Paul A. Tooney

MicroRNA expression profiling and quantitative reverse transcription-PCR analysis of the superior temporal gyrus and the dorsolateral prefrontal cortex revealed a significant schizophrenia-associated increase in global microRNA expression. This change was associated with an elevation of primary microRNA processing and corresponded with an increase in the microprocessor component DGCR8. The biological implications for this extensive increase in gene silencing are profound, and were exemplified by members of the miR-15 family and other related microRNA, which were significantly upregulated in both brain regions. This functionally convergent influence is overrepresented in pathways involved in synaptic plasticity and includes many genes and pathways associated with schizophrenia, some of which were substantiated in vitro by reporter gene assay. Given the magnitude of microRNA changes and their wide sphere of influence, this phenomenon could represent an important dimension in the pathogenesis of schizophrenia.

Analysis of global microRNA (miRNA) expression in postmortem cortical grey matter from the superior temporal gyrus, revealed significant up-regulation of miR-181b expression in schizophrenia. This finding was supported by quantitative real-time RT-PCR analysis of miRNA expression in a cohort of 21 matched pairs of schizophrenia and non-psychiatric controls. The implications of this finding are substantial, as this miRNA is predicted to regulate many target genes with potential significance to the development of schizophrenia. They include the calcium sensor gene visinin-like 1 (VSNL1) and the ionotropic AMPA glutamate receptor subunit (GRIA2), which were found to be down-regulated in the same cortical tissue from the schizophrenia group. Both of these genes were also suppressed in miR-181b transfected cells and shown to contain functional miR-181b miRNA recognition elements by reporter gene assay. This study suggests altered miRNA levels could be a significant factor in the dysregulation of cortical gene expression in schizophrenia.

MicroRNA (miRNA) are capable of regulating multitudes of target genes and are essential factors in mediating healthy neurodevelopment. We hypothesize that abnormal miRNA levels contribute to the complex global changes in gene expression that underlie the pathophysiology of schizophrenia.With a commercial bead array platform, we investigated miRNA expression in 74 samples of postmortem dorsolateral prefrontal cortex (Brodmann Area 46) (n = 37 matched pairs schizophrenia/schizoaffective disorder and control subjects). A subset of differentially expressed miRNA and genes in the miRNA biogenesis pathway was also analyzed with quantitative reverse transcription-polymerase chain reaction. Gene targets of miRNAs demonstrating significantly altered expression were predicted, and pathways analysis was performed.After correction for multiple testing, microarray analysis identified differential expression of 28 miRNA in the schizophrenia group. Significantly, 89% of these molecules were elevated in accordance with earlier work in other brain regions that showed a broad increase in miRNA expression in schizophrenia. These observations were supported by quantitative reverse transcription-polymerase chain reaction, for miR-328, miR-17-5p, miR-134, miR-652, miR-382, and miR-107 and were consistent with a schizophrenia-associated increase in miRNA processing through elevated Dicer expression. Target and pathways analysis provided insight into the potential cellular effects, with particular enrichment of miRNA targets in axon guidance and long-term potentiation.These results suggest that schizophrenia is associated with altered miRNA biogenesis and expression, which might have important implications in the complex pathophysiology of the disorder.

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are important for coordinating nervous system development and neuronal function in the mature brain. We have recently identified schizophrenia-associated alteration of cortical miRNA biogenesis and expression in post-mortem brain tissue with implications for the dysregulation of schizophrenia candidate genes. Although these changes were observed in the central nervous system, it is plausible that schizophrenia-associated miRNA expression signatures may also be detected in non-neural tissue. To explore this possibility, we investigated the miRNA expression profile of peripheral blood mononuclear cells (PBMCs) from 112 patients with schizophrenia and 76 non-psychiatric controls. miRNA expression analysis of total RNA conducted using commercial miRNA arrays revealed that 33 miRNAs were significantly downregulated after correction for multiple testing with a false discovery rate (FDR) of 0%, which increased to 83 when we considered miRNA with an FDR<5%. Seven miRNAs altered in microarray analysis of schizophrenia were also confirmed to be downregulated by quantitative real-time reverse transcription-polymerase chain reaction. A large subgroup consisting of 17 downregulated miRNAs is transcribed from a single imprinted locus at the maternally expressed DLK1-DIO3 region on chromosome 14q32. This pattern of differentially expressed miRNA in PBMCs may be indicative of significant underlying genetic or epigenetic alteration associated with schizophrenia.

A common limitation of neuroimaging studies is their small sample sizes. To overcome this hurdle, the Enhancing Neuro Imaging Genetics through Meta-Analysis (ENIGMA) Consortium combines neuroimaging data from many institutions worldwide. However, this introduces heterogeneity due to different scanning devices and sequences. ENIGMA projects commonly address this heterogeneity with random-effects meta-analysis or mixed-effects mega-analysis. Here we tested whether the batch adjustment method, ComBat, can further reduce site-related heterogeneity and thus increase statistical power. We conducted random-effects meta-analyses, mixed-effects mega-analyses and ComBat mega-analyses to compare cortical thickness, surface area and subcortical volumes between 2897 individuals with a diagnosis of schizophrenia and 3141 healthy controls from 33 sites. Specifically, we compared the imaging data between individuals with schizophrenia and healthy controls, covarying for age and sex. The use of ComBat substantially increased the statistical significance of the findings as compared to random-effects meta-analyses. The findings were more similar when comparing ComBat with mixed-effects mega-analysis, although ComBat still slightly increased the statistical significance. ComBat also showed increased statistical power when we repeated the analyses with fewer sites. Results were nearly identical when we applied the ComBat harmonization separately for cortical thickness, cortical surface area and subcortical volumes. Therefore, we recommend applying the ComBat function to attenuate potential effects of site in ENIGMA projects and other multi-site structural imaging work. We provide easy-to-use functions in R that work even if imaging data are partially missing in some brain regions, and they can be trained with one data set and then applied to another (a requirement for some analyses such as machine learning).

Background Sex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk. Methods We conducted the largest to date genome-wide genotype-by-sex (G×S) interaction of risk for these disorders using 85,735 cases (33,403 SCZ, 19,924 BIP, and 32,408 MDD) and 109,946 controls from the PGC (Psychiatric Genomics Consortium) and iPSYCH. Results Across disorders, genome-wide significant single nucleotide polymorphism–by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815, p = 3.2 × 10−8), which interacts with sodium/potassium-transporting ATPase (adenosine triphosphatase) enzymes, implicating neuronal excitability. Three additional loci showed evidence (p < 1 × 10−6) for cross-disorder G×S interaction (rs7302529, p = 1.6 × 10−7; rs73033497, p = 8.8 × 10−7; rs7914279, p = 6.4 × 10−7), implicating various functions. Gene-based analyses identified G×S interaction across disorders (p = 8.97 × 10−7) with transcriptional inhibitor SLTM. Most significant in SCZ was a MOCOS gene locus (rs11665282, p = 1.5 × 10−7), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509, p = 1.1 × 10−7) in a locus containing IDO2, a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant G×S interaction of genes regulating vascular endothelial growth factor receptor signaling in MDD (false discovery rate-corrected p < .05). Conclusions In the largest genome-wide G×S analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development and immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway levels.

About 20% to 30% of people with schizophrenia have psychotic symptoms that do not respond adequately to first-line antipsychotic treatment. This clinical presentation, chronic and highly disabling, is known as treatment-resistant schizophrenia (TRS). The causes of treatment resistance and their relationships with causes underlying schizophrenia are largely unknown. Adequately powered genetic studies of TRS are scarce because of the difficulty in collecting data from well-characterized TRS cohorts.To examine the genetic architecture of TRS through the reassessment of genetic data from schizophrenia studies and its validation in carefully ascertained clinical samples.Two case-control genome-wide association studies (GWASs) of schizophrenia were performed in which the case samples were defined as individuals with TRS (n = 10 501) and individuals with non-TRS (n = 20 325). The differences in effect sizes for allelic associations were then determined between both studies, the reasoning being such differences reflect treatment resistance instead of schizophrenia. Genotype data were retrieved from the CLOZUK and Psychiatric Genomics Consortium (PGC) schizophrenia studies. The output was validated using polygenic risk score (PRS) profiling of 2 independent schizophrenia cohorts with TRS and non-TRS: a prevalence sample with 817 individuals (Cardiff Cognition in Schizophrenia [CardiffCOGS]) and an incidence sample with 563 individuals (Genetics Workstream of the Schizophrenia Treatment Resistance and Therapeutic Advances [STRATA-G]).GWAS of treatment resistance in schizophrenia. The results of the GWAS were compared with complex polygenic traits through a genetic correlation approach and were used for PRS analysis on the independent validation cohorts using the same TRS definition.The study included a total of 85 490 participants (48 635 [56.9%] male) in its GWAS stage and 1380 participants (859 [62.2%] male) in its PRS validation stage. Treatment resistance in schizophrenia emerged as a polygenic trait with detectable heritability (1% to 4%), and several traits related to intelligence and cognition were found to be genetically correlated with it (genetic correlation, 0.41-0.69). PRS analysis in the CardiffCOGS prevalence sample showed a positive association between TRS and a history of taking clozapine (r2 = 2.03%; P = .001), which was replicated in the STRATA-G incidence sample (r2 = 1.09%; P = .04).In this GWAS, common genetic variants were differentially associated with TRS, and these associations may have been obscured through the amalgamation of large GWAS samples in previous studies of broadly defined schizophrenia. Findings of this study suggest the validity of meta-analytic approaches for studies on patient outcomes, including treatment resistance.

Progress in determining the aetiology of schizophrenia (Sz) has arguably been limited by a poorly defined phenotype. We sought to delineate empirically derived cognitive subtypes of Sz to investigate the association of a genetic variant identified in a recent genome-wide association study with specific phenotypic characteristics of Sz. We applied Grade of Membership (GoM) analyses to 617 patients meeting ICD-10 criteria for Sz (n=526) or schizoaffective disorder (n=91), using cognitive performance indicators collected within the Australian Schizophrenia Research Bank. Cognitive variables included subscales from the Repeatable Battery for the Assessment of Neuropsychological Status, the Controlled Oral Word Association Test and the Letter Number Sequencing Test, and standardised estimates of premorbid and current intelligence quotient. The most parsimonious GoM solution yielded two subtypes of clinical cases reflecting those with cognitive deficits (CDs; N=294), comprising 47.6% of the sample who were impaired across all cognitive measures, and a cognitively spared group (CS; N=323) made up of the remaining 52.4% who performed relatively well on all cognitive tests. The CD subgroup were more likely to be unemployed, had an earlier illness onset, and greater severity of functional disability and negative symptoms than the CS group. Risk alleles on the MIR137 single-nucleotide polymorphism (SNP) predicted membership of CD subtype only in combination with higher severity of negative symptoms. These findings provide the first evidence for association of the MIR137 SNP with a specific Sz phenotype characterised by severe CDs and negative symptoms, consistent with the emerging role of microRNAs in the regulation of proteins responsible for neural development and function.

Reduced mismatch negativity (MMN) in response to auditory change is a well-established finding in schizophrenia and has been shown to be correlated with impaired daily functioning, rather than with hallmark signs and symptoms of the disorder. In this study, we investigated (1) whether the relationship between reduced MMN and impaired daily functioning is mediated by cortical volume loss in temporal and frontal brain regions in schizophrenia and (2) whether this relationship varies with the type of auditory deviant generating MMN. MMN in response to duration, frequency, and intensity deviants was recorded from 18 schizophrenia subjects and 18 pairwise age- and gender-matched healthy subjects. Patients' levels of global functioning were rated on the Social and Occupational Functioning Assessment Scale. High-resolution structural magnetic resonance scans were acquired to generate average cerebral cortex and temporal lobe models using cortical pattern matching. This technique allows accurate statistical comparison and averaging of cortical measures across subjects, despite wide variations in gyral patterns. MMN amplitude was reduced in schizophrenia patients and correlated with their impaired day-to-day function level. Only in patients, bilateral gray matter reduction in Heschl's gyrus, as well as motor and executive regions of the frontal cortex, correlated with reduced MMN amplitude in response to frequency deviants, while reduced gray matter in right Heschl's gyrus also correlated with reduced MMN to duration deviants. Our findings further support the importance of MMN reduction in schizophrenia by linking frontotemporal cerebral gray matter pathology to an automatically generated event-related potential index of daily functioning.

While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression.The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene.This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia.

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n=315) and from ex-vivo blood tissues (n=578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia.

<h2>Abstract</h2><h3>Background</h3> The 1q21.1 distal and 15q11.2 BP1-BP2 CNVs exhibit regional and global brain differences compared to non-carriers. However, interpreting regional differences is challenging if a global difference drives the regional brain differences. Intra-individual variability measures can be used to test for regional differences beyond global differences in brain structure. <h3>Methods</h3> Magnetic resonance imaging data were used to obtain regional brain values for 1q21.1 distal deletion (n=30) and duplication (n=27), and 15q11.2 BP1-BP2 deletion (n=170) and duplication (n=243) carriers and matched non-carriers (n=2,350). Regional intra-deviation (RID) scores i.e., the standardized difference between an individual's regional difference and global difference, were used to test for regional differences that diverge from the global difference. <h3>Results</h3> For the 1q21.1 distal deletion carriers, cortical surface area for regions in the medial visual cortex, posterior cingulate and temporal pole differed less, and regions in the prefrontal and superior temporal cortex differed more than the global difference in cortical surface area. For the 15q11.2 BP1-BP2 deletion carriers, cortical thickness in regions in the medial visual cortex, auditory cortex and temporal pole differed less, and the prefrontal and somatosensory cortex differed more than the global difference in cortical thickness. <h3>Conclusion</h3> We find evidence for regional effects beyond differences in global brain measures in 1q21.1 distal and 15q11.2 BP1-BP2 CNVs. The results provide new insight into brain profiling of the 1q21.1 distal and 15q11.2 BP1-BP2 CNVs, with the potential to increase our understanding of mechanisms involved in altered neurodevelopment.

