The role of epigenetic mechanisms and chromatin remodeling has been implicated in both synaptic plasticity and neuronal gene expression. Studies with histone deactylase complex inhibitors like  SAHA ,  toluene , garcinol,  trichostatin A  and  sodium butyrate  have shown that acetylation is important for the synaptic plasticity of the brain; by inhibiting deactylase complexes total acetylation rates in the brain increased leading to increased rates of  transcription  and enhanced memory consolidation. [ 17 ] [ 18 ]  By using various learning assays like the  Morris water maze test  and fear conditioning assays in conjunction with acetylation influencing drugs it was shown that acetylation patterns in the hippocampus are integral to memory association and learning behavior. [ 19 ]  Studies with various  HDAC inhibitors  and neural development have shown increased learning and memory, as a result of an increased acetylation state. Conversely studies conducted with HAT inhibitors yielded impairment of memory consolidation and an overall decrease in learning. [ 20 ]


Studies have shown that the  ERK / MAPK  cascade is important for the regulation of lysine acetylation in the  insular cortex  of the brain (A part of the brain implicated in the formation of  taste  memories). The activation of the ERK/MAPK cascade was seen in mice after the introduction of a new taste, the cascade was shown to be necessary for the memory of the taste to be formed. The proposed mechanism for how this cascade works is that MAPK regulates histone acetylation and subsequent chromatin remodeling by means of downstream effectors, such as the  CREB binding protein  (which has HAT activity). [ 21 ] [ 22 ] [ 23 ]  By observing the rates of acetylation in the insular cortex researchers were able to determine which patterns of acetylation were due to deacetylase or acetylase activity and which were a result of lysine acetyltransferase activity. [ 22 ]


Long term potentiation  (LTP) is the enhancement of signal strength between neurons. LTP is the basis of  synaptic plasticity  and plays a pivotal role in memory formation. LTP is dependent on the activity of  NMDA receptors  in the brain and it has been shown that  NMDA  activity influences acetylation. When NMDA receptors are activated they cause an influx of calcium into the cell which in turn activates various signal pathways that ultimately activate the  ERK pathway  which then modulates transcription factors like  CREB . CREB then recruits a HAT to help create and stabilize the long term formation of memory, often through the self-perpetuation of acetylated histones. Studies done on Acetylation of histone H3 in the CA1 region of the hippocampus show that the activation of NMDA receptors increased the acetylation of H3 and conversely inhibition of the ERK pathway in the CA1 region resulted in a decrease in acetylation of H3. [ 23 ]  In summary:


Histone deacetylases  (HDAC) remove acetyl groups (-COCH3) from histones altering  chromatin structures  and decreasing accessibility of transcriptional factors to DNA, thereby reducing transcription of genes. HDACs have shown to play a role in learning and memory through their regulation in the  CREB-CBP pathway .


Studies conclude that HDAC inhibitors such as  trichostatin A  (TSA) increase histone acetylation and improve  synaptic plasticity  and  long-term memory  (Fig 1A).  CREB , a  cAMP response element-binding protein  and transcriptional activator, binds  CBP  forming the CREBP complex. This complex activates genes involved in synaptic formation and long-term memory.(Fig 1B) TSA treatments in the hippocampal CA1 region of mice increased acetylation levels and enhanced long-term potentiation (LTP), a mechanism involved in learning and memory (Fig 1B). However, TSA treatments in CBP mutants lacking  KIX domains  did not effect LTP in mice (Fig 1D). The KIX domain allows for interaction between CREB and CBP, so knocking out this region disrupts formation of the CREBP complex. Knock outs of CREB produced similar results to those of mutant CBP mice (Fig 1C). Therefore, HDAC inhibition and CREBP association are both necessary for memory development. TSA treatments showed increased expression levels of  Nr4a1  and  Nra2  genes while other CREB regulated genes were unaffected. HDAC inhibitors improve memory through activation of specific genes regulated by CREBP complex. [ 24 ]


The role of individual HDACs in learning and memory is not well understood, but  HDAC2  has been shown to negatively regulate memory formation and synaptic plasticity. [ 19 ]


Overexpression (OE) of  HDAC1  and HDAC2 in mice resulted in decreased levels of acetylated lysines. After exposing these mice to context and tone-dependent fear conditioning experiments, HDAC1 OE mice did not change, but HDAC2 OE mice showed a decrease in freezing behavior, suggesting impairment in memory formation. On the other hand, mice with HDAC2 knockouts (KO) illustrated increased freezing levels compared to wild-type (WT) mice while HDAC1 displayed similar freezing behaviors to WTs.  In summary, Guan  et al. [ 19 ]  have shown that:


HDAC3  is also a negative regulator of long term potentiation formation. McQuown  et al. [ 25 ]  have shown that:


Research has shown that HDACs and HATs play a crucial role in  central nervous system  (CNS) disorders such as  Rett syndrome . [ 26 ]
 Rubinstein-Tabyi syndrome  causes intellectual disability through possible mutations in  CREB-binding protein  and  p300 . However, enhancing expression of CREB-dependent genes or inhibition of HDAC activity partially restore LTP loss and ameliorate late LTP deficits. HDAC inhibitor like TSA may provide a possible therapy for Rubinstein-Tabyi syndrome.
Other memory-deficit disorders which may involve HDAC inhibitors as potential therapy are:


During a new  learning  experience, a set of  genes  is rapidly expressed in the  brain . This induced  gene expression  is considered to be essential for processing the information being learned. Such genes are referred to as  immediate early genes  (IEGs).  DNA topoisomerase II beta (TOP2B)  activity is essential for the expression of IEGs in a type of learning experience in mice termed associative fear memory. [ 27 ]  Such a learning experience appears to rapidly trigger TOP2B to induce double-strand breaks in the  promoter DNA  of IEG genes that function in  neuroplasticity .  Repair  of these induced breaks is associated with  DNA demethylation  of IEG gene promoters allowing immediate expression of these IEG genes. [ 27 ]


The double-strand breaks that are induced during a learning experience are not immediately repaired.  About 600 regulatory sequences in  promoters  and about 800 regulatory sequences in  enhancers  appear to depend on double strand breaks initiated by  topoisomerase 2-beta (TOP2B)  for activation. [ 28 ] [ 29 ]   The induction of particular double-strand breaks are specific with respect to their inducing signal.  When neurons are activated  in vitro , just 22 of TOP2B-induced double-strand breaks occur in their genomes. [ 30 ]


Such TOP2B-induced double-strand breaks are accompanied by at least four enzymes of the  non-homologous end joining (NHEJ) DNA repair pathway  (DNA-PKcs, KU70, KU80 and DNA LIGASE IV) (see Figure). These enzymes repair the double-strand breaks within about 15 minutes to two hours. [ 30 ] [ 31 ]   The double-strand breaks in the promoter are thus associated with TOP2B and at least these four repair enzymes.  These proteins are present simultaneously on a single promoter  nucleosome  (there are about 147 nucleotides in the DNA sequence wrapped around a single nucleosome) located near the transcription start site of their target gene. [ 31 ]


The double-strand break introduced by TOP2B apparently frees the part of the  promoter  at an RNA polymerase-bound  transcription start site  to physically move to its associated  enhancer  (see  regulatory sequence ).  This allows the enhancer, with its bound  transcription factors  and  mediator proteins , to directly interact with the RNA polymerase paused at the  transcription start site  to start  transcription . [ 30 ] [ 32 ]


Contextual fear conditioning  in the mouse causes the mouse to have a long-term memory and fear of the location in which it occurred.  Contextual fear conditioning causes hundreds of DSBs in mouse brain medial prefrontal cortex (mPFC) and hippocampus neurons (see Figure: Brain regions involved in memory formation).  These DSBs predominately activate genes involved in synaptic processes, that are important for learning and memory. [ 33 ]


As reviewed by Massaad and Klann in 2011 [ 35 ]  and by Beckhauser et al. in 2016, [ 36 ]   reactive oxygen species  (ROS) are required for normal learning and memory functions.


One of the most frequent  DNA oxidation  products of ROS is  8-hydroxy-2'-deoxyguanosine  (8-OHdG).  Removal of oxidized bases in DNA usually occurs in a matter of minutes, with a half-life of 11 minutes for 8-OHdG. [ 37 ]   Steady-state levels of  endogenous  DNA damages represent the balance between formation and repair.  8-OHdGs are among the most frequent DNA damages present in the steady-state, with about 2,400 8-OHdG damaged nucleotides in the average mammalian cell. [ 38 ]   The steady state 8-OHdG level in the brain is similar to that in other tissues. [ 39 ]


The occurrence of 8-OHdG in neurons appears to have a role in memory and learning.  The DNA glycosylase  oxoguanine glycosylase  (OGG1) is the primary enzyme responsible for the excision of 8-OHdG in  base excision repair .  However, OGG1, which targets and associates with 8-OHdG, also has a role in adaptive behavior, which implies a physiologically relevant role for 8-OHdG combined with OGG1 in cognition in the adult brain. [ 40 ] [ 41 ]   In particular, heterozygous OGG1+/- mice, with about half the protein level of OGG1, exhibit poorer learning performance in the Barnes maze compared to wild-type animals. [ 42 ]


In adult somatic cells, such as neurons, DNA methylation typically occurs in the context of CpG dinucleotides ( CpG sites ), forming  5-methylcytosine  (5mC). [ 34 ]   Thus, a CpG site may be methylated to form 5mCpG.  The presence of 5mC at CpG sites in gene promoters is widely considered to be an epigenetic mark that acts to suppress transcription. [ 43 ]     If the guanine at the 5mCpG site is attacked by ROS, leading to 8-OHdG formation, OGG1 binds to the 8-OHdG lesion without immediate excision of the 8-OHdG.  When OGG1 is present at a 5mCp-8-OHdG site, it recruits  TET1  to the 8-OHdG lesion and TET1 oxidizes the 5mC adjacent to 8-OHdG.  This causes the 5mC to enter the  DNA demethylation  pathway (see Figure titled "Initiation of DNA demethylation at a CpG site"). [ 34 ]   This pathway is initiated by formation of  5-hydroxymethylcytosine , which may remain in the DNA, or there may be further oxidative reactions followed by base excision repair, to return the nucleoside at that position to cytosine (see Figure "Demethylation of 5-Methylcytosine (5mC) in neuron DNA").


