidence of a Nrf2-Keap1 robust interaction. iGluRs project: AMPA and kainate receptors are ionotropic glutamate receptors (iGluRs) that are important for excitatory transmission and synaptic plasticity and are linked to several neurological disorders such as epilepsy, schizophrenia and autism. This project aimed to find novel allosteric modulators binding in the ligand-binding domain (LBD) of the GluA2 and GluK1 and GluK2 subtypes of AMPA and kainate receptors, respectively, using protein purification and X-ray crystallography methodologies. Fragment screening for GluA2 identified eight novel fragments, five of which were located at the dimer interface and three located in a novel site near the glycine-threonine dipeptide linker. As regards kainate receptors, structural information on the Gluk1 and GluK2 LBD was obtained, both proteins were soaked with in-house fragments with one compound displaying 20% occupancy in the GluK2 dimer interface. These data form the basis of future studies in the search for novel drugs for the treatment of epilepsy and schizophrenia.

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista.
It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

It is generally understood that modifications in synaptic strength are the basis for learning and memory and that the strength of a synapse is largely governed by the abundance and distribution of synaptic receptors, especially alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA receptors), which mediate most of the fast synaptic transmission in the brain. GluA1-containing AMPARs are incorporated into the synapse following activity and posttranslational modifications to the carboxyl-terminus affect which proteins interact with the receptor and determine whether the receptor is inserted or removed from the synapse. In vitro research has discovered that the phosphorylation of three serine residues contained on the carboxyl-terminus (Ser 818, Ser-831 and Ser-845) regulates GluA1 synaptic incorporation; however, in vivo research investigating the relative importance of these phosphorylation sites on long-term memory formation is currently limited to knock in studies.To block the interactions between these phosphorylation sites and their binding partners in an inducible, temporally sensitive manner, we infused interference peptides containing these residues during consolidation and reconsolidation. We hypothesized that if the synaptic incorporation of GluA1 containing AMPA receptors is required for memory formation, and if this incorporation required the residues contained on the interference peptide, we would see an impairment in long-term memory expression when the peptide was infused at the time of training, or at the time of retrieval.Infusing the interference peptide GluA1-CT, containing Ser-831 and Ser 845, 1 hour before auditory fear conditioning produced no impairment in memory expression 24 hours later. However, infusing the interference peptide GluA1-MPR, containing Ser-818, 1 hour before training did produce an impairment in memory expression 24 hours later. We did not observe an impairment in long-term memory expression when both peptides were infused 1 hour before memory reactivation.
Il est généralement accepté que les modifications de la force synaptique sont à la base de l'apprentissage et la mémoire et que la force d'une synapse est largement régie par l'abondance et la distribution de récepteurs synaptiques, en particulier de récepteurs alpha-amino-3-hydroxy-5-méthyl-4- isoxazole propionate (récepteurs AMPA), qui interviennent dans la plupart des transmissions synaptiques rapides dans le cerveau. Les récepteurs AMPA contenant la sous-unité GluA1 sont incorporés dans la synapse suite à son activation et des modifications post-traductionnelles de l'extrémité carboxy-terminale influencent quelles protéines interagissent avec le récepteur et détermine si le récepteur est inséré ou retiré de la synapse. Des recherches in vitro ont découvert que la phosphorylation de trois résidus sérine contenus sur l'extrémité carboxy-terminale (Ser-818, Ser-831 et Ser-845) régie l'incorporation synaptique de GluA1; cependant, les recherches in vivo étudiant l'importance de ces sites de phosphorylation sur la formation de la mémoire à long terme est actuellement limitée à des études utilisant des « knock in ». Pour bloquer les interactions entre ces sites de phosphorylation et de leurs partenaires de liaison de manière inductible et temporellement sensibles, nous avons infusé des peptides d'interférence contenant ces résidus lors de la consolidation et la reconsolidation. Nous émettons l'hypothèse que si l'incorporation synaptique des récepteurs AMPA contenant GluA1 est nécessaire à la formation de la mémoire, et si cette incorporation exige les résidus contenus dans le peptide d'interférence, nous verrions une déficience dans l'expression de mémoire à long terme lorsque le peptide a été infusé au moment du conditionnement ou du rappel du souvenir.L'infusion du peptide d'interférence GluA1-CT, contenant les sérines Ser 831 et Ser-845, 1 heure avant le conditionnement de peur auditive n'a produit aucune altération dans l'expression de mémoire 24 heures plus tard. Cependant, l'infusion du peptide d'interférence GluA1-MPR, contenant la sérine Ser-818, 1 heure avant le conditionnement a produit une déficience dans l'expression de mémoire 24 heures plus tard. Nous n'avons pas observé d'altération dans l'expression de mémoire à long terme lorsque les deux peptides ont été infusés 1 heure avant la réactivation.

