Literatura académica sobre el tema "GLRA2"

The human sperm surface glycine receptor (GLR) plays a role in an important fertilization event, the sperm acrosome reaction. Here, by western blot analysis, we report the presence of GLRA1, GLRA2, GLRA3, and GLRB subunits in human sperm. Immunolocalization studies showed that the GLRA1 and GLRA2 subunits are present in the equatorial region, the GLRA3 subunit in the flagellar principal piece, and the GLRB subunit in the acrosomal region of sperm. This first demonstration of isoforms of the sperm GLRA subunit and of a differential spatial distribution of the α and β subunits on the surface of mammalian sperm suggests the possibility that human sperm GLRs have more than one function.

Introns of four X-linked genes (Hprt, Plp, Glra2, and Amg) were sequenced to provide an estimate of nucleotide diversity at nuclear genes within the house mouse and to test the neutral prediction that the ratio of intraspecific polymorphism to interspecific divergence is the same for different loci. Hprt and Plp lie in a region of the X chromosome that experiences relatively low recombination rates, while Glra2 and Amg lie near the telomere of the X chromosome, a region that experiences higher recombination rates. A total of 6022 bases were sequenced in each of 10 Mus domesticus and one M. caroli. Average nucleotide diversity (π) for introns within M. domesticus was quite low (π = 0.078%). However, there was substantial variation in the level of heterozygosity among loci. The two telomeric loci, Glra2 and Amg, had higher ratios of polymorphism to divergence than the two loci experiencing lower recombination rates. These results are consistent with the hypothesis that heterozygosity is reduced in regions with lower rates of recombination, although sampling of additional genes is needed to establish whether there is a general correlation between heterozygosity and recombination rate as in Drosophila melanogaster.

ABSTRACT Glycine receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system. During development, glycine receptor alpha 2 (GlyRα2) is expressed in the retina, in the spinal cord, and throughout the brain. Within the cortex, GlyRα2 is expressed in immature cells and these receptors have been shown to be active and excitatory. In the developing retina, inhibition of glycine receptor activity prevents proper rod photoreceptor development. These data suggest that GlyRα2, the developmentally expressed glycine receptor, may play an important role in neuronal development. We have generated mice with a targeted deletion of glycine receptor alpha 2 (Glra2). Although these mice lack expression of GlyRα2, no gross morphological or molecular alterations were observed in the nervous system. In addition, the cerebral cortex does not appear to require glycine receptor activity for proper development, as Glra2 knockout mice did not show any electrophysiological responses to glycine.

A-type ganglion cells of the mouse retina represent the visual channel that transfers temporal changes of the outside world very fast and with high fidelity. In this study we combined anatomical and physiological methods in order to study the glycinergic, inhibitory input of A-type ganglion cells. Immunocytochemical studies were performed in a transgenic mouse line whose ganglion cells express green fluorescent protein (GFP). The cells were double labeled for GFP and the four α subunits of the glycine receptor (GlyR). It was found that most of the glycinergic input of A-type cells is through fast, α1-expressing synapses. Whole-cell currents were recorded from A-type ganglion cells in retinal whole mounts. The response to exogenous application of glycine and spontaneous inhibitory postsynaptic currents (sIPSCs) were measured. By comparing glycinergic currents recorded in wildtype mice and in mice with specific deletions of GlyRα subunits (Glra1spd-ot,Glra2−/−,Glra3−/−), the subunit composition of GlyRs of A-type ganglion cells could be further defined. Glycinergic sIPSCs of A-type ganglion cells have fast kinetics (decay time constant τ = 3.9 ± 2.5 ms, mean ± SD). Glycinergic sIPSCs recorded inGlra2−/−andGlra3−/−mice did not differ from those of wildtype mice. However, the number of glycinergic sIPSCs was significantly reduced inGlra1spd-otmice and the remaining sIPSCs had slower kinetics than in wildtype mice. The results show that A-type ganglion cells receive preferentially kinetically fast glycinergic inputs, mediated by GlyRs composed of α1 and β subunits.

Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3−/−) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2−/Y animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.

Mesial temporal lobe epilepsy is the most common type of epilepsy. For most patients suffering from TLE, the only treatment option is surgery. However, there is a high possibility of relapse. Invasive EEG as a method for predicting the outcome of surgical treatment is a very complex and invasive manipulation, so the search for outcome biomarkers is an urgent task. MicroRNAs as potential biomarkers of surgical outcome are the subject of this study. For this study, a systematic search for publications in databases such as PubMed, Springer, Web of Science, Scopus, ScienceDirect, and MDPI was carried out. The following keywords were used: temporal lobe epilepsy, microRNA, biomarkers, surgery, and outcome. Three microRNAs were studied as prognostic biomarkers of surgical outcome: miR-27a-3p, miR-328-3p, and miR-654-3p. According to the results of the study, only miR-654-3p showed a good ability to discriminate between patients with poor and good surgical outcomes. MiR-654-3p is involved in the following biological pathways: ATP-binding cassette drug transporters, glutamate transporter SLC7A11, and TP53. A specific target for miR-654-3p is GLRA2, the glycine receptor subunit. MicroRNAs, which are diagnostic biomarkers of TLE, and epileptogenesis, miR-134-5p, MiR-30a, miRs-143, etc., can be considered as potential biomarkers of surgical outcome, as they can be indicators of early and late relapses. These microRNAs are involved in the processes characteristic of epilepsy: oxidative stress and apoptosis. The study of miRNAs as potential predictive biomarkers of surgical outcome is an urgent task and should be continued. However, when studying miRNA expression profiles, it is important to take into account and note a number of factors, such as the type of sample under study, the time of sampling for the study, the type and duration of the disease, and the type of antiepileptic treatment. Without taking into account all these factors, it is impossible to assess the influence and involvement of miRNAs in epileptic processes.

To characterize the prevalence of viruses associated with grapevine leafroll disease in China, 249 grapevine (Vitis spp.) samples (86 popular cultivars and a rootstock) from 19 provinces and regions were collected and tested for Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, GLRaV-4, and GLRaV-4 strain 5 by SYBR Green real-time reverse-transcription polymerase chain reaction (RT-PCR), and RT-PCR and sequencing. GLRaV-3 was found in 100% of the samples while GLRaV-1, GLRaV-2, and GLRaV-4 were detected in 24.9% (62/249), 15.3% (38/249), and 0.80% (2/249) of the samples, respectively. Single infections with GLRaV-3 were found in 66.3% (165/249) of the samples, and the remaining samples were mixed infections of GLRaV-3 with one or two other GLRaVs, those with GLRaV-1 being the most common (18.5%, 46/249). The genetic variability of Chinese GLRaV-3 isolates was characterized based on the coat protein (CP) gene. In total, 153 full-length CP gene sequences (94 sequences newly generated) of Chinese GLRaV-3 isolates from different grapevine-growing regions showed 89.3 to 100.0% and 92.7 to 100.0% identity at the nucleotide and amino acid levels, respectively. The average nucleotide diversity for the population of Chinese GLRaV-3 isolates was estimated at 0.037 (standard error = 0.0032). GLRaV-3 isolates from China segregated into five distinct phylogenetic groups and two novel recombination events were found in the viral population. This is the first and most extensive report of the prevalent species of GLRaV in China, which also provides an assessment of genetic variability of GLRaV-3 Chinese isolates.

