The present invention relates generally to therapeutics. More specifically, the present invention is novel therapeutics to treat post-traumatic stress disorder using DNA methylation profiles discovered in animal models.
Post-traumatic stress disorder (PTSD) is a trauma and stress related disorder that may develop in survivors of a traumatic event, such as a military combat. PTSD is currently defined by the coexistence of four clusters of symptoms: re-experiencing, avoidance, negative cognitions and mood and arousal, persisting for at least one month. In addition, PTSD can cause intense fear, feeling of helplessness and anhedonia1. Fear and anxiety may increase over time, upon conditioning with stress-associated cues and in the absence of further stress exposure. Although cognitive behavioral therapy as well as pharmacotherapy with SSRIs, tricyclic anti-depressants2 and cognitive enhancers in combination with psychotherapy3 alleviate PTSD symptoms, there is no reliable curative treatment to date.
A dominant brain juncture that is related to motivation and hedonia (the ability to experience pleasure or reward) is the nucleus accumbens (NAc)4-6. Several studies reported alterations in NAc-related functions in PTSD patients, particularly reduced reward responsivity. A decreased NAc response to reward was demonstrated in individuals suffering from common stress-related psychopathologies, such as PTSD and depression7-9. A recent fMRI study probed the neural correlates of sensitivity to signals of risk and reward in 24 healthy soldiers at pre-exposure and post-exposure to stressful military service, demonstrating increased amygdala activation and decreased NAc activation in response to risky anticipation and rewarding outcome, respectively4-8, 10-14. Stress-induced, diminished NAc response to reward, combined with predisposed high amygdala responsivity to potential harm, may represent an underlying neural mechanism for vulnerability to stress psychopathology in humans. Neuroadaptations in the process of learning and memory have been suggested to explain the persistent distressing memories of a transient emotionally traumatic event. Since PTSD is characterized by a disordered memory, it most probably involves long-lasting changes in regulation of gene expression. This study tests the hypothesis that epigenetic alterations in the NAc play a critical role in linking traumatic exposure and presence (susceptibility) or absence (resilience) of PTSD-like behavior later in time.
Epigenetic mechanisms program the genome during cellular differentiation and embryonal development and enable a single genome to express multiple phenotypes in the different tissues that comprise a multicellular organism. Epigenetic mechanisms include covalent modification of the DNA molecule itself by methylation15 and hydroxymethylation16 as well as modifications of the histone proteins which are the building blocks around which DNA is tightly wrapped in chromatin17. DNA methylation is involved in memory formation in the adult hippocampus. For example, elevated expression of DNMT3A was implicated in memory formation in a model of reward-related associative memory18 and DNA methylation inhibitors 5-aza-2-deoxycytidine and zebularine inhibited long term potentiation19. Fear conditioning was associated with changes in methylation of candidate genes involved in memory formation20. A double conditional knockout of dnmt3a and dnmt1 in adult neurons results in reduced DNA methylation and deficits in learning and memory without neuronal loss21. DNA methylation alterations in NAc were implicated in the reward responses to cocaine22. DNA methylation was recently examined in the NAc in an animal model of incubation of cocaine craving23 and it was demonstrated that broad changes in DNA methylation are involved in cocaine craving and that they could be reversed by epigenetic therapeutics. Several studies in humans demonstrated associations between PTSD and differential DNA methylation. Since brain is inaccessible in humans, most studies were conducted in peripheral WBC. Using a “genome wide” approach Uddin et al., demonstrated association between PTSD and differentially methylated genes related to immune functions24. Mehta et al., showed DNA methylation alterations in PTSD victims that were also abused as children in comparison with either controls or PTSD victims with no childhood trauma25. Several candidate genes representing neurobiological pathways implicated in PTSD were shown to be associated with PTSD risk when differentially methylated; HPA axis (FKBP5, NR3C1), noradrenergic systems (COMT) and limbic frontal systems (SLC6A4)26. Interactions between risk alleles, DNA methylation and environment were demonstrated. FKBP5 risk allele is demethylated and associated with PTSD only in people who also suffered from child adversity, thus providing a molecular mechanism for gene environment interaction in PTSD27. SLC6A3-9 repeat allele is associated with PTSD risk only when it is methylated28.
