Fig. 5: TNT-mediated mitochondrial transfer from pericytes rescues OGD-induced apoptosis of astrocytes.

Astrocytes were stained with CM and exposed to 2% O₂ for 24 h in glucose- and serum-free medium, washed and refreshed in normal complete medium with a constant number of healthy MT-stained pericytes (OGD/R), in the presence or absence of CytoD (200 nM), as described in Fig. 4A. Twenty-four hours later, the co-culture was analyzed for mitochondria trafficking and astrocyte apoptosis by time-lapse and confocal fluorescence microscopy. A Time-lapse fluorescence microscopy analysis of mitochondria trafficking from healthy pericytes to OGD-astrocytes regenerated for 24 h (OGD/R) in the absence or presence of CytoD. TNT and actively transported mitochondria from healthy pericytes to OGD-astrocytes are indicated by yellow and white arrows, respectively (A1 and Suppl. Movie 10; A2 and Suppl. Movie 11 for OGD-astrocytes with overt morphological alterations). CytoD abolished TNT formation and strongly reduced the intercellular mitochondria trafficking to OGD-astrocytes (A3; Suppl. Movie 12) also when astrocytes are morphologically altered (A4; Suppl. Movie 13). Representative images from N = 4 independent experiments in which N ≥ 10 areas per experiment were analyzed. B Apoptosis of OGD-astrocyte regenerated for 24 h (OGD/R) with healthy pericytes in the presence or absence of CytoD. Astrocytes (orange) 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 OGD-astrocyte apoptosis. The percentage of Caspase 3/7-positive astrocytes on the total astrocyte population is shown. Pericytes rescued astrocytes from OGD-induced apoptosis only in the presence of an intact F-actin cytoskeleton. N = 3 independent experiments. ****p < 0.0001; Student’s t-test (normoxia); two-way ANOVA/Tukey’s tests (OGD/R).