Abstract Schizophrenia is a prototypical network disorder with widespread brain-morphological alterations, yet it remains unclear whether these distributed alterations robustly reflect the underlying network layout. We tested whether large-scale structural alterations in schizophrenia relate to normative structural and functional connectome architecture, and systematically evaluated robustness and generalizability of these network-level alterations. Leveraging anatomical MRI scans from 2439 adults with schizophrenia and 2867 healthy controls from 26 ENIGMA sites and normative data from the Human Connectome Project ( n = 207), we evaluated structural alterations of schizophrenia against two network susceptibility models: (i) hub vulnerability, which examines associations between regional network centrality and magnitude of disease-related alterations; (ii) epicenter mapping, which identifies regions whose typical connectivity profile most closely resembles the disease-related morphological alterations. To assess generalizability and specificity, we contextualized the influence of site, disease stages, and individual clinical factors and compared network associations of schizophrenia with that found in affective disorders. Our findings show schizophrenia-related cortical thinning is spatially associated with functional and structural hubs, suggesting that highly interconnected regions are more vulnerable to morphological alterations. Predominantly temporo-paralimbic and frontal regions emerged as epicenters with connectivity profiles linked to schizophrenia’s alteration patterns. Findings were robust across sites, disease stages, and related to individual symptoms. Moreover, transdiagnostic comparisons revealed overlapping epicenters in schizophrenia and bipolar, but not major depressive disorder, suggestive of a pathophysiological continuity within the schizophrenia-bipolar-spectrum. In sum, cortical alterations over the course of schizophrenia robustly follow brain network architecture, emphasizing marked hub susceptibility and temporo-frontal epicenters at both the level of the group and the individual. Subtle variations of epicenters across disease stages suggest interacting pathological processes, while associations with patient-specific symptoms support additional inter-individual variability of hub vulnerability and epicenters in schizophrenia. Our work outlines potential pathways to better understand macroscale structural alterations, and inter- individual variability in schizophrenia.

Schizophrenia is a heterogenous disorder that is phenomenologically characterised by a combination of negative, positive, and cognitive symptoms with variable expression in the course of illness. Here, we investigated differential gene expression in relation to age to address the heterogeneity of this disorder We used 6000 gene cDNA microarrays to generate gene expression profiles from peripheral blood lymphocytes from 14 individuals with schizophrenia and 14 non-psychiatric controls. Genes showing altered expression were identified and 18 genes with brain-related functions were altered, 4 of which, endothelial differentiation gene 2 (Edg-2), ezrin-radixin-moesin phosphoprotein 50 (EBP50), Myc-associated zinc finger protein (MAZ) and Tumor Necrosis Factor Receptor 2 (TNFR2), were confirmed by relative real-time PCR. Dendrograms were constructed using genes that showed significantly different expression (p<0.05) between groups based on median split of age dividing the matched pairs into distinct subclasses. Our findings suggest that distinct gene expression profiles in peripheral blood lymphocytes associated with schizophrenia phenotypes may provide a first step towards the biological classification of schizophrenia subtypes. The validity of this approach may lead to better methods of defining this enigmatic disease.

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.

Peripheral blood mononuclear cells (PBMCs) represent an accessible tissue source for gene expression profiling in schizophrenia that could provide insight into the molecular basis of the disorder. This study used the Illumina HT_12 microarray platform and quantitative real time PCR (QPCR) to perform mRNA expression profiling on 114 patients with schizophrenia or schizoaffective disorder and 80 non-psychiatric controls from the Australian Schizophrenia Research Bank (ASRB). Differential expression analysis revealed altered expression of 164 genes (59 up-regulated and 105 down-regulated) in the PBMCs from patients with schizophrenia compared to controls. Bioinformatic analysis indicated significant enrichment of differentially expressed genes known to be involved or associated with immune function and regulating the immune response. The differential expression of 6 genes, EIF2C2 (Ago 2), MEF2D, EVL, PI3, S100A12 and DEFA4 was confirmed by QPCR. Genome-wide expression analysis of PBMCs from individuals with schizophrenia was characterized by the alteration of genes with immune system function, supporting the hypothesis that the disorder has a significant immunological component in its etiology.

This article describes the establishment of the Australian Schizophrenia Research Bank (ASRB), which operates to collect, store and distribute linked clinical, cognitive, neuroimaging and genetic data from a large sample of people with schizophrenia and healthy controls.Recruitment sources for the schizophrenia sample include a multi-media national advertising campaign, inpatient and community treatment services and non-government support agencies. Healthy controls have been recruited primarily through multi-media advertisements. All participants undergo an extensive diagnostic and family history assessment, neuropsychological evaluation, and blood sample donation for genetic studies. Selected individuals also complete structural MRI scans.Preliminary analyses of 493 schizophrenia cases and 293 healthy controls are reported. Mean age was 39.54 years (SD = 11.1) for the schizophrenia participants and 37.38 years (SD = 13.12) for healthy controls. Compared to the controls, features of the schizophrenia sample included a higher proportion of males (cases 65.9%; controls 46.8%), fewer living in married or de facto relationships (cases 16.1%; controls 53.6%) and fewer years of education (cases 13.05, SD = 2.84; controls 15.14, SD = 3.13), as well as lower current IQ (cases 102.68, SD = 15.51; controls 118.28, SD = 10.18). These and other sample characteristics are compared to those reported in another large Australian sample (i.e. the Low Prevalence Disorders Study), revealing some differences that reflect the different sampling methods of these two studies.The ASRB is a valuable and accessible schizophrenia research facility for use by approved scientific investigators. As recruitment continues, the approach to sampling for both cases and controls will need to be modified to ensure that the ASRB samples are as broadly representative as possible of all cases of schizophrenia and healthy controls.

The interaction of genetic and environmental factors may affect the course and development of psychotic disorders. We examined whether the effects of childhood trauma on cognition and symptoms in schizophrenia were moderated by the Catechol-O-methyltransferase (COMT) Val158Met polymorphism, a common genetic variant known to affect cognition and prefrontal dopamine levels. Participants were 429 schizophrenia/schizoaffective cases from the Australian Schizophrenia Research Bank (ASRB). Cognitive performance was assessed using the Repeatable Battery for Assessment of Neuropsychological Status (RBANS), Controlled Oral Word Association Test (COWAT), Letter Number Sequencing (LNS) test, and the Wechsler Test of Adult Reading (WTAR). Hierarchical regression was used to test the main effects and additive interaction effects of genotype and childhood trauma in the domains of physical abuse, emotional abuse, and emotional neglect, on cognition and symptom profiles of clinical cases. Consistent with previous findings, COMT Val homozygotes performed worse on cognitive measures in the absence of childhood adversity. In addition, a significant interaction between COMT genotype and physical abuse was associated with better executive function in Val homozygotes, relative to those of the same genotype with no history of abuse. Finally, the severity of positive symptoms was greater in Met carriers who had experienced physical abuse, and the severity of negative symptoms in Met carriers was greater in the presence of emotional neglect. These results suggest that the possible epigenetic modulation of the expression of the COMT Val158Met polymorphism and consequent effects on cognition and symptoms in schizophrenia, with worse outcomes associated with adverse childhood experiences in Met carriers.

Distinct gene expression profiles can be detected in peripheral blood mononuclear cells (PBMCs) in patients with schizophrenia; however, little is known about the effects of antipsychotic medication. This study compared gene expression profiles in PMBCs from treatment-naive patients with schizophrenia before and after antipsychotic drug treatment. PBMCs were obtained from 10 treatment-naive schizophrenia patients before and 6 wk after initiating antipsychotic drug treatment and compared to PMBCs collected from 11 healthy community volunteers. Genome-wide expression profiling was conducted using Illumina HumanHT-12 expression bead arrays and analysed using significance analysis of microarrays. This analysis identified 624 genes with altered expression (208 up-regulated, 416 down-regulated) prior to antipsychotic treatment (p < 0.05) including schizophrenia-associated genes AKT1, DISC1 and DGCR6. After 6-8 wk treatment of patients with risperidone or risperidone in combination with haloperidol, only 106 genes were altered, suggesting that the treatment corrected the expression of a large proportion of genes back to control levels. However, 67 genes continued to show the same directional change in expression after treatment. Ingenuity® pathway analysis and gene set enrichment analysis implicated dysregulation of biological functions and pathways related to inflammation and immunity in patients with schizophrenia. A number of the top canonical pathways dysregulated in treatment-naive patients signal through AKT1 that was up-regulated. After treatment, AKT1 returned to control levels and less dysregulation of these canonical pathways was observed. This study supports immune dysfunction and pathways involving AKT1 in the aetiopathophysiology of schizophrenia and their response to antipsychotic medication.

<h3>Importance</h3> A recently published study of national data by McGrath et al in 2014 showed increased risk of schizophrenia (SCZ) in offspring associated with both early and delayed parental age, consistent with a U-shaped relationship. However, it remains unclear if the risk to the child is due to psychosocial factors associated with parental age or if those at higher risk for SCZ tend to have children at an earlier or later age. <h3>Objective</h3> To determine if there is a genetic association between SCZ and age at first birth (AFB) using genetically informative but independently ascertained data sets. <h3>Design, Setting, and Participants</h3> This investigation used multiple independent genome-wide association study data sets. The SCZ sample comprised 18 957 SCZ cases and 22 673 controls in a genome-wide association study from the second phase of the Psychiatric Genomics Consortium, and the AFB sample comprised 12 247 genotyped women measured for AFB from the following 4 community cohorts: Estonia (Estonian Genome Center Biobank, University of Tartu), the Netherlands (LifeLines Cohort Study), Sweden (Swedish Twin Registry), and the United Kingdom (TwinsUK). Schizophrenia genetic risk for each woman in the AFB community sample was estimated using genetic effects inferred from the SCZ genome-wide association study. <h3>Main Outcomes and Measures</h3> We tested if SCZ genetic risk was a significant predictor of response variables based on published polynomial functions that described the relationship between maternal age and SCZ risk in offspring in Denmark. We substituted AFB for maternal age in these functions, one of which was corrected for the age of the father, and found that the fit was superior for the model without adjustment for the father's age. <h3>Results</h3> We observed a U-shaped relationship between SCZ risk and AFB in the community cohorts, consistent with the previously reported relationship between SCZ risk in offspring and maternal age when not adjusted for the age of the father. We confirmed that SCZ risk profile scores significantly predicted the response variables (coefficient of determination<i>R2</i> = 1.1E-03,<i>P</i> = 4.1E-04), reflecting the published relationship between maternal age and SCZ risk in offspring by McGrath et al in 2014. <h3>Conclusions and Relevance</h3> This study provides evidence for a significant overlap between genetic factors associated with risk of SCZ and genetic factors associated with AFB. It has been reported that SCZ risk associated with increased maternal age is explained by the age of the father and that de novo mutations that occur more frequently in the germline of older men are the underlying causal mechanism. This explanation may need to be revised if, as suggested herein and if replicated in future studies, there is also increased genetic risk of SCZ in older mothers.

Clarifying the role of neuroinflammation in schizophrenia is subject to its detection in the living brain. Free-water (FW) imaging is an in vivo diffusion-weighted magnetic resonance imaging (dMRI) technique that measures water molecules freely diffusing in the brain and is hypothesized to detect inflammatory processes. Here, we aimed to establish a link between peripheral markers of inflammation and FW in brain white matter.All data were obtained from the Australian Schizophrenia Research Bank (ASRB) across 5 Australian states and territories. We first tested for the presence of peripheral cytokine deregulation in schizophrenia, using a large sample (N = 1143) comprising the ASRB. We next determined the extent to which individual variation in 8 circulating pro-/anti-inflammatory cytokines related to FW in brain white matter, imaged in a subset (n = 308) of patients and controls.Patients with schizophrenia showed reduced interleukin-2 (IL-2) (t = -3.56, P = .0004) and IL-12(p70) (t = -2.84, P = .005) and increased IL-6 (t = 3.56, P = .0004), IL-8 (t = 3.8, P = .0002), and TNFα (t = 4.30, P < .0001). Higher proinflammatory signaling of IL-6 (t = 3.4, P = .0007) and TNFα (t = 2.7, P = .0007) was associated with higher FW levels in white matter. The reciprocal increases in serum cytokines and FW were spatially widespread in patients encompassing most major fibers; conversely, in controls, the relationship was confined to the anterior corpus callosum and thalamic radiations. No relationships were observed with alternative dMRI measures, including the fractional anisotropy and tissue-related FA.We report widespread deregulation of cytokines in schizophrenia and identify inflammation as a putative mechanism underlying increases in brain FW levels.

// Mathew Lozinski 2 , 3 , 4 , Nikola A. Bowden 2 , 3 , Moira C. Graves 2 , 3 , 4 , Michael Fay 2 , 3 , 4 , 5 , Bryan W. Day 6 , Brett W. Stringer 7 and Paul A. Tooney 1 , 3 , 4 1 School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, NSW, Australia 2 School of Medicine and Public Health, College of Health, Medicine and Wellbeing, University of Newcastle, NSW, Australia 3 Drug Repurposing and Medicines Research Program, Hunter Medical Research Institute, New Lambton, NSW, Australia 4 Mark Hughes Foundation Centre for Brain Cancer Research, University of Newcastle, NSW, Australia 5 GenesisCare, Newcastle, NSW, Australia 6 QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia 7 Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia Correspondence to: Paul A. Tooney, email: paul.tooney@newcastle.edu.au Keywords: glioblastoma; DNA damage response; ataxia-telangiectasia and rad3-related protein; radiation therapy; temozolomide Received: November 06, 2023&emsp;&emsp;&emsp;&emsp; Accepted: December 28, 2023&emsp;&emsp;&emsp;&emsp; Published: January 16, 2024 Copyright: &copy; 2024 Lozinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Glioblastoma cells can restrict the DNA-damaging effects of temozolomide (TMZ) and radiation therapy (RT) using the DNA damage response (DDR) mechanism which activates cell cycle arrest and DNA repair pathways. Ataxia-telangiectasia and Rad3-Related protein (ATR) plays a pivotal role in the recognition of DNA damage induced by chemotherapy and radiation causing downstream DDR activation. Here, we investigated the activity of gartisertib, a potent ATR inhibitor, alone and in combination with TMZ and/or RT in 12 patient-derived glioblastoma cell lines. We showed that gartisertib alone potently reduced the cell viability of glioblastoma cell lines, where sensitivity was associated with the frequency of DDR mutations and higher expression of the G2 cell cycle pathway. ATR inhibition significantly enhanced cell death in combination with TMZ and RT and was shown to have higher synergy than TMZ+RT treatment. MGMT promoter unmethylated and TMZ+RT resistant glioblastoma cells were also more sensitive to gartisertib. Analysis of gene expression from gartisertib treated glioblastoma cells identified the upregulation of innate immune-related pathways. Overall, this study identifies ATR inhibition as a strategy to enhance the DNA-damaging ability of glioblastoma standard treatment, while providing preliminary evidence that ATR inhibition induces an innate immune gene signature that warrants further investigation.

The superior temporal gyrus (STG), which encompasses the primary auditory cortex, is believed to be a major anatomical substrate for speech, language and communication. The STG connects to the limbic system (hippocampus and amygdala), the thalamus and neocortical association areas in the prefrontal cortex, all of which have been implicated in schizophrenia.To identify altered mRNA expression in the superior temporal gyrus (STG) in schizophrenia, oligonucleotide microarrays were used with RNA from postmortem STG tissue from 7 individuals with schizophrenia and 7 matched non-psychiatric controls. Overall, there was a trend towards down-regulation in gene expression, and altered expression of genes involved in neurotransmission, neurodevelopment, and presynaptic function was identified. To confirm altered expression identified by microarray analysis, the mRNA expression levels of four genes, IPLA2gamma, PIK31R1, Lin-7b and ATBF1, were semi-quantitatively measured using relative real-time PCR. A number of genes with altered expression in the STG were also shown to have similar changes in expression as shown in our previous study of peripheral blood lymphocytes in schizophrenia.This study has identified altered expression of genes in the STG involved in neurotransmission and neurodevelopment, and to a lesser extent presynaptic function, which further support the notion of these functions playing an integral role in the development of schizophrenia.

