Tese de Doutoramento Doutoramento em Envelhecimento e Doenças Crónicas 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Abstract Skin pigmentation is achieved and sustained by the crosstalk between melanocytes and keratinocytes. Melanin is synthesized in melanocytes, within lysosome-related organelles termed melanosomes, and is then transferred to neighbouring keratinocytes. Upon internalization by keratinocytes, melanin becomes polarized and concentrated in the perinuclear region, forming the supra-nuclear melanin caps. This distribution provides the cells protection against the genotoxic effects of ultraviolet radiation, the main cause of skin cancer. Even though melanin transfer and polarization are critical steps in skin pigmentation, little is known about the molecular mechanisms involved. Therefore, this project aimed to identify new regulators of melanin internalization and determine how melanin is processed and trafficked inside keratinocytes. We confirmed the existence of two distinct routes of melanin internalization. Indeed, we observed that melanin cores, or melanocores, are internalized by phagocytosis and melanosomes by macropinocytosis. The small GTPases of the Rho family are known modulators of the assembly and organization of the actin cytoskeleton and play important roles in endocytosis. Specifically, Rac1, Cdc42 and RhoA have been shown to modulate phagocytosis and macropinocytosis, which prompted us to investigate if these Rho GTPases participated in melanin internalization. Remarkably, we showed that melanocore internalization is dependent on Rac1 and Cdc42, whereas melanosome uptake relies on RhoA. Therefore, these results indicate that the way melanin is presented to keratinocytes, namely the presence or absence of a surrounding membrane, influences the internalization route. Moreover, we found evidence that the melanin-containing phagosomes undergo a maturation process inside keratinocytes and fuse with acidic and hydrolase-active lysosomes, to form a final compartment termed melanokerasome. Considering this, we questioned whether the transport of melanokerasomes toward the perinuclear region of keratinocytes would be regulated by the same mechanisms as the ones that govern lysosomal transport. It has been previously shown that, similar to lysosomes, the retrograde transport of melanin inside keratinocytes requires the motor protein dynein. Since the small Rab GTPase Rab7 is a known modulator of the dyneinmediated transport, we assessed if this protein was involved in melanin positioning. In fact, we showed that the correct positioning of melanin, at the perinuclear region of keratinocytes, is impaired upon the depletion of Rab7 and its effector RILP. In addition, we observed that the retrograde transport of melanokerasomes is dependent on the integrity of the microtubule network and on the polarization regulator PAR3. This protein has been reported to play a role in the polarization of the microtubule-organizing center and the recruitment of lysosomes in immune cells. This led us to the conclusion that, after the fusion of melanin-containing phagosomes with lysosomes, the resulting melanokerasomes exploit the lysosomal trafficking machinery for their correct positioning at the perinuclear region of keratinocytes. In order to further investigate the melanin polarization mechanisms in a more physiological model, we developed 3D reconstructed pigmented epidermises with human epidermal melanocytes and human embryonic keratinocytes. These reconstructed epidermises were found to mimic not only the architecture, but also the physiological pigmentation processes that occur in the human skin, including melanin synthesis, transfer, processing and polarization. Indeed, we observed the presence of supra-nuclear caps in our reconstructed epidermises, which were never exposed to ultraviolet radiation. These results suggest that keratinocyte stratification alone promotes melanin polarization and the formation of the supra-nuclear caps and that, although ultraviolet radiation may play a role in this process, it is not essential. Furthermore, among the processes involved in keratinocyte stratification, we found that cell migration was especially relevant for melanin polarization. Together, these findings provide new insights about the mechanisms required for efficient skin photoprotection. Furthermore, this work identified new regulatory molecules that can potentially be targeted for the treatment of pigmentary disorders, as well as for the development of new drugs for the pharmacological and cosmetic industries.

