Article Figures & Data
Figures
- Fig. 1.
Alteration of ion channel clusters in CST-deficient mice. Double immunostaining of Na+ and K+ channels was performed in the CNS and PNS axons.a, In the spinal cord of wild-type mice, Na+ channels (green) were restricted to the nodes of Ranvier, whereas the K+channels (red) were concentrated in the juxtaparanodes.b–e, In contrast to the spinal cord of CST-deficient mice, the K+ channels were aberrantly localized to the paranodes (b, c, d). The Na+channels formed clusters in the nodes; however, the domains were longer (b) in comparison with wild type (a). d, The binary form of Na+ channel clusters is frequently observed.e, Heminode formation that labeled just one side of the juxtaparanode or paranode with the Kv1.1 antibody was also observed.f, In the spinal root of wild-type mouse, Na+ channels (green) were restricted to the nodes of Ranvier, whereas the K+channels (red) were concentrated in the juxtaparanodes.g, In contrast, in the spinal root of this mutant mouse, the K+ channel clusters were aberrantly localized to the paranode. Scale bar, 5 μm.
- Fig. 2.
CST-deficient mice revealed severely disrupted Caspr clustering both in the CNS and in the PNS. a, d, In 6-week-old wild-type mice, Caspr (green) was highly concentrated in the paranodal regions of the optic nerve (a) and in the paranodal region of the spinal root (d). b (6 weeks),c (10 weeks), e (6 weeks), In contrast, CST-deficient mice exhibited a more diffuse labeling pattern. The number of K+ channel clusters is markedly decreased in the mutant optic nerve as well as in the spinal root.f, Western blot analysis of brain homogenate revealed no differences in the amount of Caspr protein between wild-type (+/+) and mutant (−/−) mice in the CNS axons at 18 weeks of age. Scale bar, 10 μm.
- Fig. 3.
The ion channel clusters were reduced with age in CST-deficient mice. The optic nerves from 4-week-old (a, d), 10-week-old (b, e), and 22-week-old (c, f) animals were double stained by Na+ (green) and K+ (red) channel antibodies. In wild-type mice (a–c), the numbers of Na+ and K+ channel clusters were maintained without any changes throughout all ages. In the mutants (d–f), both types of ion channel clusters were reduced, and irregular forms (f) became prominent with age. Scale bar, 10 μm. g, h, Numbers of K+ channel (g) and Na+ channel (h) clusters per field of view in the optic nerves of wild-type (♦) and mutant mice (▪) plotted at different ages. An average of 12 FOVs was analyzed at each age. Single and double asterisksindicate p < 0.01 and p < 0.05, respectively. Wherever statistics are used, results are given as mean ± SD. i, The nodal lengths in the optic nerves of wild-type mice (white bar) and mutant mice (black bar), which were revealed by Na+ channel immunoreactivity. Error bars indicate SD. Single and double asterisks indicatep < 0.001 and p < 0.05, respectively. 4w, Four weeks; 6w, six weeks; 10w, 10 weeks; 14w, 14 weeks;16w, 16 weeks; 22w, 22 weeks.
- Fig. 4.
The changes of shapes and fluorescence intensities of Na+ channel clusters with age in CST-deficient mice. The optic nerves from wild-type mice (a) and 6-week-old (b), 16-week-old (c), 22-week-old (d), and 36-week-old (e) mutant mice were stained by Na+ channel antibodies. In the mutant, the numbers of Na+ channel clusters decreased significantly with age, and the clusters of longer shapes with lower intensities became more and more prominent in older mutant animals (compared or e with a).f, Typical Na+ channel clusters in wild-type mice whose intensity levels are >150 using LSM510 software (see Materials and Methods). g–i, The representative clusters with abnormal shapes (g, h, ∗) and intensities (h, i, arrowheads) observed in the mutants. Scale bars: a–e, 7 μm; f, g, 2 μm. j, An immunoblot of wild-type (+/+) and mutant (−/−) mice spinal cords at 36 weeks of age probed with anti-mouse Na+ channel antibody. The bands at ∼260 kDa represent Na+ channel α subunit. There were no differences in the amount of Na+ channel protein between wild-type and mutant mice.
- Fig. 5.
CST-deficient mice displayed no signs of demyelination. The staining pattern of MBP (a, b) exhibited no differences between wild-type (a) and CST-deficient (b) mice in optic nerves at 22 weeks of age, whereas it is apparently different in demyelinating mutant mice generated by overexpression of PLP gene (Kagawa et al., 1996) at 28 weeks of age (c). Scale bar, 10 μm. Analysis of the membrane fraction of the brain from wild-type (+/+) and mutant (−/−) mice revealed no differences in the levels of MBP (d) and PLP (e) at 18 weeks of age. Electron microscopic analysis showed no signs of demyelination. Cross-sectional analysis of optic nerves from 29-week-old wild-type (f) and CST-deficient (g) mice revealed no difference in myelin stability. In addition, common indications of demyelination, such as myeloid figures and microglial activation, were not observed in the sulfatide-deficient mice at any of the ages analyzed. Spinal cord analysis revealed similar findings (data not shown).
- Fig. 6.
Optic nerves from CST-deficient mice exhibited severe disruptions in Caspr cluster formation during development (P17). Optic nerves from control littermates (a) and CST-deficient mice (b) were double labeled to indicate the localization of Na+ channels (green) and Caspr (red). Caspr clustering was hardly observed, and this protein was distributed diffusely along the axons in CST-deficient mice (b). In contrast, Na+ channel cluster formation was comparable between mutants (b,arrows) and control littermates. Scale bar, 10 μm.
- Fig. 7.
The distribution of PSD-95-related molecule is altered in CST-deficient mice. Optic nerves from control littermates (a) and CST-deficient mice (b) were double labeled by the antibodies against Na+ channels (green) and PSD-95 (red). In the wild-type controls, the PSD-95-related molecule formed clusters primarily in the juxtaparanodal regions, where the K+ channels were usually localized. In contrast, in the mutants, the PSD-95-related molecule was concentrated in the paranodal regions rather than at the juxtaparanode and was sometimes distributed diffusely throughout the internode. Scale bar, 10 μm.
Tables
- Table 1.
Quantification of Na+ channel clusters that classified into the fluorescence intensities
Fluorescence intensity Wild type CST-deficient mice 6 weeks (n = 11) 6 weeks (n = 13) 16 weeks (n = 11) 22 weeks (n = 15) 36 weeks (n = 14) ≥150 37 ± 6 17 ± 4 10 ± 4 5 ± 3 3 ± 2 <150 2 ± 1 13 ± 5 7 ± 5 7 ± 3 4 ± 3 The Na+ channel clusters in the optic nerves were counted per FOV (1 FOV = 48.7 × 48.7 mm2). The fluorescence intensity was classified as ≥150 <150 using LSM510 software.
