Epigenetics Of Stress Adaptations In The Brain 17

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Brain Research Bulletin 98 (2013) 76–92 Contents lists available at ScienceDirect Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull Review Epigenetics of stress adaptations in the brain Adrian M. Stankiewicz a , Artur H. Swiergiel b , Pawel Lisowski c,∗ a Department of Animal Behavior, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Postepu 1, 05-552 Jastrzebiec n/Warsaw, Magdalenka , Warsaw, Poland b Department of Animal and Human Physiology, Department of Biology, Gdansk University, Wita Stwosza 59, 80-308 Gdansk, Poland c Department of Molecular Biology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Postepu 1, 05-552 Jastrzebiec n/Warsaw, Warsaw, Poland a r t i c l e i n f o Article history: Received 17 January 2013 Received in revised form 4 July 2013 Accepted 6 July 2013 Available online xxx Keywords: Epigenetics Gene expression Brain Limbic system Stress Behavior a b s t r a c t Recent findings in epigenetics shed new light on the regulation of gene expression in the central nervous system (CNS) during stress. The most frequently studied epigenetic mechanisms are DNA methylation, histone modifications and microRNA activity. These mechanisms stably determine cell phenotype but can also be responsible for dynamic molecular adaptations of the CNS to stressors. The limbic–hypothalamic–pituitary–adrenal axis (LHPA) is the primary circuit that initiates, regulates and terminates a stress response. The same brain areas that control stress also react to stress dynamically and with long-term consequences. One of the biological processes evoking potent adaptive changes in the CNS such as changes in behavior, gene activity or synaptic plasticity in the hippocampus is psychogenic stress. This review summarizes the current data regarding the epigenetic basis of molecular adaptations in the brain including genome-wide epigenetic changes of DNA methylation and particular genes involved in epigenetic responses that participate in the brain response to chronic psychogenic stressors. It is concluded that specific epigenetic mechanisms in the CNS are involved in the stress response. © 2013 Elsevier Inc. All rights reserved. Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stress-induced epigenetic modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Epigenetic mechanisms in acute stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Restraint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Forced swim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Novelty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Epigenetic mechanisms in chronic stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Chronic restraint stress (CRS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Chronic social stress (CSS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Chronic variable stress (CVS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Long-term mental and pain stress (LMPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Epigenetic regulation of stress-related gene expression levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Glucocorticosteroid receptor gene (Nr3c1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Corticotrophin-releasing hormone gene (Crh/Crf) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Glial-derived neurotrophic factor gene (Gdnf) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Brain-derived neurotrophic factor gene (Bdnf) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Vasopressin gene (Avp) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. Disks large-associated protein 2 gene (Dlgap2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Glutamic acid decarboxylase 1 and reelin genes (Gad1/Gad67, Reln) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 78 78 78 78 79 80 80 81 81 82 82 82 84 84 84 85 85 85 Abbreviations: 5hmC, 5 -hydroxymethyl-2 -deoxycytidine; 5-HT, serotonin; CA, cornu ammonis; DG, dentate gyrus; DNMT, DNA methyltransferase; GR, glucocorticoid receptor; HPA, hypothalamic–pituitary–adrenal axis; LG–ABN, lick-groom and arched-back nursing; LHPA, limbic–hypothalamic–pituitary–adrenal axis; LMPS, long-term mental-pain stress; NA, nucleus accumbens; PTSD, post-traumatic stress disorder; PVN, periventricular nucleus of hypothalamus; H3/4, histone 3 or 4; K, lysine; S, serine; ac, acetylation; me, methylation; p, phosphorylation. ∗ Corresponding author. Tel.: +48 22 736 70 56; fax: +48 22 756 14 17. E-mail addresses: [email protected] (A.M. Stankiewicz), [email protected] (A.H. Swiergiel), [email protected] (P. Lisowski). 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.07.003 3. 4. A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 77 Application and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 86 89 89 1. Introduction Any stimulus that endangers one’s integrity or function (stressors) results in a stress response, an adaptive response to solve stressful situation and determine new coping strategies (Landowski, 2007). Stress characteristics depend on various factors such as the type of stress stimuli (Herman et al., 2003; Reyes et al., 2003), age, gender, hormonal state, stressor controllability, gene polymorphisms, previous experiences of the individual (Joels and Baram, 2009), and the studied brain areas (Keeley et al., 2006). The limbic–hypothalamic–pituitary–adrenal axis (LHPA) is the brain system essential in coordinating both rapid and long-term behavioral, physiological and molecular responses to psychogenic stressors (Lopez et al., 1999). The LHPA axis acts through a number of mediators such as corticotropin-releasing hormone (CRH) or glucocorticosteroids (GC). Stress alters neurotransmission and synaptic plasticity in the brain areas involved in the LHPA axis such as the prefrontal cortex, the hippocampus or the amygdala (Cuadra et al., 1999; Dunn and Swiergiel, 2008; Gardner et al., 2009). Stimuli that are long lasting and intensive can lead to a persistent change in the stress response and mechanisms and in the function and structure of the brain itself (Armario et al., 2008; Darnaudery and Maccari, 2008; Fontenot et al., 1995). These changes can lead to cognitive deficits and behavior alterations (Radley et al., 2004; Raju et al., 2007; Wellman et al., 2011; Winocur et al., 2012) that can translate to neurodegenerative or mental illnesses such as schizophrenia (Corcoran et al., 2003), drug addiction (Sinha and Chronic Stress, 2008), Alzheimer’s disease and depression (Sotiropoulos et al., 2008) or anxiety disorders (Coplan et al., 1996). Certain cellular processes affected by stress such as apoptosis, neurogenesis and chromatin modifications may be responsible for the long-term, irreversible, stress effects (Lisowski et al., 2011). These persistent alterations in biological processes can be caused by changes in gene expression. Epigenetic mechanisms such as DNA methylation and histone modifications are strongly suspected in long-term and in rapid, dynamic gene expression regulation during stress (Tsankova et al., 2007). The role of epigenetics is strongly emphasized in synaptic plasticity, memory and cognitive processes as well as in shaping stress-vulnerable phenotypes and behavioral adaptations to chronic stress (Siegmund et al., 2007; Uchida et al., 2011). Epigenetics indicates stable heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence (Berger et al., 2009). Epigenetic mechanisms (defined here as a series of connected enzymatic reactions) can cause epigenetic modifications such as the methylation of cytosine nucleobases or histones. Specific sets of epigenetic modifications (epigenomes) can result in specific molecular changes, which can then be involved in epigenetic processes such as gene silencing, X-chromosome inactivation or imprinting. The gene sequence remains unchanged throughout life; however, environmental factors such as stress (McGowan et al., 2009), diet (Weaver et al., 2005) or maternal care (Szyf et al., 2005) act through certain chemical reactions to influence the chromatin state. These reactions can unravel the chromatin and cause stretches of DNA containing a gene to be exposed for longer or shorter periods of time, essentially turning the gene on or off and allowing for changes in protein production. This change in protein production, in turn, can affect physiological and behavioral traits and can be passed from one cell to the next as the cells multiply within an organism and can even be passed from parents to children (Roth et al., 2009). The epigenetic signal cascade begins with an “epigenator” (Berger et al., 2009). Epigenator is a concept consisting of all signals, both environmental cues and intrinsic processes, usually transient, that end with the recruitment of an “epigenetic initiator”. The epigenetic initiator acts directly on chromatin and determines the site of epigenetic modification. An example of an epigenetic initiator is the REST protein, which is specific to neurons. The protein contains a zinc finger domain that is responsible for recognizing specific DNA sequences. Furthermore, the initiator can invoke modifications without environmental cues and can persist within a cell, along with an “epigenetic maintainer”, for long periods of time. The epigenetic maintainers are recruited by the epigenetic initiator (the epigenetic initiator binding site complexes) and may act through different pathways (such as DNA methylation and histone modification) to establish epigenetic patterns (Berger et al., 2009). Epigenetic maintainers include enzymes that modify histone amino acids or cytosines in DNA such as histone acetyltransferases (HATs) or DNA methyltransferases (DNMTs). To summarize the above terminology see Fig. 1. Each cellular state and each environmental factor to which a cell is responsive can be characterized by a specific epigenetic pattern, i.e., an epigenome (Tsankova et al., 2006). The combination of the specific epigenetic pattern, their functional meaning, and the pathways that lead from pattern to function gives rise to a concept known as the “epigenetic code” (Jenuwein and Allis, 2001; Turner, 2007). The most thoroughly described epigenetic mechanisms in the context of stress are DNA methylation and histone modifications. Only these two mechanisms will be discussed in this review. The methylation of cytosines (5-methylcytosine—5mC) in DNA is considered the most stable epigenetic modification and is generally, though not always, transcription-repressive in nature Fig. 1. Epigenetic signaling cascade (Berger et al., 2009). See text for description. 78 A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 (Graff and Mansuy, 2008). 