Functional analysis of gene and microRNA expression profiling in schizophrenia: brain, blood and antipsychotics

Research Doctorate - Doctor of Philosophy (PhD)Schizophrenia is a complex and heterogeneous disorder that has strong genetic links. While no single gene of large effect size has been identified, it has been hypothesised that dysreglation of many genes could be involved in the underlying molecular biology. The expression of microRNA (miRNA), master regulators of gene expression at the post-transcriptional level, was analysed in post-mortem superior temporal gyrus (STG) and dorsolateral prefrontal cortex (DLPFC) from patients with schizophrenia and healthy controls using high-throughput microarray technology. miR-181b was up-regulated in the STG of patients with schizophrenia, and indeed a global up-regulation of miRNA was observed in both the STG and the DLPFC along with an up-regulation of components of the machinery involved in miRNA biogenesis. Luciferase reporter expression was then analysed to validate regulatory relationships between miRNA and their predicted target gene(s) in vitro. Several miR-15 family members, which share a seed region and therefore many mRNA targets, regulated schizophrenia candidate genes such as visinin-like 1 (VSNL1) and reelin (RELN). Gene and miRNA expression was analysed in a larger cohort of participants with schizophrenia and non-psychiatric controls in peripheral blood mononuclear cells (PBMCs) from the Australian Schizophrenia Research Bank (ASRB). The expression of 83 miRNA was significantly down-regulated in schizophrenia relative to controls including several brain expressed miRNA that have previously been reported to be altered in the brain in schizophrenia (miR-128, miR-134 and miR-181b). Importantly, almost a quarter of the down-regulated miRNA reside within an imprinted cluster, at chromosome at 14q32, which has previously been associated with schizophrenia. Down-regulation of the 14q32 cluster was not the result of a copy number variation suggesting other mechanisms, possibly epigenetic, may be involved. Further analysis suggested that these miRNA regulated several nervous-system related pathways and also implicated dysregulation of immune-associated pathways in schizophrenia. Functional analysis of differential gene expression in the same cohort also revealed enrichment of processes and pathways associated with immune and inflammation responses: 105 genes were down-regulated (e.g. Chemokine (C–C motif) receptor 7; CCR7) and 59 up-regulated (e.g. Defensin alpha-4; DEFA4) in participants with schizophrenia compared to controls. Since medication could potentially contribute to altered expression in these cohorts, differential gene expression was assessed in PBMCs from antipsychotic naïve schizophrenia patients and post-treatment. Several genes that were differentially expressed in the schizophrenia patients appeared to be responsive to medication including the schizophrenia candidate gene v-akt murine thymoma viral oncogene homolog 1 (AKT1), incidentally located in the 14q32 chromosomal region, which was common to several dysregulated canonical pathways implicated in this study. Again, immune and inflammation-associated pathways were also altered in schizophrenia patients both prior to and following antipsychotic treatment, consistent with the analysis of the ASRB cohort, providing further evidence of immune dysfunction in schizophrenia. Finally, assessment of the impact of acute and subacute exposure to 3 antipsychotics (chlorpromazine, clozapine and haloperidol) in JM-Jurkat T-lymphocytes on gene and miRNA expression showed minimal overlap with the differential expression observed in PBMCs, suggesting that expression patterns observed in the PBMCs were associated with schizophrenia as opposed to a consequence of medication. Moreover functional analysis indicated that antipsychotics dysregulate genes involved in oxidative stress which may reflect the molecular mechanism(s) underlying some of their side effects such as extra-pyramidal symptoms, weight gain and in the case of clozapine, agranulocytosis. The molecular profiles obtained from the gene and miRNA expression analyses in post-mortem brain, PBMCs from living schizophrenia patients and healthy controls, as well as in human tissue after antipsychotic exposure, provide insight into the underlying molecular biology of schizophrenia and antipsychotics, which could lead to targets for therapeutic intervention. Furthermore, functional analysis of the differential expression in these aforementioned studies implicated dysregulation of several biological processes and pathways such as immune and inflammation pathways