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

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

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

Additional file 1: Figure S1. of Activation of FXR pathway does not alter glial cell function

Cerebellar oligodendroglial differentiation is not affected by direct FXR activation or supernatant of FXR-activated BMMs. Addition of GW4064 during the differentiation of cerebellar oligodendrocytes over 24 and 48 h does not influence Mbp expression levels (a) or the percentages of PDGFRα+ OPCs or MBP+ oligodendrocytes (b, c). Oligodendrocytes of cerebellar origin exhibit no significant difference in their Mbp expression level when incubated with supernatants from BMM cultured either in the presence or absence of GW4064 (d). The percentage of OPCs is unchanged after incubation with BMM-conditioned medium; the percentage of MBP+ oligodendrocytes is reduced independent of additional GW4064 treatment (e, f). Activation of FXR cultures using 10 and 20 μM GW4064 during 14 days of remyelination in cerebellar slice after toxic demyelination does not alter the ratio of MBP+ and NFL+ axonal fibres (g). Remyelinated fibres are exemplarily highlighted (arrows). Note that a high amount of MBP+ debris is still present after 14 days of remyelination (h). In vitro: n = 3, 1way ANOVA with Bonferroni’s correction, 200 cells per condition were evaluated, *p < 0.05; ex vivo: n = 2, 1way ANOVA with Bonferroni`s correction, 6 slices with 3 images each per condition and preparation; all images are representative. (TIF 5564 kb

Expression of TCF7L2 in MS lesions and non-demyelinating inflammatory CNS diseases.

<p>In a subset of early MS lesions increased numbers of TCF7L2 positive cells were detected in the periplaque white matter and in remyelinating lesion areas. However, also in other inflammatory neurological diseases (OND) TCF7L2 positive cells were present. Red dots indicate a subset of tissue samples with high numbers of TCF7L2 expressing cells (see Figure 4) (<b>A</b>). There was no correlation between numbers of TCF7L2 and NOGOA positive cells in the different MS lesion areas. NOGOA positive oligodendrocytes and TCF7L2 expressing cells were quantified in periplaque white matter (PPWM), actively demyelinating (AD), demyelinated (DM) and remyelinating (RM) lesion areas; TCF7L2 expressing cells (dots) and NOGOA expressing cells (squares) from the same lesion area are labelled in the same colour (<b>B</b>). Double stainings revealed that a subset of TCF7L2 positive cells were NOGOA positive oligodendrocytes (arrows). TCF7L2 negative oligodendrocytes are indicated by arrow heads (Double immunohistochemistry for NOGOA (red) and TCF7L2 (black) (<b>C</b>). In inflammatory non demyelinating disease TCF7L2 positive oligodendrocytes and astrocytes were detected (<b>D</b> and <b>E</b>). In <b>D</b> a GFAP and TCF7L2 positive astrocyte (arrow) as well as a GFAP negative TCF7L2 positive cell (arrow head) are depicted (double immunohistochemistry for GFAP (green) and TCF7L2 (red). Additional GFAP and TCF7L2 positive cells are shown in <b>E</b> (arrows) (double immunohistochemistry for GFAP (red) and TCF7L2 (black)).</p

Myelination and expression of TCF7L2 in the human CNS.

<p>The extent of myelination in human frontal lobes was quantified using a semiquantitative score. Between 30 and 40 weeks of gestation no MBP-positive axons were found. Myelination became first obvious between 0 and 6 months after birth (<b>A</b>). First TCF7L2-positive cells were detected at the end of gestation with maximal numbers between 7 and 12 months after birth. Afterwards, the numbers of TCF7L2-positive cells decreased quickly (<b>B</b>). At 6 months after birth numerous myelinating oligodendrocytes were observed (immunohistochemistry for MBP) (<b>C</b>). Many TCF7L2 positive cells also expressed NOGOA (double immunohistochemistry for NogoA (red) and TCF7L2 (black) (<b>D</b>) but not GFAP (double immunohistochemistry for TCF7L2 (black) and GFAP (red)) (<b>E</b>). TCF7L2 was also expressed in human fetal oligodendrocytes in vitro (green O4, red TCF7L2) (<b>F</b>).</p

Expression of HDAC2 in MS lesions and control tissue samples.

<p>In control tissue samples (<b>A</b>) as well as in MS lesions (<b>B</b>) numerous NOGOA positive oligodendrocytes were seen which express abundantly HDAC2 (arrows) (double immunohistochemistry for NOGOA (red) and HDAC2 (black)).</p

CXCL12-mediated promotion of differentiation marker expression in cultured human oligodendroglial precursor cells.

<p>(A) The percentage of O4 positive cells expressing GalC was significantly increased after 7 days of CXCL12 stimulation as compared to control cells. (B-C') Representative immunostainings of GalC/O4 positive OPCs stimulated with CXCL12 and control cells. Note that in CXCL12-stimulated cultures complex cell morphologies (arrows) were more common than in control cultures (arrowheads indicating cells with fewer cellular processes). (D) Determination of the percentage of CNPase positive hOPCs under CXCL12 stimulation after 7 days. This demonstrated that the CXCL12-dependent promotion of myelin induction (white vs. gray bars) was completely abolished in the presence of CCX771 (dashed gray bar). CCX771 alone did not affect myelin expression (compare white to dashed white bar). (E-E') Double immunostaining revealed that O4 positive precursor cell express CXCR7. Data are shown as mean values +/- SEM derived from 3 independent experiments. (t-test, ***p <0,001 and ANOVA, **p < 0,01). Scale bars: 70 μm (B-C'), 30 μm (E-E') μm.</p

Expression of TCF7L2 during remyelination in the CNS of mice.

<p>Mice were fed with 0.25% cuprizone for six weeks and the numbers of oligodendroglial lineage cells were quantified at the indicated time points. Lowest numbers of OLIG2 positive cells were observed 21 days after onset of cuprizone diet; the increased numbers at the end of the cuprizone diet (at 42 days) suggest a recruitment of OPCs during ongoing demyelination (<b>A</b>). NOGOA is expressed by mature oligodendrocytes; lowest numbers of NOGOA positive cells were as well found at day 21 (B). High numbers of TCF7L2 expressing cells were only detected at day 42 (C).</p