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

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

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

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

(A) Differentiated Oli- cells or primary oligodendrocytes were treated with the indicated concentrations of L1-Fc

Equal amounts of cell lysates were analyzed by Western blotting using the indicated antibodies. Numbers on top indicate L1-Fc concentration in nM. (B, left) Oli- cells were incubated with control Fc (C-Fc, human IgG) or L1-Fc in the presence of control or Fyn siRNA. Tyrosine-phosphorylated proteins were immunoprecipitated (P-Tyr IP) and analyzed by Western blotting for hnRNP A2 (P-Tyr A2). Levels of tyrosine-phosphorylated A2 are strongly increased in L1-Fc–treated cells compared with control Fc–treated cells, and this effect is reduced in cells treated with Fyn siRNA. Total lysates (before IP) were analyzed by Western blotting with hnRNP A2, Fyn, and GAPDH antibodies and demonstrated unchanged levels of total hnRNP A2 and a reduction of Fyn protein by Fyn siRNA. GAPDH served as a loading control. (B, right) The diagram represents the data from three such experiments. Protein bands of tyrosine-phosphorylated hnRNP A2 were densitometrically quantified and the values of control Fc–treated cells were set to 1. The relative increase of tyrosine-phosphorylated hnRNP A2 in L1-Fc–treated cells in the presence of control and Fyn siRNA was plotted. Error bars indicate SEM; = 3.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p

(A) Oli- cells and primary oligodendrocytes were transfected with full-length hnRNP A2 and immunostained for hnRNP A2 or MBP

Images were acquired by confocal microscopy, and either a single slice (Oli-) or the complete stack (primary oligodendrocytes) is depicted. HnRNP A2–containing granules are present in the processes of Oli- cells as well as primary oligodendrocytes. Insets show an enlarged view of the boxed sections. Bars, 10 μm. (B) A granule-free supernatant was analyzed by Western blotting for hnRNP E1 and A2 proteins after RNase A treatment or L1-Fc binding. Western blot bands were analyzed densitometrically from 8 and 15 experiments for RNase treatment and L1-Fc binding, respectively. The control values (RNase A and C-Fc) were set to 1 and the mean relative increase of hnRNP E1 and A2 in the granule-free fraction was plotted in response to RNase A treatment or L1-Fc binding. Error bars indicate SEM; significance was tested with tests: *, P ≤ 0.05; **, P ≤ 0.01. = 8 (RNase A) and = 15 (L1-Fc). (C) The model illustrates the proposed events: During initial axon–glial contacts, neuronal L1 binds glial F3 (1), leading to an activation of Fyn (2), which phosphorylates hnRNP A2 (3). This leads to a release of hnRNP A2 and E1 from the granule and liberation of MBP mRNA (4) at the axon–glial contact site, allowing localized synthesis of the MBP protein (5) required for generation of the myelin sheath. The dotted lines illustrate potential alternative activation pathways of Fyn kinase mediated by L1 binding.<p><b>Copyright information:</b></p><p>Taken from "Activation of oligodendroglial Fyn kinase enhances translation of mRNAs transported in hnRNP A2–dependent RNA granules"</p><p></p><p>The Journal of Cell Biology 2008;181(4):579-586.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386098.</p><p></p