Blood–brain biomarkers for stress susceptibility

Posttraumatic stress disorder (PTSD) is a debilitating condition that arises in the aftermath of a highly traumatic event and was initially described as “shell shock” in combatants (1). Patients display three primary sets of symptoms: repeated flashbacks of the trauma, hyperarousal, and hyperanxiety (2). The condition was subsequently documented to occur in victims of sexual violence and natural disasters, among others (3). Further, there is extensive evidence that certain individuals have a higher predisposition to developing the condition (4), and there is also a higher prevalence of PTSD in women (5). In PNAS, Daskalakis et al. (6) explore the question of individual and sex differences using a combination of behavioral analyses and high-throughput genomics in two brain areas involved in the pathophysiology of PTSD: the hippocampus and amygdala. The authors report that glucocorticoid receptor (GR) signaling is a convergent signaling pathway associated with individual differences in both sexes. Indeed, corticosteroid treatment after trauma exposure is shown to prevent the onset of PTSD-like symptoms. They also find a similar genomic profile for individual and sex variation in blood samples, highlighting the value of blood-based biomarkers as a diagnostic tool for changes in brain signaling.

Two complementary strategies have facilitated our current understanding of PTSD. On the one hand, structural and functional imaging of the human brain, along with rigorous clinical assessments of PTSD etiology and psychopathology, has helped identify brain regions implicated in the behavioral symptoms. These top-down clinical studies in turn have inspired a bottom-up strategy that combines a range of neurobiological techniques and models to analyze the effects of trauma across biological scales: from molecular and cellular correlates at one end to behavioral and circuit-level analyses at the other (Fig. 1). Together, these approaches have contributed to animal models of stress that not only capture salient features of the disorder at the behavioral level but also provide insights into the underlying neuronal, endocrine, and genetic mechanisms. For instance, neuroimaging studies in patients show contrasting functional/structural modulation of the hippocampus and amygdala. Reduced hippocampal volumes, compared with either trauma-exposed control subjects or trauma-unexposed healthy subjects, have been reported in PTSD patients (7). Further, hippocampal volumes have been inversely associated with PTSD symptom severity (7). Amygdala activation, by contrast, is positively correlated with the severity of PTSD symptoms (7). Consistent with these clinical observations, accumulating evidence from animal models shows that stress has opposite effects on these brain structures. In the hippocampus, stress causes dendritic atrophy, loss of spines, and impairs synaptic plasticity mechanisms like long-term potentiation (8). However, chronic stress strengthens the structural basis of synaptic connectivity through dendritic growth and spinogenesis in the basolateral amygdala (BLA) (9). Repeated stress also enhances long-term potentiation (LTP) in the BLA and facilitates fear and anxiety (9). Another striking characteristic of PTSD lies in the temporal domain. Although PTSD is triggered by a single intensely traumatic, often life-threatening event, some symptoms persist well beyond the original event. This aspect has been captured by several animal models wherein a single, brief exposure to severe stress, followed by extended periods of stress-free recovery, leads to a delayed and gradual buildup of behavioral abnormalities that is manifested well after the acute stressor and is accompanied by cellular changes in the brain (10–12). The animal model used by Daskalakis et al. (6) represents one such paradigm wherein enhanced arousal and anxiety behavior is assessed 7 d after a brief exposure to predator scent stress.

Fig. 1.

Exposure to chronic stress triggers contrasting effects in the amygdala and hippocampus at multiple levels of neural organization (8, 9). In addition to the brain region-specific differences, various models of acute stress are known to elicit delayed increase in anxiety and fear. However, relatively little is known about the molecular/genetic basis of these unique spatiotemporal patterns of stress-induced plasticity in the two brain areas. Daskalakis et al. (6) used a rat model of PTSD wherein a single exposure to an ethologically relevant stressor (predator odor) leads to the development of anxiety-like behavior and increased startle responses after 7 d. Using high-throughput genomics, this study identifies genes that confer individual variability and sex differences in the pathophysiology of PTSD.

