CD200R1 and CD200 expression are regulated by PPAR-γ in activated glial cells.

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.

CCAAT/enhancer binding protein δ regulates glial proinflammatory gene expression.

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-1ß, 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.

Inhibition of CD200R1 expression by C/EBP ß in reactive microglial cells.

BACKGROUND: 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. METHODS: Murine primary microglial cultures, mixed glial cultures from wild-type and CCAAT/enhancer binding protein ß (C/EBPß)-deficient mice, and the BV2 murine cell line overexpressing C/EBPß were used to study the involvement of C/EBPß transcription factor in the regulation of CD200R1 expression in response to a proinflammatory stimulus (lipopolysaccharide (LPS)). Binding of C/EBPß 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/EBPß was also determined by co-immunoprecipitation and qChIP. RESULTS: 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/EBPß. C/EBPß overexpression in BV2 cells resulted in a decrease in basal CD200R1 mRNA and protein expression. In addition, C/EBPß binding to the CD200R1 promoter was observed in LPS-treated but not in control glial cells, and also in control BV2 cells overexpressing C/EBPß. Finally, we observed that histone deacetylase 1 co-immunoprecipitated with C/EBPß and showed binding to a C/EBPß 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. CONCLUSIONS: 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/EBPß. Histone deacetylase 1 may mediate C/EBPß inhibition of CD200R1 expression, through a direct effect on C/EBPß transcriptional activity and/or on chromatin structure.

C/EBPß expression in activated microglia in amyotrophic lateral sclerosis.

Neuroinflammation is thought to play a pathogenic role in many neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). In this study we demonstrate that the expression of nitric oxide (NO) synthase-2 (NOS2), and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) with interferon-γ is higher in microglial-enriched cultures from G93A-SOD1 mice, an ALS animal model, than from wild type mice. The levels of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor that regulates proinflammatory gene expression, are also upregulated in activated G93A-SOD1 microglial cells. In vivo, systemic lipopolysaccharide also induces an exacerbated neuroinflammatory response in G93A-SOD1 mice versus wild type mice, with increased expression of glial fibrillary acidic protein (GFAP), CD11b, nitric oxide synthase-2, cyclooxygenase-2, proinflammatory cytokines, and C/EBPß. Finally, we report that C/EBPß is expressed by microglia in the spinal cord of ALS patients. This is the first demonstration to our knowledge of microglial C/EBPß expression in human disease. Altogether these findings indicate that G93A-SOD1 expression results in an exacerbated pattern of neuroinflammation and suggest that C/EBPß is a candidate to regulate the expression of potentially neurotoxic genes in microglial cells in ALS.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein ß.

BACKGROUND: 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 ß (C/EBPß) 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/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. METHODS: Due to fertility and mortality problems associated with the C/EBPß-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/EBPß-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/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. RESULTS: C/EBPß 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/EBPß 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-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFNγ-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia. CONCLUSIONS: These findings show involvement of C/EBPß in the regulation of pro-inflammatory gene expression in glial activation, and demonstrate for the first time a key role for C/EBPß in the induction of neurotoxic effects by activated microglia.

CCAAT/enhancer binding protein-alpha is down-regulated by toll-like receptor agonists in microglial cells.

The transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPalpha is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down-regulates C/EBPalpha mRNA at 2 hr and all C/EBPalpha protein isoforms at 4 hr. This effect is elicited by LPS concentrations >/=100 pg/ml. LPS-induced C/EBPalpha down-regulation occurs in microglial cells both in mixed glial and in microglial-enriched cultures. As seen with LPS, other toll-like receptor agonists (polyinosinic-polycytidylic acid, peptidoglycan from Staphylococcus aureus, and the oligonucleotide CpG1668) also down-regulate C/EBPalpha whereas cytokines such as interleukin-1beta, interleukin-6, macrophage-colony stimulating factor, and interferon-gamma do not. These findings suggest that C/EBPalpha down-regulation in activated microglia could play an important role in the increased expression of genes that are potentially pathogenic in a variety of neurological disorders.

Upregulation of CCAAT/enhancer binding protein beta in activated astrocytes and microglia.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPbeta in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPbeta expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPbeta in untreated mixed glial cultures. C/EBPbeta is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS-induced upregulation of glial C/EBPbeta is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPbeta nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS-induced increase in microglial C/EBPbeta is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPbeta as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPbeta expression in activated glial cells in vivo. These findings support a role for C/EBPbeta in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPbeta-regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation. (c) 2006 Wiley-Liss, Inc.

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia.

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.

Effects of beta-AP peptides on activation of the transcription factor NF-kappaB and in cell proliferation in glial cell cultures.

The effect of two beta amyloid peptides (Abeta 25/35 and Abeta 1/42) on the activation of the transcription factor kappaB (NF-kappaB) in pure astroglial, pure microglial and mixed glial cell cultures was compared by means of single or double immunofluorescence and Western blot techniques. We also studied the effect of both peptides in cell proliferation in mixed glial cultures and pure astrocytes. The Abeta 1/42 peptide induced the activation of NF-kappaB in all studied cell cultures and its effect was potentiated by interferon-gamma (IFN-gamma). Abeta 25/35 alone did not induce NF-kappaB activation, but Abeta 25/35 plus IFN-gamma induced the activation of the transcription factor in the mixed and pure microglial cultures, although not in pure astroglia. The Abeta 1/42 peptide, but not Abeta 25/35, induced proliferation in pure astroglial and mixed glial cell cultures. The results suggest that the state of peptide aggregation is related to their ability to activate glial cells.

Structural, kinetic and cytotoxicity aspects of 12-28 beta-amyloid protein fragment: a reappraisal.

A chemical, structural and biological study on the beta-amyloid peptide beta12-28 is reported which was carried out in order to assess the feasibility using this peptide fragment as a model of the natural beta-amyloid protein. The aggregation properties of beta12-28 have been investigated by pulse field-gradient NMR spectroscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. The results obtained suggest that beta12-28 behaviour is comparable to that of the natural beta-amyloid protein although kinetically slower. Translational diffusion coefficients obtained by NMR on an aged beta12-28 solution suggest that the soluble peptide fraction is composed of oligomeric intermediates adopting an extended ellipsoidal assembly rather than a spherical one. The beta12-28 peptide proved to be cytotoxic in PC12 cell cultures as monitored by the MTT assay, although a lack of reproducibility was observed in the dose-response experiments.

Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells.

Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin-treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.

Relationship between beta-AP peptide aggregation and microglial activation.

We compared the relationship between the state of aggregation of two peptides (beta-AP 25-35 and beta-AP 1-42) and microglial activation. After 7 days at 37 degrees C beta-AP 25-35 was in an amorphous state and did not activate microglial cells. In the same conditions, aggregated beta-AP 1-42 activated these cells and caused changes in microglial ramification, increasing the proliferation index and inducing tumor necrosis factor alpha (TNF alpha) release. Neither peptide induced a release of nitric oxide (NO). As the toxicity of beta-AP peptides in cell culture is associated with the formation of amyloid fibrils, we also examined the toxicity of both peptides in microglial cell cultures and in PC 12 cell cultures. The results suggest that the two beta-AP fragments studied have similar neurotoxic effects but different pro-inflammatory activities.