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

Physical activity measured by implanted devices predicts atrial arrhythmias and patient outcome: Results of IMPLANTED (Italian Multicentre Observational Registry on Patients With Implantable Devices Remotely Monitored)

Background--To determine whether daily physical activity (PA), as measured by implanted devices (through accelerometer sensor), was related to the risk of developing atrial arrhythmias during long-term follow-up in a population of heart failure (HF) patients with an implantable cardioverter defibrillator (ICD). Methods and Results--The study population was divided into 2 equally sized groups (PA cutoff point: 3.5 h/d) according to their mean daily PA recorded by the device during the 30- to 60-day period post-ICD implantation. Propensity score matching was used to compare 2 equally sized cohorts with similar characteristics between lower and higher activity patients. The primary end point was time free from the first atrial high-rate episode (AHRE) of duration 656 minutes. Secondary end points were: first AHRE 656 hours, first AHRE 6548 hours, and a combined end point of death or HF hospitalization. Data from 770 patients (65\ub115 years; 66% men; left ventricular ejection fraction 35\ub112%) remotely monitored for a median of 25 months were analyzed. A PA =3.5 h/d was associated with a 38% relative reduction in the risk of AHRE 656 minutes (72-month cumulative survival: 75.0% versus 68.1%; log rank P=0.025), and with a reduction in the risk of AHRE 656 hours, AHRE 6548 hours, and the combined end point of death or HF hospitalization (all P < 0.05). Conclusions--In HF patients with ICD, a low level of daily PA was associated with a higher risk of atrial arrhythmias, regardless of the patients' baseline characteristics. In addition, a lower daily PA predicted death or HF hospitalization

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

Novel Biomarkers in Heart Failure: New Insight in Pathophysiology and Clinical Perspective

Heart failure (HF) is a complex clinical syndrome with a huge social burden in terms of cost, morbidity, and mortality. Brain natriuretic peptide (BNP) appears to be the gold standard in supporting the daily clinical management of patients with HF. Novel biomarkers may supplement BNP to improve the understanding of this complex disease process and, possibly, to personalize care for the different phenotypes, in order to ameliorate prognosis. In this review, we will examine some of the most promising novel biomarkers in HF. Inflammation plays a pivotal role in the genesis and progression of HF and, therefore, several candidate molecules have been investigated in recent years for diagnosis, prognosis, and therapy monitoring. Noncoding RNAs are attractive as biomarkers and their potential clinical applications may be feasible in the era of personalized medicine. Given the complex pathophysiology of HF, it is reasonable to expect that the future of biomarkers lies in the application of precision medicine, through wider testing panels and “omics” technologies, to further improve HF care delivery

Adsorption and thermal transformation of lignin model compound (ferulic acid) over HY zeolite surface studied by temperature programmed desorption mass-spectrometry, FTIR and UV–Vis spectroscopy

The adsorption of an aqueous solution of ferulic acid (FA) on protonic zeolites was studied. Three different types of zeolites and adsorption parameters (time, solid/liquid ratio, temperature and pH) were analyzed and optimized. The HY zeolite (SiO2/Al2O3 = 12) showed an adsorption percentage of 90% at 30 °C, 5 min, 10 g L−1 and pH ≤ 3. The adsorption complexes structures on the molecular adsorbates were determined by UV–Vis DRS and FTIR spectroscopies. The FA molecule interacted through phenol and carboxyl group forming monodentate and bidentate surfaces complexes (bridge and chelate). Temperature programmed desorption mass-spectrometry (TPD MS) has been used to study the decarboxylation, demethoxylation, and transalkylation processes during pyrolysis of FA surface complexes with the formation of carbon dioxide, 4-vinylguaiacol, methanol, methane, polyaromatic hydrocarbons, phenols and others.Fil: Pazo Cepeda, Maria Victoria. Universidad de Valladolid; EspañaFil: Nastasiienko, N. S.. National Academy Of Sciences Of Ukraine; UcraniaFil: Kulik, T. V.. National Academy Of Sciences Of Ukraine; UcraniaFil: Palianytsia, B. B.. National Academy Of Sciences Of Ukraine; UcraniaFil: Alonso, E.. Universidad de Valladolid; EspañaFil: Aspromonte, Soledad Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera". Universidad Nacional del Litoral. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera"; Argentin