apoptotic cell fate. Loss of mitochondrial activity will have a negative impact on the metabolic function, and the cell will operate below its threshold.

One of the most prevalent theories of aging is the mitochondrial theory, which proposes that oxidative damage can eventually lead to dysfunctional or defective mitochondria. As highlighted by Liang and Godley, damage to mtDNA probably has more relevance to the mitochondrial theory of aging than damage to lipid or protein [65]. The latter can be repaired, while the former can be propagated during replacement of mitochondria.

Mitochondria redox function in macular RPE cells has a greater susceptibility to oxidative damage compared to peripheral RPE, and this vulnerability appears to increase with age [66]. Moreover, this appears to correlate with mtDNA damage, which is significantly higher in macular RPE cells compared to those in the periphery [67]. It appears that RPE mtDNA is particularly susceptible to oxidative damage, and that such damage is only poorly repaired. Thus, mitochondria, while essential for cell metabolism, may make a significant contribution to RPE aging and dysfunction. It further appears that the age-related loss of mitochondria reported by Feher et al. is accelerated in AMD patients [25].

Bruch’s Membrane Aging

The overall flow of nutrients and waste products across the RPE is likely to be significantly impaired with increasing age [26, 68]. This will be a combination of a decrease in basal interdigitations, thus reducing the area of RPE plasma membrane available for transport; a reduction in the activity of enzymes involved in transepithelial transport; and a decrease in the hydraulic conductivity of Bruch’s membrane. The deposition of lipid-rich membranous debris [69, 70] together with diminishing membrane porosity due to the accumulation of AGEs results in a significant decline in resistance to water movement and permeability to small solutes and macromolecules [71].

Bruch’s membrane aging alters the normal gene expression profile of RPE cells, including the upregulation of transforming growth factor-α and downregulation of vitronectin and the membrane transporter ATP-binding cassette, sub-family C, 5 (ABCC5) [72]. This may in part be due to the accumulation of AGEs in Bruch’s membrane with increasing age and the expression of the AGE receptors receptor for advanced glycation end products (RAGE), AGE R1, and AGE R3 by RPE cells [73]. This is supported by the observation that AGE-induced aging of the RPE was associated with a transcriptome response of early inflammation, matrix expansion, and aberrant lipid processing and later downregulation of energy metabolism genes and upregulation of crystalline genes [74]. RPE cell survival is significantly impaired on aged submacular Bruch’s membrane, further confirming the impact of matrix aging on RPE function [75].

OXIDATIVE STRESS AND RPE AGING

Reactive oxygen species (ROS) are highly reactive molecules that can cause oxidative damage to proteins, nucleic acids, and lipids (Fig. 6) [76]. ROS can be free radicals (i.e., species capable of independent existence that contain one or more unpaired electrons), oxygen species that have been elevated to a higher energy level (e.g., singlet oxygen), or strong oxidizing agents (e.g., hydrogen peroxide). The most important

Fig. 6. Cellular generation of reactive oxygen species and antioxidant defenses. Fe2+ ferrous ion, GPx glutathione peroxidase, H2O2 hydrogen peroxide, NO+ nitric oxide, O2− superoxide anion, OH hydroxyl radical, onoo− peroxynitrite, SOD superoxide dismutase (Modified from [98].

ROS of pathophysiological relevance in the eye are the superoxide anion (O2.-), hydroxyl radical (OH.), singlet oxygen, nitric oxide (NO), lipid peroxyl radicals (LOO.), and peroxynitrites (ONOO.) (see [76]). The hydroxyl radical and superoxide anion are highly reactive, have short half-lives of 10−9 and 10−5 s, respectively, and normally react with molecules in their immediate vicinity [53].

Mitochondria account for the bulk of endogenously formed ROS in most cells [76– 78]. An unavoidable respiratory electron leak results in the formation of superoxide anions, which are toxic to mitochondrial enzymes and can undergo the Fenton reaction, generating the most reactive and harmful of ROS, the hydroxyl radical. In addition, the superoxide anion can be reduced by SOD to form hydrogen peroxide, which can itself undergo the Fenton reaction to form hydroxyl radicals. It is the reaction of these ROS with lipids and proteins that leads to the formation of lipid hydroperoxides and lipid–protein adducts (e.g., Schiff bases), which while not as reactive as superoxide anions and hydroxyl radicals, have a significantly longer half-life and can diffuse through the cell to cause oxidative damage at distant sites [76].

Nitric oxide synthase (NOS) is present in most cells and converts L-arginine to citrulline and nitric oxide. Nitric oxide is itself a contradiction since on the one hand it can act as an intracellular signaling molecule, while on the other it can react with the superoxide anion to form peroxynitrite, which can cause lipid peroxidation [79, 80]. Lipid peroxidation, irrespective of the ROS responsible for its initiation, leads to the formation of lipid hydroperoxides capable of propagating a lipid peroxidation chain reaction.

