Implicación del receptor AhR en neuroprotección y neurorreparación en ictus isquémico experimental

231 p.-81 fig.-17 tab.[EN] AhR (Aryl hydrocarbon receptor) is a transcription factor that belongs to the bHLH (basic helix-loop-helix)/PAS(Per-Arnt-Sim homology domain) family of highly conserved proteins. One of the main known functions of AhR is to mediate the toxic and carcinogenic effects of xenobiotics. Furthermore, several evidences indicate a physiological role of this receptor in normal cell physiology and function. Although AhR is widely expressed in the CNS (Central Nervous System), its physiological and pathological roles are stillunclear. To further define the roles of this receptor in normal and pathologic brain function, we decided to explore the contribution of AhR to cerebral ischemic damage caused by permanent middle cerebral artery occlusion (pMCAO) and oxygen-glucose deprivation (OGD). The results presented in this Thesis show for the first time that AhR is induced after different experimental models of cerebral ischemia with specific temporal and regional profiles. In this context, AhR is mainly located in neurons placed in peri-infarct areas surrounding core region. Pharmacological or genetic loss-offunction approaches using the AhR antagonists 2',4',6-trimethoxyflavone (TMF) and CH-223191 (CH) (Murray et al., 2010a; Zhao et al., 2010) or AhR heterozygous mice (Fernández-Salguero et al., 1995) resulted in neuroprotection in our in vitro and in vivo models of brain ischemia and excitotoxicity.Furthermore, cerebral ischemia induced AhR nuclear translocation and its transcriptional activity. Since AhR is a ligand activated transcription factor, we decided to determine whether L-kynurenine (L-Kyn), a metabolite of tryptophan degradation through the so called “Kynurenine pathway” and recently described as an AhR agonist in different contexts (Adams et al., 2012; DiNatale et al., 2010; Hao et al., 2013; McBerry et al., 2012; Opitz et al., 2011), was responsible for AhR activation after cerebral ischemia. Indeed, cerebral and plasmatic L-kynurenine levels were induced as a consequence of ischemic damage in mouse (Graphical abstract, 1). This result suggests the possibility that, after cerebral ischemia, AhR activation in neurons is mediated by this tryptophan metabolite. Different experimental approaches in our in vitro and in vivo models confirmed L-kynurenine as an endogenous ligand of AhR after brain ischemia. In addition, exogenous L-kynurenine treatment or, inhibition of endogenous L-kynurenine synthesis by administration of TDO (tryptophan-2,3-dioxygenase) inhibitor resulted in AhR-dependent signaling modulation. In our experimental models of cerebral ischemia, AhR activation by L-kynurenine or endogenous L-kynurenine synthesis inhibition resulted in increased brain damage or neuroprotection, respectively. Therefore, our data demonstrate that AhR activation by L-kynurenine participates in deleterious mechanisms that mediate neuronal death in the acute phase of cerebral ischemia. The mechanisms through which AhR activation participates in ischemic damage include transcriptional processes (Graphical abstract, 2) but also a novel mechanism by which AhR is recruited to the neuronal membrane (Graphical abstract, 4).In the first ones, nuclear AhR translocation produces an increase in AhR target genes such as Cyp1a1, AhRR and Cyp1b1 (Graphical abstract, 3) but, in addition, promotes AhR interaction with CBP (CREB binding protein) and CREB (Cyclic adenosine monophosphate response element binding). This AhR/CBP/CREB complex decreases CREB transcriptional activity and therefore the expression of the CREB survival target genes BDNF (Brain derived neurotrophic factor) and NPAS4 (Neuronal PAS 4). In the second mechanism, AhR activation by an agonist (Graphical abstract, 4) induces the recruitment of this receptor to the neuronal plasma membrane. There, AhR interacts with the MAGUK (Membrane associated guanilate kinase) protein Sap102 (synapse associated protein 102) and, as a consequence, total and membrane Sap102 protein levels are decreased. Although we do not know exactly the mechanism responsible of this AhR dependent regulation of Sap102, it is possible that, in this context, AhR acts like a ligand activated E3 ubiquitin ligase, a AhR function that has been previously demonstrated (Barouche et al., 2007; Bock y Kohl, 2006; Harper et al., 2006; Oesch-Bartlomowicz et al., 2005; Ohtake et al., 2007), promoting Sap102 degradation by 26S proteosome system (Graphical abstract, 5). Decreased Sap102 levels allow the movement of NR2B receptors from synaptic to extrasynaptic sites (Graphical abstract, 6) or modulate Sap102/NR2B-dependent endocytosis and exocytosis processes (Rudinskiy et al., 2009; Sans et al., 2005; 2000). The final effect is an increase in NMDA receptors composed by NR1/NR2B subunits in extrasynaptic sites. This effect is dependent of AhR activation. Furthermore, overactivation of these extrasynaptic NMDA receptors mediates an increase in intracellular [Ca2+] (Graphical abstract, 7) that cause a decrease in CREB phosphorylation (Graphical abstract, 8). Therefore, the inhibition of NMDA-dependent survival pathways together with the potentiation of NMDA-induced deleterious mechanisms would lead to neuronal death after AhR activation in cerebral ischemia.