Fig. 2

Age-dependent motor dysfunction and muscle atrophy of heterozygous male N390D/+ mice. a Rotarod test of heterozygous knock-in mouse lines. Both lines of N390D/+ male mice (left panel) exhibited motor dysfunction at the age of 6 months, but not the two A315T/+ mouse lines (right panel). The average time periods of mice staying on the rotarod (mean ± SEM) at different ages are shown in the line graphs. *p < 0.05; **p < 0.01, n.s., not significant. The numbers (N) of mice analyzed per group are listed in the figure. b Hind-limb clasping test. Exemplified are the abnormal clasping behavior of 8-month old N390D/+ male mice (right 2 panels) and their +/+ littermates (left 2 panels). c ALS-like kyphosis phenotype of N390D/+ male mice (right panels) and the +/+ littermates (left panels) at the ages of 8 months and 18 months, respectively. d Cartoon illustration of the standard measurement position (arrow) of the calf muscle by red I symbol. Photos of the hind-limb muscles of 3-, 6-, 12- and 24-month old N390D/+ male mice, and 24-month old +/+ and A315T/+ male mice, respectively. The statistical results are shown on the lower left side. The red lines point to the measured sites of the muscle. Scale bars 1 cm. *p < 0.05; **p < 0.01; #p < 0.001. e The soleus muscles of hind-limb were isolated from +/+, A315T/+ and N390D/+ male mice at different ages. *p < 0.05. f The NMJ of soleus from +/+ and N390D/+ male mice were analyzed by whole-mount IF staining using α-Bungarotoxin-555 (α-BTX, red) and anti-SV2A (green, a synaptic vesicle marker) plus anti-NF (green, a nerve marker). The white box indicates the magnified region. N = 3 (randomly chosen from each of the two independent lines) per group. Scales bars 50 μm. g The percentages of denervation of NMJs of the soleus muscles of hind-limb at the ages of 3 and 6 months, respectively. The numbers of NMJ analyzed are list in the bottom of the table