A2E as a Marker of Lipofuscin Accumulation

Experiments on ABCR−/− animals indicated that A2E, once accumulated over a period of 12 months when animals are kept in a light/dark cycle, is not further metabolized by the RPE over a period of 16 months when animals are kept in the dark [55]. It suggests that A2E may be used as a quantitative marker of lipofuscin accumulation.

The content of A2E/iso-A2E has been estimated at about 7.8 × 10−20 mol per lipofuscin granule isolated from human donors 60–70 years old [36]. Thus, based on A2E content determined by Sparrow and colleagues in RPE cells obtained postmortem from human donors 58–79 years old [56], it can be estimated that RPE cells with 34–134 ng of accumulated A2E per 105 cells contains 7,400 and 29,000 lipofuscin granules per cell, respectively. Assuming the average diameter of a lipofuscin granule is 0.5 m and the cuboidal shape of the RPE cell has a side length of 14 m, these estimates give a range of 17–69% of RPE cell volume occupied by lipofuscin granules.

Another estimate of RPE volume occupied by lipofuscin can be obtained from data of Eldred and Lasky, who reported an average of 400 ng (0.675 nmol) of A2E per retina from donors 40 years old and older [32]. This corresponds to about 8.7 × 109 lipofuscin granules per retina. Assuming the average diameter of a lipofuscin granule is 0.5 m, the retinal surface is 1,000 mm2, and the RPE cells are densely packed in a monolayer 14 m thick, the RPE volume occupied by lipofuscin accounts for 3.9%.

Morphometric measurements of RPE cell volume occupied by lipofuscin gave an upper limit of 19% in donors above 80 years old [11], which is an intermediate value in comparison to values obtained based on A2E content.

It needs to be kept in mind that these estimates refer to content of lipofuscin averaged over a great number of RPE cells and retinas. There is a great deal of heterogeneity in lipofuscin accumulation in different RPE cells within the same retina and even greater heterogeneity for different retinas, particularly in cases of retinal dystrophies [11, 14, 17, 52, 57].

Also, it needs to be borne in mind that exposure to light or other sources of oxidative stress results in photodegradation of A2E and possibly other chromophores contributing to the absorption of light and fluorescence of lipofuscin. The content of A2E in lipofuscin was determined in lipofuscin obtained from donors 60–70 years old with no reported pathology of the retina [36]. It remains to be determined whether the A2E content per lipofuscin granule changes with age or retinal degeneration. Therefore, while monitoring A2E by its fluorescence in vivo or quantification by high-performance liquid chromatography (HPLC) in RPE extracts seems convenient for lipofuscin quantification, it requires further elucidation of A2E content in lipofuscin formed under specific conditions accompanying different retinal degenerations.

FACTORS AFFECTING ACCUMULATION OF RPE LIPOFUSCIN

Lipofuscin accumulation in the RPE can be accelerated, as in other cells, under oxidative stress conditions or due to inhibition of lysosomal enzymes [3, 4]. RPE cells are very active phagocytes, and phagocytosed POSs appear to be the main substrate for lipofuscin formation. In addition, the phototransduction cascade involving hydrolysis and removal of isomerized visual pigment chromophore, ATR, from opsin, is a critical

factor responsible for lipofuscin formation. The conditions under which ATR transiently accumulates in POSs accelerate lipofuscin accumulation (reviewed in [58]). Next, we discuss the roles of different factors in lipofuscin accumulation that allow proposing a hypothetical scenario for biogenesis of lipofuscin in the RPE.

Phagocytosis and Autophagy

Molecular composition of lipofuscin indicates that several components originate from POSs, lysosomes, and mitochondria [28–30]. In particular, proteomic analysis indicated that lipofuscin includes peptides of opsin [29]. In comparison with POSs, lipofuscin contains about three times less PE and phosphatidylserine and about seven times more free fatty acids [27]. The most unsaturated fatty acid, DHA with six double bonds, accounts for about 30–35% of total fatty acids (including acyl chains of phospholipids) in POSs, whereas in lipofuscin DHA is four to seven times less abundant than in POSs. These discrepancies in the lipid content between POSs and lipofuscin may be due to partial hydrolysis of phospholipids by lysosomal phospholipases and a contribution to the lipid content of phagolysosomes from the RPE plasma membrane and lysosomes. Moreover, unsaturated fatty acids may exhibit preferential loss in lipofuscin due to oxidation.

