miR-137, schizophrenia and sleep regulation in Drosophila melanogaster.

Abstract

Introduction – microRNAs are non-coding DNA sequences, which regulate gene expression by promoting the destruction of target mRNAs. The evolutionarily conserved miR-137 is expressed in invertebrate and mammalian neuronal tissue and there is experimental evidence for mir-137 regulating neurological development, synaptic plasticity, and cognitive function. Other genes likely regulated by miR-137 include members of the dopamine signalling pathway. Furthermore, genome wide association studies (GWAS) have linked SNPs in mir-137 with schizophrenia (SZ). Modulation of dopaminergic signalling and SZ are associated with abnormal sleep quality, suggesting that miR-137 may mediate sleep-wake behaviour. Method – This prediction was tested by assessing the diurnal and circadian sleep-wake behaviour of Drosophila melanogaster homozygous for a null miR-137 allele (miR-137KO) or in flies where miR-137 expression had been silenced (miR-137sponge). Further investigation involved reintroduction of miR-137 expression in selected brain regions through use of the Drosophila UAS/Gal4 genetic construct. Sleep-wake behaviour was monitored and quantified using a well characterised Drosophila activity monitoring system (DAMS). Locomotor behaviour was assessed by video tracking flies and quantifying climbing ability in a negative geotaxis assay. For gene expression, qualitative PCR (qPCR) Taqman assays were used, and PCR genotyping was completed with custom designed primer sets and gel electrophoresis. A total transcriptomics assay was conducted and analysed using bioinformatical tools, then compared to GWAS datasets. Results – miR-137 null and knockdown genotypes had an extreme sleep phenotype characterised by increased total sleep amount. The phenotype was attributed to the homeostatic sleep control pathway through activation of the fan- shaped and mushroom bodies in the brain. Successful knockdown of miR-137 expression in the brain by the miR-137sponge was verified in qPCR, and PCR genotyping confirmed the replacement of miR-137 locus with an inserted wmW.hs sequence. There was also a moderate locomotor defect, though this did not account for the severity of the sleep phenotype. Additionally, there was a developmental delay along with an increased mortality in the pupal stage. There was no evidence for circadian disruption or shortening of lifespan because of mir-137 loss of function. Comparison of the transcriptome expression changes with sleep and psychiatric disorder related GWAS identified a select set of genes which provide putative mechanisms for miR-137 function. Conclusion – miR-137 is an important conserved microRNA with alleles already significantly associated with a major psychiatric disorder in humans. Currently, this research demonstrates that miR-137 is responsible for regulating pathways ultimately controlling sleep amount, and causing a slight locomotor phenotype, both of which are synonymous with some symptoms in the human SZ disorder. The project also acts to validate the high efficiency and ability of using Drosophila melanogaster as a model for future research into conserved microRNAs.