Mechanisms involved in the remyelinating effect of sildenafil

Remyelination occurs in demyelinated lesions in multiple sclerosis (MS) and pharmacological treatments that enhance this process will critically impact the long term functional outcome in the disease. Sildenafil, a cyclic GMP (cGMP)-specific phosphodiesterase 5 inhibitor (PDE5-I), is an oral vasodilator drug extensively used in humans for treatment of erectile dysfunction and pulmonary arterial hypertension. PDE5 is expressed in central nervous system (CNS) neuronal and glial populations and in endothelial cells and numerous studies in rodent models of neurological disease have evidenced the neuroprotective potential of PDE5-Is. Using myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) as a MS model, we previously showed that daily administration of sildenafil starting at peak disease rapidly ameliorates clinical symptoms while administration at symptoms onset prevents disease progression. These beneficial effects of the drug involved down-regulation of adaptive and innate immune responses, protection of axons and oligodendrocytes (OLs) and promotion of remyelination. In this work we have investigated mechanisms involved in the remyelinating effect of sildenafil. Using demyelinated organotypic cerebellar slice cultures we demonstrate that sildenafil stimulates remyelination by direct effects on CNS cells in a nitric oxide (NO)-cGMP-protein kinase G (PKG)-dependent manner. We also show that sildenafil treatment enhances OL maturation and induces expression of the promyelinating factor ciliary neurotrophic factor (CNTF) in spinal cord of EAE mice and in cerebellar slice cultures. Furthermore, we demonstrate that sildenafil promotes a M2 phenotype in bone marrow derived macrophages (BMDM) and increases myelin phagocytosis in these cells and in M2 microglia/macrophages in the spinal cord of EAE mice. Taken together these data indicate that promotion of OL maturation directly or through induction of growth factor expression, regulation of microglia/macrophage inflammatory phenotype and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue and further support the contention that this well tolerated drug could be useful for ameliorating MS pathology

La "Viagra" podria reduir els símptomes de l'esclerosi múltiple

Investigadors de l'IBB han descobert que la "Viagra" redueix dràsticament els símptomes de l'esclerosi múltiple en un model animal de la malaltia. L'estudi, publicat a Acta Neuropathologica, demostra una recuperació pràcticament total en el 50% dels animals en només vuit dies de tractament. Els investigadors confien que es podran dur a terme aviat assajos clínics en pacients, donat que es tracta d'un fàrmac ben tolerat i ja ha estat utilitzat per al tractament de la disfunció erèctil en alguns malalts d'esclerosi múltiple.Investigadores del IBB han descubierto que la "Viagra" disminuye drásticamente los síntomas de la esclerosis múltiple en un modelo animal de la enfermedad. El estudio, publicado en Acta Neuropathologica, demuestra una recuperación prácticamente total en el 50% de los animales al cabo de ocho días de tratamiento. Los investigadores confían en que puedan realizarse pronto ensayos clínicos en pacientes ya que es un fármaco bien tolerado y ya ha sido utilizado para el tratamiento de la disfunción eréctil en algunos enfermos de esclerosis múltipleIBB researchers have discovered that "Viagra" drastically reduces multiple sclerosis symptoms in animal models with the disease. The research demonstrates that a practically complete recovery occurs in 50% of the animals after eight days of treatment. Researchers are confident that clinical trials soon will be carried out in patient

Smelling in the dark: Phylogenomic insights into the chemosensory system of a subterranean beetle

The chemosensory system has experienced relevant changes in subterranean animals, facilitating the perception of specific chemical signals critical to survival in their particular environment. However, the genomic basis of chemoreception in cave-dwelling fauna has been largely unexplored. We generated de novo transcriptomes for antennae and body samples of the troglobitic beetle Speonomus longicornis (whose characters suggest an extreme adaptation to a deep subterranean environment) in order to investigate the evolutionary origin and diversification of the chemosensory gene repertoire across coleopterans through a phylogenomic approach. Our results suggested a diminished diversity of odourant and gustatory gene repertoires compared to polyphagous beetles that inhabit surface habitats. Moreover, S. longicornis showed a large diversity of odourant-binding proteins, suggesting an important role of these proteins in capturing airborne chemical cues. We identified a gene duplication of the ionotropic coreceptor IR25a, a highly conserved single-copy gene in protostomes involved in thermal and humidity sensing. In addition, no homologous genes to sugar receptors or the ionotropic receptor IR41a were detected. Our findings suggest that the chemosensory gene repertoire of this cave beetle may result from adaptation to the highly specific ecological niche it occupies, and that gene duplication and loss may have played an important role in the evolution of gene families involved in chemoreception. Altogether, our results shed light on the genomic basis of chemoreception in a cave-dwelling invertebrate and pave the road towards understanding the genomic underpinnings of adaptation to the subterranean lifestyle at a deeper level

