Transcriptional effects of mood stabiliser drugs in a serotonergic cell line

Abstract

Sodium valproate (VPA) and lithium are drugs of different chemical classes that are widely prescribed in the treatment of bipolar disorder (BD). Molecular and pharmacological studies have revealed some relevant properties and targets, but the precise modes of action are not yet understood. In this thesis, the effects of lithium and VPA on gene expression in a cell line (RN46A) were examined. This cell line is derived from the rat medullary raphe nucleus, and represents a relevant cellular model to study the molecular effects of lithium and VPA. RNA-Seq analysis of cells treated with 0.5mM lithium for 72 hrs failed to detect significant gene expression changes. However, exposure of cells to 0.5mM VPA for 72 hrs induced widespread changes in expression of many genes, with more than 700 significantly upregulated and ~400 downregulated genes. The NanoString platform was used to validate a subset of 23 observed gene expression effects, in independent cell culture experiments. High correlation (r2=0.75) was observed between RNA-Seq and NanoString for the selected VPA-regulated genes. Clear validation was observed for ADAM23, LSP1, MAOB, MMP13, PAK3, SERPINB2, SNAP91, WNT6 and ZCCHC12. Differential alternative splicing was also observed with VPA exposure. Some of the genes were both differentially expressed and alternatively spliced. VPA is recognized as an inhibitor of chromatin modifying histone deacetylase (HDAC) enzymes, and part of its therapeutic action in mood disorders may relate to modification of gene expression via this chemical property. To this aim, the effects of the non-HDAC inhibitory analogue of VPA, valpromide (VPD), and HDAC inhibitors, including trichostatin A (TSA) and CI994, on the selected genes was explored. Expression of eight genes was modified by HDAC inhibition (HDACi): ZCCHC12 and SHANK3 were upregulated by VPA and v CI994 but downregulated by TSA; CDKN1C, MAOB, NGFR and WNT6 were upregulated by CI994 only, and MMP13 and VGF were upregulated by TSA only. The complex regulatory effects of different HDAC inhibitors or VPD on the selected genes suggest VPA can exert its regulatory effects via both HDACi-dependent and independent properties. VPA and lithium are drugs of different chemical classes. Genes displaying specific regulation by either or both of these drugs may be relevant to the mechanism of action, and co-regulated genes highlighting the common pathways are of greatest interest. We failed to detect any significant changes at 0.5mM lithium so, increased the concentration to 1mM and 2mM. At these exposure levels, four genes were co-regulated by VPA and lithium; CDKN1C and LSP1 at 1mM and LSP1, SERPINB2 and WNT6 at 2mM. We observed extensive gene expression changes in response to VPA with several genes and pathways involved in neuronal function or nervous system development. Understanding the broader gene regulatory effects of VPA in this serotonergic cell model has provided insights into how this drug may work, and identified new candidate genes or biochemical pathways that may be of relevance to the biology and treatment of mood disorders.