Mizuki Hino

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.

High-Speed and Scalable Whole-Brain Imaging in Rodents and Primates

Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of light-sheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.

Decreased VEGFR2 expression and increased phosphorylated Akt1 in the prefrontal cortex of individuals with schizophrenia

The Akt signaling pathway involves various cellular processes and depends on extracellular stimuli. Since Akt signaling participates in cytoprotection, synapse plasticity, axon extension, and neurotransmission in the nervous system, alteration in Akt signaling might be a potential cause of schizophrenia. In this study, we performed multiplex fluorescent bead based immunoassays for members of the Akt signaling pathway in postmortem brains of controls and patients with schizophrenia. Vascular endothelial growth factor receptor 2 (VEGFR2/KDR) was significantly decreased in the prefrontal cortex (PFC) of patients with schizophrenia, and the expression level of VEGFR2 was inversely correlated with the positive symptom subscale of the Diagnostic Instrument for Brain Studies (DIBS) in patients with schizophrenia. There was also an increase in phosphorylated Akt1 in the PFC in the patients, though the ratio of phospho/total Akt1 is not significantly different. In the nucleus accumbens (NAcc) there was no significant difference in expression and phosphorylation levels of Akt signaling proteins. Genetic analysis revealed a significant correlation of a SNP of KDR (rs7692791) with ERK1/2 and Akt1 phospho/total rates. Since VEGFR2 participates in angiogenesis and neurotrophic activation, either or both functions might be responsible for onset of schizophrenia.

Increased ratio of calcineurin immunoreactive neurons in the caudate nucleus of patients with schizophrenia.

Calcineurin (CaN) has been investigated extensively in numerous biochemical, behavioral, and genetic studies in schizophrenia because its function is closely related to dopamine-glutamate signal transduction, which is thought to be associated with pathophysiological changes in schizophrenia. Although evidence has suggested that dysfunction of CaN may be a risk factor for schizophrenia, there have been few reports focusing on the expression of CaN mRNA and CaN protein levels in the brains of schizophrenic patients. In addition, findings on CaN expression in postmortem brains from patients with schizophrenia have been inconsistent. Here, we conducted immunohistochemical examinations of several regions in postmortem brains, including the dorsolateral prefrontal cortex (DLPFC), hippocampus, caudate nucleus, and putamen, using specific antibodies, and compared the results from the brains of nine schizophrenic subjects to nine age- and sex-matched control subjects. There was no significant difference in the ratio of CaN immunoreactive (IR) neurons between schizophrenia and control groups in the DLPFC or hippocampus, and a significantly increased ratio of CaN-IR neurons was seen in the caudate nucleus in the brains from schizophrenia patients. As the striatum contains most of the brain dopamine, the results of the present study have critical implications and suggest that alterations in CaN signaling in the caudate contribute to the pathogenesis of schizophrenia. This is the first report of caudate CaN abnormalities in schizophrenia.

Elevated postmortem striatal t-DARPP expression in schizophrenia and associations with DRD2/ANKK1 polymorphism.

BACKGROUND Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN) have been implicated in the pathogenesis of schizophrenia because they function as molecular integrators of dopamine and glutamate signaling. DARPP-32 and CaN are mainly expressed in the caudate nucleus and putamen; however, a few postmortem brain studies have focused on DARPP-32 expression in striatum from patients with schizophrenia. METHODS We used immunoblotting techniques and postmortem tissue samples from patients with schizophrenia and from normal control individuals to examine the expression of two major DARPP-32 isoforms, full-length (FL-DARPP) and truncated (t-DARPP), and of CaN in the striatum. We also assessed whether there was any significant correlation between the expression levels of either protein and the A1 allele of Taq1A genotype in the dopamine D2 receptor (DRD2) gene/ankyrin-repeat containing kinase 1 (ANKK1) gene. RESULTS We found that the mean t-DARPP expression level in the caudate was higher in patients with schizophrenia than in control individuals (P<0.05) and the A1 allele of Taq1A genotype in DRD2/ANKK1 was significantly associated with elevated expression of t-DARPP in the caudate. Also, the A1 allele was significantly correlated with the total score of antemortem psychiatric symptoms. CONCLUSION These results may reflect potential molecular mechanisms important to the pathogenesis of schizophrenia.

Influence of -141C Ins/Del Polymorphism in DRD2 Gene on Clinical Symptoms and Plasma Homovanillic Acid Levels in the Treatment of Schizophrenia With Aripiprazole.

FIGURE 1. Mean change in PANSS total (A), positive (B), negative (C), and plasmaHVA levels (D) of 2 genetic subgroups. There were no time genotype interactions on PANSS scores (total: P = 0.63, positive: P = 0.92, negative: P = 0.29) or plasma HVA levels (P = 0.61). B ecause the dopamine D2 receptor (DRD2) plays an important role in schizophrenia, genetic studies have been focused on this gene. A meta-analysis demonstrated a significant association between the −141C Ins/Del polymorphism in DRD2 and the response to antipsychotics, whereas the Taq1A polymorphism was not associated with the response. The −141C Ins/Del represents a deletion of cytosine at position −141, located in the 5′ promoter region of DRD2. The −141C Ins/Del polymorphism has effects on DRD2 density in the brain; however, the function of this polymorphism in schizophrenia remains unclear. Homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are major metabolites of dopamine and noradrenaline, respectively. In this study, we investigated the effect of the −141C Ins/Del polymorphism on clinical symptoms and plasma levels of HVA and MHPG during treatment with aripiprazole in antipsychoticfree patients with schizophrenia. Subjects were 34 Japanese patients (men, 22; women, 12; mean [SD] age, 41.2 [12.6] years) whose conditions were diagnosed with schizophrenia or schizoaffective disorder according to theDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Thirty of 34 patients were the same subjects from our previous study, and a previous report contains detailed descriptions of the study design. The −141C Ins/Del genotype was determined with the polymerase chain reaction method as previously described. This study was approved by the ethics committee of FukushimaMedical University, and the patients consented to participate after having been informed of the purpose of the study. Student t test (unpaired) and Pearson χ test were used for comparisons between the 2 genotype groups. Student t test (paired) was used to compare measures from before and after the aripiprazole treatment. Analysis of variance with repeated measures was used to evaluate changes in the Positive and Negative Syndrome Scale (PANSS) score and