L’Esclerosi Lateral Amiotròfica (ELA o ALS) és una malaltia neuromuscular caracteritzada per la degeneració selectiva de les motoneurones (MN) superiors e inferiors del còrtex motor, el tronc de l’encèfal i la medul•la espinal, que resulta en una debilitat, espasticitat i atròfia progressives de la musculatura. Menys del 10% dels casos corresponen a la forma familiar de la malaltia, i un 20% d’aquests estan relacionats a mutacions en el gen de l’enzim superòxid dismutasa 1 (mSOD1). La resta de casos corresponen a la forma esporàdica. Les causes implicades en la degeneració selectiva de les MN en la ELA són encara desconegudes. La seva patogènesi s’ha atribuït a diversos mecanismes com serien l’estrès oxidatiu, l’agregació proteica anormal, la disfunció mitocondrial, el transport axonal aberrant, la neuroinflamació, l’autoimmunitat o l’excitotoxicitat per glutamat. En el present estudi hem treballat amb dues d’aquestes hipòtesis en avaluar l’efecte dels sèrums de pacients amb ELA i altres malalties de la MN sobre l’activitat del receptor ionotròpic de glutamat de tipus N-metil-D-Aspartat (NMDAR), expressat en el model d’oòcit de Xenopus laevis. Mitjançant assaigs de ELISA hem analitzat la presència d’autoanticossos associats a ELA en el sèrum de pacients. L’acció dels sèrums control i patològics en els oòcits de Xenopus produïa la generació de corrents oscil•latoris de clorur (Cl-). Aquests corrents havien estat prèviament descrits en aquestes cèl•lules i són deguts a l’activació dels canals de Cl- dependents de calci (Ca2+), endògens en els oòcits de Xenopus, a causa de la mobilització de Ca2+ intracel•lular. L’alliberació de Ca2+ dels compartiments intracel•lulars es activada per l’acció d’un factor sèric, anomenat àcid lisofosfatídic o lisofosfatidat (LPA), sobre el seu receptor, present en la membrana dels oòcits, i a través d’una via de senyalització de segons missatgers. Així doncs, en aquest model, la generació de corrents oscil•latoris de Cl- és una mesura indirecta de la mobilització intracel•lular de Ca2+. En presència del NMDAR, les respostes generades pel sèrum ELA eren significativament superiors a les activades pel sèrum d’individus sans i d’altres malalties de la MN. La resposta generada pel sèrum ELA presentava una dependència respecte de la presència de les dues subunitats del NMDAR i era sensible al bloqueig del receptor amb MK-801, un antagonista no competitiu. Vàrem reproduir els experiments amb sèrums del model de rata transgènica mSOD1 G93A, considerat un model de la forma familiar de la malaltia. Les mostres de sèrum mSOD1 G93A generaven, en presència del NMDAR, respostes significativament superiors a les activades pel sèrum de rata WT. En analitzar l’acció de la fracció de IgG purificada dels sèrums control i patològics en el model d’oòcit de Xenopus, es generaven corrents transitoris d’entrada de tipus no oscil•latori, els quals diferien dels generats en el cas del sèrum complet. La resposta activada per IgG de pacients amb ELA en presència del NMDAR era també significativament superior a la generada per les IgG d’individus sans. En la segona part d’aquest estudi s’ha comprovat la presència d’anticossos contra la proteïna Semaforina 3A (Sema3A) en alguns sèrums de ELA i Lower Motor Neuron Disease (LMND), una altra forma comuna de malaltia de la MN. La Sema3A és una molècula quimiotàctica de guia axonal recentment relacionada amb la patologia de la ELA en detectar-se una sobreexpressió d’aquesta proteïna en cèl•lules de Schwann terminals del model de ratolí mSOD1 G93A. Tot i descartar-se que els anticossos contra Sema3A siguin un marcador específic de la ELA, al no detectar-se en tots el sèrums de pacients, i alhora, al estar presents també en algunes mostres LMND, aquests autoanticossos podrien tenir un efecte defensiu contra les senyals nocives exercides per Sema3A sobre els axons de les MN.
Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease, characterized by the selective degeneration of the superior motor neurons in the motor cortex and of the inferior motor neurons in the brain-stem and spinal cord. The familial form of the illness is associated with the mutation of the superoxide dismutase enzyme (SOD-1). This and other mutations accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form. In this study we tested the effect of sera from sporadic ALS patients and from mutated human SOD-1 (mSOD1 G93A) transgenic rats on N-methyl-D-aspartate receptors (NMDAR). We hypothesize that an endogenous excitotoxic factor is implicated in neuronal death in ALS, mediated by the activation of NMDAR noncanonical signalling pathways. Sera from ALS patients or healthy subjects were pretreated to inactivate complement pathways and dialysed to remove glutamate. Sera from mSOD1 G93A rats were obtained at different stages of the neurodegenerative progression. Sera from transgenic rats were also pretreated to eliminate complement system and glutamate. Immunoglobulins G (IgGs) from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and glutamate-induced currents were recorded using the two electrode voltage clamp technique. We observed that sera from sporadic ALS patients induced transient oscillatory currents in Xenopus oocytes expressing NMDAR with a total electric charge significantly higher than the electric charge carried by currents induced by sera from healthy subjects. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. Results of sera from mSOD1 G93A transgenic rats were similar to those of sera from ALS patients; samples from patients with another type of neuromuscular disease did not exert this effect. IgG from ALS patients have a significant effect on NMDAR-injected oocytes and that response was doubled respect to the observed in the case of IgG from healthy subjects. Our data agree with the view that ALS patients sera contain some soluble factors that activates NMDAR, not opening directly the ionic conductance, but activating a non-canonical pathway.

Neocortical and hippocampal synaptic long-term potentiation (LTP) initially requires the AMPA receptor subunit GluR1, while late-phase LTP depends upon nitric oxide synthase (NOS). To investigate whether this was relevant to in vivo ED potentiation, GluR1 and/or NOS knockout mice were deprived of all whiskers but the D1 to induce barrel cortex synaptic potentiation, which was quantified by single unit recordings. In deprived cortex, D1 whisker responses potentiated approximately 40% less in GluR1 and NOS3 knockout mice than wild-type mice. Potentiation in the NOS1 knockout was influenced by gender; female NOS1 knockout potentiation was similar to wild-types, yet was absent in male NOS1 knockout mice. The ED potentiation in GluR1 knockout mice was dependent upon NOS, supporting LTP studies. However, NOS1 was more important for potentiation. Thus, while potentiation occurred in the GluR1/NOS3 double knockout mice, it was completely absent in the GluR1/NOS1 double knockout. To determine the interaction between GluR1 and NO activity in memory, behavioural studies examined their impact on spatial and contextual memory. The results partly confirmed earlier findings that retention of contextual fear conditioning was sensitive to GluR1 deletion. However, this was only the case in male GluR1 knockout mice. Female GluR1 KO mice were unimpaired. In a spatial radial arm watermaze task, GluR1 knockout mice acquired the location of a submerged platform more slowly than wild-types. Nevertheless, spatial reference memory was comparable to wild-type mice at the end of training and was not influenced by gender. In contrast to predictions, GluR1-indepdnent reference memory was not dependent upon NOS. Therefore while emotional learning requires GluR1 in male mice, spatial reference memory can form in its absence in both genders and is insensitive to NOS antagonism.

Early-life stress is associated with maladaptive long-lasting brain effects. Such alterations increase the likelihood of developing several psychiatric disorders. However, the molecular consequences of early-life stress are poorly understood. Here, we evaluated the impact of maternal separation with early weaning (MSEW) at different phases of cocaine self-administration (SA) and the subsequent molecular alterations in brain reward regions of male and female mice. Our findings show that MSEW affects males while females appear to be resilient to this kind of stress. Maternally separated mice evidence higher percentage of acquisition, more cocaine intake and decrease capacity to extinguish the SA behaviour. Moreover, molecular analyses of the brain areas studied reveal sex-induced alterations in the AMPA receptor composition and MSEW-induced changes in the expression of GluA1, GluA2, pCREB and CREB. Cocaine also alters the expression of these molecules. Together, results suggest that MSEW induces molecular alterations in areas related to reward processing, potentiating the vulnerability to cocaine-seeking behaviour and depression.
