Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome–like phenotypes
Janine M. Lamonica, … , Sigrid Veasey, Zhaolan Zhou
Janine M. Lamonica, … , Sigrid Veasey, Zhaolan Zhou
Published April 10, 2017
Citation Information: J Clin Invest. 2017;127(5):1889-1904. https://doi.org/10.1172/JCI90967.
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Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome–like phenotypes

Abstract

Mutations in the X-linked gene encoding methyl-CpG–binding protein 2 (MeCP2) cause Rett syndrome (RTT), a neurological disorder affecting cognitive development, respiration, and motor function. Genetic restoration of MeCP2 expression reverses RTT-like phenotypes in mice, highlighting the need to search for therapeutic approaches. Here, we have developed knockin mice recapitulating the most common RTT-associated missense mutation, MeCP2 T158M. We found that the T158M mutation impaired MECP2 binding to methylated DNA and destabilized MeCP2 protein in an age-dependent manner, leading to the development of RTT-like phenotypes in these mice. Genetic elevation of MeCP2 T158M expression ameliorated multiple RTT-like features, including motor dysfunction and breathing irregularities, in both male and female mice. These improvements were accompanied by increased binding of MeCP2 T158M to DNA. Further, we found that the ubiquitin/proteasome pathway was responsible for MeCP2 T158M degradation and that proteasome inhibition increased MeCP2 T158M levels. Together, these findings demonstrate that increasing MeCP2 T158M protein expression is sufficient to mitigate RTT-like phenotypes and support the targeting of MeCP2 T158M expression or stability as an alternative therapeutic approach.

Authors

Janine M. Lamonica, Deborah Y. Kwon, Darren Goffin, Polina Fenik, Brian S. Johnson, Yue Cui, Hengyi Guo, Sigrid Veasey, Zhaolan Zhou

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Figure 6

Tg elevation of MeCP2 T158M expression increases DNA binding.

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Tg elevation of MeCP2 T158M expression increases DNA binding. (A) IHC fo...
(A) IHC for MeCP2 in brain sections from animals of the indicated genotype at 12 weeks. Scale bar: 20 μm. Original magnification: ×63 with digital zoom. (B) Western blot of nucleosolic (Nuc) and chromatin-enriched (Chr) proteins in cortices from Mecp2+/y, Mecp2T158M/y, and Mecp2T158M/y T158M–Tg animals at 12 weeks. Graph shows the quantification of MeCP2 levels in nucleosolic and chromatin fractions normalized to TBP. Values are represented as the fold-change relative to nucleosolic MeCP2 from Mecp2+/y animals (n = 3 per genotype). **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA, followed by Sidak’s multiple comparisons test. (C) MeCP2 ChIP-qPCR in cortices from 12-week-old Mecp2+/y, Mecp2T158M/y, and Mecp2T158M/y T158M–Tg animals at the indicated loci revealed significantly increased MeCP2 binding in Mecp2T158M/y T158M–Tg animals at several loci (n = 3–5 biological replicates). *P < 0.05, by 1-way ANOVA, followed by Tukey’s post-hoc test. (D) FLAG-MeCP2-Dendra2 protein levels following treatment with inhibitors of protein degradation pathways. MG132 increased the levels of MeCP2 to the greatest extent. Values were normalized to β-actin and are represented as the fold change relative to vehicle-treated cells (n = 3–5 per condition). (E) Time course of MG132 treatment in FLAG-T158M-Dendra2 and FLAG-MeCP2-Dendra2 N2a cell lines (n = 3 biological replicates). (F) FLAG-T158M-Dendra2 levels following treatment with increasing concentrations of MG132 (n = 3 per condition). (G) P0 plus 3-DIV cortical cultures from Mecp2+/y and Mecp2T158M/y animals treated with vehicle or 20 μM MG132 for 4 and 8 hours (n = 3 per genotype). Quantifications in E–G were performed by normalizing to the TBP loading control; values are shown as the fold-change relative to t0. Comparisons in D–G were done using 1-way ANOVA, followed by Dunnett’s multiple comparisons test. *P < 0.05 and **P < 0.01. All error bars represent the mean ± SEM.