The total number of  CpG sites  in the human genome is approximately 28 million and the average frequency of CpG sites in the genome is about 1 per hundred base pairs. [ 44 ]  An intense learning situation can be applied to rats, referred to as contextual  fear conditioning . [ 45 ]  This can result in a life-long fearful memory after a single training event. [ 45 ]  While the  long-term memory  of this event appears to be first stored in the hippocampus, this storage is transient and does not remain in the hippocampus. [ 45 ]  Much of the long-term storage of contextual fear conditioning memory appears to take place in the anterior cingulate cortex. [ 46 ]  (See Figure: Brain regions involved in memory formation, and also this reference. [ 47 ] ) When contextual fear conditioning is applied to a rat, more than 5,000  differentially methylated regions  (DMRs) of 500 nucleotides each occur in the rat  hippocampus  neural genome both one hour and 24 hours after the conditioning in the hippocampus. [ 48 ]  This causes about 500 genes to be up-regulated (often due to hypomethylation of CpG sites) and about 1,000 genes to be down-regulated (often due to newly formed 5mC at CpG sites in a promoter region). The pattern of induced and repressed genes within neurons appears to provide a molecular basis for forming this first transient memory of this training event in the hippocampus of the rat brain. [ 48 ]  When similar contextual fear conditioning is applied to a mouse, one hour after contextual fear conditioning there were 675 demethylated genes and 613 hypermethylated genes in the hippocampus region of the mouse brain. [ 49 ]  These changes were transient in the hippocampal neurons, and almost none were present after four weeks.  However, in mice subjected to conditional fear conditioning, after four weeks there were more than 1,000 differentially methylated genes and more than 1,000 differentially expressed genes in the anterior cingulate cortex, [ 49 ]  where long-term memories are stored in the mouse brain. [ 46 ]


Embryonic stem cells  are capable of self-renewing and differentiating to the desired fate depending on their position in the body. Stem cell  homeostasis  is maintained through  epigenetic  mechanisms that are highly dynamic in regulating the  chromatin  structure as well as specific  gene transcription  programs. [ 1 ]  Epigenetics has been used to refer to changes in  gene expression , which are heritable through modifications not affecting the  DNA  sequence.
 [ 2 ]


The  mammalian   epigenome  undergoes global remodeling during early stem cell development that requires commitment of cells to be restricted to the desired lineage. There has been multiple evidence suggesting that the maintenance of the lineage commitment of stem cells is controlled by epigenetic mechanisms such as  DNA methylation ,  histone modifications  and regulation of  ATP -dependent remolding of  chromatin structure . [ 1 ] [ 3 ]  Based on the  histone code  hypothesis, distinct  covalent   histone  modifications can lead to functionally distinct chromatin structures that influence the cell's fate.


This regulation of chromatin through epigenetic modifications is a molecular mechanism that determines whether the cell continues to  differentiate  into the desired fate. A research study by  Lee et al.  examined the effects of epigenetic modifications on the chromatin structure and the modulation of these epigenetic markers during stem cell differentiation through  in vitro  differentiation of  murine  embryonic stem (ES) cells. [ 4 ]


Embryonic stem cells exhibit dramatic and complex alterations to both global and site-specific  chromatin structures .  Lee et al.  performed an experiment to determine the importance of de acetylation  and  acetylation  for stem cell differentiation by looking at global acetylation and  methylation  levels at certain site-specific modification in  histone  sites  H3K9  and  H3K4 . Gene expression at these histones regulated by epigenetic modifications is critical in restricting the embryonic stem cell to desired cell lineages and developing  cellular memory .


For mammalian cells, the maintenance of  cytosine  methylation is catalyzed by  DNA methyltransferases  and any disruption to these methyltransferases will cause a lethal  phenotype  to the embryo.  Cytosine methylation  is examined at  H3K9 , which is associated with inactive  heterochromatin  and occurs mainly at  CpG dinucleotides  while global acetylation is examined at  H3K4 , which is associated with active  euchromatin . The mammalian  zygotic  genome undergoes active and passive global cytosine demethylation following  fertilization  that reaches a minimal point of 20% CpG methylation at the  blastocyst  stage to which is then followed by a wave of methylation that reprograms the chromatin structure in order to restore global levels of CpG methylation to 60%. [ 4 ]  Embryonic stem cells containing reduced or elevation levels of methylation are viable but unable to differentiate and therefore require critical regulation of cytosine methylation for mammalian development.


Histones modifications in  chromatin  were analyzed at various time intervals (along a 6-day period) following the initiation of  in vitro  embryonic stem cell differentiation. The removal of  leukemia inhibitory factor  (LIF) triggers differentiation. Representative data of the histone modifications at the specific sites after LIF removal, assessed using  Western blotting , confirms strong deacetylation at the  H3K4  and  H3K9  positions on  histone H3  after one day, followed by a small increase in acetylation by day two. [ citation needed ]


The methylation of histone  H3K4  also decreased after one day of LIF removal but showed a rebound between days 2–4 of differentiation, finally ending with a decrease in methylation on day five. These results indicate a decrease in the level of active  euchromatin  epigenetic marks upon initiation of embryonic stem cell differentiation which is then followed immediately by reprogramming of the epigenome.


Histone modifications of  H3K9  position show a decrease in di- and tri-methylation of undifferentiated embryonic stem cells and had a gradual increase in methylation during the six-day time course of in vitro differentiation, which indicated that there is a global increase of inactive  heterochromatin  levels at this histone mark. [ citation needed ]


As the embryonic stem cell undergoes differentiation the markers for active euchromatin (histone acetylation and  H3K4  methylation) are decreased after the removal of LIF showing that the cell is indeed becoming more differentiated. The slight rebound in each of these marks allows for further differentiation to occur by allowing another opportunity to decrease the markers once again, bringing the cell closer to its mature state. Since there is also an increase throughout the six-day period in  H3K9me , a marker for active heterochromatin, once differentiation occurs it is concluded that the formation of heterochromatin occurs as the cell is differentiated into its desired fate making the cell inactive to prevent further differentiation.


Global levels of 5-methylcytosine were compared between undifferentiated and differentiated embryonic stem cells in vitro. The global cytosine methylation pattern appears to be established prior to the reprogramming of the histone code that occurs upon in vitro differentiation of embryonic stem cells.


As the embryonic stem cell undergoes differentiation the level of DNA methylation increases.  This indicates that there is an increase in inactive heterochromatin during differentiation.


In mammals, DNA methylation plays a role in regulating a key component of  multipotency —the ability to rapidly self-renew. Khavari et al. discussed the fundamental mechanisms of DNA methylation and the interaction with several pathways regulating differentiation. [ 5 ]  New approaches studying the genomic status of DNA methylation in various states of differentiation have shown that methylation at CpG sites associated with putative  enhancers  are important in this process. DNA methylation can modulate the binding affinities of  transcription factors  by recruiting repressors such as  MeCP2  which display binding specificity for sequences containing methylated CpG dinucleotides. DNA methylation is controlled by certain  methyltransferases ,  DMNTs , which perform different functions depending on each one.  DNMT3A  and  DNMT3B  have both been linked to a role in the establishment of DNA methylation pattern in the early development of the stem cell, whereas  DNMT1  is required to methylate a newly synthesized strand of DNA after the cell has undergone replication in order to sustain the epigenetic regulatory state. Numerous proteins can physically interact with  DNMTs  themselves, which help target  DNMT1  to hemi-methylated DNA.


Several new [ when? ]  studies point to the central role of DNA methylation interacting with the regulation of  cell cycles  and  DNA repair  pathways in order to maintain the  undifferentiated  state. In embryonic stem cells,  DNMT1  depletion within the undifferentiated progenitor cell compartment led to cell cycle arrest, premature differentiation and a failure of tissue self-renewal. The loss of  DNMT1  occurred from profound effects associated with activation of differentiation genes and loss of genes promoting cell cycle progression, thus indicating that  DNMT1  and other  DNMTs  do not continuously suppress differentiation and thus maintain the  pluripotent  state.


These studies point to the importance of the interaction of DNMTs in order to maintain stem cell states allowing for further differentiation and formation of heterochromatin to occur.


Okamoto et al.  previously documented the expression level of the  Oct4  gene decreasing with embryonic stem cell differentiation. [ 6 ]   Lee et al.  performed a  ChIP  analysis of the Oct4 promoter, associated with undifferentiated cells, region to examine the epigenetic modifications of regulated genes undergoing development during embryonic stem cell differentiation.  This promoter region decreased at  H3K4  methylation and  H3K9  acetylation sites and increased at the  H3K9  methylation site during differentiation. Analysis of a CpG motif of the  Oct4  gene promoter revealed a progressive increase of DNA methylation and was completely methylated at day 10 of differentiation as previously reported in Gidekel and Bergman. [ 7 ]


These results indicate that there was a shift from the active eurchromatin to the inactive heterochromatin due to the decrease of acetylation of H3K4 and an increase of H3K9me. This means that the cell is becoming differentiated at the Oct4 gene, which is coincident with the silence of Oct4 gene expression.


Another site specific gene tested for histone modification was a  Brachyury  gene, a marker of  mesoderm  differentiation and is only slightly expressed in undifferentiated embryonic stem cells. "Brachyury" was induced at day five of differentiation and completely silencing by day 10, corresponding to the last day of differentiation. [ 8 ]  The ChIP analysis of the "Brachyury" gene promoter revealed increase of expression in mono- and di-methylation of  H3K4  at day 0 and 5 of embryonic stem cell differentiation with a loss of gene expression at day 10.  H3K4  trimethylation coincides with the time of highest Brachyury gene expression since it only had gene expression on day 5.  H3K4  methylations in all forms are absent at day 10 of differentiation, which correlates with the silencing of Brachyury gene expression.  Mono-methylation of both histones produced expression at day 0 indication a marker that is not useful for chromatin structure. Acetlyation of H3K9 does not correlate to  Brachyury  gene expression since it was down regulated at the induction of differentiation.
Upon examining of DNA methylation expression, there was no formation of intermediate sized bad in the  Southern analysis  suggesting that CpG motifs upstream of the promoter region are not methylated in the absence of cytosine methylation at this site.


It is demonstrated from these studies that both  H3K9  di-and tri-methylation correlate with the DNA methylation and gene expression while  H3K4  tri-methylation is associated the highest gene expression stage of the  Brachyury  gene. A previous report from Santos-Rosa is in agreement with these data showing that active genes are associated with  H3K4  tri-methylation in yeast. [ 9 ]


This data indicated the same results as for the  Oct4  gene, in that heterochromatin is forming as differentiation occurs again coinciding with the silence of  Brachyury  gene expression.