PKA activation and endothelial claudin-5 breakdown in the schizophrenic prefrontal cortex

Schizophrenia is thought to be caused by a combination of genetic and environmental factors; however, its pathogenesis remains largely unknown. Here, we focus on the endothelial tight-junction protein claudin-5 (CLDN5), because the CLDN5 gene is mapped to the schizophrenia-associated 22q11.2 deletion region, and a single nucleotide polymorphism in the CLDN5 locus is also linked to schizophrenia. We show, by RT-qPCR and immunohistochemistry, that the expressions of CLDN5 mRNA and protein are significantly increased and decreased, respectively, in the schizophrenic prefrontal cortex (PFC) compared with control PFC. These changes were not observed in the schizophrenic visual cortex (VC), and neither the density nor diameter of the CD34-positive microvessels was altered in the schizophrenic PFC or VC. Interestingly, protein kinase A (PKA) was activated in the microvascular and perivascular regions of the schizophrenic PFC, and the pPKA-positive microvascular endothelial cells occasionally exhibited focal loss of CLND5. Since we previously demonstrated that cAMP induced CLDN5 mRNA expression and size-selective loosening of the endothelial barrier in PKA-independent and -dependent manners, respectively, a similar mechanism could contribute to the discrepancy between mRNA and protein expression of CLDN5 in the schizophrenic PFC. Taken collectively, these findings provide novel insights into the pathophysiology of schizophrenia.

Effects of the −141C insertion/deletion polymorphism in the dopamine D2 receptor gene on the dopamine system in the striatum in patients with schizophrenia

The relationships between -141C insertion/deletion (Ins/Del) polymorphisms in the dopamine D2 receptor gene and the two dopamine system integrators, i.e., dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) and calcineurin (CaN), are still unclear. In this study, we assessed the effect of this polymorphism on DARPP-32 and CaN protein expression in the postmortem striatum of patients with schizophrenia and control individuals. The expression levels of truncated DARPP and CaN were lower in Del allele carriers. These findings provide important insights into the mechanism by which this genotype could result in a poor response to antipsychotic drugs.

COMT Val 108/158 Met polymorphism and treatment response to aripiprazole in patients with acute schizophrenia

Introduction The COMT Val 108/158 Met polymorphism (rs4680) may affect treatment response to antipsychotics, as well as metabolism and dynamics of neurotransmitters during the treatment of schizophrenia. We investigated the effects of the COMT Val 108/158 Met polymorphism on treatment response to aripiprazole and plasma monoamine metabolite levels in patients with acute schizophrenia. Materials and methods Forty patients with schizophrenia were treated with aripiprazole for 6 weeks. We measured Positive and Negative Syndrome Scale (PANSS) and plasma levels of homovanillic acid (HVA) and plasma MHPG (3-methoxy-4-hydroxyphenethyleneglycol) at baseline and endpoint. The COMT Val 108/158 Met polymorphism was genotyped with the polymerase chain reaction and restriction fragment length polymorphism. Results There were significant genotype–time interactions on PANSS total and general psychopathology scores, with Met/Met genotype showing greater improvement. The response rate to aripiprazole did not differ between COMT Val 108/158 Met genotype groups. We found a significant time effect on plasma MHPG levels, but no time effect on plasma HVA levels or time–genotype interactions in the plasma levels of HVA and MHPG. Although the responder rate did not differ among the 3 genotype groups. Conclusion Our results suggest that individuals with the Met/Met genotype had greater improvement in PANSS score after the treatment with aripiprazole. On the other hand, the Val 108/158 Met polymorphism may not induce changes in plasma levels of monoamine metabolites during aripiprazole treatment. Because of the small sample size, further studies are needed to confirm and to extend our results.

DNA methylation of ANKK1 and response to aripiprazole in patients with acute schizophrenia: A preliminary study

Epigenetic modification including DNA methylation may affect pathophysiology and the response to antipsychotic drugs in patients with schizophrenia. The objective of the present study was to investigate the effect of the DNA methylation of ANKK1 (ankyrin repeat and kinase domain containing 1) on the response to aripiprazole and plasma levels of monoamine metabolites in antipsychotic-free acute schizophrenia patients. The subjects were 34 Japanese patients with schizophrenia who had been treated with aripiprazole for 6 weeks. Comprehensive DNA methylation of ANKK1 was determined using a next-generation sequencer. DNA methylation levels at CpG site 387 of ANKK1 were higher in responders to treatment with aripiprazole and correlated with the changes in Positive and Negative Syndrome Scale scores, although the associations did not remain significant after Bonferroni correction. In responders, methylation at all CpG sites was significantly correlated with plasma levels of homovanillic acid (r = 0.587, p = 0.035) and 3-methoxy-4hydroxyphenylglycol (r = 0.684, p = 0.010) at baseline. Despite our non-significant results after multiple correction, our preliminary findings suggest that methylation levels at CpG site 387 of ANKK1 may be associated with treatment response to aripiprazole. Furthermore, methylation of ANKK1 may affect dopaminergic neural transmission in the treatment of schizophrenia, and may influence treatment response. Caution is needed in interpreting these findings because of the small sample size, and further studies are needed to confirm and expand our preliminary results.