Expression of PTGDS and Nptx2 in primary OPC.

<p><b>A</b> Expression of cell-type specific markers was analyzed by Western-Blot of total cell lysates of pOPC and the negative (neg.) sort fractions, HEK cells and primary cortical neurons (DIV5 in culture). <b>B-D</b> show the expression of proteins in pOPC over time, related to DIV0. In <b>B</b> a peak of the OPC protein NG2 is shown at DIV1, PLP indicates differentiation into oligodendrocytes starting at DIV2, GFAP shows astrocyte differentiation starting at DIV2. <b>C</b> PTGDS expression peaks together with NG2 at DIV1. <b>D</b> Nptx2 expression increases together with NG2 at DIV1, peaks at DIV2 and returns to basal levels at DIV4. <b>E</b> Expression of PTGDS and Nptx2 mRNA in pOPC at DIV1, as revealed by <i>in situ</i> hybridization, OPC were identified by antibody staining of NG2 protein. (A-D: 3 sorts were analyzed for each time point. Two tailed student’s t-test was applied.)</p

Primary OPC culture.

<p><b>A</b> Cell-type specific staining of magnetically sorted (MACS) primary OPC (pOPC) at different timepoints of culture. DIV0, 1 and 2: 2h, 24h and 48h of culture respectively (DIV: days in vitro). OPC were identified by co-expression of NG2 and Olig2 (NG2+/Olig2+), Scale bar = 30μm and 15μm for the zoom. The percentage of the identity of each cell-type of the total DAPI+ cells is given in <b>B</b>. Differentiated oligodendrocytes were identified by the expression of PLP, astrocytes by expression of GFAP, microglia by expression of F4/80 and neurons by expression of ß-III-tubulin (TUJ-1). Pericytes were not detected in in the pOPC culture, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.s001" target="_blank">S1 Fig</a> for pericyte staining. (A&B: 200 cells were analyzed for each staining/time-point from 2 independent MACS sorts).</p

NG2 fragments exhibit distinct subcellular localization including the nucleus.

<p>Fluorescent staining of OPC expressing NG2_del_Myc or the NG2_ICD_Myc construct (in red) and GFP tagged Histone2B (H2B-GFP, only A&B). <b>A&B</b> Confocal laser scanning microscope (cLSM) pictures of Oli-neu (OPC cell line) show NG2_del_Myc (red) staining at the plasma membrane and intracellular membranes, but nuclei are largely spared. Expression of the NG2 ICD (NG2_ICD_Myc, red) results in an almost homogeneous cytoplasmic staining including an intense staining of the nuclei. <b>A</b> Standard deviation (STD) projection is shown for the total LSM stack. NG2 ICD (Myc staining) shows much stronger overlap with nuclear stainings (H2B-GFP, DAPI) than the one of NG2_del (scale bar = 20μm). <b>B</b> Three dimensional orthogonal views (ImageJ3DViewer) are shown for cell bodies of the two transfection conditions. NG2 ICD reveals a striking intranuclear staining (overlap with H2B-GFP), while NG2_del only overlaps at the outer layer of the H2B stained nucleus. <b>C</b> Transfected pOPC showed a much lower expression of both NG2 constructs. The strongest staining of Myc-tagged constructs is observed close to the nucleus in NG2_del_Myc transfected cells. In NG2_ICD_myc transfected cells, many cells additionally show a staining of nuclear substructures. Images show one plane from the center of a confocal z-stack (pAB = primary antibody, scale bar = 15μm). For staining of pOPC also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.s002" target="_blank">S2B Fig</a>. <b>D</b> Cartoon of the intracellular domain (ICD) of NG2. <b>E</b> Selected NG2 ICD sequences with predicted functions. ELM database analyses show 2 NLS sequences indicating nuclear transport and a predicted WW4 binding motif, a binding site for protein complexes which localize to nuclei. (A&B: 20 double-transfected cells from 3 independent transfections per condition were analyzed; 100% of the NG2_del and over 70% of the NG2_ICD transfected cells showed the described effects. cLSM stacks had a z-stack depth of at least 12 μm, with a z-increment of < 0.3 μm per single image.)</p

NG2 intracellular fragments influence PTGDS protein levels.

