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

The purpose of the present study was to investigate the accumulation o f rod outer segment (ROS)-derived debris in cultured human retinal pig ment epithelial cells (RPE). The RPE cell layer is responsible for the phagocytosis and digestion of photoreceptor outer segments. Due to th e immense volume of photoreceptor-derived material processed by the RP E cells, even minor changes in the efficiency of ROS processing may ca use the accumulation of lipofuscin and photoreceptor derived debris. I n this work, 17 RPE cultures were established from the globes of eye b ank donors whose ages ranged from 18 to 79 years. Third passage cultur es were challenged with bovine ROS and the accumulation of an autofluo rescent debris was quantified using a now cytometer. It was demonstrat ed that ROS challenge greatly increased the rate of autofluorescent de bris accumulation. The accumulation of autofluorescent debris varied s ignificantly from culture to culture. This variation was independent o f the phagocytosing capacity of individual cultures and was not age de pendent. To further investigate the factors which may be responsible f or these differences, the presence of cathepsin D, an aspartic proteas e responsible for 80% of proteolysis of rhodopsin, was analysed by Wes tern 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 cultur es having a singlet active form (mean 18.8, S.D. +/- 5.5) at 34 kDa (S tudent's t-test, DF = 15, t = 6.834, P < 0.001). The former cultures i ncluded one from a donor with age related macular degeneration, the la tter cultures included one from a donor with diabetic retinopathy. Thi s study demonstrates that the rate of autofluorescent debris accumulat ion in cultured RPE cells is not age dependent, but is an intrinsic pr operty of the donor RPE cells that is possibly related to the presence of a multimer form of the lysosomal enzyme cathepsin D. (C) 1996 Acad emic Press Limited