TITLE: Post traumatic stress syndrome (PTSD) and epigenetics
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Posttraumatic stress disorder (PTSD) is a severe disorder that is related to exposure to trauma. It remarkably impairs the quality of life and hampers normal functioning of the human system. Epigenetic modifications for example the DNA methylation can be actuated by external environmental factors or gene expressions in a propagative manner that can be transmissible. Due to this, epigenetics can explain the differential variations of responses to trauma exposure. Epigenetic mechanisms have been seen to alter the risk of PTSD as they show both genetic and environmental factors. In this study, evidences for epigenetic variations in the animal models for PSTD has been described. Regulation of stress hormones has been used to show how epigenetics varies in the animal models (rodents) for PTSD. Studies for epigenome related studies which depict associations with PTSD have also been summarized.
Table of Contents
DNA methylation; Epigenetics; PTSD; Trauma
HPA Axis – Hypothalamic–pituitary–adrenal axis
BAM – Bacteriological Analytical Manual
SAM – Systolicanteriormotion
DMR – differentiallymethylatedregion
PTSD -Post traumatic stress syndrome
NR3CI – Nuclear Receptor Subfamily 3 Group C Member 1
DNA – Deoxyribonucleic acid.
Genetic disorders are caused when there are changes in the DNA of a person. This change could be due to mutation in one’s DNA. Mutations can be caused by either errors in replication of the DNA or by changes on the external environment for example lifestyle habits like smoking, radiation or variations in the DNA sequence (Roth, et al., 2011). Epigenetics involves the study of changes in the expression of genes that does not necessarily involve changes in the sequence of the DNA, that is, changes in the phenotypical characteristics that does not affect genotypic structure (Zannas, Provençal & Binder 2015). It through the epigenetic processes, that the cell function and structure are determined. Epigenetic variations can also be caused by changes in the environment factors such as exposure to toxicans (Marsit, 2015). Research has shown that there are at least three processes that may initiate and propagate epigenetic variations in the human system. These processes are: histone modification, non-coding RNA- associated gene silencing and DNA methylation (Pitman et al., 2012). However, more research studies are continually discovering the contribution of epigenetics in the pathogenesis of human disorders and diseases. Post-traumatic stress disorder (PTSD) is a psychiatrist disorder that is stress-related, and is presumed to be caused by interactions between traumatic experiences and a number of genetic factors (Skelton et al., 2012). This disorder is mainly characterized by signs of intrusion, avoidance, and cases of vigorous arousal following a traumatic incident. Recently, there has been an association between epigenetic mechanisms and risk of PTSD. Proofs from a number of human studies support the role that epigenetics play in the pathogenesis of PTSD. Studies involving fear conditioned models also support the role of epigenetics in the occurrence of PTSD. Many studies that have been done in humans and animals show that an epigenetic adjustment of distinctive genes contributes to the pathogenesis of PTSD. Using blood samples, Uddin et al. (2010) evaluated the methylation patterns of PTSD patients and compared it with that of the patients without PTSD. The study established that subjects with PTSD had altered DNA methylation level. Additionally, there is substantial evidence on controlled epigenetics in lab animals subjected to fear-instilling situations (Kwapis & Wood, 2014). According to the study, new learning can be achieved by exposing an individual to the frightening stimulus, but with no aversive outcome. Therefore, if the individual no longer relates the reminder with danger, then the individual’s fear for the given stimulus will lessen. In the study, histone acetylation, DNA methylation, and histone methylation was implicated for the development of extinction learning. However, the later research focused more on histone acetylation than the other two factors. The research in focus therefore, will aim to establish the role of histone methlyation in promoting extinction learning. This research is based on the hypothesis that histone methylation diminish response to fear stimuli. Therefore, if this hypothesis will be true, then it will be concluded that fear-controlled stimuli enhances healing of PTSD. The knowledge obtained from this study will significantly contribute to the management of PTSD cases.
This research is going to use animal models (rodents) since it is very complex to use human models.
