Both laminin and Schwann cell dystroglycan are necessary for proper clustering of sodium channels at nodes of Ranvier

Abstract

Nodes of Ranvier are specialized axonal domains, at which voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is mostly unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axonglial surface. Because dystroglycan is a laminin receptor, and laminin 2 mutations [merosin-deficient congenital muscular dystrophy (MDC1A)] cause reduced nerve conduction velocity, we asked whether laminins are involved. Here, we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin 2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting that other laminins compensate for the lack of laminin 2. Thus, although laminins are located at a distance from the axoglial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder.

Figure 1.

Specific laminin isoforms are present in the basal laminas above nodes of Ranvier. Images of teased fibers isolated from wild-type and dystrophic (dy^2J/dy^2J) adult mice. A-I, Double staining for the paranodal protein Caspr (red) and laminin chains (green). Laminin α2 (A, arrows) and laminin γ1 (G, arrows) are abundant in the region of basal laminas surrounding nodes. Lamin in α 5 (D, arrows) is absent along internodes (D, arrowheads) but is specifically localized in the basal laminas around nodes and paranodes (E). J-O, Laminin α1 expression is upregulated at nodes in the absence of laminin α2 chain. Teased fibers from sciatic nerves of dystrophic (dy^2J/dy^2J) and wild-type mice are stained with an anti-laminin α1 (green; J, M) and an anti-Caspr antibody (red; K, N). The immunostaining reveals the presence of the α1 chain in the nodes of Ranvier of dystrophic mice (J, arrows) where this laminin is normally absent (M). Lam, Laminin. Scale bar, 16 μm.

Figure 2.

Specific members of the dystrophin- glycoprotein complex are present at nodes. Teased fibers of adult rat sciatic nerves stained with an antibody against the paranodal marker Caspr (green in B, E, H, K; red in N). The picture shows the localization of α-dystroglycan (A, arrow) and β-dystroglycan (D, arrow) at the nodes of Ranvier. The monoclonal antibody against the dystrophin isoform, Dp116, shows a striking microvillar enrichment (G, arrow). Utrophin is also present at nodes (J, arrow), whereas dystrophin-related protein 2 and periaxin are absent at nodes (M; data not shown). The merged confocal images are shown at the right (C, F, I, L, O). DG, Dystroglycan. Scale bar, 16 μm.

Figure 3.

Proximal defects in radial sorting and reduced nerve conduction velocity in peripheral nerves of dystrophic dy^2Jdy^2J mice. Semithin cross sections of spinal roots (A) and sciatic nerve (B) from adult dy^2Jdy^2J mice and wild-type mice (C, D, respectively). Defect in radial sorting is prevalent in spinal roots of dystrophic mice, whereas in sciatic nerves few bundles of amyelinated axons are present (B, asterisks). Scale bar, 80 μm. E, Traces show the control and dystrophic profiles of compound motor action potentials recorded after stimulation at the ankle (distal) and at the sciatic notch (proximal). The onset and end of the compound motor action potential and the onset of the F-wave are indicated by flags. F, The table shows an increase in the F-wave latency and a decrease in nerve conduction velocity in dystrophic peripheral nerves (n = 7) compared with wild type (n = 3). SDs are indicated in parentheses. sn, Sciatic nerve.

Figure 4.

Ultrastructural abnormalities in microvilli and the nodal region of dystrophic dy^2Jdy^2J mice. Electron micrographs of longitudinal sections of mouse sciatic nerves show increased width of some nodes in dystrophic mice (B, D). Microvilli (m in wt) are hypotrophic in dystrophic nodes (D, enlargement from B). E-G, Immunofluorescent analysis of dystrophic teased fibers from adult sciatic nerves double stained with Caspr (E, F, mouse monoclonal in red; G, rabbit antiserum in green) and microvilli markers. The ERM proteins ezrin (F, arrows) and radixin (data not shown) and the dystrophin isoform Dp116 (G, arrow) are correctly concentrated in most Schwann cell microvilli. The phosphorylation of ERM proteins, as determined by staining with a phosphospecific antibody (E, arrow), appears normal in some dystrophic nodes and reduced in others (E, arrowhead). Scale bars: A, B, 2 μm; C, D, 1 μm; E-G, 16 μm. ERMp, Phosphorylated ERM.

