*2.3. Biology of BDNF*

Synthesis and maturation of BDNF is a multistage process, involving the formation of several precursor isoforms. The BDNF protein, discovered in 1982 [66], is a highly conserved protein of 247 amino acids, synthesized and folded in the endoplasmic reticulum as preproBDNF (32–35 kDa). Upon translocation to the Golgi apparatus, the signal sequence of the preregion is rapidly cleaved, and the isoform proBDNF (28–32 kDa) is generated [67]. The proBDNF is further cleaved to reach the mature isoform (mBDNF, 13 kDa) [67,68]. Intracellular proteolytic cleavage of proBDNF may occur by the subtilisin-kexin family of endoproteases such as furin, or in intracellular vesicles by convertases [69,70] (Figure 1).

**Figure 1.** Brain-derived neurotrophic factor (BDNF) protein structure. The preproBDNF consists of three sequences: signal sequence (s.s), pro-domain, and mature domain. The intra- or extracellular cleavage of preproBDNF generates functionally active isoforms: BDNF pro-peptide and mature BDNF (mBDNF), each of which exhibits a characteristic affinity to a specific type of receptor. Arrowheads indicate known protease cleavage sites involved in the processing of mature BDNF. The position of the single nucleotide polymorphism (rs6265, Val66Met) and the substitution of valine (Val) in methionine (Met) at codon (aa) 66 in the human BDNF gene is indicated by an arrow.

Extracellular cleavage of proBDNF is determined by plasmin [71] and matrix metalloproteases 2 and 9 (MMP2 and MMP9) [72,73]. Depending on the cell type, BDNF can be secreted in a constitutive or activity-dependent manner [74]. In neuronal cells, both proBDNF and mBDNF are released following cell membrane depolarization [75–77]. The balance of proBDNF and mBDNF depends on the particular stages of brain development and regions. In the early postnatal period, the concentration of proBDNF is higher and may be considered as an important factor modulating brain function; while mBDNF prevails in adulthood and is important for processes occurring in adulthood, such as neuroprotection and synaptic plasticity [78]. Both proBDNF and mBDNF are active, eliciting opposing effects via the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor (TNF) receptor family and TrkB receptor, respectively. In resting form, both types of receptor are located in the membrane of intracellular vesicles. Stimulation with cAMP, Ca2+, or electrical impulse initiates their transfer and fusion with the cellular membrane [79,80].

The mature domain of proBDNF interacts preferentially with p75NTR, mediating synaptic pruning in the prenatal brain [81]. ProBDNF, through its pro-domain, can also interact with the sortilin receptor or other vacuolar protein sorting 10 protein (Vps10p) (Figure 2). Thus, proBDNF binding to specific receptors triggers signaling pathways, which can determine neuronal fate via promoting their death or survival [82,83]. The proBDNF/p75NTR/sortilin binding complex initiates signaling cascades leading to the activation of c-Jun amino terminal kinase (JNK). This pathway is involved in neuronal apoptosis [82,83]. High levels of p75NTR expression are detected during brain development and post-traumatic recovery [84]. When mature domain of BDNF binds to p75NTR, the RIP2 (serine/threonine-protein kinase 2)/TRAF6 (tumor necrosis factor receptor associated factor 6)-mediated pathway is initiated, which leads to NF-kB activation [82,85]. The activation of NF-kB promotes neuronal survival and maintenance during brain development [85]. In addition, p75NTR interacts also with the Ras homologous (Rho) protein family. This pathway is reported to regulate neuronal growth cone development and motility [85].

**Figure 2.** Intracellular signaling cascades activated by interaction of BDNF isoforms with its receptors. proBDNF and mBDNF bind to different receptors, respectively. The mBDNF isoform exhibits highest affinity for the tyrosine kinase B receptor (TrkB) receptor, which when stimulated undergoes homodimerization and autophosphorylation, but also binds the low affinity neurotrophin receptor p75NTR. The interaction between the TrkB receptor and the p75NTR receptor in a complex increases the ligand binding affinity to BDNF. Sortilin is considered a co-receptor for p75NTR. The proBDNF isoform, consisting of two sequences (pro-domain and mature domain), interacts with specific receptors, sortilin and p75NTR, respectively. The binding of proBDNF to a p75NTR/sortilin-complex induces signaling pathways that are specific for proBDNF. The binding of proBDNF in combination with sortilin causes the involvement of neurotrophin receptor-interacting factor (NRIF), tumor necrosis factor receptor-associated factor 6 (TRAF6), and neurotrophin receptor-interacting MAGE homologue (NRAGE) proteins. This pathway activates the JNK-associated pathway that promotes programmed cell death, or the receptor-interacting serine/threonine-protein kinase 2 (RIP2) /TRAF6-mediated pathway is initiated. Multi-subunit IκB kinase (IKK) phosphorylates (orange dot) the inhibitor of kB (IkB) protein, which results in dissociation of IkB from NF-κB. The activated nuclear factor kappa B (NF-kB) is then translocated into the nucleus where it binds to specific sequences of DNA and promotes neuronal survival and maintenance. In addition, p75NTR interacts with the Rho family of proteins, whose activation mediates the activity of Rho-associated protein kinase (ROCK), which subsequently leads to activation of the AKT pathway, involved in cytoskeletal remodeling. The mBDNF/TrkB receptor complex triggers signaling pathways associated with activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC-γ), and GTP-ases of the Rho family, involved in survival, plasticity and neurite outgrowth, transcription regulation, and synaptic plasticity.