5-methylcytosine is most abundant in cytosine-rich, approximately 200-nucleotide long, “CpG (cytosine-phosphate-guanine) islands”. The two main mechanisms of cytosine methylation through which the 5mC acts involve (a) physically preventing the binding of transcription factors to DNA and (b) recruiting proteins that contain methylated CpGbinding domain (MBD proteins) such as MeCP2. MBD proteins contain domains with histone deacetylating (HDAC) (Dhasarathy and Wade, 2008) and methylating (HMT) activity (Tsankova et al., 2007) that induce chromatin condensation and gene silencing. Recently, another type of modified cytosine was identified in brain cells: minute amounts of 5 -hydroxymethyl-2 -deoxycytidine (5hmC) present in the genome that are thought to participate in epigenetic processes, especially demethylation (Dahl et al., 2011) and may constitute another functional class of nucleobases (Yu et al., 2012). Although epigenetic modifications apply to all nucleosomal histones, the H3 and H4 histones have been studied the most frequently (De Ruijter et al., 2003). Generally, epigenetic histone modifications are located at the amino-terminal tail of histones and present two modes of action: altering electrostatic charge, and thus the level of chromatin condensation; or recruiting epigenetic readers, which are histone-binding proteins that alter the transcription process. There are several different epigenetic maintainers responsible for specific epigenetic histone modifications. Epigenetic maintainers include histone acetyltransferases (HATs), methyltransferases (HMTs), and protein kinases (PKs); these are so-called histone writers that induce acetylation, methylation and phosphorylation, respectively. Moreover, other maintainers (called “erasers”) can remove epigenetic modifications; these erasers include histone deacetylases (HDACs), demethylases (HDMs), and phosphatases (PPs). Histone acetylation usually leads to chromatin relaxation and upregulation of gene expression. Histone methylation can induce or repress gene expression depending on which residue is methylated and to what degree (http://www.actrec.gov.in/histome/index.php). Histone phosphorylation can translate to expression activation or repression and can co-locate with histone acetylation to form a complex, expression-inducing histone modification called phosphoacetylation (Tsankova et al., 2007). There are also other, less studied histone modifications such as ubiquitination, ADPribosylation, and the addition of a small ubiquitin-like modifier (SUMO) protein or biotin that can have various effects on transcription. Stress produces long-lasting, epigenetic changes in gene expression in various brain structures that can result in CNS pathology. It is thus important that the epigenetic basis of stress adaptation and pathology are understood. Below, we review research findings regarding the role of epigenetics in brain responses to stress. 2. Stress-induced epigenetic modifications There are several reports regarding the general trends in epigenetic modifications in various acute and chronic stress paradigms. Differing results from these studies suggest a very complex and specific web of factors influencing epigenetic patterns. Table 1 summarizes the data presented in this section. 2.1. Epigenetic mechanisms in acute stress 2.1.1. Restraint Various paradigms of acute stress have been employed for studying the immediate biological effects of different stressors. Acute restraint has been suggested to be the most potent and harmful psychogenic stressor. In a study conducted in adult male rats (Sprague–Dawley) by Hunter et al. (2009), 45 min from an acute 30-min restraint stress, the dentate gyrus and the cornu ammonis 1 (CA1) zone of the hippocampus displayed epigenetic responses in the primarily expression-suppressing histone H3: (1) an increase in lysine 9 trimethylation (H3K9me3); (2) a decrease of the H3K9me3 prerequisite, lysine 9 monomethylation (H3K9me1); and (3) a decrease in lysine 27 trimethylation (H3K27me3). Acute injections of corticosterone had no effect on histone methylation (Hunter et al., 2009). Hunter et al. (2009) concluded that histone methylation, at least during acute restraint stress, may be mediated by agents other than glucocorticosteroids such as catecholamines or glutamate, which are known to respond to acute stress. 2.1.2. Forced swim Bilang-Bleuel et al. (2005) reported that various stressors differently influenced a number of cells containing histone H3 phosphorylated at the 10th serine of (H3S10p) in the dentate gyrus (DG) of the adult male mouse (C57BL/6) or rat (Wistar) hippocampus. Acute forced swim in 25 ◦ C or 19 ◦ C water (both mice and rats were subjected to the stress) and predator stress increased the immunoreactivity of neurons showing histone H3 phosphorylation (H3S10p) as well as immobility in a forced swim re-test, which was conducted 24 h after the initial test. The change in H3S10p immunoreactivity was absent when other stressors were used such as acute exposure to ether (rats) or chronic exposure to cold (rats) (Bilang-Bleuel et al., 2005). In contrast, voluntary exercise, as would its role in stress alleviation suggest (Binder et al., 2004), significantly reduced the number of neurons showing high histone H3 phosphorylation (H3S10p) in mice. Interestingly, a number of neurons showing histone hyperphosphorylation and the time of immobility appeared to rise along with the intensity of the swim stress. After swimming in 25 ◦ C water, histone H3 phosphorylation (H3S10p) was observed only in a fraction of adult cells of the dentate gyrus; however, when rats swam in 19 ◦ C water, immature neurons also begun to show histone H3 phosphorylation. Both histone hyperphosphorylation and decreased immobility time were completely attenuated by subcutaneous (s.c.) injections of glucocorticoid receptor (GR) antagonists (RU 38486 and ORG 34517) administered subcutaneously. Apart from explaining the shared molecular mechanisms that evoke the abovementioned changes, the results suggest that histone phosphorylation may regulate the expression of genes involved in stress-induced immobility (and not the other way around, as will be argued below). In vitro histone phosphorylation and Fos gene induction are known to be related (Clayton et al., 2000). c-Fos, a protein product of the Fos gene, is an indicator of acute neuronal activation (Dragunow and Faull, 1989). Fos is also a target gene of the cAMP response element-binding protein (CREB), a transcription factor implicated in the regulation of epigenetic patterns (Tsankova et al., 2007). CREB functions at various molecular levels and is known to be regulated in the hippocampus after both chronic and acute stress (Alboni et al., 2011; Boer et al., 2007; Gronli et al., 2006). It is thus noteworthy that H3 phosphorylation did not appear to co-occur with increased cFos expression after acute forced swim in Bilang-Bleuel’s work (Bilang-Bleuel et al., 2005). It is important to observe that the abovementioned changes were elicited only by the stressors that may be qualified as psychogenic (forced swim, predator exposure) and not by stressors purely physical (ether, cold). This may point to the important role of limbic structures and cortical areas in inducing histone phosphorylation after acute stress in the dentate gyrus. In a related study by the same group (Chandramohan et al., 2008), acute forced swim in 25 ◦ C water increased the number of neurons positive for the chromatin-relaxation epigenetic modification of phosphoacetylation of histone H3 (acetylation of the 14th lysine and phosphorylation of the 10th serine; H3K14acS10p) in A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 79 Table 1 List of modifications of genome-wide epigenetic patterns induced by various stressors. Research were conducted on adult male animals, unless stated otherwise in “Effect”. Stress Brain area Organism Effects Refernces Acute forced swim Hippocampus (dentate gyrus—DG) Hippocampus (DG) Acute forced swim Hippocampus (DG) R. norvegicus (Wistar)/M. musculus (C57BL/6) Acute novelty stress Acute restraint stress Hippocampus (DG) R. norvegicus (Wistar) Increased number of H3S10p+ (phosphorylation of serine 10 of histone 3) neurons Increased number of H3S10p+ (phosphorylation of serine 10 of histone 3) neurons Increased number of H3K14acS10p+ (acetylation of lysine 14 and phosphorylation of serine 10 of histone 3) neurons Increased number of H3K14acS10p+ neurons Bilang-Bleuel et al. (2005) Acute predator exposure Rattus norvegicus (Wistar)/Mus musculus (C57BL/6) M. musculus (C57BL/6) Hippocampus (DG, CA1) R. norvegicus (Sprague-Dawley) Hunter et al. (2009) Chronic variable stress Hippocampus (DG, CA3) R. norvegicus (Wistar) Subchronic restraint stress Hippocampus (DG) R. norvegicus (Sprague-Dawley) Subchronic restraint stress Chronic restraint stress Chronic social defeat Hippocampus (CA1, CA3) Infralimbic medial prefrontal cortex R. norvegicus (Sprague-Dawley) Subchronic social defeat Chronic social defeat/social isolation Hippocampus Nucleus accumbens R. norvegicus (Sprague-Dawley) M. musculus (C57BL/6ByJ) Increased H3K9me3 (trimethylation of lysine 9 of histone 3), decreased number of H3K9me1 (monomethylation of lysine 9 of histone 3 and H3K27me3 (trimethylation of lysine 27 of histone 3) immunoreactivity of brain cells Decreased H4K12ac (acetylation of lysine 12 of histone 4) and H3K9acS10p (phosphorylation of serine 10 and acetylation of lysine 9 of histone 3) content Decreased H3K27me3 (Post-stress levels) and increased H3K9me3 (Basal level) immunoreactivity of brain cells Increased H3K9me3 (Basal level) immunoreactivity of brain cells Increased H3K4me3 immunoreactivity of brain cells Increased number of H3K9ac+ (acetylation of lysine 9 of histone 3) or K14a+ (acetylation of lysine 14 of histone 3) neurons and glial cells Increased H3ac (histone 3 acetylation) content Wilkinson et al. (2009) Hippocampus (CA3) R. norvegicus (Wistar) Mostly increased H3 methylation at gene promoters, H3 methylation pattern of mice resistant to stress similar to vulnerable mice after antidepresant treatment, similar H3 methylation pattern in social defeat and isolation Increased number of H4ac+ (histone 4 acetylation) neurons after 2 weeks of recovery in mice of high excitability threshold Long-term mental and pain stress Hippocampus (DG) the dentate gyrus but not in the neocortex, amygdala or striatum of male adult Wistar rats. The phosphoacetylation was prevented by intraperitoneal (i.p.) injections of (1) MK-801, an antagonist of NMDA receptors, (2) SL-327, an inhibitor of extracellular-signalregulated kinases 1 and 2 (ERK1/2) kinases, which are part of MAPK/ERK signaling pathway, (3) knockout of mitogen and stressactivated kinases (MSK 1 and 2-knockout male adult C57BL/6 mice) (Chandramohan et al., 2008), which are downstream targets of ERK1/2 and are crucial factors in biochemical pathways leading to histone H3 phosphorylation (Soloaga et al., 2003). Behavioral responses to antagonists were more divergent. Intraperitoneal treatment with MK-801 resulted in reduced immobility in rats; however, SL-327 in rats and MSK-knockout mice reduced immobility only in a re-test that was 24 h after the initial pre-swim. These findings exclude the possibility that it is the immobility during the forced swim that elicits changes in histone phosphorylation. Nevertheless, the results confirmed that at least some histone modifications in the dentate gyrus (DG) depend on NMDA-ERK-MSK pathway activity (Chandramohan et al., 2008). As histone hyperphosphorylation after forced swim is glucocorticosteroid dependent (Bilang-Bleuel et al., 2005), and hyperphosphoacetylation after novelty stress (see below) depended on both NMDA and GR action (Chandramohan et al., 2007), it is proposed that GR activation plays an important role in these processes, likely by forming a histone–phosphorylating complex with ERK/MSK (Chandramohan et al., 2008). Bilang-Bleuel et al. (2005) Chandramohan et al. (2008) Chandramohan et al. (2007) Ferland and Schrader (2011) Hunter et al. (2009) Hunter et al. (2009) Hunter et al. (2009) Hinwood et al. (2011) Hollis et al. (2010) Sokolova et al. (2006) This hypothesis was supported by Gutièrrez-Mecinas (Gutierrez-Mecinas et al., 2011). The authors used GR and NMDA antagonists (s.c. injection of RU486 and i.p. injection of MK-801, respectively) to study the effects of forced swim test on histone phoshoacetylation DG in adult male Wistar rats. This response results in long-term elevation of immobility in forced swim re-test conducted 24 h or 4 weeks after initial forced swim. NMDA antagonists decreased: (1) phoshorylation (and thus activation) of both ERK and two epigenetic initiators – Elk-1 and MSK1 – lying downstream of ERK; (2) histone H3 phosphoacetylation (H3K14S10) of c-Fos promoter in DG; (3) elevated immobility in re-test. GR antagonist-induced effects were similar, except that GR was not involved in ERK phosphorylation in DG. Despite this apparent lack of interaction, GR and pERK actually formed complexes which were crucial in consolidation of behavioral responses. Thus, NMDA-GR pathway is critically involved in stress-dependent: regulation of phosphoacetylation in DG and long-lasting behavioral changes. 