these complexes across the fundus mirrors that of lipofuscin, and the spectral characteristics are intermediate between those of melanin and lipofuscin [12].

Mitochondria

The RPE, typically for a highly metabolically active cell, contains large numbers of mitochondria [3]. These organelles are located toward the base of the cell, where the majority of active transport takes place (Fig. 1). Feher and colleagues demonstrated a significant decrease in number and area of RPE mitochondria with increasing age as well as loss of cristae and matrix density [25]. Alterations of mitochondria were accompanied by proliferation of peroxisomes and lipofuscin granules.

Bruch’s Membrane

Bruch’s membrane is an acellular membrane that separates the RPE from the underlying choroid. Entrapment of molecules and cellular debris occurs within Bruch’s membrane throughout life [26–28]. This results in an age-related increase in thickness and lipid content of Bruch’s membrane and appears to be greatest in the macular region. The accumulation of material within Bruch’s membrane acts to reduce or prevent the free flow of molecules between the choroidal circulation and the photoreceptors [29]. Bruch’s membrane also becomes more brittle with age due to loss of the elastin layer [30, 31] and the formation of cross-links such as advanced glycation end products (AGEs) [32]. To what extent the RPE contributes to these changes is unclear. However, the RPE is almost certainly the source of basal linear deposits that form on the innermost margin of Bruch’s membrane. Over the age of 40 years, focal aggregation of subpigment epithelial deposits is associated with Bruch’s membrane; these deposits are termed drusen [33]. Drusen are usually concentrated in the macular region and can be predominantly either lipid or protein.

FUNCTIONAL CONSEQUENCES OF RPE CELL AGING

Phagocytic Load

It has been postulated that loss of RPE cells occurs at a greater rate than overlying photoreceptors, and that this increases the phagocytic burden on the RPE. However, this would not appear to be the case for the macula, in which photoreceptor loss is greater than RPE loss [10, 34, 35]. However, decreased activity in many of the intrinsic functions of an RPE cell may place an added burden on the RPE, leading to loss of overlying photoreceptors cells.

The Effect of Lipofuscin on the RPE

Analysis of blue light photoreactivity of isolated human RPE cells demonstrates that the rate of photoinducible oxygen uptake increases with donor age, the uptake of oxygen being predominantly due to lipofuscin [36]. Lipofuscin is a photoinducible generator of superoxide anion, singlet oxygen, hydrogen peroxide, and lipid peroxides [36–38]. The generation of these radical species is strongly wavelength dependent, with, for example, efficiency increasing with wavelength by a factor of ten when excitations of

Fig. 4. The effect of lipofuscin on mitochondrial DNA (mtDNA) (A) and nuclear DNA (nDNA) damage (B). Confluent human retinal pigment epithelium (RPE) cultures were fed lipofuscin and exposed to blue light for 1, 3, and 6 h. Control cultures were maintained in the dark. The graphs represent the number of lesions per 10 kb as determined by quantitative polymerase chain reaction (QPCR) of mitochondrial and nuclear genes in the presence of lipofuscin. The vertical bars indicate the standard error of the mean (SEM). (Reproduced from [41] courtesy of the Journal of Biological Chemistry.)

520 and 420 nm are compared. Given the photoreactivity of RPE lipofuscin, it is not surprising that exposure of RPE cells containing lipofuscin to short-wavelength visible light (390–550 nm) results in wavelength-dependent lipid peroxidation (malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, DNA damage, and RPE cell death [39–41]. Lipofuscin was able to photodamage both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in blue light-exposed RPE cells, with greatest damage occurring to nDNA (Fig. 4) [41].

The most studied of the potential photosensitizers of RPE lipofuscin is A2E (N-retinylidene-N-retinylethanolamine), which can provoke an apoptotic form of cell death [42–44]. However, the potency of A2E is at least an order of magnitude less than lipofuscin, suggesting the presence of other, more reactive chromophores, which may be nonretinoid in origin [45, 46]. However, A2E can form epoxides, which are significantly more photoreactive than A2E [47]. Furthermore, A2E has been shown to be a photoinducible upregulator of vascular endothelial growth factor (VEGF) [48] and

complement activation [49] in RPE cells, both of which are implicated in the pathogenesis of AMD. In addition to its photoreactivity, A2E has been shown to have lysosomotropic properties. Exogenous A2E localizes predominantly to lysosomes in cultured RPE cells, causing an increase in lysosomal pH and exerting an inhibitory effect on protein and glycosaminoglycan catabolic pathways.

Interestingly, both lipofuscin and melanin granules can be found in early drusen [33], and lipofuscin distribution shows a characteristic distribution within RPE cells overlying drusen (Fig. 3A,C,D).

Melanosomes

The blue light photoreactivity of melanosomes increases significantly with age [50], and this can result in toxicity to the RPE [51]. Cultured RPE cells containing human melanosomes from aged eyes exposed to blue light exhibit vacuolation, membrane blebbing, and cell death [51]. By contrast, melanosomes from young eyes do not exhibit a substantial phototoxic effect. The phototoxicity of aged melanosomes is at least one order of magnitude less than lipofuscin.

Antioxidant Capacity of the RPE

Even though the neural retina and RPE are particularly rich in a range of antioxidants [52–54], the levels of these decrease in the macular RPE after 70 years of age, while levels in peripheral cells remain constant throughout life [55]. Catalase activity in the human RPE has been shown to decrease with age and AMD, while superoxide dismutase (SOD) activity does not appear to show a correlation with donor age [56].

Castorina et al. demonstrated an age-related correlation between lipid peroxidation and antioxidant enzyme activity [57]. Decreased levels of carotenoids are associated with aging and AMD [58]. Microsomal glutathione S-transferase-1, an enzyme that displays significant reduction activity toward peroxides, oxidized RPE lipids, and oxidized retinoids, decreases threeto fourfold with increasing age in the mouse RPE [59]. Heat shock proteins and chaperones such as crystallins may also protect proteins from oxidative damage [60]. Crystallin becomes truncated with age, and this reduces its ability to protect proteins against oxidative damage [61]. Those ROS that do escape detoxification will contribute to an insidious buildup of oxidative damage within the RPE throughout life that will manifest itself as pathology in the aged eye and is likely to contribute to the pathogenesis of such diseases as AMD. Interestingly, a pathology with similarity to AMD including drusen, geographic atrophy, RPE dysfunction, and choroidal neovascularization presents when CuZn–SOD is knocked out in mice [62].

Lysosomal Enzyme Activity

Any decrease in the degradatory capacity of lysosomal enzymes within the RPE would affect the careful balance in the breakdown of ingested photoreceptors by the RPE. It is clear that there is a regional distribution of lysosomal enzyme activity, with highest activities found in the macular region. While the effect of aging on RPE lysosomal enzyme activity is equivocal, an age-related increase in acid phosphates and cathepsin D has been reported (Fig. 5) [63]. Ogawa and colleagues similarly reported

Fig. 5. The activity of acid phosphatase as a function of age in human retinal pigment epithelium (RPE) cells taken from different regions of the fundus. (Modified from [63].)

an increase in cathepsin S in the RPE of aged mice [64]. This increase is perhaps not surprising since lysosomes are associated with pigment granules, and these granules increase with age. Thus, the net lysosomal enzyme activity available to break down ingested photoreceptors may actually be reduced in aged eyes and contribute to the buildup of lipofuscin granules.

Mitochondrial Damage in the RPE

The RPE cells contain high numbers of mitochondria, typical for a cell with high metabolic needs. Mitochondria not only provide a steady supply of energy in the form of adenosine triphosphate (ATP) but also regulate the cellular redox state and influence