El estrés en la etapa temprana de la vida se asocial con efectos cerebrales maladaptativos y duraderos. Dichas alteraciones pueden aumentar la probabilidad de desarrollar diversos trastornos psiquiátricos. Sin embargo, las consecuencias moleculares del estrés en la vida temprana son poco conocidas. En este trabajo evaluamos el impacto de la separación maternal con destete temprano (MSEW, por sus siglas en inglés) en diferentes fases de la autoadministración de cocaína, así como las posteriores alteraciones moleculares en regiones cerebrales asociadas al sistema de recompensa, en ratones machos y hembras. Nuestros resultados muestran que la MSEW afecta a los machos, mientras que las hembras parecen ser resistentes a este tipo de estrés. Los ratones separados maternalmente muestran un mayor porcentaje de adquisición, más consumo de cocaína y una menor capacidad para extinguir el comportamiento de autoadministración. Además, los análisis moleculares de las áreas cerebrales estudiadas revelan alteraciones inducidas por el sexo en la composición del receptor AMPA y cambios inducidos por la MSEW en la expresión de GluA1, GluA2, pCREB y CREB. La cocaína también altera la expresión de estas moléculas. Los resultados en conjunto sugieren que la MSEW induce alteraciones moleculares en áreas relacionadas con el procesamiento de la recompensa, potenciando la vulnerabilidad al comportamiento de búsqueda de cocaína y la depresión.

Ionotropic glutamate AMPA-type receptors mediate fast excitatory neurotransmission in the central nervous system and are essential for synaptic plasticity. Expression of the receptor subunits varies with cell type, stage of development and brain region. Subunit composition determines functional properties of the receptor, including gating kinetics and synaptic trafficking. The research aimed to selectively disrupt the GluA1 subunit abundantly expressed in the hippocampus of the wild-type mouse, so as to examine its role in learning and memory. Site-directed monoclonal antibodies were engineered to target the extracellular amino-terminal domain of GluA1 for subunit-selective manipulation. The antibody-binding region was selected for heterogeneity and accessibility based on the amino acid sequences and crystal structures solved for the AMPA receptor subunits. Immunisations of peptide antigen in mice generated serum antibodies that recognise the equivalent epitope on the fully folded GluA1 subunit. The antigen-binding Fab fragment of the monoclonal anti-GluA1 antibody was cloned from hydridoma mRNA and purified from large-scale transient expression in mammalian cells. Biophysical characterisations of anti-GluA1 Fab immunoglobulin showed high specificity and affinity for the target subunit. Acute bilateral intrahippocampal administration of anti-GluA1 Fab protein into awake, behaving wild-type mice produced dissociations in spatial memory performance that resembled GluA1-/- knockout mice. Impaired short-term spatial working memory but intact long-term spatial reference memory observed with anti-GluA1 Fab infusions suggested that the immunoglobulin reagent exerted an acute, reversible, localised, GluA1-specific antagonism in the brain. The findings argue for a critical involvement of the hippocampal GluA1 subunit in certain short-term memory processes, but not in other distinct long-term memory processes. Temporal resolution of the antibody-mediated disruption revealed novel fractionations of short-term memory performance never before observed in the GluA1-/- knockout mice, demonstrating the strength of the monoclonal anti-GluA1 antibodies as an investigative tool.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Os recetores de cainato (KARs) são recetores de glutamato ionotrópicos tetraméricos, com caraterísticas biofísicas distintas, que regulam a atividade dos circuitos sinápticos a nível pré-sináptico e pós-sináptico através de ações ionotrópicas e/ou metabotrópicas. Estes recetores podem formar combinações homoméricas ou heteroméricas constituídas por diferentes subunidades do tipo GluK1–GluK5. Em mamíferos, o KAR heteromérico GluK2/GluK5 é o subtipo de KAR mais abundante no cérebro. Aquando da sua ativação por glutamato libertado endogenamente, os KARs GluK2/GluK5 medeiam correntes pós-sinápticas excitatórias (EPSCs) de cinética lenta e baixa amplitude, tendo um papel importante na transmissão sináptica excitatória e na integração sináptica.A necessidade atual de validar e caracterizar novos compostos farmacológicos que possam atuar seletivamente nos KARs surgiu naturalmente de forma a melhor entender as funções fisiológicas desta família de recetores de glutamato e também para melhor explorar o seu impacto na patofisiologia de algumas doenças neurológicas, tais como a epilepsia e a dor neuropática. Esta tese de Mestrado tem como objetivo validar e caracterizar novos antagonistas ortostéricos putativos e moduladores alostéricos putativos dos KAR GluK2/GluK5 usando eletrofisiologia em fatias de cérebro e aplicando diferentes protocolos de eletrofisiologia. Estes compostos farmacológicos foram sintetizados e fornecidos por um colaborador do projeto KARTLE (Laboratório do Dr. Bernard Pirotte – Universidade de Liège, Bélgica). Todo o conjunto de experiências foi realizado num contexto sináptico, especificamente nas sinapses das fibras musgosas com os neurónios da região Cornu ammonis 3 (sinapses mf-CA3) no hipocampo. Entre os dois grupos principais de compostos farmacológicos testados, o grupo de antagonistas ortostéricos putativos mostrou ter um impacto maior na redução quer da amplitude máxima quer da carga elétrica sináptica das EPSCs mediadas por KARs (KAR-EPSCs) nas sinapses mf-CA3. Neste grupo, verificou-se que o composto experimental P03, na concentração testada, foi mais potente que o composto de referência UBP310 em reduzir a carga elétrica sináptica mediada por KARs, indicando que este poderá ser um novo composto líder a atuar nos KARs GluK2/GluK5. Antecipamos que este e outros potenciais novos compostos farmacológicos que atuem nos KARs GluK2/GluK5 possam servir como ferramentas para melhor caraterizar as suas funções fisiológicas e representar opções adicionais para novas abordagens terapêuticas.
