Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Abstract

Impairment of mitochondria function 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 that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9-20)-, mid-age (48-60)-, and >60 (62-76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H(2)O(2) toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca(2+) [Ca(2+)](c), and mitochondrial Ca(2+) [Ca(2+)](m) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5',6,6'-tetrachloro 1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca(2+)](c), and increased sequestration of [Ca(2+)](m) in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.

Keywords: ATP; Aging; Ca2+; Mitochondria; Mitochondrial membrane potential; Oxidative Stress; RPE cells.

Fig. 1

Phase-contrast micrographs showing examples of primary cultures of RPE cells from the young, mid-age, and >60 age groups (top panel). Cells appear larger and more elongated in the oldest age group. Scale bar 30 μm. Dot plots from flow cytometry analyses indicating increase in cell size (forward scatter) and cytoplasmic complexity (side scatter) with increased chronological age

Fig. 2

Greater than 99% of cells in all primary RPE cultures express the RPE specific marker, RPE-65 (green fluorescence). Scale bar 30 μm

Fig. 3

a Phase-contrast light micrographs and PI staining (red labeling) show that cultures from the oldest age groups are more sensitive to H₂O₂ toxicity. PI staining was carried out on unfixed cells after H₂O₂ treatment. Scale bar 30 μm. b Age-related increase in LDH release at all concentrations of H₂O_2. Data are expressed as percentage of cell death as the mean ± SE of all samples within an age group (n = 7; p < 0.05)

Fig. 4

In the control groups grown in standard growth medium, cytochrome c (green) colocalizes with mitochondrial staining (Mitotracker red) in RPE cells of all age groups (merged images) indicating that the cells in all control groups were healthy. With H₂O₂ treatment, however, colocalization of cytochrome c was only evident in the young and mid-age cells (merged images). The discrete green and red labeling in cells of the >60-year-old group suggest that cytochrome c was released from the mitochondrial into the cytoplasm of these cells. Scale bar 30 μm

Fig. 5

Expression of antioxidant and apoptotic genes in RPE cells with increasing chronological age. Real-time PCR shows that there is a significant increase in transcripts for SOD 2 and decrease for mtHsp70, UCP2, SOD3, Bcl-2, and Bax genes with increasing age. The histogram represents mRNA fold changes to the mean of the “young” group. Results are expressed as the mean ± SE. *Significantly different from young group (p < 0.05)

Fig. 6

a Electron micrographs of primary RPE cultures. The mitochondria in the two youngest age groups are regular in shape and size and contain intact membranes with visibly distinct inner and outer membranes and cristae. Those from the >60-year-old samples are fewer, larger, irregular in size, tubular in shape, have highly electron dense matrices and show architectural disruption in the membranes and cristae. Scale bar 1.5 μm. b Length/width ratio of these organelles is almost sevenfold different compared to young and mid-age samples (using NIH image J software)

Fig. 7

a The mitochondria in young and mid-age samples are in discrete perinuclear regions of the cells and appear more tightly packed compared to those in the >60-year-old samples. Scale bar 10.5 μm. b The histogram confirms visual observation of decreased numbers of individual mitochondria with increased aging. The data represent the mean of the average counts in all three areas of 20 cells per sample within the group

Fig. 8

a Fluorescence intensity of the mitochondria indicator, Mito Tracker Red, is decreased is decreased with increasing age. The confocal images confirm discrete mitochondrial labeling in the perinuclear region in young and mid-age samples and branching labeled structures throughout cells of the >60-year-old samples. Scale bar 30 μm. b, c The histograms generated from flow cytometry (10,000 cells) show decrease fluorescence intensity with both Mitotracker red and green. Data are expressed as a fold change in fluorescence levels to the young group and as the mean ± SE of all samples in the group (n = 7). *Significantly different from young group (p < 0.05)

Fig. 9

a Relative amount of total H₂-DCF-DA fluorescence. There is a 0.30-fold (±0.13) decrease in ROS levels in the >60-year-old RPE group compared to young group. Data are expressed as a fold change in fluorescence levels to the young group and as the mean ± SE all samples within the group (n = 7). *Significantly different from young group (p < 0.05). b Similar comparisons are shown for ATP levels when the mean for the young samples were arbitrarily set at 100%. Results are expressed as a mean percentage of the ATP levels ± SE. *Significantly different from young samples (n = 7; p < 0.05). c Comparative ΔΨm measurements using the fluorescence indicator JC-1 and flow cytometry. The ΔΨm is 0.85-fold (±0.15) and 0.48-fold (±0.17) lower in mid-age, and >60 RPE cells, respectively, compared to the young samples. Results are expressed as the mean fold decrease in fluorescence levels to the young group, arbitrarily set at 100% ± SE. *Significantly different from young group (n = 7; p < 0.05)

Fig. 10

a Relative amounts of total fluo-3 AM fluorescence ([Ca²⁺]_c). b Relative amount of total Rhod-2 fluorescence ([Ca²⁺]_m) using flow cytometry. Data are expressed as a fold changes in fluorescence levels to the young samples and as the mean ± SE of all samples within the group. *Significantly different from young group (n = 7; p < 0.05)