MBP and sncRNA715 Expression in Schwann cells.

<p><b>A</b>, Reverse transcription PCR (RT-PCR) on RNA extracted from Oli-<i>neu</i> or IMS32 cells using <i>Mbp</i>-specific primers. The 88nt long amplicon for <i>Mbp</i> was visualized in an ethidium bromide-stained 4% agarose gel. <b>B</b>, Western Blots of lysates from P18 mouse brain (brain lysate), primary oligodendrocytes (pOL, 7DIV), IMS32 and Oli-<i>neu</i> cells using MBP and GAPDH (loading control) specific antibodies. <b>C</b>, Reverse transcription PCR (RT-PCR) on RNA extracted from Oli-<i>neu</i> or IMS32 cells using a sncRNA715-specific primer assays. PCR products (~60-nt long due to the use of hairpin primers in the RT reaction) were visualized in an ethidium bromide stained 4% agarose gel. <b>D</b>, Northern Blots with RNA from IMS32 and undifferentiated primary Schwann cells (pSC) shows expression of sncRNA715 in IMS32 and a lower expression in pSC. Synthetic sncRNA715 (715-mimic) and U6 snRNA were used as positive control and loading control, respectively. <b>E</b>, RT-PCR on RNA from IMS32 and undifferentiated pSC confirms lower expression of sncRNA715 in pSC compared to IMS32 cells shown in D. 715-mimic was used as positive control and snoRNA135 as loading control.</p

Inverse correlation of MBP and sncRNA715 in the sciatic nerve.

<p><b>A&B</b>, The sciatic nerve was lysed from mice at postnatal day 1, 4 and 9 and myelin proteins as well as sncRNA715 expression was analyzed by Western blotting (<b>A</b>) and qPCR (<b>B</b>), respectively. MBP and CNP Western blots show increasing levels in differentiating sciatic nerves (<b>A</b>) while sncRNA715 levels decrease during differentiation, P-values P4: 0,0313, P9: 0,0313 (<b>B</b>, log2 values are plotted, sncRNA715 levels at P4 and P9 were quantified relative to P1 using snoRNA135 as a reference gene). Number of experiments (n) are indicated and bar graphs represent mean values ± s.e.m. (Wilcoxon signed-rank test, *P< 0.05, GraphPad Prism5 was used for statistical analysis).</p

Inverse correlation of MBP and sncRNA715 in primary Schwann cells.

<p><b>A</b>, Primary Schwann cells derived from sciatic nerves of P3 Wistar rats were cultured in non-differentiating (untreated) or differentiating (+NRG1 +dbcAMP) conditions. MBP protein can only be detected by immunocytochemistry in differentiated Schwann cells. Scale bar represents 50μm. <b>B</b>, Western Blots of undifferentiated and differentiated primary Schwann cells show MBP protein only present in differentiated Schwann cells. CNP is expressed in both maturation stages of primary Schwann cells. GAPDH serves as loading control. <b>C</b>, MBP and sncRNA715-specific RT-PCR on RNA extracted from undifferentiated or differentiated primary Schwann cells. <i>Mbp</i> mRNA is present at both differentiation states while sncRNA715 is detectable in undifferentiated and hardly in differentiated Schwann cells. SnoRNA135 and <i>G6pdh</i> mRNA were used as loading controls.</p

The absence PrP^c increases OPC proliferation in cultured embryonic ONs.

<p><b>A</b>) Western blots of embryonic ONs (E16.5) from <i>Prnp^0/0</i> and <i>Prnp</i>^+/+ mice probed for Nestin and Olig2. Expression of both proteins was increased in <i>Prnp^0/0</i> versus <i>Prnp</i>^+/+ ONs. <b>B-C</b>) Representative immunofluorescence images of a 2 DIV ON explant culture from <i>Prnp</i>^+/+ mice in three dimensional collagen matrices after staining with anti-AB25 and counterstaining with DAPI. Cells that migrated radially were identified as OPCs by the expression of the A2B5 epitope (see also lower box). <b>D-G</b>) Immunofluorescence photomicrographs of 2 DIV ON explant cultures from <i>Prnp</i> (D-E) and <i>Prnp^0/0</i> (F-G) mice in three dimensional matrices after double staining with anti-BrdU and anti-A2B5. <b>H</b>) Scheme illustrating the method to quantify A2B5 and BrdU staining in explant cultures. Yellow boxes to quantify each quadrant are shown. <b>I</b>) Histogram showing relative percentage of A2B5 and BrdU-positive cells in <i>Prnp^0/0</i> and <i>Prnp^+/+</i> mice. The absence of PrP^c significantly increased the number of BrdU-labeled OPCs. <b>J</b>) Quantification of the maximal distance migrated by OPCs in ON explants. <i>Prnp</i>^+/+ OPCs migrated significantly further than their <i>Prnp^0/0</i> counterparts. Values in I and J represent the mean ± standard deviation, and the asterisks indicate statistical significance (<i>P</i> < 0.01, Student´s <i>t</i>-test). Scale bars: in B and D  =  200 µm also apply to A, and E-G, respectively.</p

