Alteration of GABAAR trafficking during cerebral ischemia: the role of Huntingtin-associated protein 1

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.A isquémia cerebral resulta de um fornecimento insuficiente de sangue ao cérebro, levando a uma desregulação no equilíbrio entre a neurotransmissão excitatória/inibitória e consequente morte celular por excitotoxicidade. No sistema nervoso central (SNC) a regulação deste equilíbrio é determinada principalmente pelo balanço entre a neurotransmissão glutamatérgica e GABAérgica e diversos estudos têm mostrado que a neurotransmissão glutamatérgica e GABAérgica está aumentada e reduzida, respectivamente, nas lesões isquémicas. Ao contrário das alterações na neurotransmissão glutamatérgica na isquémia cerebral que têm sido amplamente investigadas, poucos estudos têm abordado os mecanismos moleculares que contribuem para as alterações na neurotransmissão GABAérgica. Resultados recentes do nosso laboratório, obtidos utilizando o modelo de isquémia cerebral baseado na privação de oxigénio e glicose (OGD), mostraram que o insulto isquémico induz a desfosforilação e consequente internalização dos receptores de GABA do tipo A (GABAAR), contribuindo para a morte neuronal. Após a internalização os GABAAR são rapidamente reciclados e voltam para a membrana plasmática ou são encaminhados para os lisossomas a fim de serem degradados. O rumo que os GABAAR endocitados tomam depende da interacção das subunidades β1-3 com a proteína associada à huntingtina 1 (HAP1). Estudos anteriores do nosso laboratório mostraram que a OGD transitória também reduz a reciclagem e o regresso para a membrana plasmática dos GABAAR, e diminui a interacção dos receptores com a proteína HAP1 em neurónios do hipocampo em cultura. A proteína HAP1 existe em duas isoformas, HAP1-A e HAP1-B, que compartilham a mesma região central (aminoácidos 277-445). A HAP1 está associada a microtúbulos e a diversos tipos de organelos, incluindo as mitocôndrias, lisossomas e vesículas sinápticas. Tendo em consideração estas observações, no presente trabalho investigámos o papel da HAP1 na redução da expressão à superfície e reciclagem dos GABAAR em neurónios de hipocampo em cultura após OGD. Os resultados obtidos mostram que a exposição transitória de neurónios de hipocampo a OGD (90 min) reduz os níveis da proteína HAP1, quando testado por western blot duas horas após o insulto isquémico. Este efeitodependente do tempo de incubação, não foi observado em neurónios incubados na presença do inibidor das calpaínas MDL28170. A inibição das fosfatases PP1/PP2A com ácido ocadáico também diminuiu a redução de HAP1 induzida pela OGD. A diminuição dos níveis da proteína HAP1 foi também observada em neurónios corticais expostos a OGD, à semelhança dos resultados obtidos em neurónios do hipocampo em cultura. Porém, a oclusão transitória da artéria cerebral média (MCAO), um modelo in vivo de isquémia cerebral, teve o efeito oposto sobre os níveis da proteína HAP1 no núcleo isquémico, localizado na região cortical. Esta discrepância pode ser devida ao efeito do insulto isquémico sobre os níveis da proteína Hap1 em células da glia, presentes no tecido cerebral mas ausentes nas culturas neuronais. De acordo com essa hipótese, a análise por western blot realizada com extractos de células da glia em cultura expostas a 90 min de OGD seguido de 12 h de pós-incubação mostraram um aumento dos níveis de proteína HAP1. Para investigar o papel modulador de HAP1 nas alterações do tráfego dos GABAAR induzidas pelo OGD, foram realizadas experiências em culturas de neurónios de hipocampo transfectados com as isoformas HAP1-A ou-1B. A sobre-expressão das duas isoformas de HAP1, em neurónios de hipocampo em cultura, preveniu a redução da expressão superficial da subunidade β3 do GABAAR induzida pela OGD. Este efeito foi devido ao aumento da reciclagem da subunidade β3 do receptor GABAAR, como mostrado através do ensaio de reciclagem. Em resumo, os nossos resultados sugerem que a proteína HAP1 desempenha um papel fundamental na redução da neurotransmissão GABAérgica durante a isquémia cerebral.Cerebral ischemia is a pathological condition characterized by a reduction of blood flow to the brain leading to an imbalance between excitatory and inhibitory neurotransmission and consequent neuronal cell death. In the CNS this balance is mostly regulated by glutamate and GABA meurotransmitters. Several studies have shown that during an ischemic insult the glutamatergic and GABAergic neurotransmission is up- and down-regulated respectively. However, few studies have addressed the molecular mechanisms contributing to the alterations in GABAergic neurotransmission in brain ischemia. Recent data from our laboratory using the oxygen and glucose deprivation (OGD) model of brain ischemia showed that the ischemic insult induces the dephosphorylation and consequent internalization of GABAA receptors (GABAAR), contributing to the death of cultured hippocampal neurons. Following internalization, GABAAR are rapidly recycled back to the plasma membrane or targeted for lysosomal degradation. The sorting of endocytosed GABAAR depends on the interaction of GABAAR β1-3 subunits with huntingtin-associated protein 1 (HAP1). Previous studies from our laboratory also showed that transient OGD reduces the recycling of GABAAR back to the plasma membrane and decrease the interaction of the receptors with the HAP1 protein in cultured hippocampal neurons. HAP1 consists of two isoforms, HAP1-A and HAP1-B, which share the same middle part (amino acids 277-445). The protein is associated with microtubules and with various types of membranous organelles, including mitochondria, lysosomes and synaptic vesicles. Taking into consideration these observations, in the present work we investigated the putative role of HAP1 in the reduction of the surface expression and recycling of GABAAR in cultured hippocampal neurons subjected to OGD. Our results show that exposure of hippocampal neurons to OGD (90 min) downregulates HAP1 protein levels when tested 2 h after ischemic insult by western blot analysis. This effect was time dependent and was inhibited in the presence of the calpain inhibitor MDL28170. Inhibition of PP1/PP2A phosphatases with okadaic acid also reduced the OGD-induced downregulation of HAP1. A decrease in HAP1 protein levels was also observed in cortical neurons exposed to OGD, but transient middle cerebral artery occlusion (MCAO), an in vivo model of cerebral ischemia, had the opposite effect on HAP1 protein levels in the ischemic core located incortical region. This discrepancy may be due to the effect of the ischemic insult in HAP1 protein levels in glial cells present in the brain tissue but not in neuronal cultures. Accordingly, western blot analysis performed with extracts of cultured glial cells exposed to 90 min of OGD followed by 12 h of post-incubation showed an increase of HAP1 protein levels. To investigate the modulatory role of HAP1 in OGD-induced changes in the traffic of GABAAR, experiments were performed in cultured hippocampal neurons transfected with HAP1-A or -1B isoforms. Overexpression of the two isoforms of HAP1 in cultured hippocampal neurons decreased the OGD-induced downregulation of the surface expression of GABAAR β3 subunits. This effect was due to the increased recycling of GABAAR β3 as shown with receptor recycling assay. Taken together, our results suggest that HAP1 protein has a key role in the down-modulation of GABAaergic neurotransmission during cerebral ischemia

