title: Correlation between autofluorescent debris accumulation and the presence of partially processed forms of cathepsin D in cultured retinal pigment epithelial cells challenged with rod outer segments.
abstract: The purpose of the present study was to investigate the accumulation of rod outer segment (ROS)-derived debris in cultured human retinal pigment epithelial cells (RPE). The RPE cell layer is responsible for the phagocytosis and digestion of photoreceptor outer segments. Due to the immense volume of photoreceptor-derived material processed by the RPE cells, even minor changes in the efficiency of ROS processing may cause the accumulation of lipofuscin and photoreceptor derived debris. In this work, 17 RPE cultures were established from the globes of eye bank donors whose ages ranged from 18 to 79 years. Third passage cultures were challenged with bovine ROS and the accumulation of an autofluorescent debris was quantified using a flow cytometer. It was demonstrated that ROS challenge greatly increased the rate of autofluorescent debris accumulation. The accumulation of autofluorescent debris varied significantly from culture to culture. This variation was independent of the phagocytosing capacity of individual cultures and was not age dependent. To further investigate the factors which may be responsible for these differences, the presence of cathepsin D, an aspartic protease responsible for 80% of proteolysis of rhodopsin, was analysed by Western blot. Although the 34 kDa active form of cathepsin D was found in all cultures, in 41% of the cultures higher-molecular-weight forms of cathepsin D were additionally present, thus providing a multimer form of cathepsin D in these cultures. The rate of autofluorescent debris accumulation in cultures possessing a multimer form of cathepsin D was significantly greater (mean 42.3, S.D. +/- 19.8) than those in cultures having a singlet active form (mean 18.8, S.D. +/- 5.5) at 34 kDa (Student's t-test, DF = 15, t = 6.834, P < 0.001). The former cultures included one from a donor with age related macular degeneration, the latter cultures included one from a donor with diabetic retinopathy. This study demonstrates that the rate of autofluorescent debris accumulation in cultured RPE cells is not age dependent, but is an intrinsic property of the donor RPE cells that is possibly related to the presence of a multimer form of the lysosomal enzyme cathepsin D.
authors.affiliation: Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands.
authors.forename: P E
authors.initials: PE
authors.lastname: Rakoczy
authors.forename: M
authors.initials: M
authors.lastname: Baines
authors.forename: C J
authors.initials: CJ
authors.lastname: Kennedy
authors.forename: I J
authors.initials: IJ
authors.lastname: Constable
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D006801:Humans; D008062:Lipofuscin / Q000032:analysis; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010587:Phagocytosis / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000378:metabolism*
chemical_list: D008062:Lipofuscin; D002402:Cathepsin D