In a study done by Mierlo et al., data was obtained that suggests unique patterns of  H3K27me3  and  PCR2  polycomb repressive complexes shield ESCs from leaving the ground state or entering a primed state. [ 10 ]
They started from the observation that ground state ESCs are maintained by LIF or 2i, and began to investigate what the ground state epigenome looked like. Using ChIP-seq profiling, they were able to determine that the baseline levels of H3K27me3 were higher in 2i ESCs as opposed to those in serum (primed). This conclusion was further supported by the finding in the same paper that 2i ESCs gained primed-like qualities after removing a functional PRC2 complex, which rendered H3K27me3 useless. [ 10 ]


Leukemia inhibitory factor  (LIF) was removed from all the  cell lines . LIF inhibits cell differentiation, and its removal allows the cell lines to go through cell differentiation.
The cell lines were treated with  Trichostatin A  (TSA) - a  histone deacetylase inhibitor  for 6 days. One group of cell lines was treated with 10nM of TSA.  The  western analysis  showed the lack of initial deacetylation on Day-1 which, was observed in the control for the embryonic stem cell differentiation.  The lack of  histone deacetylase  activity allowed the acetylation of  H3K9  and  histone H4 . Embryonic stem cells were also analyzed morphologically to observe the formation of  embryoid body  formation as one of the measures of cell differentiation. The 10nM TSA treated cells failed to form the embryoid body by Day-6 as observed in the control cell line. This implies that the ES cells treated with TSA lacked the deacetylation on Day-1 and failed to differentiate after the removal of LIF.
Second group,’-TSA Day4’ was treated with TSA for 3days. As soon as the TSA treatment was stopped, on day 4 the deacetylation was observed and the acetylation recovered on Day-5. The morphological examination showed the formation of embryoid body formation by Day-6. In addition, the embryoid body formation was faster than the control cell line. This suggests that the ‘-TSA Day4’ lines were responding to the removal of LIF but, were unable to acquire any differentiation phenotype. They were able to acquire the differentiation phenotype after the cessation of TSA treatment and at rapid rate.
The morphological examination of the third group,’ 5 nM TSA’ showed the intermediate effect between the control and 10nM-TSA group. The lower dose of TSA allowed the formation of some embryoid body formation.
This experiment shows that TSA inhibits histone deacetylase and the activity of histone deacetylase is required for the embryonic stem cell differentiation. Without the initial deacetylation on Day-1, the ES cells cannot go through the differentiation. [ citation needed ]


Alkaline phosphatases found in humans are membrane bound glycoproteins, which function to catalyze the hydrolysis of monophosphate esters. McKenna et al. (1979) found that there are at least three varieties of alkaline phosphatases, kidney, liver, and bone alkaline phosphatases, that are all coded by the same structural gene, but contain non-identical carbohydrate moieties. The alkaline phosphatase varieties, therefore, express a unique complement of in the enzymatic processes in post-translational glycosylation of proteins. [ 11 ]


In normal stem cells, the activity of  alkaline phosphatase  activity is lowered upon differentiation. Trichostatin A causes the cells to maintain the activity of alkaline phosphatase. Trichostatin A can cause reversible inhibition of mammalian histone deacetylase, which leads to the inhibition of cell growth Significant increase in alkaline phosphatase extinction was observed when Trichostatin A was withdrawn after three days. Alkaline phosphatase activity correlates with the morphology changes. Initial deacetylation of histone is required for embryonic stem cell differentiation. [ 12 ]


Dovery et al. (2010) used  HDAC   knockout mice   to demonstrate whether  HDAC1  or  HDAC2  was important for the embryonic stem cell differentiation. Examination of  global histone acetylation  in the absence of HDAC 1 showed an increase in acetylation. Global histone acetylation levels were unchanged by the loss of HDAC2. In order to analyze the process of HDAC knockout mouse in detail, the knockout mice embryonic stem cells were used to generate embryoid bodies. It showed that just before or during  gastrulation , embryonic stem cells lacking HDAC1 acquired visible developmental defects. The continued culture of HDAC1 knockout embryonic stem cells showed that the embryoid bodies formed became irregular and reduced in size rather than uniformly spherical as in normal mice. Embryonic stem cell proliferation was unaffected by the loss of either HDAC1 or HDAC2 but the differentiation of embryonic stem cells were affected with that lack of HDAC 1. This shows that HDAC1 is required for cell fate determination during differentiation. [ 13 ]


In a study done by Chronis et al. (2017), it was found that during the reprogramming of somatic cells into  pluripotent stem cells , OSK (three of the four Yamanaka factors – Oct4,  Sox2 ,  KLF4 ,  c-Myc ) works together with other transcription factors to change the enhancer landscape, leading to the loss of differentiation and the gain of pluripotency.
They were able to determine this by mapping OSKM-binding chromatin states in reprogramming stages and doing loss and gain of function experiments. They were also able to conclude that OSK silenced ME ( MEF -enhancers) partially through Hdac1, which suggests that Hdac1 plays a role in the process of reprogramming somatic cells. [ 14 ]


Epigenetics is known to be important in the regulation of gene expression of differentiation. As such, studies done on small molecules or drugs that inhibit epigenetic mechanisms during differentiation can have great potential for clinical applications such as bone regeneration from  mesenchymal stem cells . [ 15 ]  One example of such an application is the use of  decitabine  (a  deoxycytidine  analog) to deplete DNA methyl transferase 1 (DNMT1) in the context of  melanomas . [ 16 ]  Alcazar et al. did this with the intent to cause gene expression changes (as a result of the DNMT1 depletion) that would lead to the differentiation of  melanocytes , leading to inhibited tumor growth. Small molecules such as these (epidrugs) – while harboring great potential for  therapeutic  use – are also associated with risk. Circling back to the DNMTi (decitabine), there are known side effects such as anemia that can occur – in addition to other unexpected off-target effects. [ 15 ]  This underscores the importance of improving the specificity of the epidrug target, and of devising new and improved methods of drug delivery. [ 15 ]


C. Waddington coined the metaphor of the “ epigenetic landscape ”, in which a marble rolls down a landscape, its course determined by the topography of the land, eventually working towards a lower point. At different points, these paths branch out, and the marble goes down one path. In this metaphor, the ball is a cell during development, and this metaphor can be extended to the dynamics of gene regulation that underlie cell differentiation (epigenetics). [ 17 ]  Wang et al (2011). developed a theoretical/mathematical framework for this, which is significant because it is one of the first attempts to link the Waddington landscape idea with gene regulatory networks in a data-driven way. This, in their own words, provides a “framework for studying the global nature of the binary fate decision and commitment of a multipotent cell into one of two cell fates”, which can be an extremely valuable tool for cell reprogramming or just learning more about what could drive differentiation.


Any disturbance of a stable epigenetic regulation of  gene expression  mediated by  DNA methylation  is associated with a number of human disorders, including cancer as well as  congenital diseases  such as  pseudohypoparathyroidism type IA ,  Beckwith-Wiedemann ,  Prader-Willi  and  Angelman syndromes , which are each caused by altered methylation-based imprinting at specific loci.


Perturbations of both global and gene-specific patterns of cytosine methylation are commonly observed in cancer while histone deacetylation is an important feature of nuclear reprogramming in  oocytes  during  meiosis . [ 18 ]


Recent studies have revealed that there is an array of different pathways that cooperates with one another in order to bestow proper epigenetic regulation by DNA methylation. Future studies will be needed to further clarify the certain mechanism pathways such as  DNA binding proteins ,  DNA repair  and  noncoding RNAs  serve in order to regulate DNA methylation to suppress differentiation and sustain self-renewal in somatic stem cells in the epidermis and other tissues. Addressing these questions will help extend insight into these recent findings for a central role in epigenetic regulators of DNA methylation in controlling embryonic stem cell differentiation. [ 5 ]


Epigenetics of anxiety and stress–related disorders  is the field studying the relationship between  epigenetic  modifications of genes and  anxiety  and  stress-related disorders , including mental health disorders such as  generalized anxiety disorder  (GAD),  post-traumatic stress disorder ,  obsessive-compulsive disorder  (OCD), and more. These changes can lead to  transgenerational stress inheritance . [ 1 ]


Epigenetic modifications play a role in the development and heritability of these disorders and related symptoms. For example, regulation of the hypothalamus-pituitary-adrenal axis by  glucocorticoids  plays a major role in stress response and is known to be epigenetically regulated.


As of 2015 most work has been done in  animal models  in laboratories, and little work has been done in humans; the work is not yet applicable to clinical  psychiatry . [ 2 ]  Stress-induced epigenetic changes, particularly to genes that effect the  hypothalamic–pituitary–adrenal  (HPA) axis, persist into future generations, negatively impacting the capacity of offspring to adapt to stress. Early life experiences, even when generations removed, can cause permanent epigenetic modifications of DNA resulting in changes in  gene expression ,  endocrine  function and  metabolism . [ 1 ]   These  heritable  epigenetic modifications include  DNA methylation  of the  promoter  regions of genes that affect sensitivity to stress.


Epigenetic modification in response to stress results in molecular and genetic alterations that in turn results in mis-regulated or silenced genes. Heterochromatin is the protein that controls the silencing of these genes epigenetically. For example, epigenetic modifications to the gene  BDNF  (brain derived neurotrophic factor), as well as Drosophila ATF-2 (dATF-2), as a result of stress can be passed on to offspring. Chronic variable stress induces offspring hypothalamic gene expression modifications, including elevated  methylation  levels of the BDNF  promoter  in the  hippocampus . [ 3 ]  This methylation will also occur in the heterochromatin, causing a disrupted heterochromatin to be passed on to the child. Maternal separation and postnatal maternal abuse also increases DNA methylation at regulatory regions of BDNF genes in the  prefrontal cortex  and hippocampus, leading to potential stress vulnerability in future generations. [ 4 ]


Stress can also result in inheritable changes DNA methylation in the promoter regions of the  estrogen receptor alpha  (ERα), [ 5 ]   glucocorticoid receptor  (GR), [ 1 ]  and  mineralocorticoid receptor  (MR). [ 6 ]   These changes lead to altered expression of these genes in offspring that in turn leads to decreased stress tolerance.


Gene regulation as it relates to the HPA axis has been implicated in transgenerational stress effects. Environmental prenatal stress exposure, for example, alters  glucocorticoid receptor  gene expression, gene function, and future stress response in F1 and F2 generations. [ 5 ] [ 1 ]  Maternal care  likewise contributes to HPA-related epigenetic modifications. Epigenetic re-programming of gene expression alters stress response in offspring later in life when exposed to decreased maternal care. Inattentive mothering has led to increased levels of gene methyl marks, compared to attentive mothers. [ 5 ]  Female offspring with low licking-grooming mothers have decreased promoter methylation and increased histone acetylation, leading to increased glucocorticoid receptor expression. [ 5 ]  Epigenetic modifications as a result of absent maternal care lead to decreased  estrogen receptor alpha  expression, due to increased methylation at the gene's promoter. [ 5 ]


Epigenetic changes are performed by enzymes known as writers, which can add epigenetic modifications, erasers, which erase epigenetic modifications, and readers, which can recognize epigenetic modifications and cause a downstream effect. Stress-induced modifications of these writers, erasers, and readers result in important epigenetic modifications such as DNA methylation and acetylation.


DNA methylation  is a type of epigenetic modification in which methyl groups are added to  cytosines  of DNA. It is located on the fifth position of cytosine which has importance in the development of mammals. [ 7 ]  DNA methylation is an important regulator of  gene expression  and is usually associated with gene repression. DNA Methylation is a mechanism which can suppress gene expression. It can be inherited through cell divisions in development, and is involved with cell memory. Changes in methylation occur due to mutated or deregulated chromatin regulators. This process is also used in marking cancers for diagnosis.