A critical question that needs to be addressed is whether or not alterations in DNA methylation in response to trauma are triggered in the brain and whether or not they play a causal role in PTSD. Although risk factors for PTSD illness have been examined29 resilience in adults has often been neglected30. The idea that PTSD involves neuro-adaptations in the processes of learning and memory begs the question of whether resilience results from a process of protective neuro-adaptation rather than absence of neuro-maladaptation. An important question is therefore whether or not resilience is associated with a characteristic DNA methylation profile that is different from either control or susceptible individuals or whether resilience is characterized by absence of DNA methylation changes relative to control. In other words, does “resilience” reflect an active epigenetic process and a particular DNA methylation profile that plays a role in adapting the individual to PTSD? The present invention is founded on the idea that an analysis of the DNA methylation of PTSD resilient and susceptible rats in the brain will lead to critical pathways that should be targeted with therapeutics.
In the present invention, an established rat model for post-traumatic stress disorder (PTSD) was used, which simulates several types of prevalent PTSD symptoms, including re-experiencing, avoidance, and hyperarousal31 after the exposure to severe trauma (predator scent). This model enables identification of animals that develop lasting symptoms which mimic the PTSD-like disorder after exposure to a traumatic experience. The animals are analyzed according to three clinically-relevant clusters of symptoms:
Only animals that show abnormal score in all three clusters, one month from the exposure to the trauma, are identified as susceptible rats (PTSD-like). This animal model allows the arrival of the following discoveries. First, DNA methylation alterations in the NAc triggered by “incubation of fear” and associate with PTSD-like behavior were discovered. Second, a unique “adaptive” DNA methylation pattern that is different from controls and the PTSD animals was discovered in resilient animals. Third, DNA methylation modulators such as the methyl donor S-adenosyl-methionine (SAM) reverse two of the three PTSD behaviors social interaction and re-experiencing and could therefore be used as a therapeutic for PTSD. Fourth, pathways that are enriched with genes that are differentially methylated in PTSD animals were discovered. Fifth, these pathways are used to identify new therapeutic targets for PTSD such as the retinoic acid or steroid antagonists and agonists. Sixth, retinoic acid reverses the hyperarousal behavior of PTSD. Seventh, a combination of retinoic acid (vitamin A) and SAM reverses all pathological behavior of PTSD animals and is therefore a new therapy for PTSD. The utility of the present invention is demonstrated that by analysis of the differentially methylated pathway, a novel combination of therapeutic agents SAM and retinoic acid (vitamin A) to treat PTSD is derived.
All illustrations of the drawings are for the purpose of describing selected versions of the present invention and are not intended to limit the scope of the present invention.
Adult male Sprague-Dawley rats (250-270 gr; Harlan, Rehovot, Israel) were used and housed under conditions of constant temperature (22° C.) and 50% humidity, with a 12-h light-12-h dark cycle. The rats were allowed to habituate to the animal house for 7 days (acclimation). They were housed three per cage, two experimental rats together with a third companion rat. The same three rats remained together until the end of the study. Food and water were provided ad libitum. All experiments were performed between 08:00 and 14:00 in daylight. All animal procedures were approved by the Bar-Ilan University Animal Care Committee and were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
The model used in this research is based on Kesner et al., 200932. This model consists of several stages encompassing 10 weeks (56 days), detailed below and depicted in
Each experimental or naive rat was habituated for 7 days to their home cage (the first week of the habituation). For the first stage of the experiment each experimental or naive rat was habituated for 7 days to an open field apparatus together with a companion rat from their home cage, 5 min per day for 7 days (the second week of the experiment). The open field apparatus was a 90×90×30 cm Plexiglas box, which was placed under a camera. The video and computer equipment were situated in a separate room where all video and observation analyses were performed.
After 14 days (Day 1) of habituation, baseline freezing levels of experimental and naive rats were measured prior to stress exposure. Behavioral parameters and measurements are described below (Behavioral Measurements section).
One week later (Day 7), experimental rats were exposed to stress, i.e. predator scent. Individual rats were placed for 30 min in a clean plastic cylinder (diameter: 30 cm, height: 33 cm) containing 125 ml of well-soiled cat litter, used by a cat during the 24 h prior to the experiment. After exposure to stress, each rat was transferred to the open field, and three behavioral parameters were measured (as described in the Behavioral Measurements section below). The cylinder was cleaned between testing of each rat. Temperature and humidity conditions in the stress exposure area were identical to those in the housing cages and open field. The companion rats were not exposed to stress, and were housed in separate cages in a separate housing area during all experiments. The same cat was used for the litter introduced during initial exposure.
