title: Accumulation of A2-E in mitochondrial membranes of cultured RPE cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Ophthalmology, University of Heidelberg, INF 400, 69120, Heidelberg, Germany. florian_schuett@med.uni-heidelberg.de
authors.forename: Florian
authors.initials: F
authors.lastname: Schutt
authors.forename: Marion
authors.initials: M
authors.lastname: Bergmann
authors.forename: Frank G
authors.initials: FG
authors.lastname: Holz
authors.forename: Stefan
authors.initials: S
authors.lastname: Dithmar
authors.forename: Hans-Eberhard
authors.initials: HE
authors.lastname: Volcker
authors.forename: Jurgen
authors.initials: J
authors.lastname: Kopitz
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002458:Cell Fractionation; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: C112040:A2-E (N-retinylidene-N-retinylethanolamine); D015415:Biomarkers; D012176:Retinoids; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D005700:Galactosyltransferases; D000469:Alkaline Phosphatase; D001619:beta-N-Acetylhexosaminidases