[Show abstract][Hide abstract] ABSTRACT: The mechanisms that control microglial activation are of interest, since neuroinflammation, which involves reactive microglia, may be an additional target in the search for therapeutic strategies to treat neurodegenerative diseases. Neuron-microglia interaction through contact-dependent or independent mechanisms is involved in the regulation of the microglial phenotype in both physiological and pathological conditions. The interaction between CD200, which is mainly present in neurons but also in astrocytes, and CD200R1, which is mainly present in microglia, is one of the mechanisms involved in keeping the microglial proinflammatory phenotype under control in physiological conditions. Alterations in the expression of CD200 and CD200R1 have been described in neurodegenerative diseases, but little is known about the mechanism of regulation of these proteins under physiological or pathological conditions. The aim of this work was to study the modulation of CD200 and CD200R1 expression by peroxisome proliferator-activated receptor gamma (PPAR-γ), a transcription factor involved in the control of the inflammatory response. Mouse primary neuronal and glial cultures and neuron-microglia cocultures were treated with the PPAR-γ endogenous ligand 15-deoxy-Δ(12, 14) -prostaglandin J2 (15d-PGJ2 ) in the presence and absence of lipopolysaccharide plus interferon-γ (LPS/IFN-γ)-induced glial activation. We show that 15d-PGJ2 inhibits the pro-inflammatory response and prevents both CD200R1 downregulation and CD200 upregulation in reactive glial cells. In addition, 15d-PGJ2 abrogates reactive-microglia induced neurotoxicity in neuron-microglia cultures through a CD200-CD200R1 dependent mechanism. These results suggest that PPAR-γ modulates CD200 and CD200R1 gene expression and that CD200-CD200R1 interaction is involved in the anti-inflammatory and neuroprotective action of PPAR-γ agonists. GLIA 2014.
[Show abstract][Hide abstract] ABSTRACT: The eicosanoid prostaglandin E2 (PGE2) plays important roles in neuroinflammation and it is produced by the sequential action of the enzymes cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PTGES). The expression of both enzymes and the production of PGE2 are increased in neuroinflammation. The objective of this study was to elucidate whether the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates the expression of prostaglandin synthesis enzymes in neuroinflammation. To this aim, the expression of these enzymes in wild-type and C/EBPβ-null mice was analyzed in vitro and in vivo. In mixed glial cultures, lipopolysaccharide (LPS) ± interferon γ (IFN-γ) induced C/EBPβ binding to COX-2 and PTGES promoters. LPS ± IFN-γ-induced increases in PTGES expression and in PGE2 production in mixed glial and microglial cultures were abrogated in the absence of C/EBPβ. Also, increased brain PTGES expression induced by systemic LPS administration was markedly reduced in C/EBPβ-null mice. In contrast to PTGES, the induction of COX-2 expression in vitro or in vivo was not markedly affected by the absence of C/EBPβ. These results demonstrate that C/EBPβ regulates PTGES expression and PGE2 production by activated microglial cells in vitro and point to C/EBPβ as a regulator of PTGES expression in vivo in the inflamed central nervous system. Altogether, these findings strengthen the proposed role of C/EBPβ as a key player in the orchestration of neuroinflammatory gene response.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ) is expressed in activated astrocytes and microglia and can regulate the expression of potentially detrimental proinflammatory genes. The objective of this study was to determine the role of C/EBPδ in glial activation. To this end, glial activation was analyzed in primary glial cultures and in the central nervous system from wild type and C/EBPδ−/− mice. In vitro studies showed that the expression of proinflammatory genes nitric oxide (NO)synthase-2, cyclooxygenase-2, and interleukin (IL)-6 in glial cultures, and the neurotoxicity elicited by microglia in neuron–microglia cocultures, were decreased in the absence of C/EBPδ when cultures were treated with lipopolysaccharide (LPS) and interferon γ, but not with LPS alone. In C/EBPδ−/− mice, systemic LPS-induced brain expression of NO synthase-2, tumor necrosis factor-α, IL-1β, and IL-6 was attenuated. Finally, increased C/EBPδ nuclear expression was observed in microglial cells from amyotrophic lateral sclerosis patients and G93A-SOD1 mice spinal cord. These results demonstrate that C/EBPδ plays a key role in the regulation of proinflammatory gene expression in glial activation and suggest that C/EBPδ inhibition has potential for the treatment of neurodegenerative disorders, in particular, amyotrophic lateral sclerosis.
