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

<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

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

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

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