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Miguel Seabra, M. Helena Cardoso, Michael Hall, A. Sofia Falcão, Cristina Escrevente, Pedro Antas, Gabriela V. Santos, Sandra Tenreiro, Clare E. Futter; Modeling lysosomal dysfunction in the retinal pigmented epithelium to study pathogenesis of age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1246.
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© ARVO (1962-2015); The Authors (2016-present)
Age-related Macular Degeneration (AMD) is the leading cause of vision impairment in developed countries. In AMD, photoreceptor degeneration appears to occur as a consequence of Retinal Pigment Epithelium (RPE) dysfunction. A crucial function of RPE is the ability to degrade protein- and lipid- content which result from daily processing of Photoreceptor Outer Segments (POS). Degradation and recycling of cellular components are the main responsibilities attributed to lysosomes and, remarkably, many age-related diseases stem from improper lysosomal health. The present work focuses on the development and characterization of a human RPE in vitro model, derived from pluripotent stem cells, with the ultimate goal of studying early events in age-related RPE disease. This model was used to explore the importance of lysosomes in intracellular cargo processing and the role of lysosomal dysfunction in AMD.
A drug-based model featuring chloroquine-induced hESc-RPE lysosomal dysfunction was established and characterized. Cells were subjected to extended chloroquine treatments in acute (1d), continued (3d) and chronic (7d) protocols. At the different times, several read-outs related to lysosomal function were developed using flow cytometry, light and electron microscopy (EM) methods, and immunoblot.
Chloroquine treatment of RPE cells leads to an impairment of the degradative ability, as assessed by the presence of reminiscent POS which accumulate over time. Deficiency of cargo processing was further demonstrated, through the decline of DQ-BSA proteolytic breakdown. Autophagy markers were also found to increase over the course of treatment, indicating impaired flux. Morphological changes observed by EM shared characteristics described in AMD. These changes occurred despite nuclear translocation of the lysosomal transcription factor, TFEB and the upregulation of expression and activity of cathepsins.
Chloroquine-treatment of hESc-RPE induced lysosomal dysfunction and thus recapitulates some characteristics of AMD in vitro. Identification and characterization of defective pathways responsible for dysfunctional lysosomal activity will contribute to a better understanding of AMD pathogenesis and identification of new therapeutic targets.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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