A major target of antidepressant therapy is the serotonin transporter (5-HTT). It is thought that antagonists of the transporter (SSRIs) manifest their therapeutic effect by alterations in gene expression, among them the BDNF gene. BDNF has featured prominently in animal models of depression. Therefore, it is an appropriate to examine how sequence variation at the genes for 5-HTT (SLC6A4) and BDNF may affect outcome of patients receiving SSRI therapy. A large number of studies have suggested that genetic variation at the SLC6A4 gene may be involved in vulnerability to affective disorders, including MDD, and in treatment response, although the overall conclusions are inconsistent. A nucleotide sequence repeat polymorphism in the SLC6A4 promoter region (HTTLPR) has itself been the focus of many studies on the pharmacogenomics of antidepressant response in mood disorders. We recently described a common, functional, A>G variation within the L allele of SLC6A4 HTTLPR. The LG allele reduces SLC6A4 mRNA expression to levels nearly equivalent to that of the S allele, while the LA allele confers higher SLC6A4 expression, producing a gain-of-function phenotype (Hu et al., 2006, Am J Hum Genet 78: 815-826). Prediction of SLC6A4 expression is significantly improved with knowledge of the LG allele proportional to its frequency in the populations under study. The Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial (www.star-d.org) collected DNA from 1953 participants in a clinical study who received the selective serotonin reuptake inhibitor (SSRI) citalopram in primary and psychiatric care settings followed by regular assessment of outcome and side effects. To better understand the potential influence of variation within HTTLPR on outcome following antidepressant treatment, we performed a genetic association study of phenotypes measuring treatment outcomes and side effect burden. We found that the HTTLPR polymorphism was associated with citalopram side effects. In particular, the LA allele was associated with reduced side effect burden. Because the LA allele confers increased SLC6A4 transcription, increased serotonin transporter levels in brain and other tissues may lead to fewer side effects for antidepressant medications that target the transporter.? In a second study, we sought to understand the molecular mechanisms underlying transcriptional regulation of the BDNF gene. Activity-dependent transcription is thought to be the mechanism through which neurons convert brief cellular changes to long-lasting alterations in brain function. It is well understood that BDNF plays an essential role in modulating the strength of existing synaptic connections and acts in the formation of new synaptic contacts. Two activation-dependent promoters of BDNF have been described. One major promoter is within the flanking region of exon 4 and is activated by membrane depolarization in cultured cortical and hippocampal neurons. Other activators of BDNF exon 4-specific transcription include NMDA, kainate, and dopamine. We discovered that a sequence bridging the activation-responsive NF-kappaB and CRE sites in BDNF promoter 4 contains a class B E-box (Figure 7) that regulates transcription. The bHLH transcription factor, BHLHB2, binds specifically to the E-box region. To determine the role of endogenous BHLHB2, chromatin immunoprecipitation assays (ChIP) assays were performed. Cross-linked chromatin from hippocampal neuron cultures treated with NMDA (50 microM) or untreated neurons was sheared by sonication and subsequently incubated with antibodies specific to the p65 subunit of NF-kappaB, CREB, BHLHB2, or RNA polymerase II (RNA pol II) to immunoprecipitate (IP) the proteins bound to the chromatin. The IPDNA had cross-linked proteins removed, followed by semi-quantitative PCR using primers that amplified the region of the BDNF promoter 4 flanking the E-box. IP of chromatin with anti-BHLHB2 resulted in decreased amplification of BDNF promoter 4 sequences after the neurons were treated with NMDA. In contrast, chromatin recovered with anti-p65, anti-CREB, or RNA polymerase II antibodies resulted in increased amplification products from BDNF promoter 4 after the neurons were treated with NMDA. Quantitative PCR showed that promoter occupancy by BHLHB2 of promoter 4 occurred only under basal conditions before treatment of neurons with NMDA. Following NMDA receptor activation a 3.5-fold increase in CREB binding and a 9-fold increase in NF-kappaB binding occurred. These changes in transcription factor occupancy were consistent with induction of BDNF exon 4 mRNA levels that we observed previously following NMDA treatment and support the idea that BHLHB2 acts as a transcriptional repressor in hippocampal neurons. These findings define a new role for this transcription factor, whose regulation may offer a new pathway for modulating BDNF expression in the brain.
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