Faculty Bibliography

Albinism affects about 1 in 4000 people in southern Africa. People with albinism (PWA) face physical, social and psychological challenges including high risk of skin cancer and visual deficits due to ocular hypopigmentation and optic tract maldevelopment. PWA need to avoid the sun, preventing them taking employment that necessitates being outside, while poor vision limits their independence (for example restricting their capacity to drive) and propagates the misconception that PWA have reduced intellectual capacity. Social challenges include negative self-perception, discrimination and more recently a dramatic rise in murders of PWA. While infanticide has long been reported, witchcraft-related killings have sharply increased, especially in Tanzania. Non-governmental organizations such as Under the Same Sun have raised awareness and some efforts have been made by southern African authorities to address the threat. In February 2017, a traditional healer was sentenced to life imprisonment in South Africa for the murder of a PWA whose body was used to make traditional medicine for good luck and increasing wealth. Psychological challenges include delayed mother-affected child bonding, ostracism and difficulty finding marriage partners. Outreach programs, such as those of the Albinism Society of South Africa, play an important role in educating the public, promoting advocacy and social acceptance. Ensuring that PWA are provided with the knowledge and help to face these challenges is thus critical. A valuable education mechanism for PWA is genetic counselling, which has been provided in Johannesburg since the early 1970s. Identification of the genes involved in albinism has made carrier testing and prenatal diagnosis a reality. To date seven prenatal tests have been performed in Johannesburg, with one fetus found to be affected (parents chose not to terminate the pregnancy). Addressing the needs of PWA albinism therefore ranges from countering ancient myths to education about the latest technologies for diagnosis and potential therapies

The mechanisms that initiate vitiligo are poorly understood. Vitiligo triggers, such as monobenzone (MB) exposure, induce stress. Understanding the survival responses that combat this stress is key to determining why melanocytes become immune targets. MB induces oxidative and endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR). PERK, a UPR initiator, phosphorylates eIF2alpha and master antioxidant regulator, NRF2. Here, we investigated the impact of PERK-eIF2alpha/-NRF2 activation on sensitivity to MB. Basal phospho-eIF2alpha and NRF2 levels are higher in melanocytes compared to fibroblasts or keratinocytes. PERK downregulation significantly reduced melanocyte viability (implicated in several autoimmune disorders) may link exposure to vitiligoinducing triggers with onset of autoimmunity

Vitiligo, characterized by progressive melanocyte death, can be initiated by exposure to vitiligo-inducing phenols (VIPs). VIPs generate oxidative stress in melanocytes and activate the master antioxidant regulator NRF2. While NRF2-regulated antioxidants are reported to protect melanocytes from oxidative stress, the role of NRF2 in the melanocyte response to monobenzone, a clinically relevant VIP, has not been characterized. We hypothesized that activation of NRF2 may protect melanocytes from monobenzone-induced toxicity. We observed that knockdown of NRF2 or NRF2-regulated antioxidants NQO1 and PRDX6 reduced melanocyte viability, but not viability of keratinocytes and fibroblasts, suggesting that melanocytes were preferentially dependent upon NRF2 activity for growth compared to other cutaneous cells. Furthermore, melanocytes activated the NRF2 response following monobenzone exposure and constitutive NRF2 activation reduced monobenzone toxicity, supporting NRF2's role in the melanocyte stress response. In contrast, melanocytes from individuals with vitiligo (vitiligo melanocytes) did not activate the NRF2 response as efficiently. Dimethyl fumarate-mediated NRF2 activation protected normal and vitiligo melanocytes against monobenzone-induced toxicity. Given the contribution of oxidant-antioxidant imbalance in vitiligo, modulation of this pathway may be of therapeutic interest

Purpose: GPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein-coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators. Methods: GPR143 interacts with beta-arrestin; we therefore established a beta-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation. Results: GPR143, which showed high constitutive activity in the beta-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity. Conclusions: X-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

Developmental eye defects in X-linked Ocular Albinism type I (OA1) are caused by G-Protein Coupled Receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most GPCRs, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for OA1. Many GPCRs require association with other proteins to function. These GPCR-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 co-immunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when co-expressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using Fluorescence Resonance Energy Transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis.

Vitiligo, characterized by progressive skin depigmentation, results from autoimmune-mediated melanocyte loss. The mechanisms underlying disease onset are poorly delineated. Triggers, including exposure to phenols such as monobenzone (MB), are thought to disrupt melanocyte homeostasis and ultimately instigate an autoimmune reaction. We have shown that MB disrupts cellular homeostasis and induces endoplasmic reticulum (ER) stress that leads to activation of the unfolded protein response (UPR). Three proteins, including PERK, each activate UPR arms that orchestrate the restoration of homeostasis. When activated, PERK phosphorylates eIF2a, a translation initiation factor, thus reducing protein synthesis and ER stress. In this study, we investigated the impact of the PERKeIF2a cascade on melanocyte viability and sensitivity to MB. Basal levels of phospho-eIF2a are higher in melanocytes compared to cutaneous fibroblasts or keratinocytes. When PERK expression is downregulated by RNAi, there is a significant reduction in melanocyte viability (88% decrease, p < 0.05 shPERK versus non-target/ shNT; n = 3). Some melanocytes (shPERKLT) can however survive despite prolonged PERK downregulation. Survival correlated with a paradoxical increase in phospho-eIF2a levels and reduced sensitivity to MB (Cleaved/c-PARP levels lower in shPERKLT cells treated with 400 muM MB compared to shNT cells). Chemical inhibition of PERK kinase activity, using GSK2606414, prevented eIF2a phosphorylation and sensitized melanocytes to MB (c-PARP observed with 250 muM MB+ GSK2606414, compared to 400 muM MB + vehicle). PERK-eIF2a axis activity contributes to melanocyte viability and determines sensitivity to MB. Pathways, such the UPR, which has also been implicated in autoimmune diabetes, may link exposure to vitiligo-inducing triggers and onset of autoimmunity. These pathways represent novel therapeutic targets to prevent vitiligo progression or improve efficacy of repigmentation protocols

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either White or African American (AA) skin. Two dimensional gel electrophoresis (2-DGE) and mass spectrometry demonstrated that Heat Shock Protein 70-1A (Hsp70-1A) protein levels were significantly higher in AA melanocytes compared to White melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70-family members in tyrosinase post-translational modification. Additionally, pharmacologic inhibition and siRNA-mediated down-regulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.

Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.