Glutamic acid promotes hair growth in mice

Abstract

Glutamic acid is the main excitatory neurotransmitter acting both in the brain and in peripheral tissues. Abnormal distribution of glutamic acid receptors occurs in skin hyperproliferative conditions such as psoriasis and skin regeneration; however, the biological function of glutamic acid in the skin remains unclear. Using ex vivo, in vivo and in silico approaches, we showed that exogenous glutamic acid promotes hair growth and keratinocyte proliferation. Topical application of glutamic acid decreased the expression of genes related to apoptosis in the skin, whereas glutamic acid increased cell viability and proliferation in human keratinocyte cultures. In addition, we identified the keratinocyte glutamic acid excitotoxic concentration, providing evidence for the existence of a novel skin signalling pathway mediated by a neurotransmitter that controls keratinocyte and hair follicle proliferation. Thus, glutamic acid emerges as a component of the peripheral nervous system that acts to control cell growth in the skin. These results raise the perspective of the pharmacological and nutritional use of glutamic acid to treat skin diseases.

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
Scientic Reports | (2021) 11:15453 | 
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Glutamic acid promotes hair
growth in mice
Carlos Poblete Jara 1,3,5,7*, Beatriz de Andrade Berti 1,3,5, Natália Ferreira Mendes 1,3,5,
Daiane Fátima Engel 2,3,5, Ariane Maria Zanesco 2,3,5, Gabriela Freitas Pereira de Souza
4,5, Renan de Medeiros Bezerra 1,3,5, Julia de Toledo Bagatin 6, Silvya Stuchi Maria‑Engler
6, Joseane Morari 2,3,5, William H. Velander7, Lício A. Velloso 2,3,5 & Eliana Pereira Araújo
1,3,5
Glutamic acid is the main excitatory neurotransmitter acting both in the brain and in peripheral
tissues. Abnormal distribution of glutamic acid receptors occurs in skin hyperproliferative conditions
such as psoriasis and skin regeneration; however, the biological function of glutamic acid in the
skin remains unclear. Using ex vivo, in vivo and in silico approaches, we showed that exogenous
glutamic acid promotes hair growth and keratinocyte proliferation. Topical application of glutamic
acid decreased the expression of genes related to apoptosis in the skin, whereas glutamic acid
increased cell viability and proliferation in human keratinocyte cultures. In addition, we identied the
keratinocyte glutamic acid excitotoxic concentration, providing evidence for the existence of a novel
skin signalling pathway mediated by a neurotransmitter that controls keratinocyte and hair follicle
proliferation. Thus, glutamic acid emerges as a component of the peripheral nervous system that
acts to control cell growth in the skin. These results raise the perspective of the pharmacological and
nutritional use of glutamic acid to treat skin diseases.
Glutamic acid (GA) is the major excitatory neurotransmitter in the mammalian central nervous system1. GA
receptors (Grin1, Grin2a, Gria2, and Grm1) and transporters (Slc1a1 and Slc1a2) have also been identied in
the skin across dierent species, such as mice, rats, and humans2–6. Moreover, in histological analyses, glutamate
has been identied in the epidermis, hair follicles and sebaceous glands7.
Several studies had shown that the skin performs as neuro-endocrine organ8–10 and its activities are mainly
regulated by local cutaneous factors9. is interaction between skin and environment factors can regulate Central
Nervous System (CNS) functions11. For instance, ultraviolet light absorption by the skin can upregulate neuroen-
docrine axes10,11 and it is suggested to modulate body weight12–14 and depression-like behaviour15. Specically,
UVB skin exposure stimulate corticotropin-releasing hormone protein production and gene expression in the
hypothalamus10.
Previous reports have been identied both the glutamate receptors and specic glutamate transporters in
epidermal keratinocytes2. Physiologically, glutamatergic signalling through N-methyl-D-aspartate (NMDA)
receptor was previously shown to occur in hair follicle cells. GA signalling is essential for the innervation and
dierentiation of Grin1 positive Schwann cells during piloneural collar development in hair follicles4. Specically,
NMDA receptors are highly expressed in type I and type II terminal Schwann cells. ese cells are circumferen-
tially localized in the bulge border and cover most outer root sheath keratinocytes in the isthmus4.
In cell culture studies, NMDA induced an increase in the number of keratinocytes and in the intracellular
calcium concentration16; whereas, invivo studies have shown that the topical application of GA to wounded skin
in diabetic rats increases the rate of wound closure by inducing collagen synthesis and crosslinking17. In addi-
tion, 1% L-glutamic acid-loaded hydrogels accelerated vascularization and macrophage recruitment in diabetic
wound17. D-glutamic acid has also been shown to act on damaged skin by accelerating the barrier recovery3,
altogether suggesting a positive eect in skin repair.
OPEN
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Because of the preliminary data suggesting that GA could act in the skin, we performed a search in patent
databases and found ve patent requests for the use of topical GA and derived molecules for hair growth stimula-
tion (patent numbers: CN106580722A, KR20150110149A, USOO58O1150A, FR2939038B1, and PI9302024A).
