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1.  Modelling Neuroinflammation In Vitro: A Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents 
PLoS ONE  2012;7(9):e45227.
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.
doi:10.1371/journal.pone.0045227
PMCID: PMC3447933  PMID: 23028862
2.  Inhibition of CD200R1 expression by C/EBP beta 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 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.
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/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.
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/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.
doi:10.1186/1742-2094-9-165
PMCID: PMC3414764  PMID: 22776069
Neuroinflammation; Reactive microglia; CD200R1; C/EBPβ; Neuron-microglia communication; In vitro
3.  PCAF regulates the stability of the transcriptional regulator and cyclin-dependent kinase inhibitor p27Kip1 
Nucleic Acids Research  2012;40(14):6520-6533.
P27Kip1 (p27) is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors. Recently, a new function of p27 as transcriptional regulator has been reported. It has been shown that p27 regulates the expression of target genes mostly involved in splicing, cell cycle, respiration and translation. We report here that p27 directly binds to the transcriptional coactivator PCAF by a region including amino acids 91–120. PCAF associates with p27 through its catalytic domain and acetylates p27 at lysine 100. Our data showed that overexpression of PCAF induces the degradation of p27 whereas in contrast, the knockdown of PCAF stabilizes the protein. A p27 mutant in which K100 was substituted by arginine (p27-K100R) cannot be acetylated by PCAF and has a half-life much higher than that of p27WT. Moreover, p27-K100R remains stable along cell-cycle progression. Ubiquitylation assays and the use of proteasome inhibitors indicate that PCAF induces p27 degradation via proteasome. We also observed that knockdown of skp2 did not affect the PCAF induced degradation of p27. In conclusion, our data suggest that the p27 acetylation by PCAF regulates its stability.
doi:10.1093/nar/gks343
PMCID: PMC3413142  PMID: 22547391
4.  Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein β 
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 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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1742-2094-8-156
PMCID: PMC3223504  PMID: 22074460

Results 1-4 (4)