Progressive changes in the transcriptome are evident in hair miniaturization

Follicular units from occipital and vertex scalp were extracted in patients and healthy volunteers as previously described24 and full transcriptome sequencing of mRNA and miRNA was performed from the hair root region. Clinical classification of AGA according to Hamilton-Norwood scale, location of area sampled and hair follicle morphology (Supp. Fig. 1A–D) resulted in loosely clustered groups. However transcriptome based unsupervised hierarchical clustering (Fig. 1A) resulted in four distinct groups (Fig. 1B). Group 1 (red in Fig. 1A) comprised mostly occipital and vertex samples from the normal healthy volunteers (CO, CV), (control group). Group 2 (green) contained patients’ occipital (PO) region, group 3 (blue) involved patients’ occipital and patients’ vertex (PV) area and they resembled normal hair, but were predominantly shorter. Group 4 (purple) comprised of follicular units taken from PV exclusively, and all of these are miniaturized hair follicles (Fig. 1C). In the differential gene expression analysis between group 1 and group 2, 3 and 4; there were no differentially expressed genes between group 1 and 2, while we identified 44 and 3170 genes differentially expressed in Group 1 vs Group 3 and Group 1 vs Group 4 respectively (Fig. 1D). It is interesting that follicles under group 3 show transcriptomic changes compared to Group 1 follicles prior to major morphological changes. All up-regulated genes in the Group 3 vs Group 1 comparison were expressed in group 4 samples at much higher levels than those in group 3 (compared to group 1); indicating a transition in group 3 from a healthy hair follicle (group 1 and 2) to a affected, miniaturized hair follicle in group 4 (Fig. 1E,F). Ingenuity Pathway Analysis (IPA) analysis implicated involvement of PPAR/RXRα pathway, death receptors and mechanisms for viral-host response in group 3 (Fig. 2A). A myriad of pathways were enriched in group 4 follicles including eicosanoid signaling, antigen presentation and LXR/RXR activation and particularly PPAR signaling (Fig. 2B). PPAR is controlling fatty acid metabolism and its up-regulation in hair bulb was closely related to hair miniaturization and concurrent AR up-regulation. Furthermore, IPA analysis of metabolic processes identified the up-regulation of processes including fatty acid oxidation, stearate biosynthesis and prostanoid synthesis (Fig. 2C).

Figure 1 Sample clustering by transcriptome profiling implicates transition in AGA severity. (A) Principal component analysis (PCA) plot of mRNA transcriptome profile of FUE samples classified by unsupervised hierarchical clustering. (B) Hierarchical clustering of samples according to transcriptome profile. (C) Representative images of hair follicles from group 1–4. Scale: 1 mm. (D) List of differentially expressed genes between G1 vs G2, G1 vs G3 and G1 vs G4. (E) Venn Diagram of differentially expressed genes overlapping between G1 vs G2, G3 and G4. (F) Heat map representing expression of genes differentially expressed in G1 vs G3 across G1 to G3 and G1 to G4 samples. Gene expression level are represented by intensity of red color in pixels across different groups of samples. Full size image

Figure 2 IPA analysis summary of genes were differentially expressed between groups. (A) Canonical pathways of genes differentially expressed between G1 and G3. (B) Canonical pathways of genes differentially expressed between G1 and G4. (C) Metabolic pathways of genes differentially expressed between G1 and G4. The most statistically significant canonical pathways were listed according to −log(p-value) of significance. Orange line represents ratio of genes in enriched pathway against number of genes in the input dataset. Full size image

PGC1a expression is elevated in the IRS and ORS of patient vertex hair

We identified PGC1a, a master regulator for mitochondrial biogenesis25, up-regulated in the hair bulb of progressively miniaturized hair samples. PGC1a has been shown to interact with PPARγ and the retinoid receptor RARa in thermogenesis26. Interestingly, the transcript levels of PGC1a, PPARγ and RAR-a were found to be concomitantly up-regulated with AR expression (Fig. 3A and Supp. Fig. 2A,B). Validation of PGC1a expression in control and AGA patients by RT-qPCR confirmed the up-regulation in patients’ vertex follicular hair root compared to patients’ occipital and control samples but with high inter-individual variation (Fig. 3B). In situ hybridization showed PGC1a and AR expressions (red) in the epithelial IRS and ORS cells of patient vertex (PV) samples were elevated compared to all other samples (CO, CV and PO), where they could hardly be detected (Fig. 3C,C’).

Figure 3 Identification of PGC1α as candidate gene involved in AGA. (A) AR and PGC1α transcript expression in FUE samples, black bar represents AR transcript read count, white bar represents PGC1α transcript read count. (B) Validation of PGC1α expression by RT-qPCR, fold change between values are normalized to CO samples (PO: 1.08, PV: 47.77, CV: 1.27), p = 0.22 (PV vs CO), results are depicted as mean ± SE, n = 3 per group. (C) In situ hybridization of AR and PGC1a in PV, PO, CV and CO samples. Hair follicle sectioned across matrix cells, scale: 20 µm. Arrowhead indicate in situ hybridization signals. Square indicate magnified area. Ma: matrix, Me: melanocyte, IRS: inner root sheath, ORS: outer root sheath, CTS: connective tissue sheath. the brown color represents native melanin present in the hair shaft. (C’) Higher magnification of AR and PGC1a staining in PV and CV samples. Scale: 10 µm. (D) In situ hybridization of Pgc1α expression in hair follicles of mice at morphogenesis, anagen, catagen and telogen phase of the hair cycle. Scale: 20 µm. Arrowhead indicate in situ staining signals in brown. Full size image

