Mizuho Ishiwata

Impaired feedback regulation of XBP1 as a genetic risk factor for bipolar disorder.

The pathophysiology of bipolar disorder is still unclear, although family, twin and linkage studies implicate genetic factors. Here we identified XBP1, a pivotal gene in the endoplasmic reticulum (ER) stress response, as contributing to the genetic risk factor for bipolar disorder. Using DNA microarray analysis of lymphoblastoid cells derived from two pairs of twins discordant with respect to the illness, we found downregulated expression of genes related to ER stress response in both affected twins. A polymorphism (−116C→G) in the promoter region of XBP1, affecting the putative binding site of XBP1, was significantly more common in Japanese patients (odds ratio = 4.6) and overtransmitted to affected offspring in trio samples of the NIMH Bipolar Disorder Genetics Initiative. XBP1-dependent transcription activity of the −116G allele was lower than that of the −116C allele, and in the cells with the G allele, induction of XBP1 expression after ER stress was markedly reduced. Valproate, one of three mood stabilizers, rescued the impaired response by inducing ATF6, the gene upstream of XBP1. These results indicate that the −116C→G polymorphism in XBP1 causes an impairment of its positive feedback system and increases the risk of bipolar disorder.

The role of brain-derived neurotrophic factor (BDNF)-induced XBP1 splicing during brain development.

Accumulation of unfolded proteins in the endoplasmic reticulum initiates intracellular signaling termed the unfolded protein response (UPR). Although Xbp1 serves as a pivotal transcription factor for the UPR, the physiological role of UPR signaling and Xbp1 in the central nervous system remains to be elucidated. Here, we show that Xbp1 mRNA was highly expressed during neurodevelopment and activated Xbp1 protein was distributed throughout developing neurons, including neurites. The isolated neurite culture system and time-lapse imaging demonstrated that Xbp1 was activated in neurites in response to brain-derived neurotrophic factor (BDNF), followed by subsequent translocation of the active Xbp1 into the nucleus. BDNF-dependent neurite outgrowth was significantly attenuated in Xbp1-/- neurons. These findings suggest that BDNF initiates UPR signaling in neurites and that Xbp1, which is activated as part of the UPR, conveys the local information from neurites to the nucleus, contributing the neurite outgrowth.

Mechanisms of altered Ca2+ signalling in transformed lymphoblastoid cells from patients with bipolar disorder.

Altered Ca2+ signalling has been reported in the platelets and lymphoblastoid cells of patients with bipolar disorder. Recent genetic studies have suggested possible pathophysiological roles for mitochondria and endoplasmic reticulum, both of which are essential for the regulation of intracellular Ca2+ signalling. The goal of this study was to determine molecular mechanisms of altered intracellular Ca2+ signalling in bipolar disorder. Lymphoblastoid cell lines were established from patients with bipolar I disorder (n=13) and controls (n=11). Using Ca2+ indicators, cytosolic and mitochondrial Ca2+ responses to the following three reagents were examined: platelet-activating factor; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler that abolishes mitochondrial Ca2+ uptake; and thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. The 10-5 M thapsigargin-induced cytosolic Ca2+ response was significantly higher in patients with bipolar disorder (p&0.05). Such difference was not seen when the effects of Ca2+ influx from outside the plasma membrane was eliminated using Ca2+-free measurement buffer. On the other hand, response to 10-7 M thapsigargin tended to be higher in patients with bipolar disorder when at the Ca2+-free conditions. CCCP-induced Ca2+ responses differed significantly between mitochondrial DNA 5178/10398 haplotypes (p=0.001) that had been previously reported to be associated with bipolar disorder. These results suggest that all components, i.e. the store-operated calcium channel (SOCC), endoplasmic reticulum, and mitochondria, somehow contribute to the altered Ca2+ signalling in bipolar disorder.

XBP1 induces WFS1 through an endoplasmic reticulum stress response element-like motif in SH-SY5Y cells

XBP1 is a key transcription factor in the endoplasmic reticulum (ER) stress response pathway. In a previous study, we suggested a possible link between XBP1 and bipolar disorder, but its role in neuronal cells has not yet been clarified. Here we examined the target genes of XBP1, using DNA microarray analysis in SH‐SY5Y cells transfected with an XBP1‐expressing vector. Among the genes up‐regulated by XBP1, the most significant p‐value was observed for WFS1, which is an ER stress response‐related gene. Examining the promoter region of WFS1, we found a conserved sequence (CGAGGCGCACCGTGATTGG) that is highly similar to the ER stress response element (ERSE). A promoter assay showed that this ERSE‐like motif is critical for the regulation of WFS1 by XBP1. An electrophoretic mobility shift assay suggested that XBP1 does not directly bind to this sequence. Our results demonstrate that WFS1 is one of the target genes of XBP1 in SH‐SY5Y cells.

Association of the XBP1-116C/G polymorphism with schizophrenia in the Japanese population.

Abstract  Schizophrenia and bipolar disorder share some clinical features and linkage studies have shown that several loci are common. Recently, the authors found that the −116C→G substitution in the promotor region of XBP1, a pivotal gene in endoplasmic reticulum (ER) stress response, causes the impairment of ER stress response, and that the −116C/C genotype is a protective factor; in other words the presence of the G allele increases the risk for bipolar disorder. The gene is located on 22q12.1, which is also linked with schizophrenia. The polymorphisms were investigated in 234 schizophrenic patients as compared with controls. Significant difference of genotype distribution was observed, which suggested that the −116C/C genotype is a protective factor for both of the major mental disorders.

