By the end of Romanian dictator Nicolae Ceauşescu’s regime in December of 1989, an estimated 100,000 Romanian children were living in state-run orphanages under some of the bleakest conditions imaginable (1). Children were malnourished, neglected, and clothed in rags; infectious diseases were rampant. One survivor called the orphanages “slaughterhouses of souls” (2).

While some of these children were later adopted into loving homes, most bore the mental scars of their time in the orphanages for decades. As adults, they showed higher rates of abnormal social behaviors, inattention and overactivity, and self-rated emotional problems compared to other adoptees who did not experience this type of childhood deprivation (3).

This is not an isolated data point: Authors of a study across 21 countries estimated that childhood adversity accounted for approximately 30 percent of all mental disorders (4). In addition to subjective influences, adverse childhood experiences associate with objective differences in brain activity and connectivity (5,6). Animal studies have revealed that early life adversity even leaves its mark at the molecular level, considerably altering gene transcription throughout the brain (7,8).

Exactly how transient experiences cause long-term changes in the brain and in behavior is an open question. Increasingly, scientists turn to epigenetics to help answer it. While geneticists examine the precise patterns of nucleotides that make up an organism’s genetic code, researchers studying epigenetics focus on factors that change gene expression without altering the DNA code. These include changes in DNA methylation, histone modifications, and noncoding RNA that influence whether genes are turned on or off (9).

Unlike the genome, which remains stable throughout life, the epigenome can be altered by environmental factors, especially early in life. “Epigenetics could provide a mechanism for long-lived changes in gene expression in the brain in response to lived experiences,” said Eric Nestler, a neuroscientist at the Icahn School of Medicine at Mount Sinai.

Now, researchers are investigating how early life adversity leaves epigenetic marks in brain regions related to stress, emotional processing, and reward, as well as how these changes may confer increased risk for depression and anxiety later in life. Although this field is in its infancy, researchers are beginning to explore how pharmaceuticals and epigenetic editing might be used to heal these epigenetic scars.

Of rats and men

In the late 1990s, Moshe Szyf, an epigeneticist at McGill University, studied the regulation of DNA methylation in cancer.  When he met neurobiologist Michael Meaney, also at McGill University, at a conference in Madrid, everything changed. “It was pure serendipity,” said Szyf.

At the time, Meaney studied how maternal care in infancy affected rats’ brains in adulthood. After discussing Meaney’s work, Szyf became curious about the molecular mechanisms through which these postnatal experiences might produce long-lasting changes.

Szyf believed that these kinds of changes would be recorded in the genome. “The only mechanism that could record things in the genome that I knew of at the time was DNA methylation,” Szyf said.

The idea that DNA methylation could change after birth was considered a complete heresy.
- Moshe Szyf, McGill University

At that time, said Szyf, the field of epigenetics was largely understood in the context of development. Essentially every cell in the human body has the same DNA code, but epigenetic differences tell cells whether to become heart cells or brain cells or liver cells. Because everyone needs a liver,  and because it would be problematic if a liver cell suddenly turned into a brain cell, researchers thought that these changes were highly stable and did not vary from person to person.

“The idea that DNA methylation could change after birth was considered a complete heresy,” said Szyf. “But cancer gave us the first example that this is possible.”

Meaney’s team had already observed that rats that had received more licking and grooming as pups showed greater glucocorticoid receptor (GR) expression in the hippocampus in adulthood (10). These receptors are part of an important negative feedback loop that helps shut down the stress response when it senses high levels of stress hormones. Thus, this increased GR expression likely contributed to the nurtured rats’ relatively milder responses to stressors as adults.

Szyf and Meaney teamed up to determine whether epigenetic changes might be responsible for the influence of maternal care on stress responses and gene expression later in life (11). They discovered that the offspring of more nurturing mothers had decreased methylation at a promoter region in the GR gene in the hippocampus. Importantly, decreased methylation is generally associated with greater gene transcription, so this discovery provided a link between maternal behavior and GR expression. Maternal genetics did not seem to contribute to this phenomenon. When researchers switched the pups to different mothers shortly after birth, this methylation pattern was determined by the behavior of the “adoptive” mother, not the genetic mother.

This was a major shift for the field, said Szyf. “Before that, nobody thought that behavior could induce these kinds of chemical manifestations in the hardwiring of the brain.”

In this way, early life experiences may help prepare the organism for the environment in which it will spend its life. “I see epigenetic programming as the way by which the genome adjusts itself to a dynamic world,” said Szyf.

