Figure 1. AAV9-Mediated Stasimon Gene Delivery in SMA Mice Improves Motor Function and Rescues the Loss of Proprioceptive Synapses on Motor Neurons.
(A) qRT-PCR analysis of full-length human SMN (SMN FL) mRNA in the spinal cord of SMA mice injected with AAV9-GFP, AAV9-STAS, or AAV9-SMN as well as uninjected WT mice at P11. Data represent means and SEM (n = 6). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. ***p < 0.001; ns, not significant.
(B) Western blot analysis of SMN protein levels in the spinal cord of AAV9-injected SMA mice and uninjected WT controls at P11.
(C) Righting time of uninjected (n = 10) and AAV9-STAS-injected (n = 10) WT mice as well as SMA mice injected with AAV9-GFP (n = 10), AAV9-STAS (n = 24), and AAV9-SMN (n = 15). Data represent means and SEM.
(D) Immunostaining of VGluT1+ synapses (green) and ChAT+ motor neurons (blue) in L1 spinal cord sections from SMA mice injected with AAV9-GFP, AAV9-STAS, or AAV9-SMN as well as uninjected WT mice at P11. Scale bar: 50 μm. Bottom panels show a magnified view of VGluT1 synaptic density on proximal dendrites. Scale bar: 5 μm.
(E) NMJ staining with bungarotoxin (BTX, red), Synaptophysin (Syp, green), and Neurofilament M (NF-M, blue) of QL muscles from the same groups as in (D) at P11. Scale bar: 15 μm.
(F) Number of VGluT1+ synapses on the somata of L1 motor neurons from the same groups as in (D) at P11. Data represent means and SEM from WT (n = 22), SMA+AAV9-GFP (n = 35), SMA+AAV9-STAS (n = 32), and SMA+AAV9-SMN (n = 21). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. **p < 0.01, ***p < 0.001.
(G) Density of VGluT1+ synapses on the proximal dendrites of L1 motor neurons from the same groups as in (D) at P11. Two dendritic compartments were defined based on distance from the soma. Number of dendrites scored from the 0–50-μm compartment: WT (n = 27), SMA+AAV9-GFP (n = 21), SMA+AAV9-STAS (n = 20), and SMA+AAV9-SMN (n = 15). Number of dendrites scored from the 50–100-μm compartment: WT (n = 12), SMA+AAV9-GFP (n = 14), SMA+AAV9-STAS (n = 10), and SMA+AAV9-SMN (n = 8). Data represent means and SEM. Statistics were performed with one-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.
(H) Percentage of fully denervated NMJs in the QL muscle from the same groups as in (E) at P11. Data represent means and SEM (n = 5). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. *p < 0.05, ***p < 0.001; ns, not significant.