Abstract Skin pigmentation is achieved and sustained by the crosstalk between melanocytes and keratinocytes. Melanin is synthesized in melanocytes, within lysosome-related organelles termed melanosomes, and is then transferred to neighbouring keratinocytes. Upon internalization by keratinocytes, melanin becomes polarized and concentrated in the perinuclear region, forming the supra-nuclear melanin caps. This distribution provides the cells protection against the genotoxic effects of ultraviolet radiation, the main cause of skin cancer. Even though melanin transfer and polarization are critical steps in skin pigmentation, little is known about the molecular mechanisms involved. Therefore, this project aimed to identify new regulators of melanin internalization and determine how melanin is processed and trafficked inside keratinocytes. We confirmed the existence of two distinct routes of melanin internalization. Indeed, we observed that melanin cores, or melanocores, are internalized by phagocytosis and melanosomes by macropinocytosis. The small GTPases of the Rho family are known modulators of the assembly and organization of the actin cytoskeleton and play important roles in endocytosis. Specifically, Rac1, Cdc42 and RhoA have been shown to modulate phagocytosis and macropinocytosis, which prompted us to investigate if these Rho GTPases participated in melanin internalization. Remarkably, we showed that melanocore internalization is dependent on Rac1 and Cdc42, whereas melanosome uptake relies on RhoA. Therefore, these results indicate that the way melanin is presented to keratinocytes, namely the presence or absence of a surrounding membrane, influences the internalization route. Moreover, we found evidence that the melanin-containing phagosomes undergo a maturation process inside keratinocytes and fuse with acidic and hydrolase-active lysosomes, to form a final compartment termed melanokerasome. Considering this, we questioned whether the transport of melanokerasomes toward the perinuclear region of keratinocytes would be regulated by the same mechanisms as the ones that govern lysosomal transport. It has been previously shown that, similar to lysosomes, the retrograde transport of melanin inside keratinocytes requires the motor protein dynein. Since the small Rab GTPase Rab7 is a known modulator of the dyneinmediated transport, we assessed if this protein was involved in melanin positioning. In fact, we showed that the correct positioning of melanin, at the perinuclear region of keratinocytes, is impaired upon the depletion of Rab7 and its effector RILP. In addition, we observed that the retrograde transport of melanokerasomes is dependent on the integrity of the microtubule network and on the polarization regulator PAR3. This protein has been reported to play a role in the polarization of the microtubule-organizing center and the recruitment of lysosomes in immune cells. This led us to the conclusion that, after the fusion of melanin-containing phagosomes with lysosomes, the resulting melanokerasomes exploit the lysosomal trafficking machinery for their correct positioning at the perinuclear region of keratinocytes. In order to further investigate the melanin polarization mechanisms in a more physiological model, we developed 3D reconstructed pigmented epidermises with human epidermal melanocytes and human embryonic keratinocytes. These reconstructed epidermises were found to mimic not only the architecture, but also the physiological pigmentation processes that occur in the human skin, including melanin synthesis, transfer, processing and polarization. Indeed, we observed the presence of supra-nuclear caps in our reconstructed epidermises, which were never exposed to ultraviolet radiation. These results suggest that keratinocyte stratification alone promotes melanin polarization and the formation of the supra-nuclear caps and that, although ultraviolet radiation may play a role in this process, it is not essential. Furthermore, among the processes involved in keratinocyte stratification, we found that cell migration was especially relevant for melanin polarization. Together, these findings provide new insights about the mechanisms required for efficient skin photoprotection. Furthermore, this work identified new regulatory molecules that can potentially be targeted for the treatment of pigmentary disorders, as well as for the development of new drugs for the pharmacological and cosmetic industries.
