FMRP and Pumilio co-regulate synaptogenesis by controlling Neuroglian expression

FMRP 和 Pumilio 通过控制 Neuroglian 表达共同调节突触发生

• 批准号: 9068676
• 负责人: Tyler J. Kennedy
• 金额: $ 2.79万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068676
• 关键词: Adult; Autistic Disorder; Axon; Behavioral; Binding; Binding Proteins; Biochemical; Biological Assay; Brain; Cell Adhesion Molecules; Cell Surface Receptors; Chemical Synapse; Complex; Confocal Microscopy; Cytoplasmic Granules; Defect; Dendrites; Development; Disease model; Dominant-Negative Mutation; Drosophila genus; Electrophysiology (science); Elements; Engineering; FMR1; Failure; Fiber; Fluorescence; Fragile X Syndrome; Genetic; Genetic Recombination; Growth; Heterozygote; Homologous Gene; Human; Image; Intellectual functioning disability; Intervention; Joints; Label; Light; Link; Maintenance; Measurement; Mediator of activation protein; Membrane; Messenger RNA; Methods; Microscopy; Modeling; Molecular; Monitor; Movement; Mutation; Neurologic; Neurons; Patients; Phenocopy; Phenotype; Process; Protein Dynamics; Proteins; RNA; Regulation; Reporter; Reporting; Ribonucleases; Role; Signal Transduction; Specificity; Staging; Structure; Synapses; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Time; Transcript; Transgenic Organisms; Translations; Western Blotting; Work; autism spectrum disorder; critical period; frontier; in vivo; induced pluripotent stem cell; insight; loss of function; molecular assembly/self assembly; mutant; neural circuit; neuroglian; new therapeutic target; postsynaptic; presynaptic; public health relevance; relating to nervous system; response; sensor; synaptic failure; synaptic function; synaptogenesis; therapeutic target; trafficking

 DESCRIPTION (provided by applicant): Silencing of the fragile X mental retardation 1 (FMR1) gene, resulting in loss of its protein product Fragile X Mental Retardation Protein (FMRP), causes Fragile X syndrome (FXS), the leading heritable cause of intellectual disability and autism spectrum disorders. FMRP is an mRNA-binding translational regulator important for establishing correct synaptic connectivity in the brain, but the mechanism for the specificity of is regulatory function remains largely unknown. This work employs the powerful Drosophila FXS disease model to test interactions between FMRP and a proposed partner mRNA-binding protein in regulating expression of a key cell adhesion molecule (CAM) involved in synapse stabilization during brain neural circuit refinement. First, this study investigates FMRP interactions with the conserved Pumilio (Pum) mRNA-binding protein. Accumulated evidence shows these proteins co-localize and manifest common neural phenotypes in mutant and genetic interaction tests. Understanding the cooperative interaction between these translational regulators should shed light on the specificity of their mRNA targeting mechanism and provide an avenue for therapeutic intervention. Second, this study investigates a candidate common mRNA target of FMRP/Pum, the CAM Neuroglian (Nrg), as a mechanism to explain the synapse immaturity and failure of stability characterizing FXS patients and models. Nrg mRNA contains putative FMRP and Pum binding elements, Nrg protein is progressively lost in Drosophila FMR1 null mutants, the Nrg CAM is an established mediator of synapse stability, and mutations in human Nrg homologs cause intellectual disability and autism. Thus, the core hypothesis is that FMRP and Pum jointly regulate Nrg synaptogenic protein levels in response to developmental signals, and that loss of appropriate Nrg regulation by a FMRP/Pum complex leads to a failure of synapse stabilization. Biochemical approaches will be employed to test interactions between FMRP, Pum and Nrg mRNA. Confocal microscopy in the well-defined Giant Fiber (GF) central circuit will be used to study changes in synaptic connectivity, both at architectural and molecular levels, using transgenic labeling techniques. Electrophysiology recording throughout development will test synaptic functional maturation and stabilization. Multiphoton excitation microscopy will be used to chart synapse dynamics in real time in the intact brain in vivo, including FMRP/Pum/Nrg mRNA trafficking, measurements of synapse molecular assembly rates, and fluorescence reporter synaptic activity recordings. Together, this work will provide new insights into the cooperative interaction of mRNA-binding translational regulators (FMRP and Pum), joint regulation of a common mRNA target (Nrg) and the in vivo dynamics of synapse assembly, pruning and stabilization during brain neural circuit refinement. This study should expose potential therapeutic avenues for FXS and related autistic conditions.

 描述（由申请人提供）：脆性X智力迟钝1（FMR 1）基因的沉默，导致其蛋白产物脆性X智力迟钝蛋白（FMRP）的丢失，导致脆性X综合征（FXS），这是智力残疾和自闭症谱系障碍的主要遗传原因。FMRP是一种mRNA结合的翻译调节因子，对于在大脑中建立正确的突触连接非常重要，但其调节功能特异性的机制在很大程度上仍然未知。这项工作采用了强大的果蝇FXS疾病模型来测试FMRP和一种拟议的伴侣mRNA结合蛋白在调节一种关键细胞粘附分子（CAM）表达中的相互作用，该分子参与脑神经回路完善过程中的突触稳定。首先，本研究调查FMRP与保守的Pumilio（Pum）mRNA结合蛋白的相互作用。累积的证据表明，这些蛋白质共定位和突变体和遗传相互作用测试中表现出共同的神经表型。了解这些翻译调节因子之间的协同作用，应有助于阐明其mRNA靶向机制的特异性，并为治疗干预提供途径。其次，本研究调查了FMRP/Pum的候选共同mRNA靶点，CAM神经胶质细胞（Nrg），作为解释FXS患者和模型特征的突触不成熟和稳定性失败的机制。Nrg mRNA含有推定的FMRP和Pum结合元件，Nrg蛋白在果蝇FMR 1无效突变体中逐渐丢失，Nrg CAM是突触稳定性的既定介质，人类Nrg同源物的突变导致智力残疾和自闭症。因此，核心假设是FMRP和Pum共同调节Nrg突触发生蛋白水平以响应发育信号，并且FMRP/Pum复合物的适当Nrg调节的丧失导致突触稳定的失败。将采用生物化学方法来测试FMRP、Pum和Nrg mRNA之间的相互作用。在明确的巨纤维（GF）中央回路的共聚焦显微镜将被用来研究突触连接的变化，无论是在建筑和分子 水平，使用转基因标记技术。整个发育过程中的电生理学记录将测试突触功能的成熟和稳定。多光子激发显微镜将用于绘制突触动力学在真实的时间在完整的大脑在体内，包括FMRP/Pum/Nrg mRNA运输，测量突触分子组装率，和荧光报告突触活动记录。总之，这项工作将提供新的见解的mRNA结合的翻译调节（FMRP和Pum），共同的mRNA靶点（Nrg）和突触组装，修剪和稳定在脑神经回路细化过程中的体内动力学的联合调节的合作相互作用。这项研究应该揭示FXS和相关自闭症疾病的潜在治疗途径。