Article Figures & Data
Figures
- Figure 1.
Micro-CT of cuprizone-treated mouse brains. A, B, Coronal plane reconstruction of micro-CT scans of Histodenz-immersed brains derived from wild-type (+/+) and Ptprz-deficient (−/−) mice. Images show the whole region (A) and dorsal corpus callosum corresponding to the square region in A (B). The CT number increases from black to white. Mice were fed a cuprizone-containing diet for 6 weeks (0) to induce demyelination, followed by spontaneous remyelination with the removal of cuprizone (1, 3, and 5 weeks). Normal mice were maintained on the regular diet. Scale bars, 1 mm. C, Scatterplot showing differences between the CT number of the dorsal corpus callosum and that of the cerebral cortex (each circle corresponds to an individual mouse). Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. *p < 0.05, **p < 0.01, two-way ANOVA with Bonferroni post hoc test.
- Figure 2.
Accelerated remyelination in the corpus callosum of Ptprz-deficient mice after cuprizone-induced demyelination. A, B, LFB staining of wild-type (+/+) and Ptprz-deficient (−/−) mouse brains. Images show the whole region (A) and dorsal corpus callosum (B), respectively. After micro-CT scans, the Histodenz-immersed brains shown in Figure 1 were paraffin embedded and serially sectioned for histological examinations. Scale bars, 1 mm. C, Quantification of LFB staining of the dorsal corpus callosum. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. **p < 0.01, two-way ANOVA with Bonferroni post hoc test.
- Figure 3.
A, Accelerated reexpression of MBP in the corpus callosum of Ptprz-deficient mice after cuprizone-induced demyelination. Images show the dorsal corpus callosum. Coronal brain sections from wild-type (+/+) and Ptprz-deficient (−/−) mice were immunohistochemically analyzed using a specific polyclonal antibody against MBP (anti-MBP). Scale bars, 100 μm. B, Quantification of anti-MBP staining in the dorsal corpus callosum. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. *p < 0.05, two-way ANOVA with Bonferroni post hoc test.
- Figure 4.
A, Distribution of oligodendrocyte-lineage cells in the corpus callosum. Images show the dorsal corpus callosum. Coronal brain sections from wild-type (+/+) and Ptprz-deficient (−/−) mice were stained with anti-Olig2, a marker of oligodendrocyte-lineage cells. Scale bars, 50 μm. B, Scatterplot showing the number of anti-Olig2-stained nuclei in the dorsal corpus callosum. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. No genotypic differences were detected by two-way ANOVA.
- Figure 5.
A, Distribution of reactive astrocytes in the corpus callosum after the cuprizone treatment. Images show the dorsal corpus callosum. Brain sections from wild-type (+/+) and Ptprz-deficient (−/−) mice were stained with anti-GFAP, a marker of reactive astrocytes. Scale bars, 50 μm. B, Scatterplot showing the signal intensity of anti-GFAP staining. Relative values to the average value of the normal wild-type group. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. No genotypic differences were detected by two-way ANOVA.
- Figure 6.
A, Axonal damage induced in the dorsal corpus callosum by the cuprizone treatment. Stained images of damaged axons are shown. Brain sections from wild-type (+/+) and Ptprz-deficient (−/−) mice were stained with a specific antibody against APP (anti-APP) and then stained with hematoxylin. Scale bars, 50 μm. B, Scatterplot of the anti-APP-stained puncta in the dorsal corpus callosum. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. No genotypic differences were detected by two-way ANOVA.
- Figure 7.
Increase in pleiotrophin, but not midkine or IL-34, expression in the brain by cuprizone-induced demyelination. A, B, Brain sections of normal mice and mice fed with a cuprizone-containing diet for 6 weeks were stained with a specific antibody against pleiotrophin (anti-PTN), midkine (anti-midkine), or IL-34 (anti-IL-34). Staining images of the dorsal corpus callosum (A) and cerebral cortex (B) of wild-type (+/+) and Ptprz-deficient (−/−) mice are shown. Scale bars, 25 μm.
- Figure 8.
A, Pleiotrophin transiently expressed in the corpus callosum by the cuprizone treatment. Images show the dorsal corpus callosum. Brain sections from wild-type (+/+) and Ptprz-deficient (−/−) mice were stained with anti-PTN. Mice were fed a cuprizone-containing diet and then returned to a normal diet. Scale bars, 25 μm. B, Scatterplot showing the number of anti-PTN-stained puncta in the corpus callosum. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. No genotypic differences were detected by two-way ANOVA.
- Figure 9.
A, Pleiotrophin expression upregulated in the cerebral cortex by the cuprizone-induced demyelination. Anti-PTN staining of wild-type (+/+) and Ptprz-deficient (−/−) mouse brains. Scale bars, 25 μm. B, Scatterplot showing the percentage of anti-PTN positive neuron in the cortex. Open circles, Wild-type mouse; closed circles, Ptprz-deficient mouse. n = 10 for each group. No genotypic differences were detected by a two-way ANOVA.
- Figure 10.
