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. 2014 Feb 1;28(3):290-303.
doi: 10.1101/gad.230045.113.

Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination

Maria E Sheean  1 , Erik McShane, Cyril Cheret, Jan Walcher, Thomas Müller, Annika Wulf-Goldenberg, Soraya Hoelper, Alistair N Garratt, Markus Krüger, Klaus Rajewsky, Dies Meijer, Walter Birchmeier, Gary R Lewin, Matthias Selbach, Carmen Birchmeier
Affiliations

Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination

Maria E Sheean et al. Genes Dev. .

Abstract

Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTOR-independent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination.

Keywords: MAPK; myelin; pSILAC; translational control.

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Figures

Figure 1.
Figure 1.
Protein synthesis and Nrg1 signaling are down-regulated in maturing nerves. (A) Growth of myelinating Schwann cells at P8, P15, and P30 is shown schematically. Axon diameter, myelin thickness, and internodal distance are scaled to display average changes (cf. Court et al. 2004). (B) Western blot analysis investigating phosphorylation of signaling molecules in sciatic nerves of P8, P15, and P30 mice. Tuj1 was used as loading control. (C) Outline of the pSILAC experiment performed to estimate protein synthesis in nerves of “old” and “young” mice. SILAC labeling was achieved by feeding Lys8-labeled chow or providing Lys6-labeled animals as foster mothers. (D) Kernel density estimation plots showing the fold change of newly synthesized proteins in the nerves of mice at P30 (old) versus P8 (young) as assessed by pSILAC and mass spectrometry; overall proteins or selected protein groups (ribosome, myelin) (cf. Supplemental Table 1) are displayed. A shift to the left indicates decreased protein production in old mice. (E) qPCR analysis of myelin gene expression from P0 to P62, normalized to an external control RNA from Caenorhabditis elegans (eif-3.D) and plotted as fold change of the expression observed at P0. Error bars indicate SEM. (F) Correlation plot, comparison of fold changes in protein production, and mRNA levels between old and young animals. Protein synthesis was assessed comparing P8 and P30 mice pulse-labeled for 1 wk. RNA expression was compared between P11 and P33 by microarray analysis, normalized to ERCC RNA spike-in. We observed poor correlation between changes in protein synthesis and steady-state mRNA levels.
Figure 2.
Figure 2.
Activation of MAPK signaling results in continuous myelin growth. (A) Western blot analysis of total and phosphorylated Erk1/2 and Akt in sciatic nerves of control (c) and Mek1DD (m) mice at P8, P15, and P30. Neuronal tubulin (Tuj1) was used as loading control. (B) Analysis of nerves from control and Mek1DD mice (P0–P90) by electron microscopy. Representative axons of similar diameters were false-colored. (C) Mean g-ratios of fibers in sciatic nerves of control and Mek1DD mice at the indicated ages. (D) Binning analysis of myelin thickness of groups of axons with the indicated diameters; the nerves of control and Mek1DD mice at P30 were analyzed. (E) Distribution of axon diameters in sciatic nerves of control and Mek1DD mice at P30 (n = 90 units total, three mice per genotype). (F) Conduction velocities of rapidly adapting mechanoreceptors (RAM), slowly adapting mechanoreceptors (SAM), D-hair mechanoreceptors (DH), and Aδ mechanonociceptors (AM) in saphenous nerves at P30. (G) Conduction velocities of motoneurons in ventral roots at P30. (H) Western blot analysis of sciatic nerve extracts from control and PI3KDA P15 animals; expression of PI3KDA enhanced Akt but not Erk1/2 phosphorylation. Electron microscopy analysis (I) and quantification of myelin thickness (g-ratio) (J) of control and PI3KDA mice (P15). (C,D,F,J) Two-tailed unpaired t-test. (G) Two-way ANOVA (129–145 ventral root fibers tested from L4–6, three mice per genotype). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Error bars indicate SD (C–E,G,J) and SEM (F). Bars: B,I, 2 µm.
Figure 3.
Figure 3.
Prolonged activation of MAPK signaling results in the formation of aberrant myelin structures and in physiological deficits. (A) Aberrant myelin structures in sciatic nerves of Mek1DD mice at the indicated ages; asterisks indicate compressed axons, the arrow indicates a myelinated axon with <1 μm axonal diameter, and the arrowhead indicates an “onion bulb” structure. (B) Percentage of misfolded myelin structures identified in control and Mek1DD mice at the indicated ages. (C) Visualization of Schmidt-Lanterman incisures using β-catenin staining of teased nerve fibers. Nerves from Mek1DD (Mek1) and control (con) animals are displayed. (D) Quantification of total axon numbers and axons displaying a severe compression in saphenous nerve. (E) Ratio of A/C compound action potential in nerves of control and Mek1DD mice (five to six mice per genotype) (left) and quantification of active units per axon filament detected in conduction velocity tests (right). Bars, A, 2 μm; C, 50 μm. (B,D,E, left) Two-tailed unpaired t-test. (E, right) Mann-Whitney U-test. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Error bars indicate SEM.
Figure 4.
Figure 4.
