Overexpression of hPLS3 corrects TrkB surface translocation after BDNF stimulation in Smn-deficient motoneurons. (A) Control and Smn^−/−;SMN2 growth cones unstimulated, 5 min BDNF stimulated and 5 min BDNF stimulated followed by a 10 min recovery phase (recovery assay) stained against surface TrkB (gray) and Synaptophysin-1 (SYP, magenta). Normalized mean gray values of surface TrkB (N = 3, n = 90; ANOVA Kruskal–Wallis, **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (B) Normalized mean gray values of surface TrkB in axon terminals of control motoneurons that were treated with 0.5 µM Cytochalasin D (CytoD), 10 µM Nocodazole and 20 µM Cycloheximide prior to the recovery assay (N = 4, n = 90; ANOVA Kruskal–Wallis, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (C) shLuci and shPls3 growth cones subjected to the recovery assay and stained against surface TrkB (gray) and SYP (magenta). Normalized mean gray values of surface TrkB (N = 3, n = 85; ANOVA Kruskal–Wallis, **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (D) LV-mCh and LV-hPLS3 transduced control and Smn^−/−;SMN2 growth cones were subjected to the recovery assay and stained against surface TrkB (gray) and SYP (magenta). Normalized mean gray values of surface TrkB (N = 3, n = 80; ANOVA Kruskal–Wallis, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (E) Schematic of the TrkB recycling assay. For details, see Materials and methods section. (F) Growth cones of control and Smn^−/−;SMN2; as well as LV-hPLS3-Smn^−/−;SMN2 motoneurons stained against surface TrkB (red = receptors that have been on the cell surface prior to BDNF stimulation, green = receptors located at the cell surface after recovery, yellow = co-localizing). Quantification (N = 3, n = 98) of (green), the number of recovered receptors (percentage of total TrkB) and (yellow) the number of recycled receptors (percentage of recovered TrkB). Data are presented as scatter dot plot with bar/bar. Bar represents the mean ± SEM. Scale bars: 5 µm.