*2.1. BDNF Transcripts*

The *BDNF* gene codes for a neurotrophin that is highly expressed in the CNS [26]. At the beginning of 2000s, the only data available about the structure and regulation of the *BDNF* gene were from Timmusk and colleagues, which identified in rats four 5' exons linked to separate promoters and one 3' exon encoding the preproBDNF protein [27–29]. These four *BDNF* promoters owned multiple points of *BDNF* mRNA regulation and suggested an activity-dependent regulation [28–31]. Further studies, published in 2007, clarified that *BDNF* has a complex gene structure with 11 different exons in humans, nine different exons in rodents, and nine alternative promoters for both groups [32,33]. Despite this complexity, the coding sequence is located in exon IX in both human and rodents. The latter includes the common sequence that encodes for the proBDNF protein. All other exons are untranslated regions with a start codon present in exons I, VII, VIII, and IX of the human *BDNF* gene. Exon IX is present in all BDNF mRNA isoforms. It is supposed that the nine alternative promoters can regulate the complex spatio-temporal expression of *BDNF* gene and allow BDNF to respond to a greater variety of stimuli. For instance, in human brain tissues, all exons are expressed, but to different degrees and in different brain structures [33]. Human heart tissue, instead, expresses high levels of *BDNF* isoforms containing exon IV and exon IX [33].

Currently, two *BDNF* promoters, promoter I and promoter IV, have been well characterized for their response after the activation of the L-type voltage gated calcium channel (L-VGCC) or the n-methyl-d-aspartate (NMDA) receptor. Activation of L-VGCC and NMDA receptors mediate intracellular Ca2<sup>+</sup>-signaling and regulate several aspects of brain functions (for review [34,35]). Promoter I is more responsive to neuronal activity and induces activity-dependent expression of BDNF in vitro and in vivo. It contains calcium-responsive elements (CaREs) and cyclic adenosine monophosphate (cAMP)/calcium response element (CRE) [29,36–38]. Deletion of CRE or overexpression of dominant negative of CREB (cAMP-response element-binding protein) significantly impairs rat BDNF promoter I response to neuronal depolarization [38]. Human BDNF promoter I is similar to rat promoter, since an orthologous CRE-like element is also present [39]. However, mutation of this site did not affect human *BDNF* promoter I response to depolarization [39]. Human BDNF promoter I also contains an activator protein 1 (AP1) -like element and an asymmetric E-box-like element [39]. Mutation in E-box-like element reduces human *BDNF* promoter I induction, impairing the response to neuronal depolarization [39].

Another highly characterized *BDNF* promoter is the *BDNF* promoter IV that contributes significantly to activity-dependent *BDNF* transcription. Human and rat *BDNF* promoter IV are similar. In this promoter, three CaREs and three other regulatory elements involved in regulating rat *BDNF* promoter response to NMDA receptor activation have been identified [40]. NMDA receptor activation is capable of triggering *BDNF* exon IV transcription through a protein-signaling cascade requiring extracellular signal-regulated kinase (ERK), Ca<sup>2</sup>+/calmodulin-dependent protein kinase (CaMK) II/IV, phosphoinositide 3-kinases (PI3K), and phospholipase C (PLC). *BDNF* exon IV expression also seems capable of further stimulating its own expression through TrkB activation [41]. Additionally to the CaREs, two positive regulators have been identified: the NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) [42] and NFAT (nuclear factor of activated T-cells) binding sites [30,43]. In contrast to these positive regulators, *BDNF* promoter IV also contains a negative regulatory element, the class B E-box. This is a binding site for a basic helix-loop-helix protein, BHLHB2, a suppressor of the bHLH gene superfamily [43,44]. NMDA treatment is able to remove BHLHB2 binding to the E-box and to increase rat *BDNF* promoter IV activity [43,44]. Disruption of *BDNF* promoter IV in mice significantly reduced the number of parvalbumin GABAergic neurons in the prefrontal cortex and impaired GABAergic activity [45]. These mice displayed depression-like behavior such as anhedonia-like behavior and increased latency to escape in the learned helplessness test [45]. Further evidence suggests a relationship between stress exposure and epigenetic regulation of *BDNF* promoter IV with the development of psychiatric disorders. Specifically, changes in *BDNF* promoter IV methylation levels are implicated in depression [46,47]. Preliminary evidence has demonstrated that patients with major depressive disorder (MDD) present a hypomethylation of the CpG-87 site of the promoter IV region of *BDNF* gene and are less likely to benefit from antidepressants [47,48]. In addition, *BDNF* disruption from promoter IV-derived transcripts impairs fear expression in mice, suggesting that cells expressing *BDNF* from promoter IV critically regulate hippocampal-prefrontal plasticity during fear memory [49,50].