One week later (Day 14), rats were re-exposed for 30 minutes to litter with the same texture, but without the cat scent. Behavioral parameters were subsequently tested (as described below).
Three weeks later (Day 35), rats were again exposed for 30 min to litter with the same texture and without cat scent. Behavioral parameters were subsequently tested (as described below). Behavioral data for this testing stage were analyzed according to the division criteria detailed below. Based on the obtained results, experimental rats were defined as either susceptible or resilient.
After exposure to cat litter, each rat was placed in the open field for measurement of PTSD-like behavior. In the open field, each rat was consecutively tested under three behavioral conditions: alone (termed “exploration”; 5 min), with their habituated companion (“social interaction”; 5 min), and then during post-startle response after exposure to a loud noise (“hyperarousal”; 5 min). The loud noise was a 36.57±0.3 dB pick/scale A over baseline noise of 55±0.5 dB measured by Quest instrument, Quest Technologies, model 2900, calibrated by QC 10 calibrator 114 dB at 1000 Hz, which was sounded to the tested rats during the first 5 min of hyperarousal. The following behavioral parameter was measured during each of the three conditions: freezing (amount of time that the animal remained completely immobile, for at least 2 s). The tested rats were not removed from the open field nor touched during the three testing conditions. Behavioral parameters were videotaped and monitored using Observer apparatus and software (Noldus, The Netherlands).
Baseline behavioral data was analyzed by the Explore procedure in SPSS 11 (IBM Software), to define the interquartile range of the population for each behavioral parameter. Results inside the upper range and lower range limits were treated as ‘normal baseline’. Alterations from this range were used to retrospectively define extreme behavior (after Re2 testing was concluded). According to the determined range, animals were divided into two sets: ‘susceptible’ (exhibiting PTSD-like behavior above ‘normal baseline’ in all three conditions) and ‘resilient’ (animals exhibiting at least one behavior in the ‘normal baseline’ range) (1).
Exploration, social interaction and hyperarousal data were analyzed by a two-way analysis of variance (ANOVA) (with a Bonferroni correction), followed-up by one-way ANOVAs with repeated measures followed by Bonfferoni post-hoc test to compare the different time points in each group (susceptible vs. resilient vs. control) during each behavioral condition (exploration, social interaction and hyperarousal). For analysis of PTSD-like behavior in rats treated with SAMe, a two-way ANOVA with repeated measures with Bonfferoni correction. p<0.05 was considered significant.
A total of 50 Sprague-Dawley rats were exposed to the traumatic event (litter with cat odor) and stress-associated reminders. Posttraumatic stress disorder-like behavior of each animal was compared both with its own baseline and with the range in the population to ensure that two unambiguous subpopulations “resilient” and “susceptible” are identified. The incidence of susceptible rats was approximately 20% (14 susceptible and 26 resilient animals). 10 animal were used as a control group (did not go under the cat odor exposure). Analysis of all baseline behavioral samples revealed that the upper level for excluding outliers (95% confidence) in the exploration, social interaction and hyperarousal conditions was twice the interquartile range. Animals were separated by 3 behavioral tests (
Brains were removed immediately after exposure to litter with cat scent immediately after Re3. Each brain was placed in a Perspex brain matrix and sliced into 1.0 mm segments. The NAc was punched out using a micro dissecting needle of 13-14 G. Brain tissue was frozen at −70° C. until DNA and RNA extraction.
