Molecular Pathogenesis Studies of Rett Syndrome

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ABSTRACT The long-term goal of this proposal is to understand the molecular activities of methyl CpG-binding protein 2 (MeCP2) in order to develop viable treatment options for Rett syndrome (RTT) and other MeCP2-related disorders, which range from severe neonatal encephalopathy to autism, juvenile onset schizophrenia and other neuropsychiatric conditions. Three key discoveries in the past four years have changed the way we think about MeCP2. First, we and others have shown that MeCP2 binds to non-CpG methylated dinucleotides (“mCH”) as well as methylated CpG dinucleotides (“mCG”), and that this binding correlates with transcriptional changes in mouse models of RTT and MECP2 duplication syndrome. Second, we discovered a functional AT-hook domain in MeCP2 and evidence that suggests it remodels chromatin. Third, we have shown that the brain is sensitive to levels of MeCP2 expression and that antisense oligonucleotides (ASOs) can reduce MeCP2 levels in a mouse model of the MeCP2 duplication syndrome and reverse the disease. In this proposal we capitalize on these and other recent discoveries to gain deeper insight into RTT pathophysiology and the role of MeCP2 in maintaining healthy neuronal responsiveness. In Aim 1, we will delineate the contributions of non-CpG methylation to RTT pathogenesis by deleting the mCH “writer”, Dnmt3a, from GABA-expressing neurons (to ablate mCH) and comparing the resulting phenotype and gene expression changes to those of mice lacking Mecp2 (our hypothesized mCH “reader”) in precisely the same neurons. In Aim 2, we will find what happens once MeCP2 binds its genomic targets, whether mCG or mCH, by using the newly developed in situ Hi-C approach to ascertain the 3D chromatin structure in the cerebellum and dentate gyrus in tissue from wild-type, MeCP2 null, and MeCP2 overexpressing mice. Because neuronal activity leads to a multitude of epigenetic changes, as well as altering the interactions of MeCP2, we will also perform in situ Hi-C in the dentate gyrus both before and after neuronal stimulation. In Aim 3a, we will expand on our successful ASO studies to prepare for translation by testing them in a MeCP2 duplication syndrome mouse that expresses two human MECP2 alleles (just like the patients) to titrate the ASO dose that will restore the protein levels from 2X to 1X, and identify the boundaries of safe MeCP2 levels. In Aim 3b, we will apply a novel forward genetic screening strategy that we developed for finding molecules that alter levels of other disease-related proteins to identify druggable targets that either decrease or increase MeCP2 levels. (Some Rett-causing MeCP2 mutations reduce the protein's level.) Our shRNA screen targets a 7,787 druggable gene collection using a DsRed- IRES-MeCP2-EGFP reporter cell line that allows high-throughput monitoring of MeCP2 levels. Targets will be validated and those with most promising safety profiles will be advanced for in vivo studies. In sum, the proposed studies will greatly advance our understanding of MeCP2 function, the function of chromatin changes during neurodevelopment, and provide new treatment approaches to MeCP2-related diseases.