2.1.3. Novelty Chandramohan et al. (2007) reported that acute novelty stress (new cage) in male adult Wistar rats increased the number of neurons positive for histone H3 (H3S10pK14ac) phosphoacetylation in the dentate gyrus. Interestingly, the number of phosphoacetylated neurons peaked just after 0.5–2 h, revealing the rapid formation of epigenetic patterns, an observation repeated in Chandramohan’s subsequent work (see above) on the effects of forced swim 80 A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 in rats (Chandramohan et al., 2008). After an acute forced-swim test, the phosphorylation of DG histone H3 peaked after 8–24 h and was fully blocked by GR antagonists in Bilang-Bleuel’s work (Bilang-Bleuel et al., 2005), therefore mechanisms and timings of these two epigenetic modifications are different. Novelty stress produced effects only in a small subpopulation of mature dentate gyrus cells, which is in agreement with observations from Bilang-Bleuel et al. (2005) and Chandramohan et al. (2008). The number of neurons with increased levels of phosphoacetylation correlated with the light intensity of the stressing procedure. Rats are nocturnal, and light is known to determine the intensity of their stress response (Garcia et al., 2005). In addition to the differing temperatures for swimming, this is another example of the functional relationship between the scope of epigenetic modifications and the stressor intensity. Histone H3 hyperphosphoacetylation in the dentate gyrus was strongly linked spatially and temporally with expression of Fos, contrary to previous reports on H3 phosphorylation-c-Fos co-localisation (Bilang-Bleuel et al., 2005). In addition, blocking NMDA (i.p. injection of MK-801) and GR (s.c. injection of ORG 34517) receptors inhibited post-stress c-Fos mRNA expression as well as the increase in H3 phosphoacetylation. Moreover, it was found that the number of cells showing phosphoacetylation was equal to the number of cells showing post-stress expression of Arc (Chandramohan et al., 2007). ARC protein is a mediator of learning and memory (Rosi, 2011); whether histone phosphoacetylation and Arc expression are in fact linked remains to be investigated. In more recent work (Papadopoulos et al., 2011), it was shown that pre-treatment with the indirect, full GABA-A receptor agonist, lorazepam (i.p.), inhibited the novelty stress-related increase in histone H3 (H3K14acS10p) phosphoacetylation and c-Fos expression in the DG of male adult Wistar rats. Basal levels of phosphoacetylation and c-Fos were not affected. A partial GABA-A agonist, FG–7142 (i.p.), in turn, significantly increased the number of phosphoacetylation- and c-Fos-positive neurons in the DG under stress and in a non-stressed control. When co-administered, the irreversible NMDA antagonist MK-801 abolished the ability of FG7142 to upregulate H3 phosphoac etylation levels. Hence, it is postulated that the GABA-A receptor affects epigenomic patterns through NMDA receptor pathway modulation (Papadopoulos et al., 2011). The discussed literature, originating from Reuls group, outlines mechanisms governing stress-related histone phosphorylation and phosphoacetylation, most importantly, the involvement of dual GR and NMDA-ERK-MSK pathways action and NMDA–GABA-A receptor interplay. Yet, there is still a need to discover the exact genes affected by this mechanism. Acute stress-induced phosphoacetylation pathway is presented in Fig. 2. Fig. 2. Acute stress-induced phosphoacetylation pathway in dentate gyrus cells. This figure is composed of 2 interconnecting sections Section 1: Proper pathway. Light blue boxes correspond to components of pathway. broad black arrows show interactions. Legend: (1) acute stress (swim (Bilang-Bleuel et al., 2005) or novelty (Chandramohan et al., 2007)) activates NMDA and GABAA receptors in dentate gyrus; (2) GABA—A regulates NMDA function; (3) NMDA receptors induce phosphorylation of ERK1/2; (4) GR forms a complex with ERK1/2 and MSK1 enhancing its activity; (5) pERK induces phosphorylation of epigenetic initiators—Elk-1 (HAT activator) and MSK1 (histone phoshorylase); (6) Elk-1 and MSK1 sets genome-wide H3 phosphoacetylation; (7) Acute stress-induced changes in behavior and gene expression depend on phosphoacetylation pathway and most likely, the phosphoacetylation itself. Section 2: Evidence for pathway activity. Violet boxes correspond to treatments regulating functions of pathway components. Thin red, green or grey arrows show decrease, increase or no effect on function of component, respectively. Intermediate orange lines show which pathway constituents are regulated by treatment. Legend: (a) MK-801 is a NMDA antagonist. I.p. injection before stress blocked increases in phosphorylation of ERK1/2, Elk-1, MSK1, H3 phosphoacetylation, immobility in forced swim re-test, elevation of c-Fos protein expression (Chandramohan et al., 2008; Gutierrez-Mecinas et al., 2011), and NMDA antagonism inhibited effect of GABA-A partial antagonism (Papadopoulos et al., 2011); (b) RU486 is a GR antagonist. Subcutanous injection of RU486 but not spironolactone (MR antagonist) resulted in decreased phosphorylation of Elk-1 and MSK1, but not ERK1/2, H3 phosphoacetylation, immobility in forced swim re-test, and elevation of c-Fos expression (Gutierrez-Mecinas et al., 2011); (c) Lorazepam is a full GABA-A antagonist. I.p. injection resulted in decrease of H3 phosphoacetylation (Papadopoulos et al., 2011); (d) FG 7142 is a partial GABA-A antagonist. I.p. injection resulted in elevation of H3 phosphoacetylation and c-Fos; (e) SL-327 is a ERK blocker. I.p. injection resulted in decrease of H3 phosphoacetylation and immobility in forced swim re-test (Chandramohan et al., 2008); (f) MSK1/2 KO resulted in decrease of H3 phosphoacetylation and immobility in forced swim re-test (Chandramohan et al., 2008). 2.2. Epigenetic mechanisms in chronic stress 2.2.1. Chronic restraint stress (CRS) Experiments employing acute stressors are not sufficient to understand changes in the neuronal network in response to chronic stress relevant to the pathogenesis of stress-related human diseases (Lisowski et al., 2011). For such understanding, it is more helpful to describe the adaptive and maladaptive responses to chronic stress. Chronic restraint is a stress paradigm widely used in biomedical research to induce behavioral and molecular changes in the brain similar to those reported in brain disorders (Chiba et al., 2013). In adult male Sprague–Dawley rats, a seven-day restraint resulted in a significant decrease in the number of histones showing H3 trimethylation at the 27th lysine (H3K27me3) in the dentate gyrus 45 min after the last stress episode. The histone modification levels were also slightly reduced in the DG under basal conditions (analyzed just before the last stress episode) and in the CA1 and CA3 areas in both basal and post-stress conditions; however, the differences failed to reach significance. The basal number of neurons positive for histone H3 trimethylation at the 9th lysine (H3k9me3) increased significantly in the CA1, CA3 and DG. When examined 45 min after the last episode of restraint, no significant differences between the stressed and non-stressed control groups were found. The levels of expression-activating histone H3 trimethylation at the 4th lysine (H3K4me3) did not vary significantly after 7 days of stress, although a trend towards a decrease in this modification in the DG and CA1 was observed (Hunter et al., 2009). Moreover, chronic restraint stress lasting for 21 days increased H3K4me3, while having no significant effect on H3K9me3 in the DG. Simultaneous chronic stressing and treatment with antidepressant (injected s.c. immediately before restraint) fluoxetine resulted in significantly increased H3K9me3 levels in DG compared with both the non-stressed control and the chronically stressed A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 group without antidepressant treatment. The results suggest that H3K9me3 may be a target of antidepressive drugs (Hunter et al., 2009). As the authors noted, acute stress caused more pronounced changes in repressive (methylation at the 27th and 9th lysines) histone methylations then chronic stress. This would be in agreement with literature showing a gradual decrease in the magnitude of gene expression changes during chronic restraint, possibly as a result of habituation (Hunter et al., 2009). The changes mentioned above appeared stress specific, as a 21-day treatment with high doses of corticosterone (dissolved in drinking water) reduced both H3K9me3 and H3K27me3 levels in the DG, an effect different than was produced by a 21-day restraint (Hirao et al., 1998). 