Laboratory studies have found that early life stress in rodents can cause phosphorylation of methyl CpG binding protein 2 ( MeCP2 ), a protein that preferentially binds CpGs and is most often associated with suppression of gene expression. Stress-dependent phosphorylation of MeCP2 causes MeCP2 to dissociate from the promoter region of a gene called  arginine vasopressin  ( avp ), causing  avp  to become demethylated and upregulated. This may be significant because arginine vasopressin is known to regulate mood and cognitive behavior. Additionally, arginine vasopressin upregulates  corticotropin-releasing hormone  (CRH), which is a hormone important for stress response. Thus, stress-induced upregulation of  avp  due to demethylation might alter mood, behavior, and stress responses. Demethylation of this locus can be explained by reduced binding of DNA methyl transferases (DNMT), an enzyme that adds methyl groups to DNA, to this locus. [ 2 ]


MeCP2 is known to have interactions with several other enzymes that modify chromatin (for example, HDAC-containing complexes and co-repressors) and in turn regulate activity of genes that modulate stress response either by increasing or decreasing stress tolerance. For example, epigenetic upregulation of genes that increase stress response may cause decreased stress tolerance in an organism. These interactions are dependent on the phosphorylation status of MeCP2, which as previously mentioned, can be altered by stress. [ 2 ] [ 8 ]


DNA methyltransferase 1 (DNMT1) belongs to a family of proteins known as DNA methyltransferases, which are enzymes that add methyl groups to DNA. DNMT1 is specifically involved in maintaining DNA methylation; hence it is also known as the maintenance methylase DNMT1. DNMT1 aids in regulation of gene expression by methylating  promoter  regions of genes, causing transcriptional repression of these genes.


DNMT1 is transcriptionally repressed under stress-mimicking exposure both  in vitro  and  in vivo  using a mouse model. Accordingly, transcriptional repression of DNMT1 in response to long-term stress-mimicking exposure causes decreased DNA methylation, which is a marker of gene activation. In particular, there is decreased methylation of a gene called  fkbp5 , which plays a role in stress response as a glucocorticoid-responsive gene. Thus, chronic stress may cause demethylation and hyperactivation of a stress-related gene, causing increased stress response. [ 2 ] [ 9 ]


Additionally,  DNMT1  gene locus has increased methylation in individuals who were exposed to trauma and developed  post-traumatic stress disorder  (PTSD). Increased methylation of  DNMT1  did not occur in trauma-exposed individuals who did not develop PTSD. This may indicate an epigenetic phenotype that can differentiate PTSD-susceptible and PTSD-resilient individuals after exposure to trauma. [ 2 ] [ 10 ]


Transcription factors  are proteins that bind DNA and modulate the transcription of genes into RNA such as  mRNA ,  tRNA ,  rRNA , and more; thus they are essential components of gene activation. Stress and trauma can affect expression of transcription factors, which in turn alter DNA methylation patterns.


For example, transcription factor nerve growth-induced protein A (NGFI-A, also called NAB1) is up-regulated in response to high maternal care in rodents, and down-regulated in response to low maternal care (a form of early life stress). Decreased NGFI-A due to low maternal care increases methylation of a  glucocorticoid receptor  promoter in rats.  Glucocorticoid  is known to play a role in downregulating stress response; therefore, downregulation of glucocorticoid receptor by methylation causes increased sensitivity to stress. [ 2 ] [ 11 ] [ 12 ]


Histone acetylation and deacetylation  is a type of epigenetic modification in which acetyl groups are added to lysine on histone tails. Histone acetylation, performed by enzymes known as  histone acetyltransferases  (HATs), removes the positive charge from lysine and results in gene activation by weakening the histone's interaction with negatively-charged DNA. In contrast, histone deacetylation performed by  histone deacetylases  (HDACs) results in gene deactivation.


Transcriptional activity and expression of HDACs is altered in response to early life stress. [ 2 ] [ 13 ] [ 14 ]  For animals exposed to early life stress, HDAC expression tends to be lower when they are young and higher when they are older. This suggests an age-dependent effect of early life stress on HDAC expression. These HDACs may result in deacetylation and thus activation of genes that upregulate stress response and decrease stress tolerance. [ 2 ] [ 15 ]


Genome-wide association studies  have shown that psychiatric disorders are partly heritable; however, heritability cannot be fully explained by classical  Mendelian genetics , but rather epigenetics. There are many components in understanding the heritability of psychiatric disorders. [ 16 ]  Understanding epigenetic modifications and its ability to impact epigenomes over generations is vital in analyzing potential behavioral disorders. [ 16 ]  But we must acknowledge the concept of transgenerational epigenetics (epigenetic inheritance) which is the occurrence in which parents are able to transfer traits not present in their DNA sequencing to their offspring; it is the passing of environmentally manipulated traits for two or more generations without direct DNA alteration. [ 17 ]  For example, one study found transmission of DNA methylation patterns from fathers to offspring during spermatogenesis. Similarly, several studies have shown that traits of psychiatric illnesses (such as traits of PTSD and other anxiety disorders) can be transmitted epigenetically [ 18 ] [ 19 ]  Parental exposure to various stimuli, both positive and negative, can cause transgenerational epigenetic and behavioral effects.


Trauma and stress experienced by a parent can cause epigenetic changes to its offspring. This has been observed both in  population  and experimental studies. [ 20 ]


Biological vulnerability and HPA axis alterations may be observed after maternal epigenetic programing during pregnancy, leading to similar modifications in future generations. [ 21 ]  Child abuse exposure, for example, is associated with lower baseline infant cortisol levels as well as modified HPA axis function. Human studies investigating  posttraumatic stress disorder  (PTSD) and its effects on offspring have illustrated similar molecular and HPA axis modification and function. [ 6 ]  PTSD patients who experienced trauma from genocides or terrorist attacks frequently exhibited aggressive or neglectful behavior toward offspring during critical developmental periods, possibly contributing to permanent glucocorticoid deregulation in offspring. [ 21 ]  PTSD mothers and children illustrate lower basal cortisol levels and glucocorticoid receptors and increased  mineralocorticoid receptors  when exposed to stress. [ 6 ]  Therefore, developmental experiences, such as stress exposure, may have critical effects on neuromodulatory mechanisms transgenerationally. [ 22 ]


Strong relationships between maternal care and subsequent epigenetic modification in offspring, similar to that found in animal models, has been observed in humans. Severe emotional trauma in the mother, for example, often leads to modified methylation patterns of DNA in subsequent offspring generations. [ 23 ]  PTSD exposed offspring illustrate epigenetic modifications similar to that seen in PTSD mothers, with an increased  NR3C2  methylation in exon 1 and increased  CpG  methylation in the NR3C2 coding sequence, leading to alterations in mineralocorticoid receptor gene expression. [ 6 ]  Additionally, investigation of post mortem  hippocampal  tissue indicates decreased levels of neuron-specific glucocorticoid receptor mRNA and decreased DNA methylation in promoter regions among suicidal individuals with lifelong stress or abuse exposure.


Epigenetic mechanisms as a result of early life stress may be responsible for neuronal and synaptic alterations in the brain. Developmental stress exposure has been shown to alter brain structure and behavioral functions in adulthood. Evidence of decreased complexity in the  CA1  and CA3 region of the hippocampus in terms of dendritic length and spine density after early-life stress exposure indicates transgenerational stress inheritance. [ 4 ]  Therefore, environmental and experience-dependent synaptic reorganization and structure modifications may lead to increased stress vulnerability and brain dysfunction in future generations. [ 4 ]


Human models illustrating transgenerational stress effects are limited due to relatively novel exploration of the topic of epigenetics as well as lengthy follow-up intervals required for multi-generational studies. Several models, however, have investigated the role of epigenetic inheritance and transgenerational stress effects. Transgenerational stress in humans, as in animal models, induces effects influencing social behavior, reproductive success, cognitive ability, and stress response. [ 3 ]  Similar to animal models, human studies have investigated the role of epigenetics and transgenerational inheritance molecularly as it relates to the HPA system. Prenatal influences, such as emotional stress, nutrition deprivation, toxin exposure,  hypoxia , increased maternal HPA activity, and cortisol levels may activate or affect HPA axis activity of offspring, despite placental barrier. [ 22 ]


Paternal stress is an important factor in the determination of inheritance of genes as well as maternal stress inheritance. Factors such as environment and experiences can alter the epigenetic of paternal genes as well as in sperm. Epigenetic changes to the DNA in sperm ("epigenetic tags") prior to conception can be passed to offspring. The paternal phenotype will be inherited into the offspring due to genetic information being stored in the sperm. In studies, it is shown as rodent offspring are fostered mono-parentally and have no direct exposure with their fathers, offspring born of stressed male rodents provide a good model for transgenerational stress inheritance. Direct injection of sperm RNAs to wild type oocytes results in reproducible stress-related modifications. [ 24 ]   Small non-coding RNAs may serve as a potential mechanism for stress-related genetic changes in offspring. Mouse models of traumatic early life stress exposure result in microRNA modifications and subsequent differences in gene expression and metabolic function. [ 24 ]  This effect was reproducible by sperm RNA injection, leading to similar gene modifications in future generations. The novelty of this research suggests direct mechanisms capable of altering epigenetics by stress-related factors.


