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

Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations

<div><p>Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire <i>MECP2</i> locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of <i>Drosophila</i> to provide evidence that expression of p.<i>Arg294*</i> (more commonly identified as <i>R294X</i>), a human <i>MECP2</i> E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons <i>in vivo</i>. We confirm this novel finding in HEK293T cells and then use <i>Drosophila</i> to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. <i>In vitro</i> studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in <i>Drosophila in vivo</i>, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.</p></div

(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

S80 phosphorylation mediates caspase activity in full-length but not truncated MeCP2.

<p>a-c. Representative images of cPARP reporter activity in MN5. Intact MN5 cell bodies are outlined in white. b. Phosphomimicking mutation S80E increases caspase activity in MN5 compared to controls (a), while phosphoblocking mutation S80A (c) has no effect on the toxicity of the R294X truncation. d. Percentage of preparations examined 0–24 hours post-eclosion with either 0, 1, or both MN5s (numbers in bars indicate number of animals with the respective phenotype) e-f. Quantification of caspase activity visualized by immunocytochemistry. Individual MN5 somata (white dashed lines in a-b) were traced and mean grey values were calculated using ImageJ. Expression of <i>MECP2FLS80E</i> increased cPARP reactivity in comparison to expression of normal <i>MECP2FL</i> (f). No differences in VENUS reactivity were observed (e). ** p <0.005, Pearson’s chi-square (d) or Student’s two-tailed t-test (f). Six flies from two independent crosses were observed and analyzed for each genotype. Scale bar depicts 10 μm. Error bars show mean +/- SEM.</p

Co-expression of <i>slp1</i> improves cellular and behavioral consequences of R294X at one day post-eclosion.

<p>a-b. Representative images of cPARP reactivity in MN5s at 1–2 days post-eclosion. Single asterisks mark intact MN5s, while double asterisks denote absent MN5s. c. Distribution of preparations with 0, 1, or both MN5s is presented for flies expressing <i>MECP2FL</i>, <i>R294X</i>, or co-expression of <i>slp1; R294X</i> (* p < 0.05, *** p < 0.0001 Pearson’s chi square). d. Percentage of flies failing to initiate flight in the cylinder drop test. All flies tested were collected from at least three independent crosses for each genotype. N’s are overlaid in parentheses on histogram bars for each experimental group. Scale bar depicts 10μm.</p

R294X truncation causes caspase mediated apoptosis in <i>Drosophila</i> MN5.

<p>a. Structural schematics of the <i>MECP2FL</i> and <i>R294X</i> alleles used for experiments. Only the <i>MECP2</i> E2 isoform is shown and was used for experiments. b. Schematic of <i>Drosophila</i> central nervous system showing localization of motoneuron 5 (MN5) within the ventral nerve cord (VNC). GFP labeling with the C380-GAL4 motoneuron driver line is shown overlaying the VNC, and confocal projections views of GFP labeled MN5 somata are shown as selective enlargements at right. c-e. Representative images of flies at pupal stage P15 expressing either <i>MECP2FL</i> (c), <i>R294X</i> (d), or both alleles (e) under the control of C380-GAL4. Histograms show the percentage of preparations with either 0, 1, or 2 MN5 somata present for each genotype. Exact number of flies corresponding to each bar is reported in parentheses. Both MN5 somata and no cPARP reactivity was found with control (b) or <i>MECP2FL</i> (c). Most MN5 somata expressing either <i>R294X</i> alone (d) or together with <i>MECP2FL</i> (e) were missing, and high cPARP reactivity was found in the remaining somata and neuronal projections. The distribution of intact MN5s in (d) and (e) significantly differed compared to expression of <i>MECP2FL</i> alone (c) (*** <i>p</i> < 0.0001, Pearson’s chi-square). Single asterisks indicate intact MN5 somata, double asterisks indicate absent or severely deteriorated somata. Scale bar depicts 10 μm.</p

(A) An A2RE-containing region of the 3′ UTR of MBP14 mRNA was cloned downstream of the luciferase coding sequence (CDS) and used as a translational reporter in the DualGlo luciferase assay

(B) Oli- cells were nucleofected with an A2RE containing luciferase reporter, a luciferase control, and either wild-type Fyn (Fyn WT), constitutively active Fyn (Fyn), or EGFP. was normalized to luciferase activity for every measurement in all experiments ( = 3). (C) Total RNA was isolated from the cells used in the luciferase assay shown in B. qRT-PCR was performed to compare A2RE luciferase mRNA from Fyn (WT and Fyn)-transfected cells with EGFP-transfected cells ( was used for normalization). Error bars indicate SEM. Significance was assessed with tests: *, P < 0.05; **, P < 0.01; ns, not significant.<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

<i>R294X</i> transfection promotes cell death in mammalian cell culture to a higher degree than <i>MECP2FL</i>.

<p>a. Representative images of HEK293T cells transfected with GFP tagged <i>MECP2FL</i> or <i>R294X</i>. Examples of healthy transfected cells are at left, while the condensed and/or fragmented nuclei at right were counted as apoptotic. b. Quantification of apoptotic cells following transfection of <i>MECP2FL</i>:<i>GFP</i>, <i>R294X</i>:<i>GFP</i>, or GFP control. n = 6 independent transfections/group, with > 400 cells counted for each independent transfection. Percentage data was transformed using the arcsine square root function to meet the assumptions for a one way ANOVA (F(2,15) = 46.81, p < .0001). * p < .05, ** p < .01, *** p < .001 Tukey post-hoc test. c. Relative <i>MECP2</i> expression 24 hours following transfection into HEK293T cells. No differences were observed in relative protein levels at 24 or 48 hours post-transfection (Mann-Whitney U test). Scale bar depicts 5μm. pi = propidium iodide, NS = not significant.</p