However, there are no studies reporting on the mechanisms mediating the actions of GA to stimulate hair growth
or epidermal cell proliferation. Here, we hypothesized that GA could induce proliferation and promote skin cell
viability. Using invivo, exvivo and in silico models, we show that GA promotes keratinocyte proliferation and
hair follicle growth by mechanisms that involve the control of vascularization and apoptosis.
Results
Glutamic acid increases human keratinocyte viability and proliferation. Firstly, we tested the
hypothesis that GA could stimulate proliferation and survival of HaCaT, primary keratinocytes, and broblast,
even in conuent culture conditions (Fig.1a). All traces of foetal calf serum were removed from the medium
to mitigate the eect of the growth factors present in the bovine serum. Aer two days of GA exposure, and
even under 100% conuence conditions, keratinocyte viability and proliferation were increased. We showed
that keratinocytes undergo a Gaussian distribution pattern of viability aer GA exposure (Fig.1b,c). Aer one
day of treatment, GA (100M and 10mM) increased HaCaT-keratinocyte viability (Supplementary Fig.1a).
ese dierences were higher aer two days of GA exposure: the 100M, 1mM, and 10mM GA concentrations
increased HaCaT keratinocyte viability within two days of treatment (Fig.1c). Moreover, 1M and 100M GA
concentrations increased primary keratinocyte viability within two days of treatment (Fig.1b). Also, human
broblast viability increased aer one day (Supplementary Fig.1c.) two days (Fig.1d) and four days (Supple-
mentary Fig.1d.) of GA exposure under 100% conuence conditions. Conversely, we identied an excitotoxic
concentration for keratinocytes at 100mM GA but not in broblast. Keratinocytes treated with 100mM GA
decreased cell viability aer one (Supplementary Fig.1a) and four days of treatment (Supplementary Fig.1b).
As the 10mM and 100mM GA concentrations showed opposite eects in the viability test (proliferative and
excitotoxic, respectively), we evaluated whether keratinocyte proliferation could be aected aer two days of
GA exposure (Fig.1g). Consistent with the viability results, BrdU positive keratinocytes were increased in the
10mM group (Fig.1f,g).
Topical glutamic acid decreases apoptotic related genes. To determine whether the results obtained
in cultured cells could be translated into an invivo model, we employed four dierent concentrations of GA on
the dorsal skin of Swiss mice (Fig.2a). To understand how GA promotes proliferation and improves viability,
we evaluated the expression of genes involved in apoptosis. ere were reductions of Bcl2 gene expression in
cells treated with GA 0.1%, 0.5% and 10% (Fig.2c). BAX was decreased in cells treated with 10% GA (Fig.2c).
However, we found no dierences in Casp9 expression (Fig.2c). Next, we evaluated whether topical GA could
stimulate the expression of genes related to inammatory response. We found no dierences in Il1-β, Tnf-α and
Il10. However, F4/80, a macrophage marker, and Monocyte Chemoattractant Protein-1 (Mcp1) gene expres-
sion were increased aer 14days of 1% GA (Fig.2c). Additionally, topical GA 10% decreased the expression
of Glutamate Ionotropic Receptor NMDA Type Subunit 1 (Grin1) with no dierences in Glutamate Aspartate
Transporter 1 (Glast) expression (Fig.2c).
Topical glutamic acid accelerates hair growth in healthy mice. Surprisingly, 1% and 10% GA accel-
erated hair growth aer 14days of topical treatment (Fig.3a). Using photomicrographs, we also showed that GA
increased external root sheath across all GA concentrations (Fig.3b, Supplementary Fig.1f.) with no hyperkera-
tosis eect. We also consistently identied increased BrdU positive cells in the hair follicles and epidermal layer
aer 14-days of GA topical treatment (Figs.3c, 4f).
Exogenous topical glutamic acid increased vascularization. We identied macroscopic dierences
in vascularization aer 14days of GA treatment. e 0.5% and 10% GA topical application increased skin vas-
cularization (Fig.4a,b). To further explore these ndings, we evaluated whether GA could induce the expression
of genes involved in vascular regulation. We found that 1% GA topical treatment increased Hypoxia Inducible
Factor 1 Subunit Alpha (Hif1a), a master regulator of vascularization 18,19 (Fig.4c). Also, 1% GA topical treat-
ment increased the Vascular Endothelial Growth Factor A (Vegf), which induces proliferation and migration
of vascular endothelial cells and is essential for physiological angiogenesis 20–22 (Fig.4c). However, we found no
dierence in gene expression of CD31 aer 14days of topical GA treatment (Fig.4c).
