How Stress Causes Gray Hair?

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Legend has it that Marie Antoinette’s hair turned gray overnight just before her beheading in 1791.

Though the legend is inaccurate – hair that has already grown out of the follicle does not change color – a new study from researchers at Columbia University Vagelos College of Physicians and Surgeons is the first to offer quantitative evidence linking psychological stress to graying hair in people.

And while it may seem intuitive that stress can accelerate graying, the researchers were surprised to discover that hair color can be restored when stress is eliminated, a finding that contrasts with a recent study in mice that suggested that stressed-induced gray hairs are permanent.

The study, published June 22 in eLife, has broader significance than confirming age-old speculation about the effects of stress on hair color, says the study’s senior author Martin Picard, Ph.D., associate professor of behavioral medicine (in psychiatry and neurology) at Columbia University Vagelos College of Physicians and Surgeons.

“Understanding the mechanisms that allow ‘old’ gray hairs to return to their ‘young’ pigmented states could yield new clues about the malleability of human aging in general and how it is influenced by stress,” Picard says.

“Our data add to a growing body of evidence demonstrating that human aging is not a linear, fixed biological process, but may, at least in part, be halted or even temporarily reversed.”

Studying hair as an avenue to investigate aging

“Just as the rings in a tree trunk hold information about past decades in the life of a tree, our hair contains information about our biological history,” Picard says. “When hairs are still under the skin as follicles, they are subject to the influence of stress hormones and other things happening in our mind and body. Once hairs grow out of the scalp, they harden and permanently crystallize these exposures into a stable form.”

Though people have long believed that psychological stress can accelerate gray hair, scientists have debated the connection due to the lack of sensitive methods that can precisely correlate times of stress with hair pigmentation at a single-follicle level.

Splitting hairs to document hair pigmentation, Ayelet Rosenberg, first author on the study and a student in Picard’s laboratory, developed a new method for capturing highly detailed images of tiny slices of human hairs to quantify the extent of pigment loss (graying) in each of those slices. Each slice, about 1/20th of a millimeter wide, represents about an hour of hair growth.

“If you use your eyes to look at a hair, it will seem like it’s the same color throughout unless there is a major transition,” Picard says. “Under a high-resolution scanner, you see small, subtle variations in color, and that’s what we’re measuring.”

The researchers analyzed individual hairs from 14 volunteers. The results were compared with each volunteer’s stress diary, in which individuals were asked to review their calendars and rate each week’s level of stress.

A greying transition followed by complete reversal in a single hair shaft, imaged from bulb to tip. Pigmentation intensity was dynamically captured on a motorized stage microscope at ×10 magnification. Credit: Columbia University Irving Medical Center/eLife

The investigators immediately noticed that some gray hairs naturally regain their original color, which had never been quantitatively documented, Picard says.

When hairs were aligned with stress diaries by Shannon Rausser, second author on the paper and a student in Picard’s laboratory, striking associations between stress and hair graying were revealed, and in some cases, a reversal of graying with the lifting of stress.

“There was one individual who went on vacation, and five hairs on that person’s head reverted back to dark during the vacation, synchronized in time,” Picard says.

Blame the mind-mitochondria connection

To better understand how stress causes gray hair, the researchers also measured levels of thousands of proteins in the hairs and how protein levels changed over the length of each hair.

Changes in 300 proteins occurred when hair color changed, and the researchers developed a mathematical model that suggests stress-induced changes in mitochondria may explain how stress turns hair gray.

“We often hear that the mitochondria are the powerhouses of the cell, but that’s not the only role they play,” Picard says. “Mitochondria are actually like little antennas inside the cell that respond to a number of different signals, including psychological stress.”

The mitochondria connection between stress and hair color differs from that discovered in a recent study of mice, which found that stress-induced graying was caused by an irreversible loss of stem cells in the hair follicle.

“Our data show that graying is reversible in people, which implicates a different mechanism,” says co-author Ralf Paus, Ph.D., professor of dermatology at the University of Miami Miller School of Medicine. “Mice have very different hair follicle biology, and this may be an instance where findings in mice don’t translate well to people.”

Hair re-pigmentation only possible for some

Reducing stress in your life is a good goal, but it won’t necessarily turn your hair to a normal color.

“Based on our mathematical modeling, we think hair needs to reach a threshold before it turns gray,” Picard says. “In middle age, when the hair is near that threshold because of biological age and other factors, stress will push it over the threshold and it transitions to gray.

“But we don’t think that reducing stress in a 70-year-old who’s been gray for years will darken their hair or increasing stress in a 10-year-old will be enough to tip their hair over the gray threshold.”


Hair greying (referred to during ageing as ‘canities’) is one of the earliest and most visible indicators of ageing in humans. The social significance of greying persists across cultures, geographical locations, and ethnicities, alongside a now-widespread interest in its reversal (Trüeb & Tobin, 2010; Stenn, 2016). The modulation of greying by genetic, psychoemotional, oxidative, senescence-associated, (neuro-)endocrine, metabolic and nutritional factors has increasingly fascinated pigment and skin biologists, clinicians and industry.

This is not least because the study of greying permits one to observe and interrogate gerontobiology of a uniquely accessible and experimentally tractable human mini-organ found throughout the human integument. Moreover, an increasing number of animal and human studies have identified grey hair as an age-independent predictor of serious extracutaneous pathology, including Alzheimer’s disease (Mendelsohn & Larrick, 2020), Parkinson’s disease (Jucevičiūtė et al., 2019) and cardiovascular disease (Elfaramawy et al., 2018), raising suspicion that hair greying may indeed act as an important indicator of systemic ageing-associated pathology. As we grow closer to explaining this poorly understood phenomenon, it is timely to revisit critically what is known about the biology of hair greying today.