Twenty five male rats weighing between 222 and 278g and 11 female rats of Sprague-Dawley species weighing between 220 and 278 g will be used in this research. The rats are to be conditioned for a period of one week in a closed experimental room at a non-varying temperature of 21 degrees Celsius, for a 10-hour darkness period, availing water and food, throughout the duration. After the conditioning period, the males and the females will be allowed to copulate. Pregnancy will be ascertained through virginal plugs’ examination. After that, 20 pregnant candidates be isolated into two groups; control group and the PTSD group, with each group containing 12 rats (N=12). During parturition period, all the rats will be exposed to a stress of an equal level. On day 5 of observation, the difference between the two groups of candidates will be examined. Particularly, the body weight and gestation period will be evaluated. All the off springs will be taken care and given food together with their mothers until the 30th day after parturition. This experiment will be conducted as per the standards and guidelines of the relevant regulatory standards on the use of laboratory animals. Every measure will be put to control discomfort and pain, and minimize the number of rats that will be used in this study. Euthanasia will be applied through decollation.
Extended stress will be applied to pregnant rats to arouse PTSD. The extended stress method was invented by Israel Liberzon and its use revealed neuro-endocrinological discrepancies in PTSD patients (Yamamoto et al., 2009).
The lab animals in the PTSD step will be maintained for 2 hours, and later floated in water maintained at 225oC in a 45 by 65 cm tube. 5 rats will be made to swim at once, and this procedure will be conducted between day 7 and 13 following confirmed pregnancy of the rats. These pregnant rats will be observed to complete the whole study process successfully; without any participant rat dying or experiencing fatigue during the simulation process.
Gene expression will be profiled through; RNA extraction and purification, followed by amplification and results. Eventually, the acquired results will be recorded.
Methylated DNA sequencing will be done in a stringent condition using commercially manufactured ELISA-based kits described by Kurdyukov & Bullock (2016). The ELISA method will enable a quick assessment of DNA methylation status. The rapid ELISA method is preferred because it is quick and easy to conduct hence will be suitable for the identification of large alteration in global DNA methylation.
DNA extraction and purification will be done using Tissue Kit and DNeasy Blood as per the manufacturer’s manual, after which it will be measured by the use of a NanoDrop spectrophotometer. The entire samples will be validated for use in the subsequent procedure Chavez et al., 2010).
Genomic DNA Fragmentation will be done as per the instructions of the preparation guide. Augmentation of the DNA deposits will be attained by surfacing onto a Methylated DNA Precipitation kit.
Analysis of Data
The analysis of data will be conduction following the sequenced outlined below:
- Raw reads
- Reading preprocesses
- Genome mapping
- DMR detection
- Related gene hormones
The t-test results of the weights of the PTSD group offspring and the controlled group offspring will be recorded. It is expected that the weights of the PTSD group of offspring will be significantly lower than the weight of the control group offspring.
HPA reaction to stress by the offspring will be recorded. The level of reaction to stress by the offspring will be ascertained by the level of grooming attention received from their mothers. High levels of grooming or licking of the offspring by the mothers will imply high response to stress.
The PTSD group will be evaluated against the control group so as to ascertain the up regulated and down regulated genes. All the involved genes will be interpreted using the Gene Oncology database.
The total genes will be screened, and then all the differentially expressed genes (mythlated loci distribution) will be done for all the chromosomes; sex chromosomes excluded.
Out of the total genes profiled, the number of differentially expressed genes will be identified and recorded. Amongst the differently expressed genes, it is expected that hyper-methylation will be observed to have down regulated gene expression while hypo-methylation is likely to up regulate gene expression.
An association between the epigenetic expressions of the HPA with stress has been established by various studies (Videlock et al., 2009). Immune instability is an epigenetic field which has received increased attention in the modern days. PTSD has been associated with the destabilization of the peripheral immune functions owing to the long-term impacts of the HPA alignment (Uddin et al., 2013).
