To Baldly Go – The Proteomics of Androgenetic Alopecia

Male-pattern baldness, or androgenetic alopecia, is characterized by progressive and sometimes psychologically debilitating hair loss. Men are predominantly affected by this type of alopecia, and its androgen dependency is recognized. The role of androgen receptors in disease progression is well studied. Therapeutic options exist, but these mainly comprise treatments that halt or slow hair loss rather than reverse follicular change.

In androgenetic alopecia, the follicular cell cycle moves more quickly from active growth to apoptotic regression. Follicular cells are supported and regulated by mesenchymal dermal papilla cells (DPCs). Many research teams have also shown that gene expression differs in balding DPCs, as compared to non-balding follicles in vivo. Furthermore, balding DPCs secrete inhibitory agents and are responsive to exogenous androgens in vitro, retaining their donor characteristics in culture.

Moon et al.(2013) cultured DPCs from two patients, from balding and non-balding scalp areas, using cells harvested from the third passage for proteomic analysis and comparison.¹Balding-scalp punch biopsies from four additional patients were used for validation and confirmation of methods. Proteins were harvested using gel-assisted digestion, then sequentially extracted. 1D- and 2D-LC methods were compared to separate the proteins before liquid chromatography–tandem mass spectrometry (LC–MS/MS) label-free quantification. Reverse transcriptase–polymerase chain reaction analysis of androgen-receptor, type-2 5α-reductase and β-actin expression was used to establish cultured DPC findings consistent with other studies. Protein expression in cell lysates was confirmed through SDS-PAGE and immunoblotting, in addition to immunofluorescence of hair follicle preparations.

LC–MS/MS analysis identified 690 proteins in harvested cell lysates, of which 140 showed differences in expression between balding and non-balding DPCs; 128 proteins were upregulated and 12 were decreased. Further pathway analysis showed that the upregulated proteins were associated mainly with protein homeostasis (transport, folding, degradation), cell adhesion, cytoskeleton and energy metabolism; these included gelsolin, plectin, non-erythroid α-spectrin (SPTAN1) and Ras GTPase-activating-like protein (IQGAP1). These proteins were also identified immunologically, using cell lysate immunoblot and hair follicle cryostat preparation immunofluorescence.

Network analysis of the upregulated proteins suggests an increase in protein turnover in balding follicles, along with indications that the cells are under higher levels of stress, leading to apoptosis, as shown in other studies. The authors also speculate that upregulation of DNA repair proteins is perhaps due to UV-induced DNA damage as a consequence of lack of hair cover.

Although gene expression in balding DPCs and secretion of potentially inhibitory substances has been investigated, the authors observe there is very little known about protein secretion by these cells. Using cultured DPCs, they identify proteins altered in balding follicles. Their study also validates 2-D chromatography with MS as a method of investigating the proteomics of balding, and of identifying potential therapeutic targets.

Reference

1. Moon, P-G., et al. (2013) “Proteomic analysis of balding and non-balding mesenchyme-derived dermal papilla cells from androgenetic alopecia patients using on-line two-dimensional reversed phase-reversed phase LC–MS/MS,” Journal of Proteomics, 85 (pp. 174–191).