This review focuses on the mosaically cycling human scalp hair follicles (HFs), whilst cautiously drawing from greying-related mechanistic concepts generated in studies of rapidly cycling mouse HFs (Mus musculus, L.) (Paus & Foitzik, 2004; Bernard, 2012, 2017; Oh et al., 2016; Zhang et al., 2020). Hair cycling pattern and rate, as well as other fundamental aspects of pigmentation and hair biology, differ between humans and other mammals, including mice (Stenn & Paus, 2001; Bernard, 2012).

Therefore, caution is advised when extrapolating concepts on the biology of greying generated in mouse pelage HFs to human scalp HFs. Here, we purposely focus on the latter to highlight why human hair greying, besides its obvious relevance for human melanocyte biology, also represents an under-appreciated interdisciplinary model for human gerontology, immunology, perceived stress, stem cell, and neuroendocrine research.

Greying provides an instructive model for mouse and human tissue ageing, because the HF is highly susceptible to oxidative, inflammatory, nutritional and psychoemotional stressors (Peters et al., 2006; Peters, Arck & Paus, 2006a; Paus et al., 2014). Human scalp HFs are not only the largest HFs but also have a long growth phase (anagen) of the hair cycle, thus facilitating the interrogation of greying processes over several years.

The study of long-term pigmentation loss is likely to promote our understanding of other systemic ageing controls (Peters et al., 2013). In addition, human scalp HFs can easily be obtained, dissected and organ-cultured under serum-free conditions following facelift or hair transplantation surgery. During ex vivo culture, pigmented or white hair shaft production continues at almost the normal speed and anagen HFs continue spontaneously to undergo their cyclic organ involution process called catagen (Philpott, Green & Kealey, 1990; Langan et al., 2015).

In this review, we first summarise the functional anatomy of human HF pigmentation and the strict coupling of hair shaft pigmentation by highly specialized melanocytes in the HF pigmentary unit (HFPU) to the active growth stage of HF cycling (anagen) (Fig. 1).

This is followed by synthesizing key characteristics of human hair greying before focusing ondocumented and proposed mechanisms that underlie it. We expand this to discuss how functionally important controls of the human HFPU, such as neurohormones, thyroid hormones, adhesion molecules, growth factors and peripheral clock activity might impact upon the initiation of greying.

Thereafter, we critically discuss the contribution of melanocyte stem cells (MSCs) to human hair greying, arguing that MSCs are late-comers to this event, which do not initiate but ultimately determine the potential for reversibility of greying. Further, we discuss the key role of oxidative stress in the greying process, in the context of dysregulated intrafollicular increases in free radicals, from both melanocyte-intrinsic and -extrinsic sources. Finally, we define key remaining questions in greying research.

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FIGURE 1 :Hair cycle-dependent changes in pigmentation. The human hair follicle (HF) undergoes cyclic regeneration from growth phase (anagen) to regression (catagen) to resting phase (telogen). In Anagen VI, both hair fibre production and pigmentation is maximal, with the highest density of glycoprotein 100 (gp100)-expressing melanocytes present in the bulb. Amelanotic melanocytes are present in the matrix and outer root sheath, and those in the proximal tip of the matrix express several propriomelanocortin (POMC) peptides and corticotropin-releasing hormone receptor (CRHR). Hair greying can begin only during anagen, because the HF engages in hair shaft production and pigment synthesis (melanogenesis) exclusively during anagen III–VI. During HF regression, hair matrix (HM) keratinocytes and many mature melanocytes of the hair follicle pigmentary unit (HFPU) undergo cell death and regress, while the proximal hair shaft becomes depigmented and serrated (‘club hair’) without any new hair shaft being produced. In telogen, no mature (differentiated) melanocytes exist in the HM and melanocyte stem cells (MSCs) in the bulge region remain quiescent (undifferentiated). Upon anagen re-entry, MSCs initiate migration and differentiation down the outer root sheath (ORS) to give rise to the differentiated melanocytes of the HFPU. Major positive (green) and negative (red) regulators of human HF pigmentation are listed. α-MSH, alpha melanocyte-stimulating hormone; BMAL1, brain and muscle ARNT-like 1; CTS, connective tissue sheath; HFSC, hair follicle stem cell; HGF, hepatocyte growth factor; HS, hair shaft; IRS, inner root sheath; MITF, micropthalmia-associated transcription factor; PER1, period circadian regulator 1; SCF, stem cell factor; T3, triiodothyronine; T4, tetraiodothyronine; TRH, thyrotropin-releasing hormone.

HAIR CYCLE-COUPLED REGULATION OF PIGMENTATION AND GREYING
The human HF contains several sub-populations of melanocytes (Tobin, 2008a) that are distinct from those in the epidermis (Tobin & Bystryn, 1996). These are divided across different histologic compartments based on differentiation status, pigment content and function (Fig. 1).

In pigmented Anagen VI HFs, melanocytes expressing active tyrosinase and its major product, the melanogenic intermediate dihydroxyphenylalanine (DOPA), are found in the basal infundibulum as well as in the hair matrix surrounding the mid to upper dermal papilla (those in the latter region constitute the HFPU) (Kukita & Fitzpatrick, 1955). Melanogenesis occurs within unique lysosome-derived, membrane-bound organelles termed ‘melanosomes’ that are transferred to the surrounding keratinocytes of the hair shaft via dendritic and filopodial processes.

The mechanism of transfer is not yet entirely clear, and is suspected to vary by tissue and species (Wu & Hammer, 2014). Human melanoblasts, or MSCs, are located in the bulge region of the human HF and are poorly or un-pigmented, containing few dendrites and low/no expression of glycoprotein 100 (gp100, also known as Pmel17/Silv) (Tobin & Bystryn, 1996). The hair bulb also contains an immature subpopulation of amelanotic (unpigmented) melanocytes in the proximal and peripheral regions (Tobin & Paus, 2001; Tobin & Kauser, 2005). Amelanotic melanocytes that are negative for DOPA oxidase activity of tyrosinase (but which may express inactive tyrosinase at the protein level) are also found in the mid to lower outer root sheath (Horikawa et al., 1996). It has been suggested that such amelanotic melanocytes are progenitors differentiated from MSCs (Tobin, 2008b). However, this role has yet to be confirmed.

