Constitutively active Akt induces enhanced myelination in the CNS

Abstract

The serine/threonine kinase Akt regulates multiple cellular functions. The current studies identify a new role for Akt in CNS myelination. In earlier studies on cultured oligodendrocytes, we showed that neuregulin signals through phosphatidylinositol-3'-OH kinase and Akt to enhance survival of oligodendrocytes. However, when transgenic animals were generated that overexpressed constitutively active Akt in oligodendrocytes and their progenitor cells, no enhanced survival of oligodendrocytes or progenitors was found. No alteration in the proliferation or death of progenitors was noted. In contrast, the major impact of Akt overexpression in oligodendrocytes was enhanced myelination. Most interestingly, oligodendrocytes in these mice continued actively myelinating throughout life. Thus, expression of constitutively active Akt in oligodendrocytes and their progenitor cells generated no more oligodendrocytes, but dramatically more myelin. The increased myelination continued as these mice aged, resulting in enlarged optic nerves and white matter areas. In older animals with enlarged white matter areas, the density of oligodendrocytes was reduced, but because of the increased area, the total number of oligodendrocytes remained comparable with wild-type controls. Interestingly, in these animals, overexpression of Akt in Schwann cells did not impact myelination. Thus, in vivo, constitutively active Akt enhances CNS myelination but not PNS myelination and has no impact developmentally on oligodendrocyte number. Understanding the unique aspects of Akt signal transduction in oligodendrocytes that lead to myelination rather than uncontrolled proliferation of oligodendrocyte progenitor cells may have important implications for understanding remyelination in the adult nervous system.

Figure 1.

Active Akt is overexpressed in oligodendrocytes in Plp-Akt-DD mice. A, Cerebrum samples from littermate WT and Plp-Akt-DD9 mice were analyzed for Akt expression by Western blot at P10, P14, P21, and P30, and a representative blot is presented. The number of pairs of animals analyzed at each age varied from 5 to 21. B, Akt was overexpressed in oligodendrocytes of Plp-Akt-DD9 mice (DD) as shown by representative immunostained sections for Akt at P14, P21, and P30. The number of pairs of animals analyzed at each age varied from 6 to 32. Scale bars, 200 μm. C, Akt kinase assay (using GSK-3 as substrate) was used to confirm that the overexpressed Akt was active.

Figure 2.

Oligodendrocyte progenitor cell number, proliferation, and survival are unaffected in Plp-Akt-DD mice. A, Plp-Akt-DD9/Plp-EGFP littermate cerebrum samples were analyzed at P6 for expression of Akt. Akt overexpression was observed only in EGFP-positive cells (arrows) in these samples, which, as shown by Mallon et al. (2002), are oligodendrocyte progenitors. n = 4. B, Plp-Akt-DD9 and WT littermates were injected with BrdU on P5 and analyzed on P6 for PDGFRα-positive or BrdU/PDGFRα-positive cells. n = 3. C, The three areas in motor cortex (M1, primary motor cortex; M2, secondary motor cortex) and cingulate cortex (Cg1, cingulate cortex area 1; Cg2, cingulate cortex area 2) that were studied to quantify progenitors undergoing cell proliferation are boxed [Franklin and Paxinos (2008), their Fig. 24, modified with permission]. D, The number of PDGFRα cells and the number of BrdU-positive PDGFRα cells were quantified in cortical areas as shown in C. The total number of cells for the three combined areas was obtained from one slide each from three animals per genotype. The average of the three samples per genotype is presented. There was no difference in either the number of progenitors or the number of proliferating cells. Scale bars: A, 25 μm; B, 50 μm. Error bars represent SEM.

Figure 3.

Myelination was early and enhanced in Plp-Akt-DD mice with no increase in oligodendrocyte cell number. A, Cerebrum samples from WT and Plp-Akt-DD9 littermates were immunostained for PLP/DM20 at P14, P21, and P30, and enhanced myelination was seen at all ages in Plp-Akt-DD9 compared with WT. Note increased premyelinating oligodendrocytes at P14 and increased myelinated areas at older ages. This image is representative of different experiments, in which between 3 and 10 pairs of animals were analyzed per age. Scale bars, 100 μm. B, Samples were analyzed for PLP and DM20 by Western blot, at P10, P14, P21, and P30. This is a representative image of blots of between three and five pairs of animals per age. PLP/DM20 expression was increased as early as P10, compared with WT. C, RNA was prepared from cerebrum samples at P10, P21, and P30, and quantified for PLP, MOG, and MBP by real-time PCR. Samples were quantified, relative to a standard RNA, GAPDH, and expressed relative to WT samples at P21, which were given an arbitrary value of 1.0. Thus, changes in WT RNA levels were analyzed over time, along with differences between WT and transgenic samples. n = 3. *p ≤ 0.05; **p < 0.005. D, To quantify the number of oligodendrocytes, the Plp-Akt-DD9 transgene was crossed into Plp-EGFP mice. Coronal sections were imaged for EGFP-expressing oligodendrocytes in cortical areas of WT/Plp-EGFP and Plp-Akt-DD9/Plp-EGFP littermates at P14, P21, and P30. Scale bars, 100 μm. E, The area of motor and cingulate cortex analyzed for quantifying oligodendrocytes in WT/Plp-EGFP and Plp-Akt-DD9/Plp-EGFP littermates is boxed [Franklin and Paxinos (2008), their Fig. 24, modified with permission]. It includes part of M1 (primary motor cortex), M2 (secondary motor cortex), Cg1 (cingulate cortex area 1), Cg2 (cingulate cortex area 2), and the associated subcortical white matter. F, Quantification of oligodendrocytes at P14, P21, and P30, in the area outlined in E, showed no difference in the number of oligodendrocytes between WT and Plp-Akt-DD9 mice at all ages studied. n = 3 per age per genotype. Error bars represent SEM.