2.2.2. Chronic social stress (CSS) As most of stressors acting upon humans in everyday life are social in nature, chronic social stress is suggested to be especially relevant for modeling the pathological effects of chronic stress in humans (Martinez et al., 1998). An increase in the immunoreactivity of neurons and glial cells for acetylation (H3 K9ac/K14ac) but not phosphoacetylation of histone H3 (H3S10pK14ac) was found in the infralimbic medial prefrontal cortex of adult male Sprague–Dawley rats subjected to chronic social defeat but not chronic noise. Chronic noise stress lasted as long as social stress and consisted of half-hour episodes of white noise (105 dB) (Hinwood et al., 2011). Histone acetylation was accompanied by an increase in glutaminergic neurons positive for deltaFosB, a splice variant of the FosB protein that is implicated as an indicator of chronic neuronal activity (Chen et al., 1997). Nevertheless, as a result of the technical inability to doublestain the same tissue for FosB and histone acetylation, it is still uncertain whether epigenetic modifications and chronic cell activation, measured by deltaFosB upregulation, coexist in the same cells (Hinwood et al., 2011). Subchronic, 4-day long, social defeat stress induced post-traumatic stress disorder (PTSD)-like behavioral changes and increases in acetylated histone H3 in the rat hippocampus but not in the amygdala or dorsal prefrontal cortex (Hollis et al., 2010). Histone H4 acetylation was also tested, but no changes were evident. The lack of H4 acetylation could be due to the reduced sensitivity of the histone H4 acetylation antibodies. Alternatively, histone H4 may react in the hippocampus primarily to acute stressors as suggested earlier by Tsankova et al. (2006). Histone H3 hyperacetylation was reported 30 min after last stressful episode, lasted for 24 h, and was not present after 72 h. Behavioral changes were much more stable, persisting for 6 weeks. Nevertheless, acetylation might set the basis for establishing more stable epigenetic modifications that result in long-lasting behavior alterations. Neither a i.p. injections of GR antagonist (mifepristone) nor an NMDA antagonist (MK-801) appeared to specifically affect the reversal of stress-induced acetylation, although blocking the NMDA receptor increased acetylation in both control and stressed animals. The lack of effect of manipulating these receptors, in contrast with the work of Chandramohan et al. (2007) and Bilang-Bleuel et al. (2005), might be due to differences in the stressing procedure, acute novelty and forced swim versus chronic social stress. Another explanation may be that the neuronal reactivity of NMDA and GR is specific only to the dentate gyrus and, therefore, is difficult to uncover when investigating the entire hippocampus (Hollis et al., 2010). The nucleus accumbens (NA) is a brain region responsible for reward and is involved in the development and treatment of depression (Krishnan and Nestler, 2008). Global and stable changes, mostly increases, of histone H3 dimethylation at the 9th and 27th lysine were found in the NA of adult male C57BL/6ByJ mice subjected to 10-day chronic social defeat stress or 8-week social isolation, both of which are considered mouse depression models (Wilkinson et al., 2009). The authors used a chromatin 81 immunoprecipitation microarray technique that allowed for the characterization of patterns of methylated and demethylated histones associated with specific genes. A subgroup of mice subjected to social defeat but determined to be behaviorally resistant to stress showed a histone methylation pattern similar to mice behaviorally vulnerable to stress treated with the antidepressant daily i.p. imipramine. Thus, it might be reasonable to state that resistance to stress may be an active process of specific alterations in gene expression and the epigenome rather than a simple absence of a response. As the authors suggest, the genes regulated in stress in the resistant animals but not after imipramine treatment may constitute promising targets for new therapeutics. Social defeat and social isolation evoked similar changes in behavior and histone methylation in genes implicated in cellular plasticity, inflammation or gene regulation. This suggests that these stressors may act through similar mechanisms to induce similar phenotypes. In contrast, both stressors induced different changes in phospho-CREB (pCREB) gene promoter binding. CREB (cAMP response element-binding) binding protein contains domains with histone acetyltransferase activity (Chen et al., 2001) and thus may be considered an “epigenetic initiator”. Gene promoters of mice subjected to social defeat showed increased pCREB binding close to a start site and decreased binding upstream, while promoters of mice subjected to social isolation showed the opposite pattern (Wilkinson et al., 2009). The authors argue that active forms of stress like acute forced swim (they also include social defeat in this group) increase (Pliakas et al., 2001), while passive stress (social isolation) decreases (Wallace et al., 2009) pCREB content in the nucleus accumbens. CREB is a downstream factor in the MAPK signaling cascade, which was previously confirmed as crucial in setting epigenetic patterns (Chandramohan et al., 2007, 2008). It would be thus interesting to verify whether changes in CREB activity, reported here, influence the cellular epigenome. 2.2.3. Chronic variable stress (CVS) Results of studies from our laboratory (Lisowski et al., 2011) argue for the significance of epigenetic mechanisms in the regulation of chronic stress. Microarray studies have revealed alterations in mRNA expression levels of seven factors involved in chromatin modification in adult male Swiss-Webster mice subjected to various stressors for five weeks. Three transcripts that encode histones were found to be upregulated (H2afj, Hist1h2bm, and Hist1h2bg), and four were down-regulated. These four down-regulated genes encoded histones (Hist1h2bn, Hist1h2bh), a silencing factor known to recruit histone methyltransferases and deacetylases (Satb1) (Pavan Kumar et al., 2006), and a protein involved in histone acetylation (Hmgn2) (Ueda et al., 2006). In another work published the same year, Ferland and Schrader (2011) reported changes in the levels of epigenetic histone modifications after chronic variable stress in adult male Wistar rats. The authors found a decrease in the content of histone H4 acetylated at the 12th lysine (H4K12ac), and phosphoacetylated histones H3 (H3K9acS10p) were evident in the hippocampal areas CA3 and DG. This effect was linked with activity but not content of a histone deacetylase (HDAC) called sirtuin. Sirtuins are a group of proteins with histone deacetylase activity, differing from other HDACs by their zinc independence (Lawson et al., 2010). Sirtuin 1 activity was increased in both CA3 and DG, but not in CA1, where no histone modifications were found (Ferland and Schrader, 2011). In DG and CA3 of hippocampal slices from stressed animals treated with sirtinol (a sirtuin inhibitor) reversed histone hypoacetylation (H4K12ac) and hypophosphoacetylation (H3K9acS10p). Under the same conditions, an inhibitor of HDAC I/II (NaB) also raised histone H4 acetylation levels (H4K12ac), but this effect was not specific to stressed animals, as it was evident also in controls. In addition, the observed increase in HDAC5 protein content was specific to the CA1 82 A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 area, which did not respond to stress. Hence, only sirtuins are proposed to significantly contribute to epigenetic modifications in the hippocampus after chronic variable stress (Ferland and Schrader, 2011). It is worth noting that the mechanisms of histone acetylation may be different in other parts of the brain. For example, in another chronic variable stress paradigm, HDAC5 was regulated during stress in the extended amygdala of male and female rats (Tsankova et al., 2006). 2.2.4. Long-term mental and pain stress (LMPS) Randomly administered electric shocks have been used to induce severe LMPS in adult male Wistar rats (Sokolova et al., 2006). This stress paradigm generates persistent behavioral and hormonal changes similar to those of human PTSD (Sokolova et al., 2006). It was found that LMPS causes different histone acetylation patterns in hippocampal CA3 fields of rats with a high and low excitability threshold of the tibial nerve to electric current (Sokolova et al., 2006). At 24 h after the final episode of a 15-day stress, histone H4 acetylation levels were not altered in either group of rats; however, two weeks later, the animals with a high excitability threshold showed a significant decrease in this epigenetic modification (Sokolova et al., 2006). In an earlier study, it was found that LMPS decreased methyl-CpG-binding protein 2 (MeCP2) levels in the CA3 area of the hippocampus in rats with a high excitability threshold at 24 h and two weeks after the last stress (Dyuzhikova et al., 2006). MeCP2 is a mediator of epigenetic modifications that, most interestingly in this case, recruits histone deacetylases (Jones et al., 1998). Therefore, the biochemical events involving MeCP2 might underlie stress-induced changes in hippocampal acetylated histone levels (Dyuzhikova et al., 2006). 2.3. Epigenetic regulation of stress-related gene expression levels In addition to research on global changes in epigenetic patterns, stress-induced epigenetic modifications of specific genes important in the stress response were studied. The results shed light on how the stress response is initiated, maintained and regulated. Only knowledge of how specific genes act during stress can result in a full understanding of the mechanisms underlying the stress response. Table 2 summarizes the data presented in this section. 2.3.1. Glucocorticosteroid receptor gene (Nr3c1) Glucocorticosteroid receptor (GR) is one of the most widespread and important mediators of the stress response (Reul and de Kloet, 1985). Disturbances of GR processes can lead to brain disorders such as depression (Holsboer, 2000). Szyf et al. provided evidence that in adult Long-Evans rats, maternal behavior can have a large impact on the offspring stress response in adulthood (Szyf et al., 2005). The effects may be mediated by epigenetic modification of the functions of the glucocorticosteroid receptor gene. The researchers (Weaver et al., 2004) compared two groups of rats displaying different levels of maternal behaviors: high or low lick-groom and arched-back nursing (LG–ABN). Significant differences in the DNA methylation of exon 17 Nr3c1 promoter in neurons and glial cells of the hippocampus of adult rats were found. The offspring of high-LG–ABN mothers showed lower DNA methylation levels and an increase in the acetylation of histone H3 (H3K9ac) of the 17 exon promoter at 17 promoter. These epigenetic changes correlated with higher hippocampal GR protein content, decreased fearfulness and post-stress corticosterone concentrations. This effect was primarily dependent on maternal behavior, as cross-fostered pups showed methylation pattern similar to the foster mother. The 17 promoter is a site of consensus binding of NGFI-A transcription factor. As suspected, binding of this factor to the 17 promoter was higher in the offspring of high-LG–ABN mothers (Weaver et al., 2004). Modulation of GR content could be due to a higher level of nursing resulting in increased serotonin (5-HT) turnover in the hippocampus (Smythe et al., 1994). Serotonin, likely acting through 5-HT7 receptors, has been reported to upregulate GR expression (Laplante et al., 2002; Seckl et al., 1990) and induce NGFI-A transcription in vitro (Szyf et al., 2005). It has been postulated that increased levels of NGFI-A recruit acetylases at the GR promoter site (Szyf et al., 2005). Acetylated histones loosen chromatin and enable DNA demethylation, which set stable methylation patterns on the Nr3c1 gene that endure to adulthood. In the adult offspring of low-LG–ABN mothers, a histone deacetylase inhibitor intracerebroventricular (i.cv.) infusions (trichostatin A) increased histone acetylation, NGFI-A-promoter binding and GR expression, while reducing DNA methylation and post-stress plasma corticosterone, making both groups virtually indistinguishable (Weaver et al., 2004). The opposite effects can be achieved by treating adult rats with methionine, which is a donor of methyl groups and thus an inducer of hypermethylation. The low methylation of the hippocampal Nr3c1 gene promoter in the adult male offspring of high-LG–ABN