A putative scheme of PrP^c involvement in oligodendrocyte proliferation and maturation.

<p><b>A-B</b>)<b>.</b> Scheme summarizing the main <i>in vitro</i> findings of the present study. The absence of PrP^c in cultured OPCs from the ON and in the isolated cortical OPCs prolonged the proliferative stage in these precursors, and the modification of intrinsic factors modulating cell proliferation and oligodendrocyte differentiation (e.g., <i>Sox17, cdk2)</i> may delay their normal maturation. <b>C-D</b>) No differences in CNS myelination were observed between <i>Prnp^0/0</i> and <i>Prnp^+/+</i> mice, although increased proliferation was observed in adult NG2 cells in <i>Prnp^0/0</i> mice. The putative changes in intrinsic factors are overcome by extrinsic (neuronal and astroglial) factors to establish normal myelination. These extrinsic signals (<i>e.g.,</i> PDGF-AA, FGF-2, IGF-1, NT-3 or CNTF) may help to control the proper timing of OPC differentiation, ensuring adequate myelination and its maintenance. Surplus NG2 cells are eliminated by cell death in this balanced system.</p

Cultured <i>Prnp^0/0</i> oligodendrocytes remain in an undifferentiated state for longer than the <i>Prnp</i>^+/+ controls.

<p><b>A</b>) Low power photomicrograph of a primary <i>Prnp</i>^+/+ mixed culture showing NG2-positive oligodendrocytes (arrows) grown and differentiated over a GFAP-positive astrocyte monolayer. <b>B-C</b>) Phase contrast (PhC) images of a <i>Prnp</i>^+/+ (B) and <i>Prnp^0/0</i> (C) primary oligodendrocyte mixed cultures showing the morphology after 3 days in SFM. Numerous ramified oligodendrocytes (arrows in B) can be seen in <i>Prnp^0/0</i>-derived cultures, in contrast to the bipolar oligodendrocyte morphologies (arrowheads in C) seen over the astrocyte monolayer (asterisk in B and C). <b>D-E</b>) High magnification images of B and C showing the greater ramification (arrows) of <i>Prnp^+/+</i> versus <i>Prnp^0/0</i> (arrowheads) oligodendrocytes. <b>F-K</b>) Immunofluorescence images of Olig2 (F,I) and CNPase (G,J) expression in mixed cultures (5 days in SFM) derived from <i>Prnp</i>^+/+ and <i>Prnp^0/0</i> mice. Cultures were counterstained with DAPI and the arrows indicate the CNPase-Olig2-positive cells in both cultures. Note the absence of double labeled (CNPase/Olig2) cells in <i>Prnp^0/0</i> derived cultures. <b>L</b>) Histogram illustrating the mean number of Olig2 and CNPase cells per mm² in cultures of both genotypes. Values represent mean ± standard deviation and the asterisks indicate statistical significance (<i>P</i> < 0.01 Student´s <i>t</i>-test). <b>M-N</b>) MBP labeling in <i>Prnp^+/+</i> (M) and <i>Prnp^0/0</i> (N) in mixed cultures after 5 days in SFM. <b>O</b>) Histogram showing RT-qPCR analysis of RNA samples extracted from <i>Prnp^+/+</i> and <i>Prnp^0/0</i> purified oligodendrocytes from mixed cultures after 5DIV in SFM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033872#s2" target="_blank">methods</a> for details). Data represent the mean induction of three independent experiments where GAPDH was used as the reference gene. <b>P-Q</b>) Immunofluorescence images of double labeled CNPase-Olig2-positive cells in isolated OPCs cultures (5 days in SFM, without astrocytes) derived from <i>Prnp^+/+</i> (P) and <i>Prnp^0/0</i> (Q) mice. Cultures were counterstained. In the <i>Prnp^0/0</i> cultures there were less oligodendrocytes (Olig2-positive arrowheads) expressing CNPase with reduced ramifications (Q) compared to wild-type oligodendrocytes (P). Scale bar: A  =  50 µm; B  =  50 µm also applies to C; D  =  20 µm also applies to E; F  =  50 µm also applies to G-K. M  =  50 µm also applies to N. P  =  40 µm also applies to Q.</p

PrP^c is expressed in oligodendrocyte precursor cells.

