Influence of environmental factors in depression

MDD is a complex, debilitating, and prevalent psychiatric disorder with a set of diverse symptoms including depressed mood, hopelessness, worthlessness, loss of interest in normally pleasurable activities (anhedonia), sleep or eating disturbances, low energy, reduced concentration, impaired cognition, excessive feeling of guilt or loss, and even suicidal thoughts, for at least two weeks (American Psychiatric Association, 2013, Schulz and Arora, Reference Schulz and Arora2015, Otte et al., Reference Otte, Gold, Penninx, Pariante, Etkin, Fava, Mohr and Schatzberg2016).

Depression affects on average 20% of the global population (Vigo et al., Reference Vigo, Thornicroft and Atun2016) and is associated to high levels of morbidity and mortality (Fredman et al., Reference Fredman, Weissman, Leaf and Bruce1988, Bijl and Ravelli, Reference Bijl and Ravelli2000, Kessler et al., Reference Kessler, Akiskal, Ames, Birnbaum, Greenberg, Hirschfeld, Jin, Merikangas, Simon and Wang2006, Kessler and Bromet, Reference Kessler and Bromet2013, Chirita et al., Reference Chirita, Gheorman, Bondari and Rogoveanu2015, Laursen et al., Reference Laursen, Musliner, Benros, Vestergaard and Munk-Olsen2016). It estimates that by 2030 depression will be the leading cause of disability in developed nations (WHO, 2017; 2018). However, its aetiology is still not fully understood.

Genetic factors increase in around 30% the risk of depression (Kendler and Gardner, Reference Kendler and Gardner2016, Smoller, Reference Smoller2016, Shadrina et al., Reference Shadrina, Bondarenko and Slominsky2018, Pettersson et al., Reference Pettersson, Lichtenstein, Larsson, Song, Agrawal, Borglum, Bulik, Daly, Davis, Demontis, Edenberg, Grove, Gelernter, Neale, Pardinas, Stahl, Walters, Walters, Sullivan, Posthuma and Polderman2019). Epidemiologic studies have identified the important genetic contribution to MDD, expressed by the high concordance between monozygotic twins (Sullivan et al., Reference Sullivan, Neale and Kendler2000), and polymorphisms that increase the vulnerability to the development of this disorder (Fabbri et al., Reference Fabbri, Di Girolamo and Serretti2013).

Several lines of evidence indicate that genes are modulated by environmental factors, especially exposure to stress (Higuchi et al., Reference Higuchi, Uchida, Yamagata, Otsuki, Hobara, Abe, Shibata and Watanabe2011). Of note, studies have shown that the first episode of depression is preceded by a stressful event in about 60% of the cases (Post and Silberstein, Reference Post and Silberstein1994). In addition, a higher prevalence of stressful episodes is observed in the life of depressed compared to healthy individuals (Peyrot et al., Reference Peyrot, Middeldorp, Jansen, Smit, De Geus, Hottenga, Willemsen, Vink, Virding, Barragan, Ingelman-Sundberg, Sim, Boomsma and Penninx2013, Klengel and Binder, Reference Klengel and Binder2015, Lopizzo et al., Reference Lopizzo, Bocchio Chiavetto, Cattane, Plazzotta, Tarazi, Pariante, Riva and Cattaneo2015, Richter-Levin and Xu, Reference Richter-Levin and Xu2018).

Although it has been possible to treat depression for decades, depressive symptoms are only reversed after chronic treatment (2–4 weeks) with the currently available antidepressant drugs. However, about 30% of the patients treated with conventional antidepressants do not respond to any medication (Berton and Nestler, Reference Berton and Nestler2006, Johnston et al., Reference Johnston, Powell, Anderson, Szabo and Cline2019). Even among those responsive, about 2/3 do not present complete remission (Rosenzweig-Lipson et al., Reference Rosenzweig-Lipson, Beyer, Hughes, Khawaja, Rajarao, Malberg, Rahman, Ring and Schechter2007, Shelton et al., Reference Shelton, Osuntokun, Heinloth and Corya2010). Overall, these data highlight the need for a better understanding of the neurobiology of depression and to develop faster and more effective treatments, reducing associated health costs, and patient suffering.

Neurobiology of depression and antidepressant action

The discovery that the first antidepressants act by blocking noradrenaline or serotonin reuptake led to the emergence of the monoaminergic theory of depression (Schildkraut, Reference Schildkraut1965, Coppen, Reference Coppen1972, Schildkraut, Reference Schildkraut1995, Castren, Reference Castren2005). This theory postulated that monoaminergic neurotransmission is impaired in depressed individuals, and it is restored after chronic treatment with antidepressants. Even today, most of the antidepressants clinically used, such as tricyclic and selective serotonin reuptake inhibitors, facilitate this neurotransmission. However, the monoaminergic theory is overly simplistic since it does not explain the latency period for the therapeutic response (Castren, Reference Castren2005, Papakostas and Ionescu, Reference Papakostas and Ionescu2015, Otte et al., Reference Otte, Gold, Penninx, Pariante, Etkin, Fava, Mohr and Schatzberg2016, Ferrari and Villa, Reference Ferrari and Villa2017).