Recently, research has confirmed that the PTSD of a mother can confer negative traits to the offspring. Research findings have also established that epigenetic factors define the relations between maternal stress and the phonotypical characteristics of the postnatal phase. Maternal stress is the progenitor of a significant number of the offspring’s physical and hyper arousal indicators. Findings from the research in focus will show the potential that PTSD has on the physical and behavioral outcome of their offspring; and this will be evaluated by low body weight and low score in the OFT test. Mulligan et al. 2012) observed that maternal stress potentially alter the offspring’s epigenetic marks. The study also observed established a correlation between prenatal stress and the offspring’s body weight as the enhancers of methylation of the NR3C1 receptor.
Together with other stressors, maternal PTSD is capable of influencing the hormonal levels and behavioral outcome of the offspring (Uddin et al., 2010). Maternal PTSD promotes changes in the stress-response of the HPA alignment, influence neo-transmitters distribution, and contributes to the intrauterine growth retardation.
The study results will indicate the rodent’s expressedgenetic changes. Therefore, depending on the findings, severe stress could be caused by histone variations in the human system due to remarkable changes in histone modifications, more especially in the hippocampus. For example, elevated stress levels will be seen to lead to increased alterations in gene expressions. When adult rodents were exposed to chronic stress, epigenetic changes will be expected to be observed.
Inducing chronic stress in adult animals will be seen to lead to epigenetic modifications, involving key genes that are involved in the signaling of the HPA axis, such as Hsp 9097 and Chr.
The study indicates a strong correlation between stress and epigenetic variations. This study will not use human humans as models as this would be complex to carry out; considering the ethical and procedural measures required when dealing with humans. Studies that have been done are likely to agree with the findings of this study, evidencing that epigeneticchanges have a direct effect on the central nervous system. Relate studies have shown that repeated exposure to traumatic events has a strong correlation to the disease course of PTSD in adults.
The PTSD lifetime is estimated to be between 6% and 11% (Zannas, Provençal & Binder 2015). Stress and environmental factors increase the risk of developing PTSD, especially when a personexposed to experiences that is likely to trigger trauma. In the research in focus, epigenetic mechanisms is associated with risk of PTSD as the studies that have been conducted depict genetic and environmental influences as the main factors contributing to PTSD.
Results from this study are likely to show that the PTSD of a mother, during pregnancy; can the case of pregnancy can hamper or slow down physical characteristics and development of the CNS. The underline factor for this phenomenon can be linked to the gene expressions and alterations related to the neurotransmitters, which may occur due to the genome methylation deregulation. Additionally, circulatory system disorders in offspring could be owed to the PTSD of the mother affecting particular genes such as Epn3, F5, and Itgb6, andMyh2.
Based on the proposed study and the previous related studies, the differential responses to trauma exposure by different people may be explained as either the influence of acquired or inherited genetic alterations. Factors that can induce changes in the gene structure are also likely to trigger epigenetic changes, especially when induced by environmental factors such as trauma, or fear inducing experiences.
Differential responds to trauma experiences by different individuals can only be explained by the effect of epigenetic to PTSD. Epigenetic changes are also more likely to affect genes, either by altering them or permanently disorient them. However, more research would need to be conducted to establish how epigenetics is related to the pathogenesis of PTSD. Researching in this field can also lead to discovery of the sub-types of PTSD.
Byun HM, Benachour N, Zalko D, Frisardi MC, et al. (2015). Epigenetic effects of low perinatal doses of flame retardant BDE-47 on mitochondrial and nuclear genes in rat offspring. Toxicology 328: 152-159. http://dx.doi.org/10.1016/j. tox.2014.12.019
Chavez L, Jozefczuk J, Grimm C, Dietrich J, et al. (2010). Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage. Genome Res. 20: 1441-1450. http://dx.doi.org/10.1101/gr.110114.110
Davidson S, Prokonov D, Taler M, Maayan R, et al. (2009). Effect of exposure to selective serotonin reuptake inhibitors in utero on fetal growth: potential role for the IGF-I and HPA axes. Pediatr. Res. 65: 236-241. http://dx.doi.org/10.1203/ PDR.0b013e318193594a
Kotozaki Y and Kawashima R (2012). Effects of the Higashi-Nihon earthquake: posttraumatic stress, psychological changes, and cortisol levels of survivors. PLoS One 7: e34612. http://dx.doi.org/10.1371/journal.pone.0034612
Kurdyukov S and Bullock M (2016). DNA Methylation Analysis: Choosing the Right Method. PubMed Central. 5(1). doi: 10.3390/biology5010003.