Best practices for the manual curation of Intrinsically Disordered Proteins in DisProt

The DisProt database is a significant resource containing manually curated data on experimentally validated intrinsically disordered proteins (IDPs) and regions (IDRs) from the literature. Developed in 2005, its primary goal was to collect structural and functional information into proteins that lack a fixed three-dimensional (3D) structure. Today, DisProt has evolved into a major repository that not only collects experimental data but also contributes significantly to our understanding of the IDPs/IDRs roles in various biological processes, such as autophagy or the life cycle mechanisms in viruses, or their involvement in diseases (such as cancer and neurodevelopmental disorders). DisProt offers detailed information on the structural states of IDPs/IDRs, including state transitions, interactions, and their functions, all provided as curated annotations. One of the central activities of DisProt is the meticulous curation of experimental data from the literature. For this reason, to ensure that every expert and volunteer curator possesses the requisite knowledge for data evaluation, collection, and integration, training courses and curation materials are available. However, biocuration guidelines concur on the importance of developing robust guidelines that not only provide critical information about data consistency but also ensure data acquisition.This guideline aims to provide both biocurators and external users with best practices for manually curating IDPs and IDRs in DisProt. It describes every step of the literature curation process and provides use cases of IDP curation within DisProt. Database URL: https://disprot.org