To explain this latency, the molecular or neurotrophic hypothesis of depression assumes that the stress triggers intracellular signalling cascades that would result in reduced expression of genes important for plasticity in limbic structures, such as BDNF(Duman et al., Reference Duman, Heninger and Nestler1997, Agid et al., Reference Agid, Buzsaki, Diamond, Frackowiak, Giedd, Girault, Grace, Lambert, Manji, Mayberg, Popoli, Prochiantz, Richter-Levin, Somogyi, Spedding, Svenningsson and Weinberger2007). Chronic administration of antidepressant drugs in stressed animals facilitates monoaminergic signalling, restores neurogenesis, and increases dendritic arborisation in the hippocampus and prefrontal cortex (Malberg and Duman, Reference Malberg and Duman2003, Rodrigues et al., Reference Rodrigues, Ledoux and Sapolsky2009, Castren and Rantamaki, Reference Castren and Rantamaki2010, Kimpton, Reference Kimpton2012, Liu et al., Reference Liu, Ge, Leng, Pan, Fan, Yang and Cui2017). Therefore, gene expression regulation has been proposed as an important molecular mechanism mediating the long-term adaptations related to stress, depression, and antidepressant effect (Tsankova et al., Reference Tsankova, Renthal, Kumar and Nestler2007, Krishnan and Nestler, Reference Krishnan and Nestler2008, Harmer et al., Reference Harmer, Duman and Cowen2017).

The recent discovery of fast-acting antidepressants such as ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has renewed the hope in more rapid and effective therapies (Cipriani et al., Reference Cipriani, Furukawa, Salanti, Chaimani, Atkinson, Ogawa, Leucht, Ruhe, Turner, Higgins, Egger, Takeshima, Hayasaka, Imai, Shinohara, Tajika, Ioannidis and Geddes2018). The exact mechanisms behind ketamine effect are not fully understood, but evidence suggests that it activates the mechanistic target of rapamycin (mTOR) and BDNF signalling, increasing the expression of synaptic proteins (e.g. postsynaptic density protein-95, PSD-95, and synapsin, Syn) in the hippocampus and prefrontal cortex (Gerhard and Duman, Reference Gerhard and Duman2018). These changes would result in a rapid, robust, and sustained antidepressant effects (Price et al., Reference Price, Nock, Charney and Mathew2009, DiazGranados et al., Reference Diazgranados, Ibrahim, Brutsche, Ameli, Henter, Luckenbaugh, Machado-Vieira and Zarate2010, Li et al., Reference Li, Lee, Liu, Banasr, Dwyer, Iwata, Li, Aghajanian and Duman2010, Li et al., Reference Li, Liu, Dwyer, Banasr, Lee, Son, Li, Aghajanian and Duman2011b, Duman and Voleti, Reference Duman and Voleti2012, Zhou et al., Reference Zhou, Wang, Yang, Li, Zhou and Yang2014, Pazini et al., Reference Pazini, Cunha, Rosa, Colla, Lieberknecht, Oliveira and Rodrigues2016, Zhang et al., Reference Zhang, Wang, Lv, Liu, Sun and Tian2018, Abdallah et al., Reference Abdallah, Roache, Averill, Young-Mccaughan, Martini, Gueorguieva, Amoroso, Southwick, Guthmiller, Lopez-Roca, Lautenschlager, Mintz, Litz, Williamson, Keane, Peterson and Krystal2019). Currently, a great body of studies has investigated other compounds that might share similar fast-acting antidepressant action, including glutamatergic neuromodulators (riluzole, agmatine) and glycine-binding site ligands (GLYX-13 and rapastinel) (Zomkowski et al., Reference Zomkowski, Hammes, Lin, Calixto, Santos and Rodrigues2002, Neis et al., Reference Neis, Bettio, Moretti, Rosa, Ribeiro, Freitas, Goncalves, Leal and Rodrigues2016a, Neis et al., Reference Neis, Moretti, Bettio, Ribeiro, Rosa, Goncalves, Lopes, Leal and Rodrigues2016b, Chen et al., Reference Chen, Almeida, Fiori and Turecki2018, Neis et al., Reference Neis, Bettio, Moretti, Rosa, Olescowicz, Fraga, Goncalves, Freitas, Heinrich, Lopes, Leal and Rodrigues2018, Kadriu et al., Reference Kadriu, Musazzi, Henter, Graves, Popoli and Zarate2019).

Brain areas related to depression

Several brain structures have been associated with stress circuits and the therapeutic action of antidepressants, including the amygdala, hypothalamus, ventral tegmental area, nucleus accumbens, hippocampus, prefrontal cortex, among others (Nestler et al., Reference Nestler, Barrot, Dileone, Eisch, Gold and Monteggia2002, Berton and Nestler, Reference Berton and Nestler2006, Krishnan and Nestler, Reference Krishnan and Nestler2008, Price and Drevets, Reference Price and Drevets2010). Plastic, neurochemical, and functional changes in these structures have been associated with the neurobiology of depression (Andrade and Rao, Reference Andrade and Rao2010).