Single cell RNA sequencing analysis showed dierences in glutamate receptor and transporter
localization between mice and human. We evaluated glutamate receptor expression using immu-
nostaining, quantitative PCR, and single-cell RNA sequencing techniques. We identied that NMDA receptor
subunits Grin1, Grin2a, Grin2b and Grin2c are expressed in the skin (Fig.4d,e), and Grin2b is expressed speci-
cally in keratin 14 + cells (Fig.4d,e). Due to the wide number of subunits (5 GA receptor families with 26 subu-
nits), we used a single cell RNA sequencing approach to improve accuracy (Fig.5a). Using public transcriptome
libraries of skin tissue, we analysed ~ 73,000 mice and human epidermal cells from back (mice), foreskin, trunk,
and scalp (human). is cross-species analysis showed a similar percentage (5%) of glutamatergic epidermal pop-
ulation in the skin (Fig.5b,d). In humans, we identied NMDA receptors as the highest expressed subunits in the
basal layer and hair follicular cell clusters, specically the GRIN2A subunit (Fig.5b). In addition, we identied
melanocytes expressing Glutamate Ionotropic Receptor Delta Type Subunit 1 (GRD1) (Fig.5b), granular cells
expressing Excitatory Amino Acid Transporter 4 (SLC1A6) and basal layer cells expressing Excitatory Amino
Acid Transporter 1 (SLC1A3) (Fig.5c). In mice, we identied Grin2d (in the sebaceous gland) and Grik1 (in the
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hair follicle bulge) as the most expressed subunits (Fig.5d). Additionally, we identied the expression of Excita-
tory Amino Acid Transporter 1 and 3 (Slc1a3 and Slc1a1) (Fig.5e) in 50% of all sebaceous gland cells (Fig.5e).
To predict a GA-mediated cell signalling pathway between GA pathway and hair follicle-related genes, we
used computational interaction network analysis STRING23. In this way, we used glutamate receptor pathway
genes and hair cycle genes ontologies (Supplementary Fig.2). We found that GA receptors interact with hair cycle
genes through the tyrosine-protein kinase Fyn, Ca 2 + /calmodulin-dependent protein kinase II (CaMKII) and
protein kinase B (Akt) (Fig.5f). Additionally, we found Bcl2 as a common apoptotic regulator between both hair
cycle and GA pathways (Fig.5f). To conrm, we evaluated the protein expression of Fyn, CaMKII and Akt in the
14-day topical GA-treated mice (Fig.5g–i). We found no dierences in Fyn quantication (Fig.5i). However,
we conrmed that AKT2 phosphorylation increased aer 14days of topical 1% GA treatment (Fig.5g). Also,
pCaMKII increased aer 14days of topical 10% GA treatment (Fig.5h).
Figure1. Eects of GA treatment in cell culture and invivo. Experimental design of cell culture experiment
using HaCaT, Primary human keratinocytes, and human broblast treated with dierent concentration of GA
(a). MTT viability test results of Primary human keratinocytes with 2days of Glutamic acid exposure (b). MTT
viability test results of HaCaT with 2days of Glutamic acid exposure (c). MTT viability test results of huma
broblast with 2days of Glutamic acid exposure (d). MTT viability test of primary keratinocytes in triplicate in
2 dierent experiments. MTT viability test of HaCaT keratinocytes in quadruplicate in 4 dierent experiments.
MTT viability test of human broblast in quadruplicate in 2–3 dierent experiments. Immunostaining results
of HaCaT keratinocytes treated with Glutamic acid 10mM or 100mM in DMEM compared to Control group
treated with DMEM (g). Immunostaining results of HaCaT keratinocytes in triplicate in 4 dierent experiments,
GA for 48h and, nally, 3h with BrdU, scale bar 50m. e proportion of BrdU-immunoreactive increased
aer exposure to GA 10mM (f). Data is presented as mean ± SEM. N = 4 per group. p = 0.03 t-test Control versus
GA 10mM; p = 0.03 one-way ANOVA.
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Discussion
Currently, there are no studies describing GA treatment or even the eect of GA on hair growth or epidermal
cell proliferation. However, upon searching through major patent agencies, we found ve patents/patent requests
claiming the benets of topical GA (or derived molecules) for hair growth. One of these patents described the
use of GA as a hair conditioner (patent number CN106580722A, China) for hair restoration and the prevention
of alopecia. Another patent showed a Poly-Gamma-GA composition for preventing hair loss and promoting hair
growth (KR20150110149A, Korea). In addition, there were synthetic compounds of GA attached to minoxidil
for keratinocyte growth and hair growth in humans (USOO58O1150A, USA), a 2 to 12% GA topical cream for
combating hair loss or alopecia in humans (FR2939038B1, France) and, nally, 42 dierent molecules derived
from L-glutamic acid were described as hair growth promoters (PI9302024A, Brazil). However, reviewing all
these patents/patents requests, we could nd no description of the cellular mechanisms responsible for the
stimulation of hair growth in response to GA application. us, our work provides experimental proof of a
mechanistic link between GA and hair growth.
Here, we evaluated some of the potential mechanisms involved in eect of GA on hair growth. First, using a
cell culture approach, we challenged 100% conuent primary human keratinocytes, HaCaT-keratinocytes, and
human Fibroblast (in medium depleted of foetal bovine serum (FBS)/growth factor supply) to continue growing.