Des variants pathogènes du gène GLRA2 codant pour la sous-unité α2 du récepteur glycinergique ont été récemment impliqués dans le trouble du spectre autistique (TSA) et la déficience intellectuelle. Notre groupe a précédemment montré que les souris mâles déficientes en Glra2 (Glra2–/Y) présentent un déficit de mémoire lors du test de reconnaissance d’objets (TRO) et une altération de la plasticité synaptique du cortex préfrontal (CPF), une région fortement impliquée dans le TSA. Par ailleurs, des études développementales des souris Glra2–/Y ont décrit un défaut de migration des interneurones et une perte de neurones de projection corticaux associée à une microcéphalie. Dans ce projet, nous avons recherché les altérations cellulaires et fonctionnelles qui sous-tendent les défauts comportementaux et synaptiques des souris Glra2–/Y, en se focalisant sur le CPF. Contrairement à ce qui a été rapporté précédemment, les souris Glra2–/Y ne sont pas microcéphales et ne présentent ni perte globale de neurones excitateurs ou inhibiteurs, ni perte de sous-populations d’interneurones à parvalbumine, calrétinine ou cholécystokinine dans le CPF ou le cortex somatosensoriel. Cependant, le nombre d'interneurones corticaux à somatostatine est augmenté chez les souris Glra2–/Y. Ces résultats démontrent un rôle plus subtil de Glra2 dans le développement du cortex que ce qui avait été suggéré auparavant, cohérent avec le phénotype des patients mâles porteurs de mutations dans GLRA2. Au moyen d’approches anatomiques et électrophysiologiques, nous avons également mis en évidence chez les souris Glra2–/Y des altérations caractéristiques des troubles neurodéveloppementaux, retrouvées dans d'autres modèles murins du TSA. Dans le CPF, ces souris mutantes présentent une diminution du nombre de synapses inhibitrices, une augmentation de la densité des épines et de la complexité dendritique des neurones pyramidaux, et une augmentation de l’activité synaptique excitatrice des neurones pyramidaux, sans effet sur la transmission synaptique inhibitrice. L'ensemble de ces résultats révèle l’existence d’un déséquilibre de la balance excitation/inhibition dans le cortex des souris Glra2–/Y. Afin d'identifier les régions cérébrales impliquées dans le déficit cognitif des souris Glra2–/Y, nous avons quantifié les neurones exprimant c-Fos, un marqueur d’activation neurale, après le TRO. Nous avons mis en évidence une hypoactivation sélective du CPF infralimbique rostral chez les souris Glra2–/Y après cette tache. Par colocalisation de c-Fos avec des marqueurs neuronaux, nous avons montré que cette hypoactivation était due à un déficit d’activation des neurones glutamatergiques. Pour évaluer plus finement l'activité neuronale dans le CPF des souris Glra2–/Y lors de l'apprentissage, nous avons mesuré l’activité des neurones glutamatergiques du cortex infralimbique par photométrie de fibre à l’aide d’un senseur calcique, pendant le TRO. Chez les souris sauvages, l'exposition répétée à des objets pendant la phase d'entraînement induit une réduction progressive de l'activité calcique, alors que cette atténuation est absente chez les souris Glra2–/Y, renforçant l’implication d’une altération de l'activité des neurones excitateurs dans le déficit cognitif de ces souris. En outre, malgré l'absence de déficits sociaux apparents, la réponse calcique des neurones glutamatergiques à la nouveauté sociale est atténuée chez les souris Glra2–/Y. Dans leur ensemble, ces résultats montrent que des altérations subtiles des circuits préfrontaux chez les souris Glra2–/Y engendrent une altération de la balance excitation/inhibition et un dysfonctionnement des neurones glutamatergiques dans le CPF lors du TRO, entraînant un déficit de mémoire. Ils suggèrent que la sous-unité α2 est cruciale pour le développement normal du CPF, et que des défauts des circuits préfrontaux pourraient sous-tendre le dysfonctionnement neurocognitif observé chez les patients présentant une perte de GLRA2