Stress and MDD are associated with hyperactivation of the hypothalamic–pituitary–adrenal (HPA) axis and consequent release of corticotropin-releasing factor (CRF) from paraventricular neurons (PVN) of the hypothalamus, triggering the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary and leading to the releasing of glucocorticoids (cortisol in human and corticosterone in rodent) from the adrenal cortex into the blood. Glucocorticoids, through the glucocorticoid receptors (GRs), mediate negative feedback that regulates the HPA axis and stress endocrine responses (Heim and Binder, Reference Heim and Binder2012). They influence the metabolic, physiological, and immunological functions regulated by several stress-associated brain structures such as the hypothalamus, amygdala, prefrontal cortex, and hippocampus (Gillespie and Nemeroff, Reference Gillespie and Nemeroff2005, Pariante and Lightman, Reference Pariante and Lightman2008, Ulrich-Lai and Herman, Reference Ulrich-Lai and Herman2009, Bonfiglio et al., Reference Bonfiglio, Inda, Refojo, Holsboer, Arzt and Silberstein2011, Belda et al., Reference Belda, Fuentes, Daviu, Nadal and Armario2015).

The amygdala is a limbic structure responsible for the regulation of emotions, behaviour, responses to stress, and memory formation (Bhatnagar et al., Reference Bhatnagar, Vining and Denski2004, Li et al., Reference Li, Pleil, Stamatakis, Busan, Vong, Lowell, Stuber and Kash2012, Gilpin et al., Reference Gilpin, Herman and Roberto2015). MDD patients with low serotonin transporter binding potential present hyperactivation of the amygdala (Drevets, Reference Drevets2001, Sheline et al., Reference Sheline, Barch, Donnelly, Ollinger, Snyder and Mintun2001, Siegle et al., Reference Siegle, Steinhauer, Thase, Stenger and Carter2002, Parsey et al., Reference Parsey, Hastings, Oquendo, Huang, Simpson, Arcement, Huang, Ogden, Van Heertum, Arango and Mann2006). It is intimately connected to other brain regions involved in depression such as hippocampus and cortices (Amaral and Insausti, Reference Amaral and Insausti1992). Moreover, chronic mild stress increases the dendritic length and neurite density in this structure (Vyas et al., Reference Vyas, Mitra, Shankaranarayana Rao and Chattarji2002, Khan et al., Reference Khan, Chuhutin, Wiborg, Kroenke, Nyengaard, Hansen and Jespersen2016a,b).

The hippocampus is a subcortical limbic brain structure, closely related to learning, memory, adaptation to stress, and depression. Depressed patients present reduced hippocampal volume (Sheline et al., Reference Sheline, Gado and Kraemer2003, Lorenzetti et al., Reference Lorenzetti, Allen, Fornito and Yucel2009). In animal models, exposure to inescapable and chronic stress causes several hippocampal alterations such as a decrease in number and length of dendritic spines, synaptic loss, and impaired adult neurogenesis (Pittenger and Duman, Reference Pittenger and Duman2008, Serafini, Reference Serafini2012), suggesting a decreased connectivity in this structure (Andrade and Rao, Reference Andrade and Rao2010). These changes are attenuated by chronic antidepressant treatment (Castren and Rantamaki, Reference Castren and Rantamaki2010, Kimpton, Reference Kimpton2012). Recent studies suggest that the dorsal and ventral portions of hippocampus are molecularly and functionally distinct (Leonardo et al., Reference Leonardo, Richardson-Jones, Sibille, Kottman and Hen2006, Fanselow and Dong, Reference Fanselow and Dong2010, Tanti and Belzung, Reference Tanti and Belzung2013, Grigoryan and Segal, Reference Grigoryan and Segal2016, Diniz et al., Reference Diniz, Casarotto, Resstel and Joca2018). This difference should be considered when investigating the role of the hippocampus in stress and depression.

Another structure closely related to the neurobiology of depression is the prefrontal cortex. The prefrontal cortex plays an important role in cognition, learning, memory, decision making, and modulation of behavioural, autonomic, and endocrine in stress responses (Barbas, Reference Barbas1995, Liston et al., Reference Liston, Miller, Goldwater, Radley, Rocher, Hof, Morrison and Mcewen2006, Quirk et al., Reference Quirk, Garcia and Gonzalez-Lima2006, Resstel and Correa, Reference Resstel and Correa2006, Resstel et al., Reference Resstel, Joca, Guimaraes and Correa2006, Girotti et al., Reference Girotti, Adler, Bulin, Fucich, Paredes and Morilak2018). Dysfunctions in this structure are related to the development of mental disorders associated with stress, such as depression and posttraumatic stress disorder – PTSD (Geuze et al., Reference Geuze, Westenberg, Heinecke, De Kloet, Goebel and Vermetten2008, Pereira et al., Reference Pereira, Casarotto, Hiroaki-Sato, Sartim, Guimaraes and Joca2013). Similar to the hippocampus, stressful insults result in plastic modifications in the prefrontal cortex such as apical dendritic atrophy and reductions of synaptogenesis and cortical volume. Antidepressant drugs reverse these morphological changes (Duncan et al., Reference Duncan, Knapp, Johnson and Breese1996, Cook and Wellman, Reference Cook and Wellman2004, Rocher et al., Reference Rocher, Spedding, Munoz and Jay2004, Brown et al., Reference Brown, Henning and Wellman2005, Czeh et al., Reference Czeh, Perez-Cruz, Fuchs and Flugge2008). In line with these data, depressed patients present functional and volume cortical changes, which are sensitive to chronic antidepressant treatment (Harmer et al., Reference Harmer, Mackay, Reid, Cowen and Goodwin2006, Fitzgerald et al., Reference Fitzgerald, Laird, Maller and Daskalakis2008, Koenigs et al., Reference Koenigs, Huey, Calamia, Raymont, Tranel and Grafman2008, Yoshimura et al., Reference Yoshimura, Okamoto, Onoda, Matsunaga, Ueda, Suzuki and Shigetoyamawaki2010). In addition, the antidepressant effect of fast-acting drugs, such as ketamine, is associated with neuroplastic changes in the prefrontal cortex such as the rapid recovery of dendritic arborisation and synaptogenesis (Dwyer and Duman, Reference Dwyer and Duman2013, Abdallah et al., Reference Abdallah, Adams, Kelmendi, Esterlis, Sanacora and Krystal2016).