[ES] El receptor de hidrocarburos de arilo (AhR) es un factor de transcripción que pertenece a la familia de proteínas altamente conservadas bHLH (dominio hélice-bucle-hélice)/PAS(Per-ARNT-Sim). Una de las principales funciones de AhR es mediar los efectos tóxicos y carcinogénicos de los xenobióticos. Sin embargo, cada vez son más las evidencias que indican un importante papel del receptor en funciones fisiológicas. Aunque AhR se expresa ampliamente en el SNC (Sistema Nervioso Central) sus funciones a nivel fisiológico y patológico en esta estructura son aún desconocidas. Con el fin de profundizar en la función fisiológica y patológica del receptor AhR en el cerebro, decidimos explorar la contribución de AhR al daño isquémico ocasionado por la oclusión permanente de la arteria cerebral media (pMCAO) y la privación de oxígeno y glucosa (OGD). Los resultados presentados en esta Tesis doctoral muestran por primera vez cómo tras modelos experimentales de isquemia cerebral, la expresión del receptor AhR se incrementa siguiendo un patrón temporal y espacial concreto. Este incremento en la expresión del receptor presenta una localización principalmente neuronal. La inhibición del receptor AhR mediante los antagonistas específicos 2',4',6-trimetoxiflavona (TMF) y CH-223191 (CH) (Murray et al., 2010a; Zhao et al., 2010) o a través del uso de ratones heterocigotos para AhR (Fernández-Salguero et al., 1995) resultó neuroprotectora en nuestros modelos de isquemia cerebral y excitotoxicidad in vitro e in vivo.AhR es un receptor activado por ligando, y ya que tras la isquemia el receptor incrementa no solo su traslocación al compartimento nuclear sino también, su actividad transcripcional, decidimos evaluar si la L-kinurenina, un metabolito derivado de la degradación del triptófano a través de “la ruta de la kinureninas” recientemente descrito como agonista de AhR en diferentes contextos (Adams et al., 2012; DiNatale et al., 2010; Hao et al., 2013; McBerry et al., 2012; Opitz et al., 2011), era el responsable de la activación del receptor AhR tras la isquemia cerebral. En efecto, los niveles cerebrales de L-kinurenina en el cerebro y a nivel plasmático se inducían como consecuencia de la isquemia (Sumario gráfico, 1), lo que sugería la posibilidad de que la activación de AhR tras la isquemia cerebral estuviese mediada por este metabolito. Los experimentos realizados in vitro e in vivo confirmaron a la L-kinurenina como ligando endógeno de AhR tras la isquemia. De hecho, la modulación de la ruta de las kinureninas mediante la adición de L-kinurenina exógena o mediante la inhibición de la síntesis endógena a través del bloqueo selectivo de la enzima TDO (Triptófano- 2,3-dioxigenasa), modularon la señalización mediada por el receptor AhR. En nuestros modelos de isquemia experimental, la activación de AhR por Lkinurenina, y la inhibición de AhR mediante antagonistas específicos o por la inhibición de la síntesis endógena de L-kinurenina, se tradujeron en un incremento del daño producido por la isquemia y en neuroprotección, respectivamente. Por tanto la activación de AhR a través de L-kinurenina en la fase aguda del ictus isquémico participa en los mecanismos asociados al daño que median la muerte neuronal.Dentro de los mecanismos a través de los cuales la activación del receptor participa en el daño isquémico se incluyen, por un lado, mecanismos a nivel transcripcional (Sumario gráfico, 2) y, por el otro, un novedoso mecanismo que implica el reclutamiento de AhR a la membrana neuronal (Sumario gráfico, 4). En el primero de ellos, la traslocación de AhR al compartimento nuclear, además de incrementar la transcripción de sus genes diana típicos Cyp1a1, AhRR y Cyp1b1 (Sumario gráfico, 3), interaccionaría con CBP y CREB. La formación de los complejos AhR/CBP/CREB disminuiría la actividad transcripcional de CREB (Sumario gráfico, 3) y, por tanto, la expresión de sus genes diana, entre los que se incluyen BDNF y NPAS4. En el segundo mecanismo, gracias al reclutamiento de AhR a la membrana al ser activado por ligando (Sumario gráfico, 4) y a la interacción del receptor con la proteína MAGUK Sap102, AhR reduciría los niveles totales y de membrana de la proteína Sap102. Aunque no hemos esclarecido el mecanismo a través del cual se produce esta regulación, es probable que, en este caso, AhR actúe a modo de E3 ubiquitina ligasa activada por ligando (Barouki et al., 2007; Bock y Köhle, 2006; Harper et al., 2006; Oesch-Bartlomowicz et al., 2005; Ohtake et al., 2007), promoviendo la degradación de Sap102 a través del proteasoma 26S (Sumario gráfico, 5). Esta disminución de los niveles de Sap102 permitiría el movimiento de los receptores NMDA de tipo NR2B desde los sitios sinápticos hasta los extrasinápticos (Sumario gráfico, 6), o bien modularía los procesos de endocitosis/exocitosis dependientes de la interacción de Sap102 y NR2B (Rudinskiy et al., 2009; Sans et al., 2005; 2000). El efecto final sería un incremento de los niveles del receptor NMDA de tipo NR1/NR2B en los sitios extrasinápticos tras la activación de AhR. La sobreactivación de estos receptores extrasinápticos mediaría un incremento en la [Ca2+] intracelular (Sumario gráfico, 7), disminuyendo la fosforilación del factor de transcripción CREB (Sumario gráfico, 8), y dando por tanto lugar a la inhibición de las rutas de supervivencia y la potenciación de las rutas de señalización que conducirían, de manera inevitable, a la muerte de la neurona.Peer reviewe

