Purchase this article with an account.
Carolina Brandstetter, Jürgen Kopitz, Eicke Latz, Frank G. Holz, Tim U. Krohne; Effects Of Lipofuscin Photoreactivity On RPE Lysosomal Membrane Stability And Activation Of The NALP3 Inflammasome. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1673.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Lipofuscin accumulation in the retinal pigment epithelium (RPE) secondary to impaired lysosomal function represents a hallmark of age-related macular degeneration (AMD). Both lipofuscin photoreactivity and local inflammation have been implicated in the progressive RPE dysfunction and degeneration in AMD. This study employs an in vitro model of lipid peroxidation-mediated lipofuscinogenesis to investigate the effects of lipofuscin photoreactivity on RPE lysosomal membrane stability and activation of the NALP3 inflammasome.
Isolated porcine photoreceptor outer segments (POS) were modified with the lipid peroxidation products 4-hydroxynonenal (HNE) or malondialdehyde (MDA). Confluent monolayers of the human RPE-derived cell line ARPE-19 were incubated with modified POS for 7 days and subsequently exposed to blue light (wavelength 455-460 nm; irradiance 0.8 mW/cm2). Cytotoxicity was assessed by LDH release and DNA fragmentation assays. Lysosomal membrane permeabilization was analyzed by acridine orange staining and activity of lysosomal marker enzyme N-acetyl-glucosaminidase (NAG) in isolated cytosolic fractions. Following priming of cells with IL-1α, NALP3 inflammasome activation was investigated by release of IL-1β.
Phagocytosis of HNE- or MDA-modified POS induced pronounced cellular lipofuscinogenesis. Subsequent blue light irradiation for 9 hours resulted in a reduction of cell viability to 17%. In contrast, control cells incubated with unmodified POS were unaffected by light treatment (viability 97%). Lipofuscin-associated cytotoxicity was mediated by apoptosis. Lysosomal membrane permeabilization occurred after blue light irradiation in a dose-dependent manner and was associated with activation of the NALP3 inflammasome as evident from expression and release of mature IL-1β. Inhibition of caspase activity using Z-VAD-FMK suppressed IL-1β release.
Lipofuscinogenesis induced by lipid peroxidation-related protein modifications renders RPE cells susceptible to light-induced lysosomal destabilization and apoptotic cell death. Lipofuscin photoreactivity-mediated lysosomal membrane permeabilization is associated with activation of the NALP3 inflammasome and release of IL-1β. Via this mechanism, lipofuscin accumulation in the RPE may contribute to local inflammation in AMD.
This PDF is available to Subscribers Only