mothers can be reversed by 7 days of i.cv. infusions of methionine (Weaver et al., 2005). The capacity for reversal of early epigenetic modifications is therefore present in the adult central nervous system (Weaver et al., 2004, 2005). Surprisingly, Daniels et al. (2009) reported that alterations in DNA methylation levels of exon 17 Nr3c1 promoter were not found in the adult male Sprague–Dawley rat hippocampus after maternal separation. The lack of methylation contrasting with previous studies might be due to differences in rat strains (Long-Evans by Szyf et al. vs. Sprague–Dawley by Daniels et al., 2009), laboratory conditions. Furthermore, Mueller and Bale (2008) reported that in adult mice of mixed strain C57Bl/6:129, the male offspring of mothers stressed during pregnancy using the chronic variable paradigm showed decreased GR mRNA expression levels in the hippocampus and increased methylation of the NGFI-A-binding region 17 of the Nr3c1 promoter in the hypothalamus (Mueller and Bale, 2008). It is remarkable that similar effects of early stress were observed in humans. Hypermethylation of the NR3C1 gene promoter was found in the hippocampus of male suicide victims exposed to child abuse (McGowan et al., 2009) and in blood leukocytes of adults with a history of childhood adversities (Tyrka et al., 2012) but not in the hippocampi of suicide victims without an abuse history (McGowan et al., 2009). It is noticeable that in blood leukocytes, the degree of NR3C1 promoter methylation correlated with the number of various childhood adversities. Furthermore, no correlation was found for NR3C1 methylation in major depression (Alt et al., 2010), which may suggest that this epigenetic modification is specific to early stress. The hypermethylation of the binding site of NGFI-A of the NR3C1 (CpG3 island of exon IF) promoter gene was observed in the umbilical cord blood of children whose mothers suffered from depression during the third trimester of pregnancy (Oberlander et al., 2008). This epigenetic modification was associated with elevated post-stress salivary cortisol in the 3-month old infants. An increase in methylation was also present in the CpG2 island of the NR3C1 promoter in another group of mothers depressed during the second and third trimester of pregnancy. This island is associated with the binding site of transcription repressor neural restrictive silencer factor (NRSF) (Oberlander et al., 2008). Methylation changes were specific to NR3C1, as methylation of retroposon LINE-1 did not vary between the depressed and control groups. Global hypomethylation of long interspersed nuclear element-1 (LINE-1) retrotransposons is considered an indicator of genomewide DNA hypomethylation (Yang et al., 2004). It is now known that synthetic glucocorticosteroids administered during pregnancy can influence both DNA methylation patterns and protein levels of the regulated epigenome in various organs (Crudo et al., 2013). As blood cortisol is well known to be increased (Christensen et al., 1985), it Table 2 List of genes which expression is changed by various stressors. Research were conducted on adult male animals, unless stated otherwise in“Effect”. Gene Gene description Brain area Organism Effects References Maternal behavior Nr3c1 Nuclear receptor subfamily 3, group C, member 1 gene (Glucocorticosteroid receptor gene) Hippocampus R. norvegicus (Long-Evans) Higher methylation of NGFI-A binding site and decrease in histone H3 (H3K9ac) content of Nr3c1 promoter in offspring of both sexes of mothers with low levels of nursing behavior Szyf et al. (2005) Hypothalamus M. musculus (mixed C57Bl/6:129) Homo sapiens M. musculus (C57/BL) Hypermethylation of GR promoter at NGFI-A binding site in males Hypermethylation of GR promoter at NGFI-A binding site After 2 weeks of recovery hypomethylation of Crh promoter at CRE-site in stress vulnerable mice Mueller and Bale (2008) McGowan et al. (2009) Elliott et al. (2010) R. norvegicus (Wistar-R Amsterdam) Hypermethylation of Crh promoter in females, no effect in males Sterrenburg et al. (2012) R. norvegicus (Sprague-Dawley) Hypomethylation of Crh promoter in both sexes, hypermethylation of intronic region in males. Functionallity of these changes is unknown Hypomethylation of Crh promoter at CRE-site. Functionallity of these change is unknown Early prenatal stress Childhood abuse Chronic social defeat Crh Corticotropinreleasing hormone gene Chronic variable mild stress Maternal deprivation Early prenatal stress Chronic ultra mild stress Gdnf Glial cell derived neurotrophic factor gene Early maltreatment Bdnf Brain-derived neurotrophic factor gene Chronic social defeat Chronic social and predator stress Chronic social and predator stress Mental state resulting in sucide Early life stress Avp Arginine vasopressin gene Acute predator stress Dlgap2 Maternal behavior Gad1/67 Discs large-associated protein 2 gene Glutamate decarboxylase 1 gene Prenatal stress Prenatal stress Reln Reelin Hippocampus Hypothalamus (Periventricular nucleus) Hypothalamus (Periventricular nucleus) Extended amygdala Hypothalamus (Periventricular nucleus) Hypothalamus/Central nucleus of amygdala Nucleus accumbens M. musculus (mixed C57Bl/6:129) M. musculus (BALB and C57BL/6) Hypomethylation of Crh promoter in males Hypoacetylation of histone 3 of Gdnf promoter in stress vulnerable (BALB), hyperacetylation in stress resistant (C57BL/6) mice Decrease of H3K4me3 in both lines, decrease of H3K27me3 in stress-resistant (C57BL/6) mice Hypermethylation of Gdnf promoter at MeCP2 binding site in both lines. MeCP2 induced Gdnf expression in stress-resistant (C57BL/6) mice, and repressed Gdnf expression in stress-vulnerable (BALB) mice Transgenerational hypermethylation of Bdnf promoter Chen et al. (2013) Mueller and Bale (2008) Uchida et al. (2011) R. norvegicus (Long-Evans) Hippocampus Hippocampus (ventral CA3) Hippocampus (dorsal CA1, dorsal dentate gyrus) Cortex—Wernicke area M. musculus (Bl6/C57) R. norvegicus (Sprague-Dawley) R. norvegicus (Sprague-Dawley) Increase of H3K27me2 in promoter of variants III and IV Hypomethylation of exon IV promoter Tsankova et al. (2007) Roth et al. (2011) Hypermethylation of exon IV promoter Roth et al. (2011) H. sapiens Hypermethylation of exon IV promoter Keller et al. (2010) Hypothalamus (Periventricular nucleus) Hippocampus M. musculus (C57BL/6 N) Hypomethylation of Avp enhancer Murgatroyd et al. (2009) R. norvegicus (Sprague-Dawley) Hypomethylation of Dlgap2 intronic site in rats of high stress reactivity Chertkow-Deutsher et al. (2010) Hippocampus R. norvegicus (Long-Evans) Zhang et al. (2010) Frontal cortex M. musculus (Swissalbino-ND4)? M. musculus (Swissalbino-ND4)? Lower DNA methylation and higher histone 3 acetylation at lysine 9 of Gad1 promoter at NGFI-A binding site in offspring of mothers with high levels of nursing behavior 5-mC and 5-hmC hypermethylation of Gad1 promoter Frontal cortex 5-mC and 5-hmC hypermethylation of Reln promoter Roth et al. (2009) Matrisciano et al. (2013) Matrisciano et al. (2013) 83 Prefrontal cortex A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 Stress 84 A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 appears possible that this heightened cortisol could be involved in the regulation of post-natal GR expression. It would be interesting to establish a potential relationship between NR3C1 expression levels in umbilical cord blood, the brain, and the HPA axis stress response (Oberlander et al., 2008). 2.3.2. Corticotrophin-releasing hormone gene (Crh/Crf) The paraventricular nucleus of the hypothalamus (PVN) CRH is the main activator of the HPA axis (Dunn and Swiergiel, 1999) and is responsible for at least some behavioral changes after stress (Elliott et al., 2010). Elliott et al. (2010) studied DNA methylation of the Crh gene in the PVN in adult male C57/BL mice. It was found that basal methylation levels of the mouse Crh were low in intronic regions and high in promoter regions including the CREB-binding site (cAMPresponsive element—CRE). The expression-modulating character of basal methylation of Crh promoter was confirmed by CRH mRNA upregulation after treatment of PVN cells in vitro with a DNMT inhibitor (5-Aza). Next, the mice were subjected to 10 days of social defeat and divided according to a degree of stress-induced social avoidance into stress-resilient and stress-vulnerable individuals. Molecular procedures were performed two weeks after the last stress episode. It was revealed that four out of 10 CpG islands of the Crh promoter, including the transcription-activating CRE-site, were demethylated in stress-vulnerable but not in stressresistant mice, and this demethylation co-occurred with increases in CRH mRNA levels. Three weeks of daily i.p. injection imipramine treatment reversed the changes in Crh promoter methylation, mRNA expression and behavior (Elliott et al., 2010). Therefore, stress vulnerability and some post-stress behavioral responses can be mediated by epigenetic, possibly CREB-dependent, modulation of paraventricular CRH mRNA expression. In addition, significant changes were found in the mRNA levels of several epigenetic maintainers 1 h after the last episode of social chronic stress (Elliott et al., 2010). Expression of DNA methyltransferase DNMT3b and histone deacetylase HDAC2 were decreased, while GADD45 mRNA levels were upregulated. The former factors may be considered chromatin-condensation inducing; the latter is associated with demethylation. Thus, it is likely that these factors are involved in stress-induced modifications of the CpG island of Crh. Furthermore, HDAC2 mRNA showed downregulation after 2 weeks from the last stressor, both in mice resistant and vulnerable to stress (Elliott et al., 2010). Similar results were reported by Sterrenburg et al. (2012). After subjecting adult Wistar-R Amsterdam rats, males and females, to chronic variable mild stress (CVMS), the hypothalamic PVN and extended amygdala were examined for epigenetic changes. In male PVN, chronic variable stress induced increases in CRH mRNA expression levels. Interestingly, in females, no such change was found. This discrepancy may be because of differential response of epigenetic mechanisms to CVMS, as the lack of enhanced CRH mRNA expression was accompanied by Crh promoter hypermethylation in the female PVN. This female-specific mechanism of stress regulation may be induced by higher levels of glucocorticosteroids known to function as epigenators (Lisowski et al., 2011). In both sexes, stress decreased the methylation levels of the Crh promoter in the bed nucleus of stria terminalis, and in males, it increased the intronic methylation of the Crh gene. Stressed females showed decreased Crh methylation in central nucleus of amygdala in comparison to non-stressed females. No significant differences in CRH mRNA were found in the extended amygdala between the experimental and control groups. Chen et al. performed additional work comparing the effects of stress in adult Sprague–Dawley rat males and females (Chen et al., 2013). Eight-week old animals subjected to early maternal deprivation showed some changes in stress endocrine markers and slight hypomethylation of the Crh promoter at CREsite in the hypothalamus PVN. Although no effects of stress were found in either sex in basal CRH mRNA levels, there was a marked increase in the expression of post-stress CRH hnRNA (pre-mRNA). Despite this increase, concentrations of post-stress blood ACTH tended to decrease. Therefore, the functionality of Crh promoter hypomethylation in early maternal deprivation stress remains to be proven. Prenatal chronic variable stress was used in adult male C57Bl/6:129 mice by Mueller and Bale (2008) to evoke depressionlike behavior using tail suspension, forced swim and sucrose intake tests in male, but not female offspring as well as increase poststress plasma corticosterone levels in males (corticosterone was not measured in females). The effects correlated with Crh promoter hypomethylation in both the hypothalamus and the central nucleus of amygdala. In the amygdala, this hypomethylation was accompanied by an increase in CRH mRNA expression. Prenatal stress was found to significantly upregulate DNA methyltransferase 1 (DNMT1) in female offspring placenta only (Mueller and Bale, 2008). Moreover, the placenta of non-stressed females showed higher levels of DNMT1 than non-stressed males. DNMT1 is involved mainly in maintaining existing DNA methylation patterns (Svedruzic, 2011). Mueller and Bale (2008) concluded that the greater efficiency of methylation maintenance might underlie female resistance to prenatal stress. 