The molecular mechanisms underlying schizophrenia remain largely unknown. Although schizophrenia is a mental disorder, there is increasing evidence to indicate that inflammatory processes driven by diverse environmental factors play a significant role in its development. With gene expression studies having been conducted across a variety of sample types, e.g., blood and postmortem brain, it is possible to investigate convergent signatures that may reveal interactions between the immune and nervous systems in schizophrenia pathophysiology. We conducted two meta-analyses of schizophrenia microarray gene expression data (N=474) and non-psychiatric control (N=485) data from postmortem brain and blood. Then, we assessed whether significantly dysregulated genes in schizophrenia could be shared between blood and brain. To validate our findings, we selected a top gene candidate and analyzed its expression by RT-qPCR in a cohort of schizophrenia subjects stabilized by atypical antipsychotic monotherapy (N=29) and matched controls (N=31). Meta-analyses highlighted inflammation as the major biological process associated with schizophrenia and that the chemokine receptor CX3CR1 was significantly down-regulated in schizophrenia. This differential expression was also confirmed in our validation cohort. Given both the recent data demonstrating selective CX3CR1 expression in subsets of neuroimmune cells, as well as behavioral and neuropathological observations of CX3CR1 deficiency in mouse models, our results of reduced CX3CR1 expression adds further support for a role played by monocyte/microglia in the neurodevelopment of schizophrenia.

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)-present in some but not all cells-remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e-4), with recurrent somatic deletions of exons 1-5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5' deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk.

Glioblastoma, the most common primary malignant brain tumour in adults, is a highly vascular tumour characterised by abnormal angiogenesis. Additional mechanisms of tumour vascularisation have also been reported in glioblastoma, including the formation of tumour cell-derived vessels by vasculogenic mimicry (VM) or the transdifferentiation of tumour cells to endothelial cells. VM and endothelial transdifferentiation have frequently been reported as distinct processes, however, the use of both terms to describe a single process of vascularisation also occurs. Some overlapping characteristics have also been reported when identifying each process. We therefore aimed to determine the markers consistently attributed to VM and endothelial transdifferentiation in the glioblastoma literature.Ovid MEDLINE and Ovid Embase were searched for studies published between January 1999 and July 2021 that assessed VM or tumour to endothelial transdifferentiation in human glioblastoma. The online systematic review tool Covidence was used for screening and data extraction. Extracted data included type of tumour-derived vasculature reported, methods and techniques used, and markers investigated. Studies were grouped based on type of vasculature reported for further assessment.One hundred and thirteen of the 419 unique records identified were included for analysis. VM was reported in 64/113 studies, while tumour to endothelial transdifferentiation was reported in 16/113 studies. The remaining studies used both terms to describe a single process, did not define the process that occurred, or concluded that neither VM nor endothelial transdifferentiation occurred. Absence of CD34 and/or CD31 in vascular structures was the most common indicator of VM, while expression of CD34 and/or CD31, in addition to various other endothelial, stem cell or tumour cell markers, indicated tumour to endothelial transdifferentiation.Cells derived from tumour to endothelial transdifferentiation express typical endothelial markers including CD34 and CD31, while tumour cells contributing to VM lack CD34 and CD31 expression. Additional tumour markers are required to identify transdifferentiation in glioblastoma tissue, and this process requires further characterisation.

MicroRNA are small RNAs that provide specificity for the RNA induced silencing complex, which forms the basis of an exquisite combinatorial system for posttranscriptional regulation. This system, essential for complex metazoans, is exemplified in the development of the cerebral cortex. To explore the complexity of human cortical miRNA expression in detail, we analyzed RNA from postmortem prefrontal cortex from 97 subjects aged 2 months to 78 years using miRNA microarray. Global miRNA expression was highest in the early years before declining significantly after adolescence (n = 140 decreased, n = 32 increased). Late adolescence was also marked by an inflection point between miRNA on an upward trajectory vs the majority going down. Functional annotation of target genes displaying inverse mRNA expression patterns in the same tissue were overrepresented in neurodevelopmentally significant pathways including neurological disease (most significantly schizophrenia), nervous system development, and cell-to-cell signaling. As mature miRNA expression is largely posttranscriptionally regulated, miRNA biogenesis gene expression was also examined. Dicer and Exportin-5 displayed significant associations with age; however, neither correlated with global miRNA expression across the lifespan. This investigation of cortical miRNA expression provides a framework for understanding the complex posttranscriptional regulatory environment during development and aging that may form a substrate for changes observed in neurodevelopmental disorders.

Since their discovery, microRNAs (miRNA) have been implicated in a vast array of biological processes in animals, from fundamental developmental functions including cellular proliferation and differentiation, to more complex and specialized roles such as long-term potentiation and synapse-specific modifications in neurons. This review recounts the history behind this paradigm shift, which has seen small non-coding RNA molecules coming to the forefront of molecular biology, and introduces their role in establishing developmental complexity in animals. The fundamental mechanisms of miRNA biogenesis and function are then considered, leading into a discussion of recent discoveries transforming our understanding of how these molecules regulate gene network behaviour throughout developmental and pathophysiological processes. The emerging complexity of this mechanism is also examined with respect to the influence of cellular context on miRNA function. This discussion highlights the absolute imperative for experimental designs to appreciate the significance of context-specific factors when determining what genes are regulated by a particular miRNA. Moreover, by establishing the timing, location, and mechanism of these regulatory events, we may ultimately understand the true biological function of a specific miRNA in a given cellular environment.

With an estimated 80% heritability, molecular genetic research into schizophrenia has remained inconclusive. Recent large-scale, genome-wide association studies only identified a small number of susceptibility genes with individually very small effect sizes. However, the variable expression of the phenotype is not well captured in diagnosis-based research as well as when assuming a 'heterogenic risk model' (as apposed to a monogenic or polygenic model). Hence, the expression of susceptibility genes in response to environmental factors in concert with other disease-promoting or protecting genes has increasingly attracted attention.The current review summarises findings of microarray gene expression research with relevance to schizophrenia as they emerged over the past decade.Most findings from post mortem, peripheral tissues and animal models to date have linked altered gene expression in schizophrenia to presynaptic function, signalling, myelination, neural migration, cellular immune mechanisms, and response to oxidative stress consistent with multiple small effects of many individual genes. However, the majority of results are difficult to interpret due to small sample sizes (i.e. potential type-2 errors), confounding factors (i.e. medication effects) or lack of plausible neurobiological theory.Nevertheless, microarray gene expression research is likely to play an important role in the future when investigating gene/gene and gene/environment interactions by adopting a neurobiologically sound theoretical framework.

Changes in the cortical expression of small non-coding microRNA (miRNA) have been observed in postmortem analysis of psychotic disorders. Antipsychotic drugs (APDs) are the most effective treatment option for these disorders and have been associated with changes in gene expression. MicroRNA regulate numerous genes involved in brain development and function. It is therefore plausible to question whether miRNA expression is also altered and hence whether they take part in the neuroleptic mechanism of action.We sought to investigate whether treatment with APDs induces changes in miRNA expression and query the functional implications of such changes. Furthermore, we investigated the possible functional interplay of miRNA-gene regulatory interactions.High-throughput miRNA profiling of the whole brain of C57BL/6 mice treated with haloperidol, olanzapine or clozapine for 7 days was performed. Functional analysis was conducted on the putative targets of altered microRNA. Significant miRNA-gene regulatory interactions were evaluated by the integration of genome-wide mRNA expression analysis using the Bayesian networks with splitting-averaging strategy and functional analysis conducted.Small subsets of miRNA were altered with each treatment with potential neurologically relevant influence. Metabolic pathways were enriched in olanzapine and clozapine treatments, possibly associated with their weight gain side effects. Neurologically and metabolically relevant miRNA-gene interaction networks were identified in the olanzapine treatment group.This study is the first to suggest a role for miRNA in the mechanism of APD action and the metabolic side effects of the atypical ADPs, and adds support for their consideration in pharmacogenomics.

Schizophrenia has a strong genetic basis, and genome-wide association studies (GWAS) have shown that effect sizes for individual genetic variants which increase disease risk are small, making detection and validation of true disease-associated risk variants extremely challenging. Specifically, we first identify genes with exons showing differential expression between cases and controls, indicating a splicing mechanism that may contribute to variation in disease risk and focus on those showing consistent differential expression between blood and brain tissue. We then perform a genome-wide screen for SNPs associated with both normalised exon intensity of these genes (so called splicing QTLs) as well as association with schizophrenia. We identified a number of significantly associated loci with a biologically plausible role in schizophrenia, including MCPH1, DLG3, ZC3H13, and BICD2, and additional loci that influence splicing of these genes, including YWHAH. Our approach of integrating genome-wide exon intensity with genome-wide polymorphism data has identified a number of plausible SZ associated loci.

Increased neuronal density, cortical thinning, and alterations of GABAergic interneurons in the prefrontal cortex have been associated with the pathophysiology of schizophrenia. This study used antibodies directed against the calcium-binding proteins, calretinin (CR), parvalbumin (PV), and calbindin (CB) to compare the relative density of subpopulations of GABAergic interneurons in BA9 of the prefrontal cortex from six subjects with schizophrenia and six control subjects matched for age, gender, and postmortem interval. The relative density of interneurons expressing CR, PV, or CB did not differ significantly between subjects with schizophrenia and control subjects. In addition, no change in somal size of immunoreactive (IR) neurons or cortical thickness was observed between the two groups. This study supports previous reports consistently demonstrating no change in the relative density of interneurons expressing CR in the dorsolateral prefrontal cortex in schizophrenia but does not support previous inconsistent findings that the relative density of interneurons expressing PV and CB might be altered in this disorder.

The amygdala is implicated in the pathophysiology of schizophrenia through its function in the processing of emotions. However, the genes involved in the dysfunction of the amygdala in schizophrenia are yet to be identified. This study examined gene expression in the amygdala in postmortem tissue from seven matched pairs of schizophrenia and non-psychiatric control subjects, using oligonucleotide-microarrays representing 19,000 gene transcripts and real-time PCR confirmation of gene expression changes in eleven matched pairs. Genes involved in presynaptic function, myelination and cellular signalling were identified as being consistently dysregulated in this cohort of subjects with schizophrenia. In particular, the expression of three genes involved in the cytomatrix active zone, Regulating membrane exocytosis 2, Regulating membrane exocytosis 3 and Piccolo, was up-regulated. These results implicate for the first time the dysfunction of the cytomatrix active zone of synapses in the amygdala in the pathophysiology of schizophrenia.

To identify altered mRNA expression of regulator of G-protein signalling 4 (RGS4) in the superior temporal gyrus (STG) in schizophrenia mRNA expression of RGS4 was measured from post-mortem STG tissue from 13 individuals with schizophrenia and 13 matched non-psychiatric controls using relative real-time PCR. Significantly decreased expression of RGS4 mRNA in the STG in schizophrenia was identified in 10 of the 13 matched pairs. Altered expression of RGS4 in cortical regions previously implicated in schizophrenia, such as the STG further supports the notion of RGS4 as a potential genetic and functional biological marker of schizophrenia.

The FOXP2 gene is involved in the development of speech and language. As some single nucleotide polymorphisms (SNPs) of FOXP2 have been found to be associated with auditory verbal hallucinations (AVHs) at trend levels, this study set out to undertake the first examination into whether interactions between candidate FOXP2 SNPs and environmental factors (specifically, child abuse) predict the likelihood of AVHs.Data on parental child abuse and FOXP2 SNPs previously linked to AVHs (rs1456031, rs2396753, rs2253478) were obtained from the Australian Schizophrenia Research Bank for people with schizophrenia-spectrum disorders, both with (n = 211) and without (n = 122) a lifetime history of AVHs.Genotypic frequencies did not differ between the two groups; however, logistic regression found that childhood parental emotional abuse (CPEA) interacted with rs1456031 to predict lifetime experience of AVH. CPEA was only associated with significantly higher levels of AVHs in people with CC genotypes (odds ratio = 4.25), yet in the absence of CPEA, people with TT genotypes had significantly higher levels of AVHs than people with CC genotypes (odds ratio = 4.90). This interaction was specific to auditory verbal hallucinations, and did not predict the likelihood of non-verbal auditory hallucinations.Our findings offer tentative evidence that FOXP2 may be a susceptibility gene for AVHs, influencing the probability people experience AVHs in the presence and absence of CPEA. However, these findings are in need of replication in a larger study that addresses the methodological limitations of the present investigation.

Cognitive deficits are a core feature of schizophrenia and contribute significantly to functional disability. We investigated the molecular pathways associated with schizophrenia (SZ; n=47) cases representing both 'cognitive deficit' (CD; n=22) and 'cognitively spared' (CS; n=25) subtypes of schizophrenia (based on latent class analysis of 9 cognitive performance indicators), compared with 49 healthy controls displaying 'normal' cognition. This was accomplished using gene-set analysis of transcriptome data derived from peripheral blood mononuclear cells (PBMCs). We detected 27 significantly altered pathways (19 pathways up-regulated and 8 down-regulated) in the combined SZ group and a further 6 pathways up-regulated in the CS group and 5 altered pathways (4 down-regulated and 1 up-regulated) in the CD group. The transcriptome profiling in SZ and cognitive subtypes were characterized by the up-regulated pathways involved in immune dysfunction (e.g., antigen presentation in SZ), energy metabolism (e.g., oxidative phosphorylation), and down-regulation of the pathways involved in neuronal signaling (e.g., WNT in SZ/CD and ERBB in SZ). When we looked for pathways that differentiated the two cognitive subtypes we found that the WNT signaling was significantly down-regulated (FDR<0.05) in the CD group in accordance with the combined SZ cohort, whereas it was unaffected in the CS group. This suggested suppression of WNT signaling was a defining feature of cognitive decline in schizophrenia. The WNT pathway plays a role in both the development/function of the central nervous system and peripheral tissues, therefore its alteration in PBMCs may be indicative of an important genomic axis relevant to cognition in the neuropathology of schizophrenia.