Our results showed that GA increases the proliferation and viability of keratinocytes, even under these extreme
conditions. us, GA could represent an interesting approach as a cell growth media supplement, replacing tradi-
tional supplements that are more expensive. is nding is further supported by data published previously, which
shows that MK-801, an antagonist of the GA receptor (NMDA receptor), decreases the proliferation of primary
human keratinocytes2 and also prevents hyperplasia induced by acetone3, suggesting an anti-proliferative eect.
e skin is a critical peripheral neuro-endocrine-immune structure that interact to central regulatory
systems24. As a response, the skin can trigger cutaneous nerve endings to inform the CNS on changes in the
epidermal or dermal environments to produce neural or immune responses at the local and systemic levels24.
Human skin reacts to several neuropeptides and neurotransmitters by paracrine, autocrine, vasculature and
nerves stimulus25. Primary and HaCaT keratinocytes are sources of Glutamic acid secreting ≈ 1mM -glutamic
Figure2. Topical glutamic acid in mice. Experimental design of Swiss mice treated topically one at day with
dierent concentration of GA for 14days (a). Dierent GA concentrations (Control, 0.1%, 0.5%, 1% and 10%
GA) for topical animal treatment were equal to 5.5 pH (b). RT-PCR of Bcl2, Bax, Casp9, F4/80, Mcp1, Il1β,
Tnfα, Il10, Grin1 and Glast genes from skin samples aer 14days of GA treatment (c). GAPDH was used as
endogenous control. Data is presented as mean ± SEM * < p 0.05 ANOVA. 5–6 animals per group.
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acid to the culture medium when 100% conuent26 suggesting a paracrine or autocrine stimulation. Indeed,
major aspect of neuroendocine regulation in the skin, specically the hypothalamic–pituitary–adrenal axis and
melatoninoergic system in the skin was previously discussed8. GA has potent neurotoxic eects, and this could
represent a challenge for either experimental or clinical use. Elevated amounts of GA lead to neuronal death in
a process described as excitotoxicity27–29. GA transporters are a potent GA uptake system, acting as a neuronal
compensatory response for excitotoxicity. GA transporters are known to prevent disproportionate activation of
Figure3. GA stimulates hair growth and increased BrdU + cells. Dose–response results of topical GA
application on the dorsal region of mice with Vaseline (CTL) or 0.1%, 0.5%, 1% and 10% GA for 14days
(a–c). 5–6 animals per group. Hair growth eect aer 14days of GA treatment on the back of Swiss mice (a).
Haematoxylin and Eosin (H&E) staining sample of the back of 14day treated mice (hair follicle pointed with
yellow arrows), scale bar 250m, samples from 3 dierent animals (b). Immunostaining results of skin samples
treated 0.1%, 0.5%, 1%, and 10% Glutamic acid compared to Control group (Vaseline) (c). Immunostaining
results of skin samples from 3 dierent animals, GA topical treatment for 14days and, nally, 2h with
intraperitoneal BrdU. Yellow arrows indicate BrdU + cells.
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Figure4. Topical GA and blood vessel. Skin samples treated for 14days with topical GA topical. Upside-
down back skin samples showing vessel dierences between Vaseline (CTL) or 0.1%, 0.5%, 1% and 10% GA
treatments (a). Quantication of blood vessel area aer 14days of vaseline (CTL) or 0.1%, 0.5%, 1% and 10%
GA treatment (b). Gene expression of Hypoxia Inducible Factor 1 Subunit Alpha (Hif1a), Vascular Endothelial
Growth Factor A (Vegf ) and the Platelet and Endothelial Cell Adhesion Molecule 1 (Cd31) from full-thickness
back skin aer 14days of GA treatment (c). GA receptor characterization in mice skin of dierent GA subunits
(d-e). Immunostaining against NMDA Grin1, Grin2b, Grin2a and Grin2c GA receptor expressed in the
epidermal layer of the skin of untreated mice (d–e). Yellow arrows indicate colocalization of K14 + Grin2b + cells
(d). Quantication of positive BrdU epidermal and hair follicle cells of mice skin treated with vaseline (CTL)
or 0.1%, 0.5%, 1% and 10% of GA (f). BrdU and quantitative PCR data are presented as mean ± SEM * < p 0.05
ANOVA. 5–6 animals per group.
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GA receptors by constantly removing GA from the extracellular space30–32. Here, we determined the excitotoxic
GA concentration. Invitro, 100mM GA decreased keratinocyte viability, and topical GA decreased Bcl2 and
Bax expression. Altogether, our results support the excitotoxic eect of higher concentrations of pH-neutralized
GA in keratinocytes.
To understand the exogenous GA eect on the skin, we explored the GA transporter landscape at single-cell
resolution in human and mice skins (Fig.5b,d). Additionally, we showed Slc1a3 expression using quantitative
PCR, and a similar Slc1a3 (Glast) expression aer exogenous GA application (Fig.2c). Future research could
help to identify the role of GA-induced excitotoxicity and the GA uptake system in the skin.