Recently, the lateral habenula nucleus has attracted interest as a relevant brain structure that is also associated with stress coping responses. Stressors led to hyperactivation of neurons in this structure following by negative effects on serotonergic function and an imbalance between glutamatergic and GABAergic signalling (Shabel et al., Reference Shabel, Proulx, Piriz and Malinow2014, Metzger et al., Reference Metzger, Bueno and Lima2017, Tchenio et al., Reference Tchenio, Lecca, Valentinova and Mameli2017, Wang et al., Reference Wang, Li, Feng, Guo, Zhou and Luo2017). These changes contribute to depressive-like behaviours (Aizawa et al., Reference Aizawa, Cui, Tanaka and Okamoto2013, Li et al., Reference Li, Zhou, Liao, Yang, Wong, Henn, Malinow, Yates and Hu2013, Cui et al., Reference Cui, Mizukami, Yanagisawa, Aida, Nomura, Isomura, Takayanagi, Ozawa, Tanaka and Aizawa2014). The deep brain stimulation of the lateral habenula nucleus prevents depressive-like behaviour in animals (Li et al., Reference Li, Piriz, Mirrione, Chung, Proulx, Schulz, Henn and Malinow2011a) and alleviates depressive symptoms in patients (Sartorius et al., Reference Sartorius, Kiening, Kirsch, Von Gall, Haberkorn, Unterberg, Henn and Meyer-Lindenberg2010).

Epigenetic overview

The human genome encompasses about 20,000 genes containing essential information on the normal growth, development, and survival of the organism. The long DNA strand (2 m) is packed in the compact nucleus of eukaryotic cells (10 μm in diameter). This process depends on the association of the DNA with histone and non-histone proteins resulting in a highly complex and organised structure called chromatin. The fundamental and functional unit of chromatin is the nucleosome consisting of a 146 base pairs (bp) envelope of DNA surrounded by an octameric histone complex with four histone proteins (H2A, H2B, H3, and H4). Mechanisms of repair, replication, recombination, transcription, dynamic opening, and closure of the chromatin structure are fundamental for homoeostasis (Hauer and Gasser, Reference Hauer and Gasser2017).

In the 1940s, Conrad Hal Waddington, a leading embryologist and geneticist, sought to explain how different cell types could be generated from cells containing the same common genome, thus generating different phenotypes that would result from the interaction between both genes and the environment. He believed that genes are important in determining the development of the organism, but environmental-induced modifications could occur and alter this development. He called this process causal embryology or “epigenetic” (Slack, Reference Slack2002). The definition of epigenetics, a word that means “above the genome”, has been modified over the years. It is now broadly understood as the process by which environmental factors can lead to stable changes in chromatin structure, altering gene expression but changing the primary sequence of bases in the DNA (Holliday, Reference Holliday2006, Allis and Jenuwein, Reference Allis and Jenuwein2016, Gayon, Reference Gayon2016). Epigenetic alterations have been related to the neurobiology of diverse illnesses, including psychiatric disorders (Tsankova et al., Reference Tsankova, Renthal, Kumar and Nestler2007, Mitchelmore and Gede, Reference Mitchelmore and Gede2014, Cattaneo et al., Reference Cattaneo, Macchi, Plazzotta, Veronica, Bocchio-Chiavetto, Riva and Pariante2015, Bartlett et al., Reference Bartlett, Singh and Hunter2017, Uchida et al., Reference Uchida, Yamagata, Seki and Watanabe2018, Bhandari et al., Reference Bhandari, Paliwal and Kuhad2020, Forero and Gonzalez-Giraldo, Reference Forero and Gonzalez-Giraldo2020, Jackson, Reference Jackson2020).

The biological properties of chromatin have been extensively studied since the 19th century. In 1884, Albrecht Kossel identified the histones and determined that these basic proteins were associated with DNA. Thanks to this pivotal contribution, he received the Nobel Prize in Physiology and Medicine in 1910. Since then, a large number of studies have investigated the structure and function of the histones (Grayson and Guidotti, Reference Grayson and Guidotti2013). Transitions between euchromatin and heterochromatin are associated with active or inactive transcription, respectively, and are mediated by modifications in the structure of the histones that constitute the nucleosome (Jenuwein and Allis, Reference Jenuwein and Allis2001).