Figure 4.

Optic nerves from Plp-Akt-DD mice were larger and had increased myelin, relative to WT mice. A, Optic nerves from WT and Plp-Akt-DD9 mice at 2 months of age were imaged (n = 15 per genotype), and Plp-Akt-DD9 nerves were dramatically larger than WT. B, Electron micrographs of WT and Plp-Akt-DD9 optic nerves at 2 months of age. Relatively circular axons, such as those labeled with asterisks, were used for calculating g ratio, whereas those with irregular shape, e.g., those labeled with x, were excluded from the analysis. Three samples were analyzed per genotype, and representative images are shown. Magnification, 6300×. C, Scatter diagram showing the distribution of the g ratio as a function of axon diameter in WT and Plp-Akt-DD9 optic nerve at 2 months of age. g ratios and axon diameters were plotted for individual axons. WT data points are in black; Plp-Akt-DD data points are in gray. D, Quantification of the number of axons in different size distribution for the two genotypes. Three pairs of optic nerves per genotype were used for calculation of g ratio and axon diameter.

Figure 5.

Enhanced myelination continued as Plp-Akt-DD mice aged. A, Cerebrum samples were analyzed for PLP/DM20 expression by Western blot at 3, 6, and 10 months of age (n = 3–5 samples per age per genotype), and both were upregulated in Plp-Akt-DD9 at all ages. B, Cerebrum samples were analyzed for PLP/DM20, MAG, MBP, and MOG mRNA expression by real-time PCR at 2, 5, and 8 months of age, and all were upregulated in Plp-Akt-DD9 at all ages. Samples were quantified, relative to a standard RNA, GAPDH, and then expressed relative to WT samples at 2 months of age, which were given an arbitrary value of 1.0. Thus, changes in WT RNA levels with age are presented, as well as changes in transgenic RNA levels, relative to WT. n = 3. *p ≤ 0.05; **p < 0.005. Error bars represent SEM. C, Tissue sections were analyzed for MBP expression by immunohistochemistry at 5 months of age, which was significantly increased. In addition, enlargement of the corpus callosum (arrows) was noted (n = 16 animals per genotype at 5–6 months of age). Scale bars, 200 μm. Representative images of the immunocytochemistry are shown. D, WT (circles) and Plp-Akt-DD9 (triangles) samples were analyzed for myelin content by x-ray diffraction at 2 (open symbols) and 6 (closed symbols) months of age. At 2 months of age, four optic nerves were analyzed for WT mice and three for Plp-Akt-DD9 mice; at 6 months of age, two nerves each were analyzed. Individual data points are presented. Inset, Raw diffraction spectra used to obtain quantitative data of the relative amount of myelin. No data points are obscured beneath this inset. E, Electron micrographs of WT and Plp-Akt-DD9 optic nerves at 6 months of age. Magnification, 10,000×. At least three samples were analyzed per genotype, and a representative image is shown.

Figure 6.

Enlarged area and reduced oligodendrocyte density in 6-month-old Plp-Akt-DD corpus callosum. A, B, Confocal images showing corpus callosum per hemisphere in WT and Plp-Akt-DD9 mice. The area occupied by corpus callosum in Plp-Akt-DD9 (B) appeared larger than that in WT (A). Scale bars, 500 μm. C, D, Representative confocal images taken at the central corpus callosum used for quantifying oligodendrocytes in WT (C) and Plp-Akt-DD9 (D) samples. Scale bars, 50 μm. E, F, Higher-magnification images showing that the number of oligodendrocytes per field of view is more in WT (E) than in Plp-Akt-DD9 (F) samples. Scale bars, 25 μm. Quantification of oligodendrocyte density (expressed as cells per cubic millimeter) at the central corpus callosum and the total number of oligodendrocytes in the corpus callosum are described in Table 1. n = 3 per genotype.

Figure 7.

PNS myelination was not impacted by Akt-DD overexpression. A, Sciatic nerves from WT and Plp-Akt-DD9 mice were analyzed for Akt (top), P0 (middle), and actin (bottom) by Western blotting. Despite Akt overexpression in sciatic nerve, no increase in P0 was detected in Plp-Akt-DD9 mice. At least three samples were analyzed per age per genotype. B, Sciatic nerves from WT (circles) and Plp-Akt-DD9 (triangles) were analyzed by x-ray diffraction and showed no difference in myelination. Four nerves from each genotype were analyzed at 2 (open symbols) or 6 (closed symbols) months of age. C, Epon sections (1 μm) of WT and Plp-Akt-DD9 ventral roots at 2 months of age. Scale bars, 20 μm.