![In vivo hPLS3 overexpression improves TrkB phosphorylation and TrkB and Cav2.1 localization in NMJs of SMNΔ7 mice. (A) Ratio of the area of the presynapse (through Synaptophysin-1 [SYP]) vs. postsynapse (ACh receptors through Bungarotoxin [BTX]) of NMJs in the Transversus abdominis anterior (TVA) of control and SMNΔ7 P10-P11 animals that were injected with AAV9-EGFP and AAV9-hPLS3 (N = 10/10/5/7, n = 185/185/130/185; ANOVA Kruskal–Wallis, *P ≤ 0.05; ****P ≤ 0.0001). (B) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against G-actin (through Vitamin D-binding protein [DBP], gray) and SYP (magenta). Dotted line depicts outline of the presynapse (SYP signal). Normalized mean gray values of G-actin (N = 3/3/3/4, n = 60/60/60/60; ANOVA Kruskal–Wallis, *P ≤ 0.05; ****P ≤ 0.0001). (C) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against TrkB (gray), SYP (magenta), and BTX (green). Dotted line depicts outline of the presynapse (SYP signal). Normalized mean gray values of TrkB (N = 5/5/3/4, n = 78/78/52/75; ANOVA Kruskal–Wallis, *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001). (D) Unstimulated and 30 min BDNF stimulated NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP and AAV9-hPLS3 and stained against p-TrkB (gray), SYP (magenta), and BTX (green). Dotted line depicts outline of the presynapse (SYP). Normalized mean gray values of p-TrkB (N = 6/4/3/3, n = 54/46/46/54; ANOVA Kruskal–Wallis, *P ≤ 0.05; ***P ≤ 0.001 ****P ≤ 0.0001). (E) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against Cav2.1 (gray), neurofilament (NF, red), and BTX (green). Dotted yellow line depicts outline of the postsynapse (BTX signal). Normalized mean gray values of Cav2.1 (N = 3/4/3/4, n = 40/50/54/62; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). Data are presented as scatter dot plot with bar. Bar represents the mean ± SEM. Scale bars: 10 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/222/3/10.1083_jcb.202204113/2/jcb_202204113_fig10.png?Expires=1771602369&Signature=tCty2EzLKYq~QT~6wcPPP56s4kXsbrJV-eMA5KhX4HLLN5Ip4RggRjSRgIgUHlJi9TA11ofKCfaYSU1K7-seYM0fWB9kJ46FmgW0ZQayFWfyJXw5yrGi4jeU8rD70lPrFUYXiPtqQ-riaCrzaV5-auXW3nQYZrJgsM1VYSpysbaFdVyHi71sGiqEBNX3SQ2JwrQpI2hxp1Knt8DtU5m~WT69erGqFJ7~rUBIl46Ng~cXrSoAiT6iqAQWs0eyPNSbyPLS4klY7ltYkPDycOe9KlYaIHdytCFzYKOXaW5LjdcjPKlzibEON6WJkfszKbFc8HMEpPG3dPyjB5GgKomhAQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In vivo hPLS3 overexpression improves TrkB phosphorylation and TrkB and Ca v 2.1 localization in NMJs of SMNΔ7 mice. (A) Ratio of the area of the presynapse (through Synaptophysin-1 [SYP]) vs. postsynapse (ACh receptors through Bungarotoxin [BTX]) of NMJs in the Transversus abdominis anterior (TVA) of control and SMNΔ7 P10-P11 animals that were injected with AAV9-EGFP and AAV9-hPLS3 (N = 10/10/5/7, n = 185/185/130/185; ANOVA Kruskal–Wallis, *P ≤ 0.05; ****P ≤ 0.0001). (B) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against G-actin (through Vitamin D-binding protein [DBP], gray) and SYP (magenta). Dotted line depicts outline of the presynapse (SYP signal). Normalized mean gray values of G-actin (N = 3/3/3/4, n = 60/60/60/60; ANOVA Kruskal–Wallis, *P ≤ 0.05; ****P ≤ 0.0001). (C) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against TrkB (gray), SYP (magenta), and BTX (green). Dotted line depicts outline of the presynapse (SYP signal). Normalized mean gray values of TrkB (N = 5/5/3/4, n = 78/78/52/75; ANOVA Kruskal–Wallis, *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001). (D) Unstimulated and 30 min BDNF stimulated NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP and AAV9-hPLS3 and stained against p-TrkB (gray), SYP (magenta), and BTX (green). Dotted line depicts outline of the presynapse (SYP). Normalized mean gray values of p-TrkB (N = 6/4/3/3, n = 54/46/46/54; ANOVA Kruskal–Wallis, *P ≤ 0.05; ***P ≤ 0.001 ****P ≤ 0.0001). (E) NMJs in the TVA muscle of control and SMNΔ7 P10 animals that were injected with AAV9-EGFP or AAV9-hPLS3 stained against Cav2.1 (gray), neurofilament (NF, red), and BTX (green). Dotted yellow line depicts outline of the postsynapse (BTX signal). Normalized mean gray values of Cav2.1 (N = 3/4/3/4, n = 40/50/54/62; ANOVA Kruskal–Wallis, **P ≤ 0.01; ****P ≤ 0.0001). Data are presented as scatter dot plot with bar. Bar represents the mean ± SEM. Scale bars: 10 µm.