Kainate receptors (KARs) are tetrameric ionotropic glutamate receptors, with distinct biophysical features, which regulate the activity of synaptic circuits at presynaptic and postsynaptic levels through ionotropic and/or metabotropic actions. They can co-assemble as homomeric or heteromeric combinations of different GluK1–GluK5 subunits. In mammals, heteromeric GluK2/GluK5 KAR is the most abundant KAR subtype in the brain. Upon its activation by endogenously released glutamate, GluK2/GluK5 KARs participate in excitatory postsynaptic currents (EPSCs) with slow kinetics and small amplitude, having an important role in the overall excitatory synaptic transmission and synaptic integration.The current need of validating and characterizing new pharmacological compounds which can selectively target KARs came naturally in order to better understand the physiological functions of this family of glutamate receptors and also to further explore their impact on the physiopathology of some neurological disorders, such as epilepsy or neuropathic pain. This Master project aims to validate and characterize new putative orthosteric antagonists and allosteric modulators of GluK2/GluK5 KARs using brain slice electrophysiology and applying different electrophysiology protocols. These pharmacological compounds were synthesized and provided by a KARTLE project collaborator (Dr. Bernard Pirotte’s Lab – University of Liège, Belgium). The whole set of experiments was performed in a synaptic context, specifically at hippocampal mossy fiber to Cornu ammonis 3 (mf-CA3) synapses. Among the two main groups of pharmacological compounds tested, the group of putative orthosteric antagonists showed a higher impact reducing both peak amplitude and synaptic charge of EPSCs mediated by KARs (KAR-EPSCs) at mf-CA3 synapses. Within this group, we found that the experimental compound P03, in the tested concentration, was more potent in reducing the synaptic charge than the reference compound UBP310, indicating that it may be a new lead compound targeting GluK2/GluK5 KARs. We anticipate that potential new pharmacological compounds targeting GluK2/GluK5 KARs will be able to serve as tools to better characterize these KARs subtype physiologically and may represent additional options for novel therapeutic approaches.
Outro - Universidade de Bordéus, Centre National de la Recherche Scientifique (CNRS) e Network of European Funding for Neuroscience Research (Projeto KARTLE).

No
The surface expression and regulated endocytosis of kainate (KA) receptors (KARs) plays a critical role in neuronal function. PKC can modulate KAR trafficking, but the sites of action and molecular consequences have not been fully characterized. Small ubiquitin-like modifier (SUMO) modification of the KAR subunit GluK2 mediates agonist-evoked internalization, but how KAR activation leads to GluK2 SUMOylation is unclear. Here we show that KA stimulation causes rapid phosphorylation of GluK2 by PKC, and that PKC activation increases GluK2 SUMOylation both in vitro and in neurons. The intracellular C-terminal domain of GluK2 contains two predicted PKC phosphorylation sites, S846 and S868, both of which are phosphorylated in response to KA. Phosphomimetic mutagenesis of S868 increased GluK2 SUMOylation, and mutation of S868 to a nonphosphorylatable alanine prevented KA-induced SUMOylation and endocytosis in neurons. Infusion of SUMO-1 dramatically reduced KAR-mediated currents in HEK293 cells expressing WT GluK2 or nonphosphorylatable S846A mutant, but had no effect on currents mediated by the S868A mutant. These data demonstrate that agonist activation of GluK2 promotes PKC-dependent phosphorylation of S846 and S868, but that only S868 phosphorylation is required to enhance GluK2 SUMOylation and promote endocytosis. Thus, direct phosphorylation by PKC and GluK2 SUMOylation are intimately linked in regulating the surface expression and function of GluK2-containing KARs.

