Although some transcripts implicated in glial activation were upregulated in cultured cells treated with 8Br-cAMP i.
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C3 and Il-6 , our results suggest that cAMP signaling essentially represses astroglial activation. This conclusion may diverge from the assumption by some authors that cAMP analogs in cultured astrocytes promote activation [ 12 — 16 ]. However, this consideration was taken on the basis of changes in shape stellation and GFAP expression, which are also typical attributes of maturing astrocytes [ 8 , 9 , 30 ].
Noteworthy, our results showing an opposite transcriptome regulation by cAMP and glial-activating cytokines are consistent with previous studies demonstrating that elevated cAMP in astrocytes antagonizes the cytokine-induced expression of adhesion molecules involved in neuroinflammation [ 31 ]. A repressive role for cAMP in glial activation is also consistent with the observations that cAMP pathways are involved in the reduction and promotion of the glial activation triggered by anti-inflammatory and pro-inflamatory extracellular agents, respectively [ 32 — 34 ].
Moreover, the observation of cAMP-induced upregulation of repressors of the glial inflammatory response, such as Socs1 2. In contrast to other neural cell types, such as neurons and oligodendrocytes, astroglial cells grown in culture do not accurately reflect their attributes in vivo. It has been revealed that in vitro astrocytes show an immature, somewhat reactive, transcriptional profile [ 21 , 37 ] see also overlap between developing and in vitro sets in Fig. Therefore by comparing our results with gene sets of Cahoy et al. The full gene lists are shown in Additional file 3 : Table S7 and S8.
Adra2a , Grin2c , and Kcnn2 and in vitro i. Our observation that the transcriptome of cultured astrocytes acquires a greater resemblance to that of in vivo cells when these cells are exposed to 8Br-cAMP may indicate that cAMP signaling confers astrocytes an in vivo-like phenotype by repressing activation and promoting differentiation. Because astrocytes cultured in serum-free conditions and in a bioactive3D system more closely resemble astrocytes in vivo [ 38 , 39 ], an involvement of cAMP signaling in the environment-dependent acquisition of a in vivo phenotype of cultured astrocytes could be suggested.
We have contrasted our gene profiles with previous studies describing the transcriptome signatures of acutely isolated astrocytes [ 21 , 28 ]. Next, we evaluate expression of representative genes regulated by cAMP in situ in cortical astrocytes in different states. In agreement with the proliferative state of cells, many nuclei of GFAP- and Nestin-positive astrocytes were immunolabeled for Mki67 in histological sections of postnatal and injured adult cortex, but not in the intact adult brain Fig.
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Although recent studies have shown that NR2C mRNA increases with age in cortical astrocytes in situ [ 40 , 41 ], the location of NMDA receptor protein in glial cells in vivo is unknown. Here we show the protein expression of NR2C subunit in many astrocytes in the adult cerebral cortex Fig. In conclusion, the present GSEA results may provide a comprehensive database of transcripts involved in the acquisition of a mature astrocyte phenotype regulated by the cAMP pathway.
Abstracts of papers
In situ expression of astroglial genes controlled by cAMP in the cerebral cortex in different states. Arrows point to identified astrocytes expressing only GFAP. Arrowheads identify double labeled cells. Crosses and the asterisk indicate the lateral ventricle and the brain surface, respectively. The induction of target genes and cellular programs regulated by the cAMP pathway is highly dependent on cellular contexts [ 42 ].
For instance, elevated cAMP results in cell-cycle arrest in astrocytes but induces proliferation of neurons [ 9 , 43 ].
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Moreover, the recruitment of specific CREB regulatory partners, the activation of PKA-independent pathways and a secondary gene regulation may also collaborate to shape the individual astrocyte transcriptome elicited by cAMP signaling [ 43 , 44 ]. We here show that the net effect of cAMP signaling is to restrict developmental and activation features of astrocytes and promote their maturation. Therefore, we propose that controlled positive modulation of cAMP signaling could be used to repress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes to support and protect neuronal populations.
Cultures were prepared from the cerebral cortex of 2-day-old mice [ 17 ]. Briefly, the cerebral cortex was isolated and the meninges were carefully dissected out. Cortical tissues were then minced and incubated in 0. At confluence days 10—12 , the flasks were shaken overnight and the cells were rinsed, detached and subcultured onto poly-L-lysine-coated plastic culture dishes and coverslips.
RNA was isolated from triplicate cultures from the four individual experiments. Reactions containing nuclease-free water instead of enzyme served as a negative control.
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They were then chilled on ice. Data were extracted using Genepix 6. Log2ratio values were computed for all pairs of control and cAMP-stimulated samples. Per-probe log2ratios were aggregated to per-gene values by taking median values.
Analysis for differential expression on a gene-by-gene basis was performed by limma [ 46 ] while distinguishing biological from technical replicates dye-swap hybridizations. Also, for cross-study comparisons GSEA, was applied. This approach was used to test gene sets of interest defined as lists of differentially expressed gene symbols derived from other studies for significant enrichment among differentially expressed genes within our study.
Only studies performed on mouse from which astrocyte cultures were obtained and maintained under similar conditions to those in the present study were considered. To compare the gene sets by Venn diagrams, we fixed the same fold change cut-off at 1. To control the specificity of the reaction, melting-curve analysis was performed after amplification.
In addition, PCR products were analyzed by agarose gel electrophoresis to confirm the size of the amplified targets. Levels of Tbp were used as normalization controls and relative mRNA levels were calculated as indicated by Pfaffl et al. Adult and postnatal P5 animals were perfused transcardially under deep anesthesia with the same fixative. Forty-micrometer-thick frozen sections were obtained with a cryostat and collected in PBS.
Cell cultures and brain sections were processed for immunofluorescence using secondary fluorochrome-conjugated antibodies Alexa Fluor and Alexa Fluor , Molecular Probes, Eugene, OR.
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Cell-containing coverslips and histological sections were mounted with Mowiol. Double immunofluorescences were performed by using primary antibodies raised in different species. The specificity of the immunostaining was tested by omitting the primary antibodies or by replacing them with an equivalent concentration of nonspecific IgG. No immunostaining was observed in these conditions. Animal experimental procedures were approved by the Ethics Committee of the Universitat de Barcelona, in compliance with Catalan and European legislation All efforts were made to minimize the number used and animal suffering.
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Two distinct inwardly rectifying conductances are expressed in long term dibutyryl-cyclic-AMP treated rat cultured cortical astrocytes. FEBS Lett.
Mol Pharmacol. Astroglial differentiation of cortical precursor cells triggered by activation of the cAMP-dependent signaling pathway. J Neurosci. Curr Pharm Des. Astrocyte cell lineage. Similarity of astrocytes that form in the presence of dBcAMP in cultures to reactive astrocytes in vivo.
J Neurosci Res. Dibutyryl cAMP, interleukin-1 beta, and macrophage conditioned medium enhance the ability of astrocytes to promote neurite growth. Expression profiling identifies a molecular signature of reactive astrocytes stimulated by cyclic AMP or proinflammatory cytokines. Exp Neurol.
Differentiation of reactive-like astrocytes cultured on nanofibrillar and comparative culture surfaces. Nanomedicine Lond. J Neurochem. Oxidative stress regulates type 3 deiodinase and type 2 deiodinase in cultured rat astrocytes.