Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOM-ER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.

Circular RNAs (circRNAs) are a novel category of covalently-closed non-coding RNAs mainly derived from the back-splicing of exons or introns of protein-coding genes. In addition to their inherent high overall stability, circRNAs, have been shown to have strong functional effects on gene expression via a multitude of transcriptional and post-transcriptional mechanisms. Furthermore, circRNAs, appear to be particularly enriched in the brain and able to influence both prenatal development and postnatal brain function. However, little is known about the potential involvement of circRNAs in the long term influence of prenatal alcohol exposure (PAE) in the brain and their relevance for Fetal Alcohol Spectrum Disorders (FASD). Using circRNA-specific quantification, we have found that circHomer1, an activity-dependent circRNA derived from Homer protein homolog 1 (Homer1) and enriched in postnatal brain, is significantly down-regulated in the male frontal cortex and hippocampus of mice subjected to modest PAE. Our data further suggest that the expression of H19, an imprinted embryonic brain-enriched long non-coding RNA (lncRNA), is significantly up-regulated in the frontal cortex of male PAE mice. Furthermore, we show opposing changes in the developmental- and brain region specific- expression of circHomer1 and H19. Lastly, we show that knockdown of H19 results in robust increases in circHomer1 but not linear HOMER1 mRNA expression in human glioblastoma cell lines. Taken together, our work uncovers notable sex- and brain region-specific alterations in circRNA and lncRNA expression following PAE and introduces novel mechanistic insights with potential relevance to FASD.

Identifying genetic factors that influence electrophysiological signatures associated with disordered behavior will reveal mechanisms of cognitive dysfunction relevant to disease. Homer1 is a reproducible gene candidate for synaptic and cognitive function. Alternative splicing produces activity-dependent isoforms including the circular RNA, circHomer1. While dysregulation of circHomer1 expression has been identified across multiple psychiatric and neurodegenerative disorders and is associated with impaired cognitive flexibility, it is unknown whether circHomer1 can induce electrophysiological signatures relevant to cognitive dysfunction in these disorders. Using in vivo microarray recordings within the orbitofrontal cortex (OFC) of awake, behaving mice during a translational touchscreen task, we demonstrate that reduction of circHomer1 within the OFC induces robust changes in multiunit firing rate and local field potential (LFP) coordination and power to salient stimuli. Further, these electrophysiological changes are associated with transcriptional downregulation of glutamatergic signaling effectors and behavioral alterations leading to impaired cognitive flexibility.

BackgroundCircHomer1 is an activity-dependent circular RNA (circRNA) isoform produced from back-splicing of the Homer1 transcript. Homer1 isoforms are well-known regulators of homeostatic synaptic plasticity through post-synaptic density scaffold regulation. Homer1 polymorphisms have been associated with psychiatric diseases including schizophrenia (SCZ) and bipolar disorder (BD). Postmortem tissue from patients with SCZ and BD showed reduced circHomer1 levels within the orbitofrontal cortex (OFC), a region associated with behavioral flexibility. While dysregulation of circHomer1 expression has recently been identified across multiple psychiatric and neurodegenerative disorders and is associated with impaired behavioral flexibility in mice, it is unknown whether circHomer1 can induce electrophysiological signatures relevant to cognitive dysfunction in these disorders.MethodsCircHomer1 was knocked down in bilateral orbitofrontal cortex of C57BL/6J male mice and in vivo microarray recordings were captured throughout a touchscreen reversal learning task to identify electrophysiological changes associated with reduction of circHomer1. Following task completion, qRT-PCR was used to quantify transcriptional changes following circHomer1 knockdown.ResultsKnockdown of circHomer1 within the OFC induced robust changes in multiunit firing rate and local field potential coordination and power to salient stimuli during reversal learning. Further, these electrophysiological changes were associated with transcriptional downregulation of glutamatergic signaling effectors and behavioral alterations leading to impaired cognitive flexibility.ConclusionsCircHomer1 is a stable biomolecule, whose knockdown in rodent OFC produces lasting electrophysiological and transcriptional changes important for efficient reversal learning. This is, to our knowledge, the first demonstration of a psychiatric-associated circRNA contributing to electrophysiological, transcriptional, and behavioral alterations relevant to psychiatric phenotypes.

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