The pathways to better skin disease treatments

Attendees at UCD’s Charles Institute Seminar Series heard a presentation by Dr Vivek T Natarajan on the behaviour
of melanocytes in conditions such as vitiligo, melasma and melanoma

The Charles Institute, Ireland’s national dermatology research and education centre, hosts a range of guest speakers who cover a variety of topics ranging from skin cancer to psoriasis, among others. The series, which is sponsored by RELIFE (part of the A.Menarini group), is designed to provide expert advice from a range of distinguished national and international experts in their respective fields and is chaired by Prof Desmond Tobin, Full Professor of Dermatological Science at UCD School of Medicine and Director of the Charles Institute of Dermatology.

The seminars are broadcast to attendees with a special interest in dermatology and cutaneous science in other
locations, who access the talks remotely via an audio-visual link.

The seminar was held using a hybrid model, combining in-person attendance with interactive online access.Attendees heard a presentation by Dr Vivek T Natarajan, Senior Principal Scientist at the CSIR Institute of Genomics and Integrative Biology in New Delhi, India. Dr Natarajan spoke on the topic ‘Decoding Cell Fate Decisions in Pigment Cell Lineage: From Cues to Codes’. Dr Natarajan and his team use cultured cells and zebrafish to examine fate decisions in pigment cell lineage, as well as state-of the-art genomics tools to better understand state transitions in melanocytes.

All these research efforts are directed towards improving knowledge of the role of melanocytes in skin homeostasis and pigmentary disorders of the skin.

He explained that melanocytes assume various forms that are discernible through the development of homeostasis and skin diseases. Understanding this continuum, said Dr Natarajan, is crucial to elucidate the behaviour of melanocytes and therefore arrive at a meaningful treatment solution for conditions such as melanoma, melasma and vitiligo. Dr Natarajan presented examples of the ongoing work in his laboratory on the specification of melanocytes, including the directed migration that they require to establish the stem cell pool.

He also provided an outline of recently-published research by him and his team describing the role of a histone variant in melanocyte specification. He also discussed the ongoing work to examine the role of the plexin-semaphorin signalling axis that not only catalyses melanocyte migration, but also fine-tunes melanocytes to stem cell establishment and maturation cues.

Unmet needs

The motivation behind his research, Dr Natarajan explained, is to address unmet clinical needs in skin disease. Vitiligo, in particular, requires cell-based approaches to treatments: “There is an unmet need to understand the etiopathogenesis of vitiligo and to provide a meaningful solution for repigmentation,” he said. “Currently, there are several methods where the immune activation is curtailed and there are ways in which melanocytes are repopulated.

One common method in the Indian subcontinent is transplantation, where autologous melanocytes are put back into the skin. That’s currently done, but what we would like to achieve eventually is repigmentation by stimulating stem cells and repopulating stem cells as an adjunct methodology to repopulate the skin, in addition to curtailing the immune response.”

He provided an outline of the cells relevant to this treatment ambition and explained that zebrafish have melanocytes in their hypodermis, whereas in mice, they are present in the hair follicles and in humans, they are located in the epidermis and hair follicles. “Despite these differences across organisms, the core melanocyte transcription network seems to be conserved.

Therefore, meaningful insights can be gained to investigate their involvement in pathogenesis in the human context,
and understand their involvement in the much more complex tissue we have in humans,” he told the seminar.

Zebrafish

Dr Natarajan provided an overview of his research and discussed the zebrafish model system. “It comes in quite handy because it is an early vertebrate system where the gene networks are fairly conserved, particularly with respect to pigmentation,” he said. “We have been able to produce a number of lines that are able to report faithfully about the various stages of melanocyte maturation.”

This model allows pigmented cells to be seen easily. “It’s amazing to see how the whole organism develops when the pigmentation comes to its rudimentary form within three days of fertilisation,” he added.