Histone modifications occur mainly in histones 3 (H3) and 4 (H4) and comprise phosphorylation, ubiquitination, acetylation and deacetylation, and methylation and demethylation (Mersfelder and Parthun, Reference Mersfelder and Parthun2006, Bannister and Kouzarides, Reference Bannister and Kouzarides2011). Regarding the amino acids present along the histone tails, lysine (K) and arginine (R) are preferentially subject to methylation (Jenuwein and Allis, Reference Jenuwein and Allis2001). Histones are acetylated in K-residues on the N-terminal branching as part of the genetic regulation, being catalysed by the histone acetyltransferases (HATs) enzymes (Fig. 2). In deacetylation, in turn, acetylated histones lose the acetyl group by the action of histone deacetylase enzymes (HDACs, (Grayson and Guidotti, Reference Grayson and Guidotti2013). Histone acetylation is primarily associated with decondensed chromatin followed by increased accessibility of DNA to transcriptional factors and gene activation, being able to facilitate the expression of genes important for cellular plasticity and involved with the neurobiology of depression and antidepressants effects (Strekalova et al., Reference Strekalova, Spanagel, Bartsch, Henn and Gass2004, Krishnan and Nestler, Reference Krishnan and Nestler2008).

Figure 2. Summary of stress-induced epigenetic changes including histone acetylation and DNA methylation processes that can occur in mammalian tissues including brain. Stress is related with increased glucocorticoid levels and activation of signalling cascades that can result in the epigenetic enzymes modulation (activation or inhibition followed by alterations in the epigenetic mechanisms and transcription gene. Histone modifications can add various groups (methyl, acetyl, phosphoryl and ubiquitin) to the tails of the histone proteins. The histone acetyl transferase (HAT) enzyme catalyses the transfer of the acetyl group from acetyl-CoA to the histone protein. DNA methylation (DNAm) is the addition of methyl group from S-adenosyl-L-methionine (SAMe), catalysed by DNA methyltransferase (DNMT) enzymes, to the cytosine resulting in 5-methyl cytosine. These epigenetic mechanisms can affect the gene transcription and protein expression. H, histone protein; Acetyl-CoA, acetyl coenzyme A; HAT, histone acetyltransferase; SAMe, S-adenosyl-L-methionine; DNMT, DNA methyltransferase; DNAm, DNA methylation. Figure designed using imagens from BioRender.com.

In addition to post-translational modifications in histone tails (e.g., methylation, acetylation, phosphorylation, and ubiquitination), epigenetic mechanisms encompass covalent modifications (e.g., DNAm, detailed below) and nontranslational mechanisms of gene modulation (e.g., microRNAs, miRNAs, ribonucleic acid) (Issler and Chen, Reference Issler and Chen2015, Luoni and Riva, Reference Luoni and Riva2016, Ivanova et al., Reference Ivanova, Bozhilova, Kaneva and Milanova2018). These mechanisms have been extensively discussed elsewhere (O’Carroll and Schaefer, Reference O’carroll and Schaefer2013, Correia de Sousa et al., Reference Correia De Sousa, Gjorgjieva, Dolicka, Sobolewski and Foti2019, Tolsma and Hansen, Reference Tolsma and Hansen2019) and are not the scope of the current review.

Epigenetic mechanisms could promote long-lasting effects on mature neurons, interfering with complex neural mechanisms such as plasticity synaptic and neurogenesis (Tsankova et al., Reference Tsankova, Renthal, Kumar and Nestler2007, Karpova et al., Reference Karpova, Sales and Joca2017). The increase or decrease of chromatin condensation makes it more difficult or easier, respectively, the access of the transcriptional machinery to specific promoter regions, resulting in messenger RNA (mRNA) and protein levels changes (Tsankova et al., Reference Tsankova, Berton, Renthal, Kumar, Neve and Nestler2006, Tsankova et al., Reference Tsankova, Renthal, Kumar and Nestler2007, Krishnan and Nestler, Reference Krishnan and Nestler2008).

Although less understood, epigenetic mechanisms have recently been proposed to also play a crucial role in the rapid stress coping responses (Chandramohan et al., Reference Chandramohan, Droste, Arthur and Reul2008, Saunderson et al., Reference Saunderson, Spiers, Mifsud, Gutierrez-Mecinas, Trollope, Shaikh, Mill and Reul2016, Mifsud et al., Reference Mifsud, Saunderson, Spiers, Carter, Trollope, Mill and Reul2017) (Fig. 2). For example, stress and antidepressants modify cochaperones (mainly FK506-binding protein 51 and 52, FKBP51 and 52), which proteins that participate in the function of other chaperone proteins that act facilitating the folding proteins (Saibil, Reference Saibil2013). FKBP51 and 52 modulate the phosphorylation and activity of DNA methyltransferase (DNMT, an enzyme that catalyses DNAm), altering the DNAm levels (Gassen et al., Reference Gassen, Fries, Zannas, Hartmann, Zschocke, Hafner, Carrillo-Roa, Steinbacher, Preissinger, Hoeijmakers, Knop, Weber, Kloiber, Lucae, Chrousos, Carell, Ising, Binder, Schmidt, Ruegg and Rein2015). Still, the exposure to acute stressful events results in DNA demethylation of c-Fos (FBJ murine osteosarcoma viral oncogene homolog), an immediate-early gene, leading to its increased expression in the dentate gyrus of the hippocampus (Saunderson et al., Reference Saunderson, Spiers, Mifsud, Gutierrez-Mecinas, Trollope, Shaikh, Mill and Reul2016).