Full-text · Article · Mar 2013 · Neurobiology of Aging
[Show abstract][Hide abstract] ABSTRACT: Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.
[Show abstract][Hide abstract] ABSTRACT: In physiological conditions, it is postulated that neurons control microglial reactivity through a series of inhibitory mechanisms, involving either cell contact-dependent, soluble-factor-dependent or neurotransmitter-associated pathways. In the current study, we focus on CD200R1, a microglial receptor involved in one of these cell contact-dependent mechanisms. CD200R1 activation by its ligand, CD200 (mainly expressed by neurons in the central nervous system),is postulated to inhibit the pro-inflammatory phenotype of microglial cells, while alterations in CD200-CD200R1 signalling potentiate this phenotype. Little is known about the regulation of CD200R1 expression in microglia or possible alterations in the presence of pro-inflammatory stimuli.
Murine primary microglial cultures, mixed glial cultures from wild-type and CCAAT/enhancer binding protein β (C/EBPβ)-deficient mice, and the BV2 murine cell line overexpressing C/EBPβ were used to study the involvement of C/EBPβ transcription factor in the regulation of CD200R1 expression in response to a proinflammatory stimulus (lipopolysaccharide (LPS)). Binding of C/EBPβ to the CD200R1 promoter was determined by quantitative chromatin immunoprecipitation (qChIP). The involvement of histone deacetylase 1 in the control of CD200R1 expression by C/EBPβ was also determined by co-immunoprecipitation and qChIP.
LPS treatment induced a decrease in CD200R1 mRNA and protein expression in microglial cells, an effect that was not observed in the absence of C/EBPβ. C/EBPβ overexpression in BV2 cells resulted in a decrease in basal CD200R1 mRNA and protein expression. In addition, C/EBPβ binding to the CD200R1 promoter was observed in LPS-treated but not in control glial cells, and also in control BV2 cells overexpressing C/EBPβ. Finally, we observed that histone deacetylase 1 co-immunoprecipitated with C/EBPβ and showed binding to a C/EBPβ consensus sequence of the CD200R1 promoter in LPS-treated glial cells. Moreover, histone deacetylase 1 inhibitors reversed the decrease in CD200R1 expression induced by LPS treatment.
CD200R1 expression decreases in microglial cells in the presence of a pro-inflammatory stimulus, an effect that is regulated, at least in part, by C/EBPβ. Histone deacetylase 1 may mediate C/EBPβ inhibition of CD200R1 expression, through a direct effect on C/EBPβ transcriptional activity and/or on chromatin structure.
Full-text · Article · Jul 2012 · Journal of Neuroinflammation
[Show abstract][Hide abstract] ABSTRACT: Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPβ in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPβ-null glial cultures.
Due to fertility and mortality problems associated with the C/EBPβ-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPβ-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPβ DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation.
C/EBPβ mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon γ (IFNγ). Quantitative chromatin immunoprecipitation showed binding of C/EBPβ to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFNγ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1β and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPβ. In addition, neurotoxicity elicited by LPS+IFNγ-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPβ in microglia.
These findings show involvement of C/EBPβ in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBPβ in the induction of neurotoxic effects by activated microglia.
