**3. Neuro-biological perspective**

### **3.1 Focus on genomics and Epigenomics in post-traumatic stress disorder**

Post-traumatic stress disorder (PTSD) is a multifactorial disease characterized by structural, metabolic, and molecular changes in various brain regions and neural circuits, such as the limbic system, hippocampal region, and prefrontal cortex (in **Figure 2**), which regulate neurobehavioral functions [10]. Epigenetic and genetic current studies are included in this section. PTSD can occur at the organic, cellular, and molecular level due to the effect of an external event such as psychological trauma, as

#### **Figure 2.**

*Current candidate genes associated with different brain regions and neuro-behaviors in PTSD [10]. Abbreviations: post-traumatic stress disorder (PTSD), catechol-O-methyltransferase (COMT), Cordon-Bleu WH2 Repeat Protein (COBL), Solute Carrier Family 6 Member 4 (SLC6A4), pituitary adenylate cyclaseactivating polypeptide 1 receptor (ADCYP1R1), Opioid-Related Nociceptin Receptor 1 (OPRL1), FK506 binding proteins (FKBPS), Spindle And Kinetochore Associated Complex Subunit 2 (SKA2), Brain-Derived Neurotrophic Factor (BDNF), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1).*

well as being inherited from generation to generation. In PTSD, genetic and epigenetic studies are prioritized based on biological research because they are promising in elucidating molecular functioning and finding biomarkers. The goal of these studies is to lay the groundwork for new and preventive treatments to ameliorate the symptoms that cause the disease. In this context, there is the current evidence for the potential of current genetic and epigenetic studies from the biological risk factors of PTSD.

Thousands or even hundreds of thousands of single nucleotide polymorphisms (SNPs) with a polygenic background are the genetic basis of PTSD according to Genome-wide association study (GWAS) summary statistic [11]. Considering the studies on twins with a traumatic history for hereditary dimensions, PTSD is inherited from 30% males and 70% females, SNPs play a major role in this hereditary process from women [12]. The heritability of PTSD following trauma has been demonstrated, but biological variations have not yet been fully defined. Elucidating the biological mechanisms underlying PTSD may contribute to a more accurate diagnosis and development of swelling-specific treatment interventions. Among the biological processes involved in PTSD and related conditions, this section focuses on epigenetic and genetic mechanisms. Genomic and epigenomic studies in large groups are valuable. Loci most recently examined in large-scale GWAS and Epigenome-wide association studies (EWAS) became candidate biological markers for PTSD.

#### *3.1.1 Current genome-wide association studies*

Specific PTSD genetic variants that contribute to genetic studies have been most extensively researched and are currently known in the monoaminergic neurotransmission and hypothalamic-pituitary-adrenal (HPA) axis [13, 14]. The most frequently studied serotonin transporter gene (SLC6A4) polymorphisms in the

monoaminergic system were associated with PTSD and identified with a prevalence of 45% in Europeans is the S allele frequency of SLC6A4. Association studies of *5*-hydroxytryptamine *5*-HT (5HTTLPR) and PTSD have been inconclusive, and a recent meta-analysis of 12 studies found no evidence of association overall, but the S allele was associated with PTSD in samples classified as having high trauma exposure [15]. Nominally significant associations between PTSD symptoms and many neurotransmitter-related genes, including 5-hydroxytryptamine (serotonin) 2A receptor gene (HRT2A), Solute Carrier Family 6 Member 3 (SLC6A3), Dopamine Receptor D3 (DRD3), Neuropeptide Y (NPY) Cannabinoid Receptor 1 (CNR1), and Regulator of G Protein Signaling 2 (RGS2) have been investigated [16–18].