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.Fil: Quaglia, Federica. Università di Padova; Italia. Consiglio Nazionale delle Ricerche; ItaliaFil: Mészáros, Bálint. European Molecular Biology Laboratory; AlemaniaFil: Salladini, Edoardo. Università di Padova; ItaliaFil: Hatos, András. Università di Padova; ItaliaFil: Pancsa, Rita. Research Centre for Natural Sciences; HungríaFil: Chemes, Lucia Beatriz. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Pajkos, Mátyás. Eötvös Loránd University; HungríaFil: Lazar, Tamas. Vlaams Instituut voor Biotechnology; Hungría. Vrije Unviversiteit Brussel; BélgicaFil: Peña Díaz, Samuel. Universitat Autònoma de Barcelona; EspañaFil: Santos, Jaime. Universitat Autònoma de Barcelona; EspañaFil: Ács, Veronika. Research Centre for Natural Sciences; HungríaFil: Farahi, Nazanin. Vlaams Instituut voor Biotechnology; Bélgica. Vrije Unviversiteit Brussel; BélgicaFil: Fichó, Erzsébet. Research Centre for Natural Sciences; HungríaFil: Aspromonte, Maria Cristina. Università di Padova; Italia. Città della Speranza Pediatric Research Institute; ItaliaFil: Bassot, Claudio. Stockholms Universitet; SueciaFil: Chasapi, Anastasia. Centre for Research & Technology Hellas; GreciaFil: Davey, Norman E.. Chester Beatty Laboratories; Reino UnidoFil: Davidović, Radoslav. University of Belgrade; SerbiaFil: Laszlo Holland, Alicia Verónica. European Molecular Biology Laboratory; Alemania. Research Centre for Natural Sciences; HungríaFil: Elofsson, Arne. Stockholms Universitet; SueciaFil: Erdős, Gábor. Eötvös Loránd University; HungríaFil: Gaudet, Pascale. Swiss Institute of Bioinformatics; SuizaFil: Giglio, Michelle. University of Maryland School of Medicine; Estados UnidosFil: Glavina, Juliana. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Iglesias, Valentín. Universitat Autònoma de Barcelona; EspañaFil: Kálmán, Zsófia. Pázmány Péter Catholic University; HungríaFil: Lambrughi, Matteo. Danish Cancer Society Research Center; DinamarcaFil: Leonardi, Emanuela. Università di Padova; Italia. Pediatric Research Institute Città della Speranza; ItaliaFil: Rodriguez Sawicki, Luciana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The Gene Ontology knowledgebase in 2023

The Gene Ontology (GO) knowledgebase (http://geneontology.org) is a comprehensive resource concerning the functions of genes and gene products (proteins and noncoding RNAs). GO annotations cover genes from organisms across the tree of life as well as viruses, though most gene function knowledge currently derives from experiments carried out in a relatively small number of model organisms. Here, we provide an updated overview of the GO knowledgebase, as well as the efforts of the broad, international consortium of scientists that develops, maintains, and updates the GO knowledgebase. The GO knowledgebase consists of three components: (1) the GO-a computational knowledge structure describing the functional characteristics of genes; (2) GO annotations-evidence-supported statements asserting that a specific gene product has a particular functional characteristic; and (3) GO Causal Activity Models (GO-CAMs)-mechanistic models of molecular "pathways" (GO biological processes) created by linking multiple GO annotations using defined relations. Each of these components is continually expanded, revised, and updated in response to newly published discoveries and receives extensive QA checks, reviews, and user feedback. For each of these components, we provide a description of the current contents, recent developments to keep the knowledgebase up to date with new discoveries, and guidance on how users can best make use of the data that we provide. We conclude with future directions for the project

Caractérisation des périodes de sécheresse sur le domaine de l'Afrique simulée par le Modèle Régional Canadien du Climat (MRCC5)