Resumo A pigmentação da pele é alcançada e sustentada pela comunicação entre melanócitos e queratinócitos. A melanina é sintetizada nos melanócitos, em organelos especializados denominados de melanossomas, e é posteriormente transferida para os queratinócitos adjacentes. Ao ser internalizada pelos queratinócitos, a melanina é polarizada e concentrada na região perinuclear, formando as capas de melanina supra-nucleares. Essa distribuição proporciona às células proteção contra os efeitos nocivos da radiação ultravioleta, considerada como a principal causa do cancro de pele. Embora a transferência e a polarização da melanina sejam etapas fundamentais da pigmentação da pele, pouco se sabe sobre os mecanismos moleculares envolvidos. Deste modo, este projeto teve como objetivo identificar novos reguladores da internalização da melanina e determinar como a melanina é processada e transportada dentro dos queratinócitos. O nosso grupo confirmou a existência de duas vias distintas de internalização de melanina. De facto, observámos que os núcleos de melanina, ou melanocores, são internalizados por fagocitose e os melanossomas por macropinocitose. Por outro lado, as GTPases da família Rho têm como principal função a regulação da síntese e organização do citoesqueleto de actina e desempenham papéis importantes na via endocítica. Em particular, o Rac1, Cdc42 e RhoA foram identificados como reguladores essenciais dos processos de fagocitose e macropinocitose, o que nos fez questionar sobre um potencial papel das Rho GTPases no processo de internalização de melanina. Notavelmente, mostrámos que a internalização de melanocores é dependente de Rac1 e Cdc42. Em contrapartida, os melanossomas são internalizados por um processo dependente de RhoA. Por conseguinte, estes resultados indicam que a forma como a melanina é apresentada aos queratinócitos, nomeadamente, a presença ou ausência de uma membrana envolvente, influencia a sua via de internalização. Além disso, determinámos que os fagossomas onde a melanina reside passam por um processo de maturação dentro dos queratinócitos, o qual culmina na sua fusão com lisossomas com propriedades acídicas e degradativas. Esta fusão resulta na formação de um compartimento final designado de melanokerassoma. Posto isto, interrogámo-nos se o transporte de melanina até à região perinuclear dos queratinócitos seria regulado pelos mesmos mecanismos do transporte de lisossomas. Estudos anteriores demonstraram que, tal como o transporte intracelular de lisossomas, o transporte retrógrado de melanina requer a proteína motora dineína. Como a pequena Rab GTPase Rab7 é um conhecido modulador do transporte mediado pela dineína, avaliámos se esta proteína estaria envolvida no posicionamento da melanina. De facto, mostrámos que o silenciamento da Rab7 e do seu efetor RILP inibiu o posicionamento correto da melanina, na região perinuclear dos queratinócitos. Além disso, observámos que o transporte retrógrado de melanokerasomes é dependente da integridade da rede de microtúbulos e do regulador de polarização PAR3. Esta proteína foi identificada como sendo relevante para a polarização do centrossoma e no recrutamento de lisossomas em células do sistema imunitário, o que nos levou a especular que, após a fusão dos fagossomas que contêm melanina com lisossomas, os melanokerasomes resultantes utilizam a maquinaria de transporte lisossomal para o seu posicionamento na região perinuclear dos queratinócitos. De forma a investigar os mecanismos de polarização da melanina num contexto mais aproximado do fisiológico, desenvolvemos um modelo 3D de epidermes pigmentadas reconstruídas. Para tal, utilizámos melanócitos queratinócitos primários humanos. Observou-se que o modelo resultante mimetizava não só arquitetura, mas também os processos fisiológicos de pigmentação que ocorrem na pele humana, tal como a síntese, transferência, processamento e polarização de melanina. Efetivamente, identificámos a acumulação de melanina na zona supra-nuclear das epidermes reconstruídas, as quais nunca foram expostas à radiação ultravioleta. Os resultados indicam que a estratificação dos queratinócitos promove intrinsecamente a polarização da melanina e a sua acumulação na zona supra-nuclear dos queratinócitos e que, embora a radiação ultravioleta possa desempenhar um papel nesse processo, não é essencial. De todos os processos envolvidos na estratificação dos queratinócitos, determinámos que o processo de migração é particularmente relevante para a polarização de melanina. Em suma, este estudo fornece uma nova perspetiva sobre os mecanismos reguladores da fotoproteção da pele. Adicionalmente, este estudo identificou novas moléculas reguladoras que podem potencialmente ser direcionadas para o tratamento de distúrbios de pigmentação, e para o desenvolvimento de novos medicamentos pelas indústrias farmacêutica e cosmética