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.
Loss of the Fragile X Mental Retardation Protein (FMRP) causes Fragile X syndrome (FXS), the leading cause of heritable intellectual disability and autism spectrum disorders (ASDs). FMRP is an mRNA-binding translational regulator, which I propose partners with Pumilio, a protein of similar function, to regulate expression of the downstream target Neuroglian, a cell adhesion molecule (CAM) directing synapse stability during neural circuit refinement. My work will test this hypothesis and investigate Pumilio and Neuroglian as therapeutic targets for FXS treatments and ASD intervention more generally.
|Kennedy, Tyler; Broadie, Kendal (2017) Fragile X Mental Retardation Protein Restricts Small Dye Iontophoresis Entry into Central Neurons. J Neurosci 37:9844-9858|