LAUSR

Background Brain-Derived Neurotrophic Factor (BDNF) is important for the survival of existing neurons and promotion of the growth and differentiation of new neurons and synapses. BDNF protein expression has been associated with various psychiatric disorders including major depressive disorders. The gene contains eight exons with their individual promoters that initiate transcription of multiple mRNA transcripts, each of which contains an alternative 5′ exon spliced to a common 3′ coding exon IX. The use of different promoters allows a diverse, temporal and spatial regulation of the gene. We were particularly interested in exon IV as it has been shown to play an important role in response to monoaminergic anti-depressant drugs which mainly exert their effects via downstream signaling leading to phosphorylation of CREB (calcium/cAMP response element binding protein) and possibly a transcriptional repressor, MeCP2. The pCREB binds to the response element in the promoter region and enhances exon IV transcription. Our aim is to investigate the interaction between antidepressant-mediated transcriptional regulation and BNDF exon IV methylation. Methods We treated the SH-SY5Y neuroblastoma cell line with Venlafaxine, Fluoxetine, and Mirtazapine and standardized the concentrations and time points at which these drugs increase the phosphorylation of CREB as well as MeCP2 using western blot analysis. We cloned BDNF exon IV promoter region upstream of the luciferase gene in a CpG free reporter vector and reporter assays were performed in the presence of Venlafaxine, Fluoxetine, and Mirtazapine at desired concentrations. Immunofluorescence was used to study the drug-mediated changes in localization of MeCP2 and CREB (phosphorylated and unphosphorylated). We used site-directed mutagenesis to replace the CpGs of interest in the reporter construct by methylated CpGs. Results An increased phosphorylation of both CREB and MeCP2 as early as 0, 5 h and up to 2hrs treatment of SHSY-5Y cells by Venlafaxine (10 μM /50 μM), Fluoxetine (5 μM/10 μM) and Mirtazapine (10 μM /50 μM) was observed. We did not see any increase in the exonIV transcriptional activity under these conditions in the unmethylated plasmid whereas methylated plasmid showed a statistically significant upregulation with all the three drugs. Immunofluorescence data shows that Fluoxetine changes the localization of only unphosphorylated MeCP2 to nucleo-cytoplasmic from nuclear. Venlafaxine and Mirtazapine showed no change in localization of MeCP2. Discussion A previous study from our group has shown that methylation of the CpG residues present on exon IV promoter complements the increase in serum BDNF levels by Venlafaxine and Fluoxetine, at physiological doses. The response to the drugs can be predicted by the methylation of the CpGs present in the proximal promoter, -35 bp to -147 bp. We hypothesize, kinases that get activated downstream of the 5-HT receptor, phosphorylate methyl binding protein (MeCP2) and thus abrogate its binding to the methylated CPGs. Contrary to MeCP2, phosphorylated MeCP2 can bind to unmethylated CpGs. As MeCP2 is a transcriptional repressor, it counteracts the upregulation of BDNF exon IV transcript by pCREB binding and thereby determines the efficacy of the drugs. This work commences a profound understanding of the epigenetic regulation of exon IV by antidepressants. Furthermore, it emerges to be in support with previous clinical findings regarding the use of CPG methylation as a predictive marker for antidepressant therapy.