Junya Matsumoto

Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry

Schizophrenia is one of the major psychiatric disorders, and lipids have focused on the important roles in this disorder. In fact, lipids related to various functions in the brain. Previous studies have indicated that phospholipids, particularly ones containing polyunsaturated fatty acyl residues, are deficient in postmortem brains from patients with schizophrenia. However, due to the difficulties in handling human postmortem brains, particularly the large size and complex structures of the human brain, there is little agreement regarding the qualitative and quantitative abnormalities of phospholipids in brains from patients with schizophrenia, particularly if corresponding brain regions are not used. In this study, to overcome these problems, we employed matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS), enabling direct microregion analysis of phospholipids in the postmortem brain of a patient with schizophrenia via brain sections prepared on glass slides. With integration of traditional histochemical examination, we could analyze regions of interest in the brain at the micrometric level. We found abnormal phospholipid distributions within internal brain structures, namely, the frontal cortex and occipital cortex. IMS revealed abnormal distributions of phosphatidylcholine molecular species particularly in the cortical layer of frontal cortex region. In addition, the combined use of liquid chromatography/electrospray ionization tandem mass spectrometry strengthened the capability for identification of numerous lipid molecular species. Our results are expected to further elucidate various metabolic processes in the neural system.

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.

Mental disorders that exacerbated due to the Fukushima disaster, a complex radioactive contamination disaster

The Fukushima disaster was caused by an earthquake that occurred on 11 March 2011. Following the serious damage caused by the earthquake and the subsequent tsunami, radioactive contamination occurred because of the meltdown accident at the Fukushima nuclear power plant. We investigated what mental disorders were likely to become exacerbated under these conditions.

Worsening of manic state in patients with bipolar I disorder following the Fukushima disaster

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.

Profiles of DARPP-32 in the insular cortex with schizophrenia: A postmortem brain study

In patients with schizophrenia, various physical disorders are sometimes discovered only when they have reached a later and more severe stage. This phenomenon is believed to be caused, at least in part, by an increase in pain threshold. The gamma-aminobutyric acid (GABA)-ergic and glutamatergic systems in the rostral agranular insular cortex (RAIC) are thought to be involved in the regulation of pain threshold. However, no postmortem studies of the cerebral cortex have previously been published. Dopamine and cAMP-regulated phosphoprotein 32 kD (DARPP-32), which is involved in the GABAergic and glutamatergic systems, is considered to be crucial for elucidating the pathogenesis of schizophrenia. Using specific antibodies, we conducted immunohistochemical examinations of the RAIC in 10 subjects from a healthy control group, and 11 subjects from a schizophrenia group. The sex, age, and postmortem interval (PMI) of the schizophrenia group were matched to those of the healthy control group. We revealed that the density of DARPP-32-immunoreactive (IR) neurons in the II and III layers of the RAIC was significantly decreased (p<0.05) in the schizophrenia group compared with the healthy control group. Our findings could partially explain the molecular basis of the pain threshold abnormalities found in patients with 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.