Genomic DNA was extracted using All Prep DNA/RNA Micro Kit, Qiagen (Hilden, Germany)
The SeqCap Epi Enrichment System was used for target bisulfite sequencing of promoters and enhancers in the rat genome. The rat (rn4) target probes were custom designed by utilizing H3K4me1 and H3K4me3 (indicating enhancers and promoters respectively) signals from mouse samples as there is no currently available ChIP data of H3K4me1 and H3K4me3 with respect to the rat species. PuttivH3K4me1 and me3 were selected by mapping and aligning genomic fragments from mouse to the rat and retaining any fragments that share a high similarity and the subsequent associated signals. A total of 53374 target probes have been designed spanning 48072248 base pairs of sequence with a mean probe size of 900 bp. Biotinylated target probes were designed for both strands of bisulfite converted genomic DNA. Bisulfite treated genomic DNA is ligated to methylated NGS adaptors, hybridized to the biotinylated oligonucleotide probes which is followed by a series of washes of off-target DNA sequences and unbound DNA. The resulting isolated specific DNA then undergoes PCR amplification for downstream sequencing on Illumina Hiseq 2000 sequencer. Capture and hybridization is performed as a service for fee by ICRM in Montreal licensed by Roche/Nimblegen. The following bioinformatics workflow is used in the lab. Quality control is done using FastQC to assess sequencing scores and other quality metrics. If necessary, trimming of raw file is performed using Trimmomatic to remove low scoring sequencing results and/or adapter contents. Sequences are aligned to rn4 rat reference genome using Bsmap v2.89. A typical alignment rate of 90%-95% is achieved (30× coverage). The resulting sequencing alignment file (SAM) is converted to binary alignment format (BAM) for downstream analysis and filtering. BAM files are split, merged, and sorted by strand for additional downstream filtering and analysis. Duplicate reads within the BAM files are removed via Picard Tools (v1.93, BROAD institute) strand-specifically and the separate BAM files are merged back together and examined by Bamtools to assess whether the sequences are properly mapped to genome, whether both paired end reads exist (for paired end sequencing), and whether paired end reads are properly paired to each other (for paired end sequencing). Using the final BAM file, methylation ratios and coverage is recovered by provided scripts within Bsmap as well as methylation context. Annotation of genomic locations is performed by HOMER. Computation of differential methylation sites and tiled windows of differentially methylated regions was performed using a logistic regression method with the Methylkit R package in Bioconductor1. FDR adjusted P value significance threshold is held at <0.2.
Whether trauma triggers a distinct DNA methylation profile in the NAc that differentiates traumatized animals from controls, whether resilient or susceptible animals exhibit DNA methylation responses to trauma and whether these responses are different between the resilient and susceptible groups were tested. NAcs were isolated from the animals (n=4-7 per group) following the third reminder and DNA and RNA were extracted as described in the methods. DNA was bisulfite converted, enriched using capture arrays for all predicted promoters and enhancers in the rat genome and was then subjected to next generation sequencing. The distribution of DNA methylation levels across the captured regions as described was first mapped. On average 5306667 CGs per animal was sequenced which showed an average coverage per CG of 39. The question of whether exposure to trauma triggered changes in the DNA methylation profile in the susceptible and the resilient groups was addressed by computing differential methylation sites using a logistic regression method with the Methylkit R package in Bioconductor1. First, the total CG methylation levels along the susceptible, resilient and control groups were compared. A significant methylation differences between the susceptible group to the resilient and control groups was revealed, on average the susceptible group is hypo-methylated in comparison with either the resilient or control groups (
Hierarchical clustering by One minus Pearson correlation of the 738 (excluding the ‘trauma sites’) CGs sites that were altered in response to the ‘incubation of fear’ model in either the resilient or susceptible groups revealed that these sites cluster the resilient susceptible and control animals by their DNA methylation profile. (
The pathways taught by the present invention serve as platform for discovery of new drugs to treat PTSD. Each of the pathways discovered here are targets for therapeutic interventions to convert PTSD susceptible patients to become either PTSD or resilient or better indistinguishable from controls. The broad changes in DNA methylation instructed that treatment should be done with a broad DNA methylation modulator.
The first embodiment of the present invention reveals a broad change in DNA methylation in susceptible animals, it was reasoned therefore that agents that affect DNA methylation might reverse the PTSD-like phenotype. The susceptible animals with the ubiquitous methyl donor SAM were treated which is an approved natural supplement with a good safety record that could be translated to humans.
After Re2, susceptible, resilient and control group were intraperitoneally injected with S-adenosylmethionine (SAM) (Life Science Laboratories Lakewood N.J.) or saline (25 mk/kg), 24 hours and 1 hour before the third reminder. Each group was randomly divided into two groups, each receiving an injection of SAM or saline intraperitoneally. Afterward the third reminder was performed and rats were again exposed for 30 min to litter with the same texture and without cat scent. Behavioral parameters were subsequently tested (as described below).