Early life experiences and environmental factors may lead to epigenetic modification at specific gene loci, leading to altered  neuronal plasticity , function, and subsequent behavior. [ 4 ]  As mentioned, there are genetic markers in all living organisms. There can also be the presence of epigenetically marks and this are basically areas of modification on DNA that in with gene expression. Certain exposures within the environment can lead to the expression of genes in various ways which can contribute to behavioral plasticity patterns that can potentially also change the ways in which organism functions when under normal conditions. [ 25 ]  Chromatin remodeling in rodent offspring and altered gene expression within the limbic brain regions that may contribute to depression, stress, and anxiety-related disorders in future generations. [ 4 ]  Variations in maternal care, such as maternal licking and grooming, indicates reduced HPA axis reactivity in subsequent generations. [ 5 ]  Such HPA axis modifications lead to decreased anxiety-like behavior in adulthood and increased glucocorticoid receptor levels leading to negative feedback on HPA reactivity and further behavioral modifications. [ 5 ] [ 1 ]  Rodent models of maternal separation also reveal increased depressive-like behavior in offspring, decreased stress coping abilities, and changes in DNA methylation. [ 1 ]


An  epidemiological  study investigating behavioral, physiological, and molecular changes in the children of  Holocaust  survivors found epigenetic modifications of a glucocorticoid receptor gene,  Nr3c1 . This is significant because glucocorticoid is a regulator of the hypothalamus-pituitary-adrenal axis (HPA) and is known to affect stress response. These stress-related epigenetic changes were accompanied by other characteristics that indicated higher stress and anxiety in these offspring, including increased symptoms of PTSD, greater risk of anxiety, and higher levels of the stress hormone  cortisol . [ 20 ] [ 26 ] [ 27 ] [ 28 ]  The offspring demonstrate greater risk of developing PTSD in response to their own trauma or traumas. [ 17 ]  Offspring with maternal exposure to the Holocaust during the mother's childhood has demonstrated significantly lower site 6 methylation. [ 29 ]  The site 6 methylation impacts the stress response. In addition to PTSD risks in response to individual trauma in offspring, there has also been an increase in nightmares of offspring related to persecution and torment. [ 30 ]


The effect of parental exposure to stress has been tested experimentally as well. For example, male mice who were put under early life stress through poor maternal care—a scenario analogous to human childhood trauma—passed on epigenetic changes that resulted in behavioral changes in offspring. Offspring experienced altered DNA methylation of stress-response genes such as  CB1  and  CRF2  in the  cortex , as well as epigenetic alterations in transcriptional regulation gene MeCP2. Offspring were also more sensitive to stress, which is in accordance with the altered epigenetic profile. These changes persisted for up to three generations. [ 20 ] [ 31 ]


In another example, male mice were socially isolated as a form of stress. Offspring of these mice had increased anxiety in response to stressful conditions, increased stress hormone levels, dysregulation of the HPA axis which plays a key role in stress response, and several other characteristics that indicated increased sensitivity to stress. [ 20 ] [ 31 ]


Studies have found that early life stress induced through poor maternal care alters sperm epigenome in male mice. In particular, expression patterns of  small-noncoding RNAs  (sncRNAs) are altered in the sperm, as well as in stress-related regions of the brain. [ 32 ] [ 33 ] [ 34 ]  Offspring of these mice exhibited the same sncRNA expression changes in the brain, but not in the sperm. These changes were coupled with behavioral changes in the offspring that were comparable to behavior of the stressed fathers, especially in terms of stress response. Additionally, when the sncRNAs in the fathers' sperm were isolated and injected into fertilized eggs, the resulting offspring inherited the stress behavior of the father. This suggests that stress-induced modifications of sncRNAs in sperm can cause inheritance of stress phenotype independent of the father's DNA. [ 32 ] [ 33 ]


Just as parental stress can alter epigenetics of offspring, parental exposure to positive environmental factors cause epigenetic modifications as well. For example, male mice that participated in voluntary physical  exercise  resulted in offspring that had reduced fear memory and anxiety-like behavior in response to stress. This behavioral change likely occurred due to expressions of small non-coding RNAs that were altered in sperm cells of the fathers. [ 20 ] [ 35 ]   Participation in aerobic exercise led to decreased cortisol levels in males. [ 36 ]


Additionally, exposing fathers to enriching environments can reverse the effect of early life stress on their offspring. When early life stress is followed by environmental enrichment, anxiety-like behavior in offspring is prevented. [ 20 ] [ 37 ]  Similar studies have been conducted in humans and suggest that DNA methylation plays a role. [ 38 ]  Other studies have been conducted to find drugs such as T2D and PPArG can be used as an epigenetic regulation for tissues associated with diabetes. These drugs used show evidence for the therapies that can be associated with the stress effect reversal. [ 39 ]


Early life development and childhood trauma


Healthy development early in life is critical. Early life is characterized by rapid development and increased susceptibility to modifications. Childhood trauma can severely affect the development of the brain, resulting in the alteration of neural circuits which are involved in emotional regulation and threat detection.  Childhood trauma  has been associated with a wide array of mental health disorders such as bipolar disorder, anxiety, post traumatic stress disorder (PTSD), and depression. [ 40 ]


PTSD Inheritability


Research involving  PTSD  in those who experienced childhood trauma had a 25% to 60% inheritability rate, which is a relatively low to moderate rate. This study suggests that other factors play a role in the contribution to this disorder such as gene interactions involving epigenetic modifications. These epigenetic modifications, specifically DNA methylation can lead to the phenotypic expression of mental disorders. [ 40 ]


Changes to the HPA Axis Due to Childhood Trauma


Hypothalamic-pituatary-adrenal (HPA)  axis is essential component of the neuroendocrine system that regulates stress response. Persistent dysregulation of the stress response pathway resulting from childhood trauma causes alterations in the (HPA). These alterations lead to prolonged harmful physiological and physical changes. [ 41 ] [ 40 ]


Postmortem Brain Tissue DNA Methylation


A  DNA methylation  study was done by Labonte on postmortem human brain tissue comparing humans with or without a history of childhood abuse who died by suicide. Childhood abuse and trauma was associated with increased cytosine methylation of the NR3C1 promoter resulting in the decrease of GR expression. The NR3C1 gene encodes glucocorticoid receptor (GR) which is essential for glucose regulation and managing stress response through both genetic and epigenetic pathways. [ 40 ]


Post-traumatic stress disorder  (PTSD) is an stress-related mental health disorder that emerges in response to traumatic or highly stressful experiences. It is believed that PTSD develops as a result of an interaction between these traumatic experiences and genetic factors. The signs and symptoms of PTSD can include avoidance behaviors, invasive thoughts, and significant alterations in normal behavior and thinking. [ 42 ]  There is evidence suggesting PTSD formation is associated with epigenetic changes such as DNA methylation and acetylation of histone proteins. Increased DNA methylation has been found to regulate the induction of fear conditioning behaviors associated with PTSD triggers. [ 43 ] [ 10 ]  Histone modifications, like acetylation and deacetylation, play an important role in the development of PTSD, which is related to fear memory from traumatic events. [ 44 ]


The DSM-5 asserts that PTSD manifests differently in children over six years old than in adults. Specifically that their flashbacks or intrusive memories may be explained by recreating their traumatic event(s) through their play. They may also experience reoccurring nightmares that are indirectly related to the event. Additionally, there is a separate criteria altogether for PTSD in children under six years old. [ 45 ]


Through a number of human studies, PTSD is known to affect DNA methylation of CpG islands in several genes involved in numerous activities, including stress responses and neurotransmitter activity. CpGs are used to describe cytosine-guanine adjacent nucleotides within the same strand of DNA. CpG islands are defined by computer algorithms as being made up of at least 60% CpGs and being anywhere between 200 and 3000 base pairs in size. The methylation of these CpG islands can cause histone modifications which can lead to the condensation of chromatin which can ultimately alter gene expression. [ 42 ]


DNA methyltransferase, DNMT, is an enzyme responsible for increased methylation of DNA. It has been found that DNMT and its associated increased methylation can regulate risk for  memory consolidation  and fear conditioning. [ 43 ]


The removal of methyl groups from cytosine is initiated by a TET enzyme. TET is an enzyme known to oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) within the genome. This reaction initiates active DNA demethylation to ultimately alter gene expression. It has been found that the TET enzyme exists as two isoforms which are differentially regulated and expressed across brain regions. The regulation of these isoforms can affect synaptic connections and ultimately memory formation. [ 46 ]  The manipulation of the TET enzymes' expression levels has become a potential source of interest for PTSD medication. [ 43 ] [ 10 ]


The table below identifies differentially methylated regions (DMRs) across the genome which undergo PTSD-induced epigenetic changes which alter gene expression.


Fathers with PTSD have offspring with higher NR3C1 1F promoter methylation; [ 28 ]


Lower levels of NR3C1 1F promoter methylation is associated with PTSD in combat veterans; [ 56 ]


Higher levels of NR3C1 methylation in male (but not female) Rwandan genocide survivors is associated with decreased PTSD risk [ 57 ]


Histone acetylation is performed by histone acetyl transferases (HATs) and histone deacetylation is carried out by histone deacetylases (HDACs). [ 58 ]  In rodent PTSD models, it has been found that an increase in histone acetylation is associated with fear conditioning. [ 58 ]  Histone acetylation can be involved in all parts of fear memory, including the development to memory extinction. It can also play a role in long-term potentiation (LTP). [ 47 ]  It was also observed that HDACs increase memory formation in fear extinction and HDAC inhibitors (HDACi) have shown evidence for modifying memory extinction, a possible treatment for PTSD. [ 58 ]


The hypothalamus-pituitary-adrenal (HPA) axis is a neuroendocrine system largely involved in ascertaining the levels of cortisol circulating the body at any given point in time. As cortisol plays a key role in the stress response, so does the HPA axis. The dysregulation of the HPA axis has been found to be characteristic of several stress disorders, including PTSD. This system works under a negative feedback loop structure. Hence, this HPA axis dysregulation may take the form of amplified negative inhibition and result in down-regulated cortisol levels. [ 59 ]  Epigenetic modifications play a role in this dysregulation, and these modifications are likely caused by the traumatic/stressful experience that triggered PTSD. [ 47 ]


PTSD is often linked with immune dysregulation. Traumatic experiences can induce epigenetic changes at the gene loci that are immune-related which can lead to immune dysregulation and an increased risk of PTSD. [ 47 ]  Trauma exposure can also disrupt the HPA axis, thus altering peripheral immune function. The effect of PTSD on immune function arises in at least two ways: 1) Continuous disturbances on the HPA axis can dysregulate peripheral immune function, and 2) the effects of immune dysregulation in the periphery can lead to increased development of PTSD because of alterations in brain function. [ 47 ]


PTSD-associated changes in immune cells found in blood or saliva can serve as biomarkers that trigger epigenetic changes which are involved in the pathogenesis of PTSD. [ 47 ]  These unique biomarkers serve as means of identifying PTSD subtypes. Beyond identifying subtypes, these distinct biomarkers can potentially be used to develop PTSD treatments. [ 47 ]


Epigenetic modifications have been observed in immune-related genes of individuals with PTSD. For example, deployed military members who developed PTSD have higher methylation in the immune-related gene  interleukin-18  (IL-18). [ 47 ] [ 54 ]  This has interested scientists because high levels of IL-18 increase cardiovascular disease risk, and individuals with PTSD have elevated cardiovascular disease risk. Thus, stress-induced immune dysregulation via methylation of IL-18 may play a role in cardiovascular disease in individuals with PTSD. [ 47 ] [ 60 ] [ 50 ]


Additionally, an epigenome-wide study found that individuals with PTSD have altered levels of methylation in the following immune-related genes:  TPR ,  CLEC9A ,  APC5 ,  ANXA2 ,  TLR8 ,  IL-4 , and  IL-2 . This again shows that immune function in PTSD is disrupted, especially by epigenetic changes that are likely stress-induced. [ 47 ] [ 50 ]