One of the largest polymorphism studies of the Nurse's Health Study II, which included 845 PTSD cases and 1693 trauma-exposed controls, examined 3742 single nucleotide polymorphisms (SNPs) spanning more than 300 genes, but no gene was of clinical significance [19]. Meta-analysis and GWAS studies take an agnostic approach to the discovery of risk loci by comparing the frequencies of hundreds of thousands of SNPs and other genetic markers from the whole genome with those of controls, at least an update five gene markers are promising, including Zinc Finger DHHC-Type Palmitoyltransferase 14 (ZDHHC14), Parkinson Protein 2 E3 Ubiquitin Protein Ligase (PARK2), Kazrin, Periplakin Interacting Protein (KAZN), TMEM51 antisense RNA 1 (TMEM51-AS1), and Zinc Finger Protein 813 (ZNF813) [20–22]. The latest Military cohort study (29,539 PTSD cases and 166,145 controls) reported that Zinc Finger Protein 140 (ZNF140) is upregulated in blood, and Small Nuclear Ribonucleoprotein U11/U12 Subunit 35 (SNRNP35) is downregulated in the dorsolateral prefrontal cortex in Military PTSD [23]. Duncan et al. investigated strong evidence of overlapping SNPs and multi-loci risk between PTSD and schizophrenia (from 20,730 individuals) via 11 genome-wide case–control molecular genetic studies [24]. Chen et al. found two loci including chr10\_6953246\_D and rs2311207 that were associated with the severity of PTSD symptoms [25]. Other genome-wide significant loci were Ankyrin Repeat Domain 55 (ANKRD55) (rs1595))2 and Zinc Finger Protein 626 (ZNF626) on chromosome 19, moreover, the ANKRD55 gene was also related to rheumatoid arthritis and psoriasis that are additionally seen in patients with PTSD [26]. Maihofer et al. also found loci on four genes: Gamma-Aminobutyric Acid Type B Receptor Subunit 1 (GABBR1), Forkhead box protein P2 (FOXP2), Family with Sequence Similarity 120A (FAM120A), and ADP Ribosylation Factor Guanine Nucleotide Exchange Factor 2 (ARFGEF2) which had genome-wide significant (p < 5 × 10–8) from African American ancestry and the external Million Veteran's PTSD [27]. Pooler also discovered two SNPs; rs13160949 on chromosome 5 (p = 7.33 × 10–9) and SNP rs2283877 on chromosome 22 (p = 2.55 × 10–8) which have been firstly investigated in PTSD [28]. SNP rs267943 is located on chromosome 5 in the intron of the death-associated protein 1 (DAP1) gene had the strongest association from 396 chronic PTSD patients (Thai Tsunami survivors) and 457 controls [29]. Large-scale genome studies have identified heterogeneous and numerous SNPs and genes at multiple loci. Successful polygenic prediction models can be discovered in the future by increasing the number of current and large-scale studies. Current candidate genes associated with different brain regions and neuro-behaviors in PTSD are given in **Figure 2**.

#### **3.2 Current epigenome-wide association studies**

To better observe the Gene-Trauma Correlations in PTSD, epigenetic studies are also important to investigate the effects of environmental factors. Epigenome-wide

association studies (EWAS) have identified epigenetic mechanisms for PTSD due to alteration of gene expression modifications without changing the genetic code. Epigenetic studies are carried out due to traumatic memory in the hippocampal region, frontal cortex associations, and extreme fear in the limbic system. An important regulation of gene function and phenotypic expression occurring in the understanding of PTSD occurs at the level of epigenetic regulation. Epigenetic changes include DNA methylation, histone modifications, and non-coding RNAs.

