Anti-GM1 antibodies cause complement-mediated disruption of sodium channel clusters in peripheral motor nerve fibers

Abstract

Voltage-gated Na+ (Na(v)) channels are highly concentrated at nodes of Ranvier in myelinated axons and facilitate rapid action potential conduction. Autoantibodies to gangliosides such as GM1 have been proposed to disrupt nodal Nav channels and lead to Guillain-Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness. To test this hypothesis, we examined the molecular organization of nodes in a disease model caused by immunization with gangliosides. At the acute phase with progressing limb weakness, Na(v) channel clusters were disrupted or disappeared at abnormally lengthened nodes concomitant with deposition of IgG and complement products. Paranodal axoglial junctions, the nodal cytoskeleton, and Schwann cell microvilli, all of which stabilize Na(v) channel clusters, were also disrupted. The nodal molecules disappeared in lesions with complement deposition but no localization of macrophages. During recovery, complement deposition at nodes decreased, and Na(v) channels redistributed on both sides of affected nodes. These results suggest that Na(v) channel alterations occur as a consequence of complement-mediated disruption of interactions between axons and Schwann cells. Our findings support the idea that acute motor axonal neuropathy is a disease that specifically disrupts the nodes of Ranvier.

Figure 1.

Clinical, serological, and morphological features of AMAN rabbits. A, Clinical course of each paralyzed rabbit. Eight rabbits were divided into three groups: Bg-16, Bg-17, and Bg-18 at acute progressive phase (within a few days after the neurological onset); Bg-19 and Bg-20 at early recovery phase (∼2 weeks after the onset); Bg-21, Bg-22, and Bg-23 at late recovery phase (>4 weeks after the onset). B, Thin-layer chromatography with immunostaining. Lane 1, Bovine brain ganglioside mixture; lane 2, ganglioside mixture extracted from rabbit peripheral nerves. Plasma IgG obtained from AMAN rabbits Bg-16, Bg-17, and Bg-18 strongly react with GM1 from both bovine brain and rabbit peripheral nerve. IgG from Bg-16 and Bg-18 also weakly bind to GD1b. The immunostaining patterns of IgG from paralyzed rabbits are similar to IgG from a patient with AMAN. Anti-GM1 mAb (GB2) reacts strongly and specifically to GM1. C, D, Electron microscopy showing lengthened nodes in ventral roots from a rabbit 2 d after onset (C) and Bg-20 at the early recovery phase (D). Schwann cell cytoplasm expands forming bulb-like structure filled with mitochondria. No typical microvilli of Schwann cells are obvious. E, Higher magnification of the boxed area in D. Some paranodal loops (arrowheads) are detached from axonal membrane. Scale bars: C, 5 μm; D, 2 μm; E, 0.2 μm.

Figure 2.

Lesions at nodes of Ranvier with anti-GM1 IgG and complement products in ventral roots. A–E, Immunofluorescent studies showing autoimmune lesions at nodes in longitudinal sections of ventral roots at acute progressive phase. Nerve fibers run horizontally in all panels. A, Localization of GM1 and IgG deposition. Staining of CT-B (green) colocalizes with that of rabbit IgG (red) on the nodes. Left column, In shorter lesions, the staining is mainly at nodes; right column, at longer lesions, the staining extends toward internodes. B, Complement and IgG deposition. Staining of the antibody to C3 (green) is present at the nodes with deposition of rabbit IgG (red). Left column, In shorter lesions, the staining is mainly at nodes; right column, at longer lesions, the staining extends toward internodes. C, C3 (green), MAC (blue), and CNP (red) staining in control ventral root. Neither C3 nor MAC is detected at node. CNP is present at paranodal myelin. D, Formation of C3 and MAC at lengthened nodal gap. Myelin shown with CNP staining in red or differential interference contrast image is retracted from the node. Both C3 (green) and MAC (blue) staining is present between the ends of two adjacent myelin indicated by arrows. E, Formation of C3 and MAC at lengthened nodal gap between two adjacent paranodes (arrows). Both C3 (green) and MAC (blue) staining is present mainly on the surface of naked axon at nodes shown with immunofluorescence of neurofilament-M (red). F, Frequency of nodes with C3 deposition per total nodes in ventral roots. Affected nodes are frequently present at the acute progressive phase and lessen during recovery phase. C3 deposition is never found in control rabbits. Error bars indicate 95% confidence interval. DIC, Differential interference contrast image. Scale bars: A, B, 5 μm; C–E, 10 μm.

