Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders1. MECP2 duplication syndrome is one of the most common genomic rearrangements in males2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death3,4,5. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders6,7,8, including loss of MeCP2 in Rett syndrome9, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2-overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it10,11, and have been successfully used to correct deficits in different mouse models12,13,14,15,16,17,18. We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult symptomatic transgenic MECP2 duplication mice (MECP2-TG)19,20, and corrected MECP2 levels in lymphoblastoid cells from MECP2 duplication patients in a dose-dependent manner.
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We thank R. Jaenisch for the Mecp2tm1Jae mice, S. Chun for ASOs tolerability studies in wild-type mice, L. Lombardi, M. Rousseaux, C. Alcott and V. Brandt for critical input, and C. Spencer for behavioural assays training. We are indebted to the patients and families who participated in this study. This project was funded by the National Institutes of Health (5R01NS057819 and 5P30HD024064 to H.Y.Z.), the Rett Syndrome Research Trust (401 Project), the Carl. C. Anderson, Sr and Marie Jo Anderson Charitable Foundation, the Howard Hughes Medical Institute (H.Y.Z.), NSF DMS-1263932 (Z.L.), and the Baylor Intellectual Disabilities Research Center (1U54HD083092) neurovisualization, neuroconnectivity and neurobehavioral cores.
F.R. is an employee of Isis Pharamaceuticals.
Extended data figures and tables
Extended Data Figure 1 Mice that overexpress a human MECP2 transgene over a floxed Mecp2 allele resemble the classic MECP2-TG mice at the molecular level.
a, Breeding strategy to generate mice that have a wild-type MECP2 allele and a Mecp2 allele flanked by loxP sequences. b, Western blot of MeCP2 in hypothalamus, amygdala and cerebellum. GAPDH was used as the internal control (for gel source data, see Supplementary Fig. 3). c, Densitometric analysis of western blots in b. Flox;TG mice overexpress MeCP2 at levels similar to transgenic mice. It is noteworthy that in the hypothalamus, Flox mice were 30% hypomorphic (n = 2 mice per group; two-tailed t-test) when compared to wild-type mice, but not in the other regions. d, RT–qPCR analysis in hypothalamus, amygdala and cerebellum, using primers common to mouse and human MECP2. Flox;TG mice overexpressed the MECP2 transcript at levels similar to transgenic mice. Hprt1 was used as the internal control (n = 5 mice per group; two-tailed t-test). e, RT–qPCR analysis of three selected genes know to be altered by MeCP2 overexpression. Flox;TG mice overexpressed the Sst, Crf and Prl2c2 transcripts at levels similar to those of transgenic mice. Hprt1 was used as the internal control (n = 4 for wild-type group; n = 6 for transgenic group; n = 5 for Flox and Flox;TG groups; two-tailed t-test). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 2 Mice that overexpress a human MECP2 transgene over a floxed Mecp2 allele show characteristic behavioural deficits.
a, Flox;TG mice displayed hypoactivity and anxiety in the open field test similar to transgenic mice. b, Flox;TG mice showed heightened anxiety-like behaviour in the elevated plus maze test. c, Flox;TG mice showed enhanced motor learning in the rotarod test similar to transgenic mice (n = 18 for wild-type group; n = 9 for transgenic group; n = 14 for Flox group; n = 12 for Flox;TG group (for all behavioural tests)). Data were analysed by one-way ANOVA, with the exception of the rotarod test that was analysed by two-way ANOVA repeated measures followed by Tukey HSD post hoc correction for multiple comparisons. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
a, b, RT–qPCR from the hypothalamus (a) and cerebellum (b) shows correction of altered expression of selected genes after normalization of MeCP2 levels in Flox;TG;Cre-TMX mice (n = 6 for Flox group; n = 7 for Flox;TG group; n = 7 for Flox;TG;Cre-vehicle group; n = 8 for Flox;TG;Cre-TMX group; two-tailed t-test). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Figure 4 Hippocampal basal synaptic transmission and short-term synaptic plasticity are normal in MECP2-TG mice.