2.3.3. Glial-derived neurotrophic factor gene (Gdnf) GDNF is thought to play a role in the early conditioning of stress vulnerability (Kawano et al., 2008) and has been proposed as a therapeutic agent for neurodegenerative diseases (Lapchak et al., 1996). Uchida et al. (2011) examined the impact of chronic ultra-mild stress on GDNF mRNA expression in the nucleus accumbens of males of two mouse lines. The first line, BALB, was prone to developing post-stress depression-like behaviors and was considered vulnerable to stress; the second line, B6 (C57BL/6), was stress resistant. Stress-induced behavioral changes in BALB mice were attenuated by chronic administration of antidepressant imipramine. BALB mice showed decreased post-stress mRNA and protein content of GDNF (both normalized by chronic imipramine dissolved in drinking water), while B6 mice expressed the opposite trends. In both lines, the CpG island of the Gdnf promoter II was hypermethylated compared with non-stressed mice of the same line. In BALB mice, the methylated CpG island of the Gdnf promoter II recruited the MeCP2–HDAC2 complex that displayed chromatin-condensing activity; however, in B6 mice, it was MeCP2–CREB that showed affinity at this site. MeCP2–CREB is responsible for activating gene expression. As an effect of increased HDAC recruitment, H3 showed lower acetylation levels in BALB mice than in B6 mice. Proving the functional effect of HDAC2 in Gdnf regulation, the overexpression of a hyperactive variant of HDAC2 in B6 mice resulted in reduction of GDNF mRNA and behavioral resiliency to stress. Moreover, other epigenetic histone modifications were reported to respond to stress. Histone H3 trimethylation (H3K4me3) declined in both lines after stress, and B6 displayed an additional decrease in transcription-repressing H3K27me3. Changes both in H3 acetylation and H3K4me3 were normalized by chronic imipramine in BALB mice (Uchida et al., 2011). 2.4. Brain-derived neurotrophic factor gene (Bdnf) Brain-derived neurotrophic factor (BDNF) is a protein implicated in stress response (Fanous et al., 2011; Wichers et al., 2008), long-term memory (Lu et al., 2008), Alzheimer’s disease (TapiaArancibia et al., 2008), and psychiatric disorders such as bipolar disorder (Post, 2007), depression and schizophrenia (Schumacher A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 et al., 2005). Adult Long–Evans rats, males and females, maltreated during the first week of postnatal life showed hypermethylation of the promoter of the Bdnf gene (exon IV) and decreased BDNF mRNA levels in prefrontal cortex (PFC) while BDNF mRNA in hippocampus was not changed. (Roth et al., 2009). The relationship between methylation and BDNF expression levels was proved causal, as stress-related transcriptional changes of BDNF were reversed by the 7 day long administration of a DNA methylation inhibitor ventricle (i.cv.) infusion zebularine. As the maltreated females themselves displayed increased abusive behavior towards their pups, the authors asked whether Bdnf gene methylation status is transferred to next generation. Changes in Bdnf promoter methylation were found in the PFC of the offspring of maltreated females, even when cross fostered to normal females. Changes in Bdnf methylation were present also in the hippocampus, but they were fully reversed by cross fostering. Therefore, although some of the heritable DNA methylation changes are purely due to the abusive maternal behavior (methylation in hippocampus), others are a consequence of epigenetic changes in germ cells or the prenatal environment. Similar results were reported from studies on suicide, for which stress is a risk factor (Vilhjalmsson et al., 1998). The BDNF exon IV promoter was significantly hypermethylated in Wernicke’s area of suicide victims of both sexes (Keller et al., 2010). In another study in adult male Bl6/C57 mice (Tsankova et al., 2006), chronic social defeat led to persistent downregulation of the mRNA expression levels of BDNF variant III and IV in the mouse hippocampus, which correlated with an increase in the dimethylation of the 27th lysine of histone H3 (H3K27me2) in the Bdnf promoter and an increase in avoidance behavior. This histone modification was considered stable, as it was present one month after the last stress episode and was not reversed by chronic treatment with antidepressant imipramine. Furthermore, chronic intake of imipramine reversed depressive-like behavior and altered BDNF mRNA expression, but the effects was through increasing dimethylation of the 4th lysine and acetylation of histone H3 of the Bdnf promoter and not by reversing methylation at the 27th lysine. The authors proposed that acetylation might have been altered by a reported decrease of HDAC5 deacetylase mRNA expression after imipramine treatment. Confirming that hypothesis, overexpression of HDAC5 resulted in attenuation of the imipramine effect on behavior. The i.p. imipramine effect on Bdnf epigenetic status was observed only in stressed individuals, which, as the authors noted, corresponded to observations of antidepressants having no effect in normal, non-depressed humans (Tsankova et al., 2006). Moreover, in agreement with Uchida et al. (2011), acute stress caused hypoacetylation of histone H4 and not histone H3. The authors thus hypothesized that acetylation of histone H4 is modulated in acute stressors, and acetylation of histone H3 may be specific to long-termed stress (Tsankova et al., 2006). Roth et al. (2011) reported that Bdnf also responds to chronic social-predator stress in adult male Sprague–Dawley rats. This stress paradigm induced PTSD-like behavioral changes. DNA methylation levels of Bdnf exon IV promoter were decreased in the CA3 zone of the hippocampus and increased in CA1 and DG but remained unchanged in the prefrontal cortex and amygdala. The methylation was proved molecularly relevant at least in CA1 zone, where real-time PCR analysis confirmed the decreased expression of BDNF mRNA in stressed animals (Roth et al., 2011). Although Roth et al. (2009) in their previous study on early maltreatment did not find changes in BDNF expression, such an effect might be due to a balancing of opposite changes in BDNF mRNA levels in different parts of the hippocampus. BDNF content is known to be regulated during stress by CREB (Gronli et al., 2006) and MeCP2 (Chen et al., 2003), both of which are known to be involved in epigenetic processes (Murgatroyd et al., 2009). Hence, it appears valid to confirm 85 the role of those factors in the epigenetic regulation of BDNF in stress. 2.4.1. Vasopressin gene (Avp) Hypothalamic vasopressin is known to potentiate CRH action and stress response (Volpi et al., 2004). In a study by Murgatroyd et al. (2009), adult male C57BL/6N mice subjected to early life stress showed increased post-stress blood corticosterone concentrations, immobility in a forced swim test and memory deficits. The responses correlated with a stable (at least a year) increase of vasopressin gene expression levels in parvocellular neurons of the hypothalamic PVN. A vasopressin receptor antagonist (SSR149415) partially reversed the stress-related changes in the ACTH precursor, Pomc, gene expression, corticosterone secretion and impairment in memory, proving that a change in AVP signaling exerted a functional effect on phenotype. This change in gene expression was a result of hypomethylation of the Avp enhancer containing a binding site for MeCP2, an epigenetic silencing mediator. As the authors convincingly demonstrated, an early stress activation of neurons resulted in recruitment of calcium/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated MeCP2, thus preventing it from binding to MeCP2-binding sites. Therefore, MeCP2 was not able to induce methylation of Avp enhancer and persistently decreased its activity (Murgatroyd et al., 2009). 2.4.2. Disks large-associated protein 2 gene (Dlgap2) Acute predator stress, a rat model of post-traumatic stress disorder, induced genome-wide changes in DNA methylation of the hippocampus (Chertkow-Deutsher et al., 2010). Among the genes reacting to this stress paradigm was Dlgap2, which is involved in synaptic remodeling and neuronal transmission (Hirao et al., 1998). Only a subgroup of tested animals, male adult Sprague–Dawley rats presenting PTSD-like behavioral response to stress, showed DLGAP2 mRNA upregulation and concurrent hypomethylation in intron 4 of Dlgap2 when compared with the stressed rats that presented a low stress reactivity or a non-stressed control group. Although the functionality of intronic methylation is yet uncertain, it is known that intron sequences may impact gene expression levels (Rose, 2008) and alter splicing patterns (Cooper and Mattox, 1997). It also may be that this methylation is representative of more wide-spread methylation changes in Dlgap2, picked by random choice of analyzed DNA fragments from the sequencing (ChertkowDeutsher et al., 2010). 2.4.3. Glutamic acid decarboxylase 1 and reelin genes (Gad1/Gad67, Reln) Glutamic acid decarboxylase 1 is a limiting factor in GABA synthesis and is implicated in the pathophysiology of schizophrenia and mood disorders (Thompson et al., 2009). Zhang et al. (2010) studied the effect of maternal behavior on GABA circuitry in the male adult Long–Evans rat hippocampus. High maternal pup lickgroom behavior correlated with the upregulation of GAD1 mRNA, a decrease in DNA methylation and an increase in histone H3 acetylation (H3K9ac) of the Gad1 promoter in adult male offspring. As concluded from the results of in vitro experiments (Zhang et al., 2010), the pathway leading to reported epigenetic modifications resembles the one proposed by Weaver et al. (2004) for GR expression regulation. This pathway was described in Section 2.3.1, and it involves 5-HT-dependent activation of NGFI-A that recruits histone acetylases that, in turn, relaxes chromatin and enables DNA demethylation of gene promoter in offspring of high lick-groom mothers. Corresponding with this hypothesis, a decreased level of DNA methyltransferase (DNMT1) mRNA was found in the hippocampi of the offspring of high lick-groom mothers (Zhang et al., 2010). 