Regarding the GA receptors, different subpopulations of glutamatergic cells have been extensively
described33–36. In the skin, previous reports identied the localization of GA receptors and transporters in the
epidermis from rats and mice, as well as in human keratinocytes2,3,5. ese studies showed similar cross-species
characteristics: a smaller subpopulation of cells expressing receptors and transporters2. Consistent with these
ndings, here, we showed a small subpopulation of epidermal cells expressing GA receptors along the skin,
with varying intensity (Fig.5b,d). Our results suggest that these glutamatergic keratinocytes are responsive to
exogenous GA stimulation.
Previous reports showed that vascularization increases during the anagen phase of the hair cycle and decreases
during the catagen and telogen phases. is angiogenesis process was spatially correlated with the upregulation
of VEGF37. Also, the hypoxia-inducible factor (HIF) has been shown to coordinate the up-regulation of multiple
genes controlling neovascularization, such as Vegf38. Here, we showed that Hif1a and Vegf expression increased
aer 14days of GA topical treatment on the back skin of mice with a remarkable change in angiogenesis, as
previously shown17.
e Hypoxia-inducible factor-1α, encoded by the gene Hif1a showed to stimulate hair growth39 and some
HIF-1α-stimulating agents signicantly increase dermal papilla cell proliferation40. Minoxidil 2,4-diamino-
6-piperidinopyrimidine3-oxide, a vasodilator used for the treatment of pattern hair loss, is a direct inhibitor of
PHD-2 (prolyl-hydroxylase 2) which hydroxylates HIF-1α causing its degradation. Also, Minoxidil stimulates
the transcription of hypoxia-response element genes such as VEGF39–41. Here, we showed dierent concentra-
tions of GA with hair growth and angiogenic eects. Publicly available patents proclaim benet by using high
percentage of GA concentration for skin treatment and hair growth stimulation. e 10% GA treatment showed
hair growth and angiogenesis stimulation, but no dierences in Hif1a expression aer 14day of topical GA.
Previous studies showed the ranges of GA saturation by specic GA concentration 42–44. In this way, we suggest
that 10% GA topical treatment could achieve a post-acute signal saturation secondary to the time exposure
and the Glutamic Acid concentration here presented. However, more studies are needed to elucidate a possible
saturation and angiogenic eect of 14days GA topical treatment on the back skin of mice.
A recent study supports that GA-mediated signalling could be involved in hair growth45. e authors showed
that glutamine, a molecule similar to GA, controls the fate of stem cells in the hair follicle. e capacity of the
outer root sheath cells to return to the stem cell state requires suppression of a metabolic switch from glutamine
metabolism and is regulated by the mTORC2-Akt signalling axis45. Similarly, our result suggests that GA increases
AKT phosphorylation and hair follicle cell stimulation. In this way, our results further suggest that GA activates
the hair cycle by stimulating the stem cells to dierentiate into the outer root sheath. However, future studies
should describe the hair-follicle cycle modulation aer topical GA treatments.
Taken together, the cell-based and experimental outcomes of this study provide a mechanistic advance in
the characterization of GA-induced eects on hair growth and could become an attractive approach to treat hair
growth disorders, or for aesthetic hair stimulation.
Further studies should focus on the relationship between skin disorders and GA and how GA, present in
food, could impact on skin health.
Methods
Experimental animals. Eight-week-old male Swiss mice (n = 6) were obtained from the Breeding Animal
Center of the University of Campinas. Animals were maintained under pathogen-free conditions in individual
cages on a 12–12-h dark–light cycle, at 21–23°C. Mice received food and water adlibitum. Mice were anesthe-
tized with intraperitoneal injections (according to body weight), using ketamine hydrochloride 80mg/kg and
xylazine chlorhydrate 8mg/kg. Hair was removed from the dorsal region (1.0cm × 2.5cm) of the anesthetized
mice using a mechanical razor and depilatory cream (Veet). e dorsal region of all mice was carefully cleaned
to remove any trace of Veet cream. Animal experiments were approved by e Animal Ethical Committee at
the University of Campinas, Brazil (certicate of approval no. 4930–1/2018). All experiments were performed in
accordance with the “Guide for the Care and Use of Laboratory Animals”, National Academy Press, 1996 guide-
lines of standard humane animal care. All the animal experiments have been performed following the ARRIVE
guidelines.
Topical glutamic acid treatment. e dorsal region of mice was treated once daily using dierent con-
centrations of GA. To ensure a uniform 200 L treatment, we used dierent syringes preloaded with Vaseline
(control), 0.1%, 0.5%, 1% or 10% GA (Supplementary Fig.1c). e treatment was spread manually using gloves
which were changing between each group. To avoid removal of the treatment, mice used Elizabethan collars 8
of 14 days of treatment.
Topical glutamic acid composition. We made ve dierent formulations: 0% (Control), 0.1% (6mM),
0.5% (30mM), 1% (60mM) and 10% (600mM) of GA. Table1 shows the dierent composition of each treat-
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ment (Table1). e pH of the formulations was adjusted using aqueous NaOH until the desired pH 5.5 was
achieved (Fig.1f). is pH value was chosen to resemble the skin surface pH46.
