Rett syndrome mutation MeCP2 T158A disrupts DNA binding, protein stability and ERP responses

Darren Goffin, Megan Allen, Le Zhang, Maria Amorim, I-Ting Judy Wang, Arith-Ruth S Reyes, Amy Mercado-Berton, Caroline Ong, Sonia Cohen, Linda Hu, Julie A Blendy, Gregory C Carlson, Steve J Siegel, Michael E Greenberg, Zhaolan Zhou, Darren Goffin, Megan Allen, Le Zhang, Maria Amorim, I-Ting Judy Wang, Arith-Ruth S Reyes, Amy Mercado-Berton, Caroline Ong, Sonia Cohen, Linda Hu, Julie A Blendy, Gregory C Carlson, Steve J Siegel, Michael E Greenberg, Zhaolan Zhou

Abstract

Mutations in the MECP2 gene cause the autism spectrum disorder Rett syndrome (RTT). One of the most common MeCP2 mutations associated with RTT occurs at threonine 158, converting it to methionine (T158M) or alanine (T158A). To understand the role of T158 mutations in the pathogenesis of RTT, we generated knockin mice that recapitulate the MeCP2 T158A mutation. We found a causal role for T158A mutation in the development of RTT-like phenotypes, including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those present in Mecp2 null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. The age-dependent development of event-related neuronal responses was disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials (ERPs) may serve as a biomarker for RTT and treatment evaluation.

