Masako Shibuya

The cellular and behavioral consequences of interleukin-1 alpha penetration through the blood–brain barrier of neonatal rats: A critical period for efficacy

Proinflammatory cytokines circulating in the periphery of early postnatal animals exert marked influences on their subsequent cognitive and behavioral traits and are therefore implicated in developmental psychiatric diseases such as schizophrenia. Here we examined the relationship between the permeability of the blood-brain barrier to interleukin-1 alpha (IL-1 alpha) in neonatal and juvenile rats and their later behavioral performance. Following s.c. injection of IL-1 alpha into rat neonates, IL-1 alpha immunoreactivity was first detected in the choroid plexus, brain microvessels, and olfactory cortex, and later diffused to many brain regions such as neocortex and hippocampus. In agreement, IL-1 alpha administration to the periphery resulted in a marked increase in brain IL-1 alpha content of neonates. Repeatedly injecting IL-1 alpha to neonates triggered astrocyte proliferation and microglial activation, followed by behavioral abnormalities in startle response and putative prepulse inhibition at the adult stage. Analysis of covariance with a covariate of startle amplitude suggested that IL-1 alpha administration may influence prepulse inhibition. However, adult rats treated with IL-1 alpha as neonates exhibited normal learning ability as measured by contextual fear conditioning, two-way passive shock avoidance, and a radial maze task and had no apparent sign of structural abnormality in the brain. In comparison, when IL-1 alpha was administered to juveniles, the blood-brain barrier permeation was limited. The increases in brain IL-1 alpha content and immunoreactivity were less pronounced following IL-1 alpha administration and behavioral abnormalities were not manifested at the adult stage. During early development, therefore, circulating IL-1 alpha efficiently crosses the blood-brain barrier to induce inflammatory reactions in the brain and influences later behavioral traits.

Assessment of copy number variations in the brain genome of schizophrenia patients.

BackgroundCytogenomic mutations and chromosomal abnormality are implicated in the neuropathology of several brain diseases. Cell heterogeneity of brain tissues makes their detection and validation difficult, however. In the present study, we analyzed gene dosage alterations in brain DNA of schizophrenia patients and compared those with the copy number variations (CNVs) identified in schizophrenia patients as well as with those in Asian lymphocyte DNA and attempted to obtain hints at the pathological contribution of cytogenomic instability to schizophrenia.ResultsBrain DNA was extracted from postmortem striatum of schizophrenia patients and control subjects (n = 48 each) and subjected to the direct two color microarray analysis that limits technical data variations. Disease-associated biases of relative DNA doses were statistically analyzed with Bonferroni’s compensation on the premise of brain cell mosaicism. We found that the relative gene dosage of 85 regions significantly varied among a million of probe sites. In the candidate CNV regions, 26 regions had no overlaps with the common CNVs found in Asian populations and included the genes (i.e., ANTXRL, CHST9, DNM3, NDST3, SDK1, STRC, SKY) that are associated with schizophrenia and/or other psychiatric diseases. The majority of these candidate CNVs exhibited high statistical probabilities but their signal differences in gene dosage were less than 1.5-fold. For test evaluation, we rather selected the 10 candidate CNV regions that exhibited higher aberration scores or larger global effects and were thus confirmable by PCR. Quantitative PCR verified the loss of gene dosage at two loci (1p36.21 and 1p13.3) and confirmed the global variation of the copy number distributions at two loci (11p15.4 and 13q21.1), both indicating the utility of the present strategy. These test loci, however, exhibited the same somatic CNV patterns in the other brain region.ConclusionsThe present study lists the candidate regions potentially representing cytogenomic CNVs in the brain of schizophrenia patients, although the significant but modest alterations in their brain genome doses largely remain to be characterized further.

ErbB inhibitors ameliorate behavioral impairments of an animal model for schizophrenia: implication of their dopamine-modulatory actions

Ligands for ErbB receptors, including epidermal growth factor (EGF) and neuregulin-1, have a neurotrophic activity on midbrain dopaminergic neurons and are implicated in the pathophysiology of schizophrenia. Although ErbB kinase inhibitors ameliorate behavioral deficits of the schizophrenia model that was established by hippocampal lesioning of rat pups, the antipsychotic action of ErbB kinase inhibitors and its general applicability to other models are not fully characterized. Using a different animal model, here, we examined whether and how ErbB kinase inhibitors ameliorate the behavioral endophenotypes relevant to schizophrenia. The animal model for schizophrenia was prepared by exposing neonatal rats to the cytokine EGF. Intraventricular infusion of the ErbB1 inhibitors ZD1839 and PD153035 in these animals ameliorated the deficits in startle response and prepulse inhibition in a dose-dependent manner. The deficits of latent inhibition of fear learning were also alleviated by ZD1839 with its limited effects on body weight gain or locomotor activity. ZD1839 infusion also decreased the busting activity of nigral dopamine (DA) neurons and reduced pallidal DA metabolism, a result that mimics the anti-dopaminergic profile of risperidone and haloperidol in this brain region. ErbB inhibitors appear to have anti-dopaminergic actions to alleviate some of the behavioral deficits common to animal models for schizophrenia.

