Sex-related differences of cAMP-specific PDE4B3 mRNA in oligodendrocytes following systemic inflammation

Sex-related differences have been observed in the incidence and severity of several neurological diseases and in sepsis in humans. Cyclic adenosine monophosphate (cAMP) has been shown to play an important role in modulating the inflammatory environment during neuroinflammation and importantly in protecting myelin from excitotoxic cell death. Considering the sexual dimorphism in the functional properties of oligodendrocytes and the importance of a systemic inflammation in the progression of multiple sclerosis, we focused on identifying possible sex-related differences in the alterations previously reported for the two phosphodiesterase4B (PDE4B) splice-variants (PDE4B2 and PDE4B3) mRNA expression during innate neuroinflammation. PDE4A, PDE4B, and PDE4D are present in oligodendrocytes and we have previously reported that PDE4B3 mRNA is readily expressed in both oligodendrocytes and neurons. In this study, we analyzed the influence of an intraperitoneal lipopolysaccharide injection on the distribution pattern and expression levels of the PDE4B mRNA splicing variants in both male and female mice brains. Clear differences were observed in PDE4B2 and PDE4B3 mRNA expression levels in males compared with females in a time-dependent manner. Furthermore, we observed that the clear downregulation of PDE4B3 mRNA was reflected in a lower percentage of oligodendrocytes positive for this transcript which correlated with a decrease in inducible cAMP early repressor expression in female corpus callosum. © 2012 Wiley Periodicals, Inc.Grant sponsor: Spanish Ministerio de Educación y Ciencia; Grant number: SAF2006-10243; Grant sponsor: Ministerio de Ciencia e Innovación; Grant numbers: SAF 2009-11052, PI-10/01874; Grant sponsor: Generalitat de Catalunya; Grant number: SGR2009/220; Grant sponsor: FEDER Funds (European Union).Peer Reviewe

AMPc i neuroinflamació: Identificació de proteïnes implicades en la regulació dels nivells d’AMPc en l’encefalomielitis autoimmune experimental

L'AMPc té un paper clau com a missatger intracel.lular regulant la transmissió dels senyals extracel•lulars en diferents teixits i controlant múltiples processos cel lulars. Els nivells intracel.lulars d'AMPc es controlen mitjançant la seva síntesi, catalitzada per l'enzim adenilat ciclasa, i mitjançant la seva degradació a través de l'acció de les fosfodiesterases (PDE) de nucleòtids cíclics. Hi ha 11 famílies de PDEs. La PDE4 representa a una família de fosfodiesterases específiques d'AMPc formada per quatre gens paràlegs (PDE4-D), cadascun dels quals és capaç de generar múltiples variants d’splicing. La PDE4A, la PDE4B i la PDE4D es troben expressades en diferents tipus de cèl lules inflamatòries on tenen un important paper com reguladores dels processos inflamatoris. La inhibició selectiva, tant in vitro com in vivo, de PDEs ha demostrat tenir diferents efectes antiinflamatoris. En aquest treball es mostra la implicació de la isoforma PDE4B, i en concret la seva variant de splicing PDE4B2, durant el procés neuroinflamatori del model animal d’Esclerosis Múltiple, l’Encefalomielitis Autoimmune Experimental (d’EAE). Els resultats mostren un augment de l’expressió de l’ARNm de PDE4B2 a la medul•la espinal de ratolins EAE que correlaciona amb l’expressió d’alguns marcadors inflamatoris de forma dependent a la simptomatologia clínica dels animals. També s’observa que l’enzim PDE4B es trova localitzat principalment en cèl•lules presentadores d’antigen (APCs) com les cèl•lules dendrítques i els macròfags/micròglia. A més, els ratolins PDE4B-/- mostren una aparició temprana dels símptomes clínics en comparació amb els ratolins wildtype, amb alteracions en l’expressió de l’ARNm d’algunes citocines. L’alteració selectiva de la PDE4B2 en el model d’EAE en ratolí i la seva participació en el desenvolupament de la malaltia com s’ha observat en els animals PDE4B-/- presenta noves possibilitats sobre l’ús d’inhibidors selectius per les diverses isoformes (i variants d’splicing) tant per aplicacions terapèutiques com per investigar mecanismes d’inflamació en malalties neurodegeneratives.Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that courses with neuroinflammation, axonal damage and demyelination. The model is characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein which produce a wide range of pro- and anti-inflammatory cytokines. The modulation of cAMP levels through pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses. In the EAE model, amelioration of the clinical signs and delayed onset is observed after PDE4 inhibition and the PDE4B gene has been related to the inflammatory immune response in mice. Here we analyzed post-immunization changes in the expression of mRNA coding for the PDE4B2 splice variant by semiquantitative real-time PCR and in situ hybridization. The results showed an upregulation of PDE4B2 mRNA in the spinal cord of EAE mice which correlates with FoxP3 and TGF-β mRNAs expression in a score-dependent manner. We also found that PDE4B enzyme is mainly localized in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages. PDE4B-/- mice show an earlier onset of the disease compared to wildtype mice, with alterations in some cytokine mRNA expression. The results point to a protective role of the PDE4B enzyme and PDE4B2 splice variant in particular, during EAE pathogenesis by modulating cAMP levels in APCs and controlling the cytokine environment for T-cell differentiation