Dr Natarajan presented examples of his work to track melanocytes and observe how they migrate to cover the entire embryo, and identify and characterise the genes involved in the biological transitions in vitiligo and other skin conditions. He provided a brief overview of his work using the zebrafish model to decipher the role of guidance cues in melanocyte migration, patterning and survival.

More specifically, “we wanted to identify intrinsic cellular factors that guide melanocyte movements, melanocyte-specific temporal transcriptional profiling and phenotypic validations,” he said. He also described his efforts to understand the underlying guidance modality in melanocyte migration and to delineate the molecular mechanism, including the signalling pathway, partners and mediators using gene expression analysis.

Zebrafish embryos are very transparent, which allows extremely clear imaging at different stages of development. Dr Natarajan and his colleagues found evidence to suggest that Plexin D silencing disrupts the positioning of melanoblasts and therefore affects them in some manner. “We believe that Plexin D1 is important for the consolidation of melanocytes into directional streams and hence, when you silence Plexin D1, the melanocytes are not able to migrate and pattern properly,” he told the seminar.

Dr Natarajan summarised by telling the attendees: “We have identified the Sema3e-Plxnd1 signalling pathway and we have been able to show that it excessively prompts the α-MSH-MC1R signalling by up-regulating the expression of GNAS. Melanocyte functioning is therefore affected by the plexin-mediated signalling,” he said. “There is also an activation arm of the Plexin pathway, which activates the EGFR phosphorylation at activation loop, which is responsible for the establishment of the stem cell pool and is being hindered because of the absence of Plexin D1.

“Sema3e-Plxnd1 signalling leads to decreased EGFR phosphorylation at the activation loop (Y992),” he continued.“Plexin D1 signalling in this manner regulates the ErbB activity essential for the recruitment and patterning of regenerative melanocytes. Loss of Plexin D1, however, leads to an ectopic position of melanoblasts, leading to their apoptosis,” he told the attendees.

“All of this is investigated in studies with embryonic zebrafish and some of the validation is in mechanistic investigation in cells,” he continued. Dr Natarajan and his team were successful in building a CRISPR mutant for the CRISPR-mediated, targeted disruption of Plexin D1, he added.

Diversity

During an interactive Q&A session following the presentation, Prof Tobin and Dr Natarajan discussed what was described as a long-term dilemma in tracing the different developmental stages of melanocytes in the human skin and hair follicle. Prof Tobin spoke about the stem cell reservoir in adult hair follicles, or even in the rete ridges of the epidermis, and whether there is a diversity in melanocyte differentiation states.

“For example, some people refer to melanocyte ‘precursors’, whilst other people use the term ‘melanoblasts’,” said Prof Tobin. “Other people suggest that in melanoma, you may have de-differentiation of previously differentiated melanocytes back to an immature state. So the whole area of pigment cell plasticity in adult human skin and hair follicle is an important topic for researchers— and as we now know, the eccrine sweat gland may also have skin melanocyte stem cells.

So it is extremely complicated, and currently, we don’t really have the markers to segregate all these melanocyte subpopulations. Some people suggest, as we did previously, that if any melanocytes survive in vitiligo, it is likely the ones that lack significant differentiation, and therefore may be essentially ‘invisible’ to the immune system trained on melanocyte maturation/ differentiation markers. Therefore, that issue of pigment cell plasticity in the zebrafish is quite interesting.”

Dr Natarajan commented: “That is very true, and very aptly put. The melanocytes are in fact a continuance of transitions that are present, and they are almost simultaneously present,” he commented. “Therefore, the human tissue or hair follicle becomes too difficult a model to do discovery. So I believe that discovery done in a system such as ours, and then investigated in the human skin context, is the way to go. A lot of clarity would come through looking at it through the human context; the model and mechanisms in zebrafish are going to be operational, even in humans. So, discovering here [zebrafish] and validation there [humans] would be a very easy way to identify the real perpetrators.”

RELIFE has had no input into the content of this article or series of seminars