Neutrophil Extracellular Trap Targeting Protects Against Ischemic Damage After Fibrin-Rich Thrombotic Stroke Despite Non-Reperfusion.

Stroke is one of the most prevalent diseases worldwide caused primarily by a thrombotic vascular occlusion that leads to cell death. To date, t-PA (tissue-type plasminogen activator) is the only thrombolytic therapy approved which targets fibrin as the main component of ischemic stroke thrombi. However, due to its highly restrictive criteria, t-PA is only administrated to less than 10% of all stroke patients. Furthermore, the research in neuroprotective agents has been extensive with no translational results from medical research to clinical practice up to now. Since we first described the key role of NETs (Neutrophil Extracellular Traps) in platelet-rich thrombosis, we asked, first, whether NETs participate in fibrin-rich thrombosis and, second, if NETs modulation could prevent neurological damage after stroke. To this goal, we have used the thromboembolic in situ stroke model which produces fibrin-rich thrombotic occlusion, and the permanent occlusion of the middle cerebral artery by ligature. Our results demonstrate that NETs do not have a predominant role in fibrin-rich thrombosis and, therefore, DNase-I lacks lytic effects on fibrin-rich thrombosis. Importantly, we have also found that NETs exert a deleterious effect in the acute phase of stroke in a platelet-TLR4 dependent manner and, subsequently, that its pharmacological modulation has a neuroprotective effect. Therefore, our data strongly support that the pharmacological modulation of NETs in the acute phase of stroke, could be a promising strategy to repair the brain damage in ischemic disease, independently of the type of thrombosis involved.This work was supported by grants from Instituto de Salud Carlos III and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RETICS RD16/0019/ 0009 (IL), from Regional Madrid Government B2017/BMD- 3688 (IL), from Spanish Ministry of Science and Innovation PID2019- 106581RB-I00 (MÁM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MÁM), from Fundación La Caixa HR17_00527 (MM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Conceptual analysis of digital competence models of university teacher