AMPc i neuroinflamació: Identificació de proteïnes implicades en la regulació de nivells d'AMPc en l'encefalomielitis autoimmune experimental

Memòria presentada per optar al grau de Doctor en Biologia per la Universitat de Barcelona, dins del Programa de Docotorat en Biomedicina (Departament de Neurociències, Facultat de Medicina, Universitat de Barcelona).-- Tesis elaborada en el Departament de Neuroquímica i Neurofarmacologia, Institut d'Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomèdiques August Pi i Sunyer.L'AMPc té un paper clau com a missatger intracel.lular regulant la transmissió dels senyals extracellulars en diferents teixits i controlant múltiples processos cel lulars. Els nivells intracel.lulars d'AMPc es controlen mitjançant la seva síntesi, catalitzada per l'enzim adenilat ciclasa, i mitjançant la seva degradació a través de l'acció de les fosfodiesterases (PDE) de nucleòtids cíclics. Hi ha 11 famílies de PDEs. La PDE4 representa a una família de fosfodiesterases específiques d'AMPc formada per quatre gens paràlegs (PDE4-D), cadascun dels quals és capaç de generar múltiples variants d’splicing. La PDE4A, la PDE4B i la PDE4D es troben expressades en diferents tipus de cèl lules inflamatòries on tenen un important paper com reguladores dels processos inflamatoris. La inhibició selectiva, tant in vitro com in vivo, de PDEs ha demostrat tenir diferents efectes antiinflamatoris. En aquest treball es mostra la implicació de la isoforma PDE4B, i en concret la seva variant de splicing PDE4B2, durant el procés neuroinflamatori del model animal d’Esclerosis Múltiple, l’Encefalomielitis Autoimmune Experimental (d’EAE). Els resultats mostren un augment de l’expressió de l’ARNm de PDE4B2 a la medulla espinal de ratolins EAE que correlaciona amb l’expressió d’alguns marcadors inflamatoris de forma dependent a la simptomatologia clínica dels animals. També s’observa que l’enzim PDE4B es trova localitzat principalment en cèllules presentadores d’antigen (APCs) com les cèllules dendrítques i els macròfags/micròglia. A més, els ratolins PDE4B-/- mostren una aparició temprana dels símptomes clínics en comparació amb els ratolins wildtype, amb alteracions en l’expressió de l’ARNm d’algunes citocines. L’alteració selectiva de la PDE4B2 en el model d’EAE en ratolí i la seva participació en el desenvolupament de la malaltia com s’ha observat en els animals PDE4B-/- presenta noves possibilitats sobre l’ús d’inhibidors selectius per les diverses isoformes (i variants d’splicing) tant per aplicacions terapèutiques com per investigar mecanismes d’inflamació en malalties neurodegeneratives.Peer Reviewe

