Molecular Genetics of Brain Derived Neurotrophic Factor (BDNF)

抗抑郁治疗的主要靶点是5-羟色胺转运体（5-HTT）。 据认为，转运蛋白拮抗剂（SSRIs）通过改变基因表达（其中包括BDNF基因）来显示其治疗效果。 脑源性神经营养因子在抑郁症动物模型中表现得尤为突出。 因此，研究5-HTT（SLC 6A 4）和BDNF基因的序列变异如何影响接受SSRI治疗的患者的结果是适当的。 大量研究表明，SLC 6A 4基因的遗传变异可能与情感障碍（包括MDD）的易感性和治疗反应有关，尽管总体结论不一致。 SLC 6A 4启动子区的核苷酸序列重复多态性（HTTLPR）本身已成为心境障碍抗抑郁反应的药物基因组学研究的焦点。我们最近描述了SLC 6A 4 HTTLPR的L等位基因内的一种常见的功能性A>G变异。LG等位基因将SLC 6A 4 mRNA表达降低至几乎等同于S等位基因的水平，而LA等位基因赋予更高的SLC 6A 4表达，产生功能获得性表型（Hu et al.，2006，Am J Genet78：815-826）。 SLC 6A 4表达的预测显着提高与LG等位基因的知识成比例的频率在研究中的人群。 缓解抑郁症的序贯治疗替代方案（星星 *D）试验（www.star-d.org）收集了1953名参与者的DNA，这些参与者在初级和精神病护理环境中接受了选择性5-羟色胺再摄取抑制剂（SSRI）西酞普兰，然后定期评估结果和副作用。为了更好地了解HTTLPR内变异对抗抑郁药治疗后结局的潜在影响，我们进行了一项测量治疗结局和副作用负担的表型遗传关联研究。 我们发现HTTLPR多态性与西酞普兰副作用相关。特别是，LA等位基因与副作用负担降低相关。 由于LA等位基因赋予增加的SLC 6A 4转录，增加大脑和其他组织中的5-羟色胺转运蛋白水平可能导致靶向转运蛋白的抗抑郁药物的副作用较少。 在第二项研究中，我们试图了解BDNF基因转录调控的分子机制。 活动依赖性转录被认为是神经元将短暂的细胞变化转化为脑功能长期变化的机制。 众所周知，BDNF在调节现有突触连接的强度和形成新的突触接触中起重要作用。 已经描述了BDNF的两种激活依赖性启动子。 一个主要的启动子位于外显子4的侧翼区域内，并在培养的皮层和海马神经元中被膜去极化激活。BDNF外显子4特异性转录的其他激活剂包括NMDA、红藻氨酸和多巴胺。 我们发现，在BDNF启动子4中桥接活化应答性NF-κ B和CRE位点的序列含有调节转录的B类E盒（图7）。bHLH转录因子BHLHB 2特异性结合E-box区域。 为了确定内源性BHLHB 2的作用，进行染色质免疫沉淀测定（ChIP）测定。将来自用NMDA（50 μ M）处理的海马神经元培养物或未处理的神经元的交联染色质通过超声处理剪切，随后与对NF-κ B的p65亚基、CREB、BHLHB 2或RNA聚合酶II（RNA pol II）特异性的抗体孵育，以免疫沉淀（IP）与染色质结合的蛋白质。IPDNA去除了交联蛋白，然后使用扩增BDNF启动子4侧翼E-box区域的引物进行半定量PCR。用抗BHLHB 2的染色质IP处理神经元后，BDNF启动子4序列的扩增减少。与此相反，染色质恢复与抗p65，抗CREB，或RNA聚合酶II抗体导致从BDNF启动子4的扩增产物增加后，神经元用NMDA处理。 定量PCR显示，启动子4的BHLHB 2的启动子占用仅发生在基础条件下处理前的神经元与NMDA。 NMDA受体激活后，CREB结合增加3.5倍，NF-κ B结合增加9倍。 这些转录因子占有率的变化与我们先前观察到的NMDA处理后BDNF外显子4 mRNA水平的诱导一致，并支持BHLHB 2在海马神经元中作为转录抑制因子的观点。 这些发现定义了这种转录因子的新作用，其调节可能为调节脑中BDNF表达提供新途径。

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.