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

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

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

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