Behavioral and gene expression analyses of Wfs1 knockout mice as a possible animal model of mood disorder

Wolfram disease is a rare genetic disorder frequently accompanying depression and psychosis. Non-symptomatic mutation carriers also have higher rates of depression and suicide. Because WfS1, the causative gene of Wolfram disease, is located at 4p16, a linkage locus for bipolar disorder, mutations of WfS1 were suggested to be involved in the pathophysiology of bipolar disorder. In this study, we performed behavioral and gene expression analyses of Wfs1 knockout mice to assess the validity as an animal model of mood disorder. In addition, the distribution of Wfs1 protein was examined in mouse brain. Wfs1 knockout mice did not show abnormalities in circadian rhythm and periodic fluctuation of wheel-running activity. Behavioral analysis showed that Wfs1 knockout mice had retardation in emotionally triggered behavior, decreased social interaction, and altered behavioral despair depending on experimental conditions. Wfs1-like immunoreactivity in mouse brain showed a similar distribution pattern to that in rats, including several nuclei potentially relevant to the symptoms of mood disorders. Gene expression analysis showed down-regulation of Cdc42ep5 and Rnd1, both of which are related to Rho GTPase, which plays a role in dendrite development. These findings may be relevant to the mood disorder observed in patients with Wolfram disease.

Abnormal Ca2+ Dynamics in Transgenic Mice with Neuron-Specific Mitochondrial DNA Defects

Maintenance of mitochondrial DNA (mtDNA) depends on nuclear-encoded proteins such as mtDNA polymerase (POLG), whose mutations are involved in the diseases caused by mtDNA defects including mutation and deletion. The defects in mtDNA and in intracellular Ca2+ ([Ca2+]i) homeostasis have been reported in bipolar disorder (BD). To understand the relevance of the mtDNA defects to BD, we studied transgenic (Tg) mice in which mutant POLG (mutPOLG) was expressed specifically in neurons. mtDNA defects were accumulated in the brains of mutPOLG Tg mice in an age-dependent manner and the mutant mice showed BD-like behavior. However, the molecular and cellular basis for the abnormalities has not been clarified. In this study, we investigated Ca2+ regulation by isolated mitochondria and [Ca2+]i dynamics in the neurons of mutPOLG Tg mice. Mitochondria from the mutant mice sequestered Ca2+ more rapidly, whereas Ca2+ retention capacity and membrane potential, a driving force of Ca2+ uptake, of mitochondria were unaffected. To elucidate the molecular mechanism of the altered Ca2+ uptake, we performed DNA microarray analysis and found that the expression of cyclophilin D (CyP-D), a component of the permeability transition pore, was downregulated in the brains of mutPOLG Tg mice. Cyclosporin A, an inhibitor of CyP-D, mimicked the enhanced Ca2+ uptake in mutant mice. Furthermore, G-protein-coupled receptor-mediated [Ca2+]i increase was attenuated in hippocampal neurons of the mutant mice. These findings suggest that mtDNA defects lead to enhancement of Ca2+ uptake rate via CyP-D downregulation and alter [Ca2+]i dynamics, which may be involved in the pathogenesis of BD.

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.

Exome sequencing for bipolar disorder points to roles of de novo loss-of-function and protein-altering mutations

Although numerous genetic studies have been conducted for bipolar disorder (BD), its genetic architecture remains elusive. Here we perform, to the best of our knowledge, the first trio-based exome sequencing study for BD to investigate potential roles of de novo mutations in the disease etiology. We identified 71 de novo point mutations and one de novo copy-number mutation in 79 BD probands. Among the genes hit by de novo loss-of-function (LOF; nonsense, splice site or frameshift) or protein-altering (LOF, missense and inframe indel) mutations, we found significant enrichment of genes highly intolerant (first percentile of intolerant genes assessed by Residual Variation Intolerance Score) to protein-altering variants in general population, an observation that is also reported in autism and schizophrenia. When we performed a joint analysis using the data of schizoaffective disorder in published studies, we found global enrichment of de novo LOF and protein-altering mutations in the combined group of bipolar I and schizoaffective disorders. Considering relationship between de novo mutations and clinical phenotypes, we observed significantly earlier disease onset among the BD probands with de novo protein-altering mutations when compared with non-carriers. Gene ontology enrichment analysis of genes hit by de novo protein-altering mutations in bipolar I and schizoaffective disorders did not identify any significant enrichment. These results of exploratory analyses collectively point to the roles of de novo LOF and protein-altering mutations in the etiology of bipolar disorder and warrant further large-scale studies.

Association analysis of HSP90B1 with bipolar disorder

AbstractPathophysiological role of endoplasmic reticulum (ER) stress response signaling has been suggested for bipolar disorder. The goal of this study was to test the genetic association between bipolar disorder and an ER chaperone gene, HSP90B1 (GRP94/gp96), which is located on a candidate locus, 12q23.3. We tested the genetic association between bipolar disorder and HSP90B1 by case-control studies in two independent Japanese sample sets and by a transmission disequilibrium test (TDT) in NIMH Genetics initiative bipolar trio samples (NIMH trios). We also performed gene expression analysis of HSP90B1 in lymphoblastoid cells. Among the 11 SNPs tested, rs17034977 showed significant association in both Japanese sample sets. The frequency of the SNP was lower in NIMH samples than in Japanese samples and there was no significant association in NIMH trios. Gene expression analysis of HSP90B1 in lymphoblastoid cells suggested a possible relationship between the associated SNP and mRNA levels. HSP90B1 may have a pathophysiological role in bipolar disorder in the Japanese population, though further study will be needed to understand the underlying functional mechanisms.