HF pigmentation is strictly coupled to the hair cycle. In early anagen (I–II) no pigment is produced as the HF grows and differentiates following telogen (Oh et al., 2016). Subsequently, pigmentation is initiated during anagen III, and reaches its maximum in anagen VI HFs, both in mice and humans (Slominski, Paus & Costantino, 1991; Slominski & Paus, 1993; Slominski et al., 1994, 2005b; Müller-Röver et al., 2001; Oh et al., 2016) (Fig. 1). Current evidence (mostly from the mouse) suggests that in catagen, most differentiated HFPU melanocytes undergo apoptosis, while bulge MSCs survive, possibly along with some amelanotic melanoblasts and other melanocyte progenitors in the secondary hair germ (Tobin, 1998; Tobin et al., 1999; Commo & Bernard, 2000).

This is equally evident in experimental induction of catagen using toxic agents such as cyclophosphamide and dexamethasone, as pioneered in mouse hair research (Slominski et al., 1996; Ermak & Slominski, 1997). Therefore, the prevailing view is that follicular MSCs that survive catagen contribute immature progenitors to replenish bulbar melanogenic melanocytes at the start of each new anagen phase (Tobin et al., 1999; Commo & Bernard, 2000; Nishimura et al., 2002).

In humans, it is not yet clear which MSC sub-populations repopulate the HFPU during early anagen. However, corticotropin-releasing hormone (CRH) receptor+ melanocyte progenitor cells located in the most proximal hair matrix of human anagen HFs (Ito et al., 2004b) could represent an alternative, bulge MSC-independent melanoblast pool. It is likely that these cells retreat into the secondary hair germ during catagen development, similar to HF progenitor cells (Geyfman et al., 2015; Oh et al., 2016; Panteleyev, 2018), and may have a key role in re-establishing the HFPU during the subsequent anagen. Probing this hypothesis is important for understanding both hair greying and the hair cycle.

The gradual nature of greying is evident upon histological investigation. Grey HFs contain a reduced number of differentiated, i.e. melanogenically active, bulbar melanocytes (Commo, Gaillard & Bernard, 2004; Nishimura, Granter & Fisher, 2005; Tobin, 2009) (Fig. 2). Grey HF-resident melanocytes show vacuolization and incomplete melanization of melanosomes at the ultrastructural level (Orfanos, Ruska & Mahrle, 1970), and contain aberrantly distributed melanosomes (Nordlund et al., 2008).

Mature melanocytes may exhibit a dystrophic hypertrophic state during greying, accompanied by interrupted melanin transfer, and ectopic distribution of melanin in the dermal papilla and connective tissue sheath (Tobin, 2009), possibly due to phagocytosis of melanocytes or melanocyte fragments (Tobin, 1998). Some melanocytes in the HFPU become apoptotic [as observed via Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining], while those that remain exhibit reduced or abolished dendricity, and occasionally ectopic positioning in the proximal matrix (Arck et al., 2006).

Eventually, ‘white’ HFs exhibit a complete loss of melanin transfer to the hair shaft, usually accompanied by the corresponding loss of all bulbar melanocytes. However, it is important to emphasize that even optically white scalp HFs may still contain a few isolated hair bulb melanocytes, some of which may engage in residual melanogenesis despite a lack of both dendritic morphology and transfer of melanin to the hair shaft (Arck et al., 2006) (Fig. 2).

Amelanotic melanocytes present in the bulge, outer root sheath (ORS) and proximal matrix (Horikawa et al., 1996; Ito et al., 2004b; Commo, Gaillard & Bernard, 2004; Slominski et al., 2005b; Nishimura, Granter & Fisher, 2005) persist beyond the loss of the bulbar melanocytes, but are gradually lost over time. For instance, the number of gp100+ cells drops progressively in the ORS from grey and white HFs, and in some cases gp100+ cells of white HFs are completely absent (Horikawa et al., 1996; Commo, Gaillard & Bernard, 2004). Likewise, Nishimura, Granter & Fisher (2005) noted a progressive reduction and eventual loss and differentiation of speculative MSCs expressing low levels of micropthalmia-associated transcription factor (MITF; MITFlow) in the bulge of human scalp HFs over the course of ageing.

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FIGURE 2 : The cellular composition of the human hair follicle (HF) changes during greying. (A) Illustration summarizing the compositional differences between the melanocyte compartment of white, grey and pigmented human scalp HFs, based on Horikawa et al. (1996), Commo, Gaillard & Bernard (2004), Nishimura, Granter & Fisher (2005) and Tobin (2009). In fully pigmented follicles, the anagen hair bulb is rich in pigment-producing melanocytes, but also contains amelanotic melanocytes in its peripheral epithelium. During greying, pigmented melanocytes are lost from the bulb, although some ectopic pigmentation may be observed near the bulge (Nishimura, Granter & Fisher, 2005). Ultimately, glycoprotein 100 (gp100)-positive amelanotic melanocytes and presumptive melanocyte stem cells (MSCs) in the bulge are lost as well. Histochemically grey/white HFs demonstrate a lack, or very substantial reduction, of melanin (B, F), while immunofluorescence microscopy shows reduced expression of the pre-melanosome marker gp100 (C, G) and histochemically detectable tyrosinase activity (D, H). In the bulge, the number of gp100+ amelanotic melanocytes (see arrowheads) is reduced in grey hairs (E, I). CTS, connective tissue sheath; DAPI, 4′,6-diamidino-2-phenylindole; DP, dermal papilla; HFPU, hair follicle pigmentary unit; HM, hair matrix; MITFlow, micropthalmia-associated transcription factor (low = low expression); ORS, outer root sheath.

