Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 180 (5), 947-55

Essential Role of B-Raf in Oligodendrocyte Maturation and Myelination During Postnatal Central Nervous System Development

Affiliations

Essential Role of B-Raf in Oligodendrocyte Maturation and Myelination During Postnatal Central Nervous System Development

Gergana Galabova-Kovacs et al. J Cell Biol.

Abstract

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.

Figures

Figure 1.
Figure 1.
Neurological defects and growth retardation in B-Raf–deficient mice. (A) Limb clasping reflex in P18 Mox2cre/+;b-raf f/f and b-raf Δ/Δneu mice suspended by the tail. (B) Complete conversion of the b-raf f/f to b-raf Δ/Δ alleles in brain but not in other tissues of P18 b-raf Δ/Δneu mice. PCR analysis of: T, tail; B, brain; Liv, liver; L, lung; Sp, spleen; Th, thymus; H, heart; and K, kidney. N, negative control (H2O); f/− and +/+, positive controls. (C) Immunoblots of brain lysates from P18 b-raf f/f (WT) and b-raf Δ/Δneu (KO) mice probed with antibodies against N- or C-terminal B-Raf epitopes demonstrate the complete absence of B-Raf protein. The position of the molecular weight markers is shown between the autoradiograms. The arrow indicates B-Raf. Actin immunoblot, loading control. (D) Immunoblot of brain and spinal cord lysates from P18 b-raf f/f (WT) and b-raf Δ/Δneu (KO) mice. MEK2 immunoblot, loading control.
Figure 2.
Figure 2.
b-raf ablation in neuronal precursors leads to hypomyelination of the brain. (A–C) Hypomyelination of different regions of the P18 b-rafΔ/Δneu brain revealed by immunohistochemistry (IHC) using an α-MBP antibody on paraffin sections. Brown staining indicates MBP+ fibers and cells. The sections were counterstained with hematoxylin. (A) Brain cortex. (B) Detail of the cortex. (C) Hippocampus and the dentate gyrus (arrows). (D) MBP expression determined by immunoblotting of brain lysates. The reduced MBP expression in the b-rafΔ/Δneu lysates correlates with the results obtained in the IHC.
Figure 3.
Figure 3.
Hypomyelination in the CNS of the of b-rafΔ/Δneu mice. Electron micrographs of P18 optic nerves (A) and spinal cords (B) in cross section. Note the high proportion of unmyelinated axons (plotted on the right as mean ± SD for the optic nerve; *, P < 0.05 comparing b-raf f/f and b-raf Δ/Δneu mice) and the thinner myelin sheaths in b-raf Δ/Δneu sections. The scatter plots show g ratios (diameter of the axon proper:outer diameter of the myelinated fiber) as a function of axon diameter for optic nerve and spinal cord (100 axons/mouse, three b-raf f/f, and three b-raf Δ/Δneu mice). White symbols indicate WT and gray symbols indicate b-raf Δ/Δneu mice. g ratios are higher in the b-raf Δ/Δneu mice (P < 0.0005), which indicates hypomyelination. Bars, 2.5 μm.
Figure 4.
Figure 4.
Oligodendrocytes maturation is defective in b-raf Δ/Δneu mice. Immature, PDGFRα+ oligodendrocytes, stained in brown, are present in several areas of the P18 b-raf Δ/Δneu but not b-raf f/f brain. Subjects were counterstained with hematoxylin. (A) Brain cortex (inset and bottom) and the corresponding quantification of positive cells/brain area. The plot shows means ± SD; *, P < 0.02; **, P < 0.005; and ***, P < 0.0005 comparing three b-raf f/f and three b-raf Δ/Δneu mice. A quantification of the βIV-tubulin+ cells (premyelinating, early myelinating, and myelinating oligodendrocytes) from the same areas is also shown. (B and C) b-raf Δ/Δneu oligodendrocytes fail to differentiate in vitro. Oligodendrocyte precursors from P0 b-raf f/f and b-raf Δ/Δneu pups were allowed to differentiate for 5 (B) or 6 d (C). Differentiation was analyzed by immunofluorescence staining with α-NG2, α-O4, and α-MBP antibodies. Examples of single+ and double+ cells from a WT culture are shown below in B. In C, cell morphology was visualized by staining with an α-tubulin antibody. The percentage of NG2+ cells (oligodendrocyte progenitors), O4+ cells (pro-oligodendrocytes), and MBP+ cells (terminally differentiated oligodendrocytes) present in the b-raf f/f and b-raf Δ/Δneu cultures were compared. Each culture consisted of a pool of two WT or KO mice; for the chemical inhibition of MEK, oligodendrocyte cultures were established from pools of four WT mice and kept for 6 d in differentiation medium in the absence (untreated [UT]) or presence of MEK inhibitor (U0126; 10 μM). In all cases, a minimum of 100 cells/culture were counted independently by two investigators. The experiment was repeated three times, the plots show the mean ± SD of the results obtained by assessing three separate experiments (*, P < 0.05 for b-raf f/f vs. b-raf Δ/Δneu). Bars, 30 μm.