AMPc i neuroinflamació: Identificació de proteïnes implicades en la regulació dels nivells d'AMPc en l'encefalomielitis autoimmune experimental

Peer Reviewe

Neuroanatomical distribution and neurochemical characterization of cells expressing adenylyl cyclase isoforms in mouse and rat brain

El pdf del artículo es el manuscrito de autor.Transmembrane adenylyl cyclases (Adcy) are involved in the regulation of multiple brain processes such as synaptic plasticity, learning and memory. They synthesize intracellular cyclic adenosine monophosphate (cAMP) following activation by G-protein coupled receptors. We examined the neuroanatomical distribution of the nine Adcy isoforms in rat and mouse brain by in situ hybridization, as well as their location in glutamatergic, GABAergic and cholinergic neurons in several mouse brain areas by double in situ hybridization. The Adcys are widely distributed throughout the brain in both rat and mouse, being especially abundant in cortex, hippocampus, thalamic nuclei, the olfactory system and the granular layer of the cerebellum. Double-labeling experiments showed that Adcy isoforms are differently expressed in glutamatergic, GABAergic and cholinergic neuronal cell populations. We report the neuroanatomical distribution of the nine known Adcy isoforms in rat and mouse brain and their cellular localization. © 2010 Elsevier B.V.This work was supported by grants awarded by the Spanish Ministerio de Educación y Ciencia and FEDER Funds (SAF2006-10243, SAF 2009-11052).Peer Reviewe

Critical role for PDE4 subfamilies in the development of experimental autoimmune encephalomyelitis

El pdf del artículo es la versión post-print.Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis exhibiting neuroinflammation, axonal damage and demyelination, further characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein. Pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses through modulation of cAMP levels. The PDE4B subfamily has been related to the inflammatory immune response in mice and PDE4 inhibition produces amelioration of the clinical signs and delayed onset in the EAE model. Analyses of the expression of the mRNA coding for PDE4B splice variants revealed an upregulation of PDE4B2 in the brainstem and spinal cord of EAE mice which correlated with forkhead box P3 (FoxP3) and transforming growth factor beta (TGF-β) mRNAs expression in a score-dependent manner. The increase observed for the PDE4B protein was mainly found in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages, in areas with high cellular infiltration. Unexpectedly, PDE4B-/- mice showed an earlier onset of the disease compared to wildtype mice. The results point to a possible role of the PDE4B enzyme and in particular the PDE4B2 splice variant during EAE pathogenesis, probably by modulating cAMP levels in APCs, consequently influencing the cytokine environment important for T-cell differentiation. © 2013 Elsevier B.V.This work was supported by grants awarded by the Ministerio de Ciencia e Innovación (SAF2006-10243; SAF2009-11052; PI-10/01874), FEDER Funds and by the Generalitat de Catalunya (SGR2009/220).Peer Reviewe

Lipopolysaccharide administration in vivo induces differential expression of cAMP-specific phosphodiesterase 4B mRNA splice variants in the mouse brain