Melanocyte sub-populations across the HF therefore appear to be differentially lost during ageing, with tyrosinase (DOPA) + melanocytes in the bulb disappearing first, followed by amelanotic melanocytes and presumptive MSCs in the bulge region, which remain unaffected for a long time after hair greying has already affected the HFPU.

Currently, the stimuli that initiate melanocyte loss in the HFPU remain unclear. Certainly, the concept that failure of bulge MSCs to replenish the HFPU between each hair cycle is responsible for the onset of greying in human HFs is frequently asserted (Jo et al., 2018; Clark & Deppmann, 2020), but still lacks supporting evidence. There is also no evidence that bulge MSCs replenish the human HFPU during the multi-year lifespan of anagen VI HFs (Tobin, 2011; Oh et al., 2016) (Fig. 1), despite the otherwise well-documented migration of these cells to the epidermis (Chou et al., 2013; Paus, 2013).

Thus, in light of the dystrophic changes occurring within mature melanocytes in grey follicles (Orfanos, Ruska & Mahrle, 1970; Arck et al., 2006; Nordlund et al., 2008; Tobin, 2009), the causes for greying initiation in humans must be sought within the HFPU, and possibly intra-bulbar melanocyte progenitor cells, but not the bulge. It is also important to note that progressive or temporary loss of melanin transfer from HFPU melanocytes into hair shaft keratinocytes (trichocytes) occurs exclusively during anagen because the functional HFPU exists only during anagen III–VI (Fig. 1) (Tobin et al., 1998).

ARE GREYING AND PIGMENTATION GENETICALLY CONTROLLED?
Given the considerable phenotypic overlap between mouse mutants with a hair pigmentation phenotype (Nakamura et al., 2013) and the hair pigmentation abnormalities seen in human patients with corresponding genetic abnormalities, at least some shared essential genes in melanogenesis and melanocyte differentiation, such as tyrosine protein kinase kit (c-kit), MITF, tyrosinase-related proteinase 1 (TRP-1), TRP-2/DCT, paired box gene 3 (PAX3), SRY-box 10 (SOX10), biogenesis of lysosome-related organelles complex 3 (BLOC-3), and neurofibromin 1 (NF1), have been identified (Nordlund et al., 2008; Pingault et al., 2010; Sleiman et al., 2013; Feng, Sun & Wang, 2014; Kubasch & Meurer, 2017; Saleem, 2019).

The role of genes in human greying remains poorly understood, but there are clear trends in greying onset within kinships and between populations. The age of greying onset is linked to geographic ancestry, and significant greying is considered premature when it occurs before 20 years of age in Caucasians, before 25 years in populations of Asian ancestry and before 30 years of age in populations with African ancestry (Tobin, 2009; Sonthalia, Priya & Tobin, 2017; Kumar, Shamim & Nagaraju, 2018). Men also grey faster than women, but in distinct scalp regions (Panhard, Lozano & Loussouarn, 2012).

Although ethnic and sex differences suggest a genetic component in the age of greying onset, heritability itself seems to vary regionally; in a twin-controlled study of heritability within Danish and British Caucasians, the onset of greying was highly heritable (Gunn et al., 2009). By contrast, in another larger study of facial and scalp hair features across an ethnically diverse Latin American cohort, hair greying had the lowest heritability of all traits studied (Adhikari et al., 2016), with greying-associated single nucleotide polymorphisms (SNPs) explaining only 6.7% of the observed phenotypic variation.

A comparison of individual HFs on the same scalp – which consequently share the same genetic makeup and exposome – indicates a high level of HF-to-HF heterogeneity, and typically early-greying HFs undergo depigmentation decades before other HFs on the same head (Panhard, Lozano & Loussouarn, 2012). This is perhaps most evident in so-called steel/salt and pepper-headed individuals, which can remain a life-long feature. Intra-individual and inter-follicular heterogeneity strongly argues against a solely genetic pathobiology of greying, as it necessarily implicates modifiable factors that operate at the single-HF organ level.

So far, only a single SNP has been significantly associated with greying in an admixed Latin American population with European ancestry. The SNP is in the interferon regulatory factor 4 gene (IRF4) (Adhikari et al., 2016), and mechanistically would be expected to affect the HFPU directly. Since IRF4 and MITF act cooperatively to activate transcription of tyrosinase (TYR) in cultured human melanoma cells (Praetorius et al., 2013), this presumptive driver of human greying (Adhikari et al., 2016) would be associated with reduced tyrosinase activity in the HFPU, rather than in MSC maintenance, since the bulge is constitutively tyrosinase-negative. However, functional evidence that IRF4 really playes an important role in human hair graying is still missing.

In a separate study, an HFPU-centric model of greying onset is also suggested by changing patterns of expression of melanogenesis genes during premature greying. In a small sample population, reduced expression of HFPU-resident melanogenic enzymes was accompanied by an increase in their corresponding, complementary inhibitory microRNAs (Bian et al., 2019). Overall, whilst loss of HF niche-resident MSCs is very likely responsible for the irreversibility of hair greying, the process invariably requires and begins with primary changes in melanogenesis, melanosome transfer and/or HF melanocyte survival within the anagen HFPU.

As critical as MITF and tyrosinase are for the control of melanocyte function and melanogenesis (Levy, Khaled & Fisher, 2006; Ganesan et al., 2008; Vachtenheim & Borovanský, 2010; Chen et al., 2018), neither the human nor mouse HFPU appear to be controlled by a single master gene. Instead, the importance of metabolites, co-factors, pH and other biochemical determinants of the local activity of melanogenesis enzymes, as well as reactive oxygen species (ROS) production and scavenging (Schallreuter et al., 1994; Wood et al., 2004, 2009; Slominski et al., 2005b) cannot be overemphasized. Furthermore, the HFPU is subject to a flux of numerous regulatory inputs that jointly ensure its appropriate function, mediating hair cycle-associated changes in HFPU activity and modulating melanin production, melanocyte survival, migration, and proliferation/apoptosis ratios. Currently recognized regulators of the human HFPU are summarized in Fig. 3.