Primary keratinocytes isolation. Normal human skin from healthy donors were obtained by postec-
tomy (University Hospital, University of São Paulo, São Paulo, Brazil). Keratinocytes were isolated and cul-
tured as described previously47,48. Declaration of Helsinki Principles and approved by the Ethics Committees
for Research (HU CEP Case No. 943/09 and CEP FCF/USP 534). Parents and/or guardians were informed and
signed written consent.
Cell culture MTT and BrdU. Human keratinocyte lineage (HaCaT) passage 27–30 were cultured in a 37°C,
7% CO2 incubator with Dulbecco’s modied Eagle’s medium (DMEM) medium supplemented with 5% FBS to
100% conuence in 6-well plates. Primary keratinocytes passage 3–6 were cultured in a 37°C, 7% CO2 incubator
with KGM Gold Keratinocyte Growth Medium to 100% conuence in 6-well plates. Human Fibroblast cell line
(BJ-5ta) passage 28–30 were cultured in a 37°C, 5% CO2 incubator with 4:1 mixture of High glucose Dulbecco’s
Modied Eagle’s Medium and Medium 199 supplemented with 10% FBS and 0.01mg/ml hygromycin B, to
100% conuence in 6-well plates. We replaced the culture medium 2–3 times a week. A 3-4,5-dimethylthiazol-
2-yl-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyse cell viability, as previously described49.
MTT solution was prepared in Krebs-HEPES buer (10mM HEPES, 1.2mM MgCl2, 144mM NaCl, 11mM
glucose, 2mM CaCl2 and 5.9mM KCl). Aer 100% conuence 6-wellplates, HaCaT and human broblast were
incubated with the dierent concentrations of GA in DMEM without FBS for 1, 2 and 4days. Primary keratino-
cytes were incubated with the dierent concentrations of GA in KGM Gold medium without growth factors for
2days. Aer treatment, the medium was removed, MTT solution (0.5mg/mL) was added to each well and the
plates were incubated at 37°C for 3h. e solution was then removed and 300 L of DMSO was added before
being incubated in the dark with 60rpm shaking. e absorbance was measured at a wavelength of 540nm in
a microplate reader (Globomax). HaCaT culture experiments were performed in quadruplicate in 4 dierent
experiments. Primary keratinocytes culture experiments were performed in triplicate in 2 dierent experiments.
Human broblast culture experiments were performed in triplicate in 2–3 dierent experiments. BrdU experi-
ments were performed as previously described26. Briey, to assess the eect of GA on cell proliferation, HaCaT
human keratinocytes were maintained DMEM (Gibco) containing 4.5g/L glucose, 4mM L-glutamine, 100
units/mL of penicillin, 100g/mL of streptomycin and 10% FBS. Incubation conditions were 37°C in 5% CO2/
humidied air. HaCaT cells were plated on coverslips in 24-well plates (1 × 105 cells/well) and exposed to GA for
48h (10 and 100mM) in DMEM without FBS. Aer treatment, cells were incubated with BrdU (10µM, Sigma)
for 3h, then xed with 4% PFA in 0.1M PBS for 10min at RT. For BrdU staining, cells were washed with PBS,
and DNA was denatured with 1N HCl for 1h at RT. Cells were blocked for 1h in blocking solution containing
10% goat serum and 0.2% Triton X-100 in PBS, followed by an incubation with primary (rat anti-BrdU; 1:200;
Ab6326); and secondary (goat anti-rat FITC, 1:200; sc2011) antibodies prepared in 3% goat serum/0.2% Triton
X-100 in PBS, and incubated overnight and for 2h, respectively. e nuclei were labelled with DAPI, and cover-
slips were mounted onto glass slides for microscope imaging. Images were captured on uorescence microscopy
(Olympus BX41). e results of BrdU immunopositivity cells represent the average of 3 coverslips per experi-
mental replicate, where 3 elds were imaged per coverslip and averaged. e number of immunopositive cells
was quantied per image using the ImageJ soware and are expressed as a percentage relative to the total DAPI
nuclei.
Animal photo documentation. Hair growth processes were photo documented using a D610 Nikon digi-
tal camera (Nikon Systems, Inc., Tokyo, Japan). We used a stand to secure a similar distance from the camera to
the treated skin site, and the same person took the photos.
Vessel analysis. Vascular density measurements were calculated from digital images obtained using a D610
Nikon digital camera (Nikon Systems, Inc., Tokyo, Japan). We used a stand to secure a similar distance from the
camera to the upside-down back skin samples. Vascular density ratio was calculated vascular as follow: vessel
area/total area * 100%50. e number of pixels were digitally determined by densitometry, using Image J soware
(National Institutes of Health).
