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 midage 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.

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…24 In addition, use of hfRPE cells may have an advantage over adult human RPE cells in that, adult human RPE cells show an age-dependent decline in mitochondrial function and antioxidant potency. 25 In the present study, we found that mini-chaperone peptides derived from aA and aB-crystallins exhibited antiapoptotic properties. Both aA and aB-crystallin mini-chaperone peptides protected RPE from H 2 O 2 -induced cell death and inhibited caspase-3 activation.…”

Mitochondrial network responses in oxidative physiology and disease

“…1, NADP-linked processes supporting redox switches are shown in green while representative examples of the broader spectrum of post-translational modifications linking macromolecular structure and function to bioenergetics and metabolism is shown in blue. In this simplified conceptualization, we have omitted details about production of oxidants by mitochondria, which are included within the redox code [6] and extensively studied in terms of pathology and aging [13–15]. In addition to pathologic consequences from excess production, evidence is accumulating that mitochondria produce oxidants as signaling molecules that integrate mitochondria-cell functions [16–18].…”

Daphnetin-mediated Nrf2 antioxidant signaling pathways ameliorate tert-butyl hydroperoxide (t-BHP)-induced mitochondrial dysfunction and cell death

“…Oxidative damage and mitochondrial dysfunction are strongly related to the pathogenesis of various diseases, such as age-related macular degeneration (AMD), Parkinson's disease, heart ischemia/ reperfusion injury, alcoholic hepatitis, diabetes and cardiovascular diseases [10]. Excess reactive oxygen species (ROS) generation and release induces a variety of oxidative stress responses, which further exacerbate mitochondrial dysfunction and result in cell injury and death to accelerate disease progression [11,12].…”