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FIGURE 3 : Positive and negative regulators of pigmentation in the human hair follicle pigmentary unit (HFPU). Positive regulators are indicated by arrows, negative regulators by inhibitor lines. ACTH, adrenocorticotropic hormone; α-MSH, alpha melanocyte-stimulating hormone; BMAL1, brain and muscle ARNT-like 1; CRH, corticotropin-releasing hormone; c-KIT, tyrosine protein kinase Kit; c-Met, tyrosine protein kinase Met; FGF, fibroblast growth factor; GR, glucocorticoid receptor; HGF, hepatocyte growth factor; HPA, hypothalamus–pituitary–adrenal axis; MC1R, melanocortin receptor; MITF, micropthalmia-associated transcription factor; NGF, nerve growth factor; PER1, period circadian regulator 1; POMC, propriomelanocortin; SCF, stem cell factor; T3, triiodothyronine; T4, tetraiodothyronine; TRH, thyrotropin-releasing hormone; TrkA, tyrosine receptor kinase A; TSC, tuberous sclerosis complex.

How exactly the expression/activity of these regulators is altered in greying human HFs compared to their fully pigmented counterparts in vivo remains unknown, but ex vivo studies on micro-dissected and organ-cultured human scalp HFs are revealing how these regulators may influence HFPU activity in vivo.

Additionally, given the general role of autophagy in skin ageing (Eckhart, Tschachler & Gruber, 2019), that a certain level of autophagic flux in the anagen hair matrix is required for anagen maintenance of human scalp HFs ex vivo (Parodi et al., 2018), and that functional autophagy appears crucial for melanocytes to cope with cellular stress and oxidative damage (Zhou et al., 2018; Kim et al., 2019; Qiao et al., 2020), it is conceivable that insufficient autophagic flux within the HFPU may contribute to hair greying.

Both thyroid hormones [triiodothyronine (T3) and tetraiodothyronine (T4)] (Van Beek et al., 2008) and the central neuroendocrine regulator of the hypothalamic–pituitary–thyroid (HPT) axis, thyrotropin-releasing hormone (TRH), which is produced within human scalp HFs (Slominski et al., 2002b; Gáspár et al., 2010), stimulate melanin production in the HFPU of human anagen VI HFs (Gáspár et al., 2011).

Pigmentation is also regulated by the activity of an intrinsic hypothalamus–pituitary–adrenal (HPA) axis within the follicle, such that upstream corticotropin-releasing hormone (CRH), its receptor, as well as downstream adrenocorticotropic hormone (ACTH), alpha melanocyte-stimulating hormone (α-MSH), and the melanocortin receptors are all produced within the HF itself and promote pigmentation (Slominski et al., 1999, 2004a, 2004b; Ito et al., 2005; Kauser et al., 2005, 2006; Van Beek et al., 2008; Meyer et al., 2009; Gáspár et al., 2011).

Study of hair pigmentation in knockout mice unable to synthesize downstream HPA hormones suggests that their action is secondary to TRH, which promotes pigmentation in their absence (Slominski et al., 2005a) likely via the melanocortin receptor (Schiöth et al., 1999; Slominski et al., 2002b), although this is yet to be verified in the human HF. Whilst known aspects of neuroendocrine regulation of hair pigmentation have been reviewed in detail elsewhere (Slominski et al., 2004b; Paus et al., 2014), the extent to which changes in these hormonal axes intrinsic to the HF affect the greying process has not been systematically investigated. This also applies to cytokines and growth factors such as nerve growth factor (NGF), stem cell factor (SCF) and hepatocyte growth factor (HGF) that protect human HFs from pigment loss ex vivo (Botchkareva et al., 2001; Campiche et al., 2019).

Hormonal stimulation of human melanocytes by the intrinsic HPA-like axis (Price et al., 1998), or by TRH (Gáspár et al., 2011) promotes MITF expression and it is likely that reduced intrafollicular production of and stimulation by melanotropic hormones may result in lowered MITF activity within the HFPU of greying human HFs, ultimately causing insufficient melanogenesis and melanosome transfer (Slominski et al., 2004b; Paus, 2011).

As illustrated in Fig. 4, a reduction of these melanotropic HPA hormones can be observed in the hair bulb epithelium of grey/white human scalp HFs, while hair pigmentation-stimulatory drugs such as fluoxetine have been suggested to up-regulate intrafollicular α-MSH expression in some white human scalp HFs ex vivo (Chéret et al., 2020). This provides further circumstantial support for the concept that a relative decline in the intrafollicular production of key melanotropic neurohormones contributes to the greying process (Paus, 2011; Paus et al., 2014).

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FIGURE 4 : Immunohistochemistry illustrates a reduction in hypothalamus–pituitary–adrenal (HPA) axis hormones in grey follicles. A reduction in immunoreactivity is seen for adrenocorticotropic hormone (ACTH) (A, D), corticotropin-releasing hormone (CRH) (B, E) and alpha melanocyte-stimulating hormone (α-MSH) (C, F) in grey hair follicles. Images in A and B have been reproduced with additional annotations from Ito et al. (2005) and Ito et al. (2004b), respectively. Images C–F are original immunohistochemical images. DAPI, 4′,6-diamidino-2-phenylindole; DP, dermal papilla.

A fundamentally important concept is that HF cycling controls HFPU activity, and thereby hair greying, insofar as defective hair shaft pigmentation can only occur during anagen (there is currently no evidence that isolated it changes in HFPU activity can regulate HF cycling). It therefore deserves emphasis that some recently discovered, potent intrafollicular regulators of human HF cycling also regulate the HFPU and its melanin production in a hair cycle-independent manner, namely P-cadherin (Samuelov et al., 2012, 2013), TRH (Gáspár et al., 2010, 2011), and peripheral clock activity [i.e. circadian locomotor output cycles kaput (CLOCK), brain and muscle ARNT-like 1 (BMAL1), Period1] (Al-Nuaimi et al., 2014; Hardman et al., 2015). The roles of such dual regulators that independently impact upon human anagen duration and intrafollicular melanin production have not been systematically dissected in the context of greying.