El pdf del artículo es la versión pre-print.Many inflammatory processes involve cAMP. Pharmacological manipulation of cAMP levels using specific phosphodiesterase (PDE) inhibitors provokes an antiinflammatory response. The aim of this study was to investigate changes in the pattern and levels of expression of mRNAs coding for the cAMP-specific PDE4 family and subfamilies in mouse brain during the immediate acute immune response provoked by an intraperitoneal injection of lipopolysaccharide (LPS). PDE4B, and furthermore the splice variants PDE4B2 and PDE4B3, were the only mRNAs that showed altered expression. Whereas PDE4B2 presented increased expression at both 3 and 8 hr postinjection, PDE4B3 mRNA showed decreased expression that reached a minimum 8 hr postinjection. PDE4B2 mRNA upregulation was observed mainly in endothelial and macrophage/neutrophil cell populations in the leptomeninges, and the downregulation of PDE4B3 was observed mainly in oligodendrocytes throughout the brain. Our results clearly illustrate the distinctive anatomical distribution and cellular localization of the PDE4Bs during neuroinflammation and emphasize the importance of PDE4B splice-variant-specific inhibitors as therapeutic tools. © 2011 Wiley-Liss, Inc.This work was supported by grants awarded by the Spanish Ministerio de Educación y Ciencia and FEDER Funds (SAF2006-10243; SAF2009-11052). Emily Johansson was a recipient of a fellowship from the Ministerio de Educación y Ciencia and Cristina Sanabra was a recipient of a fellowship from the IDIBAPS.Peer Reviewe

Phosphodiesterase PDE4B2 expression in an EAE mice model

Póster presentado en el: !0th ISNI Congress (International Congress of Neuroimmunology) & 10th ESNI Course (European School of NeuroImmunology), celebrado del 26 al 30 de octubre de 2010 en Sitges, Barcelona (España)Abstract publicado en: Journal of Neurommunology 228(1-2): 49 (2010). DOI 10.1016/j.jneuroim.2010.08.001Inflammatory responses involve cAMP and pharmacological manipulation of cAMP levels by using specific phosphodiesterase (PDE) inhibitors provokes an anti-inflammatory response. Experimental autoimmune encephalomyelitis (EAE) is an animal model of the chronic inflammatory, neurodegenerative demyelinating disease multiple sclerosis (MS). Previously we demonstrated that in the brain and spinal cord of EAE rats there was a dramatic increase in the mRNA expression levels of the PDE4B isoenzyme, solely due to the splicing mRNA variant PDE4B2. This expression was found in the infiltrating T-cells and macrophage/microglia in microvessels and brain parenchyma. We present our results on the alterations in the expression of the cAMP-specific PDE4 and of several inflammatory cytokine mRNAs in the brain and spinal cord of C57BL6 EAE mice model by neuroanatomical techniques (double in situ hybridization histochemistry and immunohistochemistry), and quantitative real time RT-PCR. We observed a PDE4B2 mRNA upregulation after the onset of the EAE model, being significant 30 days post-immunization. No changes on PDE4B3 mRNA splice variant could be detected. Double in situ hybridization and immunohistochemistry studies showed that cellular infiltrates in brain and spinal cord tissues of EAE mice are overexpressing PDE4B isoform. We found that PDE4B2 mRNA splice variant was overexpressed in T cells and macrophages/microglia in the inflammatory foci. These findings support the important and distinctive role of different PDE4 isoforms during the neuroinflammatory response produced by EAE model and its importance as a therapeutic target for the treatment of some neuroinflammatory diseases using subtype-selective PDE4B inhibitorsPeer Reviewe

cAMP specific PDE4B3 splice variants expression in male and female mouse brain during systemic inflammation