Interstitial neurons are located among white matter tracts of the human and rodent brain. Post-mortem studies have identified increased interstitial white matter neuron (IWMN) density in the fibre tracts below the cortex in people with schizophrenia. The current study assesses IWMN pathology in a model of maternal immune activation (MIA); a risk factor for schizophrenia. Experimental MIA was produced by an injection of polyinosinic:polycytidylic acid (PolyI:C) into pregnant rats on gestational day (GD) 10 or GD19. A separate control group received saline injections. The density of neuronal nuclear antigen (NeuN(+)) and somatostatin (SST(+)) IWMNs was determined in the white matter of the corpus callosum in two rostrocaudally adjacent areas in the 12week old offspring of GD10 (n=10) or GD19 polyI:C dams (n=18) compared to controls (n=20). NeuN(+) IWMN density trended to be higher in offspring from dams exposed to polyI:C at GD19, but not GD10. A subpopulation of these NeuN(+) IWMNs was shown to express SST. PolyI:C treatment of dams induced a significant increase in the density of SST(+) IWMNs in the offspring when delivered at both gestational stages with more regionally widespread effects observed at GD19. A positive correlation was observed between NeuN(+) and SST(+) IWMN density in animals exposed to polyI:C at GD19, but not controls. This is the first study to show that MIA increases IWMN density in adult offspring in a similar manner to that seen in the brain in schizophrenia. This suggests the MIA model will be useful in future studies aimed at probing the relationship between IWMNs and schizophrenia.

Schizophrenia-associated anomalies in gene expression in postmortem brain can be attributed to a combination of genetic and environmental influences. Given the small effect size of common variants, it is likely that we may only see the combined impact of some of these at the pathway level in small postmortem studies. At the gene level, however, there may be more impact from common environmental exposures mediated by influential epigenomic modifiers, such as microRNA (miRNA). We hypothesise that dysregulation of miRNAs and their alteration of gene expression have significant implications in the pathophysiology of schizophrenia. In this study, we integrate changes in cortical gene and miRNA expression to identify regulatory interactions and networks associated with the disorder. Gene expression analysis in post-mortem prefrontal dorsolateral cortex (BA 46) (n = 74 matched pairs of schizophrenia, schizoaffective, and control samples) was integrated with miRNA expression in the same cohort to identify gene–miRNA regulatory networks. A significant gene–miRNA interaction network was identified, including miR-92a, miR-495, and miR-134, which converged with differentially expressed genes in pathways involved in neurodevelopment and oligodendrocyte function. The capacity for miRNA to directly regulate gene expression through respective binding sites in BCL11A, PLP1, and SYT11 was also confirmed to support the biological relevance of this integrated network model. The observations in this study support the hypothesis that miRNA dysregulation is an important factor in the complex pathophysiology of schizophrenia.

Abstract Schizophrenia is considered a neurodevelopmental disorder as it often manifests before full brain maturation and is also a cerebral cortical disorder where deficits in GABAergic interneurons are prominent. Whilst most neurons are located in cortical and subcortical grey matter regions, a smaller population of neurons reside in white matter tracts of the primate and to a lesser extent, the rodent brain, subjacent to the cortex. These interstitial white matter neurons (IWMNs) have been identified with general markers for neurons [e.g., neuronal nuclear antigen (NeuN)] and with specific markers for neuronal subtypes such as GABAergic neurons. Studies of IWMNs in schizophrenia have primarily focused on their density underneath cortical areas known to be affected in schizophrenia such as the dorsolateral prefrontal cortex. Most of these studies of postmortem brains have identified increased NeuN+ and GABAergic IWMN density in people with schizophrenia compared to healthy controls. Whether IWMNs are involved in the pathogenesis of schizophrenia or if they are increased because of the cortical pathology in schizophrenia is unknown. We also do not understand how increased IWMN might contribute to brain dysfunction in the disorder. Here we review the literature on IWMN pathology in schizophrenia. We provide insight into the postulated functional significance of these neurons including how they may contribute to the pathophysiology of schizophrenia.

Thrombospondin-1 and -2 (TSP1 and TSP2) are multifunctional, multimodular extracellular matrix proteins encoded by separate genes. We compared the distributions of TSP1 and TSP2 in mouse embryos (day 10 and later) by immunohistochemistry. TSP1 was detected on day 10 in the heart and intestinal epithelium, on day 11 in megakaryocytes, and on day 14 in the lung. TSP2 was not detected until day 14, with strongest staining in mesenchymal condensation that gives rise to cartilage and bone. The distribution of TSP2 was different from but overlapped with the distribution of TSP1. TSP1 was found in cartilage proper with diminished staining around chondrocytes undergoing differentiation and hypertrophy, whereas TSP2 was restricted to the matrix surrounding chondrocytes of the growth zone cartilage. TSP2 and TSP1 were both expressed in centers of intramembranous ossification that form the skull bones, in reticular dermis, on the apical surface of nasal epithelium, in skeletal muscle, and in the sheath surrounding vibrissae. Areas of exclusive staining for TSP2 included the perichondrium surrounding the cartilage of the nasal cavities, developing bone of the lower mandible, and adrenal gland. The distinct localizations of TSP1 and TSP2 indicate that the two proteins have specific functions during mouse embryogenesis.

Common variants of the FK506 binding protein 5 (FKBP5) gene are implicated in psychotic and other disorders, via their role in regulating glucocorticoid receptor (GR) receptor sensitivity and effects on the broader function of the HPA system in response to stress. In this study, the effects of four FKBP5 polymorphisms (rs1360780, rs9470080, rs4713902, rs9394309) on IQ and eight other cognitive domains were examined in the context of exposure to childhood maltreatment in 444 cases with schizophrenia and 292 healthy controls (from a total sample of 617 cases and 659 controls obtained from the Australian Schizophrenia Research Bank; ASRB). Participants subjected to any kind of maltreatment (including physical, emotional, or sexual abuse or physical or emotional neglect) in childhood were classified as ‘exposed’; cognitive functioning was measured with Repeatable Battery for the Assessment of Neuropsychological Status, the Controlled Oral Word Association Test, and IQ was estimated with the Weschler Test of Adult Reading. Hierarchical regressions were used to test the main effects of genotype and childhood maltreatment, and their additive interactive effects, on cognitive function. For rs1360870, there were significant main effects of genotype and childhood maltreatment, and a significant interaction of genotype with childhood trauma affecting attention in both schizophrenia and healthy participants (C-homozygotes in both groups showed worse attention in the context of maltreatment); in SZ, this SNP also affected global neuropsychological function regardless of exposure to childhood trauma, with T-homozygotes showing worse cognition than other genotypes. The mechanisms of trauma-dependent effects of FKBP5 following early life trauma deserve further exploration in healthy and psychotic samples, in the context of epigenetic effects and perhaps epistasis with other genes. Study of these processes may be particularly informative in subgroups exposed to various other forms of early life adversity (i.e., birth complications, immigration).

We performed a transcriptome-wide meta-analysis and gene co-expression network analysis to identify genes and gene networks dysregulated in the peripheral blood of bipolar disorder (BD) cases relative to unaffected comparison subjects, and determined the specificity of the transcriptomic signatures of BD and schizophrenia (SZ). Nineteen genes and 4 gene modules were significantly differentially expressed in BD cases. Thirteen gene modules were shown to be differentially expressed in a combined case-group of BD and SZ subjects called “major psychosis”, including genes biologically linked to apoptosis, reactive oxygen, chromatin remodeling, and immune signaling. No modules were differentially expressed between BD and SZ cases. Machine-learning classifiers trained to separate diagnostic classes based solely on gene expression profiles could distinguish BD cases from unaffected comparison subjects with an area under the curve (AUC) of 0.724, as well as BD cases from SZ cases with AUC = 0.677 in withheld test samples. We introduced a novel and straightforward method called “polytranscript risk scoring” that could distinguish BD cases from unaffected subjects (AUC = 0.672) and SZ cases (AUC = 0.607) significantly better than expected by chance. Taken together, our results highlighted gene expression alterations common to BD and SZ that involve biological processes of inflammation, oxidative stress, apoptosis, and chromatin regulation, and highlight disorder-specific changes in gene expression that discriminate the major psychoses.

Antipsychotic drugs (APDs) can have a profound effect on the human body that extends well beyond our understanding of their neuropsychopharmacology. Some of these effects manifest themselves in peripheral blood lymphocytes, and in some cases, particularly in clozapine treatment, result in serious complications. To better understand the molecular biology of APD action in lymphocytes, we investigated the influence of chlorpromazine, haloperidol and clozapine in vitro, by microarray-based gene and microRNA (miRNA) expression analysis. JM-Jurkat T-lymphocytes were cultured in the presence of the APDs or vehicle alone over 2 wk to model the early effects of APDs on expression. Interestingly both haloperidol and clozapine appear to regulate the expression of a large number of genes. Functional analysis of APD-associated differential expression revealed changes in genes related to oxidative stress, metabolic disease and surprisingly also implicated pathways and biological processes associated with neurological disease consistent with current understanding of the activity of APDs. We also identified miRNA-mRNA interaction associated with metabolic pathways and cell death/survival, all which could have relevance to known side effects of APDs. These results indicate that APDs have a significant effect on expression in peripheral tissue that relate to both known mechanisms as well as poorly characterized side effects.

Neurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood.Here we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23 degrees C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 +/- 3.9 vs. 64.7 +/- 5.0 pA; n = 25 each), decay time was slower (8.5 +/- 0.8 vs. 5.5 +/- 0.3 ms), and frequency was lower (0.15 +/- 0.03 vs. 0.72 +/- 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 +/- 2.4, n = 14 vs. 25. +/- 2.0 pA, n = 18) and frequencies (0.21 +/- 0.08 vs. 0.18 +/- 0.04 Hz) in both regions; however, decay times were slower (23.0 +/- 3.2 vs. 18.9 +/- 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 +/- 1.6 pS, n = 11 vs. 55.7 +/- 1.8, n = 8) and GABAARs (22.7 +/- 1.7 pS, n = 10 vs. 22.4 +/- 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 muM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons.Together these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.

Abstract Background MicroRNA (miRNA) are small non-coding RNA molecules which function as nucleic acid-based specificity factors in the universal RNA binding complex known as the RNA induced silencing complex (RISC). In the canonical gene-silencing pathway, these activated RISC particles are associated with RNA decay and gene suppression, however, there is evidence to suggest that in some circumstances they may also stabilise their target RNA and even enhance translation. To further explore the role of miRNA in this context, we performed a genome-wide expression analysis to investigate the molecular consequences of bidirectional modulation of the disease-associated miRNAs miR-181b and miR-107 in multiple human cell lines. Results This data was subjected to pathways analysis and correlated against miRNA targets predicted through seed region homology. This revealed a large number of both conserved and non-conserved miRNA target genes, a selection of which were functionally validated through reporter gene assays. Contrary to expectation we also identified a significant proportion of predicted target genes with both conserved and non-conserved recognition elements that were positively correlated with the modulated miRNA. Finally, a large proportion of miR-181b associated genes devoid of the corresponding miRNA recognition element, were enriched with binding motifs for the E2F1 transcription factor, which is encoded by a miR-181b target gene. Conclusions These findings suggest that miRNA regulate target genes directly through interactions with both conserved and non-conserved target recognition elements, and can lead to both a decrease and increase in transcript abundance. They also multiply their influence through interaction with transcription factor genes exemplified by the observed miR-181b/E2F1 relationship.

Abstract The immune system is implicated in the pathogenesis of schizophrenia, with elevated proinflammatory cytokine mRNAs found in the brains of ~40% of individuals with the disorder. However, it is not clear if antibodies (specifically immunoglobulin-γ (IgG)) can be found in the brain of people with schizophrenia and if their abundance relates to brain inflammatory cytokine mRNA levels. Therefore, we investigated the localization and abundance of IgG in the frontal cortex of people with schizophrenia and controls, and the impact of proinflammatory cytokine status on IgG abundance in these groups. Brain IgGs were detected surrounding blood vessels in the human and non-human primate frontal cortex by immunohistochemistry. IgG levels did not differ significantly between schizophrenia cases and controls, or between schizophrenia cases in ‘high’ and ‘low’ proinflammatory cytokine subgroups. Consistent with the existence of IgG in the parenchyma of human brain, mRNA and protein of the IgG transporter (FcGRT) were present in the brain, and did not differ according to diagnosis or inflammatory status. Finally, brain-reactive antibody presence and abundance was investigated in the blood of living people. The plasma of living schizophrenia patients and healthy controls contained antibodies that displayed positive binding to Rhesus macaque cerebellar tissue, and the abundance of these antibodies was significantly lower in patients than controls. These findings suggest that antibodies in the brain and brain-reactive antibodies in the blood are present under normal circumstances.

The aetiology of schizophrenia is complex, heterogeneous, and involves interplay of many genetic and environmental influences. While significant progress has been made in the understanding the common heritable component, we are still grappling with the genomic encoding of environmental risk. One class of molecule that has tremendous potential is miRNA. These molecules are regulated by genetic and environmental factors associated with schizophrenia and have a very significant impact on temporospatial patterns of gene expression. To better understand the relationship between miRNA and gene expression in the disorder we analysed these molecules in RNA isolated from peripheral blood mononuclear cells (PBMCs) obtained from an Australian cohort of 36 individuals with schizophrenia and 15 healthy controls using next-generation RNA sequencing. Significant changes in both mRNA and miRNA expression profiles were observed implicating important interaction networks involved in immune activity and development. We also observed sexual dimorphism, particularly in relation to variation in mRNA, with males showing significantly more differentially expressed genes. Interestingly, while we explored expression in lymphocytes, the systems biology of miRNA-mRNA interactions was suggestive of significant pleiotropy with enrichment of networks related to neuronal activity.

Glioblastoma is a highly aggressive, invasive and treatment-resistant tumour. The DNA damage response (DDR) provides tumour cells with enhanced ability to activate cell cycle arrest and repair treatment-induced DNA damage. We studied the expression of DDR, its relationship with standard treatment response and patient survival, and its activation after treatment. The transcriptomic profile of DDR pathways was characterised within a cohort of isocitrate dehydrogenase (IDH) wild-type glioblastoma from The Cancer Genome Atlas (TCGA) and 12 patient-derived glioblastoma cell lines. The relationship between DDR expression and patient survival and cell line response to temozolomide (TMZ) or radiation therapy (RT) was assessed. Finally, the expression of 84 DDR genes was examined in glioblastoma cells treated with TMZ and/or RT. Although distinct DDR cluster groups were apparent in the TCGA cohort and cell lines, no significant differences in OS and treatment response were observed. At the gene level, the high expression of ATP23, RAD51C and RPA3 independently associated with poor prognosis in glioblastoma patients. Finally, we observed a substantial upregulation of DDR genes after treatment with TMZ and/or RT, particularly in RT-treated glioblastoma cells, peaking within 24 h after treatment. Our results confirm the potential influence of DDR genes in patient outcome. The observation of DDR genes in response to TMZ and RT gives insight into the global response of DDR pathways after adjuvant treatment in glioblastoma, which may have utility in determining DDR targets for inhibition.