Nr3c1  is a transcription factor that encodes a glucocorticoid receptor (GR) and contains many GR response elements. Npas4 is another regulatory transcription factor also responsible for the regulation of GRs. Stress-induced changes in Nr3c1 and Npas4 methylation have been shown to alter stress sensitivity. This response differs between short-lived stress exposure and chronic stress exposure. In response to short lived stress, the NR3C1 promoter is more hydroxymethylated which is a modification associated with increased transcription of GR-associated genes. Thus, short lived stress exposure increases stress sensitivity. Conversely, in response to chronic stress, the Npas4 promoter has been presumed to be increased in methylation, a modification which is associated with inhibitory regulation of GRs. Thus, chronic stress exposure decreases stress sensitivity. These distinctions are important in understanding the epigenetic patterns of stress and genetic interactions with PTSD triggers. [ 61 ]  Overall, in the hippocampus of chronically stressed animals, the 3′-UTR (untranslated region of DNA) of the glucocorticoid receptor Nr3c1 showed increased hydroxymethylation, which led to increased transcription and thus, the disruption of stress tolerance and increased risk of disorders such as PTSD. However, early life stress increases methylation of the 1 F  promoter in this gene (or the 1 7  promoter analog in rodents). Because of its role in stress response and its link to early life stress, this gene has been of particular interest in the context of PTSD and has been studied in PTSD of both combat veterans and civilians. [ 47 ]


In studies involving combat veterans, those who developed PTSD had lowered methylation of the  Nr3c1  1 F  promoter compared to those who did not develop PTSD. Additionally, veterans who developed PTSD and had higher  Nr3c1  promoter methylation responded better to long-term psychotherapy compared to veterans with PTSD who had lower methylation. These findings were recapitulated in studies involving civilians with PTSD. [ 47 ] [ 62 ]  In civilians, PTSD is linked to lower methylation levels in the T-cells of exons 1 B  and 1 C  of  Nr3c1 , as well as higher GR expression. Thus, it seems that PTSD causes lowered methylation levels of GR loci and increased GR expression. [ 47 ] [ 55 ]  The methylation of GR in T-cells are investigated because of its role in regulating cell immunity which, as such, stores cellular memory with environmental factors. T-cell fragments from individual cell populations are preferred over homogenized tissue because of the drastic variation in DNA methylation patterns between different cell fragments.


Although these results of decreased methylation and hyperactivation of GR conflict with the effect of early life stress at the same loci, these results match previous findings that distinguish HPA activity in early life stress versus PTSD. For example, cortisol levels of HPA in response to early life stress is hyperactive, whereas it is hypoactive in PTSD. Thus, the timing of trauma and stress—whether early or later in life—can cause differing effects on HPA and GR. [ 55 ]


Fkbp5  encodes a GR-responsive protein known as Fk506 binding protein 51 (FKBP5). FKBP5 is induced by GR activation and functions in negative feedback by binding GR and reducing GR signaling. [ 47 ] [ 63 ] [ 64 ] [ 65 ]  There is particular interest in this gene because some  FKBP5  alleles have been correlated with increased risk of PTSD and development of PTSD symptoms—especially in PTSD caused by early life adversity. Therefore,  FKBP5  likely plays an important role in PTSD. [ 47 ] [ 66 ] [ 67 ] [ 68 ]


As mentioned previously, certain  FKBP5  alleles are correlated to increase PTSD risk, especially due to early life trauma. It is now known that epigenetic regulation of these alleles is also an important factor. For example, CpG sites in intron 7 of FKBP5 are demethylated after exposure to childhood trauma, but not adult trauma. [ 47 ] [ 69 ]  Additionally, methylation of FKBP5 is alters in response to PTSD treatment; thus methylation levels of FKBP5 might correspond to PTSD disease progression and recovery. [ 47 ] [ 28 ]


Pituitary adenylate cyclase-activating polypeptide (ADCYAP1) and its receptor (ADCYAP1R1) are stress responsive genes that play a role in modulating stress, among many other functions. Additionally, high levels of ADCYAP1 in peripheral blood is correlated to PTSD diagnosis in females who have experienced trauma, thus making ADCYAP1 a gene of interest in the context of PTSD. [ 47 ]


Epigenetic regulation of these loci in relation to PTSD still require further investigation, but one study has found that high methylation levels of CpG islands in ADCYAP1R1 can predict PTSD symptoms in both males and females. [ 47 ] [ 51 ]


Alcohol use disorder is a type of brain disorder that requires one to have dependency on alcohol. Alcohol use disorder can vary in severity. Alcohol dependence can impact stress and other disorders in many ways. [ 70 ]  For example, stress-related disorders such as anxiety and PTSD are known to increase risk of  alcohol use disorder  (AUD), and they are often  co-morbid . Mental disorders that pair with AUD can impacts the brain in many ways. For example, AUD can have the ability to aid those diagnosed with depression by alleviating depression symptoms such as insomnia, restlessness, and/ or the ability to re-engage in normal activities, and re-engage in hobbies. Bipolar disorder can cause manic episodes ranging fro different sudden mood changes. AUD can also be used to stall the same symptoms expressed in depression as well as with bipolar disorder. In some cases AUD can cause other brain disorders to worsen itself, or the symptoms of the disorder. An example of this can be seen in some with Obessive-Compulsive Disorder (can typically include anxiety “triggers” that often cause an individual to have very specific compulsions or obsessions). With this type of disorder, although it can help in ways by relieving symptomatic stress, it can also aid in promoting addiction to alcohol which can be a negative impact if uncontrolled. [ 71 ] [ 72 ]  This may in part be due to the fact that alcohol can alleviate some symptoms of these disorders, thus promoting dependence on alcohol. Conversely, early exposure to alcohol can increase vulnerability to stress and stress-related disorders. AUD is a type of epigenetic influencing disorder, it is able to be passed down generation to generation epigenetically following a process mentioned before as transgenerational epigenetics. [ 20 ] [ 73 ]  Moreover, alcohol dependence and stress are known to follow similar neuronal pathways, and these pathways are often unable to be regulated by similar epigenetic modifications.


Histone acetylation is dysregulated by alcohol exposure and dependence, often through dysregulated expression and activity of HDACs, which modulate histone acetylation by removing acetyl groups from lysines of histone tails. For example, HDAC expression is upregulated in chronic alcohol use models. [ 74 ]  Monocyte-derived dendritic cells of alcohol users have increased HDAC gene expression compared to non-users. [ 75 ]  These results are also supported by  in vivo  rat studies, which show that HDAC expression is higher in alcohol-dependent mice that in non-dependent mice. Furthermore, knockout of HDAC2 in mice helps lower alcohol dependence behaviors. [ 76 ]  The same pattern of HDAC expression is seen in alcohol withdrawal, but acute alcohol exposure has the opposite effect;  in vivo , HDAC expression and histone acetylation markers are decreased in the amygdala. [ 74 ] [ 76 ]


Dysregulation of HDACs is significant because it can cause upregulation or downregulation of genes that have important downstream effects both in alcohol dependence and anxiety-like behaviors, and the interaction between the two. A key example is BDNF (see "BDNF" below).


Brain-derived neurotrophic factor  (BDNF) is a key protein that is dysregulated by HDAC dysregulation. BDNF is a protein that regulates the structure and function of neuronal synapses. It plays an important role in neuronal activation, synaptic plasticity, and dendritic morphology—all of which are factors that may affect cognitive function. Dysregulation of BDNF is seen both in stress-related disorders and alcoholism; thus BDNF is likely an important molecule in the interaction between stress and alcoholism. [ 74 ] [ 77 ]


For example, BDNF is dysregulated by acute ethanol exposure. Acute ethanol exposure causes phosphorylation of CREB, which can cause increased histone acetylation at BDNF loci. Histone acetylation upregulates BDNF, in turn upregulating a downstream BDNF target called activity-regulated cytoskeleton associated protein (Arc), which is a protein responsible for dendritic spine structure and formation. This is significant because activation of Arc can be associated with anxiolytic (anxiety-reducing) effects. Therefore, ethanol consumption can cause epigenetic changes that alleviate stress and anxiety, thereby creating a pattern of stress-induced alcohol dependence. [ 74 ] [ 77 ] [ 78 ]


Alcohol dependence is exacerbated by ethanol withdrawal. This is because ethanol withdrawal has the opposite effect of ethanol exposure; it causes lowered CREB phosphorylation, lowered acetylation, downregulation of BDNF, and increase in anxiety. Consequently, ethanol withdrawal reinforces desire for anxiolytic effects of ethanol exposure. Moreover, it is proposed that chronic ethanol exposure results in upregulation of HDAC activity, causing anxiety-like effects that can no longer be alleviated by acute ethanol exposure. [ 74 ] [ 77 ] [ 76 ]


The most common treatments for anxiety disorders at the moment are  benzodiazepines ,  Buspirone , and  antidepressants . However, around one/third of patients with anxiety disorders do not respond well to the current anxiolytics, and many others have treatment-resistant anxiety disorders. [ 79 ] [ 80 ]  Recent research surrounding DNA methylation changes in genes in genes encoding proteins associated with the HPA axis, histone modifications, and sncRNAs point to epigenetic drugs potentially being effective treatment methods for anxiety disorders. [ 81 ]


Histone deacetylase inhibitors  (HDACi) fall into five different classes, not to be confused with the four different classes of HDACs. The five classes of HDACi consist of (I)  hydroxamic acids , (II)  short-chain fatty acids , (III)  benzamides , (IV)  cyclic   tetrapeptides , and (V)  sirtuin  inhibitors. The three classes of HDACs are class I, consisting of  HDAC1 ,  HDAC2 ,  HDAC3 ,  HDAC8 , class II, consisting of  HDAC4 ,  HDAC5 ,  HDAC6 ,  HDAC7 ,  HDAC9 ,  HDAC10 , class III, consisting of  NAD+ -dependent HDACS, and class IV, consisting of  HDAC11 . While most HDACi inhibit only specific classes of HDACs, certain HDACi can act against all classes, making them pan-inhibitors. [ 82 ] [ 83 ]


HDACi are currently being researched as potential anxiolytics. At the moment, the mechanism of action of HDAC inhibitors in the treatment of anxiety disorders is not clear, as they affect several targets and have multiple pharmacological effects besides the inhibition of HDACs. However, they have been shown to cause DNA demethylation, possibly due to an increase in the levels of  TET1 , which is a demethylating enzyme. [ 84 ]  In the human peripheral cells of patients with anxiety disorders and in animal models of anxiety disorders, genes such as  GAD1 , NR3C1, BDNF,  MAOA , HECA, and FKBP5 are shown to be hypermethylated. As such, the mechanism of action of HDACi in anxiety disorders could, in part, be potentially explained by the demethylation of those genes.


