Abstract
Purpose: :
Dysfunction of the mitochondria and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment, partly due to elevated oxygen partial pressure from the choriocapillaris and to digestion of polyunsaturated fatty acid laden photoreceptor outer segments. Here we examined changes in the mitochondria (mt) of human RPE cells and sensitivity of the cells to oxidative stress with increased chronological age.
Methods: :
RPE cells from donor ages 9, 52, 62, and 76 were grown to confluency and the mt examined by electron microscopy and Mitotracker red fluorescent labeling. Susceptibility of RPE cells to H2O2 toxicity was determined by PI staining and LDH assay. ROS, cytoplasmic Ca2+ [Ca2+]c and mt Ca2+ [Ca2+]m levels were measured by flow cytometry using H2-DCF-DA, fluo-3/AM and Rhod-2/AM, respectively, ATP levels by a luciferin/luciferase-based assay, and mt membrane potential (ΔΨm) by JC-1 fluorescence.
Results: :
RPE cells show greater sensitivity to oxidative stress and have alterations in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity as a function of age. Lower levels of ATP (~ 30%), ROS (~3.2 fold) and [Ca2+]c (~1.5 fold), decreased ΔΨm (~1.5 fold), and increased sequestration of [Ca2+]m (~2.3 fold) are observed in RPE cells from 76 yo when compared to 9 yo donors. There is decreased expression of the antioxidant genes mtHsp70, UCP2, and SOD3 but elevated SOD2 mRNA levels with increased aging. There were no significant changes in the release of cytochrome c or expression of the antiapoptotic genes, Bcl2 and Bax in any of the primary cultures.
Conclusions: :
Our study provides evidence for mitochondrial decay, bioenergetic deficiency, and dysfunctional antioxidant defences in RPE cells with advanced aging. With increased severity, these conditions may reduce important apical and basal RPE functions in the retina and promote the progression of retinal diseases such as age related macular degeneration.
Keywords: mitochondria • aging • retinal pigment epithelium