LIPOFUSCIN OF THE RETINAL PIGMENT EPITHELIUM: A REVIEW

CHRIS J. KENNEDY, PIROSKA E. RAKOCZY and IAN J. CONSTABLE Nedlands, Australia

SUMMARY from its unique derivation, the lipofuscin of the Accumulation of lipofuscin is one of the most retinal pigment epithelium differs from that of the characteristic features of ageing observed in retinal brain, heart, testes, and other organs in both electron s pigment epithelial (RPE) cells. The lipofuscin found in density and uniformity of granule size. The RPE cells differs from that of other body tissues due to composition, origin, and possible deleterious effects the fact that it is mainly derived from the chemically of RPE cell lipofuscin have been topics of consider­ modified residues of incompletely digested photorecep­ able scientific interest and controversy in recent tor outer segments. It is a heterogeneous material years and this article reviews current knowledge in composed of a mixture of lipids, proteins, and different these areas. fluorescent compounds, the main fluorophore of which has recently been identified as a derivative of vitamin MORPHOLOGY AND TOPOGRAPHY A. Research interest has variously focussed on the roles RPE cell lipofuscin is contained within granules of of age, light damage, free radicals, antioxidants, visual relatively uniform size and homogeneous electron pigments, retinal locus, lysosomal enzymes, and density.s The granules are typically more numerous pigmentation on lipofuscin formation, as well as the in the basal half of the R,PE cell around the nucleus. effects of lipofuscin on RPE cell function and causation As noted below, with increasing age there is a of retinal disease. This article reviews the recent tendency for lipofuscin granules to fuse with advances in knowledge of the composition, origin, and melanosomes to form complex granules. These possible deleterious effects of RPE cell lipofuscin. have a high electron density core of melanin surrounded by a lipofuscin coating of lower electron Generally regarded as a sign of cellular ageing or density (Fig. 1). The topographic distribution of senescence, lipofuscin is not a distinct chemical lipofuscin across the fundus has been found to compound but the generic name given to an parallel the density of rod outer segments (ROS). autofluorescent, membrane-bound intracellular Macular RPE cells contain greater amounts of material that is distributed widely amongst the lipofuscin than non-macular RPE cells, with the post-mitotic cells of different organs of the body. exception of a focal reduction in lipofuscin concen­ With advancing age, there is a marked increase in the tration at the fovea,z,6,7 The concentration of lipofuscin granule content of human retinal pigment complex granules within the retinal pigment epithe­ epithelial (RPE) cells and there now exists substan­ lium is similarly highest at the macula and progres­ tial evidence that the bulk of this lipofuscin sively decreases through the equator to the represents the chemically modified residues of periphery.l incompletely digested photoreceptor outer seg­ --4 ments.l The origin of the lipofuscin in the retinal FLUORESCENT CHARACTERISTICS pigment epithelium therefore differs significantly Lipofuscin is composed of a mixture of different from that of the lipofuscin in other tissues. Apart fluorophores, reflecting the heterogeneous nature of From: Molecular Biology Unit, Lions Eye Institute, Centre for this cellular pigment. Ten different fluorescent Ophthalmology and Visual Science, University of Western fractions have been identified within chloroform Australia, Nedlands, Western Australia. s Correspondence to: Dr Chris J. Kennedy, 71 Davies Road, extractions of RPE cell lipofuscin. All these Claremont, Western Australia, 6010. fluorophores have common absorption peaks in the

Eye (1995) 9, 763-771 © 1995 Royal College of Ophthalmologists 764 C. J. KENNEDY ET AL.

