MCP-1/CCL2 modifies axon properties in a PMP22-overexpressing mouse model for Charcot-Marie-tooth 1A neuropathy

Abstract

Charcot-Marie-Tooth 1A (CMT1A) neuropathy, the most common inherited peripheral neuropathy, is primarily caused by a gene duplication for the peripheral myelin protein-22 (PMP22). In an accordant mouse model, we investigated the role of monocyte chemoattractant protein-1 (MCP-1/CCL2) as a regulator of nerve macrophages and neural damage including axonopathy and demyelination. By generating PMP22tg mice with reduced levels or lack of MCP-1/CCL2, we found that MCP-1/CCL2 is involved in the increase of macrophages in mutant nerves. PMP22tg mice with wild-type levels of MCP-1/CCL2 showed strong macrophage increase in the diseased nerves, whereas either 50% reduction or total absence of MCP-1/CCL2 led to a moderate or a strong reduction of nerve macrophages, respectively. Interestingly, MCP-1/CCL2 expression level and macrophage numbers were correlated with features indicative of axon damage, such as maldistribution of K+ channels, reduced compound muscle action potentials, and muscle weakness. Demyelinating features, however, were most highly reduced when MCP-1/CCL2 was diminished by 50%, whereas complete lack of MCP-1/CCL2 showed an intermediate demyelinating phenotype. We also identified the MEK1/2-ERK1/2-pathway as being involved in MCP-1/CCL2 expression in the Schwann cells of the CMT1A model. Our data show that, in a CMT1A model, MCP-1/CCL2 activates nerve macrophages, mediates both axon damage and demyelination, and may thus be a promising target for therapeutic approaches.

Figure 1

MCP-1/CCL2 is increased in peripheral nerves of PMP22tg mice. A and B: MCP-1/CCL2-expression in peripheral nerves of two-month-old PMP22-overexpressing mice. Semiquantitative RT-PCR (mean values ± SD) in sciatic (A) and femoral quadriceps nerves (B) of PMP22tg (n = 4) revealed a significantly increased expression of MCP-1/CCL2 mRNA in comparison with nerves of PMP22wt (n = 3). Representative data from two to three different experiments are shown. *P < 0.05, **P < 0.01, Student t test. C–E: Immunohistochemical detection of MCP-1 on teased fiber preparations of femoral quadriceps nerves from two-month-old PMP22tg mice. Teased fiber preparations of PMP22tg mice incubated with primary MCP-1 antibody (ab) showed a characteristic pattern indicative for immunopositive Schmidt-Lanterman incisures (arrows, C). When the primary antibody was preincubated with rMCP-1, the labeling was weak and diffuse (D) and completely absent, when the primary antibody was omitted (E). Scale bar = 30 μm.

Figure 2

The extent of MCP-1/CCL2-expression determines the number of nerve-macrophages in PMP22tg mice. Quantification of F4/80-positive macrophages in femoral quadriceps nerves of wild-type and PMP22 mutant mice at the age of two (A) and six months (B). Note that all PMP22wt exhibit comparable numbers of macrophages independent of age and MCP-1/CCL2-expression. The increase of macrophages in PMP22tg mice is diminished by the reduction and complete absence of MCP-1/CCL2 at six months of age. *P < 0.05, **P < 0.01, ***P < 0.001, Student t test.

Figure 3

Demyelination is ameliorated in peripheral nerves of PMP22tg/MCP-1^+/− and PMP22tg/MCP-1^−/− mice. Morphometric analysis of femoral nerves from wild-type and PMP22 mutant mice (n = 3 to 4). Electron microscopy of femoral quadriceps nerves of six-month-old PMP22wt/MCP-1^+/+ (A), PMP22tg/MCP-1^+/+ (B), PMP22tg/MCP-1^+/− (C), and PMP22tg/MCP-1^−/− mutants (D). Analysis of femoral quadriceps nerves revealed fewer features indicative of demyelination in PMP22tg/MCP-1^+/− and PMP22tg/MCP-1^−/− mice, such as demyelinated axons (asterisk) and thinly myelinated axons (double asterisk). Scale bar = 10 μm. E: G-ratios reflect the milder demyelinating neuropathy in nerves of PMP22tg/MCP-1 double mutant mice. F: Quantification of pathological alterations confirms the ameliorated demyelination in femoral quadriceps nerves of PMP22tg/MCP-1^+/− and PMP22tg/MCP-1^−/− mice, whereas hypermyelination is independent from MCP-1/CCL2-expression. G: Foamy macrophages containing myelin debris showed a tendency toward lower numbers in femoral quadriceps nerves of PMP22tg/MCP-1^+/− mice compared with nerves of PMP22tg/MCP-1^+/+ and PMP22tg/MCP-1^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney U test.