a, Long-term potentiation was induced by applying high-frequency stimulation to the Schaffer collateral axons, and field excitatory postsynaptic potentials (fEPSPs) were recorded at the Schaffer collateral-CA1 synapses of the hippocampus (stratum radiatum). b, MeCP2 overexpression did not affect Schaffer collateral basal synaptic transmission, as determined by the correlation between the slopes of the evoked fEPSPs and the amplitudes of the fibre volleys. c, d, MeCP2 overexpression did not affect short-term synaptic plasticity, as determined by paired-pulse facilitation (n = 7 for Flox group; n = 6 for Flox;TG group; n = 7 for Flox;TG;Cre-vehicle group; n = 7 for Flox;TG;Cre-TMX group). Data are mean ± s.e.m.
a, Location of ASOs targeting sequences on the MECP2 pre-mRNA. Boxes represent exons, and lines denote introns. Boxes in blue denote the translatable regions. b, Section schemata from the Paxinos and Franklin35 mouse brain atlas showing site of stereotactic injection. c, Western blot (c) and densitometric analysis (d) of MeCP2 from cortical samples of mice treated with saline or the indicated ASOs 2 weeks after single bolus stereotactic injection of 500 μg ASO in the right ventricle of the brain. ASO-5 was found to be the most effective. GAPDH was used as an internal control (n = 3, one-way ANOVA followed by Tukey HSD post hoc correction for multiple comparisons; for gel source data, see Supplementary Fig. 4). e, RT–qPCR analysis of MECP2 mRNA from cortical samples of mice treated with saline or the indicated ASOs, 2 weeks after single bolus stereotaxic injection of 500 μg ASO in the right ventricle of the brain. ASO-5 was found to be the most effective. The MECP2 primers are common to the mouse and human alleles. Hprt1 was used as an internal control (n = 3, one-way ANOVA followed by Tukey HSD post hoc correction for multiple comparisons). Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
a, Micro-osmotic pump connected to a cannula through a plastic catheter. b, The micro-osmotic pump was implanted in a subcutaneous pocket and the cannula stereotaxically positioned to deliver the ASO into the right ventricle. c, Section schemata from the Paxinos and Franklin35 mouse brain atlas showing site of cannula implantation. d, Injection of 1 µl of dye through the catheter to show that the dye reaches the whole ventricular system, confirming the correct positioning of the tip of the cannula in the right ventricle. e, RT–qPCR for Aif1 (a marker of activated microglia) and Gfap (a marker of astrocytes) immediately after the end of 4 weeks of gradual ASO treatment (n = 4 for wild-type group; n = 5 for transgenic group; n = 4 for TG-ASO group). Data are mean ± s.e.m.
a, Timeline of ASO treatment and behavioural tests. b, Two weeks after cessation of ASO treatment, MECP2-TG mice showed no rescue of hypoactivity, rearing behaviour or anxiety parameters in the open field test. c, No rescue was evident in any of the parameters of the elevated plus maze test at this early post-treatment stage. d, ASO treatment normalized performance in the rotarod test in MECP2-TG mice (asterisks indicate significance between MECP2-TG ASO and control-ASO groups). e, The impaired social behaviour in the 3-chamber test was not normalized in the ASO-treated group. No significant difference was found between any of the groups in the time spent investigating the inanimate object. f, No preference for the cups placed in the left or right chambers was found in the habituation phase of the 3-chamber test (n = 19 for wild-type group; n = 16 for transgenic group; n = 15 for TG-ASO group (for all behavioural tests)). Data were analysed by one-way ANOVA followed by Fisher’s LSD post hoc test, with the exception of the rotarod test that was analysed by two-way ANOVA repeated measures followed by Tukey HSD post hoc correction for multiple comparisons. Data are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001.
Representative EEG traces of all mice recorded after gradual MECP2-ASO or control-ASO treatment.
This file contains Supplementary Figures 1-4, which show the original uncropped western blots as follows: (1) Figure 1c,d; (2) Figure 3e,d; (3) Extended Data Figure 1b; and (4) Extended Data Figure 5c,d. The black frames denote how the gels were cropped for the final figure. (PDF 1748 kb)
This table contains RNA-seq results from the genetic rescue experiment. Only the genes with expression significantly different between the groups are presented. (XLSX 43 kb)
This table contains RNA-seq results from the antisense oligonucleotides experiments. Only the genes with expression significantly different between the groups are presented. (XLSX 65 kb)
This video shows a spontaneous seizure from a 30-week old male MECP2 duplication transgenic mouse during EEG recording. (MP4 8656 kb)
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Sztainberg, Y., Chen, Hm., Swann, J. et al. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides. Nature 528, 123–126 (2015). https://doi.org/10.1038/nature16159
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