|dc.description.abstract||Mood disorders such as Major Depressive Disorder and Bipolar Disorder are devastating burdens upon sufferers, their families and society as a whole. Despite their high prevalence and socioeconomic impact, understanding the aetiology of mood disorders has proved difficult, due in part to the heterogeneity observed in disorders of mental well-being. Antidepressant and mood stabiliser drugs are the main treatment for mood disorders. Although highly efficacious, the mechanisms of action of these drugs are not well understood. This project aimed to establish the effects of such drugs on gene regulation, and in doing so, advance the field by detecting specific transcripts and biochemical pathways involved in drug response. It was anticipated that this knowledge would prove valuable for developing novel therapeutic and treatment options of the future.
Initial work focused upon promoter regions of candidate genes chosen from the literature and previous results from our laboratory. Nineteen constructs were investigated in the rat serotonergic cell line RN46A, using a luciferase reporter assay and a commonly prescribed SSRI antidepressant paroxetine. Although significant differences in expression were noted between drug-exposed and control cell cultures, the system proved inconsistent, despite considerable care in experimental design and execution.
A more direct approach, real-time quantitative PCR (qPCR), was then utilised to detect drug-induced gene expression differences in RN46A cells. Five drugs were used: the antidepressants paroxetine, citalopram and nortriptyline; the antipsychotic haloperidol; and the mood stabiliser sodium valproate. Of five potential reference genes for normalization of qPCR experiments, Actb, G6pd and Rnf4 were stably expressed over differing treatments. I screened thirty-nine candidate genes for expression changes attributable to drug exposure. The most striking result was a reproducible ~1700-fold up-regulation of sepiapterin reductase (SPR), by the drug sodium valproate. SPR encodes a key enzyme for neurotransmitter synthesis. Qdpr, encoding a protein in the same pathway, was also up-regulated by valproate, but to a lesser extent. SPR and members of the biopterin biosynthesis pathway have previously been implicated in the biology of depression. Another gene, the serotonin receptor 2A (Htr2a) was significantly down-regulated by the SSRIs paroxetine and citalopram.
Having identified that valproate induced the most significant gene expression changes,I further conducted time-course and dose-response experiments to investigate the dynamics and pharmacology of valproate- induced gene expression changes. Spr and Hdac6, a member of the histone deacetylase gene family, were found to behave differently in these experiments. Subsequently, western blotting confirmed increased SPR protein expression in treated cells, and detected at least three isoforms recognised by a commercially sourced SPR antibody.
Attempts to identify signalling pathways regulated by valproate failed to detect significant changes in the phosphorylation levels of specific kinases using an ELISA array.
Collectively, these data establish SPR and a number of other proteins in the aetiology of mood disorders and their treatment, and provide a basis for further study of the effects of mood stabilisers and antidepressants in a mammalian setting.||