In addition to the ROS discussed, which are common to most, it not all, cells in the body, singlet oxygen is especially important in the retina. Singlet oxygen (1O2) can be generated chemically, enzymatically, and photochemically [81]. The daily exposure of the eye to light means that photochemical generation of singlet oxygen is a dominant pathway in the retina. The transfer of energy from activated photosensitizers to oxygen leads to the formation of singlet oxygen, which exists in an excited state. Singlet oxygen can generate ROS such as the superoxide anion due to interaction with diatomic oxygen (O2) and by reacting directly with electrons with double bonds without the formation of free-radical intermediates [81].

While the generation of ROS occurs under physiological conditions, damage is minimized by antioxidants and repair mechanisms. However, cellular stress leads to an upregulation of ROS, which overwhelms the antioxidative capacity of cells. This can lead to acute oxidative damage, culminating in cell death or chronic oxidative damage, which leads to the accumulation of oxidatively damaged molecules, particularly in postmitotic cells such as the RPE, which eventually results in cellular dysfunction. This slow buildup of randomly damaged molecules fits with the stochastic theory of aging and thus promotes oxidative damage as the major cause of cellular and tissue aging [82].

The retina provides the ideal environment for the generation of ROS: a high metabolic rate so there is a high density of mitochondria, lots of photosensitizers, a high oxygen environment, and regular exposure to light. In addition, the daily phagocytosis of photoreceptor outer segments results in the generation of ROS [83]. Thus, the constant production of ROS in the retina is likely to contribute to aging changes in the retina and may well pass a threshold at which aging changes take on pathological significance, and vision is lost [52, 53, 84].

While melanin has the ability to efficiently scavenge a wide range of radicals, including peroxyl and carotenoid cation radicals [85–87], as well as quenching electronic excited states, it is likely that any antioxidant activity is restricted to the immediate vicinity of the melanosome. However, a study indicated that it is unlikely that melanosomes play a significant antioxidant role in RPE cells [88]. An indirect antioxidant role for melanin may be its ability to sequester redox active metal ions, thus rendering them significantly less damaging to the cellular components [15].

We have recently shown that human RPE cells have greatest resistance to oxidative stress compared to many other cell types in the body, including those normally exposed to a high oxidative environment [89]. This oxidative tolerance of the RPE coincides with greater CuZn–SOD, GPX, and catalase enzymatic activity. It is clear that cells, such as the RPE, located in highly oxidizing microenvironments appear to have more efficient oxidative defense and repair mechanisms. This increased resistance to oxidative stress may in part be due to the ability of cells to adapt to oxidative stress. The adaptive response is a biological phenomenon that involves cells reacting at a molecular level to acquire greater cellular resistance against a wide range of physiological stresses, including ROS [90]. Prior exposure of RPE cells to sublethal oxidative stress confirmed an adaptive response (Fig. 7), resulting in a greater cellular resistance to subsequent toxic exposures compared to nonadapted RPE [91]. Greater catalase, glutathione peroxidase (GTX), and CuZn–SOD activity and increased nDNA protection were also observed. However, there was no adaptive benefit for mtDNA protection or repair in response

Fig. 7. The adaptive response of retinal pigment epithelium (RPE) cells exposed to sublethal concentrations of H2O2. RPE cell cultures were exposed to the indicated nontoxic concentrations of H2O2 every day for 5 days. After incubation, the adapted and nonadapted RPE were challenged with a toxic oxidative stress from 3mM H2O2 for 1h. Cell viability was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results were expressed as the percentage increase in cell survival of the adapted RPE compared to the nonadapted RPE. Significant difference in cell viability of the adapted RPE compared to the nonadapted RPE: *p < .05. (Reproduced from [91] courtesy of Free Radical Biology and Medicine.)

to oxidative stress. This suggests that the mitochondria in the RPE are a weak link in otherwise efficient oxidative stress defenses, and that this may contribute to aging and age-related disease.

THE RELATIONSHIP BETWEEN AGING AND RETINAL PATHOLOGIES

Age changes in the RPE and Bruch’s membrane have been associated with a wide variety of retinal pathologies, in particular AMD [26, 52]. AMD, which affects more than 35% of people over the age of 65 years and accounts for 50% of the blind registrations in the age group, is always associated with RPE atrophy, pigment dispersion, increased fundus autofluorescence, or drusen. There is now strong evidence to link lipofuscin accumulation with a variety of retinal degenerations, in particular both the wet and dry forms of AMD, Leber’s amaurosis, Best’s disease, and Stargardt’s disease [17]. A further age-related retinal condition, that of RPE detachment, has been postulated to occur as a direct result of the increased accumulation of lipids within Bruch’s membrane, resulting in the impedance of fluid transport out of the retina [92]. The disturbances created by increased lipid concentration, calcification, and changes in the structural integrity of Bruch’s membrane may predispose this region to invasion by macrophages or RPE cells and neovascular invasion of the sub-RPE space [93, 94]. Other diseases that are not overtly age related, such as Best’s disease, fundus flavimaculatis, retinitis pigmentosa, and Lawrence–Moon–Biedl syndrome, also exhibit abnormal accumulations of lipofuscin and subepithelial deposits [95]. Attempts at understanding these age-related diseases is