Pathogenic variants in the GLRA2 gene, which encodes the glycine receptor α2 subunit, have been recently implicated as a novel cause of autism spectrum disorder (ASD) and intellectual disability. Our group previously showed that Glra2-deficient male (Glra2 /Y) mice display impaired learning and memory in the novel object recognition (NOR) task and altered synaptic plasticity in the prefrontal cortex (PFC), a region consistently implicated in ASD. In addition, developmental studies in mice expressing the same Glra2 mutation reported deficits in interneuron migration and loss of cortical projection neurons associated with microcephaly. In this project, we investigated the cellular and functional alterations underlying the behavioural and synaptic defects of Glra2 /Y mice, focusing on the PFC. In contrast with previous reports, Glra2 /Y mice were not microcephalic and neuronal quantification showed no loss of either glutamatergic neurons or interneurons, including parvalbumin, calretinin and cholecystokinin interneuron subpopulations in the PFC or the somatosensory cortex. However, the number of cortical somatostatin interneurons was increased in these regions in mutant mice. These findings imply that Glra2 plays a more subtle role in neocortical development and assembly than previously suggested and are consistent with the phenotype of male patients with pathogenic GLRA2 variants, who are not microcephalic and have normal brain imaging. We also show that Glra2 /Y mice exhibit many of the hallmarks of neurodevelopmental brain dysfunction observed in other rodent models of ASD. In the adult PFC, Glra2 /Y mice show a decreased number of inhibitory synapses and increased spine density and dendritic complexity of pyramidal neurons, whilst young mice (P14-P21) have increased excitatory synaptic inputs to prefrontal pyramidal neurons, with no effect on inhibitory synaptic transmission. Taken together, these findings point to excitatory hyperconnectivity in the PFC of Glra2 /Y mice, and suggest an imbalance of excitatory and inhibitory neurotransmission in these mutant mice. To identify which brain regions are associated with the recognition memory deficit observed in Glra2 /Y mice, we quantified c-Fos expression as a marker of neuronal activation following NOR. We found that the rostral infralimbic PFC was hypoactivated in Glra2 /Y mice following this task, whilst other brain regions quantified showed similar levels of c-Fos expression compared to wild-type mice. c-Fos colocalization with neuronal markers revealed that the hypoactivation of the PFC was driven by impaired activation of glutamatergic neurons following the task. To further assess neuronal activity in the PFC in Glra2 /Y mice during cognition, we recorded calcium transients from infralimbic glutamatergic neurons using in vivo fiber photometry during NOR, and compared them with the calcium response induced by social interaction with a novel mouse. In wild-type animals, repeated exposure to objects during the training phase of the NOR task caused a progressive reduction in calcium-dependent neuronal activity during exploration. This attenuation of the calcium signals was absent from Glra2 /Y mice, further implicating an impairment of prefrontal glutamatergic activity in the NOR deficit observed in this model. In addition, despite a lack of apparent social deficits, Glra2 /Y mice exhibited an attenuated glutamatergic calcium response to novel social stimuli in the PFC. Overall, these findings show that subtle alterations in prefrontal circuit organization and physiology in Glra2 /Y mice result in altered inhibitory/excitatory balance and an aberrant response of prefrontal glutamatergic neurons during recognition memory leading to impaired task performance. These results suggest that the glycine receptor α2 subunit is crucial for normal PFC development, and that defects in prefrontal circuits may underlie the neurocognitive dysfunction observed in patients lacking GLRA2

