Natalie J. Beveridge

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.

The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

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.

MicroRNA dysregulation in schizophrenia

Schizophrenia is a complex neuropsychiatric disorder that involves disturbances in neural circuitry and synaptic function. The exquisite network architecture and capacity for discreet post-synaptic remodeling of neurons requires coordination by an elaborate intracellular network of molecular signal transduction systems. The redundancy of these networks means that many combinations of gene variants have the potential to cause system dysfunction that manifest as related neurobehavioural syndromes. Recent investigation has revealed that posttranscriptional gene regulation and associated small non-coding microRNA (miRNA), are likely to be important factors shaping the topography of these networks. miRNA display complex temporospatial expression patterns in the mammalian brain and have the potential to regulate thousands of target genes by functioning as the specificity factor for intracellular gene-silencing machinery. They are emerging as key regulators of many neurodevelopmental and neurological processes as their dysregulation could lead to pervasive changes in the network structure during development and in the mature brain that are highly significant in the pathophysiology of schizophrenia. This review looks at mounting evidence that mature miRNA levels are altered in both the cerebral cortex and peripheral blood mononuclear cells (PBMCs) in schizophrenia. It also examines compelling evidence that the underlying miRNA biogenesis machinery and miRNA genes themselves are subject to disease-associated genetic mutation and epigenetic influence. Significantly, these changes in miRNA expression and associated machinery may represent new targets for pharmaceutical development, and the identification of miRNA signatures in PBMCs suggest that miRNA biomarkers of schizophrenia may also provide the basis for new clinical diagnostics. These developments have tremendous potential and highlight the significance of this avenue of research.

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.

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.

Maturation of the Human Dorsolateral Prefrontal Cortex Coincides With a Dynamic Shift in MicroRNA Expression

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.

Transcriptome-wide mega-analyses reveal joint dysregulation of immunologic genes and transcription regulators in brain and blood in 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.