Characterization of MeCP2-dependent gene regulation with temporal and mechanistic precision

项目总结 Rett综合征(RTT)是一种严重的神经发育障碍，由甲基DNA突变引起。 结合蛋白MeCP2，是女性智力残疾最常见的原因之一。RTT 症状包括进行性丧失语言和社交能力，癫痫发作，刻板印象的手部动作， 以及运动系统障碍。RTT的单基因病因学使MeCP2突变体的发展成为可能 显示可相当准确地模拟RTT的突触和行为表型的小鼠模型 症状。重要的是，在成年MeCP2基因缺失的小鼠中重新表达MeCP2可以逆转突触和行为 表型，表明有可能改善RTT女孩的认知和行为困难。 然而，对MeCP2分子功能的不完全了解限制了治疗的发展。这个 拟议的研究将使用新的小鼠模型和方法来表征 MeCP2缺失，并研究MeCP2功能的关键神经元活动依赖成分。目标1： 与MeCP2的结构性丢失相关的许多分子后果，研究人员尚不清楚哪些 是MeCP2缺失的直接结果，是长期神经损害的次要后果。 博克瑟博士将使用一种新的小鼠品系，使MeCP2蛋白能够快速和特异地降解，从而 描述大脑中急性丢失MeCP2的分子、细胞和行为后果。这些 实验将揭示表型出现的相对顺序，这将使识别 MeCP2缺失的直接分子效应和对疾病突触和行为步骤的洞察 进步。目的2：MeCP2在多个部位被快速磷酸化，以响应神经元的活动，并且 MeCP2缺失的神经元在经验依赖的突触改进中显示出缺陷。然而，突触 精制表型与活性依赖的MeCP2没有直接或直接的联系 磷酸化。博克瑟博士将使用最近开发的一种小鼠品系，该品系在所有活动中都存在丙氨酸突变- 依赖的磷酸化位点来研究活性依赖的MeCP2磷酸化在脑内的作用 基因表达的调节和突触的精炼。总体而言，这些实验将提供暂时和 机械地精确回答MeCP2研究中的关键悬而未决的问题，潜在地使 RTT靶向治疗的进展。拟议的研究将为博克瑟博士提供培训 电生理学和生物信息学，以及更广泛的神经生物学和职业发展，在 她的导师迈克尔·格林伯格博士的实验室，以及她在哈佛的咨询委员会的指导 医学院。博克瑟博士打算成为一名研究人类免疫缺陷的分子基础的学术教授。 神经发育障碍。

PROJECT SUMMARY Rett syndrome (RTT) is a severe neurodevelopmental disorder that is caused by mutations in the methyl-DNA- binding protein MeCP2 and represents one of the most common causes of intellectual disability in females. RTT symptoms include a progressive loss of speech and social engagement, seizures, stereotyped hand movements, and motor-system disabilities. The monogenic etiology of RTT has enabled the development of MeCP2-mutant mouse models that display synaptic and behavioral phenotypes that reasonably accurately model RTT symptoms. Importantly, re-expression of MeCP2 in adult MeCP2-null mice reverses the synaptic and behavioral phenotypes, suggesting the possibility of ameliorating the cognitive and behavioral difficulties of girls with RTT. However, the incomplete understanding of MeCP2 molecular function limits therapeutic development. The proposed study will use new mouse models and approaches to characterize the immediate consequences of MeCP2 loss and to investigate key neuronal activity-dependent components of MeCP2 function. Aim 1: Of the many molecular consequences associated with constitutive loss of MeCP2, researchers do not yet know which are direct results of MeCP2 loss and which are secondary consequences of long-term neurological impairment. Dr. Boxer will use a new mouse line that enables rapid and specific degradation of the MeCP2 protein to characterize the molecular, cellular, and behavioral consequences of acute loss of MeCP2 in the brain. These experiments will reveal the relative order of phenotype appearance, which will enable the identification of the direct molecular effects of MeCP2 loss and provide insight into the synaptic and behavioral steps of disease progression. Aim 2: MeCP2 is rapidly phosphorylated at multiple sites in response to neuronal activity, and MeCP2-null neurons display defects in experience-dependent synaptic refinement. However, the synaptic refinement phenotype has not been directly or immediately connected to activity-dependent MeCP2 phosphorylation. Dr. Boxer will use a recently developed mouse line with alanine mutations in all activity- dependent phosphorylation sites to investigate the role of activity-dependent MeCP2 phosphorylation in regulation of gene expression and synaptic refinement. Overall, these experiments will provide temporally and mechanistically precise answers to key outstanding questions in MeCP2 research, potentially enabling the development of targeted therapeutics for RTT. The proposed research will provide training for Dr. Boxer in electrophysiology and bioinformatics, and more broadly in neurobiology and career development, in the laboratory of her mentor, Dr. Michael Greenberg, and with guidance from her Advisory Committee at Harvard Medical School. Dr. Boxer intends to become an academic professor studying the molecular basis of neurodevelopmental disorders.