Animal research generally suggests that stress-induced epigenetic modification following environmental stress may affect stress-response functions as mediated by gene expression, HPA axis. Epigenetic factors, such as DNA methylation, have been shown to modulate the influence of the environment on gene expression [30]. McNerney et al. showed that the hippocampal volume/glucocorticoid receptor (GR) gene methylation interaction is an indicator of PTSD symptoms in 67 Veteran Patients [31]. Although animal and small sample epigenetic studies give clues about multiple genes and analysis, a major challenge for these studies is controlling the wide variety of stress factors that subjects are exposed to throughout their lives, and also they must be significant in EWAS measures. Hjort et al. reported that offspring of 72% of 117 mothers with PTSD had higher cortisol levels and differential methylation in candidate genes [NR3C1, 5-Hydroxytryptamine Receptor 3A (HTR3A), and BNDF] but the level of methylation differences did not reach epigenome-wide corrected significance levels [32]. Recent Epigenome-wide meta-analysis of military and civilian PTSD reported low DNA methylation in the four CpG regions of the Aryl-hydrocarbon repressor (AHRR) from blood DNA samples of 1896 PTSD patients [33]. Epigenetic meta-analysis of civilian PTSD (545 study participants) also found differential methylations in two CpG sites including NRG1 (cg23637605) and HGS (cg19577098) [34]. Interestingly, Yang et al. conducted two new different epigenetic biotypes for PTSD (G1 and G2). The G2 biotype has been associated with an increased risk of PTSD. The G1 biotype had higher polygenic risk scores and higher DNA methylation [35]. Logue et al. reported an epigenome-wide significant association with cg19534438 in the gene G0S2 (G0/G1 switch 2) and replicated it in other military cohorts. Although cg04130728 in Carbohydrate Sulfotransferase 11 (CHST11) had no genome-wide association, was significantly associated with PTSD in brain tissue (mostly prefrontal cortex) [36]. A longitudinal epigenome-wide association study identified three epigenome-wide significant CpGs, the intergenic CpG cg05656210 and Mitotic Arrest Deficient 1 Like 1 (MAD1L1) (cg12169700) and HEXDC (cg20756026).

Interestingly, cg12169700 was located within the same linkage disequilibrium block as a recently identified PTSD-associated (rs11761270) SNP in MAD1L1 [37]. In a meta-analytical review by Wolf et al., sex and immunity were strongly associated with the age of DNA methylation. However, they noted the lack of research into the underlying biological mechanisms [38]. In a multi-ethnic meta-analysis study (30,000 PTSD cases and 170,000 controls), non-coding RNAs such as Long Intergenic Non-Protein Coding RNA 2335 (LINC02335), microRNA 5007 (MIR5007), transcribed ultra-conserved region 338 (TUC338), (Long Intergenic Non-Protein Coding RNA 2571) (LINC02571), Long Intergenic Non-Protein Coding RNA 458 (LINC00458), microRNA 1297 (MIR1297) and Long Intergenic Non-Protein Coding RNA 558 (LINC00558) and PARK2 gene are involved in dopamine regulation, is associated with PTSD [20].

These studies support epigenetic differences in those with PTSD but it is also difficult to understand how persistent epigenomic changes affect a person's response to a traumatic event, and specifically to the molecular landscape of the brain. For this *New Diagnosis and Treatment Approaches to Post-Traumatic Stress Disorder DOI: http://dx.doi.org/10.5772/intechopen.104098*

reason, it is inevitable to encounter multiple epigenetic effects in many parts of the brain and that these have not yet found their place in translational medicine. Current epigenetic studies are focused on research on blood DNA, and analysis of postmortem data from different brain regions can be used to understand how epigenetic regulation works in PTSD at a circuit, brain region, or whole-brain level [10]. Consequently, since the biological studies of PTSD are heterogeneous, it has not yet taken its place in translational medicine for a definitive diagnosis. More research with larger sample groups is needed in the biological diagnosis and treatment of PTSD.