Each subpopulation of susceptible, resilient and control groups (see methods) received either SAM (25 mg/kg) or saline (i.p., n=4-7 per group), 24 h and again 1 h before the third reminder (Re-3). SAM treatment significantly attenuated freezing behavior in two of three behavioral tests (‘exploration’ and ‘social interaction’ tests) in the susceptible group in comparison with the group that had been treated with saline (
SAM treatment alters the DNA methylation profile of susceptible animals. If the mechanism of action of SAM on PTSD-like behaviors is mediated by DNA methylation, DNA methylation profiles that are characteristic of the susceptible state should be effected by the treatment. The DNA methylation pattern of susceptible-SAM treated animals was first compared with susceptible-saline treated rats, which revealed 599 CGs sites that were de-methylated and 363 CGs sites that were hyper-methylated at a threshold >10; and Q value of 0.2 (
SAM also affected 38 CG sites that were differentially methylated in both the susceptible and the resilient rats (
Quantitative distribution of DNA methylation across individual rats correlates with the quantitative distribution of the behavioral phenotypes and is shifted with SAM treatment.
The three behavioral phenotypes are quantitatively distributed across individual animals. DNA methylation states are also quantitatively distributed amongst the individual animals. Therefore, it was hypothesized that the quantitative distribution of these phenotypes correlates with the quantitative distribution of DNA methylation levels of particular sites in the NAc. The hypothesis was tested by performing a Pearson correlation between the level of methylation of 140 CG sites that were found to be differentially methylated between the susceptible animals and the SAMe's treated animals in the three behavioral tests: exploration, social interaction and hyperarousal across the individual animals whose DNA methylation levels were mapped (n=16) at a threshold of r>0.5 and adjusted p value <0.05. 23 CG sites that were reversed by SAM's treatment exhibited linear correlation with exploration phenotype and 21 CG sites exhibited linear correlation with social interaction phenotype and only 9 CG sites are in common to the exploration and social interaction behavioral tests (
These data support the idea that inter-individual quantitative differences in behavior are associated with quantitative differences in DNA methylation and that SAM shifts the distribution of methylation of these sites and the correlated behaviors.
Importantly, none of the sites that were correlated with the hyperarousal behavior were altered with SAM which is consistent with the observation that SAM treatment does not reverse the hyperarousal behavior, suggesting that the hyperarousal behavior is associated with different DNA methylation profiles than the other two behaviors.
The discovery could be translated to humans into a new therapy of PTSD using SAM.
If DNA methylation plays a causal role in PTSD, comparative analysis of upstream regulators of genes whose DNA methylation is different in control, susceptible, resilient and SAM treated animals could reveal important putative new pathways and networks involved in PTSD molecular pathology that are candidates for therapeutic intervention (
The retinoic biosynthesis and degradation pathway was also enriched with differentially methylated genes. In the susceptible group there was an enrichment in downstream targets of the RAR-Related Orphan Receptor A (RORA), however the impact on level of activity of this pathway could not be derived from this analysis (
Utility of Discovery of Pathways Regulating Genes that are Differentially Methylated in PTSD
The pathways discovered here will be utilized for the discovery of new drug targets for treating PTSD such as estrogen receptor agonists or antagonist and retinoic acid.
Since it was discovered that retinoic acid reverses the third pathological behavior that is not reversed by SAM, whether stimulating this pathway by supplying retinoic acid will reverse PTSD “hyperarousal” feature of the PTSD pathology and whether a combination of SAM and vitamin A (retinoic acid (RA)) will have a synergistic effect on PTSD-like behavior, relieving all three PTSD symptoms, was tested. Each subpopulation of susceptible and resilient group received either SAM (25 mg/kg), retinoic acid (2 mg/kg) or saline (i.p., n=3-6 per group) twice, 24 h and 1 h before the third reminder (Re-3). It was found that the combination of SAMe and RA significantly attenuated freezing behavior in all of the three behavioral tests in the susceptible group in comparison with the groups that were not treated with this ‘cocktail’ (
The main application of the present invention is in the field of PTSD therapeutics and drug discovery. The DNA methylation profiles and pathways that were discovered provide a general platform for discovery of new drugs to treat PTSD. Moreover, two agents SAM and retinoic acid that in combination reverse PTSD and could be used in PTSD therapeutics are disclosed.
Epigenetic mechanisms offer an attractive hypothesis for explaining the lasting effects of transient exposure to trauma as well as the potential impact of life adversity and gene environment interactions. However, evidence was lacking regarding DNA methylation alterations in brain regions known to be involved in PTSD and there was no evidence of a causal relationship between epigenetic alterations and PTSD behaviors. Addressing these questions is critical for understanding the disease as well as for prevention and intervention. These questions could only be addressed using a reliable animal model with construct validity. Previously, it was reported on such an animal model that mimics PTSD32. This model demonstrates a progressive increase in fear behavior and a shift to anxiety over time to the stress itself and to its reminders in the susceptible group (PTSD-like rats).