No
Phosphorylation or SUMOylation of the kainate receptor (KAR) subunit GluK2 have both individually been shown to regulate KAR surface expression. However, it is unknown whether phosphorylation and SUMOylation of GluK2 are important for activity-dependent KAR synaptic plasticity. We found that protein kinase C-mediated phosphorylation of GluK2 at serine 868 promotes GluK2 SUMOylation at lysine 886 and that both of these events are necessary for the internalization of GluK2-containing KARs that occurs during long-term depression of KAR-mediated synaptic transmission at rat hippocampal mossy fiber synapses. Conversely, phosphorylation of GluK2 at serine 868 in the absence of SUMOylation led to an increase in KAR surface expression by facilitating receptor recycling between endosomal compartments and the plasma membrane. Our results suggest a role for the dynamic control of synaptic SUMOylation in the regulation of KAR synaptic transmission and plasticity.

Tese de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
No sistema nervoso central, a maior parte da neurotransmissão excitatória é mediada por receptores de glutamato do tipo AMPA que possuem papéis fundamentais na plasticidade sináptica, o fenómeno celular na base de processos de aprendizagem e memória. Modificações no tráfego destes receptores e na sua inserção ao nível das sinapses, bem como na estabilidade do RNA mensageiro das subunidades dos receptores ou no seu decaimento, são cruciais para induzir alterações de longo prazo na força e eficiência sinápticas, o que permite a expressão de mecanismos de plasticidade. Tendo isto em conta, torna-se particularmente importante compreender a fundo como é que estes eventos são regulados, de forma a desvendar os mecanismos que estão na base de várias formas de plasticidade. Um estudo recente realizado no nosso laboratório permitiu a identificação da proteína 1 associada à Contactina (Caspr1) como um novo interactor da subunidade GluA1 dos receptores AMPA. Esta proteína foi capaz de induzir um aumento nos níveis superficiais de GluA1, bem como mediar o seu endereçamento para a membrana sináptica. Para além disso, e duma maneira independente da transcrição, a Caspr1 foi capaz de induzir um aumento nos níveis de RNAm de GluA1, o que sugere um papel importante da Caspr1 na regulação da estabilidade destes transcriptos. Estas evidências propõem então a existência de um novo mecanismo de regulação pós-transcripcional dos receptores AMPA, desconhecido até agora. Ainda assim, o papel da Caspr1 na regulação da subunidade GluA1 está longe de ser compreendido, pelo que é importante continuar a caracterizar este mecanismo regulador. Neste trabalho procurámos, em primeiro lugar, confirmar o papel da Caspr1 na regulação da subunidade GluA1. A sobreexpressão da Caspr1, tanto num sistema heterólogo como em neurónios primários de hipocampo, resultou num aumento significativo nos níveis totais de GluA1. Para além disso, conseguimos identificar o domínio intracelular da Caspr1 rico em prolinas como sendo o responsável por estes efeitos nos níveis de GluA1. De facto, uma forma da Caspr1 sem o domínio rico em prolinas não teve qualquer efeito nos níveis totais de GluA1, em células COS7. Tendo em conta que o domínio rico em prolinas da Caspr1 é capaz de interagir com domínios SH3 de várias moléculas de sinalização, em particular com a tirosina cínase Src, colocámos a hipótese de o efeito da Caspr1 nos níveis de GluA1 ocorrer por activação, mediada pelo seu domínio de prolinas, de uma via de sinalização a jusante da Src. De facto, a expressão da Caspr1 em células COS7 foi capaz de induzir um grande aumento nos níveis de Src fosforilada, bem como nos níveis de ZBP1 (proteína 1 de ligação a ‘zipcodes’) fosforilada. Este alvo de fosforilação pela Src é uma proteína de ligação a RNAs, conhecida por regular a tradução de vários RNAm. Para além disso, a sobreexpressão da Caspr1 em neurónios de hipocampo induziu um aumento significativo e específico no número de agregados de Src e ZBP1 fosforiladas, ao nível da sinapse. Por fim, tentámos investigar quais os estímulos fisiológicos capazes de regular a expressão endógena da Caspr1. Um pormenor interessante é que o efeito que a Caspr1 exerce sobre os níveis da subunidade GluA1 assemelha-se bastante ao efeito induzido por um bloqueio crónico da actividade neuronal, bloqueio esse que induz um fenómeno de plasticidade homeostática que depende do aumento da expressão de receptores AMPA, numa tentativa de restituir os níveis de actividade neuronal. De acordo com isto, um bloqueio crónico da actividade neuronal induzido por TTX (bloqueador específico de canais de sódio dependentes de voltagem), foi capaz de promover um aumento significativo, não só nos níveis totais de GluA1, mas também nos níveis endógenos da Caspr1. Além disso, esta manipulação da actividade neuronal foi ainda capaz de induzir a activação da via de sinalização da Src, aumentando os níveis de Src e ZBP1 fosforiladas. Em conclusão, este estudo contribuiu para caracterizar os mecanismos moleculares envolvidos na sobrerregulação da subunidade GluA1 pela Caspr1, bem como para identificar estímulos fisiológicos com impacto nestes mecanismos. Por fim, este estudo propõe um papel promissor para a Caspr1 na regulação de mecanismos na base da plasticidade homeostática.
