Disruption of TRIO signaling through PDE4A5 in neurodevelopmental disorders

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Project Summary Schizophrenia (SCZ) and autism (ASD) are highly debilitating neurodevelopmental disorders that affect millions of people. An allelic series of heterozygous loss-of-function (LOF) or damaging variants spread throughout the entire TRIO gene are significantly enriched in individuals with SCZ and ASD, suggesting that reduced TRIO function causes pathology. Indeed, our lab has shown that deletion of a single TRIO allele in mouse cortical excitatory neurons (in NEX-TRIO+/– mice) yields diverse behavioral deficits and severe defects in dendritic structure and synaptic activity. TRIO acts downstream of cell surface receptors to control axon and dendrite pathfinding, and synapse development. However, the signaling mechanisms compromised by TRIO variants and how defects in these processes disrupt neuronal development remain completely unclear. Our lab identified PDE4A5 as a new candidate TRIO signaling partner due to its reduced levels in TRIO-deficient mouse cortex and its ability to co-immunoprecipitate with TRIO. My proposal will test the hypothesis that TRIO interacts functionally with PDE4A5 to regulate neuron development and function in two complementary aims. My first aim is to determine how TRIO and PDE4A5 interact functionally and how these functions are impacted by disease-associated variants. PDE4A5 coimmunoprecipitates with TRIO, but how these proteins interact and how these interactions impact each protein’s function is poorly understood. I will use purified recombinant proteins to perform quantitative binding assays and determine whether PDE4A5 and TRIO interact directly and define the minimal TRIO and PDE4A5 fragments sufficient to mediate interaction. I will engineer disorder-associated variants that lie within the TRIO:PDE4A5 interaction interface to determine whether and how these variants impact the Trio:PDE4A5 interaction. I will use my purified proteins and an arsenal of in vitro and cell-based assays to measure how interactions between the TRIO and PDE4A5 impact their catalytic activities. My second aim is to determine how TRIO:PDE4A5 interactions regulate neuronal development. Disruption of a single TRIO allele in cultured cortical neurons yields defects in dendrite and dendritic spine development similar to those observed in vivo. How disruption of TRIO:PDE4A5 interactions contributes to the pathophysiology seen in TRIO deficient neurons is unclear. I will use shRNA-mediated knockdown of PDE4A5 to determine whether PDE4A5 is necessary for dendrite and dendritic spine development. I will perform knockdown/complementation and confocal microscopy in cultured cortical neurons to determine whether and how disruptions in TRIO:PDE4A5 interaction affects PDE4A45 localization and neuron development. I will use a small-molecule PDE4A5 activator to test whether pharmacological manipulation of PDE4A5 can rescue deficits in development of TRIO+/– and TRIO–/– cortical neurons in culture and in NEX-TRIO+/– and NEX-TRIO-/– mice.