Systematic analysis of exonic germline and postzygotic de novo mutations in bipolar disorder.

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. To better understand its genetic architecture, we analyze ultra-rare de novo mutations in 354 trios with bipolar disorder. For germline de novo mutations, we find significant enrichment of loss-of-function mutations in constrained genes (corrected-P = 0.0410) and deleterious mutations in presynaptic active zone genes (FDR = 0.0415). An analysis integrating single-cell RNA-sequencing data identifies a subset of excitatory neurons preferentially expressing the genes hit by deleterious mutations, which are also characterized by high expression of developmental disorder genes. In the analysis of postzygotic mutations, we observe significant enrichment of deleterious ones in developmental disorder genes (P = 0.00135), including the SRCAP gene mutated in two unrelated probands. These data collectively indicate the contributions of both germline and postzygotic mutations to the risk of bipolar disorder, supporting the hypothesis that postzygotic mutations of developmental disorder genes may contribute to bipolar disorder.

Therapeutic implications of down-regulation of cyclophilin D in bipolar disorder.

We previously reported that neuron-specific mutant Polg1 (mitochondrial DNA polymerase) transgenic (Tg) mice exhibited bipolar disorder (BD)-like phenotypes such as periodic activity change and altered circadian rhythm. In this study, we re-evaluated two datasets resulting from DNA microarray analysis to estimate a biological pathway associated with the disorder. The gene lists were derived from the comparison between post-mortem brains of BD patients and control subjects, and from the comparison between the brains of Tg and wild-type mice. Gene ontology analysis showed that 16 categories overlapped in the altered gene expression profiles of BD patients and the mouse model. In the brains of Tg mice, 33 genes showed similar changes in the frontal cortex and hippocampus compared to wild-type mice. Among the 33 genes, SFPQ and PPIF were differentially expressed in post-mortem brains of BD patients compared to control subjects. The only gene consistently down-regulated in both patients and the mouse model was PPIF, which encodes cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. A blood-brain barrier-permeable CypD inhibitor significantly improved the abnormal behaviour of Tg mice at 40 mg/kg.d. These findings collectively suggest that CypD is a promising target for a new drug for BD.

Methylation status of the reelin promoter region in the brain of schizophrenic patients.

BACKGROUND: Hypermethylation of the reelin (RELN) promoter region and the reduced levels of its messenger RNA and protein have been implicated in the pathophysiology of schizophrenia. We intended a technical replication of recent studies that observed hypermethylation of CpG or CpNpG sites in the RELN promoter region in the brain of schizophrenic patients. METHODS: The DNA methylation status of the promoter region of RELN was examined by using the pyrosequencing method in the prefrontal cortices of 14 patients with schizophrenia and 13 control subjects. RESULTS: All of the CpG and two proposed CpNpG sites analyzed showed no detectable DNA methylation (< 5%) in both control subjects and patients with schizophrenia. No detectable DNA methylation was observed in both gray and white matter, excluding the possibility of cellular heterogeneity of start materials. CONCLUSIONS: We did not confirm the hypermethylation of the RELN promoter region in the brains of schizophrenic patients, suggested in the previous studies.

Mutation of cGMP phosphodiesterase 6alpha'-subunit gene causes progressive degeneration of cone photoreceptors in zebrafish.

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.

The hedgehog-PKA pathway regulates two distinct steps of the differentiation of retinal ganglion cells: the cell-cycle exit of retinoblasts and their neuronal maturation.

In the developing zebrafish retina, neurogenesis is initiated in cells adjacent to the optic stalk and progresses to the entire neural retina. It has been reported that hedgehog (Hh) signalling mediates the progression of the differentiation of retinal ganglion cells (RGCs) in zebrafish. However, the progression of neurogenesis seems to be only mildly delayed by genetic or chemical blockade of the Hh signalling pathway. Here, we show that cAMP-dependent protein kinase (PKA) effectively inhibits the progression of retinal neurogenesis in zebrafish. Almost all retinal cells continue to proliferate when PKA is activated, suggesting that PKA inhibits the cell-cycle exit of retinoblasts. A cyclin-dependent kinase (cdk) inhibitor p27 inhibits the PKA-induced proliferation, suggesting that PKA functions upstream of cyclins and cdk inhibitors. Activation of the Wnt signalling pathway induces the hyperproliferation of retinal cells in zebrafish. The blockade of Wnt signalling inhibits the PKA-induced proliferation, but the activation of Wnt signalling promotes proliferation even in the absence of PKA activity. These observations suggest that PKA inhibits exit from the Wnt-mediated cell cycle rather than stimulates Wnt-mediated cell-cycle progression. PKA is an inhibitor of Hh signalling, and Hh signalling molecule morphants show severe defects in cell-cycle exit of retinoblasts. Together, these data suggest that Hh acts as a short-range signal to induce the cell-cycle exit of retinoblasts. The pulse inhibition of Hh signalling revealed that Hh signalling regulates at least two distinct steps of RGC differentiation: the cell-cycle exit of retinoblasts and RGC maturation. This dual requirement of Hh signalling in RGC differentiation implies that the regulation of a neurogenic wave is more complex in the zebrafish retina than in the Drosophila eye.

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways.

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.

N-cadherin mediates retinal lamination, maintenance of forebrain compartments and patterning of retinal neurites.

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.