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Purification and characterization of dysfunctional lysosomes in an age-related macular disease cell model
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RESUMO:O declínio da função lisossomal tem sido apontado como estando envolvido numa série de
doenças crónicas relacionadas com a idade, nomeadamente em doenças neurodegenerativas, cancro,
aterosclerose e retinopatias. Neste projeto colocamos a hipótese de que lisossomas disfuncionais
adquirem propriedades únicas e marcadores específicos, distinguindo-se dos funcionais. Durante este
trabalho, usámos o epitélio pigmentar da retina humana como modelo celular para identificar as
propriedades intrínsecas de lisossomas disfuncionais. O epitélio pigmentar da retina é responsável
pela fagocitose dos segmentos externos dos fotorreceptores, necessários ao processamento visual.
Desta forma, sugerimos que a disfunção lisossomal nestas células resulta da acumulação dos
segmentos externos dos fotorreceptores que consequentemente leva a retinopatias.
Durante este trabalho, desenvolvemos três abordagens diferentes para estudar disfunção
lisossomal em contextos distintos: uma abordagem baseada na doença, um modelo genético de
disfunção lisossomal e uma estratégia farmacológica. Estas abordagens constituem modelos chave
para estudar marcadores comuns de doença ao combinar os diferentes resultados obtidos em cada
uma das estratégias. Além do mais, otimizámos a técnica de purificação de lisossomas baseada na
immunocaptura do epítopo HA em células do epitélio pigmentar da retina, o que nos permite uma
análise detalhada do proteoma de lisossomas purificados de células do epitélio pigmentar da retina e
avaliação de novos marcadores de doença. Adicionalmente, projetámos uma estratégia de
CRISPR/Cas9 para gerar células estaminais pluripotentes que expressam proteinas lisossomais
conjugadas com o epítopo HA. Este último, além de permitir o estudo da disfunção lisossomal, pode
também ser usado para estudos de dinâmica lisossomal e interações no ambiente celular.
Pretendemos assim estabelecer uma nova interpretação relativamente aos mecanismos
moleculares que estão por detrás do desenvolvimento de variadas doenças crónicas relacionadas com
a idade, ao combinar as estratégias aqui desenvolvidas com técnicas de proteómica. Deste modo
pretendemos identificar novos alvos terapêuticos bem como desvendar mecanismos comuns a
doenças degenerativas que apresentam declínio na função lisossomal.
ABSTRACT: The decline of lysosomal function is postulated to contribute to several age-related diseases, namely neurodegenerative diseases, cancer, atherosclerosis and retinopathies. We hypothesize that dysfunctional lysosomes acquire unique properties and biomarkers, distinct from fully active lysosomes. Here, we use the human retinal pigmented epithelium (RPE) as a cell model to identify the intrinsic properties of dysfunctional lysosomes. RPE cells are responsible for the daily phagocytosis of Photoreceptor Outer Segments (POS) required for the visual process. Thus, we believe that the lysosomal dysfunction in these cells results in the accumulation of POS that ultimately leads to retinopathy. We developed three different approaches to study lysosomal dysfunction in different contexts: a disease-driven approach, a genetic-based model of lysosomal dysfunction and a pharmacological strategy. These models will be key frameworks to look for common biomarkers of disease by combining the selective readouts. Furthermore, we have optimized the lysosomes HA-based immunopurification technique (LysoIP) in RPE cells, which will allow a detailed evaluation of the proteome of immunopurified lysosomes from our human RPE cell model and the search for biomarkers of dysfunction. Additionally, we have designed a CRISPR/Cas9 strategy to generate knockin human pluripotent stem cells (PSC) expressing HA-fused lysosomal proteins. The later will provide a physiological model for the study of not only lysosomal dysfunction but other studies such as lysosomal dynamics and interactions within the cell. We believe that combining our strategies with proteomics tools will offer new insights into the molecular mechanisms behind several age-related diseases presenting declining lysosomal function, allowing the identification of new therapeutic targets and unveil missing links common to several degenerative disorders.
ABSTRACT: The decline of lysosomal function is postulated to contribute to several age-related diseases, namely neurodegenerative diseases, cancer, atherosclerosis and retinopathies. We hypothesize that dysfunctional lysosomes acquire unique properties and biomarkers, distinct from fully active lysosomes. Here, we use the human retinal pigmented epithelium (RPE) as a cell model to identify the intrinsic properties of dysfunctional lysosomes. RPE cells are responsible for the daily phagocytosis of Photoreceptor Outer Segments (POS) required for the visual process. Thus, we believe that the lysosomal dysfunction in these cells results in the accumulation of POS that ultimately leads to retinopathy. We developed three different approaches to study lysosomal dysfunction in different contexts: a disease-driven approach, a genetic-based model of lysosomal dysfunction and a pharmacological strategy. These models will be key frameworks to look for common biomarkers of disease by combining the selective readouts. Furthermore, we have optimized the lysosomes HA-based immunopurification technique (LysoIP) in RPE cells, which will allow a detailed evaluation of the proteome of immunopurified lysosomes from our human RPE cell model and the search for biomarkers of dysfunction. Additionally, we have designed a CRISPR/Cas9 strategy to generate knockin human pluripotent stem cells (PSC) expressing HA-fused lysosomal proteins. The later will provide a physiological model for the study of not only lysosomal dysfunction but other studies such as lysosomal dynamics and interactions within the cell. We believe that combining our strategies with proteomics tools will offer new insights into the molecular mechanisms behind several age-related diseases presenting declining lysosomal function, allowing the identification of new therapeutic targets and unveil missing links common to several degenerative disorders.
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Palavras-chave
Degenerescência Macular da idade Grânulos autofluorescentes Doenças Crónicas relacionadas com a idade Disfunção Lisossomal Epitélio Pigmentar da Retina Age-related Macular Degeneration Autofluorescent granules Chronic age-related diseases Lysosome dysfunction Retinal Pigmented Epithelium