SILAC pulse labeling reveals increased protein synthesis in the nerves of Mek1DD mice. (A,B) Kernel density estimation plots showing the ratios of newly synthesized proteins in Egr2Cre;Mek1DD versus control mice; overall proteins or selected protein groups (ribosome, myelin, and lipid synthesis enzymes) are displayed. The shift to the right indicates an increased protein production in Mek1DD mutants. Enzymes involved in lipid synthesis were not detected when pups were analyzed. (C) Western blot analysis of selected proteins previously detected and quantified in SILAC pulse labeling experiments. Tuj1 was used as loading control. (D,E) Correlation plot comparison of fold changes in protein production and mRNA levels in Mek1DD mutant versus control nerves at P30 and P8; black dots indicate mRNAs that are changed >1.2-fold, and dashed lines indicate the average change in protein/mRNA levels. (F) Quantification of transcripts of genes encoding myelin proteins in P8, P15, and P30 sciatic nerves by qPCR. Two-tailed paired t-test: (*) P < 0.05; (**) P < 0.01. Error bars indicate SD.
Figure 5.
Figure 5.
Pharmacological inhibition of mTORC1 rescues axonal packing but not hypermyelination. (A) Staining of P30 teased fibers using antibodies specific for phosphorylated ribosomal protein S6 (p-S6), MBP, and DAPI. The specificity of the antibody directed against p-S6 was verified by treating fibers with shrimp alkaline phosphatase (SAP). (B) Western blot analysis of Erk and eIF4E (top); mTOR and its substrates, S6 and 4EBP1 (middle); PTEN, Dlg1, and their downstream effector, Akt (bottom). (C) Electron microscopy images of P30 sciatic nerves of control and Mek1DD mice treated with the mTORC1 inhibitor everolimus or vehicle. Myelin thickness (g-ratio) (D) and axonal packing (E) in sciatic nerves of control and Mek1DD mice treated with everolimus or vehicle. (F) Western blot analysis indicates that phosphorylation of mTOR and its substrates, S6 and 4EBP1, but not of the Mnk1 substrate eIF4E, is abolished by everolimus treatment. Bars: A, 10 μm; C, 5 μm. Two-tailed paired t-test: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Error bars indicate SD (D) and SEM (E).
Figure 6.
Figure 6.
Expression of Mek1DD in Schwann cells compensates for the loss of Shp2 or ErbB3. Electron microscopy analysis at P15 (A) and quantification of myelin thickness (g-ratio) of sciatic nerves (P15 and P90) (B) of Egr2Cre;Shp2f/f, Egr2Cre;Shp2f/f;Mek1DD and control mice. (C) Western blot analysis of Erk1/2, Akt, PTEN, and Dlg1 in sciatic nerves of Egr2Cre;Shp2f/f and Egr2Cre;Shp2f/f;Mek1DD mice at P8. Electron microscopy analysis at P15 (D) and quantification of myelin g-ratio (P15) (E) of sciatic nerves of Egr2Cre;Shp2f/f, Egr2Cre;Shp2f/f;PI3KDA and control mice. Electron microscopy analysis at P15 (F) and g-ratio (P15 and P90) (G) of sciatic nerves of DhhCre;Shp2f/f, DhhCre;Shp2f/f;Mek1DD, and control mice; the arrow in the inset points to Schwann cell cytoplasm surrounding an unmyelinated axon. Electron microscopy analysis at P15 (H) and g-ratio (P15 and P90) (I) of sciatic nerves of DhhCre;ErbB3f/f, DhhCre;ErbB3f/f;Mek1DD, and control mice; the arrow in the inset points toward axonal membranes not surrounded by Schwann cell cytoplasm. Note that expression of the Mek1DD allele rescues myelination and Schwann cell deficits observed in Shp2 and ErbB3 mutant mice. Electron microscopy analysis (J) and quantification (K) of nonmyelinated/myelinated axons in DhhCre;Mek1DD and control mice at P15. The arrow in the left panel indicates a myelinated axon with <1 μm axonal diameter in the DhhCre;Mek1DD mutant. The middle and right panels show Remak bundles. Bars, 2 μm. Two-tailed paired t-test: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. Error bars indicate SEM.
Figure 7.
Figure 7.
Mek1DD rescues transcriptional and translational deficits of Shp2 mutant Schwann cells. (A) Quantification of transcripts encoding myelin proteins by qPCR at P15. Changes in the levels of several myelin gene transcripts were underestimated in microarray experiments, possibly due to probe saturation. Down-regulation of myelin gene mRNA in Egr2Cre;Shp2f/f mutant nerves was largely rescued by Mek1DD expression. (B,C) Kernel density estimation plots showing the ratios of newly synthesized proteins in the nerves of Egr2Cre;Shp2f/f mutant versus control mice at P30 and P8 as assessed by pSILAC and mass spectrometry; overall proteins and selected protein groups (ribosome, myelin, and enzymes involved in lipid synthesis) are displayed. A shift to the left indicates decreased protein production in Egr2Cre;Shp2f/f mutants. Enzymes involved in lipid synthesis were not detected when pups were analyzed. (D,E) Correlation plot of fold changes in protein production and mRNA levels in Egr2Cre;Shp2f/f mutant versus control nerves at P30 and P8; black dots indicate mRNAs that are changed >1.2-fold, and dashed lines indicate the average change in protein/mRNA levels. (F) Western blot analysis of selected proteins identified as down-regulated in the nerves of Egr2Cre;Shp2f/f mutants (Shp2) using mass spectrometry. Additional expression of Mek1DD in Egr2Cre;Shp2f/f mutants rescued the levels of these proteins (Shp2/Mek1). Two-tailed paired t-test: (*) P < 0.05. Error bars indicate SD.
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