86 A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 Similarly, prenatal stress resulted in decreased GAD1 and reelin protein content in adolescent Swiss-albino-ND4 male mouse GABA cells in the frontal cortex (Matrisciano et al., 2013). 5Methylcytosine and 5-hydroxymethylcytosine were increased in the promoters of Gad1/Gad67 and Reln genes. This effect correlated with enhanced binding of DNMT1 and MeCP2 to both promoters. Reelin is strongly implicated in schizophrenia, and prenatal stress also evoked some psychotic-like behavioral (hyperactive, deficits in social interaction) and molecular changes (increase in brain DNMT1 content, enhanced response to MK-801, an NMDA antagonist (single s.c. injection of MK-801). Considering these data, it can be hypothesized that prenatal stress may induce changes in the central nervous system that evoke a psychotic phenotype (Matrisciano et al., 2013). 3. Application and perspectives Stress is a potent stimulus that is considered a vulnerability factor for a number of mental and neurological diseases and is known to significantly alter the epigenome. Likewise, many psychological diseases are inherited abnormally and are controlled by environmental or stochastic events. This points to a role for imprinting and other forms of persistent epigenetic regulation in the etiology of mental illness (Mill and Petronis, 2007). There are now reports that directly link brain disorders with changes in neuronal epigenetic patterns. For example, methylation of the reelin gene has been studied in schizophrenia (McGowan and Kato, 2008), and deregulation of methylation patterns including GABA, glutamate and Wnt signaling pathways was found in the frontal cortex of patients with major psychoses (Mill et al., 2008). Lateonset Alzheimer’s disease patients are characterized by highly altered methylation patterns compared with a control group of their peers (Wang et al., 2008). Taking into account this substantial role of epigenetics in brain disease, it can be assumed that exploration of specific stress-induced epigenetic patterns might open new possibilities for diagnosing early disease stages and progress, monitoring treatment and developing new therapies for stressrelated disorders. New genome-wide research methods such as DNA methylation microarrays, next generation sequencing or chromatin immunoprecipitation on chip (ChIP-on-chip) allow for a precise and global evaluation of epigenetic patterns (Collas, 2010; Funk and Stewart, 1996; Hurd and Nelson, 2009), thus improving our ability to understand and implement data into practice. Research performed on animal models and on cells in-vitro can prove problematic in translation to patients. It is noteworthy, therefore, that recently, another route for studying the molecular biology of brain disease has emerged. It has been shown that chromatin extracted from peripheral blood contains epigenetic marks that reflect individual life experiences (Oberlander et al., 2008). As immune cells share a similar biochemical environment and epigenetic machinery as neurons, these markers might be especially valid for brain disorders. This enables researchers to acquire easily accessible epigenetic markers of ill tissues that are difficult to obtain for study. There is hope that epigenetic biomarkers may be detected even before illness evokes its pathological effects (Michels, 2010). These markers are implicated in schizophrenia (Gavin and Sharma, 2009) and were recently found in bipolar disorder (Di Benedetto et al., 2011), depression (Uddin et al., 2011), and heightened aggression in children (Tremblay, 2008). From the perspective of stress research, most interesting work on peripheral biomarkers comes from the studies on post-traumatic stress disorder. PTSD is the only neuropsychiatric pathology whose onset is often directly linked to severe stress. In Rusiecki et al. (2012) research, US military personnel were divided into two groups according to post-deployment PTSD diagnosis. DNA samples extracted from sera collected before deployment differed between the groups in methylation levels of Alu-elements (Rusiecki et al., 2012). Koenen et al. (2011) study compared DNA samples from whole blood of PTSD sufferers and non-PTSD controls using DNA methylation microarrays. It was found that the number of traumatic events influenced PTSD risk but only in participants showing low methylation of serotonin transporter gene (SLC6A4). Therefore, the methylation patterns of Alu-elements and SLC6A4 could be considered epigenetic vulnerability factors for PTSD development. The first experiments establishing the possibilities of counteracting behavioral and cognitive changes through the use of epigenetically-active agents have been conducted (Baek et al., 2010; Maze et al., 2010; Reolon et al., 2011), and there is now solid evidence that epigenetics research can viably translate to therapy. Epigenetics have been already incorporated into cancer treatments. First, drugs influencing DNA methylation and histone modification patterns have been approved (Kelly et al., 2010). Regulators of histone acetylation are considered therapeutic agents for, among others, Rett and Rubinstein-Taybi syndrome, Fragile X syndrome and a number of mental disorders (Kazantsev and Thompson, 2008). Pharmacological alteration of both DNA methylation patterns and histone acetylation have proved to reduce Alzheimer-related changes in mouse brains and cultured human cells (Mikaelsson and Miller, 2011). Taking the abovementioned facts into consideration, it is reasonable to expect that incorporation of epigenome-shaping therapeutics will enable for great progress in eliminating diseases, even those currently incurable. 4. Conclusions Data presented in this review show that epigenetic mechanisms are strongly involved in the stress response in the central nervous system. Magnitude of this involvement was distinctly illustrated by Wilkinson et al. (2009) who found that chronic psychogenic stress paradigms can influence epigenetic patterns of approximately 2000 genes. Relatively few epigenetic studies have been conducted in the area of stress until now. Considering that epigenetics is complex enough to be regarded as a field of its own, on a par with transcriptomics or genomics, we still know very little about epigenetic effects of stress. Moreover, as a consequence of small number of publications studying vast number of epigenetic mechanisms, only a handful of results were replicated. There is also a need for more data concerning which stress mediators and biochemical pathways are responsible for epigenetic changes during stress. Although this topic is still only emerging, there are already a few interesting conclusions that can be drawn from existing data. As transcriptomic response depend on characteristics of stress (Reyes et al., 2003), it is viable to hypothesize, that the same applies to epigenetic response. The differences between epigenetic patterns elicited by psychogenic and physical stressors were welldescribed by Bilang-Bleuel et al. (2005). Differential reaction of epigenome to various psychogenic stressors, although seemingly obvious, is still to be strongly proven. All of the studies comparing effects of different stressors on the same epigenetic changes in the same brain areas, e.g. differential regulation of Crh promoter methylation in CeA by maternal deprivation (Chen et al., 2013), prenatal stress (Mueller and Bale, 2008) and chronic variable stress (Sterrenburg et al., 2012), or Bdnf promoter methylation in PFC in chronic-social-predator (Roth et al., 2011) and early maltreatment stress (Roth et al., 2009), were done on different strains or species of animals, thus can serve as a confirmation of hypothesis, but not as the strong evidence. Indirect rationales for possible psychogenic A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 87 Fig. 3. Critical stress-related genes are regulated by epigenetic mechanisms during various paradigms of psychogenic stress. Legend: Figure is color-coded. (1) Arrows: green lines represent increase, red—decrease and gray—no effect of stressor on given epigenetic mark; The male data is marked with full line; female data is marked with dotted line and the data from both sexes is marked with dashed line; (2) Boxes: light violet boxes correspond to brain areas; dark red (Crh), dark blue (Nr3c1), dark green (Bdnf) and dark violet (other) boxes correspond to specific genes; Black boxes correspond to epigenetic marks. Light red, light blue, light yellow and gray boxes correspond to different stress paradigms or stress-related pathologies. stress-specificity of at least some of the epigenetic mechanisms can be inferred from their GC-independency. Glucocorticoids are arguably the most functionally potent stress mediators, that act irrespectively of the used paradigm (although the magnitude of the response may vary), and are known to be involved in forming the epigenetic patterns (Gutierrez-Mecinas et al., 2011). Hunter et al. (2009) reported that chronic GC treatment induced vastly different changes in H3 methylation than restraint stress of the same duration, and Hollis found GR antagonist to be ineffective in modulating stress-induced H3 acetylation. Hence, although GC is involved in some epigenetic mechanisms, others are clearly dependent on different, likely more stress-specific pathways and mediators. As was mentioned, there are studies arguing, that part of epigenetic stress response may be shared amongst different psychogenic stressors. Reuls group have found, that at least two different acute psychogenic stressors recruit the same biochemical pathway leading to behavioral changes and H3 phosphoacetylation in denate gyrus cells (Chandramohan et al., 2007, 2008). Similarly, methylation of Crh promoter in male rodent PVN was decreased by 3 different stress paradigms (Chen et al., 2013; Elliott et al., 2010; Murgatroyd et al., 2009). Reuls also provided evidence that the magnitude of some stress-induced epigenetic changes correlates with intensity of the stressor. Decreased water temperature in forced swim and increased light intensity during novelty stress are known to exaggerate behavioral stress response in rodents (Bilang-Bleuel et al., 2005; Chandramohan et al., 2007). Reul found that these stress-potentiating factors enhanced H3 phosphoacetylation/phosphorylation in DG, as compared to stress conducted in absence of these factors. Epigenetic mechanisms are showed to be involved in both rapid and long-termed effects of stress. It has been proved that epigenetic modifications can be established rapidly and induce functional effect in minutes, and decline hours later (Chandramohan et al., 2007; Gutierrez-Mecinas et al., 2011; Hunter et al., 2009). Perhaps the most important finding of the summarized studies is that stress-induced epigenetic changes can persist long after the stressor has ended and can underlie functional changes in the brain. Therefore, epigenetic mechanisms are promising candidates for causative factors of persistent, pathologic effects of stress. A number of studies on adult rodents and humans have described possibly Factor Factors mode of action Method of treatment