Dmdmdx mice have defective oligodendrogenesis, delayed myelin compaction and persistent hypomyelination

ABSTRACT Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, resulting in the loss of dystrophin, a large cytosolic protein that links the cytoskeleton to extracellular matrix receptors in skeletal muscle. Aside from progressive muscle damage, many patients with DMD also have neurological deficits of unknown etiology. To investigate potential mechanisms for DMD neurological deficits, we assessed postnatal oligodendrogenesis and myelination in the Dmdmdx mouse model. In the ventricular-subventricular zone (V-SVZ) stem cell niche, we found that oligodendrocyte progenitor cell (OPC) production was deficient, with reduced OPC densities and proliferation, despite a normal stem cell niche organization. In the Dmdmdx corpus callosum, a large white matter tract adjacent to the V-SVZ, we also observed reduced OPC proliferation and fewer oligodendrocytes. Transmission electron microscopy further revealed significantly thinner myelin, an increased number of abnormal myelin structures and delayed myelin compaction, with hypomyelination persisting into adulthood. Our findings reveal alterations in oligodendrocyte development and myelination that support the hypothesis that changes in diffusion tensor imaging seen in patients with DMD reflect developmental changes in myelin architecture.


Fig. S1 .
Fig. S1.Fewer oligodendrocyte progenitor cells in the lateral wall of the Dmd mdx V-SVZ (A) Dp427 protein expression in the V-SVZ of C57BL6 mice determined by western blotting.(B) Western blotting quantification normalized to total protein by ponceau staining.n=3 littermates per age.(C) Schematic of the dorsolateral wedge (DLW; black) and lateral wall (LW; magenta) regions of the V-SVZ that were analyzed using immunohistochemistry of coronal sections to detect Olig2 + , Ki67 + , Sox2 + , Ki67 + PDGFRa + , and Olig2 + PDGFRa + cells.Boxes depict the fields of view at 40X magnification.(D) Representative images in the LW at P8 from control and Dmd mdx mice.(E) Relative cell densities of indicated cell types in the LW control and Dmd mdx mice at P8.Not significant, multiple paired two-tailed ttest; n=7.(F) Representative images from the LW from control and Dmd mdx mice at P14. (G) Relative cell densities of indicated cell types in the DLW control and Dmd mdx mice at P14.Multiple paired two-tailed ttest, n=7.P-values shown on graph.(H) Representative images in the LW at P21 from control and Dmd mdx mice.(I) Relative cell densities of indicated cell types in the LW control and Dmd mdx mice at P21.Multiple paired two-tailed t-test, n=6.P-values shown on graph.Error bars denote SEM for all graphs.* P<0.05, ** P<0.01.Scale bar 25μm for all images.Abbreviations: corpus callosum (CC); striatum (Str); lateral ventricle (LV).

Fig. S2 .
Fig. S2.Single channel views of oligodendrocyte progenitor cells in the Dmd mdx V-SVZ.Single channel views to accompany analysis of Dmd mdx V-SVZ oligodendrocyte progenitor cells in Figure 1B, D, and F are shown.Olig2 was visualized using anti-rabbit CY5, Ki67 was visualized using anti-rat Alexa488, Sox2 was visualized using anti-mouse CY3, and PDGFRa was visualized using anti-goat Alexa680.(A) Representative images in the DLW at P8 from control and Dmd mdx mice.(B) Representative images from the DLW from control and Dmd mdx mice at P14. (C) Representative images in the DLW at P21 from control and Dmd mdx mice.Scale bar 25μm for all images.

Fig. S3 .
Fig. S3.Detection of oligodendrocyte progenitor cells and oligodendrocytes in the corpus callosum at P8 (A) Representative images from coronal sections of the corpus callosum from P8 control and Dmd mdx mice reveals cells immunoreactive for PDGFRa (magenta), CC1 (green), Olig2 (yellow), and Ki67 (cyan).(B) Relative cell densities of indicated cell types in the corpus callosum from P14 control and Dmd mdx mice.n=3, paired two-tailed t-test, not significant.

Fig. S4 .
Fig. S4.Detection of TUNEL+ cells in the corpus callosum and V-SVZ of Dmd mdx mice.(A) TUNEL+ cells in the corpus callosum (CC) in control (grey bars) and Dmd mdx (pink bars) mice.(B) TUNEL+ cells in the dorso-lateral wedge (DLW) of the subventricular zone (SVZ) neural stem cell niche in control (grey bars) and Dmd mdx (pink bars) mice.n=6; Mann-Whitney rank comparison, *p<0.05 or not significant (ns)

Fig. S5 .
Fig. S5.Western blots to detect CNP, MOG, and MBP levels in the corpus callosum of P21 and P56 Dmd mdx mice.Western blots of corpus callosum lysates from P21 (A) and (C) P57.The top membrane was probed for CNP (48 kDa).The middle blot was probed for MOG (27kDa) and the bottom blot is from the same membrane as the middle, and it was probed for MBP (21.5, 18.5, 17.25, and 12 kDa).(B) Quantification of western blot analysis of corpus callosum lysates from P21 probed for CNP, MOG and MBP.(D) Quantification of western blot analysis of corpus callosum lysates from 2 months (P57) probed for CNP, MOG and MBP.Not significant, unpaired two-tailed t-test.n= 3 control and n=5 Dmd mdx (P21), and n= 4 control and n=4 Dmd mdx (P57).Error bars denote SEM.

Fig. S7 .
Fig. S7.Axon diameters in control and Dmd mdx myelinated axons.Diameters of myelinated axons at P14 (A), P28 (B), and P60 (C) in control and Dmd mdx mice.A minimum of 274 axons were measured per condition.Unpaired two-tailed t=test was performed.ns, not significant.(D) Higher magnification views of panels depicted in Figure 5. Scale bar, 1µm.