Transient increase in pleiotrophin expression in damaged neurons by cuprizone-induced demyelination. A, Double-immunofluorescence labeling of PTN (green) and β-tubulin III or SYN1 (red) in the cerebral cortex of normal mice and mice fed a cuprizone-containing diet for 6 weeks. Scale bars, 10 μm. B, Double immunostaining for PTN (green) along with APP, SYN1, Olig2, or GFAP (red) in the corpus callosum of cuprizone-treated mice. Scale bars, 10 μm.
- Figure 11.
Accelerated oligodendrocyte differentiation through the PTN-PTPRZ interaction. A, B, Double-immunofluorescence labeling of a primary mixed glial culture obtained from Ptprz-deficient (−/−) and wild-type (+/+) brains using anti-NG2 proteoglycan (OPCs; red) and anti-MBP (oligodendrocytes, OLs; green) in conjunction with DAPI labeling of nuclei (blue). Glial cells were cultured in the absence or presence of 100 nm PTN without (A) or with (B) the oligodendrocyte differentiation factors biotin and TH for 6 d. Scale bars, 100 μm. The percentage of OPCs and OLs in all cells (DAPI-positive nuclei number) is shown in the bottom panel. Data are the mean ± SEM of four independent experiments. *p < 0.05, one-way ANOVA with Bonferroni post hoc test.
- Figure 12.
Establishment of OL1 cells. A, Isolation of OL1 cells. Dissociated brain cells from p53-deficient pups were cultured in basal medium (see the Materials and Methods section). Cell spheres (OL1 cells) formed after cultivation for 14 d (Aa). They were collected with a sterilized micropipette (Ab). A sphere was placed on a poly-l-ornithine-coated dish (Ac). Oligodendrocyte-lineage cells migrated out from the cell sphere after a 24 h culture (Ad). Scale bars, 200 μm. B, OL1 cells were cultured with or without the oligodendrocyte differentiation factors biotin and TH for 10 d. Cells were stained with anti-NG2 (red) and anti-MBP (green) antibodies. Scale bars, 100 μm. PhC, Phase contrast. C, Western blot analyses using anti-NG2 and anti-MBP antibodies. D, Double-immunofluorescence labeling of OL1 cells using anti-RPTPβ (anti-PTPRZ-A/B, green) and anti-NG2 proteoglycan (red) antibodies. Scale bars, 10 μm. DIC, Differential interference contrast. E, Western blot analysis using an anti-RPTPβ antibody in OL1 cells. The protein amounts applied were verified by Coomassie brilliant blue (CBB) staining. Because NG2 proteoglycan and PTPRZ receptors were expressed in OL1 cells as chondroitin-sulfate proteoglycans, the samples were treated with chABC before SDS-PAGE.
- Figure 13.
PTN induced the oligodendrocyte differentiation of OL1 cells. A, B, OL1 cells were cultured with various combinations of TH and PTN for 10 d. Cells were analyzed by immunocytochemistry (A) and Western blotting (B) with anti-NG2 proteoglycan (OPCs; red) and anti-MBP (oligodendrocytes, OLs; green) antibodies. The percentage of OPCs and OLs in all cells (DAPI-positive nuclei, blue) are shown at the bottom in A. Scale bars, 100 μm. PhC, Phase contrast. Data are the mean ± SEM (n = 5). **p < 0.01, Student's t test. C, D, PTN-induced phosphorylation of p190 RhoGAP at Tyr-1105. OL1 cells were incubated for 30 min with or without PTN in the presence of TH. C, The overall tyrosine phosphorylation pattern and protein expression were analyzed with anti-phosphotyrosine PY20 and anti-p190 RhoGAP antibodies, respectively. D, Tyr-1105 phosphorylation of p190 RhoGAP. Extracts were immunoprecipitated with anti-p190 RhoGAP antibody-coated beads and binding proteins were analyzed by Western blotting with anti-pY1105 and anti-p190 RhoGAP antibodies. Tyr-1105 phosphorylation levels were determined by densitometric analyses. Data are the mean ± SEM (n = 7). **p < 0.01, Student's t test.
- Figure 14.
Proposed mechanism for remyelination after demyelinating lesions. OPCs, but not mature oligodendrocytes (OLs), abundantly express PTPRZ-A/B receptor proteins as chondroitin sulfate (CS) proteoglycans. The CS moiety of PTPRZ is important for achieving the high-affinity binding of PTN to the core protein (Maeda et al., 1996). PTPRZ activity is requisite to maintaining the immature state of OPCs. After cuprizone-induced oligodendrocyte death and demyelination, the expression of PTN is transiently upregulated in damaged neurons. PTN may be released from demyelinated axons and bind to PTPRZ at the cell surface of OPCs that are recruited to lesions, probably through PTPRZ-independent mechanisms. The binding of PTN results in receptor dimerization or oligomerization, thereby inhibiting its catalytic activity (Fukada et al., 2006). PTPRZ inactivation releases the block of differentiation in OPCs, so the remyelination of neighboring axons is initiated.
