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 3 a, 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 3 b, 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.

Public Health Relevance

Disruptions in normal MECP2 functioning cause a host of neuropsychiatric disorders, from Rett syndrome to autism, intellectual disability, mood disorders, and MECP2 duplication syndrome. Our studies will provide insight into how either loss or gain of MECP2 function alters neuronal activity to cause behavioral and motor problems, and will advance promising new treatment approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS057819-12
Application #
9264030
Study Section
Developmental Brain Disorders Study Section (DBD)
Program Officer
Mamounas, Laura
Project Start
2006-09-04
Project End
2021-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
12
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Pediatrics
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030
Raman, Ayush T; Pohodich, Amy E; Wan, Ying-Wooi et al. (2018) Apparent bias toward long gene misregulation in MeCP2 syndromes disappears after controlling for baseline variations. Nat Commun 9:3225
Kee, Sara E; Mou, Xiang; Zoghbi, Huda Y et al. (2018) Impaired spatial memory codes in a mouse model of Rett syndrome. Elife 7:
Pohodich, Amy E; Yalamanchili, Hari; Raman, Ayush T et al. (2018) Forniceal deep brain stimulation induces gene expression and splicing changes that promote neurogenesis and plasticity. Elife 7:
Ito-Ishida, Aya; Yamalanchili, Hari Krishna; Shao, Yingyao et al. (2018) Genome-wide distribution of linker histone H1.0 is independent of MeCP2. Nat Neurosci 21:794-798
Lombardi, Laura M; Zaghlula, Manar; Sztainberg, Yehezkel et al. (2017) An RNA interference screen identifies druggable regulators of MeCP2 stability. Sci Transl Med 9:
Ure, Kerstin; Lu, Hui; Wang, Wei et al. (2016) Restoration of Mecp2 expression in GABAergic neurons is sufficient to rescue multiple disease features in a mouse model of Rett syndrome. Elife 5:
Meng, Xiangling; Wang, Wei; Lu, Hui et al. (2016) Manipulations of MeCP2 in glutamatergic neurons highlight their contributions to Rett and other neurological disorders. Elife 5:
Lu, Hui; Ash, Ryan T; He, Lingjie et al. (2016) Loss and Gain of MeCP2 Cause Similar Hippocampal Circuit Dysfunction that Is Rescued by Deep Brain Stimulation in a Rett Syndrome Mouse Model. Neuron 91:739-747
Zoghbi, Huda Y; Beaudet, Arthur L (2016) Epigenetics and Human Disease. Cold Spring Harb Perspect Biol 8:a019497
Sztainberg, Yehezkel; Zoghbi, Huda Y (2016) Lessons learned from studying syndromic autism spectrum disorders. Nat Neurosci 19:1408-1417

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