Glioblastoma is a highly aggressive disease with poor survival outcomes. An emerging body of literature links the role of the renin–angiotensin system (RAS), well-known for its function in the cardiovascular system, to the progression of cancers. We studied the expression of RAS-related genes (ATP6AP2, AGTR1, AGTR2, ACE, AGT, and REN) in The Cancer Genome Atlas (TCGA) glioblastoma cohort, their relationship to patient survival, and association with tumour microenvironment pathways. The expression of RAS genes was then examined in 12 patient-derived glioblastoma cell lines treated with chemoradiation. In cases of glioblastoma within the TCGA, ATP6AP2, AGTR1, ACE, and AGT had consistent expressions across samples, while AGTR2 and REN were lowly expressed. High expression of AGTR1 was independently associated with lower progression-free survival (PFS) (p = 0.01) and had a non-significant trend for overall survival (OS) after multivariate analysis (p = 0.095). The combined expression of RAS receptors (ATP6AP2, AGTR1, and AGTR2) was positively associated with gene pathways involved in hypoxia, microvasculature, stem cell plasticity, and the molecular characterisation of glioblastoma subtypes. In patient-derived glioblastoma cell lines, ATP6AP2 and AGTR1 were upregulated after chemoradiotherapy and correlated with an increase in HIF1A expression. This data suggests the RAS is correlated with changes in the tumour microenvironment and associated with glioblastoma survival outcomes.

The distribution of tachykinin NK(1) and NK(3) receptors in the prefrontal (Brodmann area 9) and visual cortex (Brodmann area 17) of formalin-fixed postmortem human brain tissue was studied by immunohistochemistry. NK(1)-like immunoreactivity (NK(1)-LI) was observed as a thin band at the cortical surface and dots of NK(1)-LI localised on small non-pyramidal cells and in the neuropil (layers I-III). NK(3)-LI was found in beaded fibres and cells with astrocyte-like morphology in the superficial cortical layers and white matter. Dots of NK(3)-LI were prominent in the neuropil and on pyramidal (layers III/V) and non-pyramidal (layers V/VI) cells. The NK(3)-LI was more abundant and widespread than the NK(1)-LI. This is the first report of the distribution of the NK(1) receptor in the prefrontal and visual cortex of the human brain by immunohistochemistry.

The cytomatrix active zone (CAZ) is a specialized cellular structure regulating release of vesicles. We reported previously increased expression of three CAZ genes, piccolo, RIMS2 and RIMS3 in the amygdala in schizophrenia. This study determined the levels of gene and protein expression for components of the active zone including two additional CAZ genes in the amygdala from subjects with schizophrenia and non-psychiatric controls, as well as the effects of antipsychotic drugs. Whilst relative real-time PCR analysis did not identify significant change in the expression of six additional active zone genes, Western blot analysis showed increased piccolo and RIMS2 protein expression in the amygdala in schizophrenia. In vitro analysis suggests antipsychotic drug treatment was unlikely to have caused the changes in RIMS2, RIMS3 and piccolo expression observed in the amygdala in schizophrenia. Therefore, this study provides further evidence suggesting that piccolo, RIMS2, RIMS3, but not the entire components of the active zone are involved in the neurobiology of schizophrenia.

In schizophrenia, abnormal synaptic pruning during adolescence may be due to altered expression of the Complement component 4 (C4). Overexpression of C4 genes has been identified in the total cerebral cortex and in 6 different brain regions of schizophrenic patients compared to controls. These alterations should be replicated and extended to other brain regions relevant to schizophrenia. Moreover, it remains unknown whether cerebral and peripheral C4 expression levels are related.We explored C4 genes expression both at the cerebral and peripheral levels. Using shinyGEO application we analyzed C4 expression from eight Gene Expression Omnibus datasets obtained from 196 schizophrenic patients and 182 control subjects. First, we compared C4 expression between schizophrenic patients and controls in postmortem cerebral samples from 7 different brain regions. Then, we compared C4 expression between schizophrenic patients and controls in 4 peripheral tissues.At the cerebral level, we provide further evidence of C4 overexpression in schizophrenic patients. Consistently with a previous report, we found C4 overexpression in the dorsolateral prefrontal cortex and in the parietal cortex of schizophrenic patients. The observation of C4 overexpression was further extended to the superior temporal cortex and the associative striatum of schizophrenic patients. Conversely, no significant alteration of C4 expression was observed in peripheral tissues.Our results support the hypothesis of an excessive Complement activity in various brain regions of schizophrenic patients which may disrupt the synaptic pruning process occurring during adolescence. C4 overexpression may be specific to the cerebral tissue while other alterations of the Complement system may be detected at the systemic level.

Background: Changes in levels of substance P and substance P-binding sites have been implicated in schizophrenia. However, no studies have used receptor-specific antibodies to directly investigate the substance P (neurokinin 1) receptor in schizophrenia. Methods: We used an antibody directed against the human neurokinin-1 receptor to compare the distribution of neurokinin-1 receptors in the prefrontal cortices from six subjects with schizophrenia and six control subjects, matched for age, gender, and postmortem interval. Results: In control tissue, dots of neurokinin-1 receptor immunoreactivity were observed in layer I to upper/mid layer III only. In contrast, dots of neurokinin-1 receptor immunoreactivity were observed in all layers of the prefrontal cortex in subjects with schizophrenia, and the density of dots was significantly greater than in control subjects. Conclusions: This is the first report of increased neurokinin-1 receptor immunoreactivity in the prefrontal cortex in subjects with schizophrenia. These changes may have implications for understanding the pathophysiology of the prefrontal cortex in schizophrenia and for the treatment of this disorder.

Previous research has demonstrated that individuals with schizophrenia who are homozygous at the c.267C>A single nucleotide polymorphism (rs2067477) within the cholinergic muscarinic M1 receptor (CHRM1) perform less well on the Wisconsin Card Sorting Test (WCST) than those who are heterozygous. This study sought to determine whether variation in the rs2067477 genotype was associated with differential changes in brain structure. Data from 227 patients with established schizophrenia or schizoaffective disorder were obtained from the Australian Schizophrenia Research Bank. Whole-brain voxel-based morphometry was performed to compare regional grey matter volume (GMV) between the 267C/C (N=191) and 267C/A (N=36) groups. Secondary analyses tested for an effect of genotype on cognition (the WCST was not available). Individuals who were homozygous (267C/C) demonstrated significantly reduced GMV in the right precentral gyrus compared to those who were heterozygous (267C/A). These preliminary results suggest that the rs2067477 genotype is associated with brain structure in the right precentral gyrus in individuals with schizophrenia/schizoaffective disorder. Future studies are required to replicate these results and directly link the volumetric reductions with specific cognitive processes.

1. The tachykinins are neuropeptides found in both the central and peripheral nervous systems that play a role in inflammation and pain mechanisms and some autonomic reflexes and behaviours. 2. Although the distribution of the tachykinin receptors has been described in the brains of various animal species, little is known about the distribution of the NK1 and NK3 receptors in the human brain. 3. The present paper examines the distribution of the NK1 and NK3 receptors in the prefrontal cortex of formalin-fixed postmortem human brain tissue by immunohistochemical techniques. 4. The majority of NK1 receptor immunoreactivity appeared as a thin band of punctate staining at the pial surface, with dark brown dots of NK1 receptor immunoreactivity predominantly scattered across the mid to upper cortical layers. 5. The NK3 receptor immunoreactivity was found in the glia limitans at the pial surface, where astrocytes and beaded fibres were intensely stained. Dots of NK3 receptor immunoreactivity were scattered across all cortical layers. In the white matter, astrocytes and beaded fibres displayed NK3 receptor immunoreactivity, particularly in areas surrounding blood vessels.

MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results.

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-β and TNF-α mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring.

Alterations in GABAergic interneurons and glutamic acid decarboxylase (GAD) are observed in the brains of people with schizophrenia. Studies also show increased density of interstitial white matter neurons (IWMN), including those containing GAD and somatostatin (SST) in the brain in schizophrenia. Maternal immune activation can be modelled in rodents to investigate the relationship between prenatal exposure to infections and increased risk of developing schizophrenia. We reported that maternal immune activation induced an increase in density of somatostatin-positive IWMN in the adult rat offspring. Here we hypothesised that maternal immune activation induced in pregnant rats by polyinosinic:polycytidylic acid would alter SST and GAD gene expression as well as increase the density of GAD-positive IWMNs in the adult offspring. SST gene expression was significantly reduced in the cingulate cortex of adult offspring exposed to late gestation maternal immune activation. There was no change in cortical GAD gene expression nor GAD-positive IWMN density in adults rats exposed to maternal immune activation at either early or late gestation. This suggests that our model of maternal immune activation induced by prenatal exposure of rats to polyinosinic:polycytidylic acid during late gestation is able to recapitulate changes in SST but not other GABAergic neuropathologies observed in schizophrenia.

Large-scale genetic analysis of common variation in schizophrenia has been a powerful approach to understanding this complex but highly heritable psychotic disorder. To further investigate loci, genes and pathways associated more specifically in the well-characterized Australian Schizophrenia Research Bank cohort, we applied genome-wide single-nucleotide polymorphism analysis in these three annotation categories.We performed a case-control genome-wide association study in 429 schizophrenia samples and 255 controls. Post-genome-wide association study analyses were then integrated with genomic annotations to explore the enrichment of variation at the gene and pathway level. We also examine candidate single-nucleotide polymorphisms with potential function within expression quantitative trait loci and investigate overall enrichment of variation within tissue-specific functional regulatory domains of the genome.The strongest finding (p = 2.01 × 10-6, odds ratio = 1.82, 95% confidence interval = [1.42, 2.33]) in genome-wide association study was with rs10252923 at 7q21.13, downstream of FZD1 (frizzled class receptor 1). While this did not stand alone after correction, the involvement of FZD1 was supported by gene-based analysis, which exceeded the threshold for genome-wide significance (p = 2.78 × 10-6).The identification of FZD1, as an independent association signal at the gene level, supports the hypothesis that the Wnt signalling pathway is altered in the pathogenesis of schizophrenia and may be an important target for therapeutic development.

Immunotherapies targeting tumour-infiltrating lymphocytes (TILs) that express the immune checkpoint molecule programmed cell death-1 (PD-1) have shown promise in preclinical glioblastoma models but have had limited success in clinical trials. To assess when glioblastoma is most likely to benefit from immune checkpoint inhibitors we determined the density of TILs in primary and recurrent glioblastoma. Thirteen cases of matched primary and recurrent glioblastoma tissue were immunohistochemically labelled for CD3, CD8, CD4 and PD-1, and TIL density assessed. CD3+ TILs were observed in all cases, with the majority of both primary (69.2%) and recurrent (61.5%) tumours having low density of TILs present. CD8+ TILs were observed at higher densities than CD4+ TILs in both tumour groups. PD-1+ TILs were sparse and present in only 25% of primary and 50% of recurrent tumours. Quantitative analysis of TILs demonstrated significantly higher CD8+ TIL density at recurrence (p = 0.040). No difference was observed in CD3+ (p = 0.191), CD4+ (p = 0.607) and PD-1+ (p = 0.070) TIL density between primary and recurrent groups. This study shows that TILs are present at low densities in both primary and recurrent glioblastoma. Furthermore, PD-1+ TILs were frequently absent, which may provide evidence as to why anti-PD-1 immunotherapy trials have been largely unsuccessful in glioblastoma.

Abstract While schizophrenia is considered a prototypical network disorder characterized by widespread brain-morphological alterations, it still remains unclear whether distributed structural alterations robustly reflect underlying network layout. Here, we tested whether large-scale structural alterations in schizophrenia relate to normative structural and functional connectome architecture, and systematically evaluated robustness and generalizability of these network-level alterations. Leveraging anatomical MRI scans from 2,439 adults with schizophrenia and 2,867 healthy controls from 26 ENIGMA sites and normative data from the Human Connectome Project (n=207), we evaluated structural alterations of schizophrenia against two network susceptibility models: i) hub vulnerability, which examines associations between regional network centrality and magnitude of disease-related alterations; ii) epicenter mapping, which identify regions whose typical connectivity profile most closely resembles the disease-related morphological alterations. To assess generalizability and specificity, we contextualized the influence of site, disease stages, and individual clinical factors and compared network associations of schizophrenia with that found in affective disorders. Schizophrenia-related structural alterations co-localized with interconnected functional and structural hubs and harbored temporo-paralimbic and frontal epicenters. Findings were robust across sites and related to individual symptom profiles. We observed localized unique epicenters for first-episode psychosis and early stages, and transmodal epicenters that were shared across first-episode to chronic stages. Moreover, transdiagnostic comparisons revealed overlapping epicenters in schizophrenia and bipolar, but not major depressive disorder, yielding insights in pathophysiological continuity within the schizophrenia-bipolar-spectrum. In sum, cortical alterations over the course of schizophrenia robustly follow brain network architecture, emphasizing marked hub susceptibility and temporo-frontal epicenters at both the level of the group and the individual. Subtle variations of epicenters across disease stages suggest interacting pathological processes, while associations with patient-specific symptoms support additional inter-individual variability of hub vulnerability and epicenters in schizophrenia. Our work contributes to recognizing potentially common pathways to better understand macroscale structural alterations, and inter-individual variability in schizophrenia.

Abstract In vivo experimental analysis of human brain tissue poses substantial challenges and ethical concerns. To address this problem, we developed a computational method called the Brain Gene Expression and Network-Imputation Engine ( BrainGENIE ) that leverages peripheral-blood transcriptomes to predict brain tissue-specific gene-expression levels. Paired blood–brain transcriptomic data collected by the Genotype-Tissue Expression (GTEx) Project was used to train BrainGENIE models to predict gene-expression levels in ten distinct brain regions using whole-blood gene-expression profiles. The performance of BrainGENIE was compared to PrediXcan , a popular method for imputing gene expression levels from genotypes. BrainGENIE significantly predicted brain tissue-specific expression levels for 2947–11,816 genes (false-discovery rate-adjusted p &lt; 0.05), including many transcripts that cannot be predicted significantly by a transcriptome-imputation method such as PrediXcan . BrainGENIE recapitulated measured diagnosis-related gene-expression changes in the brain for autism, bipolar disorder, and schizophrenia better than direct correlations from blood and predictions from PrediXcan . We developed a convenient software toolset for deploying BrainGENIE , and provide recommendations for how best to implement models. BrainGENIE complements and, in some ways, outperforms existing transcriptome-imputation tools, providing biologically meaningful predictions and opening new research avenues.

