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

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 TCF7L2 during myelination in the CNS of mice.

<p>At P10 single myelinated axons are observed in the lateral parts of the corpus callosum (arrow) (<b>A</b>) whereas in adult mice myelination is complete (<b>B</b>). High numbers of TCF7L2 expressing cells were observed in P10 mice (<b>C</b>), but only few TCF7L2-positive cells were found in adult mice (<b>D</b>) as quantified in the diagrams (<b>E</b> and <b>F</b>). The percentage of OLIG2 positive cells expressing TCF7L2 decreased significantly in adult mice (<b>G</b>).</p

CXCR7 detection in MS tissue.

<p>(A-F) CXCR7 staining revealed receptor expression on cells in control brains (B) and different regions of the MS brain including demyelinated (E; DM) and remyelinating (F; RM) lesions as well as active white matter lesions (D). Most CXCR7-positive cells in the MS brain were detected in the periplaque white matter (C; PPWM) while their number was significantly decreased in actively demyelinating lesion areas (D; active) at the plaque border probably reflecting ongoing oligodendroglial cell death (one-way ANOVA, **p < 0,05). (G) NogoA staining of control and MS brains demonstrated a similar distribution as CXCR7 (A) with a correlation coefficient of 0,783 between stainings. Double immunostaining then confirmed that mature NogoA positive oligodendrocytes (H-H''; see arrows) and Nkx2.2 positive oligodendroglial precursor cells (I-I''; arrows) express CXCR7. Not all CXCR7-positive cells were of oligodendroglial origin which is in line with studies describing this receptor in other CNS cell types (see arrowhead in H-H''). Scale bars: 25μm (F), 25μm (H-H''), 10μm (I-I'').</p

CXCL12 promotes morphological maturation of cultured human oligodendroglial precursor cells.

<p>Five different morphologies were distinguished ranging from a very low number of processes in precursor cells to multiple process-bearing cells (low, medium, high) to mature cells with a very high degree of arborization or flattened appearance (membrane sheet). (A,B) Analysis of hOPC morphology distribution revealed a CXCL12-dependent shift towards more mature cells (white bars: buffer treated cells; gray bars: CXCL12-stimulated cells) after 7 and 12 days in culture. Data are shown as mean values +/- SEM derived from 3 independent experiments (ANOVA, **p < 0,01). Representative O4-expressing hOPCs stimulated with either buffer or CXCL12 at 7d (A',A'') and 12d (B',B''). Scale bars: 50μm.</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 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

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

Additional file 2: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S1. Confirming activation of human astrocytes. Astrocytes were cultured for 24 h and were either left unstimulated or were stimulated with IFNγ (10 ng/ml) and IL-1β (10 ng/ml). After 24 h, the astrocytes were washed thoroughly and fresh medium was added. After an additional 24 h in culture, at which time cultures were imaged and supernatants were collected for subsequent measurement of astrocyte-secreted IL-6 by ELISA. Compared to unstimulated astrocytes (a), stimulated astrocytes exhibited activated morphology (b) and significantly-enhanced production of IL-6 (c; p = 0.0016; paired t-test). (TIFF 3951 kb

Additional file 3: of Human central nervous system astrocytes support survival and activation of B cells: implications for MS pathogenesis

Figure S2. Effects of astrocytes cytokine neutralization on B cell survival and activation. B cells from HC were either cultured alone, or with stimulated astrocyte conditioned-medium (ACM), or with ACM pre-treated with neutralizing antibodies to IL-6 (a, b; anti-IL6: aIL-6), IL-15 (c, d; anti-IL-15: aIL-15) or BAFF (e, f; anti-BAFF: aBAFF); or pre-treated with corresponding isotype control antibodies. After 2 days of culture B cell viability was assessed using ANNEXIN V and 7AAD staining, and CD86 expression was measured by flow cytometry (representative experiment). (TIFF 4226 kb