Fig. 7: Pericytes rescue STS-induced astrocyte apoptosis by TNT-mediated transfer of mitochondria.

CM-stained astrocytes were incubated with STS for 3 h. After the treatment, cells were washed and co-cultured for 24 h with pericytes (+Peri) as described in Fig. 6A, in the presence or absence of the tubulin-depolymerizing drug vincristine (VCR; 10 nM). The co-culture was analyzed for mitochondria trafficking and apoptosis by time-lapse and confocal fluorescence microscopy. A Time-lapse analysis of mitochondrial trafficking from healthy pericytes to STS-treated astrocytes. White arrows indicate mitochondria actively transported from healthy pericytes to STS-astrocytes through TNT (yellow arrow) (panel A1, insets I-III; Suppl. Movies 14–15). VCR treatment prevents TNT formation and strongly reduces intercellular mitochondrial trafficking (panel A2, insets I-III; Suppl. Movie 16). Representative images from N = 17 independent experiments with N ≥ 10 areas per experiment analyzed. B Apoptosis of STS-treated astrocytes regenerated for 24 h with healthy pericytes in the presence or absence of VCR. Astrocytes (blue) that were positive for CellEvent Caspase 3/7 (green nuclei, white arrows) were counted under the experimental conditions reported in the images. C Analysis of STS-astrocyte apoptosis. The percentage of Caspase 3/7-positive on the total astrocyte population is shown. Pericytes strongly rescue astrocytes from STS-induced apoptosis, an effect that is significantly decrease by VCR. D High magnification images of the analysis reported in panel C. Note the presence of pericyte-derived mitochodria (white arrow) inside STS-treated caspase3/7-negative astrocytes. E Confocal analysis and 3D reconstruction of rescued astrocytes after SDS treatment. Note the presence of pericyte-derived mitochodria (white arrow) inside post-STS astrocytes. N = 4 independent experiments. ****p < 0.0001; Student’s t-test (control); two-way ANOVA/Tukey’s tests (STS/R).