<p><b>A</b> Expression of NG2_del, NG2_ICD (both red) and Mock (empty Plasmid) constructs in the OPC cell-line Oli-neu, resulted in a reduction of PTGDS protein levels in post nuclear lysates (PN). Overexpression of these constructs leads to a changed ratio of protein levels between the NG2 FL and the small NG2 (intracellular) cleavage fragments the CTF and the ICD (compare to mock, see B). <b>B</b> Cartoon of NG2, NG2 expression constructs (red) and the cleavage sites for α- and γ-secretase leading to the creation of the CTF (12 kD) and ICD (8.5 kD, both blue). (A: 4 independent transfections were analyzed per construct; two tailed student’s t-test was performed.)</p

NG2 dependent regulation of PTGDS and Nptx2 <i>in vivo</i>.

<p><b>A&B</b> mRNA levels of target-genes were directly analyzed by qRT-PCR from FACS isolated OPC and other cells (negative (neg.) sort). Single cell suspensions from total brains of postnatal day 9 (P9) NG2-KO and WT mice were used for FACS. <b>A</b> Enrichment of target mRNA in OPC in comparison to other cell-types is shown for WT (black bar) and NG2-KO (grey bar), (ΔΔCT = [ΔCT OPC]–[ΔCT other cells]). Enrichment of PDGFRα mRNA validates OPC enrichment. Nptx2 mRNA was enriched within OPC of both genotypes, while PTGDS mRNA was only enriched in WT OPC. <b>B</b> Genotype-specific mRNA enrichment in OPC (black bar) and other cells (grey bar) is plotted (ΔΔCT = [ΔCT WT cells]–[ΔCT KO cells]). PTGDS was the sole target gene analyzed exhibiting differential expression between WT and KO genotypes. Expression was highly increased in WT-derived OPC and down-regulated in the other cells (neg. sort) from these mice. <b>C</b> Western Blot of soluble protein fractions. Soluble fractions were extracted from P9 mouse brain of WT and NG2-KO mice. Total PTGDS and Nptx2 protein levels show no difference between genotypes. <b>D</b> Protein levels of post nuclear (PN) cell lysates of the OPC cell line Oli-neu were analyzed after treatment with siRNA silencing NG2 expression (siNG2) or control siRNA (siC). Full-length NG2 levels were reduced as well as PTGDS protein levels, fitting to the reduced mRNA levels of PTGDS found in NG2-KO OPC (B). <b>E</b> Expression of PTGDS mRNA by the OPC cell-line Oli-neu as revealed by <i>in situ</i> hybridization. (A&B: for NG2-KO OPC, 4 independent sorts were analyzed, for WT 3 sorts were analyzed, ΔCT = (CT target)–(CT GAPDH), ΔCT and ΔΔCT values are in log2 scale; for <b>C:</b> 4 animals were analyzed for NG2-KO (KO1-4) and BL6/N (WT1-4) mice; for <b>D</b>: 4 independent transfections were analyzed per siRNA, two tailed student´s t-test was performed.)</p

The NG2 ICD is located in the nucleus.

