Mature neurons regain regrowth capacity by enhancing mitochondrial transport. (A and B) Kymographs (A) and quantitative analysis (B) showing mitochondrial motility along microgrooves in microfluidic chambers. Time-lapse imaging was recorded in cortical neurons at DIV12 for a total of 100 frames with 5-s intervals. In kymographs, vertical lines represent stationary organelles; oblique lines or curves to the right indicate anterograde transport toward distal terminals. Note that the relative motility in control neurons expressing HA is significantly higher than the motility in neurons overexpressing HA-SNPH, but lower than the motility in neurons expressing HA-Miro1. (C and D) Representative microfluidic images (C) and quantitative analysis (D) showing regrowth capacity in mature cortical neurons. Neurons infected with lentivirus encoding SNPH, SNPH-dMTB, or Miro1 were grown on microfluidic chambers for 12 d before axotomy. Axon regeneration was evaluated 6 d after axotomy (DIV18). Note that abolishing mitochondrial transport by expressing SNPH shows failed axon regrowth, whereas enhancing mitochondrial transport by expressing Miro1 robustly increases axon regrowth capacity. (E) Partial recovery of regrowth capacity in the SNPH-expressing neurons by ATP application. The electroporated neurons were immediately plated on a microfluidic chamber with medium containing 200 µM ATP. (F) Recovery of regrowth capacity 14 h after axotomy in snph KO neurons is largely abolished by blocking mitochondrial ATP generation with 2 µM oligomycin (Oligo). The axonal chambers were briefly treated with 2 µM oligomycin for 4 h after axotomy. (G and H) Representative images (G) and quantitative analysis (H) showing enhanced axonal regrowth in snph KO adult DRG neurons. DRG neurons isolated from adult (P60) WT or snph KO mice were immunostained with βIII-tubulin at DIV1. Axon regrowth was quantified by Sholl analysis. The snph-deficient adult DRG neurons display increased axon branching as indicated by the total number of axonal intersections. Mitochondrial motility data were analyzed from the total number of microgrooves (B); axonal regrowth data were analyzed in terminal chambers where new axons grow from a total number of microgrooves (E and F), total number of terminal chambers (D), or total number of DRG neurons (H) indicated within bars or in parentheses and expressed as mean ± SE and by one-way ANOVA test (B and D), Student’s t test (F and H), or Mann–Whitney U test (E). Bars: (A) 20 µm; (C and G) 100 µm.