Decades of research have demonstrated how stress can contribute to a wide variety of diseases and disorders. In the realm of mental health, we know that anxiety disorders, depression, and PTSD, for example, can be caused or exacerbated by stress—whether chronic or traumatic.

When we are subjected to stress, the body responds by producing stress hormones, the most pervasive of which in humans is cortisol. A continuing subject of research is precisely how the proliferation of stress hormones acts upon the body and brain to initiate and/or accelerate pathological process that contribute to illness.

A new study in Molecular Psychiatry, whose first author is 2020 BBRF Young Investigator Brian F. Corbett, Ph.D., of Rutgers University, does precisely this kind of work. It explains the mechanism by which cellular receptors for cortisol and other stress hormones (glucocorticoids) act to regulate the expression of a specific gene that tends to protect us from the harmful impacts of chronic stress. Interestingly, the team, led by Seema Bhatnagar, Ph.D., also shows how this mechanism has an important protective role in females even in the absence of stress. Dr. Bhatnagar, who is affiliated with the Children’s Hospital of Philadelphia and University of Pennsylvania, was the recipient of BBRF Young Investigator grants in 2000 and 1998.

Past research has shown that cortisol and other glucocorticoids counter inflammation caused by stress when these hormones activate the body’s stress response system organized around the so-called “HPA axis” (formed by the hypothalamus, pituitary and adrenal glands).

Newly generated stress hormones bind to glucocorticoid receptors (GRs) found in cells throughout the body and brain. Once bound at GR receptors, something remarkable happens: the entire complex moves into the cell nucleus, where DNA of the genome resides. There, the complex binds to specific locations, or loci, in the genome. These genome binding sites are called GREs, or glucocorticoid response elements.

Once bound to GREs, GRs act as “transcription factors”, meaning that they have the ability to regulate gene expression—when and how much a particular gene or genes are activated. Specifically, activated GREs can suppress or reduce the activation of genes that are involved in generating inflammation; their effect is anti-inflammatory. But this process can go awry. As Drs. Corbett, Bhatnagar and their colleagues note, “when the anti-inflammatory effects of stress hormone receptors are impaired, the pro-inflammatory effects of stress can go unchecked.” This is called glucocorticoid resistance, typically caused by chronic activation of GRs. This is one way in which stress generates inflammation in body and brain, which in turn can cause illness—in the case of brain, inflammation that can promote anxiety, depression, or PTSD.

Drs. Corbett and Bhatnagar have created a breed of rats that has enabled them to learn much more about exactly how stress hormone-activated GREs alter gene expression to reduce stress-related inflammation and behavioral changes that this can cause—notably, social withdrawal when an animal is subjected to intense, chronic stress.

The rats that they use in these experiments have a specific GRE deleted from the genome. The team used the genome-editing tool called CRISPR/Cas9 to cut out this tiny element from among the billions of DNA elements that comprise the rat genome. Rats are used as a proxy for humans, as their genomes and brains are very similar to those of people due to our common evolutionary history as mammals.

There are thousands of GREs in the genome (in both rats and people), but the one deleted by the team was in a specific location: very close to a gene called S1PR3. Why this particular GRE? In past experiments, they had noticed that expression of the S1PR3 gene was significantly higher in the medial prefrontal cortex (mPFC) of rats that were resilient to stress. Expression of the equivalent human gene is known to be reduced in veterans with PTSD and is negatively correlated with symptom severity (reduced expression of the gene correlates with more severe symptoms).

Like people, some rats are unusually vulnerable to stress while others are highly resistant to its negative impacts. The team subjected both kinds of rats as well as “average” stress responders to intense social stress, caused when one animal dominates and bullies another repeatedly. “When stress-vulnerable rats are stressed in these experiments,” Dr. Corbett explains, “they show reduced sociability and depression-like behavior. On the other hand, resilient rats, when stressed, behave like non-stressed controls.” Propelling the research forward was the realization that “we didn't know why S1PR3 [expression] was high in resilient rats. Then we had the idea that maybe it isn't naturally high, but is actually increased by stress.”

The team’s prior experiments established that elevated S1PR3 expression was linked with protection from the ill effects of chronic stress. They showed that when stress hormones dock at GR receptors and activate GREs in the nucleus, this raises the expression of S1PR3—specifically, in the mPFC of resilient rats. This, in turn, promotes active coping and sociability “by mitigating stress-induced inflammatory processes in the mPFC.” Elevated mPFC levels of S1PR3 were not seen in stress-vulnerable rats.

In the new experiments, the team tested rats whose GRE near the S1PR3 gene had been deleted and compared them with typical or “wild-type” animals. The aim was to test whether this GRE locus, in particular, was the one responsible for the protection from stress seen in resilient animals. Males with the missing GRE after being subjected to “social defeat stress”—as expected—had elevated markers of inflammation and exhibited social withdrawal. What was most interesting was the fact that female rats with deleted GREs showed the same inflammatory and behavioral response, but, importantly, even when they had not been subjected to social-defeat stress.

This is a potentially powerful fact pertaining to differences in stress response long observed in males and females (rodents and people). Average, healthy adult female rats have 5 to 10 times more stress hormone in circulation than males—a level similar to that seen in males only when they are under stress. The team suggests that their naturally higher stress hormone levels make females especially dependent upon the “protective” impact generated when the S1PR3 gene is activated. This was seen in their experiments: even in the absence of stress, the normally higher stress hormone levels in females corresponds with increased S1PR3 expression. And in female rats with the key GRE deleted, inflammation and social withdrawal were observed even when the animals were not challenged by stress. More than males, they depend upon the activation of this particular GRE by stress hormones to boost S1PR3 expression and the protection it provides.

Regarding that protection, in animals with deleted GREs, the team noted elevated levels of a type of neutral activity between the brain’s locus coeruleus (LC) and mPFC in males subjected to social defeat stress, but also in non-stressed females. Might this be a pathway be involved in protection against stress? Evidence in support of this theory was obtained by the team in a final set of experiments, in which they used an approach called chemogenetics to inhibit this LC-mPFC circuit. In stressed male rats that had the critical GRE deleted, the circuit was hyperactive, but inhibiting it during social defeat had the effect of increasing the animals’ social interactions. In effect, they became resilient to the stress.

In addition to confirming results of prior experiments about mechanisms involved in resilience and vulnerability to chronic stress, the team, in noting the different responses to GRE deletion in males and females, may have discovered something with powerful implications for treating stress in females. It may make sense, they suggest, to try to treat such stress by finding a way to inhibit the LC-mPFC pathway whose activation is associated, at least in rodents, with stress resilience. This possibility can now be pursued in future studies.