<p><b>A</b> Cartoon of the NG2 full length protein and the major cleavage fragments (ectodomain, CTF, ICD). Ectodomain cleavage (indicated by the α) has been reported by others [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.ref028" target="_blank">28</a>], while intracellular cleavage (indicated by the γ) has been found by our group [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.ref021" target="_blank">21</a>]. Expression constructs NG2_del leading to high NG2 CTF and lower ICD levels by proteolytic processing and the NG2_ICD construct leading to high levels of NG2 ICD are both shown in red. <b>B</b> Cytoplasmic (cyto), crude membrane (CM) and nuclear fractions of HEK cells transfected with empty plasmid (control), NG2_ICD, or NG2_del are shown. NG2_del full-length (FL) protein, the membrane bound NG2 CTF and the NG2 ICD are shown in WB (schematically shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127222#pone.0127222.g002" target="_blank">Fig 2D</a>). The NG2 ICD shows the highest levels when expressed as a recombinant protein (NG2_ICD). This is present in all fractions but highest in the nuclear fraction. NG2_del-derived NG2 ICD (generated by proteolysis) is only detectable in the nuclear fraction and runs at the same size as the recombinant NG2 ICD.</p

PDGF-AA elicited increased directed migration (= chemotaxis) in NG2-/- OPCs.

<p>Viability was comparable between NG2-/- and NG2+/+ oligodendroglial lineage cells under proliferating (A) or under differentiating (B) conditions. Additionally, no differences could be observed comparing the proliferative activity of NG2-/- and NG2+/+ OPCs after 24 h (n = 4) (C). No differences in total movement or average speed between NG2-/- and NG2+/+ were observed in chemokinesis assays. Using PDGF-AA as chemoattractant significantly increased chemotaxis of NG2-/- compared to NG2+/+ OPCs was observed (n = 5) (F). When FGF2 was used as chemoattractant, chemotaxis was comparable between the two genotypes (n = 4) (G). NG2-/- and NG2+/+ OPCs express comparable levels of <i>Pdgfra</i> mRNA (n = 5) (E).</p

No difference in the numbers of microglia/macrophages, proliferating cells and axonal damage between NG-/- and NG2+/+ mice in the cuprizone model.

<p>After 10 weeks of demyelination and during subsequent remyelination (7 and 14 days after cessation of cuprizone diet) no differences in the numbers of microglia/macrophages (Mac3) (A-B) or proliferating cells (Ki67+) (C-D) were observed. Furthermore, the extent of axonal damage measured by APP-positive spheroids was comparable in NG2-/- and NG2+/+ mice during de- and remyelination (E-F). qRT-PCR revealed no differences in the expression levels of <i>Il1ß</i> between NG2-/- and NG2+/+ mice (G) and the expression of <i>Infg</i> was reduced in NG2-/- mice after 1 week of remyelination (H). Scale bars represent 50 μm (B, D, and F).</p

NG2 is dispensable for successful remyelination in the cuprizone model.

<p>After 10 weeks of cuprizone induced demyelination and during subsequent remyelination (7 and 14 days after cessation of cuprizone diet) no differences in the extent of myelination (LFB-PAS) (A-B), the numbers of NogoA(+) mature oligodendrocytes (C-D) or Olig2(+) cells (E-F) were detected between NG2-/- and NG2+/+ mice. Furthermore, the quantification of myelinated axons and the g-ratio by EM revealed no differences between both genotypes (G-I). The expression level of <i>Mbp</i> was similar in NG2-/- and NG2+/+ mice after 10 weeks of demyelination and 1 and 2 weeks of remyelination (J). Scale bars represent 500 μm and 100 μm respectively (B & insert), 50 μm (D, F), and 1 μm (H).</p

Loss of NG2 does not affect oligodendroglial differentiation.

<p>Using qRT-PCR no differences in the relative expression of the myelin associated genes <i>Mbp</i>, <i>Plp1</i>, or <i>Mag</i> were observed comparing NG2-/- and NG2+/+ cells (n = 5) (A). Immunocytochemistry revealed no differences in the number of PDGFRα(+) and MBP(+) cells after 48 h of differentiation (n = 6). Representative pictures of differentiated cultures are shown (B). Also the evaluation of cell morphology demonstrated comparable differentiation of the two genotypes differentiation after 6, 24, 30, or 48 h (C). Scale bars represent 200 μm.</p