Department of Ophthalmology, University of Bonn, Bonn, Germany.
Exp Eye Res. 2010 Mar;90(3):465-71. doi: 10.1016/j.exer.2009.12.011. Epub 2010 Jan 6.
Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.
有几条证据表明，视网膜色素上皮 (RPE) 的进行性功能障碍是年龄相关性黄斑变性 (AMD) 发病机制的核心。我们之前的研究表明，脂质过氧化产物如 4-羟基壬烯醛 (HNE) 和丙二醛 (MDA) 引起的蛋白质修饰会导致体外 RPE 细胞溶酶体功能障碍。在这里，我们研究了吞噬经修饰的光感受器外节 (POS) 是否会影响脂褐素生成和自噬，这两个与溶酶体功能直接相关的相互关联的过程。将 HNE 和 MDA 修饰的 POS 孵育于人 RPE 细胞中，会导致具有脂褐素样自发荧光的颗粒物质在细胞内大量积累。在用修饰的 POS 每日处理 7 天后，通过流式细胞术定量，细胞内自发荧光增加了 8.2 倍。在溶酶体抑制剂氯化铵存在的情况下，未修饰的 POS 同样会引起自发荧光增加 8.0 倍。与孵育天然 POS 相比，孵育修饰 POS 后的细胞自发荧光的光谱谱图没有变化。自噬活性，以代谢放射性标记的内源性蛋白的周转率来衡量，被 HNE 和 MDA 修饰的 POS 分别降低了 40%。即使没有 POS，3-甲基腺嘌呤的自噬抑制和氯化铵的溶酶体抑制也会诱导脂褐素生成。总之，我们的结果表明，脂质过氧化衍生的蛋白质修饰诱导溶酶体功能障碍会导致体外 RPE 细胞中脂褐素生成增加和自噬活性降低。这些机制可能导致 AMD 中 RPE 细胞功能障碍和变性。