In some cases, there may be a benefit to negative experiences saddling an organism with increased anxiety. For an animal, Szyf said, “being hyper stressed can save you in a really rough world.” In modern-day humans however, it’s a different story. Instead of providing protection, these heightened stress responses can become maladaptive, leading to worse outcomes instead of better ones.

Before that, nobody thought that behavior could induce these kinds of chemical manifestations in the hardwiring of the brain. 
- Moshe Szyf, McGill University

As interest in the field grew, researchers questioned whether similar processes were at work in humans. This question presented a conundrum: epigenetic changes are tissue-specific and except in very rare cases, it’s not possible to obtain brain tissue from living humans.

Brain banks provided a potential solution. Working with Gustavo Turecki, who studies suicide and depression at McGill University, Szyf and Meaney analyzed brain tissues that were donated to the Douglas-Bell Canada Brain Bank.

“The results in animals and in humans were very similar,” said Turecki. Suicide victims who had experienced abuse as children had lower levels of hippocampal GR expression compared to nonabused suicide victims as well as nonabused individuals who had died from other causes (12). The formerly abused suicide victims also showed increased methylation of GR promoter. In the study, scientists confirmed that this type of methylation decreased transcription of the receptor. 

While this is a neatly packaged explanation for how postnatal experiences influence adult brain function via epigenetics, the researchers knew that it likely wasn’t a comprehensive explanation. When they screened a larger section of the genome in hippocampal cells, they discovered methylation patterns at hundreds of sites varied with early life experience in both humans and rats (13).

Beyond the hippocampus

The story got even more complicated as researchers expanded their experiments beyond the hippocampus.

“Back when I was in graduate school,” recalled Nestler, “most of the research done on animal models of depression focused on the hippocampus and regions of the frontal cerebral cortex.” While those regions of the brain are undoubtedly important, Nestler’s early work on the brain mechanisms of addiction prompted him to look deeper into other brain areas that might be involved in early life adversity and depression.

“What grew out of our earlier interest in drug abuse was the realization that when a person is in withdrawal from drugs of abuse, many of their symptoms are akin to a person who suffers from depression,” said Nestler. “That raised the notion that the reward pathways would also be very likely to be involved.”

This hypothesis proved to be correct. A large body of literature now links changes in the brain’s reward regions, including the ventral tegmental area and nucleus accumbens, with depression (14, 15). Nestler wanted to know if the reward pathways were also involved in the ways that early life stress influences susceptibility or resilience to depression — or at least the rodent version of depression — later in life.

Nestler and Catherine Peña, then a postdoctoral researcher in Nestler’s research group, developed a mouse model to test this idea. For seven days, young pups were subjected to separation from their mothers for short periods each day. Another group of pups was raised normally. Without further stress, both groups performed similarly in behavioral tests.

When the mice were hit with a second stressor — social defeat stress — in adulthood, however, major differences emerged. The conventionally raised mice were stress-resilient, weathering the storm without apparent ill effects. Mice stressed in early life, however, were not able to bounce back. They displayed impaired social behavior, anhedonia, and depression-like symptoms (16).

“We're interested in that mechanism,” said Nestler. “What renders those mice more susceptible? We hypothesized that there might be ‘chromatin scars’ — epigenetic changes induced early in life that then persist a lifetime to make an animal more vulnerable later in life.”

In order to investigate, researchers analyzed levels of histone-modifying enzymes in the nucleus accumbens of the two groups of mice. One of the major differences between the groups was in the expression of Dot1l, which encodes the enzyme responsible for methylation of lysine 79 of histone 3, or H3K79me (17). This gene was especially strongly expressed in D2 medium spiny neurons, which are involved in susceptibility to stress in adulthood.

By overexpressing Dot1l  in these neurons specifically, researchers mimicked the effects of early life stress in mice that had never actually experienced it. The manipulated mice displayed anxiety- and depression-like behaviors after exposure to a stressor in adulthood. Conversely, Dot1l  knockdown in these neurons erased the scars of early life stress, normalizing behaviors even after the adulthood stressor.  

Nestler, however, emphasized that this work focused on one cell type in one brain region and is just one piece of a very complex puzzle. “We have every expectation that there's going to be additional chromatin scars in the hippocampus, frontal cortex, amygdala, and other areas.”