DNA methylation

DNAm usually occurs by a covalent attachment of a methyl group from the S-adenosyl-L-methionine (SAMe) onto the C5 position of cytosine residue of DNA, primarily occurring on cytosine-phosphate-guanine (CpG) dinucleotides clusters, resulting in 5-methyl cytosine (5mC) (Fig. 3). While showing cell- and tissue-specific differences, in mammalian cells DNAm often occurs in cytosines located on the so-named CpGs islands (CGIs, regions with a high frequency of CpG sequences). These islands are typically found in or near promoter regions resulting in 70–80% of methylated CpGs during DNAm (Moore et al., Reference Moore, Le and Fan2013, Ziller et al., Reference Ziller, Gu, Muller, Donaghey, Tsai, Kohlbacher, De Jager, Rosen, Bennett, Bernstein, Gnirke and Meissner2013, Li and Zhang, Reference Li and Zhang2014).

Figure 3. Schematic chromatin structure and DNA methylation mechanism. DNMTs catalyse the transfer of methyl group from S-adenosyl-L-methionine (SAMe) to the C5 position of the cytosine residue resulting in the S-adenosyl-L-homocysteine (SAH) and 5-methyl cytosine (5mC). This covalent modification is associated with low acessibily of the transcrition factors (TF) to the promoter gene following by gene silencing. SAMe, S-adenosyl-L-methionine; DNMT, DNA methyltransferase; SAH, S-adenosyl-L-homocysteine; TF, transcription fator. Figure designed using imagens from BioRender.com.

Classically, DNAm in promoters containing CGIs is associated with transcriptional reduction (Antequera, Reference Antequera2003, Lorincz et al., Reference Lorincz, Dickerson, Schmitt and Groudine2004, Brenet et al., Reference Brenet, Moh, Funk, Feierstein, Viale, Socci and Scandura2011). The methyl-CpG-binding domain (MBD) proteins bind preferentially to methylated CGIs. In addition, MBD proteins also form a complex with corepressor and chromatin-modifying proteins, resulting in gene silencing (Wajed et al., Reference Wajed, Laird and Demeester2001, Jones, Reference Jones2012). Moreover, the methyl binding can also mechanically block the access of transcription factors to promoter regions, also resulting in transcriptional repression followed by gene inactivation (Hark et al., Reference Hark, Schoenherr, Katz, Ingram, Levorse and Tilghman2000). Contrary to this, promoter hypermethylation has also been associated with increased transcriptional activity (reviewed in Smith et al., Reference Smith, Sen, Weeks, Eccles and Chatterjee2020), showing that the DNAm-mediated gene regulation is a very complex and unclear mechanism. It is possible that the DNAm may block the interaction between DNA and transcriptional repressors facilitating gene transcription (Nabilsi et al., Reference Nabilsi, Broaddus and Loose2009, Bahar Halpern et al., Reference Bahar Halpern, Vana and Walker2014, Jia et al., Reference Jia, Wang, Li, Tao, Chang, Hua, Yu, Wong and Feng2016). However, further studies are required for a better understanding of this mechanism.

Although DNAm occurs primarily in cytosine-guanine (CpG) dinucleotides, methylation also occurs in non-CpG sites, particularly in neuronal cells. The functions of these processes are unclear (Lister et al., Reference Lister, Mukamel, Nery, Urich, Puddifoot, Johnson, Lucero, Huang, Dwork, Schultz, Yu, Tonti-Filippini, Heyn, Hu, Wu, Rao, Esteller, He, Haghighi, Sejnowski, Behrens and Ecker2013). Additionally, DNAm in non-promoter regions has variable and unpredictable effects on gene expression (Antequera, Reference Antequera2003, Lorincz et al., Reference Lorincz, Dickerson, Schmitt and Groudine2004, Hellman and Chess, Reference Hellman and Chess2007, Ball et al., Reference Ball, Li, Gao, Lee, Leproust, Park, Xie, Daley and Church2009, Aran et al., Reference Aran, Toperoff, Rosenberg and Hellman2011, Jones, Reference Jones2012, Thomas et al., Reference Thomas, Sai and Wells2012, Kulis et al., Reference Kulis, Queiros, Beekman and Martin-Subero2013, Moore et al., Reference Moore, Le and Fan2013).

The methylation reaction is catalysed by DNA methyltransferases (DNMTs) enzymes (Fig. 3), detailed below. The first suggestion that DNAm (or demethylation) plays an important biological role was done by Griffith and Mahler, in Reference Griffith and Mahler1969. They proposed that this epigenetic mechanism could be the basis of long-term memory (Griffith and Mahler, Reference Griffith and Mahler1969). In 1975, two other groups suggested that DNAm would change gene expression, resulting in the repression of diverse genes (Holliday and Pugh, Reference Holliday and Pugh1975, Riggs, Reference Riggs1975). These studies also proposed that the methylation pattern was inherited through the action of a maintenance methylase enzyme capable of recognising hemimethylated DNA after replication, but unable to act on the unmethylated DNA.

DNAm has now been implicated in regulating gene activity in the adult brain under both normal and pathological conditions (Feng et al., Reference Feng, Zhou, Campbell, Le, Li, Sweatt, Silva and Fan2010). Intracellular signal transduction events, among other functions, can activate or inhibit transcription factors. The regulation of transcriptional activity by DNA-binding transcription factors depends on the interaction between several of these factors, including co-activators or co-repressors proteins and the underlying structure of the chromatin. The synaptic activity can activate or repress gene expression through chromatin remodeling. This mechanism regulates the expression of genes important for neural activity, survival, morphology, integration, and behaviour regulation (Zuckerkandl, Reference Zuckerkandl1975, Whitehouse et al., Reference Whitehouse, Rando, Delrow and Tsukiyama2007, Sweatt, Reference Sweatt2016, Uchida et al., Reference Uchida, Yamagata, Seki and Watanabe2018).