Association study of interleukin 2 (IL2) and IL4 with schizophrenia in a Japanese population

Interleukin 2 (IL-2) and IL-4 are pleiotropic cytokines regulating Th1/Th2 balance and have a regulatory activity in brain function. Thus these cytokines have been implicated in the pathophysiology of schizophrenia. The latest studies provided controversial results regarding the genetic associations of these cytokines. The functional polymorphisms, IL2-330T/G and IL4-590C/T, were associated with schizophrenia in a German population, although contradictory findings were also reported in a Korean population. To ascertain whether IL2 and IL4 contribute to vulnerability to schizophrenia, we conducted a moderate-scale case-control (536 patients and 510 controls) association study for seven polymorphisms in Japanese subjects. There were no significant associations of these genes with schizophrenia using either single marker or haplotype analyses. The present study suggests that IL2 and IL4 do not contribute to vulnerability to schizophrenia in the Japanese population.

Interleukin 1 beta gene and risk of schizophrenia: detailed case-control and family-based studies and an updated meta-analysis.

Interleukin‐1 beta (IL‐1β) has been implicated in the pathophysiology of schizophrenia. To assess whether the IL1B gene confers increased susceptibility to schizophrenia, we conducted case–control and family‐based studies and an updated meta‐analysis.

Oxytocin receptor (OXTR) gene and risk of schizophrenia: Case–control and family-based analyses and meta-analysis in a Japanese population

OXYTOCIN (OXT), SIGNALING through its receptor (OXTR), has been implicated in the pathophysiology of schizophrenia. Thus, OXTR is a potential candidate gene for schizophrenia. The association between OXTR and schizophrenia has been tested in only one study reporting negative results using Caucasian case–control and trio samples (n = 358 and 34, respectively). To assess whether OXTR confers increased susceptibility to schizophrenia, case–control and familybased analyses and a meta-analysis were performed using Japanese case–control and trio samples (n = 1218 and 105, respectively). The present study was approved by the Ethics Committee on Genetics of the Niigata University School of Medicine, and written informed consent was obtained from all participants. The case–control subjects consisted of 544 patients with schizophrenia (290 men and 254 women; mean age, 41.8 13.6 years) and 674 mentally healthy individuals (341 men and 333 women; mean age, 38.4 10.8 years). The family-based subjects consisted of 105 trios, made up of patients (59 men and 46 women; mean age, 28.8 9.2 years) and both parents. A psychiatric assessment of every participant was conducted as previously described. We selected 17 tagging single nucleotide polymorphisms (SNP) for OXTR (chr3:8767079.8788481) from the HapMap database (http://hapmap.ncbi.nlm.nih.gov/). All SNP were genotyped using the TaqMan 5’-exonuclease assay. Deviations from Hardy–Weinberg equilibrium (HWE) and allelic associations were tested using Haploview v4.2 (http://www.broad institute.org/scientific-community/science/programs/medicaland-population-genetics/haploview/haploview). Fixed effect model meta-analysis was performed using catmap (http:// cran.r-project.org/src/contrib/Archive/catmap/). A power calculation was performed using the Genetic Power Calculator (http://pngu.mgh.harvard.edu/~purcell/gpc/). In the case–control sample, the genotypes of rs2139184 could not be determined, and HWE deviations were observed in rs11131149 in both patients and controls and in rs237889 in controls (Table S1). These three SNP were excluded from further analysis. There were no significant associations between any of the 14 SNP examined and schizophrenia in both the case–control and trio samples (Table S2). A metaanalysis using both samples indicated a nominally significant association between rs9840864 and schizophrenia (odds ratio, 1.21; 95% confidence interval: 1.03–1.41, P = 0.0176). The statistical power of the case–control and of the trio samples was 0.56 and 0.14, respectively, assuming a disease prevalence of 0.01, a risk allele frequency of 0.3 and a genotypic relative risk of 1.2 for heterozygous risk allele carriers under the multiplicative model of inheritance. Although rs9840864 was tentatively associated with schizophrenia, the present study did not provide supportive evidence for the contribution of OXTR to susceptibility to schizophrenia. To draw a definitive conclusion, further studies using larger sample sizes should be carried out in a range of ethnic populations.

