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. 2015 Aug 19;35(33):11514–11531. doi: 10.1523/JNEUROSCI.5288-14.2015

Figure 4.

Figure 4.

Neuron-specific expression of UbG76V-GFP-Syb2 leads to adult-onset paralysis and progressive degeneration of motor nerve terminals. A, external phenotype of an 8-month-old UbG76V-GFP-Syb2 transgenic mouse, compared with a nontransgenic littermate control mouse. The UbG76V-GFP-Syb2 transgenic mouse is paralyzed at this age. B, Kaplan–Meier survival curve of UbG76V-GFP-Syb2 (N = 131), GFP-Syb2 (N = 118), and nontransgenic control mice (N = 63). The majority of the UbG76V-GFP-Syb2 mice died within 12 months of age. C, Cross sections of ventral spinal cords immunostained with anti-GFAP antibodies. GFAP-positive astrocytes (arrowheads) are markedly increased in UbG76V-GFP-Syb2 mice, compared with GFP-Syb2 or non-Tg control mice. Dashed lines mark the boundary of the ventral horn. D, Quantitative analysis of the NMJs in the fourth lumbrical muscles from hemizygous UbG76V-GFP-Syb2 mice, compared with age-matched littermate control mice. The numbers of the NMJs obtained from three pairs of mice at each age are as follows: 1 month (control, n = 356; UbG76V-GFP-Syb2, n = 420); 3 months (control, n = 411; UbG76V-GFP-Syb2, n = 327); 5 months (control, n = 301; UbG76V-GFP-Syb2, n = 357); 8 months (control, n = 325; UbG76V-GFP-Syb2, n = 320). E, More than 60% of end-plates (EPs) in UbG76V-GFP-Syb2 mice at 5 months of age, and >90% at 8 months of age, are either partially denervated or totally denervated. In contrast, 100% of the NMJs in control mice are fully innervated at 1, 3, 5, and 8 months of age. F, Motor neuron morphology (black arrowheads) revealed by Toluidine Blue-stained semithin (1 μm) sections of the ventral horn of lumbar spinal cord from UbG76V-GFP-Syb2 and nontransgenic littermate control mice (8 months of age). G, Quantification of motor neuron numbers from transverse sections (at 10 μm thickness) of lumbar spinal segments (L3–L5). At 6 months of age, motor neuron numbers (n = 3006 ± 35, N = 3 mice) in UbG76V-GFP-Syb2 mice were similar to non-Tg control mice (n = 2875 ± 120, N = 3 mice). At 8 months of age, motor number numbers were significantly (p = 0.0012) decreased in UbG76V-GFP-Syb2 mice (n = 1933 ± 106, N = 3 mice), compared with age-matched non-Tg control mice (n = 2715 ± 35, N = 3 mice). H, I, Cross section of lumbrical muscles from non-Tg control and UbG76V-GFP-Syb2 mice (8 months of age): black arrowhead in I points to a cluster of atrophic muscle fibers in UbG76V-GFP-Syb2 mice. J–O, The NMJ morphology in homozygous GFP-Syb2 and UbG76V-GFP-Syb2 mice (7 weeks of age). A small bundle of teased thigh muscle fibers (J, M, phase contrast) from GFP-Syb2 (J–L) and UbG76V-GFP-Syb2 mice (M–O) were labeled with Texas Red-conjugated α-bungarotoxin (L, O). The nerve terminals were identified by the presence of GFP fluorescence (K, N). In GFP-Syb2 mice, motor nerve terminals appear in a typical “pretzel-like” shape (arrow in K). In contrast, in UbG76V-GFP-Syb2 mice, nerve terminals appear shrunken (1 in N) or swollen (2 and 3 in N); muscle fibers appear atrophic (* in M), and two of five muscle fibers are completely denervated (4 and 5 in O). Scale bars: C, 200 μm; E, 50 μm; F, 20 μm; H, I, 50 μm; J–O, 50 μm.