mTORC1 activity negatively regulates human hair follicle growth and pigmentation

Abstract Dysregulation of the activity of the mechanistic target of rapamycin complex 1 (mTORC1) is commonly linked to aging, cancer, and genetic disorders such as tuberous sclerosis (TS), a rare neurodevelopmental multisystemic disease characterized by benign tumors, seizures, and intellectual disability. Although patches of white hair on the scalp (poliosis) are considered as early signs of TS, the underlying molecular mechanisms and potential involvement of mTORC1 in hair depigmentation remain unclear. Here, we have used healthy, organ‐cultured human scalp hair follicles (HFs) to interrogate the role of mTORC1 in a prototypic human (mini‐)organ. Gray/white HFs exhibit high mTORC1 activity, while mTORC1 inhibition by rapamycin stimulated HF growth and pigmentation, even in gray/white HFs that still contained some surviving melanocytes. Mechanistically, this occurred via increased intrafollicular production of the melanotropic hormone, α‐MSH. In contrast, knockdown of intrafollicular TSC2, a negative regulator of mTORC1, significantly reduced HF pigmentation. Our findings introduce mTORC1 activity as an important negative regulator of human HF growth and pigmentation and suggest that pharmacological mTORC1 inhibition could become a novel strategy in the management of hair loss and depigmentation disorders.

. mTORC1 overactivation does not affect melanocyte dendricity and number in human scalp hair follicles.
A Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry. Mean AE SEM; N = 21-22 HFs per group from four different donors treated with siTSC2 or nontargeting oligos for 6 days; Unpaired Student's t-test. B Representative fluorescence images of Ki-67 and bright-field microscopic images of Masson-Fontana. C Quantitative analysis of MITF expression. N = 9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos (NTO) for 6 days. D Representative images of MITF immunofluorescence. White arrows show MITF + cells. E Quantitative analysis of MITF phosphorylation (pMITF). N = 8 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days. F Representative images of pMITF immunofluorescence. G Quantitative analysis of gp100 expression. N = 10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days. H Representative images of gp100 immunofluorescence. I Quantitative analysis of gp100 + cell number. N = 9 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days. J Representative images of gp100 immunofluorescence. K Quantitative analysis of melanocyte dendricity. N = 10 anagen VI HFs from four different donors treated with siTSC2 or nontargeting oligos for 6 days.
Data information: Only anagen VI HFs (except for A and B where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean AE SEM, Student's t-test. Scale bar: 50 lm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. F Representative fluorescence images of pMITF immunofluorescence. White arrows showed pMITF + cells. G Quantitative analysis of tyrosinase activity in defined reference area in the bulb. N = 12-13 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. H Representative images of tyrosinase activity immunofluorescence. I Quantitative analysis of gp100 expression in defined reference area within the bulb. N = 15-16 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. J Representative images of gp100 immunofluorescence. K Quantitative analysis of a-MSH expression in defined reference area within the bulb. N = 10-12 gray anagen VI HFs from five different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. L Representative images of a-MSH immunofluorescence.
Data information: Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean AE SEM, Mann-Whitney Utest (A) or Student's t-test (C, E, G, I, K), *P < 0.05. Scale bar: 50 lm. Samples from each donor represented by a different color. Nuclei stained with DAPI. Figure EV4. mTORC1 inhibition does not significantly alter melanocyte number, proliferation state, and dendricity in gray/white human scalp gray hair follicles.
A Quantitative immunohistomorphometry of the number of gp100 + cells. N = 14 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. B Representative images of gp100 immunofluorescence. Arrows indicate gp100 + cells. C Quantitative analysis of gp100 + /ki-67 + cell number. N = 9 gray anagen VI HFs from three different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. D Representative images of gp100/ki-67 immunofluorescence. Arrows indicate gp100 + Ki-67 + cells. E Quantitative analysis of melanocyte dendricity. N = 10-12 gray anagen VI HFs from four different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days.
Data information: Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean AE SEM, Mann-Whitney Utest. Scale bar: 50 lm. Samples from each donor represented by a different color. Nuclei stained with DAPI.