Rare UNC13B variations and risk of schizophrenia: Whole-exome sequencing in a multiplex family and follow-up resequencing and a case-control study.

Rare genomic variations inherited in multiplex schizophrenia families are suggested to play a role in the genetic etiology of the disease. To identify rare variations with large effects on the risk of developing schizophrenia, we performed whole‐exome sequencing (WES) in two affected and one unaffected individual of a multiplex family with 10 affected individuals. We also performed follow‐up resequencing of the unc‐13 homolog B (Caenorhabditis elegans) (UNC13B) gene, a potential risk gene identified by WES, in the multiplex family and undertook a case–control study to investigate association between UNC13B and schizophrenia. UNC13B coding regions (39 exons) from 15 individuals of the multiplex family and 111 affected offspring for whom parental DNA samples were available were resequenced. Rare missense UNC13B variations identified by resequencing were further tested for association with schizophrenia in two independent case–control populations comprising a total of 1,753 patients and 1,602 controls. A rare missense variation (V1525M) in UNC13B was identified by WES in the multiplex family; this variation was present in five of six affected individuals, but not in eight unaffected individuals or one individual of unknown disease status. Resequencing UNC13B coding regions identified five rare missense variations (T103M, M813T, P1349T, I1362T, and V1525M). In the case–control study, there was no significant association between rare missense UNC13B variations and schizophrenia, although single‐variant meta‐analysis indicated that M813T was nominally associated with schizophrenia. These results do not support a contribution of rare missense UNC13B variations to the genetic etiology of schizophrenia in the Japanese population.

Rare heterozygous truncating variations and risk of autism spectrum disorder: Whole-exome sequencing of a multiplex family and follow-up study in a Japanese population

Rare heterozygous truncating variations in multiplex families with autism spectrum disorder (ASD) are suggested to play a major role in the genetic etiology of ASD. To further investigate the role of rare heterozygous truncating variations, we performed whole‐exome sequencing (WES) in a multiplex ASD family with four affected individuals (two siblings and two maternal cousins), and a follow‐up case–control study in a Japanese population.

Association analysis of putative cis-acting polymorphisms of interleukin-19 gene with schizophrenia

BACKGROUND Genome-wide association studies (GWAS) and gene expression analyses have revealed that single nucleotide polymorphisms (SNPs) associated with multifactorial diseases, such as schizophrenia, are significantly more likely to be associated with expression quantitative trait loci (eQTL). It was recently suggested that an immune system imbalance plays an important role in the pathogenesis of schizophrenia. Interleukin-19 is a novel cytokine that may play multiple roles in immune regulation and various diseases. METHOD We selected eight tag SNPs in the eQTL of the IL-19 gene. Seven of the SNPs are putative cis-acting SNPs. Then, we conducted a case-control study using two independent samples. The first sample comprised 567 schizophrenia patients and 710 controls, and the second sample comprised 677 schizophrenia patients and 667 controls. RESULT We identified the TGAA haplotype as being significantly associated with schizophrenia (p=0.0036 and corrected p=0.0264), although a combined analysis of the TGAA haplotype with the replication samples exhibited a nominally significant difference (p=0.022 and corrected p=0.235). CONCLUSIONS These results suggest that the IL-19 gene might slightly contribute to the genetic risk of schizophrenia. Thus, further research on the association of eQTL SNPs with schizophrenia is warranted.

Novel Rare Missense Variations and Risk of Autism Spectrum Disorder: Whole-Exome Sequencing in Two Families with Affected Siblings and a Two-Stage Follow-Up Study in a Japanese Population

Rare inherited variations in multiplex families with autism spectrum disorder (ASD) are suggested to play a major role in the genetic etiology of ASD. To further investigate the role of rare inherited variations, we performed whole-exome sequencing (WES) in two families, each with three affected siblings. We also performed a two-stage follow-up case-control study in a Japanese population. WES of the six affected siblings identified six novel rare missense variations. Among these variations, CLN8 R24H was inherited in one family by three affected siblings from an affected father and thus co-segregated with ASD. In the first stage of the follow-up study, we genotyped the six novel rare missense variations identified by WES in 241 patients and 667 controls (the Niigata sample). Only CLN8 R24H had higher mutant allele frequencies in patients (1/482) compared with controls (1/1334). In the second stage, this variation was further genotyped, yet was not detected in a sample of 309 patients and 350 controls (the Nagoya sample). In the combined Niigata and Nagoya samples, there was no significant association (odds ratio = 1.8, 95% confidence interval = 0.1–29.6). These results suggest that CLN8 R24H plays a role in the genetic etiology of ASD, at least in a subset of ASD patients.