Possible Involvement of Epigenetic Mechanisms in the Neurobiology of PTSD Peter PREGELJ a and Alja VIDETIČ b,1 University Psychiatric Hospital Ljubljana, Slovenia b Faculty of Medicine University of Ljubljana, Institute of Biochemistry, Slovenia a
Abstract. Exposure to a traumatic event is required for the diagnosis of posttraumatic stress disorder (PTSD). It was first suggested that PTSD represents a normative response to exposure to extreme stressors, but it soon became evident that only a minority of individuals who experience a traumatic event will develop the disorder. However, the relation between psychopathological events, the phenomenology of the trauma, and neurobiological changes related to PTSD is not totally understood. The symptoms of PTSD are believed to reflect stress-induced changes in neurobiological systems representing an inadequate adaptation of neurobiological systems to exposure to severe stressors. Attempts are made to relate different neurobiological changes to the specific features represented in PTSD. It is not clear whether certain neurobiological changes in PTSD reflect preexisting vulnerability or consequences of trauma exposure. It is known that early life environmental events have persisting effects on central nervous tissue structure and function, a phenomenon called 'developmental programming'. Further it is known that glucocorticoid hormone mediators may be involved in this process. It was suggested that changes in glucocorticoid system are mediated by tissue-specific changes in gene expression. Recent studies suggest that epigenetic mechanisms may play an important role in the interplay between stress exposure and genetic vulnerability. In preclinical studies it was first suggested that epigenetic mechanisms may be involved in the modulation of gene expression in response to stressful stimuli. Recently, epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls were found, indicating the involvement of these mechanisms in human adaptation to stress. Beside DNA methylation, histone modulation is involved in epigenetic regulation of gene expression by regulation of diverse chromatin-templated processes, including transcription. These covalent modifications of histones, including phosphorylation, acetylation, ubiquitination, deimination, and methylation, affect therefore the numerous processes involving chromatin, such as replication, repair, transcription, genome stability, and cell death. PTSD may both act as environmental challenges if present in early life and may themselves be more likely in individuals made 'vulnerable' by early life stress or even by appearance of PTSD in their parents. Keywords. Epigenetics, methylation, NR3C1, BDNF, Dlgap2
1
Corresponding Author: Faculty of Medicine University of Ljubljana, Institute of Biochemistry, Vrazov trg 2, SI-1000 Ljubljana, Slovenia; E-mail:
[email protected].
Introduction Exposure to a traumatic event is required for the diagnosis of posttraumatic stress disorder (PTSD). It was first suggested that PTSD represents a normative response to exposure to extreme stressors, but it soon became evident that only a minority of individuals who experience a traumatic event will develop the disorder [1]. However, the relation between psychopathological events, the phenomenology of the trauma, and the neurobiological changes related to PTSD is not totally understood. The diathesisstress model of mental illness suggests that variables increasing an individual’s exposure to stress events can reveal or exacerbate symptoms of mental illness. Both environmental and genetic factors can contribute to stress reactivity. Retrospective and prospective studies of patients with different mental disorders indicate that increases in environmental liability brought upon by even moderate “life stressors” such as going away to college, or the death of a loved one, tend to precede the development of symptoms [2,3,4]. Similarly, persons exposed to combat experience are at an increased risk for developing psychopathological symptoms and PTSD. Stress is inherent to PTSD, which is caused by exposure to a traumatic event that is experienced as horrific or life threatening. However, not only PTSD but also other mental disorders are more common among persons exposed to stressful events. It was reported that hurricanerelated stressors play a significant role in the increased incidence not only of PTSD in Hurricane Katrina survivors, but also in increased suicide rates, and mood and anxiety disorders were also reported more often [5,6]. Neurobiological systems that have been implicated in the patophysiology of PTSD include major neuroendocrine stress response system hypothalamic-pituitary-adrenal (HPA) axis, as well as various neurotransmitters and neuropeptides that comprise a network of interconnected brain regions that regulate stress responses, including prefrontal cortex (PFC), hypocampus, amygdala, and brainstem nuclei. Other important vulnerability factors that affect outcome of the trauma (PTSD) are sex differences, developmental exposures to stress, genetic variability [7] and nevertheless epigenetics. All these factors together influence neurobiological systems and moderate responses to trauma, likely contributing to individual vulnerability versus resilience against developing PTSD [7]. Animal studies indicate that stress results in similar biochemical, cognitive, and morphological changes to the PFC as those observed in mental illness [8]. It is known that lesions of the ventral and orbital cortex are characterized by the inability to inhibit inappropriate emotions, and the inability to flexibly regulate behavior based on the predictions of future punishment or reward [9]. These brain regions have massive projections to the amygdala, ventral striatum, and hypothalamus, regions involved in stress response [10]. For example, it is now clear that PTSD involves dysregulation of norepinephrine (NE), a neuromodulator central to PFC function and the stress response, and emerging knowledge from genetic studies indicates that genes disrupted in various mental disorders encode proteins that otherwise regulate the stress response and permit optimal central nervous system functioning. It could be suggested that, interactions between biological and genetic factors contribute to dysregulated stress reactivity among individuals vulnerable to mental illness [8].