La competencia digital o competencia TIC es un concepto que en el último lustro ha marcado una línea de investigación de gran relevancia en el ámbito de la Tecnología Educativa, tanto referido al profesorado como a otros agentes educativos o sociales. La perspectiva desde la cual abordamos el estudio de la competencia digital del profesorado parte de la idea de que esta competencia forma parte de la competencia profesional de los docentes de cualquier nivel de enseñanza. En este artículo realizamos una investigación basada en una revisión documental articulada en dos fases: 1) análisis conceptual de las aportaciones más relevantes del último lustro en torno a la competencia digital; y 2) a partir de este análisis, realizamos en una segunda fase un estudio descriptivo y comparativo de los diversos modelos de competencia digital del profesorado universitario. Este análisis pone de manifiesto que todos los modelos analizados muestran dimensiones y elementos comunes, pero también algunas particularidades que resultan de interés a la hora de abordar futuras investigaciones sobre el tema. Además nos servirá como punto de partida para una investigación en torno a la certificación de competencias TIC del profesorado universitario que actualmente estamos desarrollando desde el Grupo de Investigación de Tecnología Educativa de la Universidad de Murcia.Digital competence or ICT competence is a concept that has defined a research line of great relevance in the last five years in the field of Educational Technology, related to both teachers and any other social or educational agent. Our approach is based on the idea that digital competence is part of teachers' professional competence in every educational system level. This article presents a documental research in two phases: 1) first we focus on the conceptual analysis of the most relevant studies around digital competence and 2) based on this previous analysis, a descriptive and comparative analysis of different digital competence models of university teacher is performed in a second phase. This analysis shows that all of these models have dimensions and common elements, but also some special features that are of interest in dealing with research on the subject. In addition, this study will serve as a starting point for our research about the certification of ICT skills of university teacher. This Research is being carried out by the Research Group of Educational Technology of the University of Murcia.peerReviewe

The role of gut microbiota in cerebrovascular disease and related dementia.

In recent years, increasing evidence suggests that commensal microbiota may play an important role not only in health but also in disease including cerebrovascular disease. Gut microbes impact physiology, at least in part, by metabolizing dietary factors and host-derived substrates and then generating active compounds including toxins. The purpose of this current review is to highlight the complex interplay between microbiota, their metabolites. and essential functions for human health, ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases, and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context.Instituto de Salud Carlos III (ISCIII), Grant/Award Number: PI20/00535; Leducq Foundation for Cardiovascular Research Grant/Award Numbers: TNE-21CVD04 (MAM, IL), TNE19CVD01 (MAM, MIC), TNE21CVD04, TNE19CVD01; Ministerio de Ciencia e Innovacion (MCIN), Grant/Award Numbers: PID2020-117765RB-I00, PID2019-106581RB-I00; RICORS-ICTUS, Grant/Award Number: RD21/0006/0001.N