Fast excitatory synaptic transmission in the central nervous system is mediated by glutamate receptors of the AMPA-type, which play key roles in synaptic plasticity, the cellular correlate of learning and memory. Modulating the traffic and synaptic insertion of these receptors as well as their protein levels, e.g. through regulation of their mRNA stability and turnover, is crucial to induce long-term changes in synaptic strength and efficacy, which accounts for the expression of mechanisms of synaptic plasticity. Thus, understanding how these events are regulated is of major importance to fully unravel the mechanisms that underlie several forms of plasticity. Recent data from our laboratory identified the integral membrane protein Contactin associated protein 1 (Caspr1) as a novel interactor of the GluA1 subunit of AMPARs. This protein was able to increase the cell surface expression of GluA1 and also, mediate its traffic to the synaptic membrane. Moreover, Caspr1 presented an upregulatory effect in GluA1 mRNA levels, in a transcription-independent manner, which suggests a role for Caspr1 in the regulation of GluA1 mRNA stability. These evidences suggest a novel post-transcriptional regulatory mechanism of AMPARs, unknown until now. Thus, it became important to further characterize the regulation of the GluA1 AMPAR subunit by Caspr1. We firstly sought to confirm the role of Caspr1 in regulating the protein levels for the GluA1 subunit. Overexpression of Caspr1, both in a heterologous system and in cultured hippocampal neurons, resulted in a significant increase in the total levels of GluA1. Moreover, we identified the proline-rich region of Caspr1 as the molecular determinant responsible for its effect in GluA1 levels. Indeed, when expressing a construct specifically deleted for the proline-rich domain, Caspr1 failed to increase GluA1 total levels, in COS7 cells. Taking into account that the proline-rich domain of Caspr1 interacts with SH3 domains of various signaling molecules, particularly that of the tyrosine kinase Src, we hypothesized that the effect of Caspr1 in GluA1 levels occurs through a proline domainmediated activation of a signaling pathway downstream of Src. Expression of Caspr1 in COS7 cells resulted in a marked increase in levels of phosphorylated Src as well as phosphorylated levels of its downstream target, Zipcode binding protein 1 (ZBP1), a RNA-binding protein known to regulate mRNA translation upon Src-dependent phosphorylation. Moreover, overexpression of Caspr1 in hippocampal neurons was able to induce a specific increase in the number of both phosphorylated Src and ZBP1 puncta at the synaptic level. Furthermore, we sought to investigate physiological stimuli capable of regulating the endogenous expression of Caspr1. Interestingly, the upregulatory effect that Caspr1 exerts in levels of GluA1 subunit parallels that of chronically blocking neuronal activity, which results in a homeostatic synaptic scaling of GluA1. Accordingly, chronic blockade of activity induced by TTX, a blocker of voltage-gated sodium channels, was able to significantly increase not only GluA1 total levels, but also levels of endogenous Caspr1. Moreover, this manipulation of neuronal activity was able to induce an activation of the Src signaling pathway, with increases in phosphorylated levels of both Src and ZBP1. In conclusion, this study contributed to characterize the molecular mechanisms involved in the upregulation of the GluA1 subunit by Caspr1, as well as the physiological stimuli that impinge on those mechanisms. Moreover, it unveils a potentially promising role for Caspr1 in mediating homeostatic plasticity mechanisms.

