Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy

Abstract

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.

Figure 1

The Q215X mutation in P0 causes hypomyelination and alters myelin genes expression. (A) Semithin cross sections stained with toluidine blue from WT, P0Q215X heterozygote (Q215X/+) and P0Q215X homozygote (Q215X/Q215X) sciatic nerves at 1 month. Q215X/+ and Q215X/Q215X fibers are hypomyelinated as compared to WT. Scale bars, 10 μm. (B) g-ratio was increased in Q215X mutants from 0.66 ± 0.0005 (WT) to 0.72 ± 0.003 (Q215X/+) and 0.76 ± 0.005 (Q215X/Q215X). (C–F) mRNA relative quantification by real time PCR for Pmp22, Mag, Mbp and Erg2 in WT, Q215X/+ and Q215X/Q215X sciatic nerves at P5, P10, P15 and P30. (G–K) Western analysis and densitometries from WT, Q215X (Q/+) and Q215X/Q215X (Q/Q) sciatic nerves during development at P5, P10, P15 and P30 days. Blots were probed for P0, PMP22, MAG, MBP and EGR2. The Q215X allele causes a reduction of P0 stability (25). Calnexin was used as a loading control. Analyses were performed using three mice per genotype. Western blots were cropped. Error bars indicate s.e.m. Statistical analyses were performed using one-way ANOVA with Bonferroni’s multiple comparison. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2

Neuronal overexpression of NRG1 type III improves myelination in P0Q215X. (A) Semithin cross sections stained with toluidine blue (top panel) and electron micrographs (bottom panel) from WT, Nrg1t3 hemizygote (Nrg1t3), P0Q215X heterozygote (Q215X) and Nrg1t3 hemizygote and P0Q215X heterozygote (Nrg1t3;Q215X) sciatic nerves at 1 month. NRG1t3 and Nrg1t3;Q215X fibers are hypermyelinated as compared to WT and Q215X. Scale bars, 10 μm (top panel) and 2 μm (bottom panel). (B) g-ratio was increased in Q215X (0.72 ± 0.003) and decreased in Nrg1t3 (0.53 ± 0.02), indicating hypomyelination and hypermyelination, respectively. (C) g-ratio as a function of axonal diameter showed preferential hypermyelination of small fibers in Nrg1t3 and Nrg1t3;Q215X as compared to WT and Q215X. (D) Neurophysiological studies showed a significant reduction of NCV in Q215X mice when compared to WT (47.28 ± 0.97 m/s). A recovery of NCVs can be observed in Nrg1t3;Q215X (39 ± 1.5 m/s) as compared to Q215X (35 ± 1.28 m/s) (P = 0.06). (E) F-wave latencies were increased in Q215X (6.54 ± 0.11 m/s) when compared to WT (5.32 ± 0.08 m/s). A significant recovery of F-wave latencies is observed in Nrg1t3;Q215X (6 ± 0.12 m/s), suggesting a proximal myelination improvement. (F–G) Myelin compaction is not altered in single or double mutant as indicated in electron micrographs (F) and periodicity quantification (G). Scale bar, 50 nm. Analyses were performed using three mice per genotype. Error bars indicate s.e.m. Statistical analyses from three independent experiments using one-way ANOVA with Bonferroni’s multiple comparison test. ^**P < 0.01, ^***P < 0.001.

Figure 3

Expression of HA-NRG1 type III does not affect myelin gene expression. (A) Western blot analyses from WT, Q215X, Nrg1t3;Q215X and Nrg1t3 spinal cord at P30. Blots were probed for HA tag. HA is tagged to the NRG1 type III transgene. Calnexin was used as a loading control. We detected exogenous neuregulin precursor protein level and its cleavage products. The upper band corresponds to the full-length NRG1 type III inactive isoform (135 kDa, arrow); the 75-kDa band corresponds to the active form (arrow head) of NRG1 type III produced by BACE1-cleavage, as described in the literature (30,48). (B, C, F) Western blot and (D) densitometries analyses from WT, Q215X, Nrg1t3;Q215X and Nrg1t3 sciatic nerves at P30. Blots were probed for P-ErbB2, ErbB2 (B), P0, MBP, PMP22, MAG (C) and EGR2 (F). Calnexin and HSP90 were used as loading controls. Protein levels remain steady in all genotypes. (E) Immunogold labeling of P0 protein from cross sections of WT, Nrg1t3, Q215X and Nrg1t3;Q215X sciatic nerves at 1 month. Quantification of gold particles associated with P0 from cross sections of sciatic nerve at P30. Scale bar, 200 nm. (G) Relative quantification of luciferase reporter gene mRNA (Lucif) from Luc D hemizygotes (Luc D), Q215X;LucD, Nrg1t3;Luc D and Nrg1t3;Q215X;LucD sciatic nerves at P15. Luc D mice harbor a combinatorial EGR2/SOX10 binding site upstream of basal Hsp68 promoter. No significant differences were found at P15. Analyses were performed using three mice per genotype. Error bars indicate s.e.m. Statistical analyses were performed using one-way ANOVA with Bonferroni’s multiple comparison test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

