Figure 7.
Figure 7. Mitochondrial retrograde transport protects against OGD-Rep injuries. (A–C) OCR was assessed by a Seahorse XFe96 analyzer with sequential injections of oligomycin (Oligo), FCCP, and rotenone/antimycin A (R/A). Each value was normalized to the first data-point measurement of control. n = 6–7 repeats were included for each group. The basal respiration, mitochondrial ATP production, and the maximal respiration were calculated as described in the Materials and methods. Primary cultured cortical neurons expressing GFP or GFP-SNPH were subjected to 20 min of OGD-Rep (O-R) in DIV14 (A). Neurons expressing FKBP-GFP-Mito with either FRB-KIF5B-HA (B) or FRB-BICBN-HA (C) were subjected to 20 min of OGD plus 40 min of reperfusion in DIV8 and ethanol (vehicle control) or 500 nM rapalog was added at reperfusion. (D) Primary cultured cortical neurons expressing mCherry-Tom22 or mCherry-MTB-Tom22 were subjected to 20 min of OGD plus 40 min of reperfusion in DIV14. The cleaved Casp3, mCherry, and GAPDH levels in cultured neurons were determined by Western blot. Semiquantitative analysis of Casp3 bands are shown. (E and F) Primary cultured cortical neurons from the indicated germline mice were previously transfected with FRB-BICDN-HA and FKBP-GFP-Mito by electroporation and subjected to 20 min of OGD in DIV8. Ethanol (vehicle control) or 500 nM rapalog was added at reperfusion and the neurons were harvested after 40 min of reperfusion. The cleaved Casp3, HA, and GAPDH levels in cultured neurons were determined by Western blot. Semiquantitative analysis of Casp3 bands is shown. (G) Primary cultured cortical neurons were previously transfected with GFP or GFP-SNPH by electroporation. The cultured neurons were subjected to 20 min of OGD followed by 12 h of reperfusion in DIV14. After staining with 10 nM MitoTracker Red, live cell fluorescent images of axonal mitochondria were captured by confocal microscopy for 30 min. Images show representative kymographs from three independent experiments. Columns represent the proportion of mitochondria showing different transportation patterns. n = 16 (GFP, Ctrl, and 12 h post O-R) and n = 11 (GFP-SNPH, Ctrl, and 12 h post O-R) cells from three independent experiments for each group. (H) Primary cultured cortical neurons expressing GFP or GFP-SNPH were subjected to 20 min of OGD followed by 24 h of reperfusion in DIV14. Columns represent axonal length (left panel) and number of branches (right panel). n = 25–35 cells from three independent experiments for each group. (I) Neurons expressing FKBP-GFP-Mito with FRB-BICBN-HA were subjected to 20 min of OGD followed by 24 h of reperfusion in DIV8. Columns represent axonal length (left panel) and number of branches (right panel). n = 29–31 cells from three independent experiments for each group. 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. Scale bar, 10 µm. Ctrl, control; Mito, mitochondrial/mitochondria; Num, number; Veh, vehicle.

Mitochondrial retrograde transport protects against OGD-Rep injuries. (A–C) OCR was assessed by a Seahorse XFe96 analyzer with sequential injections of oligomycin (Oligo), FCCP, and rotenone/antimycin A (R/A). Each value was normalized to the first data-point measurement of control. n = 6–7 repeats were included for each group. The basal respiration, mitochondrial ATP production, and the maximal respiration were calculated as described in the Materials and methods. Primary cultured cortical neurons expressing GFP or GFP-SNPH were subjected to 20 min of OGD-Rep (O-R) in DIV14 (A). Neurons expressing FKBP-GFP-Mito with either FRB-KIF5B-HA (B) or FRB-BICBN-HA (C) were subjected to 20 min of OGD plus 40 min of reperfusion in DIV8 and ethanol (vehicle control) or 500 nM rapalog was added at reperfusion. (D) Primary cultured cortical neurons expressing mCherry-Tom22 or mCherry-MTB-Tom22 were subjected to 20 min of OGD plus 40 min of reperfusion in DIV14. The cleaved Casp3, mCherry, and GAPDH levels in cultured neurons were determined by Western blot. Semiquantitative analysis of Casp3 bands are shown. (E and F) Primary cultured cortical neurons from the indicated germline mice were previously transfected with FRB-BICDN-HA and FKBP-GFP-Mito by electroporation and subjected to 20 min of OGD in DIV8. Ethanol (vehicle control) or 500 nM rapalog was added at reperfusion and the neurons were harvested after 40 min of reperfusion. The cleaved Casp3, HA, and GAPDH levels in cultured neurons were determined by Western blot. Semiquantitative analysis of Casp3 bands is shown. (G) Primary cultured cortical neurons were previously transfected with GFP or GFP-SNPH by electroporation. The cultured neurons were subjected to 20 min of OGD followed by 12 h of reperfusion in DIV14. After staining with 10 nM MitoTracker Red, live cell fluorescent images of axonal mitochondria were captured by confocal microscopy for 30 min. Images show representative kymographs from three independent experiments. Columns represent the proportion of mitochondria showing different transportation patterns. n = 16 (GFP, Ctrl, and 12 h post O-R) and n = 11 (GFP-SNPH, Ctrl, and 12 h post O-R) cells from three independent experiments for each group. (H) Primary cultured cortical neurons expressing GFP or GFP-SNPH were subjected to 20 min of OGD followed by 24 h of reperfusion in DIV14. Columns represent axonal length (left panel) and number of branches (right panel). n = 25–35 cells from three independent experiments for each group. (I) Neurons expressing FKBP-GFP-Mito with FRB-BICBN-HA were subjected to 20 min of OGD followed by 24 h of reperfusion in DIV8. Columns represent axonal length (left panel) and number of branches (right panel). n = 29–31 cells from three independent experiments for each group. 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. Scale bar, 10 µm. Ctrl, control; Mito, mitochondrial/mitochondria; Num, number; Veh, vehicle.

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