1. Genetic Risk Factors of PTSD It is known that PTSD is influenced by genetic factors [7]. Evidence from family and twin studies first suggested a heritable condition in the development of PTSD. It was also suggested that hereditable contributions are involved in some endophenotypes of PTSD, such as decreased hippocampal volume [11] or exaggerated amygdala reactivity [12]. More and more literature is available about genetic variations in neurobiological systems that determine responses to stress, and consequently, risk versus resilience to develop PTSD [13]. Genetic variations in the genes that are biological candidates for triggering the development of PTSD could be linked to epigenetics. Namely, especially sites that are often modified by epigenetic mechanisms (e.g. methylation of CpG dinucleotides) represent hotspots for mutations, which could lead to aberrant gene expression. This in turn could lead to PTSD development.
2. Epigenetics The term 'epigenetics' was first defined by Conrad Waddington in 1942 as the ''the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being'' [14]. It is interesting that he proposed that definition already before the mare structure of the DNA was revealed and of course before the modern molecular-biology methods were developed. Today, with the full sequence of the human genome – and hundreds of other genomes – in hand, it is apparent that those organisms with a higher order of complexity within their central nervous system (CNS) have indeed acquired a more complex non-coding genome, the organization and regulation of which may reveal a more in-depth understanding of our own human phenotype. With limited exceptions, all cells in a multicellular organism have an identical genotype, and yet development produces a wide range of differentiated cell types with different gene expression profiles driving divergent cellular functions. This progressive cell differentiation results from the epigenetic regulation of gene expression [14]. So, not all the changes in gene function can be explained by changes in DNA sequence, making this the start point of the epigenetics. Two epigenetic mechanisms: DNA methylation and various modifications of histone proteins (acetylation, methylation, phosphorylation, etc.) work in concert and affect chromatin conformation, causing an open (transcriptionally active) or closed (transcriptionally inactive) state of chromatin [15]. To date, the role of epigenetic factors has been primarily investigated in rare pediatric syndromes and cancer, but the epigenetics can be highly relevant to various complex non-Mendelian diseases, as epigenetic mechanisms allow for integrating variety of apparently unrelated clinical, epidemiological, and molecular data into a new theoretical framework [15]. At this point we know three fundamental points that enable us to consider epigenetic factors as etiological candidates in complex disease. First, the epigenetic status of genes is more dynamic in comparison to DNA sequence and can be altered by developmental programs and the environment of the organism. Second, some epigenetic signals can be transmitted along with the DNA sequence across the germline
generations. Third, epigenetic regulation is critical for normal genomic function, such as segregation of chromosomes in mitosis and regulation of gene activity [15]. It may also be important to stress here that the relationships between genotype and specific epigenetic modification is not known. However, it is entirely possible that genotype differences may contribute to the directioning or specificity of epigenetic alternations in response to environmental stimuli/influences, but it is also very important to know that the epigenetics does not necessarily depend on the existence of the specific changes in DNA sequence (e.g. Single Nucleotide Polymorphism, SNP). Since PTSD is a complex disease, the latter being defined as ''a condition caused by a combination of genetic, epigenetic and environmental factors that do not follow Mendelian/monogenic inheritance'' [15] we have to take into consideration all these different factors that together lead to the disease phenotype. 