The findings show a time-dependent gradual intensification of fear response to recurring stress reminders over 35 days. The susceptible group's behavior corresponds with the clinical definition of PTSD in the human population, including anxiety behavior, social deficit and avoidance behavior and high levels of arousal behavior30,36. In addition, exogenous stressors, such as exposure to a live predator26,37, psychological stress37 and the predatory cue used in this research correspond with natural stressors. One of the salient characteristics of PTSD is resilience; the distribution of PTSD among trauma's victims is 5%-30%, whereas other that were exposed to the same trauma appear to be resilient to it27. Therefore, understanding the mechanisms underpinning PTSD necessitates accounting for resilience and a model that has construct validity should exhibit partial resilience as well. This also highlights both importance of individual analysis of behavior and comparison of each exposed animal both with its own baseline and with the entire population. Although this analysis yields a lower percentage of susceptible rats, it more accurately simulates the heterogeneous pattern of response to stressful events in the human population. Thus, the combination of the model components, i.e., exogenous stimuli, simulation of clinical manifestation, and individual categorization, can produce more reliable results. Despite the increased risk that is associated with trauma, some individuals develop strong coping responses which defines them as a resilient group38, 39. The ability to cope adaptively with trauma refers to the resilient characteristic40. Resilience characteristics are likely to attenuate risks of developing PTSD, perhaps through effective emotional regulation, tolerance of negative affect, or active seeking of supportive or nurturing relationships41. Although detailed information is available on risk factors for posttraumatic stress disorder29,42, resilience in adults has often been misunderstood because much of what is known about coping in trauma comes from studies of treatment seeking or distressed individuals recruited from psychiatric treatment settings43.
Since PTSD is characterized by a disordered memory44, understanding long-lasting changes in the programming of gene expression may help elucidate the factors that are involved in embedding the traumatic exposure in neural circuits well as the separation of susceptible from the resilient individuals. DNA methylation is a chemical modification of DNA that plays an important role in defining stable long lasting gene expression programs during differentiation2,3 and is also implicated in mediating the long term effects of early life adversity4. Moreover, DNA methylation varies between individuals even on the same genetic background and might provide an explanation for the inter-individual variation in the response to past traumas5. In the current discovery, the above mentioned model was used to examine at a single nucleotide resolution the known functional regulators of transcription in the rat genome, promoters 5′UTR, 3′UTR and enhancers using capture bisulfite sequencing. It was hypothesized that if changes in DNA methylation occur, they will not be limited to a small list of candidate genes but will involve several genes in functional gene pathways. Genes do not act independently but in complex networks and it stands to reason that complex behaviors involve alterations in complex neuronal circuits and in complex cellular pathways of gene expression. It also stands to reason that reversing a PTSD-like phenotype, for example, will be possible if pathways rather than single genes are targeted. Using an unbiased comprehensive approach provides an opportunity to characterize pathways that are affected in PTSD as well as those that confer resilience that could serve as new candidates for therapeutic interventions. The focus was on NAc since it is a central region in the limbic system known to mediate reward, motivation and hedonia4-6. As for PTSD, anhedonia is one of its main characteristics45, albeit less studied. Recent findings have demonstrated that the NAc may have a significant role in PTSD progress. Moreover, broad changes in DNA methylation occur in the NAc during incubation of cocaine craving supporting the hypothesis that DNA methylation might be also critical in incubation of fear in the NAc23.
First, it is shown that despite the risk to develop PTSD after a traumatic event in the animal model, the resilient rats are likely to attenuate the PTSD-like symptoms and are likely to assuage the expression of PTSD-like symptoms and exhibit behaviors similar to the control group, as in humans. This means that resilient rats are not unaffected subjects but rather individuals that develop resistance.
Second, it is shown that susceptible animals show differences in DNA methylation in the NAc as compared to control animals that involve, as hypothesized, a number of functional pathways such as neurological and psychological disorders, cellular function maintenance, anxiety, learning and memory and more. In total, 474 CG sites were differentially methylated from the control group in the susceptible group and they are associated with 379 different genes. These pathways serve as a platform for discovery of new drug targets to treat PTSD.