<p><b>A-C</b>) High magnification immunofluorescence images of double-labeled (NG2-PrP^c) cells in primary OPC cultures. <b>D</b>) PrP^c detection in Western blots of brain extracts from P0 <i>Prnp</i>^+/+ mice probed with the SAF61 antibody (lane 1) and extracts from primary postnatal oligodendrocyte cultures of <i>Prnp</i>^+/+ and <i>Prnp^0/0</i> mice (lanes 2 and 3 respectively). <b>E</b>) PrP^c detection in Western blots of embryonic mice ONs (E16.5) from <i>Prnp</i>^+/+ (lane 1) and <i>Prnp^0/0</i> mice (lane 2) probed with the SAF61 antibody. No PrP^c was detected in ONs from <i>Prnp^0/0</i> mice. <b>F-I</b>) Immunofluorescence images of <i>Prnp^0/0</i> (F-G) and <i>Prnp</i>^+/+ (H-I) SVZ neurospheres after differentiation, demonstrating the ability of both genotypes to differentiate into NG2-positive cells. No PrP^c expression was detected in NG2-positive cells in <i>Prnp^0/0</i> neurospheres. Scale bars: A  =  25 µm also applies to B-C; F  =  50 µm also applies to G-I.</p

Low SGZ proliferation and neurogenesis in the dentate gyrus of adult <i>Prnp^0/0</i> mice correlate with high numbers of double labeled NG2-BrdU cells in the molecular layer.

<p><b>A</b>) Left: Quantification of the number of PSA-NCAM-positive and BrdU-positive cells in dentate gyrus sections from <i>Prnp^+/+</i> and <i>Prnp^0/0</i> mice one day after BrdU-labeling. Right: Quantification of the number of BrdU-positive and BrdU/NeuN-positive cells per section in the dentate gyrus 15 days after administering the last BrdU pulse. Values represent the mean ± standard deviation and the asterisks indicate statistical significance (<i>P < 0.01</i>, Student´s <i>t</i>-test). <b>B-C</b>) Representative photomicrographs showing the suprapyramidal region of the adult dentate gyrus of 2 month old <i>Prnp^+/+</i> (B) and <i>Prnp^0/0</i> (C) mice injected with BrdU 15 days prior to sacrifice. Sections were incubated with antibodies for NeuN and BrdU and the arrows indicate newborn neurons in the dentate gyrus. The fluorochrome used in each case is indicated in the Figure. <b>D-E</b>) Representative low power photomicrographs of the dentate gyrus of 2 month old <i>Prnp^+/+</i> (D) and <i>Prnp^0/0</i> (E) mice injected with BrdU 1 day prior to sacrifice. Note the decrease in the number of cells that incorporate BrdU (arrows) in the subgranular zone of <i>Prnp^0/0</i> compared to <i>Prnp^+/+</i> mice. By contrast, numerous BrdU-positive cells (arrowheads) can be seen in the molecular layer of <i>Prnp^0/0</i> than in <i>Prnp^+/+</i> mice. <b>F</b>) Example of a double-labeled BrdU/NG2 cells in the molecular layer of <i>Prnp^0/0</i> mice. This image was obtained on an Olympus confocal microscope and processed with Imaris Silicon Graphics software to obtain the orthogonal 3D Z-axis projections. Orthogonal projections are shown on the right (y-axis) and at the bottom (x-axis). Abbreviations: DG: dentate gyrus: GCL: granule cell layer; ML: molecular layer; H: Hilus; SGZ: subgranular zone. Scale bars: B  =  50 µm also applies to C. D  =  200 µm also applies to E; F  =  25 µm.</p

Adult <i>Prnp^0/0</i> mice exhibit normal myelination in the CNS.