Figure 3.

Lesions at nodes of Ranvier with complement deposition and disrupted Na_v channel clusters. A, Disruption of Na_v channel clusters at the acute progressive phase. Both C3 (green) and MAC (blue) staining is present at nodes. Clusters of Na_v channels (red) are preserved at lesions restricted to the node (left column), whereas Na_v channels almost disappear at lengthened nodes with extended C3 and MAC staining (right column). Nerve fibers run horizontally in both columns. B, Relationship between Na_v channel cluster disruption and length of complement deposition. Frequency of disrupted Na_v channel immunoreactivity is shown in nodes with C3 staining with lengths of <5, 5–10, or >10 μm. Data were collected from three AMAN rabbits at the acute progressive phase. For the statistical analyses to compare the three groups, the p value from the global test was <0.001. A significant difference was found in each pairwise comparison (p < 0.001). Error bars indicate 95% confidence interval. C, Relationship between Na_v channel cluster disruption and MAC deposition. Frequency of nodes with disrupted Na_v channel clusters is shown in nodes from three control rabbits, in nodes without MAC staining in three AMAN rabbits at the acute progressive phase, and in nodes with MAC deposition. A significant difference was found in the global test (p < 0.001). Specific pairwise comparisons shows that nodes with MAC deposition have more disruption of Na_v channel clusters than nodes without MAC staining or nodes from control rabbits (p < 0.001). There was no significant difference between nodes without MAC staining in AMAN rabbits and nodes from control rabbits (p = 0.95). Error bars indicate 95% confidence interval. DIC, Differential interference contrast image. Scale bars, 10 μm.

Figure 4.

Alterations of Na_v channels and related molecules at nodal axons by autoimmune attack. All panels show longitudinal sections of ventral roots, and nerve fibers run horizontally. Sections are immunostained with antibodies to C3 (green, all panels), Na_v channels (blue, A–D), βIV spectrin (red, A–D), or pan-NF (red, E–H). Anti-pan-NF antibody specifically stains NF186 in nodal axons. Different stages of nodal disruption in AMAN rabbits at the acute progressive phase are depicted. A, E, Staining results in the control rabbit showing the Na_v channel (A), βIV spectrin (A), and NF186 (E) clusters at the nodes. No C3 staining is detected. B, F, Lesions with C3 staining restricted at nodes. The clusters of Na_v channels (B), βIV spectrin (B), and NF186 (F) are mostly preserved and colocalize with C3 staining. C, G, Lesions with C3 staining extending to paranodes. The node is lengthened (C), and the shapes of clusters of Na_v channels (C), βIV spectrin (C), and NF186 (G) are altered. Note that the staining of Na_v channels does not colocalize with that of βIV spectrin (C). There is abnormal aggregation of Na_v channel staining in the middle of axon (arrowhead). At the region with remaining Na_v channel staining, signal of C3 is relatively weak (C, arrows). D, H, Lesions with remarkably extended C3 staining. The node is lengthened (D), and clusters of Nav channels (D), βIV spectrin (D), and NF186 (H) disappear. DIC, Differential interference contrast image. Scale bars, 10 μm.

Figure 5.

Alterations of Schwann cell microvilli by autoimmune attack. All panels show longitudinal sections of ventral roots, and nerve fibers run horizontally. Sections are immunostained with antibodies to MAC (blue), moesin (green), and βIV spectrin (red). Different stages of nodal disruption in AMAN rabbits at the acute progressive phase are depicted. A, Staining results in a control rabbit showing the moesin and βIV spectrin clusters at nodes. No MAC staining is detected. B, Lesions with MAC staining restricted to nodes. The clusters of moesin and βIV spectrin are mostly preserved. MAC staining does not completely colocalize with moesin staining. C, Lesions with MAC staining extending to paranodes. The node is lengthened, and the shapes of clusters of moesin and βIV spectrin are altered. D, Lesions with remarkably extended MAC staining. Node is lengthened, and clusters of moesin and βIV spectrin disappear. DIC, Differential interference contrast image. Scale bars, 10 μm.

Figure 6.

