PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum - PubMed (original) (raw)

. 2012 Aug 29;32(35):12152-64.

doi: 10.1523/JNEUROSCI.0628-12.2012.

Vimal Selvaraj, Kouji Wakayama, Lori Orosco, Eunyoung Lee, Susan E Crawford, Fuzheng Guo, Jordan Lang, Makoto Horiuchi, Konstantinos Zarbalis, Takayuki Itoh, Wenbin Deng, David Pleasure

Affiliations

PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum

Jiho Sohn et al. J Neurosci. 2012.

Abstract

Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural precursors. In neurosphere cultures prepared from the SVZ of adult mice, we found that addition of recombinant PEDF to the medium enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFrα) and transcription factors (Olig1, Olig2, and Sox10). Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein (GFAP:GFP) transgenic mice increased the proportions of GFAP:GFP+ and GFAP:GFP- SVZ neural precursors coexpressing oligodendroglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10 double-positive cells in the adult SVZ. Immunoreactive PEDF receptor was detectable in multiple cell types in both adult SVZ and corpus callosum. Furthermore, PEDF intracerebral infusion enhanced survival and maturation of newly born oligodendroglial progenitor cells in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong candidate for pharmacological intervention in demyelinative diseases.

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Figures

Figure 1.

Figure 1.

Characterization of control and PEDF-treated neurospheres derived from the SVZ of adult wild-type or GFAP:GFP transgenic mice. A, Experimental flow chart. Secondary neurospheres derived from the SVZ of either adult wild-type or GFAP:GFP transgenic mice were grown in the absence or presence of PEDF (50 ng/ml) for 5 d, and then assayed as indicated. B, RT-PCR analysis for expression of PEDF receptor in SVZ neurospheres. C, D, Wild-type control and PEDF-treated secondary neurosphere cells were immunostained for nestin, NG2, O4, or Tuj1. Immunostaining images (C) and quantification data (D) showing that PEDF significantly increased the numbers of NG2+ and O4+ cells. E, Quantification of BrdU incorporation and Ki67 labeling in wild-type control and PEDF-treated secondary neurospheres. PEDF did not alter proliferation of neurosphere cells. F, FACS plots for GFP+ and GFP− cell fractions dissociated from adult SVZ GFAP:GFP neurospheres. GFP+ selection was set using wild-type SVZ neurospheres as a negative control. G, GFAP immunostaining acutely done on plated GFP+ and GFP− cells FACS-purified from GFAP:GFP neurospheres. H, Graphic representation of flow cytometric quantification of percentages of NG2+ cells among GFP+ cells in control and PEDF-treated secondary GFAP:GFP neurospheres, showing that PEDF promoted NG2 induction in GFP+ cells. I, Quantification of BrdU incorporation among GFP+NG2+ cells FACS-purified from control and PEDF-treated secondary GFAP:GFP neurospheres, indicating that PEDF-mediated NG2 induction in GFAP:GFP+ precursors was not due to selective proliferation of GFP+NG2+ cells. ND, Not determined. Results are means ± SEM of three or four independent experiments. *p < 0.05, **p < 0.01, Student's t test. Scale bar, 30 μm.

Figure 2.

Figure 2.

PEDF exerts oligodendrogenic effect on adult SVZ GFAP+ neural precursors. A, Experimental flow chart. GFAP:GFP+NG2− and GFAP:GFP+NG2+ single cells were isolated by FACS from primary neurospheres derived from the SVZ of adult GFAP:GFP mice. Secondary neurospheres were then produced from these two FACS-purified populations in the absence or presence of PEDF (50 ng/ml). Assays were then performed as indicated. B, C, qRT-PCR analysis showing that PEDF elevated expression levels of oligodendroglial transcription factors and PDGFrα in both GFP+NG2− and GFP+NG2+ precursor-derived neurospheres. D, Western blot analysis of Olig1, Olig2, Sox10, and PDGFrα in control and PEDF-treated wild-type secondary neurospheres. E–G, Control and PEDF-treated secondary neurospheres derived from FACS-purified GFP+NG2− or GFP+NG2+ subsets were plated in differentiation medium without mitogens or PEDF for 3 d before immunostaining for NG2, O4, or Tuj1. E, G, Greater numbers of NG2+ and O4+ oligodendroglial cells were produced from both GFP+NG2− and GFP+NG2+ cell-derived neurospheres with PEDF treatment. F, G, PEDF treatment resulted in diminished neuronal differentiation of GFP+NG2− and GFP+NG2+ cell-derived neurospheres. G, Representative images of cells differentiated from control or PEDF-treated GFP+NG2− cell-derived neurospheres. Results are means ± SEM of three or four independent experiments. *p < 0.05, and **p < 0.01, Student's t test. Scale bar, 25 μm.

