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. 2017 Sep 6;37(36):8830–8844. doi: 10.1523/JNEUROSCI.0730-17.2017

Figure 5.

Figure 5.

Calpain overactivation contributes to motor deficits and motor neuron loss in GLAST+/−/GLT1-cKO mice. A, Representative immunoblot that shows proteolysis of Nup153, Nup88, Nup62, laminB1, and α-spectrin in mouse brain homogenate that was incubated with exogenous calcium and/or a calpain inhibitor, SNJ-1945 (100 μm). B, Representative immunoblot showing proteolysis of p35 in lumbar ventral horn from control and GLAST+/−/GLT1-cKO mice at 5 and 6 weeks of age. C, Quantification of the p25/p35 ratio from control mice (P5W, n = 3; P6W, n = 4) and GLAST+/−/GLT1-cKO mice (n = 3 at each age). Band intensities of p35 and p25 were normalized to α-tubulin. **p < 0.01. n.s., Not significant (unpaired two-tailed t test). D, Measurement of calpain activity of lumbar ventral horn form control mice (P4W, n = 5; P5W, n = 6; P6W, n = 5) and GLAST+/−/GLT1-cKO mice (P4W, n = 6; P5W, n = 5; P6W, n = 6). Relative fluorescence units (RFUs) were compared by calculating the fold difference in enzyme activity. *p < 0.05; n.s., Not significant (unpaired two-tailed t test). E, TDP-43 immunofluorescence in the lumbar ventral horns from control at 7 weeks of age (the top row of a panel) and GLAST+/−/GLT1-cKO mice at 7 (the second and third rows of a panel) and 9 (the bottom row of a panel) weeks of age. Sections were double labeled with TDP-43 (red) and ChAT (white). Nuclei were visualized with DAPI. The second row of a panel shows mislocalization of TDP-43 to the cytoplasm in motor neurons of GLAST+/−/GLT1-cKO mice. The third row of a panel shows reduction of nuclear TDP-43 without cytoplasmic TDP-43 immunoreactivity in motor neurons of GLAST+/−/GLT1-cKO mice. The bottom row of a panel shows loss of TDP-43 from both the nucleus (yellow arrowhead) and the cytoplasm in motor neurons of GLAST+/−/GLT1-cKO mice. Scale bar, 10 μm. F, The percentage of motor neurons showing aberrant staining pattern of TDP-43 from 5, 6, 7, and 9 weeks of age of control mice (P5W, n = 7; P6W, n = 6; P7W, n = 5; P9W, n = 6) and GLAST+/−/GLT1-cKO mice (P5W, n = 7; P6W, n = 5; P7W, n = 6; P9W, n = 5). *p < 0.05, **p < 0.01 (unpaired two-tailed t test). G, Representative immunoblot that shows proteolysis of TDP-43 and β-actin in mouse brain homogenate that was incubated with exogenous calcium and/or a calpain inhibitor, SNJ-1945 (100 μm). Black and white arrowheads indicate a full-length and cleaved fragment of TDP-43, respectively. H, SNJ-1945 treatment delayed motor deficits in the hanging wire test (n = 8 for each groups). *p < 0.05 (post hoc unpaired two-tailed t test at corresponding time point after two-way repeated-measures ANOVA). I, ChAT immunofluorescence of the lumbar ventral horns from vehicle-treated and SNJ-1945-treated GLAST+/−/GLT1-cKO mice at 7 weeks of age. Scale bar, 100 μm. J, Quantification of ChAT-positive motor neurons at 7 weeks of age (n = 5 for vehicle-treated and n = 6 for SNJ-1945-treated GLAST+/−/GLT1-cKO mice). **p < 0.01 (unpaired two-tailed t test). K, Nup153 immunofluorescence of lumbar ventral horns from vehicle-treated and SNJ-1945-treated GLAST+/−/GLT1-cKO mice at 7 weeks of age. Sections were triple labeled with anti-Nup153 (magenta), DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in the right panel. Scale bars: left, 10 μm; right, 5 μm. L, Quantitative analysis of Nup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons decreased in GLAST+/−/GLT1-cKO mice treated with SNJ-1945 (n = 4 for each groups). **p < 0.01 (unpaired two-tailed t test). M, Nup153 immunofluorescence of lumbar ventral horns from vehicle-treated and perampanel-treated GLAST+/−/GLT1-cKO mice at 7 weeks of age. Sections were triple labeled with anti-Nup153 (magenta), DAPI (blue), and Neurotrace (fluorescent Nissl stain, white). Rectangles in the middle panel are enlarged in the right panel. Scale bars: left, 20 μm; right, 5 μm. N, Quantitative analysis of Nup153-immunostained sections. The percentage of Nup153-negative large ventral horn neurons decreased in GLAST+/−/GLT1-cKO mice treated with perampanel (n = 4 for vehicle-treated and n = 5 for perampanel-treated GLAST+/−/GLT1-cKO mice). *p < 0.05 (unpaired two-tailed t test). All data are expressed as the mean ± SEM.