Figures

Figure 1. Generation and phenotypic characterization of…
Figure 1. Generation and phenotypic characterization of MeCP2 T158A knockin mice
(a) Sequencing chromatogram of RT-PCR products from MeCP2 mRNA. Mutation of T158 codon ACG to Alanine codon GCG (first box) and creation of BstEII restriction site (second box) with a silent mutation are marked with a *. (b) Western blots probed with a site-specific MeCP2 T158 antibody, a total MeCP2 antibody and Sin3a antibody. (c) Developmental presentation of RTT-like phenotypes in male Mecp2T158A/y mice (n = 6; F1,252 = 27.75, p-value < 0.0001; two-way ANOVA) relative to WT littermates (n = 5). Symbols represent mean score ± SEM. Phenotypic score is significantly higher in Mecp2T158A/y mice compared to WT littermates at 5 weeks and thereafter; * p-value < 0.05; two-way ANOVA with Bonferroni correction. (d) Developmental presentation of RTT-like phenotypes in female Mecp2T158A/+ mice (n = 7; F1,224 = 198.6, p-value < 0.0001; two-way ANOVA) relative to Mecp2+/+ littermates (n = 6). Phenotypic score is significantly higher in Mecp2T158A/+ mice compared to WT littermates at 17 weeks and thereafter; * p-value < 0.05; two-way ANOVA with Bonferroni correction. (e) Brain weights at P30 (n = 4 for both genotypes) and P90 (n = 6 for both genotypes). Bars represent mean ± SEM. ** p-value < 0.01; two-tailed t-test with Bonferroni correction. (f) Soma size in hippocampal CA1 pyramidal neurons. Bars represent mean ± SEM (n = 100 cells from 5 animals per genotype). ** p-value < 0.01; two-tailed t-test with Bonferroni correction. (g) Survival of male Mecp2T158A/y (n = 43) and Mecp2+/y littermates (n = 43).
Figure 2. Behavioral characterization of MeCP2 T158A…
Figure 2. Behavioral characterization of MeCP2 T158A mice
(a) Locomotor activity in Mecp2T158A/y mice (n = 15), Mecp2−/y mice (n = 14) and Mecp2+/y littermates (WT; n = 33) at 9 weeks of age. Bars represent mean ± SEM. * p-value < 0.01, *** < 0.001 and ## < 0.01; one-way ANOVA with Tukey’s post hoc test. (b) Motor coordination and motor learning assessed using a rotarod assay in Mecp2T158A/y mice (n = 16; F1,645 = 447.2, p-value < 0.0001; two-way ANOVA) and Mecp2−/mice (n = 14; F1,602 = 841.46, p-value < 0.0001; two-way ANOVA) and WT littermates (n = 27) at 9 weeks of age. The deficit in Mecp2−/y mice is significantly more than that observed in Mecp2T158A/y mice (F1,437 = 83.82, p-value < 0.0001; two-way ANOVA). Symbols represent mean ± SEM. (c) Anxiety-like behavior in Mecp2T158A/y mice (n = 15) and Mecp2−/y mice (n = 11) measured using elevated zero maze compared to WT littermates (n = 32) at 9 weeks of age. Bars represent mean ± SEM. * p-value < 0.05 and ** < 0.01; one-way ANOVA with Tukey’s post hoc test. (d) Learning and memory assessed using context-and cue-dependent fear conditioning in Mecp2T158A/y mice (n = 16) and WT littermates (n = 33) at 10 weeks of age. Bars represent mean ± SEM. * p-value < 0.05 and *** < 0.001; two-tailed t-test with Bonferroni correction.
Figure 3. Decreased MeCP2 protein stability in…
Figure 3. Decreased MeCP2 protein stability in MeCP2 T158A mice
(a) MeCP2 protein levels in forebrains of Mecp2T158A/y mice at P2, P30, and P90 compared to Mecp2+/y littermates (n = 3 for each genotype). Bars represent mean ± SEM. ** p-value < 0.01; one-sample t-test with Bonferroni correction. # < 0.05; one-way ANOVA with Tukey’s post hoc test. (b) MeCP2 protein levels are significantly reduced in kidney, liver, lung and heart tissues of Mecp2T158A/y (n = 3) compared to Mecp2+/y littermates at P90 (n = 3). Bars represent mean ± SEM. * p-value < 0.05; one-sample t-test with Bonferroni correction. (c) MeCP2 protein levels in female Mecp2T158A/+ mice (n = 3) and Mecp2+/y littermates at P90 (n = 3). MeCP2 protein levels in fibroblasts derived from a female RTT patient carrying the MeCP2 T158M mutation compared to fibroblasts derived from an age-matched female control (n = 3 separate passages). Bars represent mean ± SEM. * p-value < 0.05; one-sample t-test. (d) E16 + 7 DIV cortical neurons derived from Mecp2T158A/y (n = 3) and Mecp2+/y littermates (n = 3) were treated with vehicle (0) or 100 μM Cycloheximide (CHX) for 3, 6 or 9 hours. Bars represent mean MeCP2 levels relative to vehicle ± SEM. * p-value < 0.05; two-way ANOVA with Bonferroni correction.
Figure 4. Reduced MeCP2 binding to methylated…
Figure 4. Reduced MeCP2 binding to methylated DNA in T158A mice
