The Epigenetics of Stress

Overly sensitive, aversive reactions to stress seem to run in families. The literature abounds with reports of relatives in these populations predisposed to depression, anxiety, and even suicide. Some family members present with glucocorticoid levels notched abnormally high, and in curiously deregulated concentrations. Behaviorally, they seem to exist at a permanent state of high alert.

Attempts to isolate the genetic underpinnings of this obvious hyperaroused stress sensitivity have met with mixed success. People carrying certain mutations in the serotonin transporter gene seem particularly vulnerable to the normal stresses and strains in life, although there have been difficulties in replicating all the findings. Plenty of people exist who are just as vulnerable to stress but who do not carry this mutation-or any other suspect genetic anomaly-that could explain the behavior.

It is now possible to characterize some of this seeming heritability-and accompanying statistical turbulence-without invoking heritability at all. This is the world of epigenetic transfer, the ability to pass on a trait without having to stop at a meiotic border. A recent study has demonstrated how a powerful environmental stressor can exert molecular effects that last over a long period. The mechanism is epigenetic. It is the first result to characterize a molecular mechanism, induced by early life stressors, that influences behaviors penetrating into adulthood.

To explain these findings, I will first talk about transplacental cortisol and its effects on the developing fetal brain. Then I will move to the data, which do not involve humans at all.

The starting observation

For years, we have known that stressed wombs tend to produce stressed babies. Most molecular explanations for this observation invoke the effects of transplacental glucocorticoids on fetal brain development. If mothers become too stressed (so the idea posits), too many stress hormones enter the womb, penetrate the fetal brain, and interfere with its proper development.

There is some empirical support for this notion. First, excess levels of maternal glucocorticoids have been shown to cross the placenta, targeting the fetal limbic system and causing it to develop much more slowly than in typical controls. This is thought to result in future behavioral dysfunction, particularly regarding reactions to external stressors. It specifically hampers the developing “braking system” of the hypothalamic-pituitary-adrenal (HPA) axis. (As you recall, the HPA axis is a series of biochemical reactions to threat that involve interactions between the hypothalamic, pituitary, and adrenal glands.)

This embryonic braking system is a coordinated series of responses that normally result in the inhibition of glucocorticoid production after some environmental stress has been successfully negotiated. Without this braking system, the fetal brain is wired to produce excess glucocorticoids in an increasingly unregulated fashion. The baby carries this deregulated system into adulthood. If the adult is female and becomes pregnant, her system, which is already flooded with cortisol, marinates her new baby with glucocorticoid. This once again creates a hyperaroused womb, complete with new fetal damage. The trait is thus passed along, not through the germ line but simply through womb exposure.

Although a great deal of work needs to be done to complete this admittedly depressing story, large parts have empirical support. This includes some intimate, biochemical details. Recently, a molecular mechanism has been uncovered that may explain this nongenetic inheritance. Although work has been done mostly in rats, there are broad implications for human behaviors. After a brief explanation of an epigenetic mechanism involving DNA methylation, it will be to these data that we turn next.

Social states experienced in early life can directly affect later behavior. Until now, exactly what molecular mechanisms undergird such effects have remained a complete mystery.

Early life stress

The work to be described involved inducing behavioral stress in a cohort of laboratory rodent pups, and then watching the effects of that stress on behavior as the rats matured. The standard protocol to induce developmental behavioral stress is to apply an infant-mother separation schedule early in postnatal life. Typically, the animal is separated from its mother 3 hours a day for the first 10 days of its life. The experience of early life stress (ELS) results in a lifelong elevation of glucocorticoid secretion and a disruption in normal stress responses (a permanent and heightened endocrine reaction to externally supplied stress). The animal becomes hyperaroused, presenting an abnormal regulation of the HPA axis. This arousal induces broad behaviors associated with mood and cognitive disorders.

A number of important hormones regulate the HPA axis, including 2 hypothalamic secretagogues-corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). When a stressful experience is encountered, signals arise that increase the synthesis and release of pituitary CRH and AVP. There is a rich history linking CRH and AVP to cognitive and affective disorders; their receptors are the targets of a number of psychopharmacological medications. AVP is also expressed in a specific subset of neurons within the hypothalamic paraventricular nucleus.

Understanding the molecular mechanisms behind the regulation of the HPA axis involves, in part, understanding how the AVP gene is regulated. Like all genes, AVP has a promoter region (which, as you recall from your undergraduate biochem class, functions as an on/off switch for the gene). A number of regulatory sequences surround this on/off switch, controlling its transcriptional activity. What happens to these sequences ultimately determines the role AVP plays in regulating stress responses (Figure).

One biochemical modification of the AVP promoter involves a process of methylation, the replacement of a hydrogen atom in certain nucleotide residues (generally cytosine residues) with a methyl group. This process is mediated by a class of enzymes termed “methyltransferases.” Many sites along the chromosomes are naturally methylated, but the process is in equilibrium, and many residues don’t stay that way. The amount of methylation on the regulatory region of a gene can control its transcriptional activity.

Methyltransferases are enzymes whose activities are also tightly controlled. One protein involved in regulating their activity is called MeCP2. This protein is specifically involved in recruiting proteins that will assist in the methylation of DNA. If MeCP2 activity is inhibited, the methylation of specific regions of DNA will also be inhibited, and the region is said to be hypomethylated. Because methylation affects a promoter’s ability to regulate the gene to which it is attached, one way to control gene activity is to control MeCP2 activity.

Results

With this background information in mind, we are ready to talk about the data. A typical ELS protocol was instituted immediately after the rats were born. Biochemical assessments began when the animals were 6 weeks old and continued for the next 10 months. A total of 4 findings emerged from these studies:

• ELS animals secreted abnormally high levels of stress hormone (specifically corticosterone) when compared with control groups. This excess persisted over the life of the experiment.

• ELS animals experienced a sustained rise in vasopressin expression (messenger RNA) in the hypothalamic paraventricular nucleus. That’s significant. This region of the hypothalamus is involved in regulating hormones associated with stress. Reassuringly, this elevation could be reversed with the application of an AVP receptor agonist.

• There was reduced methylation in a specific regulatory region for the AVP gene.

• The change in methylation was shown to be a result of the inactivation of MeCP2.

The behavioral changes in these mature animals were what you would expect in hyperaroused humans. They were less able to cope with stress in a wide variety of testing conditions, and they showed persistent memory impairments when compared with control groups.

Conclusions

These findings are quite stunning. For the first time, a molecular change associated with a persistent hyperaroused state in an adult animal was induced by behavioral neglect during the animal’s extreme youth. It is axiomatic from a counseling perspective that social states experienced in early life can directly affect later behavior. Until now, exactly what molecular mechanisms undergird such effects have remained a complete mystery. These data reveal strong epigenetic factors.

The normal caveats apply, of course. The experiments were done in laboratory animals, not humans-a red flag for anyone trying to apply animal-based behavioral results to human patients. Also, the stress was experienced after the pup was born, not during gestation. No measurements of transplacental glucocorticoid trafficking were made in these experiments. Nor was the post-ELS next generation examined. The behaviors induced in the offspring of these animals is an area of future research.

None of these objections hurt the findings, of course-just their interpretation. Their real value lies in what they portend for the future. Uncovering a mechanism induced in childhood that mediates a persistent behavioral experience in adults is a phenomenal achievement. Showing that these associations sometimes have epigenetic underpinnings is an added bonus. These results serve as flashlights, directing where scientists interested in human reactions should spend their next research dollars. Stress responses really do run in families. Showing that some reasons for this may be mostly environmental-with no DNA in sight-is the biggest plus of all.