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Glutamate receptors are the mediators of most excitatory neurotransmission processes in the central nervous system, acting as prominent targets for the treatment of several neurological disorders such as Epilepsy, Amyotrophic Lateral Sclerosis, Parkinson’s disease and Alzheimer’s disease. Hence an improved understanding of how glutamate and other ligands interact with the binding domain, of these receptors, can bring relevant insights to the development of new ligands. Therefore, this work aims to study the GluA2–ligand interaction using the structure of GluA2 co-crystallized with the ligands glutamate, AMPA, kainate and DNQX applying a method based on the Density Functional Theory combined with the molecular fractionation with conjugate caps scheme. To address that the dielectric constant of the GluA2 receptor is not homogeneous, a novel molecular approach was proposed and it was applied to study the interaction between the GluA2 and the ligands glutamate, AMPA, kainate and DNQX. The results obtained, considering the inhomogeneous model, were compared with those obtained using an uniform dielectric function for the GluA2 receptor and with data published in the literature establishing a more detailed description of the relevant amino acid residues for the protein-ligand binding interaction. Molecular dynamics studies and protein DFT calculations usually consider a fixed value for the protein dielectric function. In this work when ε = 1 is considered, many amino acid residues seem important, but when the dielectric constant shield was considered, they lost their relevance. The results for the GluA2-ligand total interaction energy and the D1-ligand and D2-ligand total interaction energy also shed some light on the differentiation between full and partial agonists, and between agonists and antagonists. Additionally, the results allow a hypothesis on the correlation between the Glu705-ligand interaction energy and the ligand action, paving the way for the use of the inhomogeneous dielectric function to study glutamate receptors and other protein-ligand systems. Finally, the results also suggests that for different ligands, different homogeneous dielectric constant will be able to well represent the system GluA2-ligand, making it necessary the previous analyses with the inhomogeneous dielectric constant approach.
Os receptores de glutamato são os mediadores da maioria dos processos de neurotransmissão excitatória no sistema nervoso central, atuando como alvos proeminentes para o tratamento de vários distúrbios neurológicos, como Epilepsia, Esclerose Lateral Amiotrófica, Doença de Parkinson e Doença de Alzheimer. Assim, uma compreensão aprimorada de como o glutamato e outros ligantes interagem com o domínio de interação, desses receptores, pode trazer informações relevantes para o desenvolvimento de novos ligantes. Portanto, este trabalho teve por objetivo estudar a interação GluA2-ligante utilizando a estrutura de GluA2 co-cristalizada com os ligantes Glutamato, AMPA, Cainato e DNQX utilizando método baseado na Teoria do Funcional da Densidade combinado com o esquema de fracionamento molecular com capas conjugadas. Para abordar que a constante dielétrica do receptor GluA2 não é homogênea, foi proposta uma nova abordagem molecular, que foi aplicada para estudar a interação entre a GluA2 e os ligantes Glutamato, AMPA, Cainato e DNQX. Os resultados obtidos, considerando o modelo não-homogêneo, foram comparados com aqueles obtidos usando uma função dielétrica uniforme para o receptor GluA2 e com dados publicados na literatura, estabelecendo uma descrição mais detalhada dos resíduos de aminoácido mais relevantes para a interação proteína-ligante. Estudos de dinâmica molecular e cálculos DFT de sistemas proteicos normalmente consideram um valor fixo para a função dielétrica proteica. Nesse trabalho quando ε = 1 é considerado, muitos resíduos de aminoácido parecem relevantes, mas quando a blindagem da constante dielétrica foi considerada, eles perderam sua relevância. Os resultados apresentados para a energia de interação total GluA2-ligante e a energia de interação total D1-ligante e D2-ligante contribuiu com a diferenciação entre agonistas totais e agonistas parciais e entre agonistas e antagonistas. Além disso, os resultados permitem que seja feita hipótese sobre a correlação entre a energia de interação Glu705-ligante e a ação do ligante, abrindo caminho para o uso da função dielétrica não-homogênea para estudar receptores de glutamato e outros sistemas proteína-ligante. Por fim, os resultados também sugerem que para diferentes ligantes, diferentes constantes dielétricas homogêneas serão capazes de representar bem o sistema GluA2-ligante, tornando necessária a análise prévia com a abordagem da constante dielétrica não-homogênea.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases.
Master of Science in Life Sciences