(a) MeCP2 binding to methylated DNA (methylated oligonucleotides spanning the −148 CpG site of Bdnf promoter IV) is reduced by T158A mutation relative to WT and MeCP2 S421A mutation in Southwestern assay. (b) MeCP2 binding across a 39 kb of the promoter region of the Bdnf locus in brains obtained from Mecp2T158A/y mice mice and Mecp2+/y littermates (n = 3; two-way ANOVA, F1,84 = 639.1, p-value < 0.0001). Symbols represent mean ± SEM. Alternative Bdnf exons are indicated with black rectangles. (c) MeCP2 binding to the Xist, Snrpn, and Crh loci in Mecp2T158A/y mice (n = 3) compared to Mecp2+/y littermates (n = 3). Bars represent mean ± SEM. *** p-value < 0.001; two-tailed t-test with Bonferroni correction. (d) Salt extraction of WT MeCP2 and MeCP2 T158A protein with increasing concentrations of NaCl (n = 3). Bars represent mean ± SEM normalized to MeCP2 levels extracted with 700 mM NaCl. ** p-value < 0.01 and *** < 0.001; two-way ANOVA with Bonferroni correction. (e) MeCP2 co-localization with heterochromatin-dense foci in male WT but not Mecp2T158A/y mice at P90. Representative images of neuronal nuclei shown are single confocal planes at 100X magnification. Scale bar corresponds to 20 μm. (f) MeCP2 staining in nuclei obtained from female Mecp2T158A/+ mice. Nuclei showing diffuse MeCP2 staining are marked with an *. Scale bar corresponds to 20 μm.
Figure 5. Disruption of MeCP2 methyl-DNA binding…
Figure 5. Disruption of MeCP2 methyl-DNA binding leads to deregulation of gene expression
(a)Bdnf, Crh and Sgk mRNA expression in the hypothalamus of Mecp2T158A/y mice compared to Mecp2+/y littermates (n = 4). Bars represent mean ± SEM. * p-value < 0.05; two-tailed t-test with Bonferroni correction. (b)Bdnf, Crh and Sgk mRNA expression in the striatum of Mecp2T158A/y mice and Mecp2+/y littermates (n = 4). Bars represent mean ± SEM. * p-value < 0.05; two-tailed t-test with Bonferroni correction. (c) MeCP2 T158A mutation does not impair the association of MeCP2 with HDAC1 or Sin3a. MeCP2 immunoprecipitation from brain nuclear extracts prepared from Mecp2T158A/y and Mecp2+/y littermates are probed with indicated antibodies.
Figure 6. EEG and ERP recordings in…
Figure 6. EEG and ERP recordings in MeCP2 T158A mice
(a) Representative EEG traces from awake, freely mobile mice. Scale bar corresponds to 1 second (horizontal) and 200 μA (vertical). (b) Basal EEG power measurements in P90 Mecp2T158A/y mice (n = 7) compared to Mecp2+/y littermates (n = 8). Frequency bands are represented as follows: δ (2–4 Hz), θ (4–8 Hz), α (8–12 Hz), β (12–30 Hz), low-γ (30–50 Hz), and high-γ (70–140 Hz). Insets show β and high-γ mean amplitudes across EEG recordings. Scale bars represent one oscillation cycle (horizontal) and 20 μA (vertical). Bars represent mean ± SEM. *** p-value <0.001; two-tailed t-test with Bonferroni correction. (c) Grand average event-related potential (ERP) traces following presentation of 85-dB white-noise clicks with 4-second interstimulus intervals. Traces represent mean amplitude (solid line) ± SEM (dashed lines). The characteristic polarity peaks P1, N1 and P2 are highlighted with straight lines with the length indicating latency range. Scale bar corresponds to 50 ms (horizontal) and 20 μA (on vertical). (d) Latencies and (e) amplitudes of ERP peaks. Bars represent mean ± SEM. * p-value < 0.05 and ** < 0.01; two-tailed t-test with Bonferroni correction
Figure 7. Decreased event-related power and PLF…
Figure 7. Decreased event-related power and PLF in Mecp2T158A/y mice
(a) Time-frequency plots showing changes in event-related power in response to 85-dB auditory stimulation in P90 Mecp2T158A/y mice and Mecp2+/y littermates. Time is plotted on the abscissa (where t = 0 at sound presentation) and frequency on the ordinate. Color represents mean power with warmer colors corresponding to an increased power and cooler colors representing decreased power compared to pre-stimulus baseline. (b) Changes in event-related mean power averaged across δ (2–4 Hz), θ (4–8 Hz), α (8–12 Hz), β (12–30 Hz), low-γ (30–50 Hz), and high-γ (70–140 Hz) frequencies. Scale bars represent the length of a single oscillation cycle of the lowest frequency in the range. Insets showed power traces on expanded time-scale denoted by length of single oscillation cycle. Traces represent mean power ± SEM. (c) Time-frequency plots showing changes in event-related phase locking factor (PLF) in response to 85-dB auditory stimulation. Color represents PLF with warmer colors corresponding to a higher PLF or lower circular variance in EEG phase across trials. (d) Changes in event-related PLF averaged across frequencies as above. Scale bars represent the length of a single oscillation cycle and insets show traces on expanded time-scale. Traces represent mean PLF ± SEM.
Figure 8. Age-dependent increase in event-related power…
Figure 8. Age-dependent increase in event-related power and PLF is absent in Mecp2T158A/y mice
(a) Event-related power changes in Mecp2+/y (WT) mice at P30 and P90. (b) Event-related phase-locking factor (PLF) changes in WT mice at P30 and P90. (c) Event-related power changes in Mecp2T158A/y mice at P30 and P90. (d) Event-related PLF changes in Mecp2T158A/y mice at P30 and P90. Bars represent mean ± SEM. * p-value < 0.05, ** < 0.01 and *** < 0.001; two-tailed t-test with Bonferroni correction.

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