DNMT enzymes comprise five subtypes (DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L). Three of these subtypes (DNMT1, DNMT3a, and DNMT3b) are functionally active and widely studied. DNMT1 plays a maintenance role by copying the preexisting methylation of the parent into the daughter cell DNA during the replication phase. DNMTs 3a and 3b, in turn, are responsible for the new methylations in unmethylated CpG sequences (Jeltsch, Reference Jeltsch2006). DNMT2 is usually inactive or with low activity in both in vitro and in vivo studies (Herman and Baylin, Reference Herman and Baylin2003, Liu et al., Reference Liu, Wylie, Andrews and Tollefsbol2003). DNMT3L also lacks catalytic activity but is important for methylation by recruiting the active DNMTs (Bourc’his et al., Reference Bourc’his, Xu, Lin, Bollman and Bestor2001, Bourc’his and Bestor, Reference Bourc’his and Bestor2004, Ooi et al., Reference Ooi, Qiu, Bernstein, Li, Jia, Yang, Erdjument-Bromage, Tempst, Lin, Allis, Cheng and Bestor2007, Liao et al., Reference Liao, Karnik, Gu, Ziller, Clement, Tsankov, Akopian, Gifford, Donaghey, Galonska, Pop, Reyon, Tsai, Mallard, Joung, Rinn, Gnirke and Meissner2015, Long et al., Reference Long, Smiraglia and Campbell2017, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018).

During cell proliferation, DNMT1 is located in replication loci and primarily methylates the unmethylated DNA of daughter cells, maintaining the pattern of parental methylation across generations. In divided cells, DNMT1 is concentrated in replication sites during the S phase in order to maintain DNAm profile after the DNA synthesis of the daughter cell. DNMT1 deletion in progenitor neural cells results in reduced DNAm levels in post-mitotic neurons (Feng et al., Reference Feng, Zhou, Campbell, Le, Li, Sweatt, Silva and Fan2010, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018). However, DNMT1 participates not only in maintenance but also in new methylations (Pradhan et al., Reference Pradhan, Bacolla, Wells and Roberts1999, Gowher et al., Reference Gowher, Stockdale, Goyal, Ferreira, Owen-Hughes and Jeltsch2005, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018).

DNMTs 3a and 3b (known as “de novo” DNMTs) are more actively involved in the methylation of unmethylated DNA, establishing new methylation patterns (Feng et al., Reference Feng, Zhou, Campbell, Le, Li, Sweatt, Silva and Fan2010, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018). DNMT 3b is widely expressed in the early stages of neurogenesis (Okano et al., Reference Okano, Bell, Haber and Li1999) but is also present in adults (Feng et al., Reference Feng, Chang, Li and Fan2005, Feng et al., Reference Feng, Zhou, Campbell, Le, Li, Sweatt, Silva and Fan2010, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018). DNMT 3a is present in both the mature and development brain, suggesting its involvement in embryonic development and adult neuronal function. The two enzymes are often co-localized and associated with heterochromatin regions independent of the cell cycle (Bachman et al., Reference Bachman, Rountree and Baylin2001), possibly maintaining DNAm in pre- and post-mitotic cells (Feng et al., Reference Feng, Zhou, Campbell, Le, Li, Sweatt, Silva and Fan2010, Hervouet et al., Reference Hervouet, Peixoto, Delage-Mourroux, Boyer-Guittaut and Cartron2018).

Pharmacological manipulation of DNA methylation

In the 1960s, Sorm et al. (Reference Sorm, Piskala, Cihak and Vesely1964) synthesised the first epigenetic drugs (or epi-drugs) with possible inhibitory action on DNMTs, 5-azacytidine (5-AzaC), and 5-Aza-2′-deoxycytidine (5-AzaD or decitabine; Sorm et al., Reference Sorm, Piskala, Cihak and Vesely1964). Initially, these drugs were tested as antimetabolic nucleoside inhibitors for the treatment of acute myeloid leukemia. In 1968, Sorm and Veseley demonstrated that they cause a potent antileukemic effect in mice (Sorm and Vesely, Reference Sorm and Vesely1968). The clinical studies began in the 1980s (Rivard et al., Reference Rivard, Momparler, Demers, Benoit, Raymond, Lin and Momparler1981, Momparler et al., Reference Momparler, Rivard and Gyger1985) and, corroborating the preclinical findings, indicated that the 5-AzaD treatment induces complete remission in leukemic patients (Richel et al., Reference Richel, Colly, Kluin-Nelemans and Willemze1991). In 2004, the Food and Drug Administration (FDA) approved the first DNMT inhibitor (DNMTi) drug, 5-AzaC, and in 2006 the second, 5-AzaD, for clinical use in myelodysplastic syndromes. Their mechanisms are not fully understood but it is known that they inhibit DNAm and, at high doses, can be cytotoxic.

