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

MeCP2 依赖性基因调控的时间和机械精度表征

• 批准号: 9804807
• 负责人: Lisa D. Boxer
• 金额: $ 8.94万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9804807
• 关键词: Acute; Adult; Advisory Committees; Alanine; Appearance; Behavioral; Binding; Bioinformatics; Brain; Chromatin; Cognitive; DNA Binding; DNA Binding Domain; DNA Methylation; Defect; Development; Disease; Disease Progression; Electrophysiology (science); Etiology; Exhibits; Experimental Models; Female; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomic DNA; Genomics; Hand; Histones; Impairment; Intellectual functioning disability; Investigation; Knockout Mice; Laboratories; Limb structure; Long-Term Effects; Longevity; Mentors; Methyl-CpG-Binding Protein 2; Methylation; Modeling; Molecular; Motor; Movement; Mus; Mutation; Neurobiology; Neurodevelopmental Disorder; Neurologic; Neurons; Nuclear; Phenotype; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Proteins; RNA; Regulation; Regulator Genes; Research; Research Personnel; Rett Syndrome; Role; Secondary to; Seizures; Site; Speech; Stereotyping; Stimulus; Symptoms; Synapses; System; Testing; Training; Transcription Repressor/Corepressor; Up-Regulation; Weight; career development; disability; experience; experimental study; gene repression; genome-wide analysis; girls; insight; medical schools; molecular phenotype; mouse model; mutant mouse model; neuron loss; neurotransmission; prevent; professor; protein degradation; response; small molecule; social engagement; targeted treatment; temporal measurement; therapeutic development

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.

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症状包括进行性言语和社会参与丧失、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能够开发MeCP2突变小鼠模型，其显示合理准确地模拟RTT症状的突触和行为表型。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，这表明有可能改善患有RTT的女孩的认知和行为困难。然而，对MeCP2分子功能的不完全理解限制了治疗的发展。拟议的研究将使用新的小鼠模型和方法来表征MeCP 2丢失的直接后果，并研究MeCP 2功能的关键神经元活动依赖性成分。目标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，哪些是长期神经功能障碍的继发后果。Boxer博士将使用一种新的小鼠品系，能够快速特异性降解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，这将能够鉴定MeCP2损失的直接分子效应，并提供对疾病进展的突触和行为步骤的深入了解。目标二：MeCP2在多个位点迅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性突触细化中显示缺陷。然而，突触细化表型尚未直接或立即连接到活性依赖性MeCP2磷酸化。Boxer博士将使用最近开发的在所有活性依赖性磷酸化位点中具有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细化调节中的作用。总的来说，这些实验将为MeCP2研究中关键的悬而未决的问题提供时间和机制上的精确答案，从而有可能开发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。