2.1. Epigenetic Mechanisms It is known that early life environmental events have persisting effects on central nervous tissue structure and function, a phenomenon called 'developmental programming'. Further it is known that glucocorticoid hormone mediators may be involved in this process. It was suggested that changes in glucocorticoid system are mediated by tissue-specific changes in gene expression and these can be explained by epigenetics. Epigenetic mechanisms by definition refer to the regulation of various genomic functions, including gene expression, which are not based on DNA sequence but rather controlled by heritable and potentially reversible chemical modifications of DNA molecule and/or the chromatin structure [16,17]. DNA methylation is a chemical heritable modification characterized by the covalent addition of a methyl group to cytosines. In human somatic cells, DNA methylation typically occurs at CpG dinucleotides, which accounts for approximately one percent of the total genome. Moreover, 60-90% of all disperse CpG sequences are methylated. On the other hand, CpG islands (GC dinucleotide-rich regions located at the 5’ end in 60% of human genes) posses high relative densities of unmethylated CpG dinucleotides at all stages of development and in all tissue types [18]. The most often the methylation of CpG dinucleotides occurs in the promoter regions of the genes, less frequent in the first intron of the gene, and rarely in other, subsequent introns of the same gene. DNA methylation is essential for mammalian development. This was evidenced by the ultimate lethality of the DNA methyltransferase (DNMT) knockout mice [14]. DNA methylation occurs at the C5 position of cytosine in the pyrimidine ring and is regulated by different types of DNA methyltransferases (DNMTs). There are several DNMTs in the methyltransferase family, including DNMT1, DNMT2, DNMT3a, and DNMT3b. It was suggested that DNMT1 is the main enzyme responsible for the maintenance of DNA methylation. It is also known that DNMT1 methylates hemimethylated DNA more rapidly than unmethylated DNA [19,20] indicating the possibility that methylation profiles could be inherited from mother to daughter cell [21]. For the cytosine methylation a methyl donor, S-adenosylmethionine (SAM) is required. Different factors are involved in methionine metabolism, like enzymes (betaine-homocysteine methyltransferase, methionine synthase, and methionine adenosyltransferase) and cofactors (vitamin B12, betaine, folate, and homocysteine) [22].
However, cytosines in the CpG dinucleotide are the preferred, but not the exclusive, targets for DNA methylation [23]. It is also known that not all of the CpG dinucleotides are methylated, but there is a cell-specific pattern of distribution of methylated CpG dinucleotides [24]. Methylation of CpG islands is associated with gene regulation because the density of DNA methylation at such islands is often inversely proportional to the transcriptional activity of the gene [25]. Interestingly, DNA methylation patterns, like DNA sequences, are transmitted from maternal chromatids to daughter chromatids during mitosis. This transmission from one generation of cells to another is called the epigenetic inheritance system [26]. However, in comparison to nucleotide sequences, the degree of mitotic fidelity of epigenetic patterns is lower [27]. It was first suggested that epigenetic patterns are erased in the early stages of germline cell development and that new patterns emerged after maturation [28]. Newer investigations however suggested that some epigenetic signals do survive gametogenesis and that this information can be passed on from one generation to the next [29]. Beside DNA methylation, histone modulation is involved in epigenetic regulation of gene expression by regulation of diverse chromatin-templated processes, including transcription. In association with DNA molecule nuclear proteins histones are the basic building units of nucleosomes. DNA molecule is wrapped around a protein octamer made of histones. Each histone has a ''tail'' protruding out of the nucleosome, which can be modified in different ways: phosphorylated, ubiquitinated, sumoylated, acetylated, and methylated [30]. These modifications influence gene transcription, acetyaltion being the most frequent among them. Different types of histones are known, among them four core histones, H2A, H2B, H3, and H4. Histone H3 dimethylation of aminoacid lysine on position 9 and trimethylation of lysine on position 27 have been linked to the formation of transcriptionally inactive heterochromatin [31]. On the contrary, histone H3 and H4 acetylations of lysine residues and trimethylation of lysine on position 4 in H3 are generally associated with active gene transcription [32,33]. It was suggested that methylated cytosines in transcription factor-binding sites change the affinity of DNA for the transcription factor, which in turn alters the transcriptional activity of a gene [34]. Further, methylated cytosines attract methyl-CpG-binding protein, which recruits chromatin-remodeling proteins (e.g. proteins that deacetylate the histones, resulting in transcriptional silencing) [35]. Epigenetic modifications provide all multicellular organisms with a system of gene regulation that allows clonally heritable