Molecular Pathogenesis Studies of Rett Syndrome

摘要 这项提案的长期目标是了解甲基CpG结合蛋白2的分子活性 为了开发Rett综合征（RTT）和其他MeCP 2相关疾病的可行治疗方案， 这些疾病的范围从严重的新生儿脑病到自闭症、青少年发作的精神分裂症和其他 神经精神疾病在过去的四年里，有三个关键的发现改变了我们对 MeCP2。首先，我们和其他人已经表明MeCP 2结合非CpG甲基化二核苷酸（“mCH”）， 以及甲基化的CpG二核苷酸（“mCG”），并且这种结合与以下转录变化相关： RTT和MECP 2复制综合征小鼠模型。其次，我们发现了一个功能性的AT钩结构域 有证据表明它重塑了染色质。第三，我们已经证明大脑对 MeCP 2表达水平和反义寡核苷酸（ASO）可以降低小鼠中的MeCP 2水平 MeCP 2复制综合征模型并逆转疾病。在本提案中，我们利用这些， 其他最近的发现，以获得更深入的了解RTT病理生理学和作用的MeCP 2在维持 健康的神经元反应在目标1中，我们将描述非CpG甲基化对RTT的贡献。 通过从GABA表达神经元中删除mCH“写入者”Dnmt 3a（以消融mCH）来治疗发病机制， 将所得到的表型和基因表达变化与缺乏Mecp 2的小鼠（我们的 假设mCH“读者”）在完全相同的神经元。在目标2中，我们将发现MeCP 2 通过使用新开发的原位Hi-C方法结合其基因组靶点，无论是mCG还是mCH， 确定来自野生型、MeCP 2缺失、 和MeCP 2过表达小鼠。因为神经元活动也会导致大量的表观遗传变化， 为了改变MeCP 2的相互作用，我们还将在齿状回中进行原位Hi-C， 在神经元刺激之后。在目标3a中，我们将扩展我们成功的阿索研究，为翻译做准备 通过在表达两个人类MECP 2等位基因的MeCP 2复制综合征小鼠中测试它们（就像 患者）滴定将蛋白质水平从2X恢复到1X的阿索剂量，并鉴定 MeCP 2安全水平的界限。在目标3b中，我们将应用一种新的正向遗传筛选策略， 开发用于寻找改变其他疾病相关蛋白质水平的分子，以确定可药物治疗的目标 降低或增加MeCP 2水平。(Some引起RET的MeCP 2突变减少了蛋白质的 水平。）我们的shRNA筛选使用DsRed-IRES-MeCP 2-EGFP靶向7，787个可药用基因集合。 报告细胞系，允许高通量监测MeCP 2水平。目标将得到验证， 具有最有希望的安全性特征的药物将用于体内研究。总而言之，拟议的研究将 大大推进了我们对MeCP 2功能的理解，染色质的功能在 神经发育，并为MeCP 2相关疾病提供新的治疗方法。

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.