The maize abscisic acid (ABA)-responsive rab17 mRNA and Rab17 protein distribution in maize embryo tissues was investigated by in situ hybridization and immunocytochemistry. rab17 mRNA and Rab17 protein were found in all cells of embryo tissues. Synthesis of rab17 mRNA occurred initially in the embryo axis. As maturation progressed, rab17 mRNA was detectable in the scutellum and accumulated in axis cells and provascular tissues. However, the response to exogenous ABA differed in various embryo cell types. The Rab17 protein was located in the nucleus and in the cytoplasm, and qualitative differences in the phosphorylation states of the protein were found between the two subcellular compartments. Based on the similar domain arrangements of Rab17 and a nuclear localization signal (NLS) binding phosphoprotein, Nopp140, interaction of Rab17 with NLS peptides was studied. We found specific binding of Rab17 to the wild-type NLS of the SV40 T antigen but not to an import incompetent mutant peptide. Moreover, binding of the NLS peptide to Rab17 was found to be dependent upon phosphorylation. These results suggest that Rab17 may play a role in nuclear protein transport.

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.

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.