Fig. 5: ICV injection of shDR6 in SOD1G93A mice leads to partial recovery of ALS model.
a Unfixed 20 μm-thick L4/L5 section of SC from P50 WT mice were labeled with mRNA-based fluorescent probes for DR6 and ChAT using RNAscope technique. Representative images of three experiments repeated independently with similar results. Scale bar: 50 μm. b L4–L5 of spinal cord of the P125 mice were frozen and cut into 15 µm-thick sections with a cryostat, visualized by immunohistochemistry using ChAT antibody. Scale bar: 100 μm. The sections were imaged on Leica confocal microscope. Representative images are shown. c ChAT stained neurons in each ventral horn hemi-section were manually counted and quantified. Data are means ± SEM of n = 4 independent experiments and were analyzed by one-way ANOVA (F(2,9) = 43.85; P < 0.0001) followed by a Neuman–Keuls post hoc test: ****P < 0.0001 NTg+sh-scram vs mutSOD1+sh-scram (d = 10.43) and NTg+sh-scram vs mutSOD1+shDR6 (d = 5.34); *P = 0.034 mutSOD1+sh-scram vs mutSOD1+shDR6 (d = 1.63). d Survival of NTg mice (n = 10) and Tg mutSOD1 mice injected with sh-scram (n = 13) or shDR6 (n = 13) over time. Data were analyzed by Kaplan–Meier estimator with Log rank test which revealed statistical significant difference in survival between NTg vs Tg mutSOD1 mice injected with sh-scram or shDR6 (P < 0.0001, df = 2). However, log rank test revealed no statistical significant difference in survival between Tg mutSOD1 mice injected with sh-scram compared to Tg mutSOD1 mice injected with shDR6. e Loaded grid assay was performed in NTg (n = 10 mice) and Tg mutSOD1 mice injected with sh-scram (n = 19 mice) or shDR6 (n = 16 mice) over the course of the disease. Data were analyzed by non-linear regression curve fit using least sum-of-squares method and revealed that shDR6-injected Tg mutSOD1 mice motor performance is significantly different than sh-scram injected Tg mutSOD1 mice (F(4,234) = 11.41; P < 0.0001). f Tibialis anterior muscle from NTg mice (black) and Tg mutSOD1 mice injected with sh-scram (red) or shDR6 (blue) was processed and incubated with α-bungarotoxin-594, anti-neurofilament and anti-synaptophysin antibodies. Representative images are shown. Scale bar: 50 μm. g Quantitative assessment of muscle innervation was done by counting at least 50 neuromuscular junctions (NMJs) by first imaging α-bungarotoxin-594 and then confirming innervation using anti-neurofilament. Studies were done at P90 in NTg mice injected with sh-scram (n = 3) and Tg mutSOD1 mice injected with sh-scram (n = 4) or shDR6 (n = 2) and at P125, in NTg mice injected with sh-scram (n = 3) and Tg mutSOD1 mice injected with sh-scram (n = 3) or shDR6 (n = 3). Data are means ± SEM of independent mice (n) and were analyzed by two-way ANOVA (genotype main factor F(2, 12) = 37.26; P < 0.0001 with unbalanced design followed by Sidak post hoc test: **P = 0.0064 P90 NTg sh-scram vs Tg mutSOD1 sh-scram (CI: 13.79–81.81%; d = 3.07); ****P ≤ 0.0001 P125 NTg sh-scrambled vs Tg mutSOD1 sh-scrambled (CI: 53.25–126%; d = 23.40); ***P = 0.0001 P125 NTg sh-scram vs Tg mutSOD1 shDR6 (CI: 47.23–119.9%; d = 14.84). See also Supplementary Figs. 6 and 7. Source data provided as source data file.