Aberrant promoter DNAm in tumour suppressor genes inhibits their expression and can contribute to tumourigenesis. The reactivation of these genes by inhibition of DNAm induced by DNMTi has potential antitumoural effects. Several studies in tumour cells have shown that genes involved in the development and progression of cancer are hypomethylated, whereas those associated with tumour suppression are hypermethylated (Stresemann and Lyko, Reference Stresemann and Lyko2008). DNMTi reversed this methylation pattern by reactivating tumour suppressor genes, such as p15 (Momparler, Reference Momparler2005, Yoo and Jones, Reference Yoo and Jones2006, Karahoca and Momparler, Reference Karahoca and Momparler2013).

DNMTi are divided into two groups, nucleoside and non-nucleoside, which inhibit DNMTs through different mechanisms. Nucleosidic DNMTi, such as 5-AzaC, 5-AzaD, zebularine, SGI-110, and CP-4200, are chemical analogues of cytidine, being integrated into the DNA molecule during replication (S-phase). They covalently bind to the DNMTs, causing an irreversible blockage of these enzymes and preventing DNAm (Stresemann and Lyko, Reference Stresemann and Lyko2008, Diesch et al., Reference Diesch, Zwick, Garz, Palau, Buschbeck and Gotze2016). 5-AzaC and 5-AzaD are administered as prodrugs with low oral bioavailability (Zhang et al., Reference Zhang, Sun, Gao, Jin, Xu, Lian, Sun, Sun, Liu, Fan, Zhang and He2013). They also have a short plasma half-life, approximately 30 minutes after intravenous administration (Daskalakis et al., Reference Daskalakis, Blagitko-Dorfs and Hackanson2010, Estey, Reference Estey2013, Navada et al., Reference Navada, Steinmann, Lubbert and Silverman2014). In general, the cellular uptake of these prodrugs depends on diverse transporters, including nucleoside transporter proteins (SLC28 and SLCA29 gene families; Pastor-Anglada et al., Reference Pastor-Anglada, Molina-Arcas, Casado, Bellosillo, Colomer and Gil2004, Qin et al., Reference Qin, Jelinek, Si, Shu and Issa2009). These drugs are activated by uridine-cytidine kinase and deoxycytidine kinase and are inactivated by deamination (Pastor-Anglada et al., Reference Pastor-Anglada, Molina-Arcas, Casado, Bellosillo, Colomer and Gil2004, Qin et al., Reference Qin, Jelinek, Si, Shu and Issa2009, Daskalakis et al., Reference Daskalakis, Blagitko-Dorfs and Hackanson2010, Valencia et al., Reference Valencia, Masala, Rossi, Martino, Sanna, Buchi, Canzian, Cilloni, Gaidano, Voso, Kosmider, Fontenay, Gozzini, Bosi and Santini2014). Moreover, nucleoside inhibitors are potentially nonspecific cytotoxic compounds with structural instability, which restricts their clinical therapy (Yoo and Jones, Reference Yoo and Jones2006, Stresemann and Lyko, Reference Stresemann and Lyko2008, Gros et al., Reference Gros, Fahy, Halby, Dufau, Erdmann, Gregoire, Ausseil, Vispe and Arimondo2012).

Non-nucleoside inhibitors, such as RG108, hydralazine, procaine, procainamide, IM25, and disulfiram, have a variety of action mechanisms. They are all independent of cell division. Their mechanisms include non-covalent inhibition in the DNA catalytic sites, prevention of their enzymatic activity (Lyko and Brown, Reference Lyko and Brown2005, Mai and Altucci, Reference Mai and Altucci2009), reduction of DNMTs affinity for the DNA (Zambrano et al., Reference Zambrano, Segura-Pacheco, Perez-Cardenas, Cetina, Revilla-Vazquez, Taja-Chayeb, Chavez-Blanco, Angeles, Cabrera, Sandoval, Trejo-Becerril, Chanona-Vilchis and Duenas-Gonzalez2005, Castellano et al., Reference Castellano, Kuck, Sala, Novellino, Lyko and Sbardella2008, Datta et al., Reference Datta, Ghoshal, Denny, Gamage, Brooke, Phiasivongsa, Redkar and Jacob2009), inhibition of methyl donor proteins (Cui et al., Reference Cui, Wakai, Shirai, Yokoyama, Hatakeyama and Hirano2006), or suppression of DNMTs expression (Pina et al., Reference Pina, Gautschi, Wang, Sanders, Schmitz, France, Cornell-Kennon, Sambucetti, Remiszewski, Perez, Bair and Crews2003). Usually, these drugs have a short plasma half-life (approximately 4 h for RG108 after subcutaneous administration) (Schneeberger et al., Reference Schneeberger, Stenzig, Hubner, Schaefer, Reichenspurner and Eschenhagen2016) and reduced adverse effects (Xu et al., Reference Xu, Hu, Luo and Liang2016). Additionally, natural compounds such as curcumin, genistein, EGCG, and resveratrol also act by indirect inhibition of DNMTs (revised in Lascano et al., Reference Lascano, Lopez and Arimondo2018).

DNMTi drugs have a wide distribution in body fluids, crossing the blood-brain barrier (Marcucci et al., Reference Marcucci, Silverman, Eller, Lintz and Beach2005, Schneeberger et al., Reference Schneeberger, Stenzig, Hubner, Schaefer, Reichenspurner and Eschenhagen2016) and possibly reversing situations where there is increased methylation in diverse tissues including CNS.