Cuando el trabajo se convierte en tortura. La mujer y el acoso en la empresa

El ámbito laboral ocupa un espacio, tanto en tiempo como en dedicación, muy importante en nuestras vidas. En la empresa transcurre gran parte de nuestra existencia, de ahí la importancia de cualquier contratiempo provocado en el lugar de trabajo con el objetivo de hacernos imposible la actividad. Cuando eso ocurre, probablemente estamos frente a un caso de acoso laboral o mobbing, que puede llevarnos a situaciones desesperadas que hagan tambalear todo nuestro entorno. Suelen ser muchas las situaciones que confluyen y las personas que juegan un papel definitivo en estas intrigas laborales, en donde el objetivo final es que el acosado abandone su puesto de trabajo. Este marco situacional ha marcado las directrices en el desarrollo de nuestro estudio, centrándonos específicamente en la situación actual de la mujer en el puesto de trabajo y el acoso laboral en la micro-pymes ubicadas en el municipio de Zaragoza. El análisis se ha encuadrado en aquellos aspectos que desde el punto de vista psicológico y sociológico, sin entrar a valorar las repercusiones en el ámbito judicial, están repercutiendo en la mujer y en su desarrollo labora

Diurnal Differences in Immune Response in Brain, Blood and Spleen After Focal Cerebral Ischemia in Mice.

BACKGROUND The immune response to acute cerebral ischemia is a major factor in stroke pathobiology. Circadian biology modulates some aspects of immune response. The goal of this study is to compare key parameters of immune response during the active/awake phase versus inactive/sleep phase in a mouse model of transient focal cerebral ischemia. METHODS Mice were housed in normal or reversed light cycle rooms for 3 weeks, and then they were blindly subjected to transient focal cerebral ischemia. Flow cytometry was used to examine immune responses in blood, spleen, and brain at 3 days after ischemic onset. RESULTS In blood, there were higher levels of circulating T cells in mice subjected to focal ischemia during zeitgeber time (ZT)1-3 (inactive or sleep phase) versus ZT13-15 mice (active or awake phase). In the spleen, organ weight and immune cell numbers were lower in ZT1-3 versus ZT13-15 mice. Consistent with these results, there was an increased infiltration of activated T cells into brain at ZT1-3 compared with ZT13-15. CONCLUSIONS This proof-of-concept study indicates that there are significant diurnal effects on the immune response after focal cerebral ischemia in mice. Hence, therapeutic strategies focused on immune targets should be reassessed to account for the effects of diurnal rhythms and circadian biology in nocturnal rodent models of stroke.Supported in part by the Rappaport Foundation and Leducq Foundation. The authors thank all team members of the MGH animal facility for help with light schedule switching.S

Defective hippocampal neurogenesis underlies cognitive impairment by carotid stenosis-induced cerebral hypoperfusion in mice.

Chronic cerebral hypoperfusion due to carotid artery stenosis is a major cause of vascular cognitive impairment and dementia (VCID). Bilateral carotid artery stenosis (BCAS) in rodents is a well-established model of VCID where most studies have focused on white matter pathology and subsequent cognitive deficit. Therefore, our aim was to study the implication of adult hippocampal neurogenesis in hypoperfusion-induced VCID in mice, and its relationship with cognitive hippocampal deficits. Mice were subjected to BCAS; 1 and 3 months later, hippocampal memory and neurogenesis/cell death were assessed, respectively, by the novel object location (NOL) and spontaneous alternation performance (SAP) tests and by immunohistology. Hypoperfusion was assessed by arterial spin labeling-magnetic resonance imaging (ASL-MRI). Hypoperfused mice displayed spatial memory deficits with decreased NOL recognition index. Along with the cognitive deficit, a reduced number of newborn neurons and their aberrant morphology indicated a remarkable impairment of the hippocampal neurogenesis. Both increased cell death in the subgranular zone (SGZ) and reduced neuroblast proliferation rate may account for newborn neurons number reduction. Our data demonstrate quantitative and qualitative impairment of adult hippocampal neurogenesis disturbances associated with cerebral hypoperfusion-cognitive deficits in mice. These findings pave the way for novel diagnostic and therapeutic targets for VCID.This work was supported by grants from Spanish Ministry of Science and Innovation (MCIN) PID2019-106581RB-I00 (MM), from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MM) and TNE-21CVD04 (MM and IL), and from Instituto de Salud Carlos III (ISCIII) and co-financed by the European Development Regional Fund “A Way to Achieve Europe” PI20/00535 and RICORS-ICTUS RD21/0006/0001 (IL). CNIC was supported by ISCIII, MCIN, and ProCNIC Foundation, and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). The microscopy experiments were performed in Unidad de Microscopía e Imagen Dinámica, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” (#ICTS-2018- 04-CNIC-16). Part of the research work included in this publication has been carried out in the ReDIB ICTS infrastructure BioImaC, MCIN.S

Myeloid cells in vascular dementia and Alzheimer's disease: Possible therapeutic targets?