Figure5. Cross-species skin GA receptor landscape using single-cell RNA sequencing. Generation of glutamic
acid receptor landscape using public data reveals GA distribution at single cell resolution in mice and humans
(b–e). Schematic representation of the single-cell RNA sequencing analysis using publicly available datasets
from mice and human epidermal layers (a). Human Epidermal Glutamate receptors (b) and transporter
expression (c). Mice Epidermal Glutamate receptors (d) and transporter expression (e). Glutamic acid receptor
and hair cycle Protein–Protein Interaction Network performed with STRING V11 (f). Glutamic acid and hair
cycle interactome were retrieved with the data-mining toolkit STRING. Closer interactors of glutamic acid and
hair cycle ontologies (GO:0007215 and GO:0042633) were selected and categorized by coloured nodes. Yellow
arrows indicate shared shell interactors, red nodes indicate glutamate receptor signaling pathway genes and
blue nodes hair cycle genes (f). Display of cropped blots quantied to conrm the Protein–Protein Interaction
Network prediction (g–i). Western blot analysis of AKT Phosphorylation (g), phospho-CaMKII (h) and Fyn
quantication (i). Full-length blots are presented in Supplementary Fig.3. Western blot data are presented as
mean ± SEM * < p 0.05 ANOVA. 4–5 animals per group.
◂
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Histology. Aer 14days of treatment, tissues were harvested and xed by immersion in formaldehyde over-
night. Any traces of formaldehyde were removed by 3 washes of PBS 1x. e tissues were processed in alcohol at
dierent concentrations (70%, 80%, 95% and 100%), xylol and paran, before being xed in paran blocks and
sectioned at 5.0m. In total, 3 to 5 sessions were placed on microscope slides pre-treated with poly-L-lysine. To
evaluate cell and extracellular matrix morphology, the skin sections were stained with haematoxylin and eosin
(H&E). e sections were incubated with haematoxylin for 30s, rinsed in water, incubated for 30s with eosin,
rinsed again in water, and dehydrated. e slides were mounted in Entellan® and then analysed; digital images
were captured under bright-eld microscopy.
Protein–protein interaction networks. Protein functional interaction networks were performed
using STRING v11. e default functional interaction network was congured to evidence meaning of net-
work edges, experiments, and databases in active interaction sources. Mus musculus organism was visualized by
known molecular action. We analysed two Biological Processes using the Gene Ontology Term from the Mouse
Genome Informatics database: A permalink webpage of Glutamate receptor (GO:0,007,215) and hair follicle
(GO:0,042,633). e gene ontologies interaction network is accessible through https:// versi on- 11-0. string- db.
org/ cgi/ netwo rk. pl? taskId= lKUAb EZgGk Vu for selected genes and https:// versi on- 11-0. string- db. org/ cgi/
netwo rk. pl? netwo rkId= 7i1fM IP01x qT for all genes.
Single‑cell RNA sequencing data acquisition, ltering, and processing.. In silico analyses were
performed using a HP ENVY 17 Leap Motion SE NB PC notebook with 16GB RAM and four-cores Intel i7
processor. Sample expression matrices (mice and humans) were downloaded from Gene Expression Omni-
bus and European Genome-phenome Archive: GSE67602 and EGAS00001002927. Cells were ltered by their
total number of reads, by their number of detected genes and by their mitochondrial percentage. For mice, we
used nFeature_RNA > 10 and < 6,000, nCount_RNA > 100 and < 50,000, percent.mt < 9.5 settings. For humans,
we use d nFeature_RNA > 100 and < 5,000, nCount_RNA > 100 and < 25,000 and percent.mt < 6 settings. Samples
were processed in Seurat v3.1.5 using the default Seurat workow. For clustering and visualization, we used
the default Seurat pipeline gold standard and dot plot visualization. Cluster names were annotated to cell types
according to original articles of Cheng etal. and Joost etal.51,52.
Western blotting. For protein quantication, mice were treated topically 14days with GA, once a day. Ani-
mals were anesthetized (ketamine hydrochloride 80mg/kg and xylazine chlorhydrate 8mg/kg) using Labinsane53,
placed them in ventral decubitus position for cleaning the skin. en, we harvested a 6mm full-thickness skin
sample54 from the back of each animal using a 6mm biopsy punch. Immediately aer the extraction, the tissues
were stored in − 80°C, until further analysis. e animals were sacriced by anesthetic deepening. We used 5–6
animals per group. For the immunoblot experiments, the tissues were homogenized in Radioimmunoprecipita-
tion assay buer (150mM NaCl, 50mM Tris, 5mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0,1%
sodium dodecyl sulfate, and supplemented with protease inhibitors). Insoluble materials were removed by cen-
trifugation 11,000 rpm for 40min at 4°C, and the supernatant was used for protein quantication by the biuret
reagent protein assay. Laemmli buer (0.5M Tris, 30% glycerol, 10% SDS, 0.6M DTT, 0.012 bromophenol blue)
was added to the samples. One hundred micrograms of proteins were separated by SDS-PAGE and transferred
to nitrocellulose membranes (Bio-Rad) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) for 1h at 17V
(constant) in buer containing methanol and SDS. Blots were blocked in a 5% skimmed milk powder solution in
TBST (1 × TBS and 0.1% Tween 20) for 2h at RT, washed with TBST, and incubated with the primary antibodies
for 24h at 4°C. e primary antibodies used were anti-pCaMKII (Abcam, ab32678) and anti-Fyn3 (Santa Cruz,
sc-16). HRP-coupled secondary antibodies (1:5000, ermo Scientic) were used for detection of the conjugate
by chemiluminescence and visualization by exposure to an Image Quant LAS4000 (GE Healthcare, Life Sci-
ences). Anti-β-actin (Abcam, ab8227) was used as a loading control. e intensities of the bands were digitally
determined by densitometry, using Image J soware (National Institutes of Health).
