Image comparing mice submitted to pain-inducing experiment, which resulted in loss of pigmentation [bottom], to dark-colored mice in the control group. [William A. Gonçalves]

Stress has long been anecdotally linked with prematurely graying hair. It’s said, for example, that when Marie Antoinette was captured during the French Revolution, her hair turned white overnight. Anecdote this may be, but an international research team led by Harvard University scientists has now discovered how stress may, in fact, cause hair to gray. Their studies in mice and laboratory-grown cells showed that stress activates noradrenaline-releasing sympathetic nerves that are part of the fight-or-flight response, which in turn causes permanent damage to pigment-regenerating stem cells in hair follicles.

“Everyone has an anecdote to share about how stress affects their body, particularly in their skin and hair—the only tissues we can see from the outside,” said Ya-Chieh Hsu, PhD, the Alvin and Esta Star Associate Professor of Stem Cell and Regenerative Biology at Harvard. “We wanted to understand if this connection is true, and if so, how stress leads to changes in diverse tissues. Hair pigmentation is such an accessible and tractable system to start with—and besides, we were genuinely curious to see if stress indeed leads to hair graying.” Hsu is senior author of the team’s paper, which is published in Nature, and titled, “Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.”

Empirical as well as anecdotal evidence has linked stress with accelerated hair graying, which is the formation of hairs with no pigment, the authors stated. In recent history, for example, John McCain experienced severe injuries as a prisoner of war during the Vietnam War and lost color in his hair. However, the scientists acknowledged, despite this type of evidence, “ … so far there has been little scientific validation of this link … whether stressors are the causal factors, and whether stress-related changes occur at he level of somatic stem cells, remain poorly understood.”

Hair follicles that produce new hairs cycle between phases of growth (anagen), degeneration (catagen), and rest (telogen). The hair follicle contains two types of stem cell, hair follicle stem cells (HFSCs), and pigment-forming melanocyte stem cells (MeSCs). For much of the cycle these stem cells are dormant, but they are activated during early anagen to form new pigmented hairs. The MeSCs act as a reservoir of pigment-producing cells, so when hair regenerates, some of the MeSC stem cells convert into pigment-producing cells that color the hair. “ … differentiated melanocytes synthesize melanin to color the newly regenerated hair from the root,” the scientists stated.

Stress affects the whole body, so to investigate any link between stress and hair graying, the authors first had to try to identify which body system was responsible. Their work involved a series of studies, starting with whole-body response and progressively zooming into individual organ systems, cell-to-cell interaction and then down to molecular dynamics. A range of research tools were employed, including methods to manipulate organs, nerves, and cell receptors.

The investigators’ initial hypothesis was that stress might cause an immune attack on pigment-producing cells. However, their experiments showed that mice lacking immune cells still showed hair graying. The team then looked for a link between stress, graying hair and cortisol, but this also proved negative. “Using a combination of adrenalectomy, denervation, chemogenetics, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormone,” they noted. “Stress always elevates levels of the hormone cortisol in the body, so we thought that cortisol might play a role,” Hsu said. “But surprisingly, when we removed the adrenal gland from the mice so that they couldn’t produce cortisol-like hormones, their hair still turned gray under stress.”

After systematically eliminating different possibilities, the researchers honed in on the sympathetic nerve system, which is responsible for the body’s fight-or-flight response. Sympathetic nerves branch out into each hair follicle on the skin. The team’s experiments showed that stress causes these nerves to release noradrenaline, which gets taken up by the nearby MeSC pigment-regenerating stem cells.

This image illustrates the elaborate sympathetic innervation (green) around melanocyte stem cells (red). Acute stress induces hyperactivation of the sympathetic nervous system to release large amount of norepinephrine, a neurotransmitter. Norepinephrine drives rapid depletion of melanocyte stem cells and hair graying. [Bing Zhang and Ya-Chieh Hsu]
The noradrenaline then triggers excessive activation of the stem cells, which effectively all convert into pigment-producing cells, prematurely depleting the reservoir. “Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine),” the team explained. “This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche.”

“We were conducting a study on pain using black C57 mice, a dark-furred laboratory strain,” explained co-author Thiago Mattar Cunha, PhD, a researcher affiliated with the Center for Research on Inflammatory Diseases (CRID), a Research, Innovation and Dissemination Center (RIDC) funded by São Paulo Research Foundation (FAPESP) and hosted by the University of São Paulo’s Ribeirão Preto Medical School (FMRP-USP) in São Paulo State, Brazil. “In this model, we administered a substance called resiniferatoxin to activate a receptor expressed by sensory nerve fibers and induce intense pain. Some four weeks after systemic injection of the toxin, a PhD student observed that the animals’ fur had turned completely white.”

After repeated tests the CRID researchers concluded that the phenomenon was due to the application of resiniferatoxin, a naturally occurring chemical found in resin spurge (Euphorbia resinifera), a cactus-like plant native to Morocco. “We set out to check the hypothesis that the loss of fur color resulted from pain-induced stress,” Cunha said. “We designed a very simple experiment to see if the phenomenon was dependent on activation of sympathetic nerve fibers.”

After injecting resiniferatoxin into the mice, the animals were treated using guanethidine, an anti-hypertensive that can inhibit neurotransmission via sympathetic fibers. “We observed that the process of fur color loss was blocked by the treatment,” Cunha said. In another experiment, neurotransmission was interrupted by the surgical removal of sympathetic fibers. In this case, too, fur color was not lost in the weeks following pain induction.