yet reversible alterations in gene transcription. Gene expression states are set by transcriptional activators and repressors and locked in by cell-heritable chromatin states. Inappropriate expression or repression of genes can change developmental trajectories and result in disease. So, not all the processes underlying epigenetic mechanisms lead to desirable results. Like mutations in the DNA sequence that occur because of aberrant regulation, similarly epimutations occur in epigenome because of aberrant epigenetic regulation. Epimutations could have similar effect as DNA mutations because an epimutation could lead to the abnormal expression of a gene by enhancing or silencing that gene. Epimutation can occur secondary to a DNA mutation in a cis- or trans-acting factor, or as a "true" or primary epimutation in the absence of any DNA sequence change (or defect). It has been estimated that the rate of primary epimutations is one or two orders of magnitude greater than somatic DNA mutation, and therefore is the contribution of epimutations to human disease probably underestimated [36,37].
2.2. Epigenetic Regulation of the Glucocorticoid Receptor Recent studies suggest that epigenetic mechanisms may play an important role in the interplay between stress exposure and genetic vulnerability. It has been shown that maternal care influences hypothalamic-pituitary-adrenal (HPA) function in its responses to stress in the rat through tissue specific epigenetic programming of glucocorticoid receptor (NR3C1) expression. Brain studies demonstrated a greater number of hippocampal glucocorticoid receptor, and a greater expression of the glucocorticoid receptor gene in offspring of high licking and grooming mothers. Hypomethylation within the promoter region of the hippocampal glucocorticoid receptor gene was identified as responsible for its increased expression; in contrast, rat pups receiving low maternal licking and grooming showed greater cytosine methylation at the same promoter site. The observed biological effects and maternal behaviors were transmitted from female offspring to the next (third) generation. These studies provide a clear molecular link between an early environment influence (in this case maternal behavior) and gene expression, producing functional biological correlates in endocrine and behavioral measures related to stress reactivity. As such, these studies offer proof of concept for intergenerational transmission of vulnerability to stress related consequences and detail a plausible mechanism for explaining how childhood adversity increases risk for development of PTSD following traumatic events experienced in adulthood [38,39]. The primary reason to link the early handling phenomenon in rats with transgenerational vulnerability is that it offers a mechanism through which environmental exposures can result in persisting alternations in glucocorticoid receptor expression that underlie individual differences in endocrine function that strongly resembles those observed in PTSD and PTSD risk [38]. In humans, childhood abuse is associated with altered hippocampal development, enhanced HPA stress responses and increased risk for psychopathology (e.g. increased risk for suicide). Recently, epigenetic differences in a neuron-specific glucocorticoid receptor promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls were found, indicating the involvement of these mechanisms in human adaptation to stress. Decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant (1F splice variant of NR3C1 is similar to the rat exon I7, which reveals a maternal effect on cytosine methylation and expression) and increased cytosine methylation of the NR3C1 promoter were found [40]. 2.3. Lasting Epigenetic Influence on the Brain-Derived Neurotropic Factor It has been shown that childhood abuse and neglect compromise neural structure and function, rendering an individual susceptible to later cognitive deficits and psychiatric illnesses. Brain-derived neurotropic factor (BDNF), the key mediator of neural plasticity in the prefrontal cortex and hippocampus, is strongly affected by early adverse experiences. It is well documented that both prenatal and postnatal adverse experiences yield in reduction of BDNF mRNA expression and BDNF protein levels that persist into adulthood. Furthermore, altered BDNF DNA methylation in the maltreated rat offspring was observed. Altered BDNF DNA methylation in offspring of rat females that had previously experienced the maltreatment regimen has been
