Schutt Florian, Bergmann Marion, Holz Frank G, Dithmar Stefan, Volcker Hans-Eberhard, Kopitz Jurgen
Department of Ophthalmology, University of Heidelberg, INF 400, 69120, Heidelberg, Germany.
Graefes Arch Clin Exp Ophthalmol. 2007 Mar;245(3):391-8. doi: 10.1007/s00417-006-0376-5. Epub 2006 Aug 2.
Lipofuscin occurs in association with various blinding diseases, including ARMD. Formation of lipofuscin is considered to be initiated by the inability of the RPE lysosome to degrade constituents of phagocytosed material resulting in its intralysosomal accumulation. Thus, the deposition of abnormal retinoid adducts causing the autofluorescent properties of RPE lipofuscin originates from abnormal products of the retinoid cycle contained in phagocytosed photoreceptor outer segments. The major lipofuscin retinoid conjugate A2-E was previously shown to exert toxic effects on RPE cells by directly damaging lysosomal function and structure. However, A2-E was also proposed to severely harm extralysosomal RPE cell structures during the pathogenesis of ARMD. This would require release or leakage of A2-E from the lysosomal compartment with subsequent targeting of other cellular compartments.
We therefore now investigated intralysosomal accumulation, possible biodegradation, release from the lysosomal compartment and intracellular spreading of (14)C-labelled A2-E in cultured human RPE cells. We specifically loaded lysosomes of cultured human RPE cells with [(14)C]A2-E.
A linear increase of intracellular radioactivity was observed during the 4-week loading period. Cell fractionation experiments indicated that more than 90% of loaded A2-E was specifically accumulating in the lysosomes. After loading, the fate of the radioactive label was chased over a period of an additional 4 weeks. No metabolism or secretion of A2-E to the medium was detectable. Subcellular fractionation revealed that during the chase period, about 13% were shifted from the lysosomes to mitochondrial fractions. This effect was strikingly intensified when after loading the cells with the labeled retinoid, its intralysosomal concentration was boosted by an additional load with non-labeled A2-E. Thus about 44% of the label were located in mitochondria at the end of the chase period. No significant spreading to other cell compartments was detectable.
Since A2-E was suggested to act as a proapoptotic molecule via a mitochondrial pathway, we postulate that upon reaching a critical intralysosomal concentration, A2-E is released from the lysosome and then specifically targets the outer mitochondrial membrane thereby initiating apoptosis of the RPE cell. This may also apply correspondingly to other lipofuscin-associated molecules that cause leakage of the lysosomal membrane.
脂褐素与包括年龄相关性黄斑变性（ARMD）在内的多种致盲疾病相关。脂褐素的形成被认为是由于视网膜色素上皮（RPE）溶酶体无法降解吞噬物质的成分，导致其在溶酶体内积累而引发的。因此，导致RPE脂褐素自发荧光特性的异常视黄醛加合物的沉积，源于吞噬的光感受器外段中视黄醛循环的异常产物。先前已表明，主要的脂褐素视黄醛共轭物A2 - E通过直接损害溶酶体功能和结构，对RPE细胞产生毒性作用。然而，也有人提出A2 - E在ARMD发病机制中会严重损害RPE细胞的溶酶体外结构。这将需要A2 - E从溶酶体区室释放或泄漏，随后靶向其他细胞区室。
因此，我们现在研究了在培养的人RPE细胞中，（14）C标记的A2 - E在溶酶体内的积累、可能的生物降解、从溶酶体区室的释放以及在细胞内的扩散情况。我们用[（14）C]A2 - E特异性地加载培养的人RPE细胞的溶酶体。
在4周的加载期内，观察到细胞内放射性呈线性增加。细胞分级分离实验表明，超过90%加载的A2 - E特异性地积累在溶酶体中。加载后，在另外4周的时间内追踪放射性标记的去向。未检测到A2 - E的代谢或分泌到培养基中。亚细胞分级分离显示，在追踪期内，约13%的A2 - E从溶酶体转移到线粒体部分。当用标记的视黄醛加载细胞后，再用未标记的A2 - E额外加载以提高其溶酶体内浓度时，这种效应显著增强。因此，在追踪期结束时，约44%的标记位于线粒体中。未检测到向其他细胞区室的明显扩散。
由于A2 - E被认为通过线粒体途径作为促凋亡分子起作用，我们推测当达到临界溶酶体内浓度时，A2 - E从溶酶体释放，然后特异性地靶向线粒体外膜，从而引发RPE细胞凋亡。这可能同样适用于其他导致溶酶体膜泄漏的脂褐素相关分子。
