Figure 4.

Intracellular calcium levels modulate the secretion of exosome-associated RA in NG2⁺ cells and determine their differentiation into oligodendrocytes via RARα signaling. A, Expression of RARα, OSP, and RA in NG2 cultures treated for 3 d with: vehicle, BAPTA-AM, GW4869, AM 580, retinal + BAPTA-AM, or retinal + GW4869. Scale bar, 50 μm and for the bottom 300 μm. B, C, Quantification of OSP (B) and RARα (C) expression showed that both were significantly higher in the presence of AM580 or when increased intracellular concentrations of RA occurred (retinal + BAPTA-AM and retinal + GW4869). Data are shown as mean ± SEM taken from five random fields from three independent experiments, ***p ≤ 0.001, One-way ANOVA followed by Tukey's test. D, Electron microscopy images of exosomes (arrows) isolated from vehicle-treated NG2⁺ cell cultures. Scale bar, 50 nm. E, β-galactoside staining of RARE LacZ-transfected F9 cells showing RA in exosomes isolated from NG2⁺ cell cultures treated with: vehicle, retinal, retinal + BAPTA-AM, GW4869, or retinal + GW4869. F, Quantification of RA plotted as percentage of LacZ⁺ cells shows that preventing exosome release from the NG2⁺ cells results in a significant decrease in secreted RA. Data are shown as mean ± SEM taken from five random fields from three independent experiments, ***p ≤ 0.001, one-way ANOVA, followed by Tukey's test.