THE ROLE OF MELANOCYTE STEM CELLS IN GREYING
To guide future efforts in human hair greying research, it is important to avoid conceptual confusion. Ever since the landmark discovery of bulge MSCs and their involvement in hair greying (Nishimura et al., 2002; Nishimura, Granter & Fisher, 2005), it has become a frequently repeated misconception that damage to bulge MSCs is the primary cause of human hair greying (e.g. Jo et al., 2018; Qiu et al., 2019).

As explained above, greying of human scalp HFs begins within the highly differentiated melanocytes of the HFPU within a single anagen phase. As there is no evidence to support the view that continuous replenishment of the scalp HFPU from the bulge MSC reservoir is required to support a single anagen VI phase in human HFs, the latter appears to be self-maintained.

However, the verdict that greying always results initially from major HFPU dysfunction in the anagen hair matrix, i.e. far distant from the bulge and independent of MSC activities, does not preclude the simultaneous, independent accumulation of (eventually irreversible) MSC damage over time in the bulge stem cell niche. Moreover, once a HF has become depleted of its melanocyte stem/progenitor pools, this inevitably results in an inability to generate melanin, but only after its HFPU has stopped operating during catagen. Therefore, the contribution of irreparable damage to MSCs, leading to their loss during human HF ageing (Commo, Gaillard & Bernard, 2004; Nishimura, Granter & Fisher, 2005) clearly plays a critical role in long-term irreversible human hair greying and warrants detailed coverage.

In mice, MSC progeny from the sub-bulge region repopulate the secondary hair germ before the onset of each new anagen stage (Nishimura et al., 2002). Histological snapshots from the hair cycle indicate a homologous series of events in humans, although it remains ambiguous which MSC sub-populations exactly contribute to the rebuilding of the human HFPU during anagen development (Commo & Bernard, 2000).

Failure to maintain this stem cell niche is associated with a variety of different greying phenotypes in mouse models, with some translatable insights into humans. For instance, mice deficient in B-cell lymphoma 2 (Bcl2), an anti-apoptosis factor that preserves mitochondrial membrane integrity (Kale, Osterlund & Andrews, 2018), exhibit rapid greying at 8 weeks of age, as all MSCs are lost from the niche after the first anagen phase (Nishimura, Granter & Fisher, 2005).

Reportedly, loss of MSCs in these mice was not due to apoptosis, but rather resulted from their irreversible ‘ectopic differentiation’ into pigmented, dendritic melanocytes. Likewise, evidence of ectopic differentiation within MSCs was reported in humans during middle age (Nishimura, Granter & Fisher, 2005), but not in senile white HFs (Commo, Gaillard & Bernard, 2004; Nishimura, Granter & Fisher, 2005). Therefore, understanding the molecular machinery that preserves MSC stemness and quiescence (Fig. 5) remains an important facet of understanding the entire greying process.

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FIGURE 5 : Melanocyte stem cell (MSC) depletion contributes to hair shaft greying in mice. (A) During telogen, MSCs in the sub-bulge niche are required to migrate to the secondary hair germ in order to replenish the hair follicle pigmentary unit (HFPU), similarly to humans (Tobin et al., 1998). (B) MSCs must be maintained in a quiescent state between episodes of replenishment. Transforming growth factor beta (TGF-β) is an important signal emitted from the surrounding epithelial hair follicle stem cells (eHFSCs) to promote quiescence. (C) Failure to maintain MSC quiescence due to ageing, genotoxic stress and adrenergic activity results in ectopic MSC differentiation within the niche. Differentiated melanocytes are no longer able to replenish the HFPU, and greying becomes permanent. Ectopic pigmentation within the bulge has also been reported during human hair follicle greying (Commo, Gaillard & Bernard, 2004; Nishimura, Granter & Fisher, 2005). Col17a1, collagen alpha-1(XVII) chain; SNS, sympathetic nervous system.

Stress-induced sympathetic nervous activity has been implicated in driving ectopic MSC differentiation in mice (Zhang et al., 2020). Along with radiation-induced poliosis (Gao et al., 2019) these experimental tools must not be equated and conflated with spontaneous, physiological ageing-associated greying in human HFs. However, it is instructive to highlight that increased exposure to ionizing radiation and genotoxic reagents during telogen results in increased levels of ectopically differentiated MSCs and poliosis during the next anagen phase (Inomata et al., 2009).

Additionally, compromised signal transduction upstream of DNA repair mechanisms lowers the dose of ionizing radiation required to produce both ectopic pigmentation in the bulge and a grey phenotype (Inomata et al., 2009). It is noteworthy that reactive oxygen species (ROS) are also well-known inducers of DNA damage and inhibit DNA repair (Van Houten, Santa-Gonzalez & Camargo, 2018). The effects of oxidative damage upon MSC maintenance in the human HF remains poorly understood, but this should constitute a key component of further efforts to understand the progression of greying in humans (Jo et al., 2018).

Similarly to ageing, hypomorphic mutation of Mitf elicits a more gradual process of greying (Lerner et al., 1986) associated with progressive ectopic differentiation of MSCs in the sub-bulge region of unpigmented hairs (Nishimura, Granter & Fisher, 2005; Harris et al., 2018). Current evidence in mice suggests that bulge-resident epithelial HF stem cells (eHFSCs) maintain adjacent MSCs in an immature state with quiescence-promoting transforming growth factor beta (TGF-β) signalling that is dependent on the expression of eHFSC-derived collagen alpha-1(XVII) chain (Col17a1) (Nishimura et al., 2010; Tanimura et al., 2011) (Fig. 5).

