Identifying the mechanism of action of valproic acid on phosphoinositide signalling

A third of epilepsy patients are resistant to currently available medication, highlighting a need to identify new antiepileptic drugs. To identify new drugs, researchers often explore new chemical structures with a common mechanism of action, but surprisingly the direct cellular target for many drugs remains unclear. One such drug is valproic acid (VPA), commonly used in the treatment of both epilepsy and bipolar disorder (BD). Previous research using the single celled amoeba Dictyostelium discoideum identified phosphoinositide recycling as a potential therapeutic mechanism for VPA and this has been validated in animal seizure models, however, the molecular target for this mechanism is unclear. To identify this target, research in this thesis initially focuses on assessing the role of several key enzymes involved in phosphoinositide signalling as potential VPA targets. Loss of these proteins did not confer resistance to VPA in this model, suggesting that VPA may function through targeting proteins in the phosphatidylinositol (PI) salvage pathway. In this pathway three key enzymes, cytidine-diphosphate-diacylglycerol synthase (CDS), cytidine-diphosphate- diacylglycerol-inositol-3-phosphatidyltransferase (CDIPT) and diacylglycerol kinase (DGK) were investigated, with phylogenetic analysis establishing evolutionary conservation. Attempts to ablate the single D. discoideum Cds (CdsA) and Cdipt encoding genes were unsuccessful, suggesting a vital role for these proteins. In contrast, overexpressing both proteins showed that cells with elevated expression of CDIPT, but not CDSA, were resistant to therapeutic VPA concentrations. Furthermore, deletion of the single D. discoideum DGK (DGKA) gene was successful, with the mutant resistant to VPA during both acute and chronic treatment, that was restored on reintroduction of DGKA. To investigate whether loss of DGKA is related to epilepsy and BD, a range of related compounds were investigated for an effect on cell development. These experiments suggest that in D. discoideum, DGKA may provide a common target for both epilepsy and BD treatments, supporting data provided from preclinical and clinical studies in both disorders. Together this work suggests that DGK may provide a new therapeutic target for the treatment of both epilepsy and BD.