Growing evidence supports the suggestion that the peripheral immune system plays a role in different pathologies associated with cognitive impairment, such as vascular dementia (VD) or Alzheimer's disease (AD). The aim of this review is to summarize, within the peripheral immune system, the implications of different types of myeloid cells in AD and VD, with a special focus on post-stroke cognitive impairment and dementia (PSCID). We will review the contributions of the myeloid lineage, from peripheral cells (neutrophils, platelets, monocytes and monocyte-derived macrophages) to central nervous system (CNS)-associated cells (perivascular macrophages and microglia). Finally, we will evaluate different potential strategies for pharmacological modulation of pathological processes mediated by myeloid cell subsets, with an emphasis on neutrophils, their interaction with platelets and the process of immunothrombosis that triggers neutrophil-dependent capillary stall and hypoperfusion, as possible effector mechanisms that may pave the way to novel therapeutic avenues to stop dementia, the epidemic of our time.Spanish Ministry of Science and Innovation (MCIN), Grant/Award Number: PID2019-106581RB-I00; Leducq Foundation for Cardiovascular Research, Grant/Award Numbers: TNE19CVD01, TNE-21CVD04; Instituto de Salud Carlos III (ISCIII) and cofinanced by the European Development Regional Fund “A Way to Achieve Europe”, Grant/Award Numbers: PI20/00535, RICORSICTUSRD21/0006/0001; Severo Ochoa Center of Excellence, Grant/Award Number: CEX2020-001041-S.S

Caracterización de la neurogénesis maladaptativa y el deterioro cognitivo post-ictus en un modelo experimental de isquemia cerebral inducida mediante cloruro férrico en ratón

El ictus isquémico es considerado una de las principales causas de demencia y discapacidad en el mundo. La neurogénesis hipocampal que ocurre tras ictus en diversos modelos experimentales de la enfermedad se ha asociado previamente al deterioro cognitivo a largo plazo. Sin embargo, dada la alta heterogeneidad de la patología es necesario estudiar la respuesta neurogénica en nuevos modelos experimentales de ictus isquémico. El objetivo de este estudio fue caracterizar la respuesta neurogénica hipocampal adulta y su relación con el deterioro cognitivo post-ictus en un modelo experimental de isquemia cerebral permanente por cloruro férrico en ratón. Se llevó a cabo la evaluación de la lesión en fase aguda y crónica tras la isquemia a través de imagen por resonancia magnética. Adicionalmente, se evaluó la respuesta neurogénica hipocampal tras isquemia mediante inmunofluorescencia, así como el deterioro cognitivo mediante pruebas de comportamiento. Nuestros resultados muestran que el modelo de isquemia por cloruro férrico produce atrofia e hipoperfusión cortical y no afecta directamente al hipocampo. Además, el modelo de isquemia estudiado resulta en un aumento de la neurogénesis hipocampal tras la isquemia. Concretamente, demostramos un incremento de neuroblastos y neuronas inmaduras en la zona subgranular del giro dentado 7 días tras la isquemia. Asimismo, nuestros resultados muestran que este modelo resulta en un aumento del número de nuevas neuronas con morfología aberrante en ambos hemisferios del cerebro. Además, observamos que parte de los animales desarrollan déficit cognitivo a largo plazo. Los resultados preliminares presentados en este estudio sugieren que el ictus isquémico inducido por cloruro férrico es un modelo experimental adecuado para el estudio de la neurogénesis hipocampal adulta y el deterior cognitivo post-ictus