Figure 4

Complete absence of MCP-1/CCL2 increases muscle strength in PMP22tg mice. Muscle (grip) strength of hindlimbs of wild-type and PMP22 mutants at the age of six months. PMP22tg/MCP-1^+/+ mice show an impaired muscle strength compared with PMP22wt/MCP-1^+/+ mutants, whereas PMP22tg/MCP-1^−/− mice exhibit an improvement of grip strength to a level similar to that of PMP22wt/MCP-1^+/+ mice. Each indicated value represents a tested individual. *P < 0.05, ***P < 0.001, Student t test.

Figure 5

PMP22tg mice show a maldistribution of Kv1.2 channels, which is ameliorated in the absence of MCP-1/CCL2. Ion channels (anti-Nav1.6 and anti-Kv1.2) in teased fiber preparations of femoral quadriceps nerves from six-month-old PMP22/MCP-1 mutant mice were visualized with Cy3, paranodal Caspr with FITC. (A–G, scale bar = 10 μm). Nav1.6 expression is comparable in wild-type (A, red) and PMP22tg/MCP-1^+/+ mice (B, red). Kv1.2 is symmetrically distributed in juxtaparanodal regions of fibers from wild-type mice (C, red). Whereas in addition to wild-type-like expression (D, red), an asymmetric distribution (E, red) and a diffuse expression (F, red) is also found in teased fibers of PMP22tg/MCP-1^+/+ mice. Note that some fibers do not show any Kv1.2 expression (G). The distribution of Caspr-positive profiles (green, FITC) are not altered in wild-type and PMP22tg/MCP-1^+/+ mutant mice. Quantification of Kv1.2 distribution in teased fiber preparations of MCP-1 mutant mice (H). In the complete absence of MCP-1/CCL2, PMP22tg mice (PMP22tg/MCP-1^−/−) showed a more wild-type–like expression of Kv1.2 in peripheral nerves. *Significant differences compared with PMP22wt/MCP-1^+/+ mice. **Significant differences compared with PMP22tg/MCP-1^+/+ mice. ***Significant differences compared with PMP22tg/MCP-1^+/+ and PMP22tg/MCP-1^+/− mice. P < 0.05, Mann–Whitney U test.

Figure 6

The MEK1/2/ERK1/2 signaling cascade is activated in nerves of PMP22tg mice. Western blot analyses of peripheral nerve lysates from two-month-old (A–E) and of six-month-old (F) wild-type (wt) and PMP22tg mutant mice. The diagrams below the immunoblots illustrate the corresponding densitometric analyses of three experiments comprising two to five mice. JNK1 (A, 46 kDa) and IKBα (B, 37 kDa) do not show any increased phosphorylation in femoral quadriceps nerves in PMP22tg mice relative to wild-type. In contrast, stronger phosphorylation of ERK1/2 (pERK1/2, 44/42 kDa) is visible in femoral quadriceps (C) and sciatic nerves (D) of PMP22tg mice compared with wild-types. E: The upstream kinase of ERK1/2, MEK1/2 (45 kDa), is stronger phosphorylated in femoral quadriceps nerve of PMP22tg mice. F: The stronger ERK1/2-phosphorylation is still visible in femoral quadriceps nerves of six-month-old PMP22tg mice compared with wild-types. In all blots, staining with antibodies against the corresponding total protein serves as loading controls. G: Localization of phospho-ERK1/2 in single fiber preparations of two-month-old mice. In comparison with a more non-nuclear and faint labeling in PMP22wt (n = 3), femoral quadriceps nerves of PMP22tg mice (n = 3) reveal an additional phospho-ERK1/2 immunoreactivity (red, Cy3, arrow) in nuclei (DAPI, blue) of S100β-positive Schwann cells (green, FITC). Scale bar = 10 μm.

Figure 7

Increased MCP-1/CCL2-expression in peripheral nerves of PMP22tg mice is mediated by the MEK1/2/ERK1/2 signaling cascade. In vivo inhibition of the MEK1/2/ERK1/2 cascade and MCP-1/CCL2-expression by daily treatment with CI-1040 for three weeks. A: Western blot analyses and the corresponding densitometric measurement of femoral quadriceps nerve lysates of CI-1040 and sham-treated mice at the age of two months. Data are shown as a mean value of two experiments with three to five mice in each group. Note the decreased phosphorylation of ERK1/2 (44/42 kDa) in nerves of mice with daily CI-1040 injection. B: MCP-1/CCL2 mRNA is reduced in nerves of CI-1040–treated PMP22tg mice in comparison with sham-treated mutants. Treatment of wild-type mice does not significantly change the MCP-1/CCL2-expression. *P < 0.05, Student t test, **P < 0.01.