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症状包括逐渐丧失语言和社交能力、癫痫发作、刻板的手部运动和运动系统残疾。RTT的单基因病因学使得mecp2突变小鼠模型的开发能够显示突触和行为表型，从而合理准确地模拟RTT症状。重要的是，在MeCP2缺失的成年小鼠中，MeCP2的重新表达逆转了突触和行为表型，这表明有可能改善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。该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组成性缺失相关的许多分子后果中，研究人员还不知道哪些是MeCP2缺失的直接结果，哪些是长期神经损伤的继发后果。Boxer博士将使用一种新的小鼠品系，该品系能够快速和特异性地降解MeCP2蛋白，以表征大脑中MeCP2急性丧失的分子、细胞和行为后果。这些实验将揭示表型出现的相对顺序，从而能够识别MeCP2缺失的直接分子效应，并为疾病进展的突触和行为步骤提供见解。目的2:MeCP2在多个位点快速磷酸化以响应神经元活动，并且MeCP2缺失的神经元在经验依赖的突触细化中表现出缺陷。然而，突触细化表型并没有直接或立即与活性依赖性MeCP2磷酸化联系起来。Boxer博士将使用最近开发的具有所有活性依赖性磷酸化位点丙氨酸突变的小鼠系来研究活性依赖性MeCP2磷酸化在基因表达和突触完善调节中的作用。总的来说，这些实验将为MeCP2研究中的关键问题提供时间和机制上的精确答案，有可能开发针对RTT的靶向治疗方法。拟议中的研究将为Boxer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，培训将在她的导师Michael Greenberg博士的实验室进行，并得到她在哈佛医学院咨询委员会的指导。Boxer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.