Genotoxic damage results in proteolysis of Col17a1 and ectopic differentiation of MSCs (Matsumura et al., 2016). Knocking out either Col17a1 or TGF-β in mice induces gradual greying and accompanying ectopic differentiation of MSCs in the sub-bulge. When MSCs are required to populate the hair germ during telogen, Wingless-related integration site (Wnt) signalling and endothelin originating from eHFSCs are subsequently required for MSC proliferation and differentiation (Rabbani et al., 2011), and are therefore candidate signals in the acceleration of greying.

It remains unknown to what extent these controls of quiescence and differentiation (Fig. 5) are mirrored in the maintenance of human MSCs in the bulge and other HF compartments. Characterising these surely is important to mechanistically understand the progression of greying to an irreversible stage.

THE ROLE OF OXIDATIVE STRESS IN GREYING
While a key role for oxidative stress in greying has long been appreciated (Trüeb & Tobin 2010, Paus 2011, Arck et al. 2006, Wood et al., 2009), many details on how exactly -excessive ROS levels damage the HFPU are as yet insufficiently understood.

Management of oxidative stress within the HF
Within melanocytes, tyrosine hydroxylation and DOPA-to-melanin oxidation in the melanogenesis pathway lead to high levels of ROS release, which are managed by an efficient local antioxidant system (Paus, 2011; Trüeb, 2015). This system include catalase, methionine sulfoxide reductase A and B (MSRA/MSRB) (Wood et al., 2009), Bcl-2 (Peters, Arck & Paus, 2006), nuclear factor erythroid 2-related factor 2 (Nrf2) (Haslam et al., 2017), tyrosinase-related protein-2 (TRP-2) (Michard et al., 2008), and even intrafollicular melatonin production (Kobayashi et al., 2005; Fischer et al., 2008). Eumelanin itself also effectively scavenges ROS (Wood et al., 1999), although its antioxidant activity is sequestered within and restricted to the melanosome itself. Thus, it has been proposed that impaired antioxidant systems and over-accumulation of ROS cause melanocyte damage during ageing, with external stimuli [inflammation, ultraviolet (UV), smoking and oxidizing agents] also contributing to the loss of redox balance (Tobin & Paus, 2001) (Fig. 6).

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FIGURE 6 : Oxidative stress plays a key role in hair greying. (A) In the bulge region of grey hair follicles (HFs), human melanocyte stem cells (MSCs) are reduced in number and undergo ectopic differentiation. The hair shaft displays millimolar concentrations of H2O2 and oxidation of methionine residues to methionine sulfoxide (Wood et al., 2009). (B) In pigmented HFs, the pre-cortical zone displays the ‘ring-of-fire’, an area containing high amounts of reactive oxygen species (ROS), suspected to be crucial in keratinocyte (KC) differentiation and hair shaft (HS) formation (Lemasters et al., 2017), whereas the HF bulb contains lower ROS levels. In grey HFs, an increase in ROS correlates with decreased melanocyte number, increased melanocyte death and increased mitochondrial DNA (mtDNA) deletion (a marker of accumulating oxidative stress damage) (Arck et al., 2006). Grey HFs exhibit a reduction in the antioxidant enzymes catalase and methionine sulfoxide reductase A and B (MSRA/MSRB) (Wood et al., 2009; Shi et al., 2014). It is also possible that grey HFs exhibit an accumulation of senescent melanocytes that cannot be removed through apoptosis. (C) The roles of melatonin in human HF pigmentation, both proven and hypothetical (marked ‘?’). BMAL1, brain and muscle ARNT-like 1; CLOCK, circadian locomotor output cycles kaput; Nrf2, nuclear factor erythroid 2-related factor

The HFPU is sensitive to oxidative damage, as can be seen by exposing HFs to H2O2 (Arck et al., 2006, Wood et al. 2009) or ROS-generating cytotoxic agents that induce lipid peroxidation, mitochondrial DNA (mtDNA) deletion and cell death (Bodó et al., 2007; Haslam et al., 2017). Greying hair bulbs often display vacuolated melanocytes, a common cellular response to oxidative stress (Nordlund et al., 2008; Tobin, 2009).

A recent study reported specific nuclear expression of ataxia-telangeictasia mutated (ATM; a serine/threonine protein kinase that is a major regulator of the cellular response to DNA double-strand breaks) within the HFPU. ATM expression increases in primary human scalp HF melanocytes in response to incubation with H2O2 and was essential for maintaining melanocyte viability during oxidative stress. Expression of ATM correlated positively with the pigmentary status of greying HFs (Sikkink et al., in press), suggesting that reduced capacity of melanocytes to resist genotoxic challenge is also characteristic of the greying follicle.

There is emerging evidence that human HF melanocytes, compared to epidermal melanocytes, are more susceptible to chronological ageing, as evidenced by their unique progressive loss of catalase expression and activity (Kauser et al., 2011). A comprehensive characterization of changes in prematurely grey HFs compared to pigmented ones shows downregulated gene expression of the pigmentation-associated genes TYR, DCT, TYRP1, melanocortin 1 receptor (MC1R) and MITF, as well as downregulation of CAT (catalase), GPX1 (glutathione peroxidase 1) and SOD (superoxide dismutase) (Shi et al., 2014), suggesting that the collapse of the antioxidant system within HFPU melanocytes plays a critical role in greying initiation. Grey and white human HFs also show reduced levels of MSRA/MSRB (Wood et al., 2009).

Additionally, the roles of glutathione-s-transferases (GSTs), a class of enzymes with antioxidant properties that offer protection against oxidative stress (Da Fonseca et al., 2010), should also be studied in the context of human hair greying since human HFs express GSTs (Dijkstra et al., 1986; Puerto et al., 1994); GST levels reportedly decrease by 78% with age alongside an 88% drop in glutathione reductase (Pruche, Kermici & Prunieras, 1991), suggesting a compromised capacity to protect against peroxides as HF ageing progresses.

