Abstract

DO.05.06

Impaired autophagy in RPE cells following incubation with lipid peroxidation-modified POS

Krohne T. U.¹, Stratmann N. K.¹, Holz F. G.¹, Kopitz J.^2
1Department of Ophthalmology, University of Bonn, Germany; ²Department of Molecular Pathology, University of Heidelberg, Germany

Objective: Autophagy, the continuous turnover of damaged organelles and macromolecules by lysosomal degradation, is vital for cellular homeostasis especially in long-lived cells like the retinal pigment epithelium (RPE). Impaired autophagy might contribute to lipofuscinogenesis and RPE cell dysfunction observed in age-related macular degeneration (AMD). We previously demonstrated that photoreceptor outer segments (POS) modified with lipidperoxidation products like 4-hydroxynonenal (HNE) and malondialdehyde (MDA) reduce lysosomal function in RPE cells. Here we analysed the effect of modified POS on RPE cell autophagy and lipofuscinogenesis.
Methods: Human RPE cells (ARPE-19) were challenged with purified POS previously modified with HNE and MDA. Endogenous cellular proteins were radioactively labelled with ³H-leucine and its turnover was measured in pulse-chase experiments. In control experiments lysosomal functions and autophagy were specifically inhibited by ammonium chloride and 3-methyladenine, respectively. Lipofuscinogenesis in treated cells was documented with fluorescence microscopy and quantified using flow cytometry (FACS).
Results: Phagocytosis of untreated POS had no effect on autophagy rate in RPE cells. In contrast, incubation with both HNE- and MDA- modified POS resulted in a marked reduction of autophagy activity to 59.8% and 59.7%, respectively. We observed intracellular accumulation of granular material with lipofuscin-like autofluorescence after 7 days. Mean cellular autofluorescence increased 8.2fold following incubation with modified POS as compared to 2.5fold for unmodified POS. Without addition of POS both inhibition of lysosomal functions and inhibition of autophagy were sufficient to increase autofluorescence 2.7fold and 2.0fold, respectively. Spectral analysis of cellular autofluorescence detected emission maxima at 440nm and 515nm with 380nm excitation.
Conclusions: Lysosomal dysfunction induced by phagocytosis of POS modified with lipidperoxidation products inhibits autophagy in RPE cells. Both lysosomal dysfunction and impaired autophagy result in increased cellular lipofuscinogenesis. These mechanisms may be involved in the pathogenesis of RPE changes observed in AMD.

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