Figure 3.

Figure 3.

PEDF infusion increases the numbers of SVZ cells expressing early oligodendroglial lineage markers or oligodendroglial transcription factors. Saline or PEDF (300 ng per day) was administered via osmotic pump into the lateral ventricle of adult GFAP:GFP transgenic mice for 7 d. The SVZ tissues were double-immunostained for GFP and markers for early oligodendroglial lineage (PDGFrα or NG2) or oligodendroglial transcription factors (Olig1, Olig2, or Sox10). A–F, Confocal images of coronal sections of saline- and PEDF-infused SVZ immunolabeled with antibodies against GFP and PDGFrα, NG2, or Olig1. Insets show magnified images of areas indicated by rectangles. Scale bar, 30 μm. G–K, Percentages of DAPI+ nuclei labeled with PDGFrα+, PDGFrα+GFP+, and PDGFrα+GFP− (G), NG2+, NG2+GFP+, and NG2+GFP− (H), Olig1+, Olig1+GFP+, and Olig1+GFP− (I), Olig2+, Olig2+GFP+, and Olig2+GFP− (J), and Sox10+, Sox10+GFP+, and Sox10+GFP− (K) in the saline- and PEDF-infused SVZ. PEDF administration increased the proportions of both GFP+ and GFP− cells coexpressing these oligodendroglial markers or transcription factors. Results are means ± SEM (n = 4–5 brains). *p < 0.05, **p < 0.01, and ***p < 0.005, Student's t test.

Figure 4.

Figure 4.

Immunohistochemical analysis of PEDF receptor expression in the adult SVZ and corpus callosum. A, Confocal image showing expression of PEDF receptor in GFAP:GFP+ (arrows) and GFAP:GFP− cells (arrowheads) in the SVZ. B, DCX+ neuroblasts (indicated by arrows and orthogonal view) in the SVZ expressed PEDF receptor. CP, Choroid plexus. Scale bar, 25 μm. C–H, PEDF receptor expression was detected in both Olig2+ oligodendroglial lineage cells (C–E) and GFAP:GFP+ astrocytes (F–H) in the adult corpus callosum. Scale bar, 20 μm.

Figure 5.

Figure 5.

PEDF induces DCX+/Sox10+ cells in the adult SVZ. Saline or PEDF (300 ng/ml) was infused into the lateral ventricle of adult wild-type mice for 7 d before the animals were killed. A, B, Confocal images of saline-infused SVZ double-immunostained for DCX and Sox10. No DCX+ cells expressed Sox10 in the SVZ. C, D, Orthogonal (C) and _Z_-series stack (D) confocal images of PEDF-infused SVZ. PEDF infusion induced DCX+/Sox10+ cells in the SVZ. The insets in D show magnified images for the area indicated by a rectangle. Scale bar, 20 μm.

Figure 6.

Figure 6.

PEDF does not alter cell proliferation in the adult SVZ. Saline or PEDF (300 ng per day) was administered via osmotic pump into the lateral ventricle of GFAP:GFP transgenic mice for 7 d. BrdU (100 mg/kg body weight, i.p.) was injected daily into the animals from day 5 to day 7. A–D, Confocal images of coronal section of saline- and PEDF-infused SVZ immunostained for BrdU (A, B) or BrdU and GFP (C, D). E, F, Quantification of BrdU-single positive (E) and BrdU/GFP-double positive (F) cells in the SVZ. Cell proliferation was not significantly different between saline- and PEDF-infused SVZ. Scale bar, 35 μm. Results are means ± SEM (n = 3 brains).

Figure 7.

Figure 7.

PEDF enhances oligodendrocyte production and maturation of cycling OPCs in the adult corpus callosum. A, Experimental design. Adult wild-type mice received three consecutive administrations of BrdU (100 mg/kg body weight, i.p.) at 6 h intervals to label cycling OPCs in the corpus callosum, followed by saline or PEDF (300 ng per day) intracerebral infusion for 9 d. B, C, Confocal images of cells double-immunostained for BrdU and Olig2 (B) or CC1 (C) at day 0. D–F, Confocal images of cells double-immunostained for BrdU and CC1 (D) or CNP (F) in PEDF-infused corpus callosum, and for BrdU and CNP (E) in saline-infused corpus callosum at day 9. G, Percentages of BrdU+ cells that were colabeled with Olig2 at day 0 and day 9. The vast majority of cycling cells in the corpus callosum were Olig2+. H, Quantification for cells coimmunolabeled for BrdU and CC1, a marker for mature oligodendrocytes, in the corpus callosum at day 0 and day 9. At day 0, BrdU-labeled OPCs did not express CC1. By day 9, CC1+ mature oligodendrocytes had been produced from BrdU+ OPCs after both saline and PEDF infusion, but significantly more with PEDF infusion. I, Percentages of BrdU+ cells colabeled with CNP in saline- and PEDF-infused corpus callosum demonstrating that PEDF robustly promoted terminal maturation of newly born OPCs. ND, Not detected. B–F, Arrows indicate BrdU+ cells coimmunostained with oligodendroglial markers. Scale bar, 35 μm. Results are means ± SEM (n = 3 brains). *p < 0.01, an increase in the number of BrdU+CC1+ cells in between saline- vs PEDF-infused corpus callosum at day 9, Student's t test.

