Fig. 5.
Syt13 gene therapy extends the survival of SOD1G93A ALS mice by protecting motor neurons and preserving motor axons and neuromuscular junctions. a Schematic drawing of in vivo Syt13 delivery. SOD1G93A mice were injected with AAV9::GFP, AAV9::Syt13, or AAV9::null at a total dose of 11 × 1011 vg bilaterally in the hindlimb quadriceps and thoracic muscles at 80 days of age (early symptomatic stage). b Increased levels of Syt13 expression were detected by RT-PCR in AAV9::Syt13 SOD1G93A spinal cord versus AAV9::null spinal cord. **P < 0.01 (t(4) = 5.533, t test). c Injection of AAV9::GFP leads to GFP expression within the spinal cord 2 weeks post-injection. Co-localization of GFP (green) with ChAT (red) demonstrated that motor neurons were efficiently transduced (n = 5 mice). d The inverted grid performance of AAV9::Syt13-treated mice was significantly ameliorated with respect to AAV9::null mice (at P100, P126, and P141: P < 0.05, n = 6/group). Until 90 days all the mice were able to complete the test, holding the wire for 60 s, which is the baseline. e Kaplan–Meier survival curves demonstrates significantly extended median survival (by 20 days) in AAV9::Syt13 treated mice (n = 12) compared to AAV9::null treated animals (n = 15 AAV9::Syt13 mice, 160 ± 14 days median survival; AAV9::null mice 140 ± 5 days; χ2 = 8.86, P = 0.0029, Kaplan–Meier log rank test). f, g Representative images of motor neurons with Neurotracer staining (blue) in the lumbar segment of the spinal cords of AAV9::null (f) and AAV9::Syt13 (g) treated mice at P120. h, i Quantification of motor neurons (MNs) (h) and axons (i) in the lumbar spinal cords of AAV9::Syt13 and AAV9::null mice (mean ± SEM) at P120. Motor neuron and axon counts significantly increased in the AAV9::Syt13 treated group compared to the AAV9:null treated group (MNs: ***P < 0.0001, F(2,87) = 323.96; one-way ANOVA, n = 30 slices counted/group, 3 mice/group; axons: ***P < 0.0001, F(2,33) = 224.16; n = 12 slices counted/group, 3 mice/group; one-way ANOVA). l, m Analysis of α-bungarotoxin (BTX, red) and neurofilament M (NF-M, green) in the NMJs of tibialis anterior muscles shows that AAV9::Syt13 treatment significantly increased the number of innervated NMJs in AAV9::Syt13 treated SOD1G93A mice (n, ***P < 0.0001, χ2(2) = 95.82, contingency test) compared to AAV9::null treated SOD1G93A mice (n = 100 NMJs analyzed for each animal, 6 mice/group). o Representative image of the performance of an AAV9::Syt13 treated SOD1G93A mouse in an inverted grid test at P126. Scale bar = 75 µm in b, 100 μm in f, g, and 50 μm in l, m