Each RPE cell apposes about 30–50 POSs and phagocytoses daily their adjacent tips, while new POS disks are produced at the other end of POSs [59] (Fig. 1). Taking into account the daily phagocytic load of human RPE cells, accounting for about 7–10% of POS length of 120 million photoreceptors, the average length and diameter of a POS is 24 m and 2 m [60], respectively; the total volume of POS tips ingested during 80 years is about 8.4 ml. Thus, the volume of ingested POSs is about 600 times greater than the volume of the RPE itself. Assuming 3 mM concentration of rhodopsin and about an equal ratio of lipids to proteins in POSs, the total amount of dry mass ingested by the RPE during 80 years accounts for more than 2 mg [27]. Yet, the highest estimate of lipofuscin volume, accounting for 19% of RPE cell volume in donors above 80 years old [11], corresponds to at most 8,000 granules of 0.5 m diameter of total dry weight of only 1.9–10.4 ng [29, 33], a tiny fraction of ingested POSs.

Experiments on animals demonstrated that phagocytosis of POSs is indeed the main source of RPE lipofuscin. The accumulation of RPE lipofuscin is substantially diminished in Royal College of Surgeons (RCS) rats, with a mutation in MERTK gene coding a tyrosine kinase essential for phagocytosis of shed POSs, lack of which leads eventually to photoreceptor degeneration [61, 62]. The animals accumulate autofluorescent material derived from POSs in an area between the POSs and RPE, indicating that some fluorophores, such as the product of condensation of two molecules of ATR with PE, A2PE, can be generated directly from POSs without involvement of the RPE [32, 63]. Also, a dramatically decreased accumulation of RPE lipofuscin was observed in albino rats that had their photoreceptors destroyed shortly after birth as a result of exposure to high-intensity light [64].

Modeling of lipofuscin formation was attempted in vitro by feeding RPE cells with culture medium supplemented with isolated POSs [65–67]. This resulted in accumulation of residual bodies in the RPE cells with fluorescent properties, albeit different from lipofuscin accumulated in vivo [68].

Interestingly, long-term culture of RPE cells, up to 2 years in the absence of POSs, leads to accumulation of fluorescence material, indicating that autophagy may also play a role in lipofuscin accumulation in the RPE [69]. The intracellular residual bodies, derived probably from autophagocytosis of intracellular organelles, such as mitochondria, exhibit fluorescence properties, but the excitation and emission spectra are distinctly different from lipofuscin granules isolated from RPE cells harvested postmortem from human donors [68].

Altogether, a substantial body of evidence indicates that RPE lipofuscin is mainly derived from incomplete lysosomal degradation of phagocytosed POSs. Molecular composition of lipofuscin and studies of RPE cells in vitro indicate that autophagy may also contribute to lipofuscin formation in the RPE.

Role of Lysosomal Degradation

Several lines of investigation indicated that dysfunction of lysosomes leads to accumulation of lipofuscin in the RPE. Rapid accumulation of intracellular material exhibiting autofluorescence is observed in rats and dogs on intraocular injection of an inhibitor of lysosomal proteases [70–72].

The role of dysfunction of lysosomal enzymes in lipofuscin formation is also supported by studies on transgenic mice expressing an inactive cathepsin D, an abundant RPE lysosomal enzyme involved in degradation of POS rhodopsin [73]. Mutant animals accumulate substantially greater amounts of fluorescent residual bodies within RPE cells than wild-type mice.

However, it has been demonstrated that there is no age-related decline in lysosomal enzyme activities in the normal human RPE [74]. In contrast, activities of several lysosomal enzymes increase with age [75]. Therefore, lysosomal dysfunction is not likely to be a result of dysfunction of the ability of RPE cells to produce lysosomes with active enzymes, but more likely it is related to an impairment in fusion of phagosome with lysosome, inhibition of lysosomal enzymes by phagosome components, or formation of products no longer susceptible to lysosomal degradation [76–78].

Indeed, several studies indicated that lipofuscin components 4-hydroxy-2-nonenal and A2E can inhibit lysosomal enzymes either directly [78] or via inhibition of lysosomal proton pumps, which results in an increase of lysosomal pH [79–81]. Consistently, experiments on cultured RPE cells demonstrated that A2E localizes mainly in lysosomes, causes an increase of lysosomal pH, and inhibits lysosomal degradation of endogenous proteins, sulfated glycosaminoglycans, and POS phospholipids [81, 82]. Interestingly, A2E does not affect the rate of DNA degradation of phagocytosed apoptotic HL-60 cells or POS proteins [82].

Altogether, lipofuscin components, such as A2E and aldehydic products of lipid peroxidation, are likely to contribute to lipofuscin accumulation by their inhibitory effect on degradation of phagocytosed POSs.

Role of Oxidative Stress

It has long been suggested that free radicals and lipid peroxidation are involved in lipofuscin formation [57, 83]. The outer retina is particularly at risk of oxidative damage due to exposure to a high concentration of oxygen from the choroidal blood supply, high metabolic rate related to production of superoxide by mitochodria, and high concentration