fraction which contains the retinoids such as A2E [71]. However, A2E can form epoxides which are more photoreactive than A2E [79] and which may be implicated in complement activation by RPE cells [80]. In addition to its photoreactivity, A2E has been shown to localize to lysosomal membranes where it can cause an increase in lysosomal pH and exert an inhibitory effect on protein and glycosaminoglycan catabolic pathways [81, 82]. The consequence of lipofuscindependent lysosomal dysregulation has been associated with impaired phagocytosis, dysregulated autophagy, and retinal degeneration [83Ð85]. Since most studies have been undertaken using pure A2E preparations or ABCA4(−/−) mice which accumulate high levels of A2E in the retina, an intriguing question remains as to whether A2E and its oxidized byproducts can actually induce damage when bound to lipofuscin granules or whether any potential A2E effects are prior to the incorporation of A2E into lipofuscin. This would be compatible with the evolutionary concept of the disposable soma theory [86] in that lipofuscin granules act as a trash receptacle for toxic chemicals and thus protect the retina during the critical stages of life, but these granules eventually accumulate to such high levels that they actually become toxic to the cells they are protecting.

3.5.4Melanosomes and Pigment Complexes

Concomitant with the age-related increase in lipofuscin granules is a decrease in RPE melanosomes and an increase in pigment complexes [58, 87]. Although the regional distribution of melanosomes with its peak at the macula is maintained throughout life, there is a signiÞcant decline of as much as 35% in the number of granules in all regions after age 40 [58, 88]. Melanin granules lose their cigar-shape, become less electrondense, and become associated with lysosomes. Their biophysical characteristics also change with an agedependent increase in the absorption of intact melanosomes between 250 and 450 nm and increased ßuorescent emission [65, 89]. Loss of melanosomes appears to be associated with both photoand lysosomal degradation [58, 90]. The functional relevance of a reduction in melanosomes in the RPE remains unclear but could decrease light absorption and/or reduce binding of toxic xenobiotics and metal ions in

the aged RPE [91]. The failure to sequestrate free iron could lead to ROS generation via the Fenton reaction and increase the potential for oxidative damage. It is unlikely that melanosomes act as an antioxidant since they do not offer signiÞcant protection against oxidative stress [87, 92]. In fact, aged-melanosomes appear to be phototoxic since cultured RPE cells containing human melanosomes from aged eyes exposed to blue light exhibited vacuolation, membrane blebbing, and cell death while melanosomes from young eyes did not exert a substantial phototoxic effect [93].

With increasing age, a large number of melanolipofuscin complexes are apparent which have photophysical properties intermediate between lipofuscin granules and melanosomes [65, 94]. The origin of melanolipofuscin is unclear. While a common view is that it represents fusion of melanosomes with lipofuscin, the presence of complexes with varying proportions of lipofuscin and melanin would suggest that this may be a much more dynamic process.

3.5.5Mitochondrial Changes in the Aged-RPE

The mitochondrion represents a critical organelle for cellular function and survival. Its principal roles include generation of chemical energy, compartmentalization of cellular metabolism, and regulation of programmed cell death. The RPE, typical for a highly metabolically active cell, contains a large mass of mitochondria located toward the base of the cell where the majority of active transport takes place [47]. A signiÞcant decrease in number and area of RPE mitochondria with increasing age together with loss of cristae and matrix density has been reported [95]. The implication of this observation is that metabolic activity is impaired in aged RPE cells. Accumulating evidence supports a role for mitochondrial dysfunction in ageing and disease in a wide range of tissues resulting in sporadic and chronic disorders, including neurodegeneration [96]. Evidence from a number of studies now strongly supports that mitochondrial dysfunction, initiated by mtDNA damage as a result of oxidative stress together with decreased mtDNA repair, is a feature that underlies the development of retinal ageing and AMD (Fig. 3.5a) [12, 98Ð100]. Increased mtDNA deletions have been documented in aged human and rodent

Fig. 3.5 Graphs showing age-related changes in mitochondrial mass and DNA damage and a comparison of the differential susceptibility of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) to oxidative stress. Mitochondrial mass (determined by mitotracker) (a) and mtDNA damage (assessed by long chain PCR) (b) was measured in RPE cells from young (20Ð30 years) and old (70Ð

90 years) donors and between RPE cells from the peripheral or macular retina (Lin, Boulton, Godley Ð unpublished data). A comparison of mtDNA and nDNA damage and repair was performed in cultured primary human RPE cells (c). All cells were treated with 3 mM H2O2 for 1 h at time 0 and allowed to recover for 0 min,

15 min, 30 min, 1 h, and 3 h. The cells were harvested immediately and the DNA was extracted and QPCR performed. The data are expressed as the means ± SEM from three separate experiments [97]

retina and RPE and this is likely to be due to a combination of increased oxidative damage and reduced DNA repair capability [13, 98, 101]. Changes in selected redox proteins and proteins involved in mitochondrial trafÞcking [14, 102, 103] and a decrease in RPE mitochondrial respiration [104] also correlate with ageing and AMD progression. Furthermore, knockdown of mitochondrial superoxide dismutase results in an AMD-like phenotype in mice [105, 106]. Ex vivo studies show that RPE cells exposed to high levels of ROS suffer preferential damage to mtDNA and subsequent repair is poor (Fig. 3.5b) [97, 107, 108]. Interestingly, A2E is known to localize to mitochondrial membranes [109] and A2E and mitochondrial

dysfunction synergistically impair phagocytosis by retinal pigment epithelial cells [84].