Valproate  is a drug that acts as an HDACi on class I and II HDACs. Six clinical trials surrounding its use as an anxiolytic have been performed so far. Five of the six trials were performed on patients with anxiety disorders, and one of the trials used healthy subjects with no anxiety disorders. Of the five trials performed on patients with anxiety disorders, three found that Valproate decreases panic disorder, one found that Valproate decreases social anxiety, and one found that Valproate reduces generalized anxiety. [ 85 ] [ 86 ] [ 87 ] [ 88 ] [ 89 ]  The trial performed on healthy subjects found that Valproate reduces anxiety and also acts as a nerve conduction inhibitor, which could be an explanation for some of its anxiety-reducing effects. [ 90 ]


Various preclinical drug trials using other HDAC inhibitors have also been performed, with most drugs targeting HDAC classes I and II and a select few targeting classes IV and III. The HDACi drug,  d-cycloserine , was found to reduce fear in 129S1/SvImJ mice, which are mice that show poor extinction acquisition and recovery of fear-induced suppression of heart-rate variability, enlarged  dendritic arbors  in  basolateral amygdala  neurons, and functional abnormalities in cortico-amygdala circuitry that mediates fear extinction. [ 91 ]   Trichostatin-a  was normalized BDNF and Arc expression in the central and the medial nucleus of the  amygdala  in rats experiencing alcohol withdrawal. [ 92 ]   Suberoylanilide hydroxamic acid  significantly reversed anxiety-like behaviors and stress-induced gastrointestinal hypersensitivity and fecal pellet output. [ 93 ]  Anxiety-like and depression-like behaviors caused by  immobilization stress  or nicotine addiction were also reduced in mice treated with the HDACi  sodium butyrate  and  valproic acid . [ 94 ]


Lactate, a  metabolite  that is naturally produced during exercise, was found to function as an HDAC II and III modulator in a pre-clinical trial. The trial was performed on C57BL/6 mice, which are mice that were exposed to chronic stress in the form of daily defeat by a CD-1 aggressive mouse. While control mice exhibited increased social avoidance, anxiety, and susceptibility to depression, mice that received lactate before each defeat demonstrated resilience to depression and stress and reduced social avoidance and anxiety. Lactate promoted this resilience by restoring regular hippocampal class I HDAC levels and activity. [ 95 ]


Preliminary research has been done about therapy involving small non-coding RNAs, demonstrating that they can regulate epigenetic mechanisms of gene expression and could present as  biomarkers  for disease. One therapy option is for the sncRNAs in patients with anxiety disorders to be targeted for  upregulation . Another option is to inhibit the miRNAs in order to reduce their effects, potentially using  antisense oligonucleotides  or  antagomirs  as inhibitors. [ 81 ]


The medication hydrocortisone is a synthetic form of cortisol, and is typically an anti inflammatory. [ 96 ]  In recent years, the administration of hydrocortisone has been tested as a possible preventative measure for the onset of PTSD symptoms. Ideally, it should be administered immediately after a traumatic event. The efficacy of hydrocortisone as a preventative intervention for PTSD has been confirmed by a meta-analysis of eight separate studies, and researchers believe the best results are obtained when hydrocortisone is administered within the first six hours of exposure to the traumatic event. At this time, however, no curative properties have been discovered. [ 97 ]  Hydrocortisone's potential operates on two bases: restoration of normal HPA axis functioning and interference with memory consolidation.


Our standard understandings of PTSD may suggest elevated glucocorticoid levels during and directly following events of trauma. However, multiple studies have indicated that overall HPA axis activity and cortisol levels are depleted in the critical aftermath and extended period after trauma. [ 98 ]  Moreover, research has also indicated that an appropriate release of glucocorticoids following acute stress may restore homeostatic equilibrium of the HPA axis, thereby preventing gradual sensitization, which is responsible for persistent cortisol reduction and increased PTSD susceptibility. Thus, the appropriately-dosed administration of hydrocortisone promptly following the traumatic incident would normalize the HPA axis and potentially prevent PTSD onset. [ 97 ]


In the absence of memory reactivation, hydrocortisone's effectiveness within a six-hour window supports the consolidation theory, which asserts that memory is labile even immediately after trauma. It is assumed that the medication is disrupting initial memory consolidation of the traumatic event. However, its exact mechanism within this context remains largely unknown.


Although trials have proven promising, there is much more research to be done. Further comprehensive studies are required amidst more diverse populations under different traumatic conditions in order to ascertain factors of optimal usage of the drug and clarify the PTSD subgroups hydrocortisone is beneficial to.


The observation of epigenetic modifications and their role in regulating fear learning is an active area of research. The use of stress-enhanced fear learning (SEFL) paradigms are important for forming preclinical models of PTSD because one is able to observe the epigenetic changes in rodents and PTSD associated changes in fear learning after stress exposure. [ 61 ]


The single-prolonged stress (SPS) model is a tool in which a complex stressor is consistently presented. This tool is used to explore the complexity of PTSD, particularly its impaired fear extinction. [ 61 ]


Despite high levels of individuals exposed to trauma, only about one third of exposed individuals develop PTSD. This suggest that individuals differ in their susceptibility to PTSD. This might arise from differences in the epigenetic modifications that they generate in response to traumatic experiences. Furthermore, a large area of research regarding increased susceptibility to PTSD investigates the transgenerational inheritance of epigenetic modifications resulting from trauma. [ 99 ]  A recent review of PTSD susceptibility suggested that a range as wide as 30% to 70% of susceptibility to PTSD can be attributed to heritability. [ 100 ]  From our observations of mice in transgenerational research, we have seen that the epigenetic modifications that stem from trauma can be passed down multiple generations. [ 99 ]  Epigenetic modifications due trauma are not the only heritable factors that affect PTSD susceptibility. General histories of health deficiencies, both physical and psychological, have also been associated with a higher PTSD susceptibility. Sociodemographic factors may come into play as well. Particularly, ethnic minorities and women are more susceptible to development of PTSD. [ 101 ]


Autism spectrum  disorder (ASD) refers to a variety of conditions typically identified by challenges with social skills, communication, speech, and repetitive sensory-motor behaviors. The 11th  International Classification of Diseases  ( ICD-11 ), released in January 2021, characterizes ASD by the associated deficits in the ability to initiate and sustain two-way social communication and restricted or repetitive behavior unusual for the individual's age or situation. [ 1 ]  Although linked with early childhood, the symptoms can appear later as well. Symptoms can be detected before the age of two and experienced practitioners can give a reliable diagnosis by that age. However, official diagnosis may not occur until much older, even well into adulthood. There is a large degree of variation  in  how much support a person with ASD needs in day-to-day life. This can be classified by a further diagnosis of ASD level 1, level 2, or level 3. Of these, ASD level 3 describes people requiring very substantial support and who experience more severe symptoms. [ 2 ]  ASD-related deficits in nonverbal and verbal social skills can result in impediments in personal, family, social, educational, and occupational situations. This disorder tends to have a strong correlation with genetics along with other factors. More research is identifying ways in which epigenetics is linked to autism.  Epigenetics  generally refers to the ways in which chromatin structure is altered to affect gene expression. Mechanisms such as cytosine regulation and post-translational modifications of histones. Of the 215 genes contributing, to some extent in ASD, 42 have been found to be involved in epigenetic modification of gene expression. [ 3 ]  Some examples of ASD signs are specific or repeated behaviors, enhanced sensitivity to materials, being upset by changes in routine, appearing to show reduced interest in others, avoiding  eye contact  and limitations in social situations,  as well as  verbal communication. When social interaction becomes more important, some whose condition might have been overlooked suffer social and other exclusion and are more likely to have coexisting mental and physical conditions. [ 4 ]  Long-term problems include difficulties in daily living such as managing schedules, hypersensitivities ( e.g.,  to foods, noises, fabric textures, light), initiating and sustaining relationships, and maintaining jobs. [ 5 ] [ 6 ]


Diagnosis is based on observation of behavior and development. Many, especially girls and those who have fewer social difficulties, may have been misdiagnosed with other conditions. Males are diagnosed with ASD four to five times more often than females. [ 6 ] [ 7 ]  The reasons for this remain predominantly unclear, but current hypotheses include a higher testosterone level in utero, different presentations of symptoms in females (leading to misdiagnosis or underdiagnosis) compared to males, and gender bias. [ 8 ]  Clinical assessment of children can involve a variety of individuals, including the caregiver(s), the child, and a core team of professionals ( pediatricians ,  child psychiatrists ,  speech-and-language therapists  and  clinical / educational psychologists ). [ 9 ] [ 10 ]  For adult diagnosis, clinicians identify neurodevelopmental history, behaviors, difficulties in communication, limited interests and problems in education, employment, and social relationships. Challenging behaviors may be assessed with  functional analysis  to identify the triggers causing them. [ 11 ]  The sex and gender disparity in ASD diagnostics requires further research in terms of adding diagnosis specifiers as well as female-oriented examples, which may be masked through camouflaging behaviors. Camouflaging is defined as a coping mechanism used in social situations, consisting of individuals pretending to be other people without any communication difficulties. [ 12 ]  Because of camouflaging and other societal factors, females with ASD are more likely to be diagnosed late or with a different mental health concern. In general, it is critical for people to understand that the female ASD phenotype is less noticeable, especially when they present as "higher functioning" than others with ASD. Lastly, due to the imbalance in sexes participating in ASD studies, the  literature is potentially biased towards the ways that it presents in male individuals. [ 13 ]


ASD is considered a lifelong condition and has no "cure." Many professionals, advocates, and people in the autistic community agree that a cure is not the answer and efforts should instead focus on methods to help people with ASD have happier, healthier, and, if possible, independent lives. [ 14 ]  Support efforts include teaching social and behavioral skills, monitoring, factoring-in co-existing conditions, and guidance for the caregivers, family, educators, and employers. There is no specific medication for ASD, however, drugs can be prescribed for other co-existing mental health conditions, such as anxiety. A study in 2019 found that the management of challenging behaviors was generally of low quality, with little support for long-term usage of psychotropic drugs, and concerns about their inappropriate prescription. [ 15 ] [ 16 ]   Genetic  research has improved the understanding of ASD-related  molecular pathways .  Animal research  has pointed to the reversibility of  phenotypes  but the studies are at an early stage. [ 17 ]


One of the leading theories of a potential pathogenic process in ASD is  cortical  hyperexcitability. Maintaining proper levels of cortical excitability is essential for many important cognitive functions, such as processing sensory information, [ 18 ]  communication between different regions of the brain, and  neural plasticity . [ 19 ]  Hyperexcitability can disrupt these functions and thereby alter cognitive dynamism in important ways. [ 18 ] [ 19 ]  For example,  cortical hyperexcitability can affect how the duration of sensory stimuli are perceived. [ 19 ]  A common trait of ASD is reduced somatosensory functioning, which has been linked to alterations in cortical hyperexcitability in ASD individuals. [ 20 ]  Cortical hyperexcitability can also alter how "old" and "new" stimuli are perceived by changing  habituation  and  adaptation  processes in the brain. [ 21 ]  Altered habituation processes have been linked to characteristic traits of ASD, such as under-responsiveness to some stimuli and over-responsiveness to others. [ 22 ]