Dynamic Pgc1α expression throughout the hair cycle

To assess the role of mouse Pgc1α in normal hair development, we performed in situ hybridization for Pgc1α mRNA in mice to characterize the pattern of expression in different stages of hair cycle (Fig. 3D). In post-natal day 5 mouse hair follicles at morphogenesis stage 5, Pgc1α mRNA expression was detected in the IRS of the hair follicle (Fig. 3D; see arrowhead). At P14, as follicles proceed to full maturation, Pgc1α expression shifted to the ORS (Fig. 3D; see arrowhead). In fully matured hair at P35, Pgc1α was expressed in the epithelial cells of the IRS in anagen, in the ORS of the catagen hair and was not detected in the telogen hair (arrowhead). This distribution pattern was consistent with the expression of PGC1a in our human hair follicles at the ORS. Interestingly, Pgc1α expression could not be detected in the mesenchymal, Versican positive DP cell population at all stages studied (Supp. Fig. 3).

The role of Pgc1α in hair development was studied by assessing hair morphology and progression of hair cycle in global Pgc1α KO mice27. Pgc1α KO mice display some postnatal lethality, but the hair morphology and histology in surviving mice was similar to wildtype animals (data not shown). Subsequent to hair morphogenesis, we did not observe significant differences in hair cycle progression (Supp. Fig. 4) brought by knocking out Pgc1α as assessed by hair cycle score28.

miRNAs targeting AR and PPAR signaling pathways were differentially expressed in miniaturized hair

miRNAs negatively regulate the expression of their target gene post-transcriptionally and are involved in regulating hair development (reviewed in29). To gain further insight into the miRNA signatures in AGA, we evaluated the miRNA expression profile in the hair bulb samples from patients and controls. Hierarchical clustering of samples according to miRNA expression profile revealed 4 distinct arms. Equivalent to the Group 4 population in mRNA clustering, the miniaturized follicles formed a distinct cluster according to miRNAs expression (Supp. Fig. 5).

To investigate how miRNA is associated with the corresponding differentially expressed target mRNA in hair miniaturization, we next analyzed miRNA expression in samples grouped on the basis of mRNA expression profile. No miRNA differential expression was detected in the comparison between G2 vs G1 and G3 vs G1. While G4 vs G1, G4 vs G2 and G4 vs G3 yielded 173, 161 and 131 differentially expressed miRNA respectively, where 114 miRNAs were commonly differentially expressed in all comparisons (Fig. 4A,B). To investigate the association between hair miniaturization and putative miRNA target genes, IPA comparative analysis integrating differentially expressed miRNAs and genes in the Group 4 vs Group 1 was performed. The miRNA target analysis reports the target genes for a miRNA from several sources. Those DE genes that were reported as targets by IPA and showed as expected an opposite trend of expression compared to the DE miRNA, considered as the “actual miRNA targets”. The analysis revealed involvement of target genes were enriched in adipogenesis pathway, PPAR signaling, TR/RXR activation and antigen presentation pathways (Fig. 4C). Thereby miRNA expression analysis provided consistent data and support for the differential mRNA expression in G4 samples. We were particularly interested in the “Androgen Signaling” and FXR/RXR activation pathways and found several miRNAs targeting genes involved in these two pathways (Table 1). We identified two miRNAs, his-miR138-5p and hs-miR615-3p which were preferentially bound on PGC1α and AR transcripts respectively were down-regulated in G4 samples (Fig. 4D). This supports a possible mechanism of miRNA regulating DE mRNAs in AGA. We also find high expression of miR128-3p, miR500a-3p and let-7a-5p which were differentially expressed in dermal papilla cells upon DHT treatment30.

Figure 4 miRNA seq analysis of samples reveal differentially expressed miRNA targeting AR and PGC1α. (A) List of miRNAs differentially expressed in G1 vs G4, G2 vs G4 and G3 vs G4 comparison. (B) Venn diagram of overlapping DE miRNA across groups. (C) Expression value of miR-138-5p and miR-615-5p across sample groups. Data represented as mean ± SD. *p < 0.05 in G4 compared to all other groups. (D) Summary IPA miRNA Target Filter analysis of DE genes from G1 vs G4 comparison superimposed onto experimentally verified and high-confidence targets reported for DE miRNAs. The most statistically significant canonical pathways were listed according to −log(p-value) of significance. Orange line represents ratio of genes in enriched pathway against number of genes in the input dataset. Full size image

Table 1 DE miRNAs in Group 4 vs Group 1 comparison predicted to target genes in the AR and PPAR signaling pathways. Full size table

Impaired keratinocyte and DP cell proliferation upon AICAR treatment

To investigate the mechanism of PGC1α in causing hair miniaturization, we then analyzed the impact of elevated PGC1α expression on immortalized epithelial keratinocytes and mesenchymal DP cell proliferation. To stimulate PGC1α expression in vitro, cells were treated with AICAR (1 mM), an AMPK stimulator well known to induce PGC1α in dermal fibroblasts and keratinocytes31,32. PGC1α expression was induced by 2 fold and 1.6 fold immortalized keratinocyte and DP cells respectively after AICAR treatment for 24 hours (Fig. 5A). AICAR-stimulation resulted in a significant decrease of cell proliferation from 11.36% (Untreated) to 5.185% (AICAR treated) in NTERTs and from 17.43% (Untreated) to 14.09% (AICAR treated) in DP cells as measured by DNA-incorporation of EdU label (Fig. 5B).