Les conséquences des changements climatiques sur la fréquence ainsi que sur l'intensité des précipitations auront un impact direct sur les périodes de sécheresse et par conséquent sur différents secteurs économiques tels que le secteur de l'agriculture. Ainsi, dans cette étude, l'habilité du Modèle Régional Canadien du Climat (MRCC5) à simuler les différentes caractéristiques des périodes de sécheresse est évaluée pour 4 seuils de précipitation soit 0.5 mm, 1 mm, 2 mm et 3 mm. Ces caractéristiques incluent le nombre de jours secs, le nombre de périodes de sécheresse ainsi que le maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans. Les résultats sont présentés pour des moyennes annuelles et saisonnières. L'erreur de performance est évaluée en comparant le MRCC5 piloté par ERA-Interim aux données d'analyses du GPCP pour le climat présent (1997-2008). L'erreur due aux conditions aux frontières c'est-à-dire les erreurs de pilotage du MRCC5, soit par CanESM2 et par ERA-Interim ainsi que l'évaluation de la valeur ajoutée du MRCC5 face au CanESM2 sont également analysées. L'analyse de ces caractéristiques est également faite dans un contexte de climat changeant pour deux périodes futures, soit 2041-2070 et 2071-2100 à l'aide du MRCC5 piloté par le modèle de circulation générale CanESM2 de même que par le modèle CanESM2 sous le scénario RCP 4.5. Les résultats suggèrent que le MRCC5 piloté par ERA-Interim a tendance à surestimer la moyenne annuelle du nombre de jours secs ainsi que le maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans dans la plupart des régions de l'Afrique et une tendance à sous-estimer le nombre de périodes de sécheresse. En général, l'erreur de performance est plus importante que l'erreur due aux conditions aux frontières pour les différentes caractéristiques de périodes de sécheresse. Pour les régions équatoriales, les changements appréhendés par le MRCC5 piloté par CanESM2 pour les différentes caractéristiques de périodes de sécheresse et pour deux périodes futures (2041-2070 et 2071-2100), suggèrent une augmentation significatives du nombre de jours secs ainsi que du maximum de jours consécutifs sans précipitation associé à une récurrence de 5 ans. Une diminution significative du nombre de périodes de sécheresse est aussi prévue.\ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : Modèle Régional du Climat, Changement climatique, Jours secs, Nombre de périodes de sécheresse, Événement de faible récurrence, Afriqu

Impact of Diet on Gut Microbiota Composition and Microbiota-Associated Functions in Heart Failure: A Systematic Review of In Vivo Animal Studies

Heart failure (HF) represents a cardiovascular disease with high mortality and morbidity. The latest evidence shows that changes in the composition of the gut microbiota might play a pivotal role in the prevention and management of HF. This systematic review aims at assessing the potential associations between the diet, gut microbiota, and derived metabolites with the outcomes of HF. A systematic literature search was performed up to July 2022 on the PubMed, Web of Science, and Scopus databases. The PRISMA guidelines were followed when possible. The risk of bias was assessed with the SYRCLE and ARRIVE tools. A total of nine pre-clinical studies on animal models, with considerable heterogeneity in dietary interventions, were included. High-fiber/prebiotic diets (n = 4) and a diet rich in polyphenols (n = 1) modified the gut microbiota composition and increased microbial metabolites’ activities, linked with an improvement in HF outcomes, such as a reduction in systolic blood pressure, cardiac hypertrophy, and left ventricular thickness. A high-fat diet (n = 2) or a diet rich in choline (n = 2) induced an increase in TMAO and indole derivative production associated with a decrease in cardiac function, systemic endotoxemia, and inflammation and an increase in cardiac fibrosis and cardiac remodeling. Although results are retrieved from animal studies, this systematic review shows the key role of the diet—especially a high-fiber and prebiotic diet—on gut microbial metabolites in improving HF outcomes. Further studies on human cohorts are needed to identify personalized therapeutic dietary interventions to improve cardiometabolic health

CNTNAP2 mutations and autosomal dominant epilepsy with auditory features

Autosomal dominant epilepsy with auditory features (ADEAF) is clinically characterized by focal seizures with prominent auditory or aphasic auras and absence of structural brain abnormalities. Mutations in LGI1 and RELN genes account for the disorder in about 50% of ADEAF families. In a recent paper, a heterozygous intragenic deletion in the CNTNAP2 gene has been associated to ADEAF in a single family. We screened 28 ADEAF families for mutations in CNTNAP2 by next generation sequencing and copy number variation analyses and found no likely pathogenic mutations segregating with the disease. CNTNAP2 should be screened in genetically unsolved ADEAF families, but causative mutations are expected to be infrequent in this gene

MobiDB: 10 years of intrinsically disordered proteins

: The MobiDB database (URL: https://mobidb.org/) is a knowledge base of intrinsically disordered proteins. MobiDB aggregates disorder annotations derived from the literature and from experimental evidence along with predictions for all known protein sequences. MobiDB generates new knowledge and captures the functional significance of disordered regions by processing and combining complementary sources of information. Since its first release 10 years ago, the MobiDB database has evolved in order to improve the quality and coverage of protein disorder annotations and its accessibility. MobiDB has now reached its maturity in terms of data standardization and visualization. Here, we present a new release which focuses on the optimization of user experience and database content. The major advances compared to the previous version are the integration of AlphaFoldDB predictions and the re-implementation of the homology transfer pipeline, which expands manually curated annotations by two orders of magnitude. Finally, the entry page has been restyled in order to provide an overview of the available annotations along with two separate views that highlight structural disorder evidence and functions associated with different binding modes