Figure 2.
Figure 2. Axonal mitophagy is not responsible for OGD-Rep–induced axonal mitochondrial loss. (A–E) Primary cultured cortical neurons expressing MitoQC were subjected to 20 min of OGD followed by 40 min of reperfusion (O-R) in DIV8. (B) Columns present the proportion of mCherry-positive (mCherry+) mitochondrial area to soma area. (C) Columns present mCherry-positive (mCherry+) mitochondrial area per 100-µm axon. (D) Columns present the proportion of GFP-negative–mCherry-positive (GFP-/mCherry+) mitochondrial area to soma area. (E) Columns present GFP-negative–mCherry-positive (GFP-/mCherry+) mitochondrial area per 100-µm axon. (F–H) Primary cultured cortical neurons were previously transfected with mCherry-LC3B and Mito-GFP by electroporation. The cultured neurons were treated with PBS as a vehicle control or 20 µM Baf A1 for 2 h and then subjected to 20 min of OGD. After 40 min of reperfusion, fluorescent images were captured by confocal microscopy. (F) Images show representative examples from three independent experiments. (G and H) Columns represent the number of mCherry-LC3B–positive puncta (G) and the number of mitochondria colocalized with LC3B puncta (H). n = 25–37 cells from three independent experiments for each group. (I–K) Primary cultured cortical neurons from the indicated germline mice were subjected to 20 min of OGD in DIV8. The mitochondrial marker Hsp60 and neuronal marker β-tubulin III were stained by immunocytochemistry at 40 min after reperfusion. (I) The ATG7 level in primary cultured neurons of WT and Atg7fl/fl;nes-Cre mice was measured by Western blot. (J) Images show the representative straightened cultured neurons. (K) Columns represent the somatic mitochondrial (upper panel) and axonal mitochondrial area per 100-µm axon (lower panel), respectively. n = 30–46 neurons from three independent experiments. (L) Cortical neurons from the indicated germline mice were cultured in a microfluidic platform and subjected to 20 min of OGD in DIV8. DNA and RNA in soma and axon compartments were extracted separately at 40 min after reperfusion and amplified by quantitative PCR. Columns represent the relative somatic (left panel) and axonal (right panel) mitochondrial DNA (mtDNA) levels, which are indicated, respectively, by the mt-Atp6 (mitochondria-encoded DNA) to Rpl13 (nucleus-encoded DNA) ratio and mt-Atp6 to Csf1 (axonal housekeeping) ratio. The experiments were repeated three times and the data are expressed as means ± SEM. Statistical comparisons were performed with unpaired Student t tests (B–E) or one-way ANOVA with Sidak’s multiple-comparisons test (G, H, K, and L). *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.

Axonal mitophagy is not responsible for OGD-Rep–induced axonal mitochondrial loss. (A–E) Primary cultured cortical neurons expressing MitoQC were subjected to 20 min of OGD followed by 40 min of reperfusion (O-R) in DIV8. (B) Columns present the proportion of mCherry-positive (mCherry+) mitochondrial area to soma area. (C) Columns present mCherry-positive (mCherry+) mitochondrial area per 100-µm axon. (D) Columns present the proportion of GFP-negative–mCherry-positive (GFP-/mCherry+) mitochondrial area to soma area. (E) Columns present GFP-negative–mCherry-positive (GFP-/mCherry+) mitochondrial area per 100-µm axon. (F–H) Primary cultured cortical neurons were previously transfected with mCherry-LC3B and Mito-GFP by electroporation. The cultured neurons were treated with PBS as a vehicle control or 20 µM Baf A1 for 2 h and then subjected to 20 min of OGD. After 40 min of reperfusion, fluorescent images were captured by confocal microscopy. (F) Images show representative examples from three independent experiments. (G and H) Columns represent the number of mCherry-LC3B–positive puncta (G) and the number of mitochondria colocalized with LC3B puncta (H). n = 25–37 cells from three independent experiments for each group. (I–K) Primary cultured cortical neurons from the indicated germline mice were subjected to 20 min of OGD in DIV8. The mitochondrial marker Hsp60 and neuronal marker β-tubulin III were stained by immunocytochemistry at 40 min after reperfusion. (I) The ATG7 level in primary cultured neurons of WT and Atg7fl/fl;nes-Cre mice was measured by Western blot. (J) Images show the representative straightened cultured neurons. (K) Columns represent the somatic mitochondrial (upper panel) and axonal mitochondrial area per 100-µm axon (lower panel), respectively. n = 30–46 neurons from three independent experiments. (L) Cortical neurons from the indicated germline mice were cultured in a microfluidic platform and subjected to 20 min of OGD in DIV8. DNA and RNA in soma and axon compartments were extracted separately at 40 min after reperfusion and amplified by quantitative PCR. Columns represent the relative somatic (left panel) and axonal (right panel) mitochondrial DNA (mtDNA) levels, which are indicated, respectively, by the mt-Atp6 (mitochondria-encoded DNA) to Rpl13 (nucleus-encoded DNA) ratio and mt-Atp6 to Csf1 (axonal housekeeping) ratio. The experiments were repeated three times and the data are expressed as means ± SEM. Statistical comparisons were performed with unpaired Student t tests (B–E) or one-way ANOVA with Sidak’s multiple-comparisons test (G, H, K, and L). *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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