Estudio sobre la expresión y localización de la guanilil ciclasa sensible a NO (GCno) en células nerviosas

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa guanilil ciclasa sensible a NO (GCNO) cataliza la formación de GMPc y es la principal diana del NO en el SNC. La GCNO es un heterodímero compuesto por una subunidad α y una β. Dos isoformas de cada subunidad han sido clonadas. En esta tesis estudiamos la distribución anatómica de los mRNAs de las subunidades en cerebro de rata y mono. Nuestros resultados revelan que en ambas especies el mRNA de la β1 es más abundante y ubicuo en cerebro. El mRNA de la subunidad β2 no pudo ser detectado en ninguna de las dos especies. La distribución de las subunidades α presentó similitudes en el sistema límbico y en caudado-putamen de ambas especies y diferencias en la distribución laminar en corteza. Mientras que en rata la subunidad α1 es elevada en capas externas corticales, en mono lo es en las capas internas. Además, el mRNA de α1 es más ubicuo en el mono y el mRNA de la α2 en la rata. Una elevada expresión de las tres subunidades se observó en ganglios basales, sistema olfatorio, hipocampo y corteza. También encontramos regiones donde la expresión de la subunidad β1 era alta y pero la expresión de ambas α era baja o indetectable. En la rata, esto ocurre en las islas de Calleja, la mayoría de los núcleos talámicos y el colículo superior, y en mono ocurre en los núcleos talámicos y en el claustrum. El sistema NO-GMPc ha sido implicado en plasticidad sináptica, procesamiento sensorial y comportamiento en la rata, los niveles elevados de las tres subunidades en regiones como los ganglios basales, hipocampo y cerebelo en mono sugieren que en primates también estaría involucrado en estos procesos. Estudios previos demostraban que agentes inflamatorios disminuían la actividad GCNO como consecuencia de la disminución de la subunidad β1 en astrocitos en cultivo y en cerebro de ratas adultas. En este trabajo estudiamos si la actividad GCNO también estaba alterada en cerebro de pacientes con enfermedades neurodegenerativas con un componente inflamatorio como Alzheimer (AD), Creytzfeldt-Jakob (CJ) y esclerosis múltiple (MS). Los resultados demuestran que no existen diferencias significativas entre los individuos control y los enfermos AD con distinto grado de afectación en la actividad basal GCNO o estimulada con donadores de NO, ni en los niveles de expresión de las subunidades β1 y α1. Mediante inmunocitoquímica demostramos que las neuronas y en astrocitos de sustancia blanca presentan un nivel de expresión elevado para la β1. En pacientes AD, los niveles de expresión no estan alterados en neuronas y astrocitos fibrilares mientras que se detectó disminución de β1 en astrocitos reactivos alrededor de las placas amiloides. El marcaje se ve también disminuido en astrocitos reactivos de sustancia blanca de pacientes con CJ y MS. Así, la inducción de la reactividad glial está asociada a una disminución en la capacidad de generar GMPc en respuesta a NO. Estudios previos demostraban que la disminución de la subunidad β1 inducida por LPS era dependiente de transcripción y síntesis de proteínas, pero independiente de NO. En esta tesis estudiamos el mecanismo involucrado en la degradación de esta subunidad, siendo la vía que involucra al proteosoma y la ubiquitina responsable de la disminución. También, demostramos que el tratamiento con LPS produce la formación de agregados de β1 en el núcleo, en estructuras ricas en proteosoma 20S y ubiquitina con características de clastosomas. La formación de estos agregados es inhibida por inhibidores específicos del proteosoma y de la síntesis de proteínas. Durante estos estudios observamos la presencia de β1 en células con actividad GCNO (neuronas y astrocitos), así como en células sin actividad GCNO (microglía). En ambos casos la localización era mayoritariamente citosólica pero también aparecía en el núcleo. La intensidad de marcaje aumentaba en células en división. A mayor aumento observamos que β1 se encontraba asociada periféricamente a los cromosomas durante todas las fases de la mitosis. Debido a la localización pericromosomal, nos propusimos estudiar si la β1 ejercía alguna función sobre la condensación de la cromatina. Mediante un ensayo de condensación in vitro demostramos que la inmunodepleción de la proteína nuclear y citoplasmática provocaba aumento en la condensación. También demostramos que el silenciamiento de β1 por siRNA producía un aumento del número de células que ingresaban en el ciclo celular y un aumento de la proliferación. Todos