Stressor Effect of factor Organism Reference Fluoxetine Antidepressant—SSRI Chronic restraint stress Normalisation of H3K9me3 levels in DG Antidepressant—TCA Chronic Social Defeat Imipramine Antidepressant—TCA Daily intraperitoneal injections for 28 days after end of stress Chronic Social Defeat M. musculus (Bl6/C57) Tsankova et al. (2007) Imipramine Antidepressant—TCA In drinking water during the last 3 weeks of stress Chronic Ultra Mild Stress M. musculus (BALB and C57BL/6) Uchida et al. (2011) Imipramine Antidepressant—TCA Daily intraperitoneal injections for 3 weeks after end of stress Chronic Social Defeat M. musculus (C57/BL) Elliott et al. (2010) MK-801 NMDA antagonist Intraperitoneal injection 15 minutes before stress Acute Forced Swim R. norvegicus (Wistar) Chandramohan et al. (2008) RU486 GR antagonist Intraperitoneal injection 15 minutes before stress Acute Forced Swim R. norvegicus (Wistar)/ Chandramohan et al. (2008) SL 327 ERK1/2 inhibitor Intraperitoneal injection 15 minutes before stress Acute Forced Swim R. norvegicus (Wistar)/ Chandramohan et al. (2008) Zebularine DNA methylation inhibitor HDAC inhibitor Daily infusions into lateral ventricle for 7 days at adulthood Early Maltreatment Normalisation of social interaction (Tsankova et al., 2007), H3 dimethylation, CREB binding in NA Increase of H3K4ac and Hdac5 mRNA in HP, normalisation of social interaction and Bdnf mRNA expression in HP Normalised HDAC2 protein expression, H3ac and H3K4me3 in Gdnf promoter, mRNA and protein content of GDNF in stress-vulerble mice in NA Normalised social avoidance, changes in Crf mRNA expression and promoter methylation levels in PVN of HT Normalisation of H3 phosphoacetylation in DG and immobility in forced swim-re test Normalisation of H3 phosphoacetylation in DG and immobility in forced swim-re test Normalisation of H3 phosphoacetylation in DG and immobility in forced swim-re test Decrease of Bdnf methylation and increase BDNF mRNA levels in PFC R. norvegicus (SpragueDawley) M. musculus (C57BL/6ByJ) Hunter et al. (2009) Imipramine Daily subcutaneous injections, immediately before each restraint episode Daily intraperitoneal injections for 28 days after end of stress R. norvegicus (Long-Evans) Roth et al. (2009) Daily intracerebroventricular infusions for 7 days at adulthood Maternal Behavior in the offspring of low LG-ABN mothers TSA reverses: 1) Nr3c1 promoter hypermetylation, 2) decreased NGFI-A binding to Nr3c1 promoter, 3) increased GR expression in HP, 4) TSA eliminates, 5) decreased basal plasma corticosterone. 6) increased histone acetylation R. norvegicus (Long-Evans) Szyf et al. (2005) Precursor to 5-mC Maternal behavior Daily intracerebroventricular infusions for 7 In offspring high LG-ABN mothers methionine reverses: 1) Nr3c1 promoter hypometylation and 2) increased NGFI-A binding to Nr3c1 promoter, 3) increased GR expression in HP as well as 4) immobility in forced swim test and 5) decreased corticosterone response in stress R. norvegicus (Long-Evans) Weaver et al. (2004) Acute Forced Swim Normalisation of H3 phosphoacetylation in DG and immobility in forced swim-re test Inhibition of imipramine ability to normalise social interaction and Bdnf mRNA expression in HP M. musculus (C57BL/6) Chandramohan et al. (2008) M. musculus (Bl6/C57) Tsankova et al. (2007) Methionine days at adulthood MSK1 and 2 KO HDAC5 overexpression Knockout of MSK1 and 2 genes Overexpression specific to dentate gyrus of hippocampus Chronic Social Defeat Wilkinson et al. (2009) A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 Trichostatin A 88 Table 3 Factors, that are capable of regulating epigenetic patterns, functional effects of these factors. This data provides evidence, that epigenetic mechanisms regulate stress-related phenotypes in rodents. Research were conducted on adult male animals, unless stated otherwise in “Effect”. A.M. Stankiewicz et al. / Brain Research Bulletin 98 (2013) 76–92 systemic increase in promoter methylation of Nr3c1 with concurrent decrease in its expression both induced by perinatal stress (see Fig. 3). Other important genes influencing stress and brain functions – Gad1, Reln, Avp, Bdnf – were also found to be epigenetically regulated in various brain areas of adult animals exposed to perinatal stress (see Fig. 3.). Thus perinatal stress constitutes strong programming factor responsible for life-long regulation of function of organism. It would be interesting to look into other priming factors known from different fields of biology, which may influence stress responsiveness (e.g. diet (Wang, 2013) or nicotine (Knopik et al., 2012). Noteworthy, two studies have found, that stress during adulthood increases H3 dimethylation, and this modification is stable for at least 4 weeks in C57 mice (Tsankova et al., 2006; Wilkinson et al., 2009). Although some epigenetic changes may be persistent, others are likely to fluctuate during course of chronic stress, as was shown by Hunter et al. (2009) in time-course analysis of global H3 methylation using restraint stress. It will be a challenge to identify specific combinations of histone modifications and DNA characteristics that would indicate stability of the gene regulation (Byun et al., 2012; Nozaki et al., 2011). Another critical topic which needs to be addressed is capacity for reversal of specific epigenetic changes. It was demonstrated that manipulating the epigenetic patters during adulthood may change behavioral phenotype and GR expression which were programmed during early life (Szyf et al., 2005). Studying processes that govern these phenomena may not be straightforward, as such recovery can be mediated by different mechanism than this, which induced the primed epigenetic state in the first place (Tsankova et al., 2006). A number of studies found significant differences in stressinduced epigenetic patterns in animals presenting various phenotypes (Chertkow-Deutsher et al., 2010; Elliott et al., 2010; Uchida et al., 2011), thus raising the possibility that genetic features can influence epigenome. Still, it may be argued that these differences by themselves may be caused by previous environmental cues and not the genotype. Nevertheless, it is almost certain, that search for mutations underling vulnerability to developing pathogenic epigenetic patterns will yield valuable results and constitutes natural progression of current research. This phenomenon was already found to regulate expression of gene coding mu opioid receptor (Oertel et al., 2012), which takes part in stress (Chong et al., 2006; Komatsu et al., 2011). Although males and females share some mechanisms (Chen et al., 2013; Roth et al., 2009), there is also evidence for sex-specificity epigenetic response to stress. Promoter methylation levels of Crh in PVN, BNST and CeA differed in males and females subjected to chronic variable stress (Sterrenburg et al., 2012). These, or similar differences may be result on general differences in epigenetic machinery, as described by Mueller and Bale (2008) who found that DNMT1 level in placenta is sex-specific. Although most of the studies presented in this review were focused on the hippocampus, other papers provided evidence, that stress-induced epigenetic changes are present throughout the brain and may or may not show area-specificity. Illustrating this, Roth et al. (2011) reported that chronic social-predator stress did not affect Bdnf promoter methylation in neither PFC nor amygdala, but did change hippocampal Bdnf methylation levels in opposite directions depending on studied HP zone (CA3 vs. CA1 and DG). Some of epigenetic mechanisms in stress may function in areaindependent manner, as perinatal stress induces similar promoter methylation of Nr3c1 gene in HP, HT and in blood (see Fig. 3). The critical question in epigenetics is whether the observed changes are biologically relevant. Unfortunately, only some of the studies addressed this problem. Through the use of inhibitors of epigenetic maintainers such as zebularin (Roth et al., 2009) or trichiostatin A (Weaver et al., 2005), the researchers have proved that reversing epigenetic changes simultaneously normalizes expression of stress-related genes and HPA axis reactivity. Moreover, 89 number of studies showed that chronic treatment with antidepressants may prevent forming of stable epigenetic changes over stress-related genes such as Crh or Bdnf. Thus, it seems likely, that some of the observed stress-and antidepressant-induced functional changes may be underlined by epigenetic mechanisms (for summary, see Table 3.). Epigenetic change does not imply functional effect, e.g. (Chen et al., 2013; Sterrenburg et al., 2012). Functionality of epigenetic change depends on multitude of factors such as: which CpG island is methylated in which part of gene regulatory region (Wang et al., 2011), intricate interdependency of epigenetic marks (Tsankova et al., 2006), and other non-epigenetic factors e.g. availability of activated transcription factors (Palacios et al., 2010). Therefore, it would be informative to routinely assess at least mRNA expression of a gene of interest, which is suspected to be epigenetically regulated. Nevertheless, hypothetically, even in absence of mRNA response, the epigenetic change may prime the cell or gene to respond in specific way to future cues. The epigenetics indicates a new, promising direction for treating stress-related pathologies. Studies presented in this review show that stress-induced epigenetic changes can be reversed in adulthood using agents such as HDAC inhibitors or methyl group donors (Roth et al., 2009; Weaver et al., 2005). Functional effects of such treatment have been confirmed in studies on rodents (Weaver et al., 2005) and epigenetic drugs are already being developed (Kelly et al., 2010). Additionally, at least some of the epigenetic mechanisms active during stress are shared between rodents and humans, providing rationales for translatory potential. For example, perinatal epigenetic programming of GR expression seems to follow this logic, as the same changes were found in hippocampi of both postnatally stressed adult mice and human suicidal victims with history of child abuse (McGowan et al., 2009; Szyf et al., 2005). Unfortunately, potential unspecific and undesirable effects of such systemic treatment seem troubling. Only through more comprehensive research of epigenetic mechanism we may be able to develop safe and effective epigenetic-based therapies. The complex epigenetic regulatory orchestra is just beginning to be understood. The role of many of its components such as non-coding RNA, 5hmC, histone variants and the editing of nucleic acids is still largely unknown. Without recognition of the impact of temporal dynamics, cellular diversity and systemic approach, both intra- and inter-cellular or structural, the final picture of the pathway from experience to changes in gene expression and behavior may continue to be vague and elusive. Acknowledgements This article was supported through funding from the following grants: Polish Ministry of Science and Higher Education Grant “Iuventus Plus” (IP2011 030371) and Polish Scientific Committee Grant 2011/03/N/NZ29/05222. All the figures were created using VUE software (http://www. actrec.gov.in/histome/index.php). 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