Although animal studies have identified key spatiotemporal features of stress-induced plasticity in the amygdala and hippocampus (Fig. 1), they also underscore several unresolved issues. The study by Daskalakis et al. represents significant progress in addressing two of these important issues, i.e., the role of individual variation and sex differences. One of the major questions in the field stems from the observation that not all individuals exposed to a traumatic experience develop PTSD. What factors make some individuals more vulnerable or resilient to PTSD? Thus far, one of the most tangible ways of addressing this issue has been the use of arbitrarily selected cutoff for behavioral measures to classify animals as being more vulnerable or resilient to stressors (10). Attempts to understand sex differences in animals, however, have been plagued by the fact that an overwhelming majority of studies use only males. Further, in the few studies that have been done, the evidence suggests that, unlike humans, female rats are far more resilient to external stressors (13, 14).

To address the relatively less known molecular/genetic underpinnings of these processes, the authors subjected rats to a single traumatic event (predator scent stress) and tested for delayed onset of anxiety-like behavior and arousal. Based on the cutoff behavioral criteria (10), rats were categorized as those showing PTSD-like symptoms (vulnerable) and those that did not (resilient). Hippocampal, amygdalar, and blood samples from these categories (along with naïve controls) were subjected to high-throughput behavioral genomics to identify differentially regulated genes. Extensive bioinformatics analyses were then applied to ascertain the transcription factors upstream of these genes and the canonical signaling pathways they operate in. The four main outcomes relate to stress exposure, individual variation, tissue type, and sex.

Not unexpectedly, stress exposure resulted in a distinct distribution of differentially regulated genes in both vulnerable and resilient groups compared with naïve controls. However, what is remarkable is that a large fraction of these genes (37–99%; in both sexes) is associated with individual variation, whereas very few genes appear to be involved in resilience (<3%). Many of these genes were common to both hippocampus and amygdala, which interestingly suggests that a small fraction of them may be responsible for the differential effects of stress on these two brain regions. Differences in gene regulation also depended on sex in all three groups. A detailed analysis of this dataset will be of considerable interest to the PTSD research community in understanding sex differences in animal models and their disparities with human data.

The most striking result from this study comes from the unbiased hierarchical clustering of the differentially regulated genes associated with individual differences: those from the vulnerable group were found to cluster not just together but also away from those of the resilient and control groups. This was true both in the hippocampus and amygdala, as also in males and females. This would suggest that modulation of these genes was related to vulnerability in both brain structures, presumably by similar upstream

Corticosteroid treatment after trauma exposure is shown to prevent the onset of PTSD-like symptoms.

mechanisms in the advent of the trauma.

To investigate this, the authors use gene network analysis to identify 73 unique transcription factors that were found to be involved in the modulation of the trauma-related genes in the three tissue types. Although several showed an overlap with one or both of the other tissues, only nine factors (CREB1, FOXO3, JUN, MYC, MYCN, NFE2L2, NFKBIA, NR3C1, and TP53) were shared by all three, i.e., hippocampus, amygdala, and blood. Data generated by this study will undoubtedly complement other microarray-based studies that have also reported some of these genes (15–17).

Among these nine factors, the glucocorticoid receptor (NR3C1) is of particular interest as the development of PTSD-like symptoms can be blocked by the administration of glucocorticoids shortly before (12) or after (18) stress. Indeed, analysis of the relationship between the 73 transcription factors implicated in vulnerability showed that 19 of these are involved in glucocorticoid signaling. This observation is validated in this study by showing that a single dose of corticosterone given 1 h after trauma exposure blocks the delayed increase in anxiety-like behavior and startle response. This is consistent with clinical reports that below normal cortisol levels may render individuals susceptible to PTSD and that glucocorticoids may protect against its development.

In addition to combining the powerful use of high-throughput genomics with clinically relevant animal models of PTSD to suggest new diagnostic markers that confer individual/sex related vulnerabilities, this study also opens up the possibility of generating more sophisticated animal models based on the unique transcription factors and signaling pathways identified here.