estos efectos fueron independientes de la actividad enzimática ya que un inhibidor especifico de la GCNO no mimetizaba los efectos del siRNA ni de la inmunodepleción. Estos resultados nos llevan a proponer que la subunidad β1 de la GCNO podría tener una función independiente de su actividad enzimática.Nitric oxide (NO) exerts most of its physiological effects through activation of a predominantly soluble guanylyl cyclase (sGC). In mammalian cells sGC exists as a heterodimer of α and β subunits. Currently, four subunits (α1-2 and β1-2) have been characterized. We used in situ hybridization with subunit-specific 33P-labeled oligonucleotide probes to compare the anatomical distribution of sGC subunit mRNAs in rat and monkey brains. Message for all subunits except β2 was detected in both species. The sGC subunit showing the highest expression and widest distribution was β1. High expression for all subunits was found in basal ganglia, olfactory system, hippocampus, cortex, and cerebellum. Minor species differences in the relative distribution of α subunits were observed. In general, the α1 message was more prominent in monkey brain and the α2 message in rat brain. This was more evident in limbic areas and cerebellar cortex. Some differences were also observed in subunit laminar distribution in cerebral cortex. The limited species differences in sGC subunit distribution suggest that in primates, as occurs in rodents, the NO-cGMP signaling pathway will be involved in important brain functions such as memory formation, sensory processing, and behavior. In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with β-amyloid peptides or IL-1B down-regulates GCNO in cultured astrocytes and in adult rat brain. In this work, we have examined GCNO activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated GCNO activity, nor in GCNO β1 and α1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of GCNO β1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of GCNO in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for GCNO β1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocytes reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response We previously showed that treatment with bacterial cell wall lipopolysaccharide (LPS) or proinflammatory cytokines decreases GCNO activity in astrocytes by decreasing the half-life of the obligate GCNO β1 subunit in a NO-independent but transcription- and translation-dependent process. Here we show that LPS-induced β1 degradation requires proteasome activity and is independent of NFκB activation or inhibition of β1 interaction with HSP90. Immunocytochemistry and confocal microscopy revealed that LPS promotes co-localization of the predominantly soluble β1 protein with ubiquitin and the 20S proteasome in nuclear aggregates that present characteristics of clastosomes, nucleoplasmic substructures involved in ubiquitin-proteasome-dependent nuclear proteolysis. Proteasome and protein synthesis inhibitors prevented LPS-induced clastosome assembly and nuclear colocalization of β1 with ubiquitin and 20S proteasome strongly supporting a role for these transient nuclear structures in GCNO down-regulation during neuroinflammation. Functional GCNO exists as α1/β1 and α2/β1 heterodimers and is predominantly cytosolic, although recent studies indicate that it can associate to membranes and other intracellular structures including nuclei. In the CNS, in situ hybridization studies evidenced that β1 is more widespread than α subunits and that in some areas is almost the only GCNO subunit expressed, suggesting that it may have functions other than GCNO activity. In the course of our studies on the cellular and sub-cellular distribution of GCNO in rat CNS glial cells we found that the β1 subunit is localized in the cytoplasm and the nucleus of cells expressing α subunits and GCNO activity (astrocytes, C6 glioma), as well as in cells devoid of α subunits and GCNO activity (microglia). In both cell types β1 associates peripherally to chromosomes during all phases of mitosis. In C6 cells, immunodepletion of β1 enhances chromatin condensation in an in vitro assay. Moreover, silencing β1 by siRNA increases the percentage of cells that enter the cell cycle and the proliferation rate. These actions are not mimicked by treatment with a specific GCNO inhibitor (ODQ). We postulate that the β1 subunit is a multifunctional protein that, in addition to its role as an obligate monomer in active GCNO, associates to chromosomes during mitosis and regulates chromatin condensation and cell cycle progression