Interestingly, vasoactive intestinal peptide (VIP) receptor expression is detected in the murine bulge during anagen IV (Wollina, Paus & Feldrappe, 1995). In human anagen HFs, VIP receptors vasoactive intestinal peptide receptor 1(VPAC1) and VPAC2 are also expressed, and are reduced in alopecia areata (Bertolini et al. 2016b), raising the question whether VIP signalling is also relevant in the context of hair greying and oxidative stress, since VIP has been shown to counteract ROS production through inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and stimulation of SOD2 (Fujimori et al., 2011).

Treating human HFs with VIP increases the number of c-Kit+ HF melanocytes (most of which are immature/amelanotic), gp100+, MITF+ and phospho-MITF+ HFPU melanocytes, as well as melanin synthesis; melanogenesis is also stimulated during in vitro culture of isolated HFPU melanocytes following VIP treatment (Shi et al., 2014; Bertolini et al., 2016a).

It has also been shown that methionine residue 374 (Met374) of tyrosinase, the rate-limiting enzyme of melanogenesis, constitutes a prime target for H2O2-mediated oxidation (Wood et al., 2009). Thus, insufficiently scavenged H2O2 within the HFPU could promote oxidation of methionine sulfoxide (Met-S=O), resulting in a dose-dependent inhibition of tyrosinase activity and so of HF melanogenesis which is preventable by free L-methionine application (Wood et al., 2009) (Fig. 6). Accumulation of excessive H2O2 in the presence of reduced catalase activity could therefore directly damage the HFPU’s enzymatic melanin synthesis and its capacity to protect itself from oxidative damage by melanin production, resulting in a vicious circle.

Sources of ROS in and around the HFPU
Oxidative damage of the HFPU can result from failure of the antioxidant control systems described above, but alternatively has been attributed to excessive intrinsic and extrinsic ROS production (Fig. 6). Melanogenesis itself generates substantial amounts of superoxide radical (Riley, 1988) and H2O2 (Koga, Nakano & Tero-Kubota, 1992; Nappi & Vass, 1996), and therefore represents a major source of melanocyte-intrinsic oxidative stress. If the ROS-generating enzymatic cascade culminating in melanin synthesis is stimulated, but the ROS-scavenging end-product eumelanin is insufficiently synthesized, e.g. owing to an inhibition of post-DOPA steps of melanogenesis (Slominski, Paus & Bomirski, 1989; Slominski et al., 2005b), or blunting of redox balancing capacity, this could result in major oxidative damage to the HFPU.

Within the proximal HF ORS and hair matrix, human HF keratinocytes are richly endowed with energetically active mitochondria (Vidali et al., 2014), and when HF keratinocytes terminally differentiate in the IRS and pre-cortical hair matrix, they show heightened membrane potential and ROS generation, after which they abruptly depolarize and secrete a circumferential spike of ROS, described as the HF ‘ring of fire’ (Lemasters et al., 2017).

Thus, insufficient scavenging controls of this region would be expected ultimately to damage the HFPU. Co-culturing human neonatal epidermal melanocytes with epidermal keratinocytes in which catalase is inhibited increases ROS levels within the melanocytes (Pelle et al., 2005), suggesting that compromised antioxidant systems in neighbouring keratinocytes may indeed contribute to melanocyte destruction or impairment.

It has been hypothesized that ‘bleaching’ of the melanin polymer itself by diffusible H2O2 may be responsible for the striking phenomenon of sudden greying (canities subita) (Paus, 2011). However no ‘bleached’ melanosomes have yet been identified in the hair shaft ultrastructure of typical grey/white HFs. Instead, initially, the grey hair shaft seems to be characterized by reduced density of smaller, yet still pigmented melanosomes (Cho et al., 2014).

Additional melanocyte-extrinsic modulators of ROS are human perifollicular mast cells (Jadkauskaite et al., 2018) and Substance P-mediated neurogenic inflammation (Peters et al., 2007). In human HFs, Substance P activates mast cell degranulation and induces catagen, thus turning off melanogenesis. Like the HF (Haslam et al., 2017), human perifollicular mast cells also show substantial NRF2 activity, which can counteract oxidative damage associated with substance P-induced mast cell degranulation (Jadkauskaite et al., 2018).

Lastly, UV radiation may also constitute a major and extrinsic source of ROS for HFs (Zhang et al., 1997; De Jager, Cockrell & Du Plessis, 2017; Slominski et al., 2018). When depilated mice were radiated with UV-A after applications of a gel containing the light-sensitive drug psoralen, and the antioxidant enzyme SOD, black hair re-grew on their backs; however, when no SOD, or heat-inactivated SOD was added together with the psoralen, solely grey or white hairs regenerated during anagen in 90% of treated mice (Emerit et al., 2007). Therefore, topical application of SOD, presumably penetrating via the transfollicular route, acted to prevent HFPU melanocyte-specific damage in these mice. It remains unclear whether MSCs were also affected in this study, or whether greying was reversible long term.

Irradiating human HFs ex vivo with 20 or 50 mJ/cm2 UV-B radiation impaired hair shaft elongation, HFPU melanogenesis and induced premature catagen via both apoptotic cell death (20 mJ) and necrosis (50 mJ) (Lu et al., 2009). These human HFs did not undergo greying similar to the murine study (Emerit et al., 2007), and both keratinocytes and melanocytes were affected similarly.

This is likely owing to the lack of epidermis, dermis and dermal white adipose tissue within the experimental design, the former of which is biologically designed to absorb a high amount of UV radiation with specific antioxidant mechanisms acting as protective agents (Brand et al., 2018; Dunaway et al., 2018). However, UV radiation may have a more damaging role to the HFPU than previously thought, since recent evidence suggests that transepidermally applied UV irradiation exerts substantial HF photodamage, which can be counteracted by topical caffeine ex vivo (Gherardini et al., 2019).

REFERENCE LINK : https://onlinelibrary.wiley.com/doi/full/10.1111/brv.12648


More information: Ayelet M. Rosenberg et al, Quantitative mapping of human hair greying and reversal in relation to life stress, eLife (2021). DOI: 10.7554/eLife.67437

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