The p38α mitogen-activated protein kinase is a key regulator of myelination and remyelination in the CNS - PubMed (original) (raw)

doi: 10.1038/cddis.2015.119.

S Biswas 2, V Selvaraj 3, X-B Liu 2, J Sohn 4, P Jiang 2, C Chen 2, F Chmilewsky 5, H Marzban 6, M Horiuchi 4, D E Pleasure 4, W Deng 7

Affiliations

The p38α mitogen-activated protein kinase is a key regulator of myelination and remyelination in the CNS

S-H Chung et al. Cell Death Dis. 2015.

Abstract

The p38α mitogen-activated protein kinase (MAPK) is one of the serine/threonine kinases regulating a variety of biological processes, including cell-type specification, differentiation and migration. Previous in vitro studies using pharmacological inhibitors suggested that p38 MAPK is essential for oligodendrocyte (OL) differentiation and myelination. To investigate the specific roles of p38α MAPK in OL development and myelination in vivo, we generated p38α conditional knockout (CKO) mice under the PLP and nerve/glial antigen 2 (NG2) gene promoters, as these genes are specifically expressed in OL progenitor cells (OPCs). Our data revealed that myelin synthesis was completely inhibited in OLs differentiated from primary OPC cultures derived from the NG2 Cre-p38α CKO mouse brains. Although an in vivo myelination defect was not obvious after gross examination of these mice, electron microscopic analysis showed that the ultrastructure of myelin bundles was severely impaired. Moreover, the onset of myelination in the corpus callosum was delayed in the knockout mice compared with p38α fl/fl control mice. A delay in OL differentiation in the central nervous system was observed with concomitant downregulation in the expression of OPC- and OL-specific genes such as Olig1 and Zfp488 during early postnatal development. OPC proliferation was not affected during this time. These data indicate that p38α is a positive regulator of OL differentiation and myelination. Unexpectedly, we observed an opposite effect of p38α on remyelination in the cuprizone-induced demyelination model. The p38α CKO mice exhibited better remyelination capability compared with p38α fl/fl mice following demyelination. The opposing roles of p38α in myelination and remyelination could be due to a strong anti-inflammatory effect of p38α or a dual reciprocal regulatory action of p38α on myelin formation during development and on remyelination after demyelination.

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Figures

Figure 1

Figure 1

Generation of NG2/PlpCrep38_α_ CKO mice and in vitro differentiation of p38_α_ CKO OPCs. (a) Generation of p38_α_ CKO mice by crossing NG2/Plp-Cre mice and mice with p38_α_-floxed alleles (p38_α_ fl/fl). Schematic diagram showing the targeted genetic locus of p38_α_ gene flanked by two loxP sites. p38_α_ gene deletion is driven by Cre-loxP recombination that is under the control of NG2/Plp promoter activity in targeted cells. (b and c) Schematic illustrations demonstrating the EYFP (b) and ROSA26tdTomato (c) reporter system. Under the control of NG2/Plp promoter, Cre recombination results in an excision of tdTomato (mT) and the expression of EGFP (mG) in NG2/Plp-positive cells. (d) Genotyping of NG2-Cre mice demonstrating homozygosity of p38_α_-floxed allele and positive expression of NG2-Cre transgene. Western blotting analysis confirmed that the p38a protein is absent in p38_α_ CKO OPC cultures. (e–g) Schematic diagram of in vitro differentiation of purified primary OPCs from CKO mice. Four days after culture, both p38_α_ CKO (green: e) and p38_α_ fl/fl cells (red: f) were observed. (h–l) Immunohistochemistry for anti-MBP after terminal differentiation of OPCs into myelin-forming OLs. MBP immunofluorescence labeling demonstrates that MBP expression was diminished in p38_α_ CKO cells (green cells in panels (i) and (l)), while p38_α_ fl/fl cells express MBP (red cells in panels (j) and (l)). Scale bars: panel (g)=500 _μ_m; panel (l)=50 _μ_m

Figure 2

Figure 2

No obvious gross myelination defects were observed in p38_α_ CKO mice. (a–d) Transverse sections were peroxidase stained for anti-MBP in p38_α_ fl/fl (a and c) and p38_α_ CKO (b and d) at P28. Panels (c) and (d) are higher magnification views of the regions indicated in panels (a) and (b). (e–h) Transverse sections peroxidase stained for anti-MBP in p38_α_ fl/fl (e and g) and p38_α_ CKO (f and h) at P28. Panels (g) and (h) are higher magnification views of the regions indicated in panels (e) and (f). MBP expression in some areas of the CC seemed to be relatively weak in the knockout compared with p38_α_ fl/fl, but generally the difference was not obvious at low magnification of light microscope level. (i) Statistical analysis of the MBP staining intensity in the CC (P<0.01) and St of P28 p38_α_ CKO and p38_α_ fl/fl mice. Western blotting analysis of MBP protein in the whole brains of P28 p38_α_ CKO and p38_α_ fl/fl mice. **P<0.01. Abbreviation: Cb, cerebral cortex. Scale bars: panel (b)=500 _μ_m; panel (d)=250 _μ_m; panel (f)=500 _μ_m; panel (h)=250 _μ_m