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique
Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity

L’enroulement de la vigne cause des pertes de rendement et de la qualité des vins au niveau mondial. Il est causé par quatre espèces de Grapevine leafroll-associated virus(GLRaVs) ; GLRaV-1, -2, -3, -4-like. Si le GLRaV-2 (genre Closterovirus) ne possède pas de vecteur connu, les trois autres espèces (genre Ampelovirus) sont transmises par cochenilles(Coccoidea) qui contribuent à leur dispersion dans et entre les vignobles. Les vignobles de la France septentrionale (Alsace, Bourgogne, Champagne) sont les plus impactés par l’enroulement viral. Ce travail a porté sur le rôle vecteur de Phenacoccus aceris, espèce connue pour son efficacité de transmission et de dispersion des ampélovirus, ainsi que de vitivirus souvent associés. Les interactions cellulaires et moléculaires entre virus et cochenille restent peu connues. Une approche pluridisciplinaire combinant entomologie,virologie, biologie cellulaire et moléculaire a été développée pour étudier la biologie de la vection du GLRaV-1 et du Grapevine virus A (GVA) par P. aceris. Des expériences de transmission ont montré que ces virus sont transmis selon le mode semi-persistant non circulant. L’étude préliminaire du comportement alimentaire de P. aceris sur vigne par électropénétrographie a révélé une activité similaire à celle d'autres espèces de cochenilles déjà décrites et suggère un effet de l’infection sur le comportement alimentaire. L’anatomie des pièces buccales de P. aceris, organes directement impliqués dans la transmission et la rétention de virus non-circulants a été décrite et une méthode basée sur l’acquisition de virus purifié sur membrane a été développée pour rechercher les sites de rétention virale dans le vecteur
Grapevine leafroll disease affects grape yield and wine quality worldwide. It is caused by four species of Grapevine leafroll-associated virus (GLRaVs) (GLRaV-1, -2, -3, -4-like).While GLRaV-2 (genus Closterovirus) has no known vector, the other three (genus Ampelovirus) are transmitted by mealybugs (Coccoidea) and thus prone to be dispersed within and between vineyards. In north-eastern France (Alsace, Bourgogne et Champagne),vineyards are more impacted by Grapevine leafroll disease. This thesis focusses on the vector role of the species Phenacoccus aceris, known for its efficiency in transmission and dissemination of ampeloviruses, as well as often associated vitiviruses. Molecular and cellular interactions between viruses and mealybugs remain poorly known. A multidisciplinary approach, combining entomology, virology, molecular and cellular biology, was developed to analyse the vector biology of GLRaV-1 and Grapevine virus A (GVA) by P. aceris.Transmission experiments showed that GLRaV-1 and GVA transmission follows the semipersistent non-circulative mode. A preliminary study of P. aceris feeding behavior on grape using electropenetrography revealed an activity similar to that of other mealybug species already described and suggested a potential effect of infection on Ph. aceris feeding behavior. The anatomy of mouth parts, directely implied in transmission and retention of non circulative viruses was described and a method for membrane acquisition of purified virus was developed to search for virus retention sites within the vector

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.

Les virus de l'enroulement de la vigne (Grapevine leafroll-associated virus, GLRaV) sont répandus mondialement et transmis à la vigne uniquement par cochenilles (Coccoidea). En France, l'enroulement viral affecte particulièrement les vignobles des régions septentrionales.L'approche biologique de la vection a montré la capacité de Phenacoccus aceris à transmettre à la vigne les GLRaV-1, -3, -4, -5, -6, -9 et ceux du bois strié Grapevine virus A et B. Cette étude est la première démonstration de la transmission du GLRaV-6 et confirme l'absence de spécificité des cochenilles dans la transmission des Ampelovirus. Les larves néonates de P. aceris et de Neopulvinaria innumerabilis représentent un stade de développement efficace pour la transmission de ces virus. En conséquence, leurs capacités vectrices, associées à leur fort potentiel de dissémination anémophile, impliquent un risque important de dispersion naturelle de ces virus dans un vignoble infesté. Les relevés sur quatre parcelles distinctes montrent que Parthenolecanium corni, Pulvinaria vitis, Heliococcus bohemicus et P. aceris sont communes, chaque vignoble différant par la diversité spécifique, le taux de ceps infestés et l'abondance des cochenilles. L'étude épidémiologique prouve le rôle des cochenilles dans la dispersion de l'enroulement viral dans les vignobles septentrionaux. A Bonzon, la responsabilité de P. aceris dans la diffusion rapide du GLRaV-1 est mise en évidence. Cette découverte représente la première preuve en Europe d'une dispersion naturelle du GLRaV-1. A Marsannayla-Côte, l'incidence du GLRaV-1 reste faible, la colonie de P. aceris ne semblant avoir qu'un rôle très limité dans la diffusion de la maladie. L'épidémiologie moléculaire à Bonzon révèle une diversité génétique importante du GLRaV-1 à l'échelle parcellaire et fournit pour la première fois des données sur le polymorphisme génétique d'une population de GLRaV-1 ayant été dispersée par des cochenilles.