Figure 5.
Figure 5.
B-Raf is required for ERK activation during postnatal brain development. (A) Expression/phosphorylation of components and targets of the ERK pathway in b-raf f/f (WT) and b-raf Δ/Δneu (KO) brains during postnatal development. (right) A quantification of B-Raf, Raf-1, pMEK, and pERK levels from at least three animals (mean ± SD). Levels are expressed as arbitrary units. Variation among experiments was minimized by normalizing the levels of the proteins of interest to loading controls that showed no change over time (actin). The dividing line in the βIV-tubulin panel indicates that the P18 samples were taken from a different gel. (B) Immunohistochemical analysis of ERK phosphorylation in B-Raf KO and WT brains. The brown staining indicates pERK+ cells. The sections were counterstained with hematoxylin. The vertical bar spans the neuropil-rich molecular layer of the cerebral cortex. G indicates the granular and M the molecular layer of the cerebellar cortex. Arrowheads indicate Purkinje cells.
Figure 6.
Figure 6.
B-Raf is required for MEK/ERK phosphorylation and ERK activation is required for differentiation in oligodendrocyte-enriched glial cell cultures. Immunoblot analysis of whole cell lysates (40 μg) from WT and B-Raf KO oligodendrocyte-enriched (A and C) or oligodendrocyte-depleted mixed glial cell cultures (B) left untreated or stimulated for 10 min with 100 ng/ml PDGF (A and B; 10 min), 20 ng/ml EGF (C), or 20 ng/ml FGF (C) before lysis. MEK/ERK phosphorylation is impaired in oligodendrocyte-containing but not oligodendrocyte-depleted cultures. In B, the first lane from the left is slightly underloaded. (D) ERK phosphorylation in oligodendrocyte cultures. Cells allowed to differentiate for 2 d and either left untreated or stimulated with 20 ng/ml FGF for the indicate times. Cells were colabeled with antibodies against pERK (green) and pan-tubulin (red). (E) MEK1 immunoprecipitates were prepared from 400 μg WT and B-Raf null (KO) oligodendrocyte-enriched glial cell cultures, and left untreated or stimulated with 100 ng/ml PDGF (for 10 min) before lysis and immunoblotting. C, isotype-matched irrelevant Ab control; MEK1 KO, MEK1 ips prepared from whole cell lysate of MEK1 KO MEFs. (F) ERK phosphorylation in cells of the oligodendrocyte lineage in situ. Brain sections were colabeled with antibodies against pERK (green) and βIV-tubulin (red). Cells were classified on the basis of their morphology as premyelinating oligodendrocytes (left and middle) and mature oligodendrocytes (right). Asterisks indicate autofluorescent erythrocytes. Bars, 30 μm. (G) A working model Raf/MEK/ERK complexes in differentiating oligodendrocytes. Soluble or axonal signals activate the ERK pathway in oligodendrocyte precursors. In WT cells, three complexes may be formed, two of which are B-Raf dependent: complex I, which comprises B-Raf, KSR, and MEK/ERK; and complex II, which consists of a B-Raf–Raf-1 heterodimer bound to KSR and MEK/ERK. In complex I, MEK is phosphorylated by B-Raf, whereas in complex II, the Raf heterodimer is the MEK kinase. Both complexes produce a strong, sustained ERK signal ultimately leading to oligodendrocyte differentiation. Complex III comprises Raf-1 and MEK/ERK, is B-Raf independent, and is the only complex found in B-Raf–deficient oligodendrocytes. This complex gives rise to a much weaker ERK signal, leading to defective oligodendrocyte differentiation.

Similar articles

See all similar articles

Cited by 37 articles

See all "Cited by" articles

References

    1. Aguirre, A., J.L. Dupree, J.M. Mangin, and V. Gallo. 2007. A functional role for EGFR signaling in myelination and remyelination. Nat. Neurosci. 10:990–1002. - PubMed
    1. Bhat, N.R., and P. Zhang. 1996. Activation of mitogen-activated protein kinases in oligodendrocytes. J. Neurochem. 66:1986–1994. - PubMed
    1. Brummer, T., P.E. Shaw, M. Reth, and Y. Misawa. 2002. Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling. EMBO J. 21:5611–5622. - PMC - PubMed
    1. Chen, A.P., M. Ohno, K.P. Giese, R. Kuhn, R.L. Chen, and A.J. Silva. 2006. Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory. J. Neurosci. Res. 83:28–38. - PubMed
    1. Davies, H., G.R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin, M.J. Garnett, W. Bottomley, et al. 2002. Mutations of the BRAF gene in human cancer. Nature. 417:949–954. - PubMed

Publication types

MeSH terms

Substances

Feedback