composed of lipid, only recently has more detailed knowledge of the chemical composition of lipofuscin been acquired.ll Whilst the proteinaceous content is still unknown, some of the lipid components of RPE lipofuscin have now been analysed and found to differ significantly from the lipid composition of the adjacent retina and the ROS that are phagocytosed by the RPE cellsY The major fatty acids of lipofuscin have. been shown to be palmitic, arachi­ donic, and oleic acids. Also, the major orange­ emitting fluorophore of RPE lipofuscin has recently been identified, using fast-atom bombardment tan­ dem mass spectrometry, as N-retinylidene-N-retinyl­ ethanolamine, an amphoteric quaternary amine that arises as a Schiff base reaction product of ethanola­ mine and retinaldehydeP Ethanolamine is a com­ ponent of the ubiquitous membrane lipid phosphatidylethanolamine and retinaldehyde is, of course, the oxidised form of vitamin A which is combined with opsin to form the photopigment rhodopsin of the ROS. Previous animal experiments in which vitamin A intake was manipulated had strongly indicated a role for vitamin A in lipofuscin formation. Dietary vitamin A deficiency had been Fig. 1. Transmission electron micrograph of the macular shown virtually to eliminate the orange-emitting retinal pigment epithelium of a 51-year-old Caucasian male. Phagosome (P) containing rod outer segment material, fluorophore of lipofuscin14 and to reduce the lipofuscingranules (L), melanosome (M), complex granule number of lipofuscin granules in RPE cells of (C) containing lipofuscin with a central melanin core and rats.15 It appears that vitamin A deficiency primarily Bruch's membrane (B) can be seen. (X 7255). reduces lipofuscin accumulation by diminishing photoreceptor outer segment turnover and hence ultraviolet range of the spectrum around 280-330 nm ROS phagocytosis by RPE cells.16 The remaining and fall into four broad categories based on their unidentified fluorophores contained in RPE lipofus­ fluorescent excitation and emission characteristics cin appear to be structurally related to each other (Table I). Earlier reports of RPE lipofuscin contain­ and may also consist of modified retinoidsY ing significantblue-emitting fluorophoresw ere found to be incorrect. With the use of carefully calibrated fluorescence instrumentation, both in situ lipofuscin THE ORIGIN OF RPE CELL LIPOFUSCIN granules and the chloroform extracts of lipofuscin Each RPE cell apposes around 30-50 photoreceptor granules or lipofuscin-laden tissues have a peak in outer segments in vivo and 10-15% of each outer emission spectra around 570-605 nm (golden-yellow) segment is phagocytosed and replaced each day.17,18 when excited at 366 nm.9,10 In order for RPE cells to eliminate successfully these vast quantities of discs synthesised by the photo­ THE CHEMICAL COMPOSITION OF RPE receptor inner segments, the RPE cells must CELL LIPOFUSCIN phagocytose and digest the ROS with great Although the histochemical characteristics and efficiency. Phagocytosis and digestion of ROS chloroform:methanol solubility of RPE cell lipofus­ occurs in several discrete stages (Fig. 2).19 The first cin had previously indicated that it was mainly stage, ROS binding, involves recognition and attach­ ment of ROS to the RPE cell. This is followed by the Table I. Fluorophores present in the chloroform extracts of activation of .intracellular microtubules and micro­ 8 RPE cell lipofuscin filaments which endocytose cell surface-bound ROS Excitation Emission to form phagosomes.2o Lysosomes then fuse with Flubrophores peaks (nm) peaks (nm) these membrane-bound particles to form phagolyso­ Two green-emitting fractions 330 520 somes or secondary lysosomes. Digestion of the ROS Three yellow/green-emitting 280, 330 568 by lytic enzymes then proceeds to yield compounds fractions that are small enough to diffuse out of the One golden-yellow-emitting 280, 330 585 fraction phagolysosome,z RPE cell lysosomes contain many Four orangelred-emitting 285, 335, 420 605, 633, 670 lytic enzymes and these include cathepsin D, fractions cathepsin B, a-mannosidase, N-acetyl-I3-D-glucos- REVIEW: RPE CELL LIPOFUSCIN 765

