An in vitro model of daily proteolytic burden provides insights into proteostatic resilience of the RPE

Abstract

Age-related macular degeneration (AMD) is a leading cause of vision loss in developed societies, affecting approximately 196 million individuals worldwide. AMD affects the macula, which is crucial for everyday activities such as reading, driving, and recognising faces. Late-stage AMD manifests as geographic atrophy (dry AMD) and is characterised by the progressive degeneration of the retinal pigment epithelium (RPE) and overlying photoreceptors. A hallmark of dry AMD is the accumulation of high-molecular-weight macromolecules within RPE cells which originate from the incomplete degradation of internalised photoreceptor outer segments (POS). These toxic molecules (lipofuscin) which undergo further modifications, accumulate in the terminal compartments of the phagosome/endosome and autophagy-lysosomal pathway, contributing to chronic proteolytic stress: a clinically well-defined route of RPE atrophy in dry AMD. Lipofuscin and its derivatives possess inherent autofluorescent properties which is used as a gold-standard clinical biomarker. The high photo-oxidative environment of the retina, alongside the additional deposition of pathogenic aggregate-prone molecules such as Amyloid beta (Aβ), further contributes to stress factors that drives RPE dysfunction. These converge to eventually impair the proteolytic capacity of healthy RPE cells, the precise mechanisms of which still remains poorly understood.
Herein we investigated the impact of the daily proteolytic burden of POS internalisation and degradation in the ARPE-19 cell-line. We developed a novel in vitro cell model that simulates this sustained proteolytic burden in RPE cells, allowing for detailed time-point analysis of POS trafficking and processing via the phagosome/endosome and autophagy-lysosomal pathways. A combination of confocal microscopy, immunoblotting and whole RNAseq approaches were used to gain insights into this process across different mechanistic levels.
Our findings highlight the ability of RPE cells to manage the daily POS burden via existing trafficking compartments in the proteolytic pathway. Interestingly, we found no evidence of new protein synthesis related to these events. However, POS trafficking/processing was correlated with changes at the mRNA level, consistent with a well-defined pattern of sequential gene upregulation and downregulation which supported this homeostatic function. In parallel experiments we showed that exposure to oxidative stress (100μM H2O2), Aβ (1μM) and OxPOS (which recapitulates the intracellular aggregation of molecules), resulted in the mis-trafficking of POS to compartments of the phagosome/endosome and autophagy-lysosomal pathway at key timepoints. We also showed how daily POS exposure can reproduce the increase in autofluorescence associated with ageing and disease.

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Identifiers

ORCID for Rebecca Denise Miller: orcid.org/0000-0001-8550-6921

ORCID for Arjuna Ratnayaka: orcid.org/0000-0002-1027-6938

ORCID for Andrew Lotery: orcid.org/0000-0001-5541-4305

ORCID for David Tumbarello: orcid.org/0000-0002-5169-0561

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