The known functional ionotropic glutamate receptors (iGluRs) are composed of three major subtypes: AMPA, NMDA, and kainate. In 1998, in the landmark paper discussed in this chapter, Armstrong et al. provided the first crystal structure of an iGluR-subunit ligand-binding core, the S1S2 region of the rat GluA2 ‘flop’ isoform. They solved its structure with X-ray crystallography from selenomethonine crystals. They also identified residues involved in kainate binding, analysed allosteric sites that regulate affinity and specificity of the agonist, and mapped potential subunit–subunit interaction sites. They also proposed that binding of different agonists may result in variable degrees of domain closure. This work has profound impact on the field and it has been importantly cited. Subsequently, numerous high-resolution crystal structures of ligand-binding domains of iGluRs in complex with ligands, both agonists and antagonists, have been solved.

This study determines the in vitro influence of viruses (GLRaV-1, GLRaV-3, GFLV, GVA) and the position of meristems on initial shoots on the growth and development of ‘Plavac Mali’ and ‘Pošip’ grapevine cultivars. Infected clones were inoculated on MS medium with 0.5 mg l1-1 benzylaminopurine (BAP) and 0.05 mg l-1 indole-3-acetic-acid (IAA). The positions of meristems were: a – 1st; b – 2nd and 3rd; and c – 4th and 5th. The height and number of nodes, as well as the size of callus, were measured four and eight weeks after inoculation. Clone, position of explants, and sanitary status show no significant influence on tested parameters, except in callus formation, where shoot apex formed the lowest callus. Results revealed that axillary buds on the shoot of grapevine, infected with economically important viruses, can be used for biotechnology purposes.

RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.

AlkB proteins that repair DNA via reversing methylation damage are conserved in a broad range of prokaryotes and eukaryotes including plants. Surprisingly, AlkB-domains were discovered in the genomes of numerous plant positive-strand RNA viruses, majority of which belong to the family Flexiviridae. The major goal of this research was to reveal the AlkB functions in the viral infection cycle using a range of complementary genetic and biochemical approaches. Our hypotheses was that AlkB is required for efficient replication and genetic stability of viral RNA genomes The major objectives of the research were to identify the functions of GVA AlkB domain throughout the virus infection cycle in N. benthamiana and grapevine, to investigate possible RNA silencing suppression activity of the viral AlkBs, and to characterize the RNA demethylation activity of the mutated GVA AlkBs in vitro and in vivo to determine methylation status of the viral RNA. Over the duration of project, we have made a very substantial progress with the first two objectives. Because of the extreme low titer of the virus particles in plants infected with the AlkB mutant viruses, we were unable to analyze RNA demethylation activity and therefore had to abandon third objective. The major achievements with our objectives were demonstration of the AlkB function in virus spread and accumulation in both experimental and natural hosts of GVA, discovery of the functional cooperation and physical interaction between AlkB and p10 AlkB in suppression of plant RNA silencing response, developing a powerful virus vector technology for grapevine using GLRaV-2-derived vectors for functional genomics and pathogen control in grapevine, and in addition we used massive parallel sequencing of siRNAs to conduct comparative analysis of the siRNA populations in grape plants infected with AlkB-containing GLRaV-3 versus GLRaV-2 that does not encode AlkB. This analysis revealed dramatically reduced levels of virus-specific siRNAs in plants infected with GLRaV-3 compared to that in GLRaV-2 infection implicating AlkB in suppression of siRNA formation. We are pleased to report that BARD funding resulted in 5 publications directly supported by BARD, one US patent, and 9 more publications also relevant to project. Moreover, two joint manuscripts that summarize work on GVA AlkB (led by Israeli PI) and on viral siRNAs in grapevine (led by US PI in collaboration with University of Basel) are in preparation.