This image illustrates the elaborate sympathetic innervation (magenta) around melanocyte stem cells (yellow). Acute stress induces hyperactivation of the sympathetic nervous system to release large amount of norepinephrine, a neurotransmitter. Norepinephrine drives rapid depletion of melanocyte stem cells and hair graying. [Bing Zhang and Ya-Chieh Hsu]
“These and other experiments conducted by our group demonstrated the participation of sympathetic innervation in achromotrichia and confirmed that pain is a powerful stressor in this model. But it remained to detail the mechanisms involved,” Cunha noted. “We used various methodologies to show that intense sympathetic activity speeds up differentiation significantly. In our model, therefore, pain accelerated the aging of the stem cells that produce melanin.”

Hsu added, “When we started to study this, I expected that stress was bad for the body—but the detrimental impact of stress that we discovered was beyond what I imagined. After just a few days, all of the pigment-regenerating stem cells were lost. Once they’re gone, you can’t regenerate pigment anymore. The damage is permanent.”

Cunha noted, “For the longest time it’s been said that stress makes the hair turn white but until now there was no scientific basis for this belief. Our study proved that the phenomenon does indeed occur, and we identified the mechanisms involved. In addition, we discovered a way of interrupting the process of hair color loss due to stress.”

The researchers used RNA sequencing to explore the mechanisms that promote melanocyte stem cell differentiation, by comparing the gene expression profiles of mice that received the injection of resiniferatoxin, and developed pain, stress and fur color loss, with those of control mice injected with a placebo. “We looked for genes whose expression was most altered after stress induction, and one caught our attention: the gene that encodes a protein called CDK [cyclin-dependent kinase]. This is an enzyme that participates in cell cycle regulation,” Cunha said. When the researchers repeated the pain induction procedure and treated the mice with a CDK inhibitor, they found that melanocyte stem cell differentiation was prevented, as was fur color loss. “This finding shows that CDK participates in the process and could, therefore, be a therapeutic target,” Cunha said. “It’s too soon to know whether it will actually become a target someday in clinical practice, but it’s worth exploring further.”

The researchers’ experiments demonstrated that when the sympathetic system is robustly activated, the fibers that innervate hair follicle bulbs release noradrenaline very near the melanocyte stem cells. “We showed that melanocyte stem cells express the protein ADRB2 [β2-adrenergic receptor], which is activated by noradrenaline, and we discovered that the stem cells differentiate when this receptor is activated by noradrenaline,” Cunha said. To confirm the finding, the researchers repeated their tests using mice that had been genetically modified, so as not to express ADRB2. As suspected, the fur of these animals did not turn white after they were injected with resiniferatoxin. “In another test, we injected noradrenaline directly into the skin of the mouse. As a result, the fur around the site of the injection turned white,” Cunha said.

In a final set of studies, the group showed that cultured primary human melanocytes (melanin-producing cells obtained directly from the skin of a volunteer) treated with noradrenaline showed increased expression of CDK , which was similar to the findings in mice.

According to Cunha, the researchers do not yet know if there will be future aesthetic applications for their findings, such as the development of a drug that could stop us growing gray as we age. “It would be necessary to see if a CDK inhibitor has side-effects, and if so whether they would be outweighed by the aesthetic benefit.”

Co-author Isaac Chiu, PhD, assistant professor of immunobiology at Harvard Medical School, studies the interplay between nervous and immune systems. He said, “we know that peripheral neurons powerfully regulate organ function, blood vessels, and immunity, but less is known about how they regulate stem cells. With this study, we now know that neurons can control stem cells and their function, and can explain how they interact at the cellular and molecular level to link stress with hair graying.”

The researchers suggest that their results underscore the negative side effects of an otherwise protective evolutionary response. “Acute stress, particularly the fight-or-flight response, has been traditionally viewed to be beneficial for an animal’s survival. But in this case, acute stress causes permanent depletion of stem cells,” said postdoctoral fellow Bing Zhang, first author of the study. “To go from the highest level to the smallest detail, we collaborated with many scientists across a wide range of disciplines, using a combination of different approaches to solve a very fundamental biological question.”

The scientists also acknowledged that the reason for any interaction between nerves and MeSCs isn’t known. The connection between the nervous system and pigment-producing cells is probably conserved during evolution they suggested. Squid, cuttlefish, and octopus are cephalopods that can rapidly change color for camouflage or to communicate. Their nervous system controls pigment-producing chromatophore cells, allowing very fast changes in color in response to threats or predators. “Therefore, an attractive hypothesis is that sympathetic nerves might modulate MeSC activity, melanocyte migration or pigment production in situations independent of the hair cycle—for example, under bright sunlight or UV irradiation,” the team suggested. “Under extreme stress, however, hyperactivation of neuronal activities overstimulates the pathway, which drives the depletion of MeSCs.

The findings could help to provide new insights into the broader effects of stress on various organs and tissues, which could ultimately lead to new approaches to modifying or blocking the damaging effects of stress. “By understanding precisely how stress affects stem cells that regenerate pigment, we’ve laid the groundwork for understanding how stress affects other tissues and organs in the body,” Hsu said. “Understanding how our tissues change under stress is the first critical step towards eventual treatment that can halt or revert the detrimental impact of stress. We still have a lot to learn in this area.”

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