Alterations of Na_v channels, paranodes, and juxtaparanodes by autoimmune attack. All panels show longitudinal sections of ventral roots, and nerve fibers run horizontally. Sections are immunostained with antibodies to MAC (blue), Na_v channels (red), and Caspr (green) (A–E), or C3 (green) and Kv channels (red) (F–J). Different stages of disruption in AMAN rabbits at the acute progressive phase are depicted. A, F, Staining in a control rabbit showing the Na_v channel cluster at node, Caspr clusters at paranodes (A), and Kv channel clusters at juxtaparanodes (F). Neither MAC nor C3 staining is detected at node. B, G, Autoimmune lesions restricted to nodes. Clusters of Na_v channels (B), Caspr (B), and Kv channels (G) are not affected. C, H, Autoimmune lesions extended to paranodes. The gap between Caspr staining is lengthened more than that in normal (depicted in A) and is filled with MAC staining (C). Note that the Na_v channel cluster is still preserved, and there are clear gaps between Na_v channel and Caspr clusters (arrowheads). Kv channel clusters are preserved (H). D, I, Autoimmune lesions extended to juxtaparanodes. Node is lengthened, and the clusters of Na_v channels and Caspr are remarkably disrupted (D). Kv channel cluster at juxtaparanode partially colocalizes with C3 staining (yellow) (I). E, J, Lesions with broadly extended MAC or C3 deposition. Node is lengthened, and the staining of both Na_v channels and Caspr completely disappears, whereas these molecules are preserved in adjacent fiber without MAC deposition (E, arrows). Kv channel staining is no longer apparent (J). DIC, Differential interference contrast image. Scale bars, 10 μm.

Figure 7.

Macrophage invasion in ventral roots. A, Longitudinal section of ventral root at the acute progressive phase stained with antibodies to MAC (blue), rabbit macrophages (pink), βIV spectrin (red), and Caspr (green). Both βIV spectrin and Caspr staining disappears at the node with MAC deposition (arrow), whereas these clusters are preserved at nodes without MAC deposition (arrowhead). Macrophages (asterisks) invade the ventral root, but none overlays the affected node (arrow). B, Longitudinal section of ventral root at the acute progressive phase. A macrophage (red) overlays the affected node with C3 deposition (green). C, Remarkable macrophage invasion in ventral root at the early recovery phase. D, Quantification of macrophage invasions in rabbit ventral roots. The area of macrophage staining per field (illustrative finding is depicted in C) is measured. Macrophage staining is most frequent at the early recovery phase. There are only minimal macrophages in control rabbits. Error bars indicate 95% confidence interval. Scale bars: A, B, 10 μm; C, 100 μm.

Figure 8.

Molecular architectures and structures at and near nodes during recovery phase. A–D, Longitudinal sections of ventral roots from rabbits in early (A–C) or late (D) recovery phases. Sections are immunostained with antibodies to C3 (green, A–C), MAC (blue, D), Na_v channels (blue, A; red, D), βIV spectrin (red, A), NF (red, B), moesin (red, C), and Caspr (green, D). In all panels, nerve fibers run horizontally. A, Na_v channels and βIV spectrin aggregate on both sides of lengthened nodes with C3 deposition. B, NF186 clusters on both sides of affected node. The intensity of C3 staining is reduced. C, Moesin clusters on both sides of lengthened node with C3 deposition. D, Binary Na_v channel clusters associated with Caspr on both sides of affected nodes (top). The intensity of MAC staining is reduced. Two adjacent Na_v channel clusters are present very close (middle) or appear to fuse (bottom). E, Electron microscopy showing a remarkably lengthened node in ventral root at the early recovery phase. F, Higher magnification of boxed area in E. Boxed areas G and H are shown in higher magnification in G and H, respectively. G, Schwann cell microvilli (arrowheads) on the end of remarkably lengthened node. The finding may be identical to immunofluorescence data depicted on C. Note the inside coating of electron-dense material at the axolemma in which microvilli are present, suggestive of the presence of multiprotein complex including Na_v channels and βIV spectrin. H, Paranodal myelin loops noted on the end of remarkably lengthened node. The finding may correspond to immunofluorescence data depicted on D. DIC, Differential interference contrast image. Scale bars: A–E, 10 μm; F, 2.5 μm; G, H, 0.5 μm.