Immunohistochemistry. Skin expressions of Grin1, Grin2a, Grin2b, and Grin2c were identied by immu-
nohistochemical staining. Immunohistochemistry was performed using the skin samples (n = 5). Tissue samples
Table 1. Glutamic acid-based creams used in the invivo experiments. Compound description of Control, GA
0.1%, 0.5%, 1%, and 10% w/w topical treatment.
Compounds Vaseline 6mM
(GA 0.1%) 30mM
(GA 0.5%) 60mM
(GA 1%) 600mM
(GA 10%)
L-Glutamic Acid − 0.03g 0.15g 0.3g 3g
NaOH 35 µL 160 µL 320 µL 2.25mL
Liquid Vaseline 2.77mL 2.77mL 2.77mL 2.77mL 2.77mL
Solid Vaseline 24.78g 24.75g 24.63g 24.48g 21.75g
Tween 20 200 µL 200 µL 200 µL 200 µL 200 µL
ddH20 2.24mL 2.215mL 2.09mL 1.91mL −
HCl 0.5 µL − − − −
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were immersed in 4% formaldehyde, overnight. Tissue samples were washed three times with PBS 1x, cryo-
preserved in sucrose 20% for 3days and 40% for 1week. Samples were then embedded in OCT and sectioned
using a cryostat (Leica CM1860). e sections (20m) were immunostained with the following primary anti-
bodies: Grin1 (1:100, sc1467), Grin2a (1:100, sc1468), Grin2b (1:100, sc1469), Grin2c (1:100, sc9057), BrdU
(1:200, ab6326) and keratin 14 (1:100, sc53253). VECTASHIELD with DAPI was used as a mounting medium
for nuclear visualization. Images were obtained using a confocal microscope (Leica TCS SP5 II). For the invivo
BrdU experiment, we treated the mice intraperitoneally, as previously described55. Briey, we applied one single
injection of BrdU (150mg/kg in buer citrate) 3h before skin harvest.
Real‑time quantitative polymerase chain reaction (RT‑qPCR). e total RNA content was
extracted from the tissue using TRIzol reagent (Invitrogen). For each sample, two micrograms of RNA were
reverse transcribed to cDNA, according to the manufacturer’s instructions (High-Capacity cDNA Reverse Tran-
scription Kit, Life Technologies). Gene expression analysis was performed via RT-qPCR using TaqMan Univer-
sal PCR Master Mix (7500 detection system, Applied Biosystems). e primers used were: Bcl2: Mm00477631_
m1; Bax: Mm00432051_m1; Casp9: Mm00516563_m1; F4/80: Mm00802529_m1; Mcp1: Mm00441242_m1;
Il1b: Mm00434228_m1; TNFa: Mm00443258_m1; Il10: Mm01288386_m1; Grin1: Mm00433790_m1; Glast:
Mm00600697_m1; Hif1a: Mm00468869_m1; Vegf: Mm00437306_m1; and Cd31: Mm01242576_m1 (er-
mosher). Analyses were run using 4 L (10ng/L) cDNA, 0.625 L primer/probe solution, 1.625 L H2O and
6.25 L 2X TaqMan Universal MasterMix. GAPDH (Mm99999915_g1) was employed as a reference gene. Gene
expression was obtained using the SDS System 7500 soware (Applied Biosystems).
Received: 24 October 2020; Accepted: 14 July 2021
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Acknowledgements
e authors are grateful to Marcio Alves da Cruz, Erika Anne Roman and Gerson Ferraz for technical assistance.
is study was supported, in part, by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil
(CAPES)—Finance Code 88882.434714/2019–01. is work was supported in part by the São Paulo Research
Foundation FAPESP # 2017/04926-6 and # 2019/14527-7.
Author contributions
CPJ: Conceptualization, Formal analysis, Methodology, Investigation, Writing—Original Dra, Writing—Review
& Editing. BAB: Investigation. NFM, DFE and AMZ: Investigation, Writing—Review & Editing. GFS: Meth-
odology, Writing—Review & Editing. LAV: Formal analysis, Writing—Review & Editing, Supervision. EPA:
Conceptualization, Methodology, Formal analysis, Resources, Writing—Original Dra, Writing—Review &
Editing, Supervision.
Funding
Coordination of Improvement of Higher-Level Personnel of Brazil (CAPES) and São Paulo Research Founda-
tion FAPESP.
Competing interests
e authors declare no competing interests.
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