There are many genetic and epigenetic factors that can contribute to increased excitability, but one of the mechanisms implicated in ASD is alterations in  GABAergic  systems in the cortex. [ 19 ] [ 6 ]   GABA  is the main  neurotransmitter  implicated in inhibition in the cortex of mammalian brains; [ 23 ]  changes to this cortical inhibitory system can result in increased excitability. [ 19 ] [ 6 ]  Alterations in this system have been associated not only with ASD but also with several other psychiatric disorders, such as  major depressive disorder (MDD)  and  schizophrenia . [ 21 ]


Alterations in the GABAergic system can occur through several epigenetic mechanisms, including modification of  chromosome 15q11 to q13  regions which cause reduced levels of GABA signaling. [ 19 ] [ 21 ] [ 24 ]  Cortical excitability can also be increased by modifications in the  glutamatergic  system. [ 6 ] [ 19 ]


Chromosome 15q11-13 contain genes encoding subunits of  GABA receptors , and both deletion and duplication of this region can lead to cortical hyperexcitability. [ 6 ]   Duplications  of 15q11-13 are associated with about 5% of patients with ASD [ 25 ]  and about 1% of patients diagnosed with classical Autism. [ 26 ]  15q11-13 in humans contains a cluster of genetically  imprinted genes  important for normal  neurodevelopment . [ 24 ] [ 27 ]  Like other genetically imprinted genes, the parent of origin determines the  phenotypes  associated with 15q11-13 duplications. [ 27 ]  "Parent of origin effects" cause gene expression to occur only from one of the two copies of  alleles  that individuals receive from their parents. (For example,  MKRN3  shows a parent of origin effect and is paternally imprinted. This means that only the MKRN3 allele received from the paternal side will be expressed.) Duplications in the maternal copy lead to a distinct condition that often includes autism. [ 28 ]


Genes that are deficient in paternal or maternal 15q11-13 alleles result in  Prader-Willi  or  Angelman syndromes , respectively, both of which are linked to high incidence of ASD. [ 6 ] [ 28 ]   Overexpression  of maternally imprinted genes is predicted to cause autism, which focuses attention to the maternally expressed genes on 15q11-13, although it is still possible that alterations in the expression of both imprinted and bilallelically expressed genes contribute to these disorders. [ 28 ]  The commonly duplicated region of chromosome 15 also includes paternally imprinted genes that can be considered candidates for ASD.


Members of the GABA receptor family, especially GABRB3, are attractive candidate genes for Autism because of their function in the nervous system.  GABRB3  null mice exhibit behaviors consistent with autism [ 29 ]  and multiple genetic studies have found significant evidence for association. [ 30 ]  Furthermore, a significant decrease in abundance of GABRB3 has been reported in the brains of patients with autism and Rett syndrome. [ 31 ]  Other GABA receptors residing on different chromosomes have also been associated with autism (e.g. GABRA4 and GABRB1 on chromosome 4p). [ 32 ]


Regulation of gene expression in the 15q11-13 is rather complex and involves a variety of mechanisms such as  DNA methylation , non-coding and  anti-sense RNA . [ 33 ]


The imprinted genes of 15q11-13 are under the control of a common regulatory sequence, the  imprinting control region  (ICR). The ICR is a differentially methylated CpG island at the 5' end of  SNRPN . It is heavily methylated on the silent maternal allele and unmethylated on the active paternal allele. [ 34 ]


MeCP2 , which is a candidate gene for  Rett syndrome , has been shown to affect regulation of expression in 15q11-13. Altered (decreased) expression of UBE3A and GABRB3 is observed in MeCP2 deficient mice and ASD patients. This effect seems to happen without MeCP2 directly binding to the promoters of UBE3A and GABRB3. (Mechanism unknown) [ 31 ]  However,  chromatin immunoprecipitation  and  bisulfite sequencing  have demonstrated that MeCP2 binds to methylated CpG sites within GABRB3 and the  promoter  of SNRPN/SNURF. [ 26 ]


Furthermore, homologous 15q11-13 pairing in neurons that is disrupted in RTT and autism patients, has been shown to depend on MeCP2. [ 35 ]  Combined, these data suggest a role for MeCP2 in the regulation of imprinted and biallelic genes in 15q11-13. However, evidently, it does not play a role in the maintenance of imprinting. [ 26 ]


One current theory of the pathophysiology of ASD is that it arises from a deficit in the folate-methionine pathway. [ 36 ]  Folate donates methyl groups to convert homocysteine into methionine, which is the precursor of S-adenosylmethionine. S-adenosylmethionine is the methyl group donor responsible for DNA and histone methylation. [ 37 ]  Epigenetic changes can result in changed gene expression of pathway enzymes resulting in a change in folate levels which can contribute to ASD. These changes to the epigenetic regulation interact with the pregnant woman's immune system activation and can result in an ASD phenotype in the fetus' brain. [ 38 ]  To add, low levels of folate in the pregnant woman are correlated with DNA hypomethylation in the fetus.


The gene MTHFR codes for the enzyme  methylenetetrahydrofolate reductase  which is necessary for the synthesis of 5-methyl-tetrahydrofolate, a biologically active form of folate [ 36 ] [ 37 ]  One important risk factor that has been identified for ASD is polymorphism in MTHFR. A meta-analysis demonstrated that polymorphism of the MTHFR C677T genotype is correlated with an ASD diagnosis in children from countries lacking  food fortification . [ 39 ]


While MTHFR is a proposed genetic factor for ASD, there is limited clinical evidence from testing for MTHFR gene polymorphisms in the diagnostic setting. [ 40 ]  The reason for these complications may be due to other modifiers of the folate metabolism pathway or other genes included in the pathway. Additionally, the levels of  homocysteine  (HCy) seem to result in an increased utility of the folate metabolism pathway as a predictor for ASD diagnosis. [ 41 ]


If the fetus is exposed to the mood stabilizer drug  valproate  (VPA), the risk of ASD as well as other developmental abnormalities (decreased intrauterine growth,  spina bifida , limb defects, craniofacial defects, etc.) is increased. [ 42 ]  VPA is an anticonvulsant drug commonly administered for generalized and partial seizures, but also for the treatment of migraines and bipolar mood disorder. Its mechanisms of action are varied, including enhanced GABA neurotransmission, modified  inositol  metabolism, and interaction with the ERK and Wnt/B-catenin signaling systems. [ 43 ]  If taken while pregnant, the risk of ASD is 8.9% to 10.8%. When VPA and another antiepileptic drug are taken, the risk increases to 11.7%. [ 44 ] [ 45 ]  Compared to the general population, this risk of ASD is 16 times higher. [ 46 ]


Currently, there are two proposed epigenetic mechanisms for VPA increasing the risk in ASD: alteration in folate metabolism and HDAC inhibition. VPA is a weak HDAC inhibitor. The VPA model discerns the potential pathogenesis and mechanisms of action of ASD in animal models. HDAC inhibition is the most understood. In animal models, mice prenatally exposed to VPA had transient hyperacetylation of histones H3 and H4, decreased HDACs, and developed ASD-like symptoms. [ 47 ]  However, mice prenatally exposed to  valpromide , analogous to VPA but not an HDAC inhibitor, did not experience transient hyperacetylation of histones H3 and H4 and did not develop ASD-like symptoms. [ 48 ]  An important thing to note is the time of VPA. In the animal models, the significant effects of VPA in causing ASD-like symptoms was demonstrated mainly in rats exposed to VPA on gestation day 12.5, not in other gestation days like day 9, 14.5, etc. [ 47 ] [ 48 ]  The ASD-like symptoms of mice included decreased distressed pup calls, decreased social exploration, decreased social behaviors, increased stereotypic  locomotion , decreased acoustic  prepulse inhibition , and increased sensitivity to non-painful stimuli. [ 48 ]


This same association was replicated in the longitudinal studies. Children prenatally exposed to VPA or with fetal valproate syndrome (FVS) have a higher prevalence of ASD. FVS is a rare condition in children that happens due to VPA exposure during the first trimester of pregnancy. [ 48 ]


Romidepsin  and MS-275, both HDAC inhibitors, improve social preference, which is the preference of social stimuli over non social stimuli,  and interaction times of SHANK 3 deficient mice.  Trichostatin A  (TSA) is another example of an HDAC inhibitor. It results in increased histone acetylation at the oxytocin and vasopressin receptors of the nucleus accumbens (NA) in female voles, increasing pair bonding. In a small clinical trial, beta hydroxybutyrate, a product of the  ketogenic diet  and inhibitor of class 1 HDACs, has shown promise in improving the social behavior and skills in children with ASD. [ 47 ] 'The inhibition of HDAC is correlated with overexpression of other genes. [ 49 ]  Treatment of mice with valproate also increases hippocampal histone H3 acetylation. [ 50 ]


Current candidate genes relating to ASD in mice exposed to valproate  in utero  are NRXN1, NRXN2, NRXN3, NLGN1, NLGN2, and NLGN3. In the somatosensory cortex, CA1, dentate gyrus, and hippocampus, NLGN3 is significantly downregulated in mice treated with valproate. [ 51 ]  While this evidence of NLGN3 downregulation due to valproate suggests a potential relevant mechanism for ASD, further research is needed.


SHANK proteins are scaffolding proteins at glutamatergic synapses crucial for synaptic development. The disruption of SHANK genes is associated with neurocognitive impairments and disorders. The disruptions, either from mutations or deletions, are associated with disorders such as  Phelan-McDermid syndrome  (PMS), schizophrenia, and ASD. SHANK 3 is the most studied gene from the SHANK gene family. Several studies have found that disruptions to SHANK 3 cause more severe cognitive impairments than disruptions to SHANK 1 or 2. These findings suggest that the SHANK gene that is disrupted may determine the severity of the cognitive impairments. [ 52 ]


A study on two mutant mice lines, one line with an ASD-linked SHANK 3 mutation on exon 21 and the other with a schizophrenia-linked SHANK 3 mutation on exon 21, found differences in the synaptic and behavioral impairments caused by disruptions to SHANK 3. The ASD-linked mutation results in a complete loss of SHANK 3 (like a deletion) and impaired striatal synaptic transmission. The schizophrenia-linked mutation results in a truncated SHANK 3 protein and severe synaptic impairments in the prefrontal cortex. [ 52 ]


Other studies suggest that SHANK3 knockout mice display behavioral phenotypes of ASD. These mice display self-injurious grooming, anxiety, and social deficits. Restoration of SHANK 3 in adult mice improved social deficits and self-grooming behaviors. These findings indicate the potential therapeutic effect of restoring SHANK 3. SHANK 3 restoration may alleviate some symptoms of ASD. In addition, modulators and proteins associated with SHANK 3 are potential therapeutic targets for ASD. However, the effects of targeting modulators differ depending on the specific SHANK 3 disruption. For instance, studies have shown that increasing mGluR5 activity improved self grooming and behavioral deficits. Yet, other studies have shown the opposite effect. This demonstrates that the therapeutic effects are dependent on specific SHANK 3 mutation. [ 52 ]