The role of TRIO signaling in neuronal development, synaptic function, and circuit connectivity

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Abstract Heterozygous loss-of-function (LOF) or damaging variants in the TRIO gene are associated with increased risk for schizophrenia and autism spectrum disorders. However, the functional role of TRIO in neuronal biology and circuit function are not well understood, which limits the advance of therapies for these disorders. TRIO acts downstream of cell surface receptors to control axon and dendrite pathfinding, synapse development, and synaptic transmission. Deletion of a single TRIO allele in mouse cortical excitatory neurons drives reductions in cortical neuropil and defects in dendrite and synapse development and function, yielding social and motor deficits and increased anxiety and compulsivity. However, the links between specific TRIO mutations and subsequent consequences for cortical function are unknown. Here, we will integrate a broad array of highly complementary, interdisciplinary approaches including genetics, biochemistry and proteomics, optogenetic analysis of synaptic function, and multimodal in vivo imaging of cortical network dynamics to address this question. Our first aim will identify the biochemical mechanisms by which TRIO regulates cortical neuron development. We identified several new candidate TRIO signaling partners (PDE4A5, L1CAM, and the LGI1/ADAM22/ADAM23 complex) and will elucidate how they interact with TRIO to regulate cortical neuron dendritic arbor, dendritic spine, and synapse development. We also generated CRISPR mice heterozygous for three disorder-related TRIO variants - K1431M (autism), K1918X (schizophrenia), M2145T (bipolar disorder) - that differentially impact TRIO’s biochemical activities and yield different anatomical and behavioral phenotypes. We will use mass spectrometry-based comparative proteomics to discover new signaling partners differentially impacted by these discrete TRIO alleles. Our second aim will determine how different TRIO variants impact neuronal connectivity and synaptic function. We will assess the consequences of our TRIO CRISPR variants for cortical neuron development by measuring how they impact axon, dendrite, and synapse development, synaptic transmission and plasticity. We will also use viral Cre-mediated sparse TRIO disruption and whole cell recordings to test which deficits reflect cell- autonomous versus network level effects. Our third aim will test how alterations in TRIO impact the functional organization of cortical networks in vivo, taking advantage of our recently developed strategies for combining single cell and mesoscopic imaging of GCaMP6-labeled neurons to measure circuit organization in awake, behaving mice. Our overall goal is to understand how altered TRIO function impacts neuronal function at the cellular, synaptic, and network levels, providing a broad framework for understanding how genetic dysregulation drives changes in behavior.