Trabajo presentado al 10th International Congress of Neuroimmunology (ISNI) celebrado en Sitges del 26 al 30 de octubre de 2010.PDE4 represents a family of cAMP-specific PDEs consisting of four paralog genes (PDE4A-D) each distinguishable by its unique N-terminal sequence. Publications indicate that the PDE4B gene is the predominant subtype involved in inflammatory induction by lipopolysaccaride (LPS) in mouse monocytes and macrophages. Together with previous reports of expression of the three PDE4 isoforms PDE4A, PDE4B and PDE4D in oligodendrocytes (Olgs), we show that the PDE4B3 mRNA is readily present in Olgs and neurons which imply a role for this PDE4B splice variant in the central nervous system (CNS). cAMP has been shown to play an important role in protecting myelin from excitotoxic cell death and considering the sexual dimorphism in Olgs functional properties and in disease susceptibility observed for e.g. multiple sclerosis (MS) together with the alterations in the two PDE4B splice-variants (PDE4B2 and PDE4B3) mRNA expression observed after LPS administration, we focused on possible gender differences for these enzymes during the acute immediate neuroinflammatory process observed during septic shock. Furthermore, sex-related differences have been observed in the incidence and severity of sepsis in humans and also in the response towards LPS administration in rodents for several cytokines. We have analyzed the influence of an intra peritoneal LPS injection on the distribution pattern and expression levels of the PDE4B3 mRNA splicing variant in both male and female mice brain. Characterization of the cell populations involved in the PDE alterations was established by double in situ hybridization histochemistry and immunohistochemistry. By semi-quantitative analyses of the autoradiograms we observed that PDE4B3 mRNA levels showed clear changes in females 24h post-injection, whereas, in male the altered expression was less evident and peaked 8h after treatment. Furthermore, we discovered that this downregulation was reflected in a lower percentage of oligodendrocytes positive for the PDE4B3 mRNA. Knowledge about PDE4B mRNAs expression in mouse brain in both sexes and the alterations provoked by LPS administration might help us to clarify sex-related differences in the susceptibility to autoimmune diseases.Financed by 2006-10243 & SAF2009-11052. EJ is a recipient of a fellowship from Ministerio de Educación y Ciencia (BES-2007-16588).Peer Reviewe

Role of Phosphodiesterase PDE4 isoforms in the neuroinflammatory response of an EAE mice model

Trabajo presentado a la Neuroscience celebrada en San Diego del 13 al 17 de noviembre de 2010.Inflammatory responses involve cAMP and pharmacological manipulation of cAMP levels by using specific phosphodiesterase (PDE) inhibitors provokes an anti-inflammatory response. Experimental autoimmune encephalomyelitis (EAE) is an animal model of the chronic inflammatory, neurodegenerative demyelinating disease multiple sclerosis (MS). Previously we demonstrated that in the brain and spinal cord of EAE rats there was a dramatic increase in the mRNA expression levels of the PDE4B isoenzyme, solely due to the splicing mRNA variant PDE4B2. This expression was found in the infiltrating T-cells and macrophage/microglia in microvessels and brain parenchima. We present here the study on the alterations in the expression of the cAMP-specific PDE4 and of several inflammatory cytokine mRNAs in the brain and spinal cord of C57BL6 EAE mice model by neuroanatomical techniques (double in situ hybridization histochemistry and immunohistochemistry), and quantitative real time RT-PCR. We observed a PDE4B2 mRNA upregulation after the onset of the EAE model, being significant 30 days post-immunization. No changes on PDE4B3 mRNA splice variant could be detected. Double in situ hybridization and immunohistochemistry studies showed that cellular infiltrates in brain and spinal cord tissues of EAE mice are overexpressing PDE4B isoform. Studies performed on PDE4A and PDE4B knockout (KO) mice showed an early onset of EAE in PDE4B KO mice, suggesting a “protective/retarding” role of PDE4B in this disease. RT-PCR experiments also detected a downregulation of some pro-inflammatory cytokines, like interferon-γ (IFNγ), in PDE4A KO mice and a trend to upregulation of interleukin 1β (IL1β) and IL6 in PDE4B KO mice. This differential response to EAE of PDE4A and PDE4B KO mice contrast with results obtained for EAE wt mice treated with a PDE4 inhibitor, rolipram. These findings support an important and distinctive role of different PDE4 isoforms during the neuroinflammatory response produced in a EAE model and their importance as a therapeutic target for the treatment of some neuroinflammatory diseases using subtype-selective PDE4 inhibitors.MICINN Grant SAF2006-10243; MICINN Grant SAF2009-11052; Sandler Foundation Grant to MC; CS IDIBAPS fellowship and EJ MICINN fellowship.Peer Reviewe