PROJECT SUMMARY Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Following unbiased circRNA profiling and validation in multiple postmortem brain cohorts, we have uncovered that circHomer1, a neuronal-enriched circRNA abundantly expressed in the adult frontal cortex, is significantly reduced in the prefrontal cortex of subjects with psychiatric disease. Our preliminary data suggest that circHomer1 can inhibit the synaptic localization of the long Homer1b mRNA isoform within the orbitofrontal cortex (OFC), by competing for binding to the RNA-binding protein (RBP) HuD, a known regulator of neuronal mRNA trafficking. Moreover, our pilot data suggest that circHomer1 can influence glutamatergic synaptic transmission and that restoring Homer1b levels in the OFC can rescue the alterations in cognitive flexibility observed following circHomer1 knockdown (KD). We propose to determine the mechanisms by which circHomer1 regulates the synaptic localization of Homer1b (Aim 1), to examine the role of Homer1b in circHomer1-mediated alterations in neuronal function and cognitive flexibility (Aim 2), and to identify novel upstream regulators of human neuronal circHomer1 biogenesis (Aim3). Our central hypothesis is that RBPs bind near the circHomer1 splice junction to promote its biogenesis within neurons and that circHomer1 can sequester HuD from binding to Homer1b, thereby inhibiting Homer1b synaptic localization and disrupting neuronal activity and OFC function. The rationale of the proposed research is that elucidating the unexplored role of synaptic plasticity-regulating circRNAs will uncover novel molecular mechanistic insights into the nature of neuronal function disturbances related to psychiatric disease. For this proposal we first intend to generate circHomer1 knockout (KO) mice via CRISPR-mediated deletion of antisense intronic regions required for circHomer1 backsplicing and examine the impact on synaptic Homer1b expression, as well as determine whether competition for HuD binding between circHomer1 and Homer1b is responsible for the increased Homer1b synaptic localization as a result of loss of circHomer1 (Aim 1). We will then examine the importance of altered Homer1b levels for neuronal activity and OFC function (Aim 2). Finally, using a novel circRNA sensor approach together with genome editing in mouse cortical neurons and iPSC- derived neuronal cultures we will uncover upstream regulators of neuronal circHomer1 biogenesis (Aim 3). The following three specific aims will attempt to address our hypotheses: 1) Test the hypothesis that circHomer1 inhibits Homer1b mRNA synaptic localization via competing for binding to HuD within the OFC. 2) Test the hypothesis that Homer1b OE is sufficient to impair OFC function and that normalizing its expression following circHomer1 loss can restore neuronal activity and OFC-mediated cognitive flexi...