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Identification of risk variants for neuropsychiatric diseases within enhancers underscores the importance of understanding population-level variation in enhancer function in the human brain. Besides regulating tissue-specific and cell-type-specific transcription of target genes, enhancers themselves can be transcribed. By jointly analyzing large-scale cell-type-specific transcriptome and regulome data, we cataloged 30,795 neuronal and 23,265 non-neuronal candidate transcribed enhancers. Examination of the transcriptome in 1,382 brain samples identified robust expression of transcribed enhancers. We explored gene-enhancer coordination and found that enhancer-linked genes are strongly implicated in neuropsychiatric disease. We identified expression quantitative trait loci (eQTLs) for both genes and enhancers and found that enhancer eQTLs mediate a substantial fraction of neuropsychiatric trait heritability. Inclusion of enhancer eQTLs in transcriptome-wide association studies enhanced functional interpretation of disease loci. Overall, our study characterizes the gene-enhancer regulome and genetic mechanisms in the human cortex in both healthy and diseased states.

Bipolar disorder (BD) is a neuropsychiatric illness defined by recurrent episodes of mania/hypomania, depression and circadian rhythm abnormalities. Lithium is an effective drug for BD, but 30-40% of patients fail to respond adequately to treatment. Previous work has demonstrated that lithium affects the expression of "clock genes" and that lithium responders (Li-R) can be distinguished from non-responders (Li-NR) by differences in circadian rhythms. However, circadian rhythms have not been evaluated in BD patient neurons from Li-R and Li-NR. We used induced pluripotent stem cells (iPSCs) to culture neuronal precursor cells (NPC) and glutamatergic neurons from BD patients characterized for lithium responsiveness and matched controls. We identified strong circadian rhythms in Per2-luc expression in NPCs and neurons from controls and Li-R, but NPC rhythms in Li-R had a shorter circadian period. Li-NR rhythms were low amplitude and profoundly weakened. In NPCs and neurons, expression of PER2 was higher in both BD groups compared to controls. In neurons, PER2 protein levels were higher in BD than controls, especially in Li-NR samples. In single cells, NPC and neuron rhythms in both BD groups were desynchronized compared to controls. Lithium lengthened period in Li-R and control neurons but failed to alter rhythms in Li-NR. In contrast, temperature entrainment increased amplitude across all groups, and partly restored rhythms in Li-NR neurons. We conclude that neuronal circadian rhythm abnormalities are present in BD and most pronounced in Li-NR. Rhythm deficits in BD may be partly reversible through stimulation of entrainment pathways.

De novo, heterozygous, loss-of-function variants were identified in Pou domain, class 4, transcription factor 1 (POU4F1) via whole-exome sequencing in four independent probands presenting with ataxia, intention tremor, and hypotonia. POU4F1 is expressed in the developing nervous system, and mice homozygous for null alleles of Pou4f1 exhibit uncoordinated movements with newborns being unable to successfully right themselves to feed. Head magnetic resonance imaging of the four probands was reviewed and multiple abnormalities were noted, including significant cerebellar vermian atrophy and hypertrophic olivary degeneration in one proband. Transcriptional activation of the POU4F1 p.Gln306Arg protein was noted to be decreased when compared with wild type. These findings suggest that heterozygous, loss-of-function variants in POU4F1 are causative of a novel ataxia syndrome.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Figure S1. Correlations between GIA1 and individual clinic traits stratified by AOD (age of death), gender and APOE genotypes in BM36 region. Figure S2. Correlation between Gja1 and individual transcripts of ADGWAS genes. Figure S3. GJA1-centric Bayesian causal network and GJA1 signaling pathway map. Figure S4. Enrichment of Gia1-/- gene signatures in GJA1 centric correlation networks. Figure S5. Regulation of A1/A2 astrocyte marker genes in Gja1-/- astrocytes. Figure S6. Quinine (GJA1 channel agonist) upregulates Gja1 and Apoe, and other network genes. Figure S7. A. Wildtype and Gja1-/- astrocytes were treated with fluorescently labeled Aβ1-42 oligomers for 24 hours and the number of astrocytes in association with Aβ1-42 oligomers were quantitatively estimated by counting total cells (DAPI+) and Aβ positive cells. B. Representative images of phase contrast, Hilyte Fluor488-labeled Aβ, and DAPI staining from wildtype (upper panels) and Gja1-/- (lower panels) astrocytes were shown. These images were taken by bright field microscope and the representatives of 2 independent experiments with similar results are shown. Figure S8. Complex regulation of Gja1 key drivers in neuron/astrocyte/microglia cocultures by carbenoxolone and quinine. (DOCX 1.4 kb)

Figure S1. Correlations between GIA1 and individual clinic traits stratified by AOD (age of death), gender and APOE genotypes in BM36 region. Figure S2. Correlation between Gja1 and individual transcripts of ADGWAS genes. Figure S3. GJA1-centric Bayesian causal network and GJA1 signaling pathway map. Figure S4. Enrichment of Gia1-/- gene signatures in GJA1 centric correlation networks. Figure S5. Regulation of A1/A2 astrocyte marker genes in Gja1-/- astrocytes. Figure S6. Quinine (GJA1 channel agonist) upregulates Gja1 and Apoe, and other network genes. Figure S7. A. Wildtype and Gja1-/- astrocytes were treated with fluorescently labeled Aβ1-42 oligomers for 24 hours and the number of astrocytes in association with Aβ1-42 oligomers were quantitatively estimated by counting total cells (DAPI+) and Aβ positive cells. B. Representative images of phase contrast, Hilyte Fluor488-labeled Aβ, and DAPI staining from wildtype (upper panels) and Gja1-/- (lower panels) astrocytes were shown. These images were taken by bright field microscope and the representatives of 2 independent experiments with similar results are shown. Figure S8. Complex regulation of Gja1 key drivers in neuron/astrocyte/microglia cocultures by carbenoxolone and quinine. (DOCX 1.4 kb)