Ubiquitin–Containing Protein Aggregates in Lipofuscin–Treated Light–Exposed RPE

Abstract

Abstract: : Purpose: To model lipofuscin–induced and age–related changes to ubiquitin conjugates in the RPE Methods: Confluent ARPE19 cultures were fed isolated lipofuscin granules (∼300 per cell) and maintained in basal medium for 7 days. Control cultures lacking lipofuscin were maintained under similar conditions. The medium was replaced with photosensitizer free medium and the cultures either exposed to blue light (409–490nm; 2.8mWcm2) from a sunlight source or maintained in the dark. Cultures were lysed at 0, 2, 4 and 6 h post exposure. Lysates were divided for determination of protein content, carbonyl content and levels of ubiquitin conjugates. Results: There was no significant change in total protein content with any of the treatments. Measurement of carbonyl content demonstrated a significant increase in oxidised membrane proteins in cells exposed to light and lipofuscin compared to either light alone or cultures maintained in the dark. Total carbonyl content was greatest in lipofuscin–fed cells exposed to blue light (3.8 fold compared to dark controls at 4 h) although light alone did show a lesser but significant increase in carbonyl content. There was little change in ubiquitin conjugate levels in samples which were exposed to light but not treated with lipofuscin. In contrast, treatment with lipofuscin and exposure to blue light was associated with a time–dependent increases in endogenous ubiquitin conjugates of 2, 3.3, and 3.5 fold at 2,4,and 6 h respectively compared to control. Discussion: We previously demonstrated that the ubiquitin pathway is associated with protein quality control during aging or upon stress in several systems including RPE. This data confirms that lipofuscin, a known photoinducible generator of reactive oxygen species, is associated with increased accumulation of high mass ubiquitin conjugates in the RPE. The accumulation is due to either enhanced formation or delayed deubiquitination and degradation of the conjugates and may contribute to retinal ageing and age–related macular degeneration.

Keywords: retinal pigment epithelium • retinal degenerations: cell biology • proteolysis