perpetuated from generation to generation along with the maltreatment behavior of female rats toward the offspring. However, a very important finding of reversing the maltreatment-induced deficits has been discovered. Namely, maltreated adult rats were administered a DNA methylation inhibitor, zebularine, and the results indicated that 7day treatment was sufficient to decrease methylation of BDNF exon IV DNA and rescue both BDNF exon IV mRNA and total mRNA levels in adults with history of maltreatment [41]. 2.4. Methylation of Disks Large-Associated Protein and PTSD In a study of posttraumatic stress disorder a validated PTSD rat model was applied. Rats were exposed to a traumatic stressor (scent of a predator - cat). The results showed differential global methylation pattern in the hippocampus, which is a key structure involved in explicit memory, memory consolidation and learning - cognitive functions that are impaired in PTSD. Of the differentially methylated genes that were a consequence of maladaptation to traumatic stress, global screening revealed disks large-associated protein (Dlgap2). Dlgap2 is localized at postsynaptic density in neuronal cells, and plays an important role in synaptic remodeling and neuronal transmission. Beside differential methylation of Dlgap2 in intron 4 of the gene (hipomethylation in extreme behavioral response animals, PTSD-like rats), a higher protein expression of Dlgap2 in the group of animals that had extreme (intense) behavioral response in comparison to control group or animals with minimum response was observed. Furthermore, it has been shown that increased expression of Dlgap is associated with synaptic malfunction in other psychiatric disorders as well. It is also important and interesting to stress that in the case of Dlgap2 we are talking about methylation change in an intron region (noncoding region inside the coding regions of the gene), and that usually most important changes affecting expression happen in the promoter region (region leading gene expression), which in the case of human or rat Dlgap2 has not yet been identified. Beside that, there is also only one CpG site of methylation in the intron 4 of Dlgap2, while usually there would be more. However, it has been reported before that one or two changes in the methylation status of promoter CpG are sufficient to produce a marked effect on the transcription of the related gene, as in the case of tropomyosin-related kinase B and the glucocorticoid receptor. Another supporting fact for the role of Dlgap2 modifications in PTSD is the fact that many of the SNPs located in intronic regions are associated with psychiatric disorders as well as with other disorders. Thus, the importance of non-promoter areas in the transcriptional regulation together with the significant association between Dlgap2 methylation status and its mRNA expression, suggest that there may be a functional role for the modified methylation in intron 4 of Dlgap2. Taken together, all these results imply that alternations in global methylation pattern are involved in behavioral adaptation to environmental stress and pinpoint Dlgap2 as a possible target in PTSD [42].
3. Conclusions The major advance made by the diagnosis of PTSD in 1980 was in emphasizing the importance of trauma exposure as a major etiologic factor in the development of chronic symptoms. Since that time the development of the modern techniques of molecular biology showed that there is more to come. The importance of the genetic background possessed by individual and the activity of those genes were implemented in the patophysiology of PTSD. Furthermore, also epigenetic mechanisms regulated by stress showed plausible involvement in the pathogenesis of PTSD. The application of epigenetic methods to the field of PTSD today represents an exciting frontier because of their ability to account for individual differences in response to trauma based on environmental exposures that permanently alter gene function. Integrating epigenetics into a model that permits prior experience to have a central role in determining individual differences is also consistent with a developmental perspective of PTSD vulnerability. Importantly, an appreciation of the mechanisms through which experience may alter the expression of genes regulating biological substrates critical to PTSD pathophysiology may help establish relevant biological subtypes of the disorder [38]. In order to be able to implement epigenetic drugs (e.g. methylation inhibitors) into specific treatment of PTSD it is necessary to build and accumulate a comprehensive knowledge of the epigenome to identify the key biomarkers. Development of drugs with greater specificity and better efficacy could be accomplished by better understanding of the structure and function of target molecules, as well as by elucidating the interplay between the drugs and target molecules.
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