FMRP and Pumilio co-regulate synaptogenesis by controlling Neuroglian expression

 描述(由申请人提供)：脆性X智力低下1(FMR1)基因沉默，导致其蛋白产物脆性X智力低下蛋白(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可遗传原因。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，但其调节功能的特异性机制在很大程度上仍不清楚。本工作使用强大的果蝇FXS疾病模型来测试FMRP与所提出的合作伙伴mRNA结合蛋白之间的相互作用，以调节参与脑神经回路精化过程中突触稳定的关键细胞黏附分子(CAM)的表达。首先，本研究研究了FMRP与保守的Pum(Pum)mRNA结合蛋白的相互作用。越来越多的证据表明，这些蛋白质在突变和遗传相互作用测试中共同定位并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将有助于揭示其mRNA靶向机制的特异性，并为治疗干预提供途径。其次，本研究探讨了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含有可能的FMRP和Pum结合元件，NRG蛋白在果蝇FMR1缺失突变体中逐渐丢失，NRG CAM是突触稳定的已知中介，人类NRG同源物的突变会导致智能障碍和自闭症。因此，核心假设是FMRP和Pum共同调节NRG突触生成蛋白水平以响应发育信号，并且FMRP/Pum复合体失去适当的NRG调节导致突触稳定失败。将使用生化方法来测试FMRP、Pum和NRG mRNA之间的相互作用。在明确定义的巨型纤维(GF)中央回路中的共聚焦显微镜将被用来研究突触连接的变化，包括结构和分子 水平，使用转基因标记技术。整个发育过程中的电生理记录将测试突触功能的成熟和稳定。多光子激发显微镜将被用于在活体完整的脑中实时绘制突触动力学图，包括FMRP/Pum/NRG mRNA运输，突触分子集合率的测量，以及荧光报告突触活动的记录。总之，这项工作将为研究mRNA结合的翻译调节因子(FMRP和Pum)的协同作用，共同的mRNA靶标(NRG)的联合调节，以及在脑神经回路精炼过程中突触组装、修剪和稳定的体内动力学提供新的见解。这项研究将揭示FXS和相关自闭症的潜在治疗途径。

 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.