Figure 5.

Abnormality in Nav clusters in the absence of laminins or dystroglycan. Immunofluorescence microscopy of teased fibers from adult sciatic nerves double stained with an anti-Nav1.6 antibody (black and white in A, C, E, G; green in the merged panels B, D, F, H) and an anti-Caspr antibody (red in the merged panels B, D, F, H). Comparative analysis of Nav clusters between dy^2Jdy^2J (A, B), dystroglycan null (C, D), laminin γ1-null (E, F), and wild-type nerves (G, H) shows that, in mutant nerves, Nav clusters are often smaller, have an irregular shape, lack definite corners, and are sometimes diffusive. I-K, Enlargements showing examples of the most common abnormal Nav clusters found in dy^2Jdy^2J nerves. The abnormalities are similar, but less severe than those of typical dystroglycan and laminin γ1-null nodes (enlarged in L-N and O-Q, respectively). U, Quantification of frequency of normal (R-T) and abnormal (I-Q) Nav clusters in teased fibers from (dy^2J/dy^2J), laminin γ1, and dystroglycan-null sciatic nerves show that abnormal clusters are more frequent in dystrophic nerves than in control (n = 288; 33%), but less frequent than in dystroglycan-null (n = 154; 58%) or laminin γ1-null nerves (n = 237; 59%). p < 0.001 by χ² test; N = 1732 (dy^2J/dy^2J); N = 516 (P0CreDGnull); N = 750 (P0Creγ1 null). Scale bar: A-H, 16 μm; I-T, 2 μm.

Figure 6.

Severe abnormalities of Nav clusters in naked axons of dystrophic roots. Confocal images of Nav clusters in dystrophic (A, B, E, F) and normal (C, D, G, H) teased spinal roots immunolabeled with anti-Nav1.6 (green) and anti-Caspr (red) antibodies in A-D, or anti-radixin (green) and anti-panNav (red) in E-H. Faint Nav immunoreactivity is present at discrete sites along dystrophic fibers (A, arrows and enlargement), likely corresponding to naked axons because of a lack of the paranodal Caspr (red) and nuclear 4′,6′-diamidino-2-phenylindole (DAPI) (blue) markers (B). Nav clusters found in these regions are small, irregularly shaped, and diffusive along the fiber (A, B, arrows), compared with Nav clusters from wild-type spinal roots (C, D). These clusters form in the absence of microvilli, as shown by the lack of radixin staining (E, F, and enlargements). Scale bar, 16 μm.

Figure 7.

Decrease in the area of Nav channel clusters in the absence of laminins or dystroglycan. Quantitative analysis of the area occupied by Nav clusters in nodes from dystrophic, laminin γ1-null, and dystroglycan-null sciatic nerves and dystrophic roots. Open columns represent the average areas stained with anti-Nav1.6 antibodies. Filled columns show the same data after normalization for fiber diameter. A reduction in the area of Nav clusters is evident in dystrophic dy^2Jdy^2J, laminin γ1, and dystroglycan-null sciatic nerves compared with controls. Similar quantitative analysis in dystrophic roots shows a severe reduction in the area of Nav clusters compared with wild-type roots.

Figure 8.

Nav clusters abnormalities in an MDC1A patient. Longitudinal sections of sural nerve biopsies from an MDC1A patient (A, B) and a control sural nerve (C, D) double stained with an antibody that recognizes all isoforms of sodium channels (PanNa⁺, red) and an anti-Caspr antibody (green). A, B, The image shows a reduction in the intensity of staining for Nav clusters (arrows, enlarged in insets) on MDC1A nerves compared with control nerves. Scale bar, 16 μm.