Paul A. Tooney

Schizophrenia is associated with an increase in cortical microRNA biogenesis

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.

Dysregulation of miRNA 181b in the temporal cortex in 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.

Upregulation of Dicer and MicroRNA Expression in the Dorsolateral Prefrontal Cortex Brodmann Area 46 in Schizophrenia

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Imprinted DLK1-DIO3 region of 14q32 defines a schizophrenia-associated miRNA signature in peripheral blood mononuclear cells

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.

Preliminary investigation of gene expression profiles in peripheral blood lymphocytes 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.

Genome-wide supported variant MIR137 and severe negative symptoms predict membership of an impaired cognitive subtype of schizophrenia

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.

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation

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.

Australian schizophrenia research bank: a database of comprehensive clinical, endophenotypic and genetic data for aetiological studies of schizophrenia

Objective: 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. Method: 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. Results: 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. Conclusion: 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.

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

Background 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. Methodology/Principal Findings 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. Conclusions 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.

Altered gene expression in the superior temporal gyrus in schizophrenia

BackgroundThe 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.ResultsTo 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, IPLA2γ, 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.ConclusionThis 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.