3.5.6The Lysosomal-Autophagy Axis

Lysosomes are essential for the degradation of macromolecules from phagocytosis, endocytosis, and autophagy (Fig. 3.6). The lysosomal system in the RPE is highly active due to the need to degrade the daily load of ingested photoreceptor outer segments. Therefore, any decrease in the degradatory capacity of lysosomal enzymes within the RPE would affect the careful balance in the breakdown of ingested photoreceptors

Fig. 3.6 Degradation of macromolecules from phagocytosis, endocytosis, and autophagy are dependent on the lysosomal system. Phagosomes derived from the ingestion of photoreceptor outer segments, endosomes enclosing membrane proteins, and autophagosomes containing intracellular organelles or aggregates fuse with lysosomes. The resultant degradation products can be either recycled, voided into the choriocapillaris, or deposited in Drusen/BruchÕs membrane (indicated by dotted lines). Not all material is degraded and some will accumulate as lipofuscin (L) in residual bodies

Phagocytosis

Photoreceptor

Outer Segment

Endocytosis

by the RPE. It is clear that there is a regional distribution of lysosomal enzyme activity with highest activities found in the macular region [110, 111]. While the effect of ageing on RPE lysosomal enzyme activity is equivocal, an age-related increase in acid phosphatase and cathepsin D has been reported for both humans (Fig. 3.7) and mice [111, 112]. This increase may simply reßect increased numbers of lysosomes associated with lipofuscin granules and pigment complexes rather than an increase in net lysosomal enzyme activity available to break down ingested photoreceptors. Thus, the available lysosomal capacity may actually be reduced in aged eyes and contribute to the build-up of lipofuscin granules.

Autophagy is a highly conserved housekeeping pathway that plays a critical role in the removal of aged or damaged intracellular organelles and their delivery to lysosomes for degradation. Under normal conditions, autophagy operates constitutively and serves as a housekeeping process through which cytoplasmic proteins and damaged intracellular organelles, such as dysfunctional mitochondria, are removed and nutrients recycled

for rebuilding these organelles [113, 114]. Of the three autophagic pathways (chaperone-mediated, micro-, and macroautophagy) that deliver cellular components of varying sizes to the lysosomes, macroautophagy, subsequently referred to as autophagy, is the primary route for sequestration of organelles or large aggregates and their delivery to the lysosome (see Fig. 3.6) [115, 116]. By contrast, the proteasome, the non-autophagic route for intracellular proteolytic degradation, is unable to degrade damaged organelles or large protein aggregates as they are too large to pass through the narrow pore of the proteosomal barrel. Autophagy will be critical to the cellular maintenance of highly metabolically active cells such as the RPE in which mitochondrial turnover will be high. Lipofuscin accumulates in a variety of ageing mammalian tissues and is derived from two sources, autophagy (degradation of intracellular substrates) [60, 117, 118] and phagocytosis (degradation of extracellular substrates (e.g., sperm by the cells of Sertoli and photoreceptor outer segments by the RPE)) [117, 119]. Similarly, lipofuscin in the RPE can be derived from both phagocytosis and autophagy, but the relative

Macular

Fig. 3.7 Lysosomal acid phosphatase (a) and cathepsin D (b) activity assayed in fresh retinal pigment epithelial cells isolated from donors of different ages. The values represent four assays per sample from each region. Vertical bars represent SEM [111]

contribution of each is open to debate. This is supported by a number of studies demonstrating that accumulation of lipofuscin in RPE cells can proceed in the absence of photoreceptor outer segments, indicating that the turnover of intracellular organelles by autophagy would signiÞcantly contribute toward the accumulation of RPE lipofuscin [119Ð123]. Since the RPE is a highly metabolically active cell, we suggest that exhausted mitochondria will be a major autophagy substrate. The dual origins of lipofuscin would help explain the heterogeneity of the photophysical properties of lipofuscin granules [124]. EfÞcient autophagy ßux is highly dependent on the elimination of autophagosomes by fusion with lysosomes, and this is impaired either by elevated

lysosomal pH or congested lysosomes (e.g., containing lipofuscin) [125, 126], leading to a build-up of damaged intracellular organelles and an RPE cell performing below its optimum. In the case of mitochondria, this will culminate in a loss of energy production, increased ROS generation, and lipofuscin accumulation, all of which will make RPE cells more susceptible to stochastic damage. Wang and colleagues have reported that a number of autophagy markers are increased in the RPE/ choroid of old mice (24Ð28 months) compared to young mice (4 months) [127]; however, whether this represents increased production of autophagosomes or impaired elimination has yet to be determined. What is clear is that the lysosomal-autophagosomal systems are intricately linked and highly dependent on one another and that these proteolytic pathways can become dysregulated as part of the ageing process.

3.5.7Antioxidant Capacity of the RPE

The neural retina and RPE are particularly rich in a range of antioxidants [4, 128, 129]. Based on the observations that antioxidant levels decline and ROS levels increase in a variety of tissues and a number of neurodegenerative diseases [130], it is perhaps not surprising that similar analogies have been made to RPE ageing and AMD. Indeed, it is now well accepted that oxidative damage shows a positive association with retinal ageing [4, 5, 129, 130] and that antioxidant therapy may reduce the progression of AMD [131, 132]. However, it is equivocal whether agerelated oxidative damage occurs predominantly via increased ROS generation, reduced antioxidant levels, or a combination of the two. Mouse models with elevated ROS levels due to knockdown of SOD1 or SOD2 develop an AMD-like phenotype [105, 106]. Although animal and human data support the concept of an age-related decline in antioxidant activity, the evidence is not conclusive. For example, Liles et al. reported that catalase activity, but not SOD, in the human RPE decreases with age [133] while Miyamura and colleagues were unable to observe signiÞcant agerelated changes in either catalase or heme oxygenase (HO)-1 in the RPE [134]. Interestingly, the latter study observed mosaic patterns in antioxidant activity in the RPE monolayer, suggesting considerable cell-to-cell