The aim of this prospective clinical study was to evaluate the potential of the prostate specific membrane antigen (PSMA) targeting ligand, [68Ga]-PSMA-Glu-NH-CO-NH-Lys-2-naphthyl-L-Ala-cyclohexane-DOTA ([68Ga]Ga-PSMA-617) as a positron emission tomography (PET) imaging biomarker in recurrent glioblastoma patients. Patients underwent [68Ga]Ga-PSMA-617 and O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET) PET scans on two separate days. [68Ga]Ga-PSMA-617 tumour selectivity was assessed by comparing tumour volume delineation and by assessing the intra-patient correlation between tumour uptake on [68Ga]Ga-PSMA-617 and [18F]FET PET images. [68Ga]Ga-PSMA-617 tumour specificity was evaluated by comparing its tumour-to-brain ratio (TBR) with [18F]FET TBR and its tumour volume with the magnetic resonance imaging (MRI) contrast-enhancing (CE) tumour volume. Ten patients were recruited in this study. [68Ga]Ga-PSMA-617-avid tumour volume was larger than the [18F]FET tumour volume (p = 0.063). There was a positive intra-patient correlation (median Pearson r = 0.51; p < 0.0001) between [68Ga]Ga-PSMA-617 and [18F]FET in the tumour volume. [68Ga]Ga-PSMA-617 had significantly higher TBR (p = 0.002) than [18F]FET. The [68Ga]Ga-PSMA-617-avid tumour volume was larger than the CE tumour volume (p = 0.0039). Overall, accumulation of [68Ga]-Ga-PSMA-617 beyond [18F]FET-avid tumour regions suggests the presence of neoangiogenesis in tumour regions that are not overly metabolically active yet. Higher tumour specificity suggests that [68Ga]-Ga-PSMA-617 could be a better imaging biomarker for recurrent tumour delineation and secondary treatment planning than [18F]FET and CE MRI.

The amygdala has a role in the modulation of moods and emotion, processes that are known to be affected in people with psychiatric disorders such as schizophrenia and depression. The tachykinin NK1 receptor is known to be expressed in the amygdala. However to date, there is limited knowledge of the distribution of the NK1 receptor in this region. This study used immunohistochemistry to analyse the distribution of the NK1 receptor in fixed human amygdala tissue in control subjects with no history of psychiatric illness and matched subjects with a diagnosis of schizophrenia (n = 4 pairs). The NK1 receptor was observed sparsely distributed in cell bodies in all amygdaloid nuclei with the basolateral and lateral having a greater relative density of NK1 receptor-immunoreactive cell bodies than the other nuclei. Double labelling with antibodies to microtubule associated protein and the NK1 receptor revealed that the NK1 receptor is expressed by large pyramidal, small stellate and large bipolar neurons. Interestingly, the basal nucleus of Meynert, which is just dorsal to the amygdala, was observed to have a significantly higher relative density of NK1 receptor-immunoreactive cell bodies compared to any of the amygdaloid nuclei. Preliminary analysis of the density of NK1 receptor-immunoreactive cell bodies in the major amygdaloid nuclei and the basal nucleus of Meynert revealed no significant differences between schizophrenia and control subjects. Real-time PCR showed that the mRNA for both the short and long isoforms of the NK1 receptor was expressed at low levels in fresh frozen human amygdala tissue from control subjects and that this was not different in matched subjects with schizophrenia (n = 11 pairs). In conclusion, this study has demonstrated that the NK1 receptor is widely distributed in the amygdala, and has shown for the first time a high relative density of NK1 receptor-immunoreactive cell bodies in the basal nucleus of Meynert.

Abstract BACKGROUND Sex differences in incidence and/or presentation of schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BIP) are pervasive. Previous evidence for shared genetic risk and sex differences in brain abnormalities across disorders suggest possible shared sex-dependent genetic risk. METHODS We conducted the largest to date genome-wide genotype–by–sex (GxS) interaction of risk for these disorders, using 85,735 cases (33,403 SCZ, 19,924 BIP, 32,408 MDD) and 109,946 controls from the Psychiatric Genomics Consortium (PGC) and iPSYCH. RESULTS Across disorders, genome-wide significant SNP-by-sex interaction was detected for a locus encompassing NKAIN2 (rs117780815; p =3.2×10 −8 ), that interacts with sodium/potassium-transporting ATPase enzymes implicating neuronal excitability. Three additional loci showed evidence ( p &lt;1×10 −6 ) for cross-disorder GxS interaction (rs7302529, p =1.6×10 −7 ; rs73033497, p =8.8×10 −7 ; rs7914279, p =6.4×10 −7 ) implicating various functions. Gene-based analyses identified GxS interaction across disorders ( p =8.97×10 −7 ) with transcriptional inhibitor SLTM . Most significant in SCZ was a MOCOS gene locus (rs11665282; p =1.5×10 −7 ), implicating vascular endothelial cells. Secondary analysis of the PGC-SCZ dataset detected an interaction (rs13265509; p =1.1×10 −7 ) in a locus containing IDO2 , a kynurenine pathway enzyme with immunoregulatory functions implicated in SCZ, BIP, and MDD. Pathway enrichment analysis detected significant GxS of genes regulating vascular endothelial growth factor (VEGF) receptor signaling in MDD ( p FDR &lt;0.05). CONCLUSIONS In the largest genome-wide GxS analysis of mood and psychotic disorders to date, there was substantial genetic overlap between the sexes. However, significant sex-dependent effects were enriched for genes related to neuronal development, immune and vascular functions across and within SCZ, BIP, and MDD at the variant, gene, and pathway enrichment levels.

Summary A model for cell movement is presented. It is suggested that cells do not migrate on collagen using their VLA (very late antigen) integrins that bind this extra cellular matrix protein. Rather, the cells utilize αv integrins to bind endogenously produced fibronectin, which binds to the underlying collagen. It is envisaged that cells proceed by a process of engagement and disengagement of αv integrins to the extracellular matrix, somewhat analogous to the motion of a monkey climbing a tree. Secretion of isoforms of the adhesion modulator, thrombospondin, regulates disengagement of the integrin from its ligand in migrating cells. The integrin disengagement signal is mediated by thrombospondin cross‐linking the αv integnn to an integrin accessory molecule and thus activating protein kinases. The cross‐linked receptor complex undergoes recycling back along actin stress fibres, guided by the integrin β‐subunit. After endocytosis and protein sorting the αv integrin is transported back to the leading edge off migrating cells in vesicles guided by the tubulin‐binding capabilities of an integrin accessory molecule. Direct attachment to collagen required for processes, such as matrix contraction, is mediated by VLA integrins which displace αv integrins from points of attachment during integrin recycling, possibly through an αvβ 1 , intermediary receptor.

Xeroderma pigmentosum (XP) is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4) photoproducts. Nucleotide excision repair (NER) orchestrates the removal of cyclobutane dimers and (6-4) photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G), which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined. Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E) and the severest form (XP-A) of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G) and without (XP-C, XP-E and XP-F) were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.

Schizophrenia is a complex disease believed to result from a combination of risk genes and environmental insults that change the trajectory of brain development. For decades researchers have strived to unravel the genetic complexity of schizophrenia, using a variety of approaches. If one can understand the nature of the risk genes, then we might better understand what causes schizophrenia, leading to better diagnosis and treatment strategies. One underlying theme has been to relate changes in genes to biological dysfunction in the brain. Indeed, in the late 1990s many researchers turned to microarray analyses of gene expression in attempts to understand what genes have altered expression in the brain in schizophrenia (Mirnics et al., 2000Mirnics K. Middleton F.A. Marquez A. Lewis D.A. Levitt P. Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex.Neuron. 2000; 28: 53-67Summary Full Text Full Text PDF PubMed Scopus (774) Google Scholar) and hence what biological pathways might be affected. These studies showed that the expression of large numbers of genes is altered in the brains of people with schizophrenia (Bowden et al., 2008Bowden N.A. Scott R.J. Tooney P.A. Altered gene expression in the superior temporal gyrus in schizophrenia.BMC Genomics. 2008; 9: 199Crossref PubMed Scopus (61) Google Scholar), but what caused this was unknown. The central dogma of gene regulation that DNA codes for mRNA which is translated into proteins, was challenged by the discovery of a new mechanism for regulation of protein coding genes via non-coding RNA species including microRNAs (miRNA) (Lee et al., 1993Lee R.C. Feinbaum R.L. Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell. 1993; 75: 843-854Summary Full Text PDF PubMed Scopus (9698) Google Scholar). One single miRNA could bind to and affect hundreds of mRNA transcripts and might explain why so many genes are dysregulated in the brain in schizophrenia. Subsequent studies in the post-mortem brains from people with schizophrenia identified significant changes in miRNA expression (Beveridge et al., 2008Beveridge N.J. Tooney P.A. Carroll A.P. Gardiner E. Bowden N. Scott R.J. Tran N. Dedova I. Cairns M.J. Dysregulation of miRNA 181b in the temporal cortex in schizophrenia.Hum. Mol. Genet. 2008; 17: 1156-1168Crossref PubMed Scopus (275) Google Scholar). The potential importance of miRNA to understanding the molecular basis of schizophrenia was further highlighted when the Schizophrenia Working Group of the Psychiatric Genetics Consortium conducted the largest ever genome wide association study involving 36,989 cases and 113,075 controls (Ripke et al., 2014Ripke S. the Schizophrenia Working Group of the Psychiatric Genomics Consortium Biological insights from 108 schizophrenia-associated genetic loci.Nature. 2014; 511: 421-427Crossref PubMed Scopus (5107) Google Scholar). One of the strongest findings was a link between schizophrenia and the genetic variant rs1625579 located in the intron that encodes a miRNA called miR-137 (Ripke et al., 2014Ripke S. the Schizophrenia Working Group of the Psychiatric Genomics Consortium Biological insights from 108 schizophrenia-associated genetic loci.Nature. 2014; 511: 421-427Crossref PubMed Scopus (5107) Google Scholar). Since miRNA have hundreds of potential targets, the question is which miR-137 targets are relevant to schizophrenia and does this variant have any effect on its function? Computational methods can identify the potential targets for a particular miRNA based on the seed sequence it uses to bind to usually the 3′-UTR of the target mRNA (Oulas et al., 2015Oulas A. Karathanasis N. Louloupi A. Pavlopoulos G.A. Poirazi P. Kalantidis K. Iliopoulos I. Prediction of miRNA targets.Methods Mol. Biol. 2015; 1269: 207-229Crossref PubMed Scopus (28) Google Scholar). To determine which targets are relevant to schizophrenia and worthy of further investigation, Wu et al., 2016Wu S. Zhang R. Nie F. et al.MicroRNA-137 inhibits EFNB2 expression affected by a genetic variant and is expressed aberrantly in peripheral blood of schizophrenia patients.EBioMedicine. 2016; 12: 133-142Summary Full Text Full Text PDF PubMed Scopus (40) Google Scholar searched their previous study of the Han Chinese (Zhang et al., 2010Zhang R. Zhong N.N. Liu X.G. Yan H. Qiu C. Han Y. Wang W. Hou W.K. Liu Y. Gao C.G. et al.Is the EFNB2 locus associated with schizophrenia? Single nucleotide polymorphisms and haplotypes analysis.Psychiatry Res. 2010; 180: 5-9Crossref PubMed Scopus (16) Google Scholar) to identify genes linked to schizophrenia that contain the target sequence for miR-137. EFNB2 is such a gene that was linked to schizophrenia (Zhang et al., 2010Zhang R. Zhong N.N. Liu X.G. Yan H. Qiu C. Han Y. Wang W. Hou W.K. Liu Y. Gao C.G. et al.Is the EFNB2 locus associated with schizophrenia? Single nucleotide polymorphisms and haplotypes analysis.Psychiatry Res. 2010; 180: 5-9Crossref PubMed Scopus (16) Google Scholar), is functionally relevant to the disorder and is predicted to be targeted by miR-137. Wu and colleagues used luciferase reporter assays to show biologically that miR-137 targets the 3′ UTR of EFNB2 and that the minor rs550067317 C allele variant at this site disrupted this interaction (Wu et al., 2016Wu S. Zhang R. Nie F. et al.MicroRNA-137 inhibits EFNB2 expression affected by a genetic variant and is expressed aberrantly in peripheral blood of schizophrenia patients.EBioMedicine. 2016; 12: 133-142Summary Full Text Full Text PDF PubMed Scopus (40) Google Scholar). With the interaction established, the task was then to determine whether miR-137 exerted its effects directly on the post-transcriptional levels of EFNB2 mRNA or protein. In a neuroblastoma cell line commonly used for neurobiological studies, Wu and colleagues showed that miR-137 reduced the levels of endogenous EFNB2 protein but not mRNA. This starts to tease out the mechanism used by miR-137 to influence the functional roles of EFNB2. There is also great interest to use information from schizophrenia-associated genes as markers of schizophrenia phenotypes or as biomarkers to assist in diagnosis. To do this, brain biopsies are not feasible and ethical, so blood studies have been the main focus showing that some gene expression changes in blood mimic what is seen in the brain in schizophrenia (Sullivan et al., 2006Sullivan P. Fan C. Perou C. Evaluating the comparability of gene expression in blood and brain.Am. J. Med. Gen. Neuropsych. Gen. 2006; 141: 261-268Crossref Scopus (437) Google Scholar). In 2013 a study showed that a variant in the MIR137 gene when combined with severe negative symptoms identified a subtype of schizophrenia sufferers with cognitive deficits (Green et al., 2013Green M.J. Cairns M.J. Wu J. Dragovic M. Jablensky A. Tooney P.A. Scott R.J. Carr V.J. Genome-wide supported variant MIR137 and severe negative symptoms predict membership of an impaired cognitive subtype of schizophrenia.Mol. Psychiatry. 2013; 18: 774-780Crossref PubMed Scopus (124) Google Scholar). To further this line of research, Wu and colleagues showed that miR-137 but not EFNB2 expression was increased in patients with schizophrenia compared to controls and that this had some diagnostic value in distinguishing patients from controls (Wu et al., 2016Wu S. Zhang R. Nie F. et al.MicroRNA-137 inhibits EFNB2 expression affected by a genetic variant and is expressed aberrantly in peripheral blood of schizophrenia patients.EBioMedicine. 2016; 12: 133-142Summary Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Fig. 6B in the paper by Wu and colleagues shows that a subset of patients with schizophrenia have much higher miR-137 expression leaving you wondering what was their cognitive status? Whilst the building evidence suggests miRNAs including miR-137 have a role in the development of schizophrenia and may have some capacity as biomarkers, clearly further collaborative efforts will determine how useful they are in understanding what causes schizophrenia, whether they can assist with its diagnosis and importantly, whether they provide insights on how to improve the current treatment options for the disorder. The author declared no conflicts of interest. MicroRNA-137 Inhibits EFNB2 Expression Affected by a Genetic Variant and Is Expressed Aberrantly in Peripheral Blood of Schizophrenia PatientsMicroRNAs (miRNAs) are a class of endogenous and non-coding single-stranded RNAs of approximately 22 nucleotides, many of which are evolutionarily conserved. Genome-wide association studies have identified a robust statistical association between the MIR137 gene and schizophrenia in Europeans, which was replicated in the Han Chinese population in a case-control study. In the previous study, we provided evidence for a significant association between the EFNB2 gene and schizophrenia in Han Chinese subjects. Full-Text PDF Open Access

Previous studies have observed increased tachykinin NK(1) receptor immunoreactivity (NK(1)-IR) in the prefrontal cortex in subjects with schizophrenia. Since the subjects were medicated the possibility of a treatment effect could not be excluded. Thus, the present study was undertaken to determine the effect of chronic treatment with the antipsychotic drug, haloperidol, on the distribution of NK(1)-IR neurons in the guinea-pig brain. Guinea pigs were treated each day for 21 days with either haloperidol (1mg/kg) or vehicle and the brains were then processed for immunohistochemistry using an NK(1) receptor-specific polyclonal antibody. NK(1)-IR neurons and fibres were abundant in the forebrain cortex and caudate putamen and more sparsely distributed in a number of other brain regions. The relative density of NK(1)-IR neurons was significantly increased in the forebrain cortex, but not in the caudate putamen in guinea pigs treated with haloperidol. This study has shown that haloperidol causes region-specific changes to the density of NK(1)-IR neurons. Whether these changes are related to the therapeutic effects or to the side effects of haloperidol in individuals with schizophrenia, remains to be determined.

