Glial roles in experience-dependent critical period remodeling

Title: Glial roles in experience-dependent critical period remodeling Summary We propose glia actively prune brain circuits to optimize connectivity based on early-life sensory experience. Drosophila approaches are used to identify molecular mechanisms of activity-dependent glial pruning restricted to this short critical period. We propose these glial mechanisms go awry in several newly-linked disease states of intellectual and autism spectrum disorders, including Fragile X syndrome (FXS), Noonan syndrome (NS), LEOPARD syndrome (i.e. NS with Multiple Lentigines; NSML), and Neurobeachin (NBEA) associated autism spectrum disorder (ASD). Our plan is to test the role of glia in experience-dependent circuit pruning in normal and disease states, and to order glial mechanisms of recruitment, infiltration, engulfment and phagocytosis. We employ targeted CRISPR knockout, conditional gene manipulations, and transgenic brain circuit connectivity mapping to dissect neuron-to-glia signaling and glia function in this sensory experience-dependent remodeling. We use timed olfactory cues to activate odorant receptor neurons, downstream projection neurons, and central learning/memory center Kenyon cells, to test glial phagocytosis activity during and following the critical period. In Aim 1, we block glial phagocytic function at multiple levels to test experience-dependent connectivity pruning throughout this defined brain circuitry. We use transgenic single neuron synaptic labeling to visualize glial phagocytosis via transmission electron microscopy. To test downstream activity-dependent mechanisms, we use both excitatory and inhibitory optogenetic tools, as well as transgenic blockage of neurotransmission, in hierarchical circuit studies of synaptic connectivity is sequential brain neuropils. This aim systematically tests glial pruning in normal juvenile brains. In Aim 2, we dissect glia-specific Fragile X Mental Retardation Protein (FMRP) roles in experience-dependent brain circuit pruning. We assay FMRP requirements in glial infiltration phagocytosis using combined light microscopy and ultrastructural imaging. We test the FMRP-dependent neuron-to-glia signaling mechanisms of glial recruitment and phagocytosis during experience-dependent circuit remodeling. This work distinguishes FMRP roles within glia and neurons to understand FXS disease model impairments in critical period brain circuit remodeling. In Aim 3, we test the roles of FMRP translational targets, and consequent regulation of PKA/ERK signaling pathways. We test roles of 1) the direct FMRP mRNA target Rugose/NBEA causative in autism spectrum disorder, which acts as a regulatory PKA anchor, and 2) the direct FMRP mRNA target Corkscrew/SHP2 causative in the two Noonan syndromes of intellectual disability, which is an ERK pathway regulatory phosphatase. We use separation of phases-based activity reporter of kinase (SPARK) biosensors to image experience-dependent PKA/ERK signaling during the early-life critical period. Taken together, this research program dissects gene-environment interactions in normal critical period brain circuit pruning by glial phagocytes, with translational links to new molecular mechanism intersections between Fragile X syndrome, two related Noonan syndromes, and Neurobeachin-associated autism spectrum disorder.