Figure 8.

Figure 8.

PEDF enhances cell survival in the adult corpus callosum. A, Experimental design. Adult wild-type mice received three consecutive administrations of BrdU (100 mg/kg body weight, i.p.) at 6 h intervals to label cycling OPCs in the corpus callosum, followed by saline or PEDF (300 ng per day) intracerebral infusion for 2 or 5 d. At day 5, EdU (100 mg/body weight, i.p.) was also injected into the animals 5 h before they were killed. B, Confocal images of cells colabeled for TUNEL and Olig2 or NG2 in saline-infused corpus callosum, showing oligodendroglial lineage cells undergoing apoptosis. C, D, Confocal images of cells double-assayed for BrdU and TUNEL in the saline (C)- or PEDF (D)-infused corpus callosum. C, BrdU+/TUNEL+ cell is indicated by white arrows. D, BrdU+/TUNEL− cell is indicated by yellow arrows. BrdU−/TUNEL+ cell (arrowheads) confirms the absence of cross-reactivity between BrdU and TUNEL staining. E, F, Percentages of TUNEL+ (E) and BrdU+/TUNEL+ (F) cells in saline- or PEDF-infused corpus callosum at day 2 and 5, indicating a potent cell survival effect of PEDF on the corpus callosal cells including newly born OPCs (F). G, EdU incorporation assay indicating that PEDF did not significantly alter cell proliferation in the corpus callosum. ND, Not detected. Scale bar, 15 μm. Results are means ± SEM (n = 3 brains). *p < 0.01, Student's t test.

Figure 9.

Figure 9.

PEDF promotes oligodendroglial regeneration in lysolecithin-induced demyelinative corpus callosum. Administration of lysolecithin to adult corpus callosum to induce focal demyelination was followed by saline or PEDF (300 ng per day) infusion for 3 or 7 d. A–H, Confocal images of cells in saline- or PEDF-infused corpus callosum immunolabeled with antibodies against NG2 (A–D), CC1 (E, F), or CNP (G, H) at 3 or 7 dpl. Scale bars, 25 μm. I–L, MBP immunostaining of saline (I, K)- or PEDF (J, L)-infused corpus callosum at 7 dpl. K, L, High-magnification images of the areas indicated by rectangles in I and J, respectively. Scale bars, 50 μm. M, N, Quantification for NG2+ OPCs at 3 and 7 dpl (M), and CC1+ and CNP+ mature oligodendrocytes (N) at 7 dpl. Note that PEDF infusion resulted in markedly higher numbers of NG2+ cells present in the lesional corpus callosum at 3 dpl, and substantially enhanced CNP+ mature oligodendrocyte production and CNP immunoreactivity, and MBP-positive myelin levels at 7 dpl. CC, Corpus callosum; LV, lumen of lateral ventricles. Results are means ± SEM (n = 4 brains). *p < 0.05 and **p < 0.01, Student's t test.

Figure 10.

Figure 10.

Regulation of PEDF protein expression in lysolecithin-induced demyelinative corpus callosum. Mice received no treatment, saline or lysolecithin (LPC) injections to corpus callosum (CC), or PEDF infusion (300 ng per day) to corpus callosum. Protein extracts from ipsilateral corpus callosum were analyzed for PEDF expression by Western blots. A, Representative Western blot showing robust upregulation of PEDF expression in the corpus callosum after lysolecithin injection. Specificity of the PEDF antibody used for Western blotting was confirmed by the absence of immunoreactive PEDF in homozygous PEDF knock-out (KO) mice. B, Densitometric quantification demonstrated that PEDF contents were 2.7-, 6.7-, and 4.6-fold higher in lysolecithin-lesioned than intact corpus callosum on days 2, 5, and 7 post-lysolecithin injection, respectively. In nonlesioned mice, 2 d of intracerebral recombinant PEDF infusion raised corpus callosum PEDF content by 4.9-fold. Results are means ± SEM (n = 3 mice/point). *p < 0.001 (Student's t test), significantly elevated over level in intact corpus callosum.

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