Figure 3

Figure 3

p38_α_ CKO mice show morphological defects and reduced axon diameters. (a–e) Sagittal sections were peroxidase stained for anti-MBP in p38_α_ fl/fl (a) and p38_α_ CKO (b–d) at P12. Axonopathies observed in the p38_α_ CKO: axonal swelling (b and c), changes in the density of axons (c and d) and degeneration (e) were frequently seen. (f–k) Electron microscopic analysis of myelinated bundles in CC sections from P12 p38_α_ CKO and p38_α_ fl/fl mice. Panels (h) and (i) are higher magnification views of the regions indicated in panels (f) and (g). The thickness of myelin bundle diameter surrounding the axons was significantly reduced in the p38_α_ CKO (k) compared with p38_α_ fl/fl (j). (h) and (i) are higher magnification views of the regions indicated in panels (f) and (g). (l) Quantification of axon diameters and g_-ratio (the ratio of axon diameter to fiber diameter). The axon diameters were significantly reduced in the CKO CC (P<0.0001). The g_-ratio was significantly increased from 0.693 in p38_α fl/fl to 0.818 in p38_α CKO mice (P<0.0001). ***P<0.005. Scale bars: panel (e)=50 _μ_m; panel (g)=5 _μ_m; panel (h)=2.5 _μ_m; panel (j)=1 _μ_m

Figure 4

Figure 4

Delay in the onset of myelination in the CC of p38_α_ CKO mice. (a–h) Transverse sections peroxidase stained for anti-MBP at P5, P7 and P12 in p38_α_ fl/fl and p38_α_ CKO revealed that the onset and progression of myelination were delayed in the p38_α_ CKO. Panels (g) and (h) are higher magnification views of the regions indicated in panels (b) and (e). (i) Quantitative analysis of MBP expression during different postnatal periods in the CC of the p38_α_ fl/fl and p38_α_ CKO mice, suggesting a delay in onset of myelination (P5 and P7 P<0.0001). (j–o) Immunofluorescence-stained sections with anti-EYFP (green) and MBP (red) showed that the onset and progression of myelination were delayed in the p38_α_ CKO CC. Green cells represent p38_α_ knockout cells. Abbreviations: Cb, cerebral cortex; LV, lateral ventricle. ***P<0.005. Scale bars: panel (f)=250 _μ_m; panel (h)=75 _μ_m; panel (o)=250 _μ_m

Figure 5

Figure 5

The p38_α_ knockout cells show myelination defects in vivo. Immunofluorescence-stained sections with anti-EYFP (green) and MBP (red) in the (a) St and (b) CC of p38_α_ CKO mice. Green cells represent p38_α_ knockout cells. Majority of p38_α_ knockout cells (arrows) did not express MBP. Abbreviation: Cb, cerebral cortex. Scale bar: panel (b)=100 _μ_m

Figure 6

Figure 6

p38_α_ gene deletion results in a delayed OL differentiation without affecting proliferation. (a and b) Transverse sections were peroxidase stained for anti-Olig2 at P7 in the CC of the p38_α_ fl/fl (a) and p38_α_ CKO mice (b). (c) Quantification of Olig2-immunopositive cells in the CC at P0 and P3 in the p38_α_ fl/fl and p38_α_ CKO. The numbers of Olig2-posivtive cells were unchanged. (d–h) Quantitative PCR analysis for Brg1 (d), Olig1 (e), Zfp488 (f), NG2 (g) and olig2 (h) at P0 and P7 in the p38_α_ fl/fl and p38_α_ CKO mice. Olig1 and Zfp488 expression is significantly downregulated in the P0 p38_α_ CKO mice (e and f). *P<0.05, **P<0.01 ***P<0.005. Abbreviation: Cb, cerebral cortex. Scale bar: panel (b)=100 _μ_m

Figure 7

Figure 7

p38_α_ CKO promotes remyelination after cuprizone-induced demyelination. (a–f) Transverse sections peroxidase stained with anti-MBP showing remyelination process in the Cc after 3 weeks following discontinuation of cuprizone intake in the p38_α_ fl/fl (a–c) and p38_α_ CKO (d–f) mice. Panels (a and c) and (d and f) are higher magnification views of the regions indicated in panels (b) and (e), respectively. MBP expression is significantly increased in the CC of p38_α_ CKO (d–f) compared with p38_α_ fl/fl (a–c) mice. (g and h) Quantification of MBP staining intensity (g) and thickness (h) during a recovery period in the p38_α_ fl/fl and p38_α_ CKO. p38_α_ CKO mice showed a better remyelination ability compared with p38_α_ fl/fl during the remyelination process. *P<0.05. Abbreviations: Cb, cerebral cortex; LV, lateral ventricle. Scale bar: panel (e)=500 _μ_m; panel (f)=250 _μ_m

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