Mitochondrial retrograde transport is required for OGD-Rep–induced mitophagy. (A and E) Schematic diagrams of rapalog-induced mitochondrial transportation assay. Rapalog induced the recruitment of a fusion protein of FRB with the truncated motor construct of dynein adaptor bicaudal D2 (FRB-BICDN-HA; A) or kinesin-1 (FRB-KIF5B-HA; E) to FKBP-GFP-Mito. (B–D and F–H) Primary cultured cortical neurons were previously transfected with FRB-BICDN-HA (B–D) or FRB-KIF5B-HA (F–H) and FKBP-GFP-Mito by electroporation and subjected to 20 min of OGD in DIV8. At reperfusion, ethanol (vehicle control) or 500 nM rapalog was added. (B and F) Live cell fluorescent images of axonal mitochondria were captured by confocal microscopy. Images show representative kymographs from three independent experiments. (C and G) Columns represent the proportion of mitochondria showing different transportation patterns. Seven cells from three independent experiments were included for each group. (D and H) Neurons were harvested after 40 min of reperfusion. The COX IV, Tim23, p62, HA, and GAPDH levels in cultured neurons were determined by Western blot. Semiquantitative analysis of COX IV, Tim23, and p62 bands is shown. The data are expressed as means ± SEM. Statistical comparisons were performed with one-way ANOVA with Sidak’s multiple-comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group. Ctrl, control; Mito, mitochondrial/mitochondria; O-R, reperfusion; Veh, vehicle.