Figure 6.
Neural- and skeletal muscle-specific deletion of Scyl1. A, Western blot analysis of brain, liver, and skeletal muscle extracts obtained from CkmmCre+;Scyl1+/fl, CkmmCre+;Scyl1fl/fl, NesCre+;Scyl1+/fl, and NesCre+;Scyl1fl/fl mice using antibodies against SCYL1 or AP50 as loading control. Note the selective absence of SCYL1 in the brain of NesCre+;Scyl1fl/fl mice and in the skeletal muscle of CkmmCre+;Scyl1fl/fl mice. B, Immunohistochemical staining of spinal cord cross-sections from NesCre+;Scyl1+/fl and NesCre+;Scyl1fl/fl, Scyl1+/+ and Scyl1−/− mice, using an anti-SCYL1 antibody. In NesCre+;Scyl1fl/fl sections, >90% of neuronal cells are deficient in SCYL1 expression. Note the absence of SCYL1 in all neurons of spinal sections obtained from Scyl1−/− mice compared with Scyl1+/+ mice. A nonspecific nuclear staining is detected by using this antibody. This nuclear staining was also seen in Scyl1−/− mouse embryonic fibroblasts (data not shown) and in sections obtained from Scyl1−/− mice. Scale bar, 200 μm. C, Hindlimb morphology of neural and skeletal muscle mutants of Scyl1. Representative photographs of hindlimbs obtained from 12-week-old CkmmCre+;Scyl1+/fl, CkmmCre+;Scyl1fl/fl, NesCre+;Scyl1+/fl, and NesCre+;Scyl1fl/fl mice. D, Body weight of 8-week-old male (circles) and female (squares) CkmmCre+;Scyl1+/fl (males, n = 5; females, n = 4), CkmmCre+;Scyl1fl/fl (males, n = 4; females, n = 4), NesCre+;Scyl1+/fl (males, n = 5; females, n = 3), and NesCre+;Scyl1fl/fl (males, n = 5; females, n = 6) mice. Values are expressed as the mean ± SEM. E, Early onset progressive motor deficit in neural-specific but not muscle-specific mutant of Scyl1. Score of 4- and 8-week-old CkmmCre+;Scyl1+/fl [4-week-old (wo) animals, n = 18; 8-week-old animals, n = 8], CkmmCre+;Scyl1fl/fl (4-week-old animals, n = 12; 8-week-old animals, n = 7), NesCre+;Scyl1+/fl (4-week-old animals, n = 10; 8-week-old animals, n = 15), and NesCre+; Scyl1fl/fl (4-week-old animals, n = 12; 8-week-old animals, n = 19) mice. Values are expressed as the mean ± SEM. F, Motor defects in tissue-specific mutants of Scyl1. The inverted grid test was performed on 4- and 8-week-old CkmmCre+;Scyl1+/fl (4-week-old animals, n = 25; 8-week-old animals, n = 12), CkmmCre+;Scyl1fl/fl (4-week-old animals, n = 8; 8-week-old animals, n = 7), NesCre+;Scyl1+/fl (4-week-old animals, n = 10; 8-week-old animals, n = 15), and NesCre+;Scyl1fl/fl (4-week-old animals, n = 12; 8-week-old animals, n = 9) mice to assess their motor functions as described in Materials and Methods. Values are expressed as the mean ± SEM. G, Myopathy in neural-specific but not in muscle-specific mutants of Scyl1. Representative micrographs of skeletal muscles (quadriceps femoris) from CkmmCre+;Scyl1fl/fl (n = 3) mice and NesCre+;Scyl1fl/fl (n = 3) mice. Lesions in NesCre+;Scyl1fl/fl muscles include fibers of different size, angulated (atrophied) fibers surrounded by rounded fibers, group atrophy, nuclear clumps (white arrow), and centrally localized nuclei (black arrow). Scale bars: top row, 1 mm; bottom row, 50 μm. H, Neuroinflammation in the spinal ventral horn of NesCre+;Scyl1fl/fl mice. Representative immunohistochemical staining using antibodies against Iba1 (top row) and GFAP (bottom row) on spinal ventral horn sections obtained from 8-week-old control NesCre+;Scyl1+/fl (n = 3) and NesCre+;Scyl1fl/fl (n = 3) mice. Note the increased Iba1 and GFAP staining in the ventral horn of NesCre+;Scyl1fl/fl mice compared with control animals. Scale bar, 50 μm.