Evolutionary Genomics of Panarthropoda: Study of the chemosensory gene families across phyla and the radiation of the spider genus Dysdera (Araneae, Dysderidae) in the Canary Islands

[eng] During the last decade, advances in high-throughput sequencing (HTS) technologies have increased exponentially the generation of genomic data, especially of non-model species. In this thesis, we generated and used available HTS data to shed light on relevant evolutionary processes that shape genome structure and organization, such as the origin and evolution of multigene families, and the genomic determinants of adaptive radiations. First, we contribute to the genome assembly of the spider Dysdera silvatica, the first published assembly of a member of the Dysderidae family. For that we used a hybrid assembly strategy based on short (Illumina) and long (low coverage PacBio and Nanopore) reads to generate an assembly of moderate to low continuity (N50 of 38 kb). Then, we upgraded the assembly to a high quality, chromosome-level, using Chicago and Hi-C libraries, which raised the continuity of the previous draft considerably (N50 of 174.19 Mb). This newly generated genomic resource is not only a significant contribution to the field of spider genomics, but also the backbone for many of the analyses presented in this thesis. We identified 33,275 putative protein-coding genes in the genome of D. silvatica (87% of them were functionally annotated) and estimated that 52.87% of the genome are repetitive elements. We used this high-quality assembly to perform a comprehensive study of the chromosomal location and evolutionary divergence of chemoreceptor gene families. We identified 545 chemoreceptor genes, which are highly clustered in the genome of D. silvatica and show a clear correlation between physical and evolutionary distances. Second, we used available genomics data, and newly generated transcriptomes, to perform a phylogenetically deep study of the evolution of the chemosensory gene families across Panarthropoda. We characterized the expression of chemoreceptor genes in the antenna of the velvet worm Euperipatoides rowelli and performed a comparative genomic analysis of these gene families across representatives of the phyla composing the Panarthropoda clade. Noticeably, we did not find the expression of the Ionotropic co-receptor IR25a in onychophorans, which questions the chemosensory role of this family in this lineage. Surprisingly, eutardigrades lack genes encoding the DEG-ENaC and CD36/SNMPs gene families, a feature that could be related to their extraordinary resistance to desiccation. Finally, we propose the NPC2 gene family as the candidate chemosensory soluble protein in the Panarthropoda ancestor. We also used the chromosome-level assembly of D. silvatica to study some aspects of the adaptive radiation of the spider genus Dysdera in the Canary Islands. We performed a population genomics study of a natural population of D. silvatica from La Gomera using a whole-genome re-sequencing strategy. We found unexpected high levels of nucleotide polymorphism and a lower X/autosomes polymorphism ratio than the theoretically anticipated from their corresponding effective number of chromosomes. Demographic inference based on the coalescence predicted that in the past this species likely underwent a long period characterized by a large effective population size (about 100 times greater than the estimate for the present). We hypothesize that the high levels of polymorphism currently observed in the population of D. silvatica is the molecular hallmark of a long period of ancestral population structure that dominated the history of this species for almost 200 Kya. Globally, our results provide very valuable new knowledge about the genomic bases of adaptation at different evolutionary timescales, ranging from the deep evolutionary dynamics of chemosensory repertories across Panarthropoda phyla, to the very recent origin of new copies and the early steps of their evolution within a genome, including the genomic signals of positive selection and demographic history at the scale of an adaptive radiations. This thesis also demonstrates the great advantage of having highly continuous genome assemblies to obtain reliable data when working gene families, and other repetitive elements, or to perform comprehensive chromosome-level population genomics analyses.[cat] Durant la darrera dècada, els avenços en les tecnologies de High- Throughput Sequencing (HTS) van augmentar exponencialment la generació de dades massives, proporcionant una gran quantitat d'informació genòmica d'espècies no models. L'objectiu principal d'aquesta tesi és investigar processos evolutius com l'origen i l'evolució de les famílies de gens i els mecanismes genòmics subjacents a l'adaptació en la diversificació d'espècies. Per això, en primer lloc, hem contribuït a la construcció de l’assemblatge genòmic de l’aràcnid Dysdera silvatica, el primer genoma de la família Dysderidae amb lectures curtes, amb una petita fracció de lectures llargues i un valor N50 de 38 kb. A continuació, el vam millorar a un genoma a nivell de cromosoma d'alta qualitat, utilitzant llibreries de Chicago i Hi-C, a més d'augmentar el valor de N50 a 174,19 Mb. Vam identificar 33.275 gens (el 87% amb anotacions funcionals), i determinar que el 52,87% del genoma estava representat per elements repetitius. Vàrem identificar 545 gens quimioreceptors, el 54% d'ells distribuïts en 83 clústers genòmics que es trobaven majoritàriament en els scaffolds més petits. D'altra banda, hem estudiat i caracteritzat les famílies multigèniques quimiosensorials a diverses espècies dels grup dels Panarthropoda, centrant-nos en Onychophora i Tardigrada. Hem caracteritzat les principals famílies quimiosensorials de l'onicòfor Euperipatoides rowelli i hem trobat que no tenen el gen que codifica el receptors ionotròpic IR25a, present en la majoria d’espècies de Protòstoms. A més, les famílies de gens DEG-ENaC i CD36/SNMPs són absents a les espècies de la classe Eutardigrada, mentre que la família de gens NPC2 seria l'única que codifica proteïnes solubles en l'ancestre Panartropoda. Finalment, hem estudiat la radiació adaptativa de l’aranya del gènere Dysdera a les Illes Canàries, mitjançant una anàlisi de genòmica de poblacions. Hem determinat que a pesar de ser una espècie endèmica i que viu a illes molt petites, presenta un alt nivell de polimorfisme nucleotídic. Les inferències demogràfiques suggereixen que aquesta espècie té una gran grandària efectiva poblacional, probablement causada per una estructuració poblacional ancestral. Els resultats d'aquesta tesi són un recurs valuós que ens ha permès caracteritzar detalladament les famílies multigèniques del sistema quimiosensorial, a més proporcionar nou coneixement sobre paper relatiu de la selecció positiva en les radiacions adaptatives

