The Recently Identified P2Y-Like Receptor GPR17 Is a Sensor of Brain Damage and a New Target for Brain Repair
Figure 5
Time course of GPR17 expression after MCAo in mice.
Micrographs show coronal brain slices obtained from mice 24, 48, 72 hours after MCAo. Brain drawings in insets show the areas (squares) where micrographs were taken. (A) Twenty-four h after MCAo, immunohistochemistry with an anti-GPR17 antibody (red fluorescence) show a marked up-regulation of the receptor within the ischemic lesioned area (the region delimited by the dotted line). Basal expression of GPR17 in the corresponding contralateral area is shown in C for comparison. (B) Cells showing marked GPR17 expression inside the lesioned area also express the specific neuronal marker NeuN, confirming that these cells are indeed neurons. (D) Forty-eight h after MCAo, GPR17+ neurons inside the lesioned area are markedly reduced, suggesting induction of cell death. At this time point, a marked up-regulation of GPR17 appears in neurons at the border of the lesion. (E) These cells co-localize with the neuronal damage marker HSP70 (see text for more details). Up-regulation of GPR17 in the ischemic area is also confirmed by western blot analysis in membrane (M) and cytosolic (C) fractions from intact contralateral (Con) and ipsilateral ischemic (MCAo) cortex of mice (right panel). GPR17 is identified with an anti-GPR17 antibody as an immunoreactive protein band with an approximate 50 kDa molecular weight; as expected for a membrane receptor, this band is present in membranes but not in cytosolic fractions. To confirm blot specificity, the 50 kDa protein band is abolished in the presence of the neutralizing peptide (indicated as+pept). A marked increase of the intensity of this band is found after MCAo (the quantification of this increase is shown in histograms as % of control set to 100%; results from 5 experiments). Forty-eight h (not shown) and seventy-two h after MCAo (F), there is a marked increase of GPR17+ cells at the border of the lesioned area. (G) These cells are not neurons but they are microglia/macrophages infiltrating the ischemic area, since most of them also positively stain for IB4, a microglia/macrophage marker (see also text). These GPR17+/IB4+ cells are indeed found inside the lesioned area one week after MCAo (data not shown). Scale bars: 50 µm.