Genetic and Developmental Analyses of Fragile X Mental Retardation Protein

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Early life sensory experience remodels brain circuitry to optimize behavioral output. This competitive renewal continues our genetic dissection of the molecular mechanisms through which synaptic connections are refined based on sensory experience. We use different odorant cues to activate different classes of odorant receptor neurons, downstream projection neurons, and brain learning/memory center Kenyon cells to test the directional remodeling of connectivity and function. In three parallel circuit channels, we use in vivo CRISPR knockout, conditional gene manipulations and transgenic trans-synaptic connectivity mapping to test both neuron and glia class-specific roles in activity-dependent remodeling. These mechanisms go awry in numerous disease states of cognitive and autism spectrum disorders. We directly test here genetic models of Fragile X syndrome (FXS), Noonan syndrome (NS), Noonan syndrome with Multiple Lentigines (NSML) and Neurobeachin (NBEA) autism spectrum disorder (ASD). In Aim 1, we test neuron-specific Fragile X Mental Retardation Protein (FMRP) roles in odorant sensory experience-dependent critical period brain circuit remodeling in 3 distinct circuit channels. We target tetanus toxin light chain (TeTXLC) and excitatory (CsChrimson)/inhibitory (GtAcR) optogenetic tools to odorant sensory neurons, projection neurons, and Kenyon cells to dissect activity-dependent mechanisms. Based on a preliminary screen, we identify channel-selective chloride transporters and Wnt signaling ligands to test their critical period roles. In Aim 2, we test glia-specific FMRP roles in experience-dependent critical period synaptic remodeling by blocking phagocytic pruning at three different levels. We test neuron-to-glia signaling mechanisms in the FXS disease model, including both synapse-secreted insulin like peptide (ILP) “find me” signals and surface phosphatidylserine (PS) “eat me” signals. We test the roles of direct FMRP mRNA targets 1) ESCRT-III Shrub and 2) glial-secreted matrix metalloproteinase 1 (MMP1) in glial synapse phagocytosis and critical period synaptic remodeling. We use single neuron labeling (miniSOG) to visualize glial phagocytosis via transmission electron microscopy (TEM). In Aim 3, we test FMRP translation regulation of PKA/ERK signaling pathways in sensory experience-dependent circuit remodeling. We use new transgenic cAMP reporter (cAMPr) and separation of phases-based activity reporter of kinase (SPARK) to visualize signaling in vivo in response to experience during the critical period; compartmentalized within odorant sensory neurons, projection neurons and Kenyon cells in our 3 parallel circuit channels. We test roles of direct FMRP mRNA target Rugose/NBEA acting as a PKA anchor, with downstream PKA pathway kinase Meng-Po. Using an ERK-SPARK reporter and whole-cell patch-clamp recording to monitor functional remodeling, we test roles of direct FMRP mRNA target Corkscrew/SHP2 acting as an ERK pathway phosphatase. Our ongoing program orders molecular mechanism intersections between Fragile X syndrome (FXS), Noonan syndrome (NS), Noonan syndrome with Multiple Lentigines (NSML), and NBEA-associated autism spectrum disorder in critical period brain circuit remodeling.