Third, it is shown that resilient animals also exhibit differentially methylated CG sites from control animals and these partially overlap with the susceptible DNA methylated differences. These changes occurred in the same direction of methylation (the “trauma sites”), but the other methylated CG sites of the resilient group are unique to this group. Here, the first evidence that resilience is associated with an active DNA methylation response rather than absence of a change in DNA methylation is provided. This suggests that DNA methylation might be involved in resilience phenotype. In total 581 CGs were DNA methylated in the resilient group and they are included in 475 different genes. In addition, the resilient group exhibit DNA methylation differences from the control group which were enriched in a number of functional pathways such as nervous system development and function, psychological disorders, learning and memory, anxiety, cell-to-cell interaction signaling and more. It is tempting to speculate that trauma evokes a heterogeneous continuous DNA methylation response that is potentially protective but could become maladaptive when it fails to target the complete “protective” DNA methylation program.
Examining the individual behavioral phenotypes of the animals in the study reveals that it shows a continuous inter-individual variation resembling a quantitative trait with a threshold rather than a binary trait. While classical genetics can easily explain binary traits caused by Mendelian inheritance of rare alleles, the continuous distribution of quantitative traits is more difficult to explain. The state of methylation of genes could vary from cell to cell even in the same tissue increasing the potential for a continuous variation. It is recently shown that quantitative distribution of DNA methylation in the epithelial growth factor receptor promoter (Egfr) is associated with quantitative distribution in body size in ants46. Quantitative distribution of DNA methylation is thus well poised to explain the quantitative inter-individual variations in the three behavioral traits that characterize PTSD. It is shown here that quantitative individual variations in each of the three behaviors exploration, social interaction and hyperarousal correlate with individual variation in 43 differential DNA methylated sites that were affected by SAM's treatment. Interestingly, two of the behaviors show an overlap in 9 differentially methylated CGs while hyperarousal seems to be controlled by an independent set of CGs. This is consistent with the fact that SAM's treatment does not affect hyperarousal nor does it change the state of methylation of sites that correlate with this behavior.
The data instructs first on the nature of changes in the DNA methylation landscape following ‘incubation of fear’ in NAc of resilient and susceptible animals as well as on the downstream pathways that are targeted by these processes. The wide cast of genes that are altered suggest that reversal of PTSD will probably require approaches that result in broad modulation of the DNA methylation agents and in modulating critical cellular pathways rather than single proteins or single targets of methylation. The ubiquitous methyl donor SAM was tested although it is clearly nonspecific for particular genes or group of genes and is involved in methylation of other macromolecules including histones. It was previously shown that SAM could cause broad but nevertheless selective alteration of DNA methylation in cancer cells6 as well as the brain7. SAM is particularly an attractive agent for PTSD since is an approved nutritional supplement with a known safety profile, thus positive results with this agent in animals could be translated into human studies. The data suggest that SAM's treatment modulates the DNA methylation profile of PTSD animals and is a candidate effective therapy to relieve two of the three characteristic pathological behaviors in PTSD in humans, exploration and social interaction.
If DNA methylation plays a causal role in PTSD, the differentially methylated genes, particularly those that exhibit correlation between levels of methylation and inter-individual differences in behavioral phenotypes are candidates for playing a role in the PTSD phenotypes susceptibility and resilience in the animals. The output of Capture bisulfite sequencing unravels candidate genes for intervention, which are likely to respond to either natural or synthetic chemicals (vitamins for example). Since the number of CG sites that were affected was large as is the case in many epigenetic studies (i.e. 7), whether they fall into particular functional groups was examined. It was reasoned that effective intervention should target pathways rather than particular genes that are differentially methylated. When the different upstream regulators of pathways enriched in differentially methylated genes in susceptible, resilient animals and reversed with SAM treatment were clustered, few significant networks of genes that have strong known relationship to PTSD, such as sex hormones, glucocorticoids, cytokines and others described in
The discovery highlights the value of important networks of genes involved in disease as an effective way for revealing new therapeutics. In the example studied here, the analysis points to a combination of two approved natural products as an effective “cocktail” for treating PTSD that could be translated to treating humans
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Although the invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/IL17/50180 | 2/13/2017 | WO | 00 |
Number | Date | Country | |
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62315513 | Mar 2016 | US |