Abstract The diagnosis of schizophrenia lacks a broadly accepted biological basis and its heterogeneity may well represent a group of disorders with different etiologies. Even so, brain imaging can map and quantify structural brain abnormalities in vivo as an intermediate (or endo-) phenotype of the disorder. Brain structural abnormalities occur in the prodromal phase and progress in the course of illness, and patterns of grey matter deficits appear to determine the clinical phenotype, thus addressing some of the diagnostic heterogeneity. Here we examined cerebral grey matter with cortical pattern matching in MRI scans from 18 previously untreated patients meeting DSM-IV diagnostic criteria for schizophrenia by employing a three-dimensional spoiled gradient recalled (SPGR) pulse sequence at 1.5 T. Data were compared to 18 pair-wise age and sex-matched healthy volunteers from the patient communities. We found widespread cerebral grey matter deficits in schizophrenia. Grey matter deficits in the right dorsolateral prefrontal cortex were the strongest predictor of diagnosis. Symptom severity and treatment response were associated with regional grey matter deficits in older patients with a longer history of untreated illness, while significant structure/function associations with cognitive impairment in prefrontal and temporal cortices were found across all ages. Quantitative brain maps are useful for assessing disease burden in schizophrenia, and for understanding its heterogeneity, including its changing clinical characteristics as the illness progresses.

Vasculogenic mimicry (VM), the ability of tumour cells to form functional microvasculature without an endothelial lining, may contribute to anti-angiogenic treatment resistance in glioblastoma. We aimed to assess the extent of VM formation in primary and recurrent glioblastomas and to determine whether VM vessels also express prostate-specific membrane antigen (PSMA), a pathological vessel marker. Formalin-fixed paraffin-embedded tissue from 35 matched pairs of primary and recurrent glioblastoma was immunohistochemically labelled for PSMA and CD34 and stained with periodic acid–Schiff (PAS). Vascular structures were categorised as endothelial vessels (CD34+/PAS+) or VM (CD34−/PAS+). Most blood vessels in both primary and recurrent tumours were endothelial vessels, and these significantly decreased in recurrent tumours (p &lt; 0.001). PSMA was expressed by endothelial vessels, and its expression was also decreased in recurrent tumours (p = 0.027). VM was observed in 42.86% of primary tumours and 28.57% of recurrent tumours. VM accounted for only a small proportion of the tumour vasculature and VM density did not differ between primary and recurrent tumours (p = 0.266). The functional contribution of VM and its potential as a treatment target in glioblastoma require further investigation.

Abstract BACKGROUND Patients with glioblastoma are confronted with a high likelihood of recurrence and poor prognosis despite an aggressive treatment-regime involving surgery followed by radiation therapy (RT) and temozolomide (TMZ). RT and TMZ cause extensive DNA damage and replication stress, thus activating tumour cell death pathways. Upregulation of the DNA repair mechanisms significantly reduces effective treatment response and contributes to poor patient outcomes. We investigated the effect of inhibiting ATM- and Rad3-Related protein (ATR), a crucial sensor of replication stress and initiator of cell cycle arrest in tumour cells, using the potent and selective ATR inhibitor, gartisertib. MATERIAL AND METHODS Twelve patient-derived glioblastoma cell lines were grown as monolayer cultures in serum-free media and treated with TMZ, RT and/or gartisertib. Cell viability of treated cells was assessed after a 7-day incubation using the MTT method, while cell confluence, apoptosis and cell death were examined using the Incucyte S3 Live-Cell Analysis System (Sartorius, Germany). Gene expression of glioblastoma cell lines treated with TMZ+RT and/or gartisertib was assessed 4-days post-treatment. RESULTS As a single agent, gartisertib potently reduced glioblastoma cell viability, while 8-fold less potent in human astrocyte cells. Glioblastoma cell lines with mutated DNA damage response-related genes were more sensitive to gartisertib treatment. Live-cell imaging of glioblastoma cells treated with gartisertib (1 µM) plus TMZ (35µM) and RT (2Gy) showed a significant increase in cell death and apoptosis compared to gartisertib or TMZ+RT alone. ATR inhibition by gartisertib strongly synergised with TMZ, while moderately synergising with RT in reducing glioblastoma cell growth. Gartisertib, alone and in combination with TMZ+RT, increased gene expression in pro-inflammatory cytokines, antigen presentation, and pattern recognition pathways while decreasing genes involved in hypoxia and epithelial-mesenchymal transition pathways. CONCLUSION These data suggest the potential for ATR inhibition as an effective chemo- and radiosensitiser in glioblastoma tumours. Whether the change in gene expression induced by ATR inhibition reduces hypoxia within or provokes an immunogenic response directed at glioblastoma tumours, requires further investigation.

Abstract BACKGROUND PSMA is a cell-surface protein that is highly expressed in the endothelium of the tumour neovasculature of several solid tumours, including glioblastoma (GBM), making it an excellent target for antibody-drug conjugates or peptide receptor radionuclide therapy. Several [68Ga]Ga-PSMA-based radioligands have shown utility in the diagnosis and treatment response assessment of primary GBM patients in a number of case studies. The small-molecule ligand [68Ga]Ga-PSMA-617 has the advantage of being suitable for radiolabelling with different radioisotopes, including 68Ga, 177Lu, 111In and 90Y, which raised the possibility of using it in a theranostic application in glioblastoma. The aim of this phase I/II prospective study was to evaluate the potential of [68Ga]Ga-PSMA-617 as a PET imaging biomarker and as a candidate ligand for targeted radionuclide therapy in patients with recurrent GBM. MATERIAL AND METHODS Ten patients with recurrent glioblastoma were recruited for the study between October 2018 and September 2021. Patients underwent [68Ga]Ga-PSMA-617 and [18F]F-FET PET brain scans done on two separate days. Two patients had full-body [68Ga]Ga-PSMA-617 scans. Tumour selectivity of [68Ga]Ga-PSMA-617 was assessed by measuring tumour volume on [68Ga]Ga-PSMA-617 PET scans and comparing it with tumour volume measured on [18F]F-FET PET scans, which are more widely used clinically. Tumour specificity of [68Ga]Ga-PSMA-617 was assessed by comparing its standard uptake value (SUV) and tumour-to-brain-ratio (TBR) with those of [18F]F-FET TBR, and by measuring [68Ga]Ga-PSMA-617 tumour-to-liver ratio (TLR). RESULTS PSMA-defined tumour volume was on average 1.87±1.10 times larger than the FET-defined tumour volume (p=0.084), and the two volumes had a DICE similarity coefficient of 0.578±0.175. Mean and maximum TBR was significantly (p=0.002) higher (~10x) for PSMA than for FET. Mean SUV in the tumour was significantly higher for FET than for PSMA (p=0.002), while maximum SUV was slightly higher for PSMA than for FET (p=0.08). For the two patients with full-body [68Ga]Ga-PSMA PET scan available, PSMA mean TLR was 0.91 and 0.68, and maximum TLR was 1.20 and 1.07, respectively. CONCLUSION [68Ga] Ga-PSMA PET delineates a larger tumour volumes compared to [18F]F-FET PET, suggesting there are areas of the tumour with emerging tumour neovasculature that are not yet metabolically active. The poor spatial overlap between PSMA and FET tumour volume suggests the complementary diagnostic role of the two tracers. The higher TBR of PSMA compared to FET demonstrates the higher tumour specificity of the former, and thus its potential ability as a diagnostic agent for recurrent tumour volume delineation. Relatively low values of PSMA SUV and TLR suggest that PSMA specific uptake is not sufficient to use this ligand for targeted radionuclide therapy in glioblastoma when conjugated to [177Lu].

Abstract BACKGROUND Glioblastomas are highly vascular tumours that overexpress vascular endothelial growth factor (VEGF), however the anti-VEGF antibody bevacizumab has not demonstrated significant overall survival benefit for patients. Vasculogenic mimicry (VM), the ability of tumour cells to form functional microvasculature lacking an endothelial lining, may compensate for insufficient or inhibited angiogenesis. VM has been reported in primary glioblastoma tissue, but not investigated at progression in recurrent tumours. Prostate specific membrane antigen (PSMA) is expressed in abnormal vessels within primary glioblastoma tumours. We aimed to determine whether VM occurs in primary and recurrent glioblastoma tumours and whether VM vessels also express PSMA. MATERIAL AND METHODS Thirty-five matched pairs of primary and recurrent formalin-fixed paraffin-embedded glioblastoma tissue were immunohistochemically labelled for the endothelial cell marker CD34 and PSMA, and counterstained with periodic acid-Schiff (PAS). Vascular structures were categorised as endothelial vessels (CD34+/PAS+) or VM (CD34-/PAS+). Vessels were counted in ten regions within each tumour and a vessel density/mm2 for each vessel category was determined. RESULTS Data are expressed as median values. Endothelial vessels accounted for most blood vessels observed. There was a significant decrease in endothelial vessel density in recurrent (41.34 vessels/mm2) compared to primary (86.98 vessels/mm2) glioblastomas (p &amp;lt;0.001). VM was observed in 15/35 primary tumours (42.86%) and 10/35 recurrent tumours (28.57%). VM accounted for only a small proportion of the overall vasculature and VM density was not different between the primary and recurrent groups (p = 0.266). Kaplan-Meier analyses with log rank tests indicated that VM-tumours had longer median OS time than VM+ tumours, regardless of whether VM status was determined based on the primary or recurrent tumour. However, there was no significant difference in survival distribution between VM+ and VM-tumours (primary χ2(1) = 0.381, p = 0.537; recurrent χ2(1) = 3.552, p = 0.059). PSMA is expressed in endothelia lined vessels based on serial section labelling for PSMA and CD34. PSMA expression significantly decreased in recurrent (median H-score = 0.22) compared to primary (median H-score = 0.44) tumours (one-tailed p = 0.031; n = 32) mirroring the CD34+ vessel density. CONCLUSION VM contributes to a relatively small proportion of the overall vasculature in glioblastoma tumours and was not associated with OS nor the expression of PMSA. Since VM- tumours had longer OS than VM+ tumours, assessment of VM in recurrent tumours in a larger cohort may determine if there is a clinical significance for VM in recurrent glioblastoma.

Abstract Glioblastoma cells can restrict the DNA-damaging effects of temozolomide (TMZ) and radiation therapy (RT) using the DNA damage response (DDR) mechanism which activates cell cycle arrest and DNA repair pathways. Ataxia-telangiectasia and Rad3-Related protein (ATR) plays a pivotal role in the recognition of DNA damage induced by chemotherapy and radiation causing downstream DDR activation. Here, we investigated the activity of gartisertib, a potent ATR inhibitor, alone and in combination with TMZ and/or RT in 12 patient-derived glioblastoma cell lines. We showed that gartisertib alone potently reduced the cell viability of glioblastoma cell lines, where sensitivity was associated with the frequency of DDR mutations and higher expression of the G2 cell cycle pathway. ATR inhibition significantly enhanced cell death in combination with TMZ and RT and was shown to have higher synergy than TMZ+RT treatment. MGMT promoter unmethylated and TMZ+RT resistant glioblastoma cells were also more sensitive to gartisertib. Analysis of gene expression from gartisertib treated glioblastoma cells identified the upregulation of innate immune-related pathways. Overall, this study identifies ATR inhibition as a strategy to enhance the DNA-damaging ability of glioblastoma standard treatment, while providing preliminary evidence that ATR inhibition induces an innate immune gene signature that warrants further investigation.

Lack of control of confounding variables, high attrition rate, and too few neurocognitive domains completed at each assessment point are some of the limitations identified in studies of the relationship between cognition and functional outcome in schizophrenia. In the ongoing Oslo multi-follow-up study 28 first episode schizophrenia patients and a pairwise matched control group (N=28) are assessed with the MATRICS Consensus Cognitive Battery (MCCB), a clinical interview, an inventory on social and role functioning and criteria of remission and recovery at several follow-up points. The current paper describes the rate of remission and full recovery, and investigates the relationship between neurocognition and functional outcome. At 2-year follow-up, 80.0% of the patients were in remission and 16.0% of them fulfilled the criteria for full recovery. The attrition rate was very low. In the follow-up period, there was a statistically significant decline in Verbal Learning and a significant improvement on Reasoning/Problem Solving and Social Cognition in the schizophrenia group, but not in the control group. This indicates a differentiated neurocognitive course. In the schizophrenia group, Attention/Vigilance and years of education at baseline were significant predictors of social and role functioning 2 years later.

There has been increasing interest in studying the impact of physical activity on the psychological and physical well-being and functioning in patients with first-episode psychosis. The exploration of factors which contribute to physical activity in psychosis may open up opportunities for improvement of functional outcome. The purpose of this study was to examine the association between physical activity level and functioning in psychosis. A total of 283 patients with first-episode psychosis were recruited from a specialized early intervention service for adult-onset psychosis (Jockey Club Early Psychosis Project) in Hong Kong. The level of physical activity, sociodemographics and clinical characteristics was assessed at study entry. Functioning was assessed at 6-months period. Ninety-six (33.9%) patients were categorized as physically inactive, and 187 (66.1%) of them were physically active. Being physically inactive (β = 0.163, P = 0.003), having more positive and negative symptoms [SAPS total score (β = − 0.161, P = 0.005), and SANS total score (β = − 0.202, P = 0.001)], and having lower household income (β = 0.207, P = 0.001) at baseline predicted poorer functioning at 6 months. Early intervention for psychosis should target to improve patients' physical activity level which may help subsequent functioning.