Full-text · Article · Nov 2011 · Journal of Neuroinflammation
[Show abstract][Hide abstract] ABSTRACT: C/EBPβ (CCAAT/enhancer binding protein beta) es un factor de transcripción de la familia b-zip cuya expresión aumenta en células microgliales activadas. Nuestro objetivo en este trabajo ha sido estudiar si C/EBPβ regula en la activación microglial la expresión de NOS2, un enzima inducible productor de NO y que se cree es clave en los efectos patogénicos de la respuesta neuroinflamatoria. El gen de NOS2 presenta secuencias consenso para C/EBP en sus regiones reguladoras. El trabajo se ha realizado en cultivos de glía mixta de corteza cerebral de embriones (E19) de ratoneswild-type y deficientes en C/EBPβ. Estos cultivos constan principalmente de astrocitos y microglía y han sido tratados con LPS o LPS+IFNγ para inducir su activación. Hemos observado que en la activación glial se induce la expresión de NOS2 (qRT-PCR), que se localiza en microglía y no en astrocitos (inmunocitoquímica) y que en estas condiciones C/EBPβ se une al promotor de NOS2 (qChIP). Por otro lado, en cultivos gliales deficientes en C/EBPβ tratados con LPS o LPS+IFNγ se produce una marcada atenuación en la inducción de NOS2 a nivel de mRNA (qRT-PCR), proteína (WB e icc) y producción de NO (Griess). Finalmente, al cocultivar neuronas con microglía wild-type o deficiente en C/EBPβ hemos observado que la neurotoxicidad inducida por células gliales activadas se ve totalmente abolida cuando la microglía es deficiente en C/EBPβ. Este trabajo demuestra que C/EBPβ regula la expresión de NOS2 en células microgliales activadas y apunta a C/EBPβ como un importante regulador de la expresión de genes con efectos neurotóxicos en la respuesta neuroinflamatoria.
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2010) 115, 283–295.
Adenosine A2A receptors (A2ARs) are G-protein coupled receptors that stimulate adenylyl cyclase activity. The most A2ARs-enriched brain region is the striatum, in which A2ARs are largely restricted to GABAergic neurons of the indirect pathway. We recently described how DNA methylation controls basal A2AR expression levels in human cell lines. The present report provides clues about the molecular mechanisms that promote human brain region-specific A2AR gene (ADORA2A) basal expression. The transcription factors ZBP-89 and Yin Yang-1 (YY1) have been characterized as regulators of ADORA2A in SH-SY5Y cells by means of specific expression vectors/siRNAs transient transfection and chromatin immunoprecipitation assay. ZBP-89 plays a role as an activator and YY1 as a repressor. No differences were found in ZBP-89 levels with western blot between the putamen and cerebellum of human postmortem brains. However, increased YY1 levels and DNA methylation percentage in the 5′ untranslated region of ADORA2A, using SEQUENOM MassArray, were found in the cerebellum with respect to the putamen of human brains, showing an inverse relationship with A2AR levels in the two cerebral regions.
Full-text · Article · Oct 2010 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2010) 112, 1273–1285.
Adenosine A2A receptors (A2ARs) appear to play important roles in inflammation and in certain diseases of the nervous system. Pharmacological modulation of A2ARs is particularly useful in Parkinson’s disease and has been tested in schizophrenia. However, little is known about the regulation of A2AR gene (ADORA2A). A bioinformatic analysis revealed the presence of three CpG islands in the 5′ UTR region of human ADORA2A. Next, HeLa, SH-SY5Y and U87-MG cells were treated for 48 h with 5 μM 5-azacytidine (Aza). Increased A2AR levels were demonstrated in HeLa and SH-SY5Y cells when compared with non-treated cells. No modifications were seen in U87-MG cells. The increased A2AR mRNA and protein levels were accompanied by a loss of DNA methylation pattern in HeLa and SH-SY5Y cells, as measured with the SEQUENOM MassArray platform. The Aza treatment also reduced the affinity of a methyl-CpG-binding protein for ADORA2A by quantitative chromatin immunoprecipitation in HeLa cells. Interestingly, A2AR levels were reduced by S-adenosyl-l-methionine treatment in U87-MG and methyl-CpG-binding protein affinity was increased for ADORA2A by quantitative chromatin immunoprecipitation. Therefore, these results show for the first time that DNA methylation plays a role in ADORA2A transcription and, subsequently, in constitutive A2AR cell surface levels.
Full-text · Article · Feb 2010 · Journal of Neurochemistry