<p><b>A-B</b>) <i>In situ</i> hybridization of <i>MAG</i> in coronal sections from the brains of adult <i>Prnp^+/+</i> and <i>Prnp^0/0</i> mice. <b>C</b>) Quantification of MAG-positive cells revealed no differences between genotypes in the corpus callosum or cingular cortex. Values represent the mean ± standard deviation and they were analyzed using the Student’s <i>t</i>-test. <b>D-E</b>) Western blots of brain extracts from <i>Prnp^+/+</i> and <i>Prnp^0/0</i> mice showing no differences in the expression of the MAG and MBP myelin proteins between genotypes in neocortex (D) and spinal cord (E). <b>F, H</b>) Lower magnification electron microscopy photomicrograph of <i>Prnp</i>^+/+ and <i>Prnp^0/0</i> mice. <b>G, I</b>) Higher magnification of F and H, respectively, showing no gross ultrastructural differences in the myelin sheaths of the corpus callosum. <b>J-K</b>) Representative photomicrographs of the parietal cortex of <i>Prnp^+/+</i> (J) and <i>Prnp^0/0</i> (K) mice immunostained for BrdU 1 day after the last BrdU pulse. Slides were lightly counterstained with Nissl. Numerous BrdU-positive cells can be seen in <i>Prnp^0/0</i> compared to wild-type (arrows in J and K) <b>L</b>) Quantification of BrdU incorporation and the double-labeled BrdU-NG2-positive cells 1 day after the last BrdU pulse. Both populations increased in the parietal cortex of <i>Prnp^0/0</i> mice when compared to <i>Prnp^+/+</i> mice. <b>M</b>) Photomicrograph of double-labeled BrdU-NG2 cells (arrows) in the parietal cortex of <i>Prnp^0/0</i> mice. Arrowheads indicate non-BrdU NG2-positive cells. <b>N</b>) Quantification of BrdU-positive cells 15 days after the BrdU pulse (4 + 15 protocol). Values in (L) and (N) represent the mean ± standard deviation and the asterisks indicate statistical significance (<i>P</i> < 0.01, Student <i>t</i>-test). <b>O</b>) Photomicrograph showing double-labeled NG2/TUNEL cells (arrows) in the <i>Prnp^0/0</i> parietal cortex, in which the arrowheads indicate non-NG2-TUNEL-positive cells. Abbreviations: DG: dentate gyrus: GCL: granule cell layer; ML: molecular layer; H: Hilus; SGZ: subgranular zone. CC: corpus callosum; CGC: cingulate cortex; NC: neocortex. H: hippocampus; Scale bars: A  =  300 µm also applies to B; F: 2 µm also applies to H; G  =  0.5 µm also applies to I; J and K  =  100 µm. M  =  50 µm and O  =  25 µm.</p

PrP^c expression modulates the number of oligodendrocytes in the perinatal and adult neocortex.

<p><b>A-D</b>) Low power photomicrographs showing Olig2 expression in coronal sections of <i>Prnp</i>^+/+ and <i>Prnp^0/0</i> embryonic (E16.5) brains. <b>B,D</b>) High magnification photomicrographs of A and C. <b>E-N</b>) Low power photomicrographs showing a coronal brain section immunostained for NG2 (E and J), Olig2 (F, H, K and M) and MAG. <i>In situ</i> hybridization (I and N) of the parietal cortex of adult <i>Prnp</i>^+/+ (E-I) and <i>Prnp^0/0</i> mice (J-N). G and L are adjacent Nissl stained sections to E and J. Note the increase in the number of NG2- and Olig2-positive cells in the <i>Prnp^0/0</i> neocortex compared to that of the <i>Prnp^+/+</i> mice. <b>O</b>) Histogram showing the number of Olig2-positive cells in the embryonic neocortex of <i>Prnp^0/0</i> and <i>Prnp</i>^+/+ mice. <b>P</b>) Histogram showing the number of Olig2- and NG2-positive cells in the adult neocortex of <i>Prnp^0/0</i> and <i>Prnp</i>^+/+ mice. The presence of Olig2- and NG2-positive cells increased significantly in the knockout mice when compared with their <i>Prnp^+/+</i> counterparts. <b>Q</b>) Quantification of the number of MAG-positive cells in the adult cortex of <i>Prnp^0/0</i> and <i>Prnp</i>^+/+ mice, revealing no significant differences between these genotypes. Values represent the mean ± standard deviation and the asterisks indicate statistical significance (<i>P</i> < 0.01, Student´s <i>t-</i> test). Scale bars: A  =  500 µm also applies to C; B  =  200 µm also applies to D. E  =  300 µm also applies to F,G and J-L; H  =  100 µm also applies to I and M-N.</p