Availability of individual proteins for quantitative analysis in postmortem brains preserved in two different brain banks.

AIM: Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. METHODS: We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. RESULTS: We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. CONCLUSIONS: When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.

Marked alteration of phosphoinositide signaling-associated molecules in postmortem prefrontal cortex with bipolar disorder.

AIM: The etiology of bipolar disorder (BD) remains unknown; however, lipid abnormalities in BD have received increasing attention in recent years. In this study, we examined the expression levels of enzyme proteins associated with the metabolic pathway of phosphoinositides (PIs) and their downstream effectors, protein kinase B (Akt1) and glycogen synthase kinase 3ß (GSK3ß), which have been assumed to be the targets of mood stabilizers such as lithium, in the postmortem brains of patients with BD. METHODS: The protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), Akt1, and GSK3ß were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays in the prefrontal cortex (PFC). Specifically, PTEN, Akt1, GSK3ß, and PIP5K1C were measured in seven BD patients and 48 controls. Additionally, PIK4CA was analyzed in 10 cases and 34 controls. RESULTS: PTEN expression levels were markedly decreased in the PFCs of patients with BD, whereas those of Akt and GSK3ß were prominently elevated. Moreover, patients medicated with lithium exhibited higher Akt1 expression levels and lower PTEN expression levels in comparison with the untreated group. CONCLUSION: Our results suggest that the expression levels of Akt1/GSK3ß and its upstream regulator PTEN are considerably altered.

Ethnicity-dependent effect of rs1799971 polymorphism on OPRM1 expression in the postmortem brain and responsiveness to antipsychotics.

Schizophrenia is associated with aberration of inhibitory neurons. Although the mu-opioid receptor (MOR) is an essential modulator of inhibitory neurons, the effect of rs1799971 polymorphism in the MOR gene on risk of schizophrenia is controversial. Moreover, the disturbance of opioids systems in patients with schizophrenia has not been fully examined. We firstly conducted preliminary meta-analyses integrating Asian and European populations separately over 12,000 subjects to assess the effect of rs1799971 on risk of schizophrenia. Based on the above result, we also investigated the effect on the expression levels of MOR mRNA in the prefrontal cortex (PFC) and caudate nucleus of 41 postmortem brains. In addition, we determined whether these levels were related to antemortem schizophrenia symptoms and pharmacotherapeutic effects. The rs1799971 G-allele reduced the risk of schizophrenia in Asian populations (OR: 0.56, 95%CI: 0.32-0.98, p = 0.042) but increased it in European populations (OR: 1.66, 95%CI: 1.08-2.56, p = 0.022). It decreased MOR mRNA levels in PFC in the Japanese population (p = 0.031). Increased MOR mRNA level in PFC correlated with higher total score of antemortem schizophrenia symptoms (p = 0.017). Furthermore, the pharmacotherapeutic effect of first-generation antipsychotics was higher for genotype AA than AG/GG of rs1799971 (p = 0.036). The rs1799971 affects risk of schizophrenia and MOR mRNA expression and the effect varies according to ethnicity. Overexpression of MOR might induce severe schizophrenia symptoms. Therefore, MOR modulation may be the key clue for treating antipsychotics-resistant schizophrenia, and genotyping rs1799971 may provide a better pharmacotherapeutic strategy.

Evidence for increased DNA damage repair in the postmortem brain of the high stress-response group of schizophrenia.

Background: Schizophrenia (SZ) is a disorder diagnosed by specific symptoms and duration and is highly heterogeneous, clinically and pathologically. Although there are an increasing number of studies on the association between genetic and environmental factors in the development of SZ, the actual distribution of the population with different levels of influence of these factors has not yet been fully elucidated. In this study, we focused on stress as an environmental factor and stratified SZ based on the expression levels of stress-responsive molecules in the postmortem prefrontal cortex. Methods: We selected the following stress-responsive molecules: interleukin (IL) -1ß, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, glucocorticoid receptor, brain-derived neurotrophic factor, synaptophysin, S100 calcium-binding protein B, superoxide dismutase, postsynaptic density protein 95, synuclein, apolipoprotein A1 (ApoA1), ApoA2, and solute carrier family 6 member 4. We performed RNA sequencing in the prefrontal gray matter of 25 SZ cases and 21 healthy controls and conducted a hierarchical cluster analysis of SZ based on the gene expression levels of stress-responsive molecules, which yielded two clusters. After assessing the validity of the clusters, they were designated as the high stress-response SZ group and the low stress-response SZ group, respectively. Ingenuity Pathway Analysis of differentially expressed genes (DEGs) between clusters was performed, and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was conducted on four cases each in the high and low stress-response SZ groups to validate DNA damage. Results: We found higher prevalence of family history of SZ in the low stress-response SZ group (0/3 vs. 5/4, p = 0.04). Pathway analysis of DEGs between clusters showed the highest enrichment for DNA double-strand break repair. TUNEL staining showed a trend toward a lower percentage of TUNEL-positive cells in the high stress-response SZ group. Conclusion: Our results suggest that there are subgroups of SZ with different degrees of stress impact. Furthermore, the pathophysiology of these subgroups may be associated with DNA damage repair. These results provide new insights into the interactions and heterogeneity between genetic and environmental factors.

The influence of tissue pH and RNA integrity number on gene expression of human postmortem brain.

