Heterologous expression of MECP2 does not affect electrophysiological properties of Drosophila motoneurons.

<p>(A) Comparison of typical MN5 firing responses to 300 pA of somatic current injection in a representative control animal (left trace) and following targeted expression of full-length human <i>MECP2</i> under the control of <i>C380-GAL4</i> (right trace). (B) Voltage dependent potassium currents in MN5 as induced by command voltage steps from a holding potential of −90 mV to 20 mV in increments of 10 mV and with cadmium and TTX in the bath solution to block sodium and calcium inward currents. Traces of control animals (left) and following targeted expression of MECP2 (right) reveal qualitatively similar transient A-type current and sustained delayed rectifier like potassium outward currents. (C) Current/Voltage relationships for A-type (left) and sustained delayed rectifier (right) potassium currents are quantitatively similar in controls (gray diamonds) and following expression of MECP2 (magenta diamonds). Error bars represent standard deviations.</p

MECP2-induced motoneuron defects result in specific motor behavioral deficiencies.

<p>(A) Representative extracellular recording of MN5 firing patterns during flight in a control (upper trace) and in fly expressing MECP2 in a subset of neurons, including MN5 (<i>C380-GAL4, UAS-mcd8-GFP; Cha-GAL80/UAS-MECP2</i>; lower trace). Traces above the recordings resemble spike counts. Black arrow demarks start of flight, and black asterisk demarks time point of flight stop in <i>MECP2</i> fly. (B) Average in-flight wing beat frequencies of control (white bar) and <i>MECP2</i> flies (grey bar). Error bars represent standard error. (C) Percentage of control (white bar) and <i>MECP2</i> flies (grey bar) engaging into flight upon a wind stimulus. (D) Numbers of flight bouts performed by control (white bar) and by <i>MECP2</i> flies (grey bar) in response to re-occuring wind stimuli (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031835#s4" target="_blank">methods</a>). Data are presented as median and quartiles. Error bars represent minimum and maximum values. (E and F) Total duration of all consecutive flight bouts (E) and average duration of individual flight bouts (F) in control (white bar) and in <i>MECP2</i> flies (grey bar). Data are presented as median and quartiles. Error bars represent minimum and maximum values. ** demarks p<0.01, Mann and Whitney U-test.</p

Heterologous expression of MECP2 with MBD defects does not affect Drosophila motoneuron dendrite development.

<p>(A) Schematic drawings of full-length human MECP2 (magenta) with intact menthyl-CpG-binding domain (MBD) and intact transcriptional repression domain (TRD). The R106W mutation (red) carries a point mutation (see x) that causes a non-functional MBD. The Δ166 mutation (orange) has a truncated MBD and N-terminus. TRD is intact in all three alleles. Nuclear localization sequences (nls) have been reported in the inter-domain region at residues 174 and 190 and also in the TRD domain between residues 255 and 271, and are intact in all three alleles. (B, D, F) Intracellular labeling of MN5 following R106W expression under the control of <i>C380-GAL4</i> (B) and subsequent geometric reconstruction (F) do not reveal obvious dendrite defects in MN5. (D) MECP2 immunolabeling following targeted R106W expression indicates strict nuclear localization (see also white arrow in B). (C, E, G) Intracellular labeling of MN5 following Δ166 expression under the control of <i>C380-GAL4</i> (C) and subsequent geometric reconstruction (G) do not reveal obvious dendrite defects in MN5. (E) MECP2 immunolabeling following targeted R106W expression indicates strict nuclear localization (see also white arrow in C). (H) Averages of total dendritic length in controls (gray bars), and following expression of full-length MECP2 (magenta), R106W (red), and Δ166 (orange). (I) Average numbers of dendritic branches in controls (gray bars), and following expression of full-length MECP2 (magenta), R106W (red), and Δ166 (orange). In (H) and (I) error bars indicate standard deviation, asterisks demark statistical significance at p≤0.05 (ANOVA with Newman Keuls posthoc test).</p

MECP2-caused dendrite defects are partially ameliorated by a reduction in osa dose.

<p>(A) Projection view of a representative intracellular staining of MN5 in a control animal. (B) Projection view of a representative intracellular staining of MN5 in an <i>osa</i> heterozygous mutant background does not reveal obvious differences in dendritic structure as compared to control. (C) Projection view of a representative intracellular staining of MN5 with heterologous expression of full-length MECP2 in an <i>osa</i> heterozygous mutant background does not show similar dendritic defects as compared to MECP2 expression in a wildtype <i>osa</i> background (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031835#pone-0031835-g001" target="_blank">figures 1C, D</a>). (D) MECP2 immunopositive label (magenta) was restricted to the nucleus (see also white arrow in C). (E) Quantitative metric measures of dendritic structure in MN5 from controls (dark gray bars), MN5 in an <i>osa</i> heterozygous mutant background (light gray bars), from MN5 with MECP2 expression (magenta), and from MN5 with MECP2 expression in an <i>osa</i> heterozygous mutant background. Values are normalized to mean control values. Arrows indicate statistical significance (ANOVA with Newman Keuls posthoc test, p≤0.01). Error bars indicate standard deviation. (F) Mean number of dendritic branches over branch order in controls (gray squares), following MECP2 expression (magenta circles), and following MECP2 expression in an <i>osa</i> heterozygous mutant background (blue). Error bars indicate standard deviation.</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

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

Working model.

<p>Phosphorylation of MeCP2FL at S80 induces conformational changes of MeCP2 that prevent C- and N-terminal interactions (CT-NT interactions), thus inducing conformational changes that (apoptotic state, red) underlie the apoptotic effect of MeCP2 <i>in vivo</i>. Calcium dependent de-phosphorylation at S80 returns MeCP2 to a healthy conformation (green) that allows CT-NT interactions. C-terminal truncation prevents CT-NT interactions thus favoring the apoptotic conformation of MeCP2. R249X is shown as an example, but it is expected that V312X and K431X truncations act in the same manner. The short truncation V481X removes just 5 amino acids, which does not prevent CT-NT interactions thus favoring the healthy conformation of MeCP2.</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

MeCP2 C-terminal domain is critical for preventing apoptosis in Drosophila MN5.

<p>a-e. Representative images of cPARP reporter activity in MN5 from flies expressing C-terminal truncated variants of <i>MECP2</i> at pupal stage P15. All c-terminal truncations except V481X (d) caused caspase mediated apoptosis in MN5. f. Schematic of <i>MECP2</i> E2 isoform with mapped truncations. The region between AA431-481 was found to be critical in preventing apoptosis as caused by truncated MeCP2 in MN5. Scale bar depicts 10 μm.</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