mBDNF binds with the high-affinity TrkB receptor, the receptor dimerizes, and the intracellular tyrosine residues are autophosphorylated [86]. Phosphorylated-TrkB activates several enzymes: PI3K, mitogen-activated protein kinase (MAPK), PLC-γ, and guanosine triphosphate hydrolases (GTP-ases) of the Rho gene family [87–89]. mBDNF-TrkB-signaling pathways regulate multiple events, such as apoptosis and survival of neurons [90–92], dendritic growth [93–96], spine maturation and stabilization, development of synapses [96–98], learning- and memory-processes-dependent synaptic plasticity [99,100].

PI3K/Akt-related pathway exerts antiapoptotic and pro-survival activity and modulates NMDA receptor-dependent synaptic plasticity [101–103]. The PI3K/Akt/mTOR cascade enhances dendritic growth and branching through regulation of protein synthesis and cytoskeleton development [104,105].

The MAPK/Ras-signaling cascade regulates protein synthesis during neuronal differentiation [85] and is also required for the activation of ERK 1/2 and CREB [106,107]. This pathway is crucial not only for early response gene expression (e.g., c-Fos), but also for cytoskeleton protein synthesis (e.g., Arc and cypin) [87], as well as dendritic growth and branching in hippocampal neurons [94,108].

The PLC-γ-dependent pathway evokes activation of CAM kinase and protein kinase C (PKC), which subsequently increases the 1,2-diacylglycerol (DAG) and Ca2<sup>+</sup> ion concentrations [89]. The PKC-dependent pathway is reported to enhance synaptic plasticity [85] (Figure 2).

In summary, the specific role of BDNF in the regulation of numerous brain physiological processes depends on the interaction of its isoforms with different types of receptors. This, in turn, elicits the activation of signaling pathways that are critical for processes of brain development, synaptic plasticity, and protection and/or regeneration after damage. Perturbation of the BDNF synthesis, resulting in dysfunctions of its signaling cascades, may be responsible for triggering several pathological processes.

#### *2.4. The Human BDNF Variant Val66Met*

BDNF level in the peripheral tissues, brain, and blood may be also affected by gene polymorphism. The pro-domain of BDNF is the locus of a functional human BDNF polymorphism (SNPs) Val66Met, also known as rs6265 or G196Apolymorphism [109]. This point mutation causes a substitution of Valine (Val) to Methionine (Met) at codon 66 (Val66Met) in the pro-domain of *BDNF* (Figure 1). The Val66Met polymorphism does not exist in the mouse or other model organisms. Thus, multiple studies aim to mimic the function of BDNF Val66Met in cellular models or in genetically engineered mouse models. The BDNF Val66Met variant was first identified in the late 1990s and in 2002 the first two genetic studies investigating the BDNF Val66Met polymorphism in the pathogenesis of neurodegenerative disease were published [110,111]. The functionality of BDNF Val66Met variant was only confirmed in 2003 [112], where BDNF Val66Met polymorphism was shown to disrupt the episodic memory in humans. In addition, Egan et al. [112] also demonstrated that in hippocampal cultures BDNF Val66Met polymorphism did not alter BDNF expression per se, but the perisomatic localization of BDNF. Then, in 2005 it was discovered that the BDNF Val66Met substitution also disrupts the sortilin-binding site, impairing activity-mediated secretion of BDNF [113]. Likewise, the BDNF Val66Met substitution also disrupts the translin-binding site, which impairs dendritic targeting of BDNF mRNA [114]. Thus, the principle molecular mechanism associated with the BDNF Val66Met polymorphism is the deficient activity-dependent release of BDNF, which consequently impacts the efficiency of BDNF-TrkB signaling [113]. Following the demonstration that this SNP was functionally relevant over the past 18 years, more than 1700 studies have investigated the effects of this polymorphism on brain function in health, as well as in diseases, particularly in neuropsychiatric disorders [115,116]. The BDNF Val66Met polymorphism has been associated with cerebral cortex plasticity [117,118], with gray matter structures [119,120], or white matter integrities and structural networks [121,122]. More specifically, BDNF Val66Met polymorphism is associated with cognitive processes [112,123–127], and cognitive impairment in neurodegenerative disease, such as Parkinson's disease (PD) [128,129] and AD [130,131], and even more with several brain disorders, including MDD and bipolar disorder [132–137], epilepsy [138–140], schizophrenia [125,141–144], aging and dementia [145] and stroke [117,146,147]. Met66, but not Val66, BDNF pro-domain can induce the growth cone retraction in young hippocampal neurons [148]. Although many studies have demonstrated the possible genetic effects of this BDNF polymorphism in diseases or brain function,

other articles have failed to replicate the findings. The discrepancies of BDNF Val66Met genetic studies may result from many factors such as environmental factors, ethnicity, age, and sex.

#### **3. Neuroplasticity in MDD: The E**ff**ects of Antidepressant Therapies**