(Table II). It has been theorised that if ROS are oxidatively damaged prior to phagocytosis, the RPE lysosomal system may fail to digest them adequately because the aberrant molecular species no lon§er match active sites on the degradative enzymes?,2 ,30 Such undigested oxidised residues within the RPE cells may then be further altered to form lipofuscin. The amount of oxidative damage to the ROS and hence the rate of RPE cell lipofuscin accumulation could potentially be influencedby factors such as the levels of retinal oxygenation and light exposure (potentiating free radical formation) and the anti­ oxidant systems within the retina?9 Alternatively, it is possible that primary failure of the action of the RPE lysosomal enzymes could lead to an accumula­ • tion of ROS-derived residues that then undergo further modification to form lipofuscin.4 The evi­ dence to support each of these theories is outlined below.

EVIDENCE THAT LIPOFUSCIN IS DERIVED Fig. 2. Retinal pigment epithelial (RPE) cell processing of FROM PHOTORECEPTOR OUTER rod outer segments (ROS). SEGMENTS Lipofuscin-like autofluorescent pigment has been aminidase, acid lipase, acid phosphatase, a-fucosi­ found to accumulate within cultured human RPE dase, a-galactosidase, a-glucosidase, l3-galactosidase, cells as a consequence of ROS phagocytosis in l3-glucosidase, l3-glucuronidase, and phospholipases vitro?,31 In vivo, elimination of photoreceptor A and A . I- The recently identifiedRPE protease outer segments from the retina by selective light­ l 2 2 24 cathepsin S has not been localised to the lysosome, induced destruction has been shown to reduce but appears to be important in ROS digestion?5 This lipofuscin accumulation in RPE cells?2 In the complex array of lysosomal enzyme within the RPE Royal College of Surgeons rat, the RPE cells have cell is believed to be specialised for the purpose of a genetic defect in ROS phagocytosis' and contain ROS digestion. RPE lysosomal enzymes are, for lower levels of lipofuscin than congenic age-matched example, around 7 times more efficient than the controls?3 lysosomal enzymes of the liver in degrading ROS?2 Despite all the evidence that the precursors of Nevertheless, since all current evidence indicates that RPE cell lipofuscin are supplied by ROS, it should be RPE lipofuscin represents an altered form of the stated that no experiment has yet succeeded in indigestible residues of lysosomal enzymatic diges­ tracing chemical groups all the way from ROS to tion of phagocytosed ROS,1,26-28 it appears that the lipofuscin granules. Immunohistochemical studies lysosomal enzyme system of the RPE cell is not targeting opsin have failed to confirm that any of absolutely effective in fulfilling its task of degrading this ROS-derived protein ends up in lipofuscin all the ROS. granules following the phagocytosis and lysosomal It is possible that lipofuscin is caused to accumu­ enzyme digestion of ROS by RPE cells.27 The most late by alterations in either the ROS or the RPE cell probable reason for this is that the lysosomal enzyme

Table ll. Factors that are known or suspected to influence the rate of lipofuscin formation in the retinal pigment epithelium

Factors retarding lipofuscin formation Factors promoting lipofuscin formation

Elimination of rod outer segments Rod outer segment oxidative damage Vitamin A deficiency Light exposure (photochemical effects) Ocular melanin High oxygen content of retina Retinal antioxidants Chemical pro-oxidants (e.g. iron) Vitamin C Antioxidant deficiency Vitamin E Vitamin E deficiency Superoxide dismutase Lysosomal enzyme defects Glutathione peroxidase Protease inhibitors Peroxidase Increasing age Catalase Reduction in dietary calories or food intake 766 C. J. KENNEDY ET AL.