“This highlights the special challenges of brain research and psychiatry in particular compared to say, cancer,” said Nestler. Unlike cancer, which generally arises from one cell type, “There is no evidence that depression arises from one cell type. On the contrary, mental illness is probably arising from multiple cell types and multiple circuits in the brain,” he said. “That’s what the field of psychiatry is grappling with today. How do you deal with the sheer complexity at the molecular level of each cell type in each brain region? And then how do you put all that together with the sheer complexity of the circuitry?”

Rewriting fate

Once researchers identify the epigenetic scars that early life adversity leaves behind, they hope to develop ways to heal them.

Nestler’s identification of DNA methylating enzyme DOT1L as a potential mediator of early life stress, for example, revealed a new potential target for treating adulthood psychiatric disorders stemming from childhood adversity. The team’s first experiments used viral vectors injected into the brain to reduce Dot1l  expression and while successful, this technique is generally too risky for human use except in extreme circumstances. Searching for a safer option, Nestler came upon pinometostat, a DOT1L inhibitor that is currently in clinical trials for certain forms of leukemia.

Researchers found that ten days of peripherally administered pinometostat not only reduced H3K79 methylation in the nucleus accumbens, it also restored behavioral resilience in mice subjected to early life stress (17).

Because epigenetic modifications can cause effects throughout the body, Nestler said that side effects will be an important consideration; he is currently working with a clinical collaborator to assess the suitability of DOT1L inhibitors for use in human patients with depression.

Researchers have discovered many small molecule compounds that target the enzymes responsible for making epigenetic alterations, several of which are currently in development as cancer therapies (18). Szyf predicts that after these drugs are developed for cancer, they will be tested in psychiatric contexts as well if they have acceptable safety profiles.

More than one way to skin a cat

These small molecule approaches are not the only potential ways to edit the epigenome. Subhash Pandey, a neuroscientist at the University of Illinois at Chicago, uses CRISPR/dCas9 constructs to target specific histone modifications of genomic regions.

Pandey studies how exposure to alcohol in adolescence alters the epigenome and behaviors later in life. “Adolescence is a critical window for brain maturation and epigenetics play a very important role in that maturation process,” said Pandey. His research has shown that rats exposed to alcohol as adolescents grew into adults with heightened anxiety-like behaviors (19).

In order to probe the molecular changes underlying this behavior, Pandey and his team analyzed tissue from the amygdala, a brain region implicated in processing emotions, especially fear and anxiety. They found decreased expression of Arc, a major regulator of synaptic plasticity, as well as decreased dendritic spine density (20). Importantly, these changes seemed to be epigenetically regulated; adolescent alcohol exposure caused transcription-repressing histone modifications, including a decrease in histone acetylation, at the synaptic activity response element (SARE), an Arc enhancer region.

Next, the researchers wanted to know if they could correct these alcohol-induced changes in the enhancer region, and whether this correction would also normalize Arc expression and ameliorate anxiety-like and alcohol drinking behaviors. They used a modified CRISPR/Cas9 construct for epigenome editing. They created guide RNAs to steer the construct to the enhancer region, used a “dead” version of Cas9 that is unable to cut DNA, and fused the whole thing to a component that catalyzes histone acetylation. Their editing worked as intended; while adolescent alcohol exposure decreased histone acetylation at the site H3K27 at the Arc enhancer (a change that decreases gene transcription), the construct increased acetylation at this site. Subsequently, this epigenome editing also normalized Arc expression and soothed the rats’ anxiety and drinking (21).

There’s a lot of work to be done before this technique can be tested on humans. Even if this exact method turns out to be unsuitable for humans, Pandey said that it’s still an important proof-of-concept. “It’s the beginning, but it’s a very promising area to explore whether targeted epigenomic editing can reverse disease conditions such as addiction, anxiety, or depression,” said Pandey.

Turtles all the way down

Even as scientists delve into epigenome modulation, there’s still so much left to understand. Nestler said that the field is vastly complex and referenced the problem of “turtles all the way down,” an expression that indicates that every explanation for a phenomenon requires its own explanation in turn.

It has struck me how complicated epigenetic mechanisms are. 
- Eric Nestler, the Icahn School of Medicine at Mount Sinai

These scientists are dealing with a process that differs not only with experience, but also with age, sex, cell type, and a host of other factors. They try to piece together how this affects the function of cells, circuits, and ultimately, brain function as a whole. It is not an easy task.

Nestler first began exploring the idea that epigenetic changes in the brain drive lifelong changes in behavior nearly two decades ago. “Looking back over those 20 years, I think that idea was correct,” he said. “But it has struck me how complicated epigenetic mechanisms are. And 20 years later, we’re still in the very early stages of understanding how this might be happening.”

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