#### **3.3 Neuro-biological perspective**

#### *3.3.1 Fear and stress network in animal models*

The paucity of human studies investigating the neurobiological mechanisms of PTSD mirrored the understanding of this disease in animal models. Dysregulations of fear and stress-focused inflammatory responses detected in various brain regions have emphasized the importance of central nervous system centers that regulate fear memories (i.e., amygdala) and in response to acute or chronic stress response (i.e., the hypothalamus) since it began to be detected in PTSD patients. While animal studies continue to investigate fear-related processes for the amygdala, the medial prefrontal cortex (mPFC) and the hippocampus, interactions of the lateral (LA) (acquisition of fear and extinction concerning learning) and central nuclei (CeA) (behavioral expression of conditional fear) of the amygdala's nuclei regulating the inhibitory and excitatory effects of fear have been identified [39]. Connections between the hippocampus and the amygdala, particularly the LA, appear to be essential for the acquisition and reinforcement of contextual fear. At this point, it is thought that the somatosensorial projection of the hippocampus to the amygdala triggers contextual fear memory and may trigger fear-related learning through the LA nucleus. In addition, other evidence suggests that projections from the hippocampus to the mPFC can innervate neurons in the prelimbic (PL) and infralimbic (IL) regions that are active during fear and stress in animal models. PL and IL regions were important by creating neuronal potentials after the mPFC learned stress and conditioned fear on rodents, especially PL activity is responsible for regulating fear while its expression [40]. For instance, Richter-Levin developed a PTSD model in which animals are conditioned to pair a water-associated zero maze (WAZM) with underwater trauma that might be related to Amygdala LA and CeA nuclei. The remainder of underwater trauma rather than swimming stress, additional evidence of increased ERK phosphorylation (pERK) in the ventral dentate gyrus and basolateral amygdala [41]. Considering animal models of electrocution, this model is used more in learning and memory mechanisms than in PTSD, although it is associated with contextual reminders of trauma (associative fear) and ambiguous stimuli in a new setting (non-associative fear). Likewise, single long-term stress patterns were associated with neuronal apoptosis and dysregulation of autophagy in the hippocampus, amygdala, and prefrontal cortex (PFC), consistent with the findings in PTSD patients in terms of neurobiological background [42]. The social and psychological stressors animal model was mostly used for the PTSD behavioral measurements. In contrast, the social defeat stress (SDS) model was associated with optogenetic modulation of neuron projections to/from the ventromedial prefrontal cortex, ventral tegmental area, nucleus accumbens, and dorsal raphe nucleus in parallel with the PTSD clinic. Interestingly, amygdala-mPFC neuroadaptation was discovered in

resting-state functional magnetic resonance imaging (rsfMRI) findings from Long-Evans rats exposed to the cat collar in predator-associated animal models [43].