Biosulfidogenesis (the microbial generation of H2S by reduction of more oxidized sulfur species) is a very useful technology for remediating acidic, metal-rich waste-waters, such as acid mine drainage. Many transition metals form highly insoluble sulfide phases when contacted with H2S, and bacterial reduction of sulfate to sulfide is, in acidic liquors, a proton-consuming reaction. Commercial-scale sulfidogenic bioreactors use neutrophilic species of sulfate- (or sulfur-) reducing bacteria that need to be shielded from direct contact with acidic waste waters, which is not the case with acidophilic and acid-tolerant species (aSRB). Here we report the kinetics and microbial dynamics of a low pH, laboratory-scale sulfidogenic bioreactor, operated as a continuous flow system for 462 days, at pH values between 4.0 and 5.0, and temperatures between 30 and 45 °C. While changing an operating parameter caused minor perturbations in the performance of the bioreactor, these were transient, and the system performed consistently well throughout the entire test period, with pH 4.0 and 35 °C being marginally the optimum operating conditions. Two species of aSRB mediated sulfide formation: Desulfosporosinus acididurans, preferentially at higher pH and lower temperatures, and Peptococcaceae CEB3, at lower pH and higher temperatures. The relative abundances of these two bacteria changed in response to an operational (pH, temperature) change. The results highlighted the robustness and adaptability of the low pH microbial consortium used to generate sulfide, and to precipitate transition metals both in situ and ex situ.

Neuromuscular diseases are both genetic and acquired conditions resulting in progressive muscle weakness and wasting which lead to disability and reduced survival. The availability of high-quality human biomaterial is crucial to support biomedical research with potential applications at all stages of development, from molecular pathophysiology to drug discovery, clinical trials and evaluation of biomarkers. Although significant progress has been made over the last few years in the diagnosis of these rare conditions, the genetic defect and underlying pathological abnormality remain unknown in approximately 1/3 of cases. Moreover, to date no definitive cure is available for most neuromuscular disorders, nor are there sufficiently reliable and specific biomarkers to monitor disease progression and response to treatment. This is in part due to the rarity and genetic heterogeneity of neuromuscular diseases and the lack of access to patient samples. The availability of the national MRC Centre Biobank for Neuromuscular Diseases in Newcastle and London has addressed this bottleneck and supported neuromuscular research. Nine years after the establishment of the MRC Centre Biobank, many high profile research publications have highlighted the positive impact of neuromuscular biobanking for translational research and proven this facility to be a unique repository source for diagnostics, basic science research, industry, drug development, and therapy.

In growth plate chondrocytes, loss of Dicer, a microRNA (miRNA)-processing enzyme, causes defects in proliferation and differentiation, leading to a lethal skeletal dysplasia. However roles of miRNAs in articular chondrocytes have not been defined in vivo. To investigate the role of miRNAs in articular chondrocytes and to explore the possibility of generating a novel mouse osteoarthritis (OA) model caused by intrinsic cellular dysfunction, we ablated Drosha, another essential enzyme for miRNA biogenesis, exclusively in articular chondrocytes of postnatal mice.First, to confirm that the essential role of miRNAs in skeletal development, we ablated the miRNA biogenesis pathway by deleting Drosha or DGCR8 in growth plate chondrocytes. Next, to investigate the role of miRNAs in articular cartilage, we deleted Drosha using Prg4-CreERT transgenic mice expressing a tamoxifen-activated Cre recombinase (CreERT) exclusively in articular chondrocytes. Tamoxifen was injected at postnatal days, 7, 14, 21, and 28 to ablate Drosha.Deletion of Drosha or DGCR8 in growth plate chondrocytes caused a lethal skeletal defect similar to that of Dicer deletion, confirming the essential role of miRNAs in normal skeletogenesis. Early postnatal Drosha deletion in articular chondrocytes significantly increased cell death and decreased Safranin-O staining. Mild OA-like changes, including surface erosion and cleft formation, were found in male mice at 6 months of age; however such changes in females were not observed even at 9 months of age.Early postnatal Drosha deficiency induces articular chondrocyte death and can cause a mild OA-like pathology.

Background: The Australian Schizophrenia Research Bank (ASRB) was established in 2007 to collect linked clinical, cognitive, neuroimaging and genetic data in people with schizophrenia and matched controls. The ASRB is the first of its kind developed in Australia. Demographic, clinical and neurological data is presented for the first 550 participants with schizophrenia. Methods: Participants were assessed using a comprehensive assessment battery that consists of socio-demographic questions including medical and family history, neurological evaluation (NES), neuropsychological assessment and cognitive performance measures (WTAR, WASI, RBANS, LNS, COWAT), a diagnostic interview that includes drug and alcohol history (DIP, Castle et al, 2006) to confirm diagnosis, ratings for negative symptoms (SANS), general functioning (GAF), and questionnaires of childhood adversity, personality disorder (IPDE) and psychosis proneness (SPQ). Results: A sample of 550 people with schizophrenia (mean age = 39.66 years; SD = 10.98) and 250 healthy controls (mean International Congress on Schizophrenia Research 54 age = 37.37 years; SD = 13.14) were compared across measures. The schizophrenia sample had a higher proportion of males (cases 16.80%; controls 46.40%), fewer living in married or de facto relationships (cases 15.80%; controls 53.60%) and fewer years of education (cases 12.93, SD = 2.91; controls 15.13, SD = 3.14). Schizophrenia participants also had lower premorbid IQ (cases 103.17, SD = 13.17; controls 111.83, SD = 8.76), current IQ (cases 102.29, SD = 15.62; controls 118.24, SD = 10.20) and RBANS total score (cases 82.55, SD = 15.59; controls 96.24, SD = 15.88), consistent with performance reported previously for Australian samples (Loughland et al, 2007). Conclusion: These findings are consistent with those reported previously in the Australian Low Prevalence Disorders Study (Castle, 1999), suggesting the ASRB sample is broadly representative of people with schizophrenia living in Australia. The ASRB is a unique schizophrenia resource that is accessible to approved national and international researchers.

Diffuse axonal injury (DAI) is fairly common during a traumatic brain injury (TBI) and is associated with high mortality. Making an early diagnosis, appropriate therapeutic decisions, and an accurate prognostic evaluation of patients with DAI still pose difficulties for clinicians. The detailed mechanisms of axonal injury after head trauma have yet to be clearly defined and no reliable biomarkers are available for early DAI diagnosis. Therefore, this study employed an established DAI animal model in conjunction with an isobaric tag for relative and absolute quantification (iTRAQ)-based protein identification/quantification approach. Alterations in rat cerebral protein expression were quantified using iTRAQ coupled LC–MS/MS, with differentially expressed proteins between the control groups, sham and sham-injured, and the injury groups, animals that died immediately post-injury and those sacrificed at 1 h, 6 h, 1 d, 3 d and 7 d post-injury, identified. A total of 1858 proteins were identified and quantified and comparative analysis identified ten candidate proteins that warranted further examination. Of the ten candidate DAI biomarkers, four proteins, citrate synthase (CS), synaptosomal-associated protein 25 (Snap25), microtubule-associated protein 1B (MAP1B) and Rho-associated protein kinase 2 (Rock2), were validated by subsequent Western blot and immunohistochemistry analyses. Our studies not only identified several novel biomarkers that may provide insight into the pathophysiological mechanisms of DAI, but also demonstrated the feasibility of iTRAQ-based quantitative proteomic analysis in cerebral tissue research.

Background: The Australian Schizophrenia Research Bank (ASRB) was established in 2007 to collect linked clinical, cognitive, neuroimaging and genetic data in people with schizophrenia and matched controls, and is the fi rst of its kind developed in Australia. Methods: Participants were assessed using a comprehensive assessment battery developed based on advice from a collaboration of schizophrenia research specialists. The three-hour battery consists of socio-demographic questions including medical and family history, neurological evaluation (NES), neuropsychological assessment and cognitive performance measures (WTAR,WASI, RBANS, LNS, COWAT), a diagnostic interview that includes drug and alcohol history (DIP) [1] to confirm (or screen for) diagnosis, ratings for symptoms (SANS) and general functioning (GAF), and questionnaires of childhood adversity, personality disorder (IPDE) and psychosis proneness (SPQ). Results: The sample currently comprises 550 people with schizophrenia (mean age = 39.66 years; SD = 10.98) and 250 healthy controls (mean age = 37.37 years; SD = 13.14). Compared to the controls, the schizophrenia sample had a higher proportion of males (cases 66.80%; controls 46.40%), fewer living in married or de facto relationships (cases 15.80%; controls 53.60%) and fewer years of education (cases 12.93, SD = 2.91; controls 15.13, SD = 3.14). Schizophrenia participants also had lower premorbid IQ (cases 103.17, SD = 13.17; controls 111.83, SD = 8.76), current IQ (cases 102.29, SD = 15.62; controls 118.24, SD = 10.20) and RBANS scores (cases 82.55, SD = 15.59; controls 96.24, SD = 15.88) consistent with performance reported previously for Australian samples [2]. Discussion: These fi ndings are broadly consistent with those reported previously in the Australian Low Prevalence Disorders Study [3], suggesting the ASRB sample is broadly representative of people with schizophrenia living in Australia. The ASRB is a unique schizophrenia resource that is accessible to approved Australian researchers in 2010 and international scientists in 2011.

The ventral subiculum (vSUB), a hippocampal efferent target implicated in learning and stress coping, receives cholinergic input and sends glutamatergic output to the bed nucleus of the stria terminalis (BNST). This study examined the roles of vSUB muscarinic activation and its interaction with BNST N-methyl-d-aspartate and noradrenergic receptors in formation of aversive memory. Male Wistar rats with cannulae implanted into the vSUB or BNST were trained on a step-through inhibitory avoidance task. Shortly after training, they received cholinergic drugs infused into the vSUB and/or glutamatergic or noradrenergic drugs infused into the BNST. Results of the 1-day retention tests showed that intra-vSUB infusion of oxotremorine (0.01 μg) or scopolamine (0.3 or 3.0 μg) enhanced or impaired retention, respectively. Both effects were dose- and time-dependent, and 0.001 μg oxotremorine attenuated the amnesia induced by 3.0 μg scopolamine. The oxotremorine-induced memory enhancement was blocked by intra-BNST infusion of dl-2-amino-5-phosphonovaleric acid or propranolol at a dose not affecting retention; the amnesia induced by scopolamine was blunted by intra-BNST infusion of glutamate or norepinephrine at a dose with a negligible effect on retention. These data suggest that in an inhibitory avoidance task muscarinic activation of the vSUB modulated memory formation by interacting with the BNST glutamatergic and noradrenergic functions.

Paul E. Rasser, Juanita Todd, Paul M. Thompson, Patricia T. Michie, Philip B. Ward, Patrick Johnston, Katrin Helmbold, Vanessa Case, Paul A. Tooney & Ulrich Schall Schizophrenia Research Institute, Sydney, Australia, Priority Centre for Brain & Mental Health Research, University of Newcastle, Newcastle, Australia, Laboratory of Neuro Imaging, University of California Los Angeles, USA, Schizophrenia Research Unit, Liverpool Hospital, University of New South Wales, Sydney, Australia, Brain Sciences Institute, Swinburne University of Technology, Melbourne, Australia, Department of Psychology, University of Konstanz, Germany.

Abstract Patients with glioblastoma almost always suffer a recurrence of an aggressive treatment resistant tumour and succumb within months highlighting the need to develop treatment options for recurrent glioblastoma. One potential target is prostate specific membrane antigen (PSMA) expressed on the new vessels in primary glioblastoma tumours. This study compared the expression of PSMA in primary and recurrent glioblastoma tumours. Formalin-fixed paraffin-embedded sections of primary and matched recurrent tumours from 13 patients with glioblastoma were processed for PSMA immunohistochemical labelling. PSMA expression was scored from digitally scanned sections using a categorical system (Total PSMA expression = PSMA label intensity (0–3) x percent PSMA-positive blood vessels). Little PSMA labelling was observed in any adjacent ‘unaffected’ brain tissue. PSMA was localised to blood vessels including those displaying microvascular proliferation in 12/13 primary and 9/13 recurrent glioblastoma tumours. Total PSMA expression scores ranged from 0 – 230 (maximum score = 300) and was divided into low and high expression based on a median split (median = 57) of PSMA expression across all tumours. Regression analysis showed a significant difference in PSMA expression between primary and recurrent glioblastoma (p = 0.04) with PSMA being highly expressed in ~70% of primary and only 31% of recurrent glioblastoma. Three cases displayed high expression in both primary and recurrent glioblastoma and one case had no PSMA expression at all. In conclusion, whilst higher expression of PSMA was detected in more primary tumours, 70%+ of all primary and recurrent tumours expressed PSMA to some extent adding evidence that this may be a useful target for treatment of glioblastoma. Larger cohorts and other techniques for detecting PSMA are needed to provide further evidence for PSMA’s utility as a target and to further define which glioblastoma patients are most likely to benefit from this approach.

Abstract Immunotherapy has been shown to have benefit in some solid tumours including melanoma where immune infiltration can be pronounced. Until recently the central nervous system was thought to be immune privileged. Whilst research has shown that immune cells are capable of infiltrating tumours from glioblastoma patients, to date immunotherapy trials have not consistently shown a benefit in these patients. The aim of this study was to profile the immune system in primary and recurrent tumours from glioblastoma patients. Formalin-fixed paraffin-embedded sections of primary and matched recurrent tumours from 13 patients with glioblastoma were processed for immunohistochemical labelling of a range of immune cell markers. Immune infiltration was scored on digitally scanned immunolabelled sections using a categorical system of 0 (absent), 1 (present), 2 (moderate) and 3 (marked). CD3+ cells were observed in three topographical locations within primary and recurrent glioblastoma tumours namely the tumour proper, perivascular spaces and associated with haemorrhages within the tumour. CD3+ cell infiltration into the tumour proper was present (Score = 1) in 7 of 13 primary and recurrent tumours. Only one case (case #9) had CD3+ infiltration scores &gt; 1 for both primary (score = 3) and recurrent tumour (score = 2). CD3+ cells were observed in perivascular spaces in 10 of the 13 cases of primary and recurrent glioblastoma. Only case #9 had CD3+ cells in perivascular spaces that was scored &gt;1 for both primary and recurrent tumours. In conclusion, whilst CD3+ infiltration was observed in the tumour proper and perivascular spaces within both primary and recurrent glioblastomas, the level of infiltration was quite low in this small cohort and as such requires further investigation in a larger cohort.

Supplemental material, Supplementary_files_of_GWAS_of_schizophrenia_684indiv_EUR_3covariates_v3-revised_v1 for Wnt receptor gene FZD1 was associated with schizophrenia in genome-wide SNP analysis of the Australian Schizophrenia Research Bank cohort by Xiaoman Liu, Siew-Kee Low, Joshua R Atkins, Jing Qin Wu, William R Reay, Heath M Cairns, Melissa J Green, Ulrich Schall, Assen Jablensky, Bryan Mowry, Patricia T Michie, Stan V Catts, Frans Henskens, Christos Pantelis, Carmel Loughland, Alan V Boddy, Paul A Tooney, Rodney J Scott, Vaughan J Carr and Murray J Cairns in Australian & New Zealand Journal of Psychiatry