Background: Evaluating and controlling confounders are necessary when investigating molecular pathogenesis using human postmortem brain tissue. Particularly, tissue pH and RNA integrity number (RIN) are valuable indicators for controlling confounders. However, the influences of these indicators on the expression of each gene in postmortem brain have not been fully investigated. Therefore, we aimed to assess these effects on gene expressions of human brain samples. Methods: We isolated total RNA from occipital lobes of 13 patients with schizophrenia and measured the RIN and tissue pH. Gene expression was analyzed and gene sets affected by tissue pH and RIN were identified. Moreover, we examined the functions of these genes by enrichment analysis and upstream regulator analysis. Results: We identified 2,043 genes (24.7%) whose expressions were highly correlated with pH; 3,004 genes (36.3%) whose expressions were highly correlated with RIN; and 1,293 genes (15.6%) whose expressions were highly correlated with both pH and RIN. Genes commonly affected by tissue pH and RIN were highly associated with energy production and the immune system. In addition, genes uniquely affected by tissue pH were highly associated with the cell cycle, whereas those uniquely affected by RIN were highly associated with RNA processing. Conclusion: The current study elucidated the influence of pH and RIN on gene expression profiling and identified gene sets whose expressions were affected by tissue pH or RIN. These findings would be helpful in the control of confounders for future postmortem brain studies.

Identification of schizophrenia symptom-related gene modules by postmortem brain transcriptome analysis.

Schizophrenia is a multifactorial disorder, the genetic architecture of which remains unclear. Although many studies have examined the etiology of schizophrenia, the gene sets that contribute to its symptoms have not been fully investigated. In this study, we aimed to identify each gene set associated with corresponding symptoms of schizophrenia using the postmortem brains of 26 patients with schizophrenia and 51 controls. We classified genes expressed in the prefrontal cortex (analyzed by RNA-seq) into several modules by weighted gene co-expression network analysis (WGCNA) and examined the correlation between module expression and clinical characteristics. In addition, we calculated the polygenic risk score (PRS) for schizophrenia from Japanese genome-wide association studies, and investigated the association between the identified gene modules and PRS to evaluate whether genetic background affected gene expression. Finally, we conducted pathway analysis and upstream analysis using Ingenuity Pathway Analysis to clarify the functions and upstream regulators of symptom-related gene modules. As a result, three gene modules generated by WGCNA were significantly correlated with clinical characteristics, and one of these showed a significant association with PRS. Genes belonging to the transcriptional module associated with PRS significantly overlapped with signaling pathways of multiple sclerosis, neuroinflammation, and opioid use, suggesting that these pathways may also be profoundly implicated in schizophrenia. Upstream analysis indicated that genes in the detected module were profoundly regulated by lipopolysaccharides and CREB. This study identified schizophrenia symptom-related gene sets and their upstream regulators, revealing aspects of the pathophysiology of schizophrenia and identifying potential therapeutic targets.

Elevation of EGR1/zif268, a Neural Activity Marker, in the Auditory Cortex of Patients with Schizophrenia and its Animal Model.

The family of epidermal growth factor (EGF) including neuregulin-1 are implicated in the neuropathology of schizophrenia. We established a rat model of schizophrenia by exposing perinatal rats to EGF and reported that the auditory pathophysiological traits of this model such as prepulse inhibition, auditory steady-state response, and mismatch negativity are relevant to those of schizophrenia. We assessed the activation status of the auditory cortex in this model, as well as that in patients with schizophrenia, by monitoring the three neural activity-induced proteins: EGR1 (zif268), c-fos, and Arc. Among the activity markers, protein levels of EGR1 were significantly higher at the adult stage in EGF model rats than those in control rats. The group difference was observed despite an EGF model rat and a control rat being housed together, ruling out the contribution of rat vocalization effects. These changes in EGR1 levels were seen to be specific to the auditory cortex of this model. The increase in EGR1 levels were detectable at the juvenile stage and continued until old ages but displayed a peak immediately after puberty, whereas c-fos and Arc levels were nearly indistinguishable between groups at all ages with an exception of Arc decrease at the juvenile stage. A similar increase in EGR1 levels was observed in the postmortem superior temporal cortex of patients with schizophrenia. The commonality of the EGR1 increase indicates that the EGR1 elevation in the auditory cortex might be one of the molecular signatures of this animal model and schizophrenia associating with hallucination.

Dysregulation of DPYSL2 expression by mTOR signaling in schizophrenia: Multi-level study of postmortem brain.

The mechanistic target of rapamycin (mTOR)-signaling and dihydropyrimidinase-like 2 (DPYSL2), which are increasingly gaining attention as potential therapeutic targets for schizophrenia, are connected via Cap-dependent translation of the 5'TOP motif. We quantified the expression of molecules constituting the mTOR-signaling and DPYSL2 in the prefrontal cortex (PFC) and superior temporal gyrus (STG) of postmortem brain tissue samples from 24 patients with schizophrenia and 32 control individuals and conducted association analysis to examine abnormal regulation of DPYSL2 expression by the mTOR-signaling in schizophrenia. The average ribosomal protein S6 (S6) levels in the PFC and STG were lower in patients with schizophrenia (p < 0.01). DPYSL2 expression showed a significant positive correlation with phospho-S6 expression levels, which were effectors of mTOR translational regulation, and the correlation slope between phospho-S6 and DPYSL2 expressions differed between cases and controls. Association analyses of these mTOR-signaling and DPYSL2 alterations with genetic polymorphisms and the clinical profile suggested that certain genetic variants of DPYSL2 require high mTOR-signaling activity. Thus, the findings confirmed decreased S6 expression levels in schizophrenia and supported the relationship between the mTOR-signaling and DPYSL2 via 5'TOP Cap-dependent translation, thus providing insights connecting the two major schizophrenia treatment strategies associated with the mTOR-signaling and DPYSL2.

Evidence for Altered Phosphoinositide Signaling-Associated Molecules in the Postmortem Prefrontal Cortex of Patients with Schizophrenia.

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3ß (GSK3ß) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3ß expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.