abstract: Intermittent social defeat stress induces psychostimulant cross-sensitization, as well as long-lasting social avoidance behavior. Previous data reveal heightened expression of AMPA receptor (AMPAR) GluA1 subunits in rat ventral tegmental area (VTA), which occurs concurrently with social stress-induced amphetamine (AMPH) cross-sensitization. These studies described herein examined whether VTA GluA1 AMPARs are important for the behavioral consequences of social stress and investigated the role of the infralimbic (IL) to VTA pathway in the induction of these responses. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization without affecting social avoidance behavior, while GluA1 overexpression in VTA DA neurons mimicked the effects of stress on AMPH sensitization. Female rats were more sensitive to the effects of stress on AMPH administration than males, specifically during proestrus/estrus, which is characterized by higher circulating estradiol. Fluorescent immunohistochemistry revealed that females expressed higher GluA1 in VTA DA neurons as a result of intermittent social defeat stress, independent of estrus stage; by contrast, females during proestrus/estrus displayed higher tyrosine kinase receptor type 2 (TrkB) expression, which is the receptor for brain derived neurotrophic factor (BDNF), in VTA DA neurons, independent of stress exposure. Functional inactivation of GluA1 in VTA DA neurons prevented stress-induced AMPH sensitization and overexpression mimicked the effects of stress on AMPH sensitization. This suggests that BDNF-TrkB signaling may work concomitantly with GluA1 signaling in the VTA to drive sex-dependent differences in stress-induced locomotor sensitization effects. Optogenetic inhibition of the IL-VTA pathway in male rats prevented stress-induced AMPH sensitization compared to control animals. In addition, fluorescent immunohistochemistry displayed less Fos labeling in the nucleus accumbens (NAc) of rats with IL-VTA light inhibition compared to control animals. This suggests that the IL-VTA pathway plays a critical role in the induction of stress-induced sensitivity to AMPH, and blocking this pathway prevents mesolimbic DA signaling to the NAc. We conclude that IL glutamate projections onto GluA1-homomeric AMPA receptors in VTA DA neurons play a critical role in driving the stress-induced sensitization response in males and females. Therefore, GluA1 VTA DA neurons could potentially be a therapeutic target to prevent stress-induced drug susceptibility in the future.
Dissertation/Thesis
Doctoral Dissertation Neuroscience 2020

Ionotropic glutamate receptors (iGluRs), like the NMDA receptor, are ligand-gated ion channels. These receptors make up an important part of the mammalian central nervous system (CNS) as they allow rapid excitatory signal transduction. NMDA receptors are built of four structurally identical subunits. Of the structurally identical subunits, three main types exist (GluN1, GluN2, GluN3), which are freely combined, with GluN1 always being incorporated twice. The transmembrane regions of these subunits consist of four alpha-helices named M1 – M4 domains. M1, M2 and M3 of all GluN subunits were identified to form the ion channel pore. Ligand binding at NMDA receptors leads to an opening of this pore which allows an ion flow across a membrane. The role of the M4 domain within the NMDA receptor is not yet clearly defined and was therefore investigated in this thesis. Herein approaches of M4-truncations, M4 segment coexpression and introduction of point mutations into the M4 domain were used to get an insight into the role of the M4 domain in functionality, assembly, and steroid modulation of NMDA receptors. It could be shown that the M4 domains of GluN1/GluN2A and GluN1/GluN3A NMDA receptors are not involved in receptor assembly nor in surface trafficking. However, M4-truncation lead to an impeded functionality of the analyzed ion channels that could be rescued by coexpression of the respective M4 segment indicating its importance in NMDA receptor function. Introduction of point mutations identified two residues in the upper part of the GluN1-M4 domain to be necessary for this rescue effect. These residues form interaction points of the GluN1-M4 domain with M1 or M3 domains of neighboring GluN2 subunits. The upper part of the M4 was thought to affect the modulation of NMDA receptors by neurosteroids like Pregnenolone sulfate (PS) and an involvement of this M4 part in neurosteroid modulation could be verified in this work. Furthermore, two further residues in GluN1-M4 were found to influence the affinity of PS to and the effect of PS on GluN1/GluN2 and GluN1/GluN3 receptors indicating these residues to build a negative allosteric modulation site for PS. All in all, this thesis gives new insights into the function of the M4 domain within GluN1/GluN2 and GluN1/GluN3 receptors highlighting the role in regulating receptor function. Furthermore, the herein obtained data allow a better understanding of NMDA receptor modulation by compounds with PS-like properties and therefore form a basis for further research investigating therapeutic strategies for selective NMDA receptor modulation.