LPS-induced down-regulation of NO-sensitive guanylyl cyclase in astrocytes occurs by proteasomal degradation in nuclear bodies

From 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications.-- This abstract is available from: http://www.biomedcentral.com/1471-2210/7/S1/P3[Background] We have previously shown that inflammatory agents (LPS, IL-1β, β-amyloid peptides) that induce reactivity and NOS-2 expression in glial cells down-regulate astroglial soluble guanylyl cyclase (sGC) in vitro and in vivo.[Results] Here we show that the decrease in sGC activity and β1 subunit protein induced by LPS (10 ng/ml, 24 h) in cultured rat cerebellar astrocytes is prevented by inhibitors of proteasome activity (MG132 5 μM; lactacystin 10 μM) whereas other protease inhibitors (calpain inhibitor 25 μM; ICE inhibitor II 100 μM and leupeptin 5 μM) were not effective. Furthermore, immunocytochemistry and confocal laser microscopy revealed that in LPS-treated cells a strong sGC β1 immunorreactivity is evident in aggregates in the cell nuclei where it co-localizes with 20S proteasomes and ubiquitin in clastosomes, nucleoplasmic substructures involved in ubiquitin-proteasome-dependent nuclear proteolysis, but do not colocalize with others proteasome-enriched structures include promyelocytic leukaemia bodies and splicing speckles. In contrast, in untreated astrocytes clastosomes are scarce and sGC β1 immunorectivity shows a diffuse cytoplasmic pattern, while in the nucleus it is very weak. A similar distribution is observed when cells are treated with LPS and the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide.[Conclusion] LPS orchestrates the recruitment of sGC-β1 protein and components of the ubiquitin-proteasome system to specialized nuclear bodies, clastosomes, suggesting a mechanism for inflammation-induced down-regulation of sGC in astrocytes.This work was supported by a SAF2004-01717 grant (Spain).Peer reviewe

Recerca, universitat i empresa

Amb motiu de la creació del seu patronat-consell empresarial, l’EPSEB va organitzar, el 25 de febrer, a la sala d’actes de l'escola, un acte sobre Recerca, universitat i empresa, en el marc del qual la secretària d’Estat per a l’R+D+i, Carmen Vela, va impartir la conferència titulada El momento de la investigación en España, després de la qual es va obrir un torn de preguntes. Tot seguit es va realitzar una taula rodona sobre el tema Les relacions universitat-empresa empresa: nous escenaris, amb la participació de Dídac Ramírez, rector de la UB; Salvador Alemany, president del Consell Social de la UB; Ferran Sancho, rector de la UAB; Gabriel Masfurroll, president del Consell Social de la UAB; Esther Real, vicerectora de Transferència de Coneixement de la UPC, i Ramon Carbonell, president del Consell Social de la UPC. Presentada per Josep Manuel Basáñez, president de l'òrgan recentment constituït, va presentar i participar en la taula rodona, que va estar moderada per Lluís Recoder, i va comptar amb la participació del director de l’Escola, Francesc Jordana

From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.The ORFEU program was created by the Catalan Enterprise and Knowledge Department with the Department of Health and funded by the Government of Catalonia, who trusted the expertise of research institutes to add value to the health system during the pandemic. We also extend our thanks to the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme / Generalitat de Catalunya, the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d’Empresa i Coneixement, and the co-financing by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program. We acknowledge support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, to the EMBL partnership and to the Co-financing with funds from the European Regional Development Fund corresponding to the Programa Operativo FEDER Plurirregional de España (POPE) 2014-2020. We acknowledge also support of the Centro de Excelencia Severo Ochoa and the Generalitat de Catalunya through the CERCA Programme, through Departament de Salut and Departament d’Empresa i Coneixement and the Co-financing with funds from the European Regional Development Fund by the Secretaria d’Universitats i Recerca corresponding to the Programa Operatiu FEDER de Catalunya 2014-202