Altered ion channels in an animal model of Charcot-Marie-Tooth disease type IA

Abstract

How demyelination and remyelination affect the function of myelinated axons is a fundamental aspect of demyelinating diseases. We examined this issue in Trembler-J mice, a genetically authentic model of a dominantly inherited demyelinating neuropathy of humans. The K+ channels Kv1.1 and Kv1.2 channels were often improperly located in the paranodal axon membrane, typically associated with improperly formed paranodes, and in unmyelinated segments between internodes. As in wild-type nerves, Trembler-J nodes contained Nav1.6, ankyrin-G, betaIV-spectrin, and KCNQ2, but, unlike wild-type nerves, they also contained Kv3.1b and Nav1.8. In unmyelinated segments bordered by myelin sheaths, these proteins were clustered in heminodes and did not appear to be diffusely localized in the unmyelinated segments themselves. Nodes and heminodes were contacted by Schwann cells processes that did not have the ultrastructural or molecular characteristics of mature microvilli. Despite the presence of Nav1.8, a tetrodotoxin-resistant sodium channel, sciatic nerve conduction was at least as sensitive to tetrodotoxin in Trembler-J nerves as in wild-type nerves. Thus, the profound reorganization of axonal ion channels and the aberrant expression of novel ion channels likely contribute to the altered conduction in Trembler-J nerves.

Figure 1.

Morphological aspects of myelinated fibers of Trembler-J nerves. These are images of unfixed teased sciatic nerve fibers immunostained for E-cadherin (FITC; to delimit myelin sheaths) and Caspr (TRITC; to mark paranodes) and counterstained with DAPI (blue). In A and B, note that internodes are as short as 30 μm (bars with circles) and either meet at nodes (apposed arrowheads) or are separated by unmyelinated segments (bars with arrows). Caspr-positive paranodes in Trembler-J mice (TrJ) (D) are often shorter than those in WT mice (C). The length and width of individual Caspr-positive paranodes are plotted for two Trembler-J (red; n = 360 paranodes) and two WT (blue; n = 535 paranodes) mice (E). Trembler-J paranodes are significantly shorter than those of WT mice (p < 0.001; one tailed t tests and Kolmogorov-Smirnov tests). Scale bars, 10 μm.

Figure 2.

Organization of nodes and heminodes in Trembler-J mice. These are images of unfixed teased sciatic nerve fibers from Trembler-J (TrJ) (A-D) and WT (insets in C, D) mice, labeled as indicated. Nodes and heminodes contain Nav channels (A), βIV-spectrin (B), and Nav1.6 (C) but not Nav1.2 (C). Unmyelinated segments (between heminodes) do not contain detectable levels of Nav channels orβIV-spectrin. A minority of Trembler-J nodes contain detectable amounts of EBP-50, a marker of Schwann cell microvilli (D). Paranodes are labeled using RPTPβ-Fc, which binds to contactin. Scale bar, 10 μm.

Figure 4.

Kv1.1, Kv1.2, KCNQ2, and Nav1.8 channels in Trembler-J mice. These are images of unfixed teased sciatic nerve fibers from WT and Trembler-J (TrJ) mice, labeled as indicated. In many internodes, Kv1.1 and Kv1.2 staining was normally localized to the juxtaparanodal region (A). In most fibers, however, Kv1.1 and Kv1.2 was abnormally localized in the paranodal region (double arrowheads in B-D) or in the unmyelinated segments between two heminodes (bars with arrows in C,D). Kv1. 1 and Kv1.2 flanked the nodes and heminodes but did colocalize with Nav channels (D). Even altered paranodes still segregated Kv1.1 and Kv1.2 channels (E). KCNQ2 staining was found in both WT (F) and Trembler-J (G) nodes (apposed arrowheads) and Trembler-J heminodes (K). In contrast, Nav1.8 was detected in Trembler-J nodes (I) and heminodes (J) but not in WT nodes (H). Asterisks indicate Schwann cell nuclei. Scale bars, 10 μm.

Figure 3.

Electron microscopy of Trembler-J nodes and heminodes. These are images of semithin (A) and thin (B-D) sections of Trembler-J sciatic nerves. A shows an aberrantly short internode (between the apposing arrowheads that mark the flanking nodes). In B and C, putative heminodes that border an unmyelinated segment appear to be contacted by Schwann cell processes (black arrows in B); C is an enlargement of the right heminode from B. Despite the myelin alteration, septate-like junctions were observed at Trembler-J paranodes (double arrowheads in D). Scale bars: A, 10 μm; B, C, 1.5 μm; D, 0.15 μm.

Figure 5.

Kv3.1b is a component of the PNS nodes in Trembler-J nerves. These are images of unfixed teased sciatic nerve fibers from WT and Trembler-J (TrJ) mice, labeled as indicated. In WT mice, a few nodes are Kv3.1b positive (apposed arrowheads in A), whereas the majority of the nodes (apposed arrowheads) and heminodes (arrowheads) were Kv3.1b positive in the Trembler-J mice (B-D). Contactin is found at paranodes (double arrowheads) but not nodes in both WT (F) and Trembler-J (E). Scale bars, 10 μm.

Figure 6.

Electrophysiological characteristics of Trembler-J nerves. CAPs recorded from Trembler-J (TrJ) sciatic nerves (n = 8) were delayed (A; p < 0.001), more dispersed (B; p < 0.001), and smaller (C; p < 0.001) than those in WT (n = 9) nerves. Examples of WT and Trembler-J CAPs are shown in D and E, respectively; both traces are superposed in F. Trembler-J fibers were recruited at higher stimulus intensities (G; p < 0.05 for stimulation intensities of 1-5 V) and had longer refractory periods (H; p < 0.05 for stimulus intervals between 1 and 10 ms). One-tailed t tests for two samples of equal variance were used in all of the above comparisons.

Figure 7.

Trembler-J nerves are not relatively resistant to TTX. Increasing concentrations of TTX decrease the amplitude of CAPs in WT (A; n = 5) and Trembler-J (TrJ) (B; n = 6) nerves. Examples of single nerves are shown in A and B; C shows the averages. At 10 and 20 nm, TTX decreased the CAP of Trembler-J nerves more than that of WT nerves (^*p < 0.05, ^‡p < 0.001, using a one-tailed t tests for two samples of equal variance). At 50 nm TTX, CAPS were blocked in both WT and Trembler-J nerves.