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

Rett综合征(RTT)是一种严重的神经发育障碍，由甲基DNA结合蛋白MeCP2突变引起，是女性智力残疾的最常见原因之一。RTT的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和运动系统残疾。RTT的单基因病因学使MeCP2突变小鼠模型的发展成为可能，这些突变小鼠模型显示的突触和行为表型可以合理准确地模拟RTT症状。重要的是，MeCP2在成年MeCP2缺失小鼠中的重新表达逆转了突触和行为表型，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。这项拟议的研究将使用新的小鼠模型和方法来表征MeCP2缺失的直接后果，并研究MeCP2功能的关键神经元活动依赖成分。目的1：在与MeCP2结构性缺失相关的许多分子后果中，研究人员尚不清楚哪些是MeCP2缺失的直接结果，哪些是长期神经损害的继发性后果。Boxer博士将使用一种新的小鼠品系，该品系能够快速和特定地降解MeCP2蛋白，以表征大脑中MeCP2急性丢失的分子、细胞和行为后果。这些实验将揭示表型出现的相对顺序，这将使我们能够识别MeCP2缺失的直接分子效应，并提供对疾病进展的突触和行为步骤的洞察。目的：MeCP2在多个位置被快速磷酸化，以响应神经元的活动，而MeCP2缺失的神经元在经验依赖的突触精炼中显示出缺陷。然而，突触细化表型与活性依赖的MeCP2磷酸化并没有直接或直接的联系。博克瑟博士将利用最近开发的一种小鼠品系，在所有活性依赖的磷酸化位点发生丙氨酸突变，以研究活性依赖的MeCP2磷酸化在基因表达调控和突触细化中的作用。总体而言，这些实验将在时间和机械上为MeCP2研究中的关键悬而未决问题提供准确的答案，潜在地使RTT靶向疗法的开发成为可能。这项拟议的研究将在博克瑟的导师迈克尔·格林伯格博士的实验室里，在她在哈佛医学院的咨询委员会的指导下，为博克瑟博士提供电生理学和生物信息学方面的培训，以及更广泛的神经生物学和职业发展方面的培训。博克瑟博士打算成为一名研究神经发育障碍分子基础的学术教授。

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.