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T. U. Krohne, N. K. Stratmann, J. Kopitz, F. G. Holz; Impaired Autophagy and Increased Lipofuscinogenesis Induced by POS Modified With Lipid Peroxidation Products in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):220.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that photoreceptor outer segments (POS) modified with lipidperoxidation products like 4-hydroxynonenal (HNE) and malondialdehyde (MDA) become stabilised against lysosomal degradation and reduce lysosomal enzyme activities in retinal pigment epithelium (RPE) cells. Here we analysed whether this lysosomal dysfunction also results in impaired degradation of endogenous cellular proteins (autophagy) and how these two mechanisms contribute to intracellular lipofuscin accumulation which represents a clinical hallmark of age-related macular degeneration (AMD).
Isolated porcine POS were modified with HNE and MDA. Human RPE cells (ARPE-19) were incubated daily with POS for up to 7 days. For specific inhibition of lysosomal functions and autophagy we employed ammonium chloride and 3-methyladenine, respectively. Cellular turnover of radiolabelled endogenous cellular proteins by autophagy was measured in pulse-chase experiments. Development of autofluorescent lipofuscin granules was documented using fluorescence microscopy. We quantified cellular autofluorescence by means of flow cytometry (FACS). Emission and excitation spectra of cellular autofluorescence were assessed by fluorescence spectroscopy.
Incubation of RPE cells with HNE- and MDA-modified POS reduced cellular autophagy by 48% and 35%, respectively, compared to 3% for unmodified POS. We observed intracellular accumulation of granular material with lipofuscin-like autofluorescence. Mean cellular autofluorescence increased 8.2fold following incubation with modified POS as compared to 2.5fold for unmodified POS. Similarly, incubation with unmodified POS with additional lysosomal inhibition by ammonium chloride caused an 8.0fold increase. Interestingly, even 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 and excitation maxima at 270nm, 360nm, and 445nm for 570nm emission consistently in all groups.
Lysosomal dysfunction induced by phagocytosis of POS modified with lipidperoxidation products results in impaired autophagy in RPE cells. Both lysosomal dysfunction and impaired autophagy increase cellular lipofuscinogenesis. These mechanisms may be involved in the pathogenesis of RPE changes observed in AMD.
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