Kwapis JL and Wood MA (2014). Epigenetic mechanisms in fear conditioning: implications for treating post-traumatic stress disorder. Trends Neurosci. 37: 706-720. http://dx.doi.org/10.1016/j.tins.2014.08.005
Marsit CJ (2015). Influence of Environmental Exposure on Human Epigenetic Regulation. Journal of Experimental Biology. 71: 71-79. doi:10.1242/jeb.106971
Labonté B, Azoulay N, Yerko V, Turecki G, et al. (2014). Epigenetic modulation of glucocorticoid receptors in posttraumatic stress disorder. Transl. Psychiatry 4: e368. http://dx.doi.org/10.1038/tp.2014.3
Mulligan CJ, D’Errico NC, Stees J and Hughes DA (2012). Methylation changes at NR3C1 in newborns associate with maternal prenatal stress exposure and newborn birth weight. Epigenetics 7: 853-857. http://dx.doi.org/10.4161/ epi.21180
Norrholm, S.D., Jovanovic, T., Smith, A., Binder, E.B., Klengel, T., Conneely, K., Mercer, K.B., Davis, J.S., Kerley, K., Winkler, J.A. and Gillespie, C.F., 2013. Differential genetic and epigenetic regulation of catechol-O-methyltransferase is associated with impaired fear inhibition in posttraumatic stress disorder. Frontiers in behavioral neuroscience, 7, p.30.
Pitman, R.K., Rasmusson, A.M., Koenen, K.C., Shin, L.M., Orr, S.P., Gilbertson, M.W., Milad, M.R. and Liberzon, I., 2012. Biological studies of post-traumatic stress disorder. nature Reviews neuroscience, 13(11), p.769.
Roth, T.L., Zoladz, P.R., Sweatt, J.D. and Diamond, D.M., 2011. Epigenetic modification of hippocampal Bdnf DNA in adult rats in an animal model of post-traumatic stress disorder. Journal of psychiatric research, 45(7), pp.919-926.
Skelton K, Ressler KJ, Norrholm SD, Jovanovic T, et al. (2012). PTSD and gene variants: new pathways and new thinking. Neuropharmacology 62: 628-637. http://dx.doi.org/10.1016/j.neuropharm.2011.02.013
Spinelli S, Chefer S, Carson RE, Jagoda E, et al. (2010). Effects of early-life stress on serotonin(1A) receptors in juvenile Rhesus monkeys measured by positron emission tomography. Biol. Psychiatry 67: 1146-1153. http://dx.doi. org/10.1016/j.biopsych.2009.12.030
Uddin M, Chang SC, Zhang C, Ressler K, et al. (2013). ADCYAP1R1 genotype predicts PTSD but not depression among women exposed to childhood maltreatment. Compr. Psychiatry 54: e12. http://dx.doi.org/10.1016/j. comppsych.2012.07.053
Uddin M, Aiello A E, Wildman D E, Koenen K C, Pawelec G et al. (2010). Epigenetic and immune function profiles associated with posttraumatic stress disorder. Proc Natl Acad Sci, 107: 9470-5
Videlock EJ, Adeyemo M, Licudine A, Hirano M, et al. (2009). Childhood trauma is associated with hypothalamicpituitary-adrenal axis responsiveness in irritable bowel syndrome. Gastroenterology 137: 1954-1962. http://dx.doi. org/10.1053/j.gastro.2009.08.058
Zannas, A.S., Provençal, N. and Binder, E.B., 2015. Epigenetics of posttraumatic stress disorder: current evidence, challenges, and future directions. Biological psychiatry, 78(5), pp.327-335.