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, 31 (3), 843-50

The ERK2 Mitogen-Activated Protein Kinase Regulates the Timing of Oligodendrocyte Differentiation

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The ERK2 Mitogen-Activated Protein Kinase Regulates the Timing of Oligodendrocyte Differentiation

Sharyl L Fyffe-Maricich et al. J Neurosci.

Abstract

Oligodendrocyte development is tightly controlled by a variety of extracellular growth and differentiation factors. The mitogen-activated protein kinases (MAPKs), ERK1 and ERK2, are critical intracellular signaling molecules important for transducing these extracellular signals. The extracellular signal-regulated kinases (ERKs) are ubiquitously expressed, coordinately regulated, and highly similar, but Erk2 deletion in mice is embryonic lethal whereas Erk1 deletion is not. Several studies have suggested that MAPK signaling is important for oligodendrocyte differentiation, although specific roles for the two ERK isoforms have not been investigated. In this study, we deleted Erk2 in the developing mouse cortex from GFAP-expressing radial glia that generate neurons and oligodendrocytes. In vitro analysis revealed that loss of ERK2 resulted in fewer galactocerebroside-expressing mature oligodendrocytes in cortical cultures. In vivo, a delay in the expression of the myelin protein MBP was observed in the corpus callosum at postnatal day 10 (P10). In contrast, Erk1 deletion did not affect oligodendrocyte differentiation. By P21, MBP expression was restored to wild-type levels, demonstrating that the loss of ERK2 results in a delay but not a complete arrest in the appearance of differentiated oligodendrocytes in vivo. Importantly, both the proliferation and total number of oligodendrocyte progenitor cells (OPCs) appeared normal in the Erk2 conditional knock-out cortex, demonstrating that ERK2 plays a specific role in the timing of forebrain myelination but is not critical for the proliferation or survival of OPCs. Oligodendrocyte-specific deletion of Erk2 also resulted in decreased levels of MBP, indicating a cell-autonomous effect of ERK2 in the oligodendrocyte lineage.

Figures

Figure 1.
Figure 1.
Activated ERK1/2 (pERK1/2) is expressed in both O4+ and GalC+ oligodendrocytes and is necessary for the appearance of GalC+ oligodendrocytes in vitro. A, Anti-pERK1/2 (red) and anti-O4 (green, left) or anti-GalC (green, right) immunostaining of A2B5+ pan-purified oligodendrocyte cultures after 5 DIV. B, Anti-O4 (red, top) or anti-GalC (red, bottom) immunostaining of mixed cultures from P5 rat brain treated with 10 μm U0126 or DMSO. DAPI staining (blue) marks cell nuclei. C, Anti-GalC (green) immunostaining of A2B5+ pan-purified oligodendrocyte cultures from P0 spinal cord treated with 10 μm U0126 or DMSO. D, The number of A2B5+ or O4+ OPCs was quantified and reported as a percentage of the total number of DAPI+ cells from A2B5+ pan-purified cultures treated with 10 μm U0126 or DMSO. Scale bars, 100 μm. NS, Not significant.
Figure 2.
Figure 2.
OPC differentiation is impaired in cortical cultures from GFAP–Cre; Erk2 CKO mice. A, B, Anti-GFP (green) and anti-GalC (red) immunostaining of mixed cultures from Erk2 flox/+ (A) and Erk2 CKO (B) P3 cortex after 12 DIV. C, The number of double-labeled GalC+ and YFP+ cells were counted and plotted as a percentage of the total number of YFP+ oligodendrocyte lineage cells. U0126 at 10 μm was added to a subset of the Erk2 CKO cultures to block ERK1 activity 2 d before the cells were analyzed by immunostaining. Scale bar, 100 μm. *p < 0.001, unpaired t test.
Figure 3.
Figure 3.
Selective requirement for ERK2 in the timing of forebrain myelination. A, Anti-MBP immunostaining (green) of corpus callosum from GFAP–Cre; Erk2 CKO, Erk1 −/−, and control littermate mice at P10. B, Western blot analysis and quantification of MBP from P10 cortical lysates. GAPDH levels serve as a loading control. C, Anti-NG2 immunostaining (red) of corpus callosum from P2 mice. Scale bars, 100 μm. *p < 0.05, unpaired t test.
Figure 4.
Figure 4.
The myelination phenotype in GFAP–Cre; Erk2 CKO mice appears transient. A, Anti-MBP immunostaining (green) of corpus callosum from P21 Erk2 CKO and control littermate. B, Toluidine blue staining demonstrates that Erk2 CKO animals retain a patchy hypomyelination phenotype in the midline corpus callosum at P21. WT animals have a relatively uniform distribution of myelinated axons, whereas Erk2 CKO animals have areas of normal myelination interspersed with areas of hypomyelination. C, The ultrastructure of the myelin was similar between WT and Erk2 CKO animals at P21. Scale bars: A, 100 μm; B, 15 μm; C, 200 nm.
Figure 5.
Figure 5.
ERK2 expression in oligodendrocyte progenitor cells is important for the proper timing of forebrain myelination. A, Anti-MBP immunostaining (green) of corpus callosum from NG2–Cre; Erk2 CKO and control littermate at P7. DAPI staining marks cell nuclei. B, Western blot analysis and quantification of MBP from P7 cortical lysates demonstrates a trend toward decreased levels of MBP in the NG2–Cre; Erk2 CKO mice. n = 2 animals per genotype. GAPDH levels serve as a loading control. C, Anti-MBP immunostaining (green) of corpus callosum from NG2–Cre; Erk2 CKO mice and littermate control at P21. Scale bars, 100 μm.

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