### *3.3.2 Neurochemical and synaptical background*

Serotonin (5-HT) is an important neurotransmitter for PTSD, targeting GABAergic neurons in response to fear-related acute stress in the amygdala, hippocampus, and ventromedial prefrontal cortex (vmPFC) regions. Clinical and animal studies have shown that symptomatic reduction associated with the use of antidepressants and/or anxiolytics in the treatment of PTSD is associated with stimulation and interaction of 5-HT1A, 5-HT1B, and 5-HT2A or 5-HT2C receptors. Sullivan et al. demonstrated positron emission tomography (PET) results of PTSD-like animals found higher 5-HT1A neuronal binding in all brain regions except the hippocampus and higher serotonin concentration in raphe nuclei compared to the healthy group [44]. Murrough et al. showed low 5-HT1B receptor density in the amygdala and anterior cingulate cortex (ACC) in PTSD patients [45]. The majority of the overactive noradrenergic activity associated with PTSD is due to the interaction of peripheral catecholamine (epinephrine, norepinephrine, and dopamine), transporter and receptor systems. In an animal and replicated study in humans, the high synaptic activity of norepinephrine (NE) in PTSD patients was detected in PFC projection areas. NPY also inhibits NE release and is found in high concentrations in the hippocampal and amygdala regions, it is associated with the projection of emotional values to memory and plays a role in the neurobiology of PTSD. Although intranasal NPY treatment reduces symptoms in many animal models of PTSD, efforts to develop NPY receptor-related pharmacological agents have failed [46]. Glutamate is an integral part of the learning, memory, and plasticity process. The glutamatergic system is studied as ionotropic and metabotropic. The PFC is transferred from the other to the amygdala and the bases of the whole brain regions to the amygdala are transmitted by glutamatergic contents and abnormal glutamate levels in PFC and N-methyl-D-aspartate (NMDA) receptor density in the hippocampus that is associated with synaptic plasticity underlying learning and memory, also have been reported in acute stress animal models. Especially metabotropic glutamate receptors have related with PTSD symptoms, high glutamate levels in the lateral temporal cortex and lower levels in ACC have been demonstrated. Research is ongoing that injection of subanesthetic doses of ketamine into rat brains increases glutamatergic neuronal activity in the PFC, which NMDA antagonists trigger learning and fear-related plasticity when examining the link between the glutamate system and dissociative symptomatology. Animal studies have shown that ketamine administration increases glutamate neurotransmitter levels and thus stimulates BDNF signaling, neurogenesis, and synaptogenesis [47]. GABA plays an important role in spatial and long-term memory, and directly in fear memory, in relation to neurogenesis in the hippocampal region. Fang et al. reported increased dysregulation of anxiety and fear memory with increased active GABAergic neurons in the CeA region of the amygdala in the single prolonged stress (SPS) animal model [48]. Behind the neurobiological mechanisms of PTSD, neuronal cell membrane damage due to stress and fear has also been researched. This damage is usually caused by oxidative stress-related free radicals (reactive oxygen species, for example, nitric oxide, glutathione, and hydrogen peroxide) damage to the cell membrane. In a recent study, Michels et al. found high higher levels of γ-amino butyric acid GABA and glutathione in PTSD patients via single-voxel proton magnetic resonance spectroscopy (MRS) in the dorsolateral prefrontal cortex (DLPFC) and ACC [49].

#### *New Diagnosis and Treatment Approaches to Post-Traumatic Stress Disorder DOI: http://dx.doi.org/10.5772/intechopen.104098*

PTSD is also related to abnormal activity of the dopaminergic system, which has a mesolimbic pathway that is related to fear conditions and high plasma dopamine concentration was reported in PTSD patients. However, the dopamine metabolism of PTSD is unclear, so the genetic background is more studied. Most of these neurobiological explanations are accompanied by synaptic losses underlying PTSD. The clinical behavioral reflections (i.e., social disinhibition, apathy, attention and memory disorders, etc.) of these synaptic losses in various parts of the brain are tried to be explained. As a result of stress, disruption of intracellular signaling may result in a decrease in glutamate receptors and shrinkage of dendrite horns in postsynaptic neurons. The synaptic degeneration hypothesis is the basis of many neurodegenerative psychiatric disorders. Results of a postmortem pilot study reported that PTSD patients were immature, as the dendrites evaluated in vmPFC tissues were smaller in their spines compared to the control group [50]. In addition, neuroimaging studies conducted in PTSD were associated with volumetric and neuronal connectivity deficiencies in cortical areas and their resulting loss of cognitive functions in PTSD clinics. In particular, losses in dendritic connections are predominantly in hippocampal regions associated with neuroplasticity, resulting in chronic or acute stress-related learning disabilities. In short, the perspective on neuroplasticity has been developed by investigating neurochemical and receptor interactions in various brain regions of PTSD. In this context, antidepressants used clinically for PTSD may contribute to clinical improvement by promoting synaptic plasticity with this neurobiological infrastructure. In addition, inferences about synaptic connectivity based on neuroimaging methods are still unclear but may reveal various risks. Due to the limited knowledge about the neurobiology of PTSD, the inadequacy of the findings from animal stress models for the pathophysiology prevents us from making definite conclusions about the clarity of the applications for the clinical treatment of this disease. As a result, PTSD has been scientifically investigated with behavioral consequences related to neurobiological, genetic, and epigenetic, literature discussions continue especially in terms of both neuroscientific and clinical aspects. The importance of neurochemical, biological, and brain-regional neurologic interactions in human and animal models remains a mystery, and further studies need to unlock this mystery.