Expression of HA-NRG1 type III triggers the activation of ERK and AKT. Densitometries and western blot analyses of (A) P-AKT, AKT, (B) P-ERK, ERK, (C) P-NFATc4 and NFATc4 were performed on WT, Nrg1t3, Q215X and Nrg1t3;Q215X sciatic nerves lysates at 1 month. Increased activation of AKT and ERK was observed in NRG1t3 and Nrg1t3;Q215X. NFATc4 activation level was unchanged. Western blots were cropped. Analyses were performed using three mice per genotype. Error bars indicate s.e.m. Statistical analyses were performed using one-way ANOVA with Bonferroni’s multiple comparison test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

Transcriptomic signature in Nrg1t3 sciatic nerves correlates with the signature of activation of the MAPK/ERK pathway. (A–C) Pearson’s correlation between RNA deep sequencing from 1-month-old sciatic nerve of NRG1t3 (left panel in blue), Q215X (middle panel in red) and Nrg1t3;Q215X (right panel in purple) and targets downstream of EGR2 and ERK. (A) Pearson’s correlation analysis with combined ChIP-Sequencing and arrays data sets obtained from mice homozygous for an Egr2 hypomorphic allele (Egr2^lo/lo). Pairwise correlation analysis showed no correlation between the exogenous expression of Nrg1t3 and EGR2 downstream regulated genes. (B) Pearson’s correlation analysis with arrays data sets obtained from conditional Schwann-specific constitutive active form of MEK1 (Mek1DD;Egr2Cre), which phosphorylates ERK. Pairwise correlation showed a high correlation between the exogenous expression of NRG1 type III and MEK1 downstream–regulated genes (panel B blue and purple). (C) Pearson’s correlation analysis with arrays data set generated from Schwann cell conditional knockout for Shp2 (Egr2Cre), which dephosphorylates ERK. Pairwise correlation showed an anticorrelation between exogenous expression of HA-NRG1 type III and Shp2 loss of function (panel C blue and purple). (D) Fold change of transcripts for NRG1, transcription factors regulating myelination and myelin genes obtained from mRNA deep sequencing in Nrg1t3, Q215X and Nrg1t3;Q215X in 1 month mice sciatic nerve. The quantification showed an overall unchanged level of the major regulators and components of the myelin, apart from Pmp2 (a fatty acid binding protein, Fabp8) and Omg.

Figure 6

Constitutive activation of MAPK/ERK increases peripheral myelination in normal and Q215X genetic context, but it is functionally detrimental. (A) Semithin cross sections stained with toluidine blue (top panel) and electron micrographs (bottom panel) from WT, Q215X, Mek1DD hemizygote and P0-Cre hemizygote (Mek1DD;P0Cre) and Mek1DD hemizygote, P0-Cre hemizygote and Q215X (Mek1DD;P0Cre;Q215X) sciatic nerves at 1 month. Mek1DD;P0Cre and Mek1DD;P0Cre;Q215X fibers are hypermyelinated, and Q215X is hypomyelinated. Scale bars, 10 μm (top panel) and 2 μm (bottom panel). (B) g-ratio was increased in Q215X (0.70 ± 0.008) and decreased in Mek1DD;P0Cre (0.40 ± 0.002) and Mek1DD;P0Cre;Q215X (0.39 ± 0.002) compared to WT (0.64 ± 0.002), indicating hypomyelination and strong hypermyelination, respectively. (C) g-ratio as a function of axonal diameter showed preferential hypermyelination of small fibers in Mek1DD;P0Cre and Mek1DD;P0Cre;Q215X. (D, E) Neurophysiological studies showed significantly reduced NCV in all mutants. The amplitude of compound muscle action potentials was decreased in Mek1DD;P0Cre;Q215X, suggesting axonal loss. (F, G) Myelin compaction (F) and periodicity (G) were not altered in Mek1DD;P0Cre and Mek1DD;P0Cre;Q215X sciatic nerves. Scale bar, 50 nm. Analyses were performed using three mice per genotype. Error bars indicate s.e.m. Statistical analyses were performed using one-way ANOVA with Bonferroni’s multiple comparison test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 7

Expression of HA-NRG1 type III transgene increases PMP2 protein level. (A) Western blot analysis from WT, Nrg1t3, Q215X, Nrg1t3;Q215X, Mek1DD;P0Cre and Mek1DD;P0Cre;Q215X sciatic nerves at 1 month. Expression of HA-NRG1t3 transgene in sciatic nerves or constitutive activation of the MAPK/ERK pathway in Schwann cells leads to an increase in ERK phosphorylation, a decrease in EGR2 protein levels and an increase in PMP2 protein levels. These effects are enhanced in Mek1DD;P0Cre mutants as compared to HA-NRG1t3 mutants. Western blots were cropped. (B) Semithin cross sections stained with toluidine blue (top panel) and electron micrographs (bottom panel) from WT, Nrg1t3 hemizygote (Nrg1t3), P0Q215X heterozygote (Q215X) and Nrg1t3 hemizygote and P0Q215X heterozygote (Nrg1t3;Q215X) sciatic nerves at 1 year.

Figure 8

Regulation of myelination by NRG1 type III. In WT animals, NRG1 type III/ ErbB2/3 promotes myelination by stimulating several signaling pathways, including PI3K/AKT, MAPK/ERK and CaN/NFATc4, which are thought to converge on the activation of EGR2. In Nrg1t3 animals, the exogenous expression of NRG1 type III leads to the activation of both PI3K/AKT and MAPK/ERK pathways (red arrows) and the increased transcription of Pmp2 and Omg.