The Recently Identified P2Y-Like Receptor GPR17 Is a Sensor of Brain Damage and a New Target for Brain Repair
Figure 4
Presence and activation of GPR17 in oligodendrocyte precursor cells of primary cortical cultures maintained in vitro.
Several GPR17+ ramified cells (green fluorescence) reminiscent of the precursors found in vivo were consistently found in primary neuron/glia cortical cultures. Micrographs (A–H) show their phenotype characterization. These cells (green fluorescence) did not co-express the stem cell marker nestin (A), or the astrocytic marker GFAP (B), or the neuronal markers MAP2 (C) and β-TubIII (D), but were instead positive for several pre-oligodendrocyte markers, such as NG2 (arrows in E) and O4 (arrows in F), which appear quite early during oligodendrocyte differentiation. Colocalization of GPR17 with these markers is shown in yellow-orange cells. A lower number of GPR17+ cells also expressed CNPase (arrow in G) and very few costained for MBP (arrow in H), which are markers of more mature oligodendroglia (for more details, see text). In blue, Hoechst 33258 stained nuclei. Single cell RT-PCR was used to confirm expression of GPR17 in oligodendroglial-like cells (I). For this analysis, cells were picked up from living cultures using cell morphology as a guide at the light microscope. Left micrograph: an example of a brightfield showing cells (arrows) with oligodendrocyte morphology. These cells can be distinguished from neurons based on their size (approximately 1/3 the size of neurons), phase-dark cell bodies and presence of fine ramifications. The oligodendrocyte nature of cells with this morphology was confirmed by co-immunostaining for O4 and GPR17 at a subsequent time after culture fixing (arrows in right micrograph). By following this method, one living cell or a small number of cells (up to 10) were sucked from living cultures (see MATERIALS AND METHODS) and processed for RT-PCR analysis. Significant GPR17 expression was detected in both the single-cell and the 10-cell samples, as indicated in the PCR panel. Rat cortex was utilized as a positive control (see last lane). Scale bars: 15 µm. In these primary cultures, the number of GPR17+ cells spontaneously increased as a function of time (J); DIV: days in vitro. (K) Quantification of the percentage of GPR17+ cells that also co-express the indicated oligodendrocyte markers. Total number (n) of GPR17+ cells counted from two different coverslips for each preparation: for NG2, n = 3502; for O4, n = 2685; for CNPase, n = 1738; for MBP, n = 3615. Data were obtained from five different primary culture preparations. (L) Exposure of cultures to maximal (100 µM) UDP-glucose (UDP-glc) concentrations for 72 h induced a highly significant increase in the number of mature MBP+ oligodendroglial cells. The number of GPR17+ cells was also increased. Both effects were partially counteracted in the presence of the P2Y receptor antagonist cangrelor. (M) Exposure of cultures to maximal (100 nM) LTD4 concentrations for 72 h also induced a significant increase in the number of mature MBP+ oligodendroglial cells and of GPR17+ cells. No synergistic nor additive effects were found when both agonists were combined together. There were no significant changes in the total number of cells in culture during the 72 h treatment, as shown by labeling of cells nuclei with Hoechst 33258. *p<0.05 compared to control; ** p<0.05 with respect to UDP-glc and not different from control; # p = 0.15 with respect to control and P = 0.245 with respect to UDP-glc, one-way analysis of variance (ANOVA).