digestion process rapidly alters or destroys the Several imine-Schiff bases were created from superficial hydrophilic antigen-binding sites on the malonaldehyde and amino acids as a result of lipid opsin molecule for which the antibodies are specific. peroxidation, and the investigators found them to have similar blue fluorescent emission spectra to . those they had recorded for lipofuscin. However, PHOTORECEPTOR OUTER SEGMENT instrument bias was later discovered to be respon­ OXIDATIVE DAMAGE sible for the blue emission peaks and the corrected Oxidative damage to the retinal tissues can result spectra were found to be dissimilar to the malonal­ from light and the natural by-products of oxidative dehyde imine-Schiff bases which were truly blue­ metabolism?9,34 Light energy transmitted through emittingY the ocular media can cause damage to the retina All attempts to generate lipofuscin fluorophores through both thermal effects and photochemical by polymerising malonaldehyde and by oxidising reactions. It is the photochemical effects, which are lipids and tissue preparations in vitro have been greater with shorter-wavelength light, that result in unsuccessful, indicating that formation of lipofuscin oxidative damage and these damaging effects are granules involves more than simply the direct greatly potentiated by the involvement of oxygen. oxidation of tissue constituents.4,9,39 Oxidative Transfer of an electron to oxygen results in the damage to the ROS does seem to play a role in formation of the highly reactive superoxide free lipofuscinogenesis, but it appears to be modulated by radical. Subsequent free radicals formed can include unknown RPE interactions. Non-enzymatic lipid singlet oxygen and the hydroxyl radical, and these oxidation induced by intravitreal injection of the can all initiate photodynamic chain reactions?9 Since catalyst iron sulphate in rats in vivo has been shown the outer segment discs and plasma membranes of to result in the appearance of golden-yellow-emitting the photoreceptors are rich in polyunsaturated fatty lipofuscin-like autofluorescent pigment within the acids and the ease with which fatty acids undergo RPE cells only after the phagocytosis of oxidised auto-oxidation is proportional to their degree of ROS. The oxidised ROS alone did not show these unsaturation, ROS are particularly vulnerable to characteristics prior to phagocytosis, thus supporting auto-oxidation?S,36 Also, oxygen is concentrated in the concept that the insoluble lipofuscin fluorophores the fatty acid layers of the ROS discs where it is form as a result of a further modification of ingested highly soluble?9 Peroxidation of unsaturated lipid oxidised ROS material.40 bonds, which can be initiated by several types of free radical, results in the formation of alkyl lipids. As EFFECT OF LIGHT EXPOSURE AND these alkyl lipids are also free radicals, these species MELANIN CONTENT OF FUNDUS can convert neighbouring unsaturated fatty acids into The lipofuscin content of RPE cells has been found alkyl lipids and propagate a chain reaction which will to be significantly greater in whites than in blacks. spread throughout the lipid bilayer of individual The inverse relationship between lipofuscin content ROS disc membranes.s ROS membranes containing of RPE cells and choroidal melanin suggests that such oxidatively altered lipids continue to be melanin may have a protective role in preventing ingested by the RPE cell as the phagocytic process lipofuscin formation. The fundi of Caucasians reflect is apparently unimpaired by these chemical altera­ around 5% of incident white light versus only 1% in tions. blacks? The increase in reflected photons in It was previously believed by many that lipofuscin Caucasians may lead to an increase in light-induced was simply a lysosomal residue representing the free radical formation, increased ROS oxidative combination of products of photoreceptor outer damage and hence greater rate of lipofuscin segment auto-oxidation. These products were accumulation. Animal experiments have certainly thought to be comprised of malonaldehyde, lipid correlated higher levels of light exposure with peroxides and other related chemical species. The increased rates of lipofuscin accumulation.41 prevalent lipid peroxidation theory of lipofuscino­ Melanin granules are thought to play a protective genesis was based on the papers of Chio et al. in role by absorbing radiation and possibly by directly 1969?7,38 Malonaldehyde is the principal and most scavenging free radicals and quenching excited characteristic breakdown product of lipid peroxida­ molecular states produced by the photochemical tic;m. It is highly reactive and forms irreversible cross­ and photodynamic effects of radiation.1,29 links through the formation of Schiff bases with the amino groups of a wide variety of macromolecules such" as DNA, phospholipids and proteins in a THE ROLE OF ANTIOXIDANTS nonspecific manner.s These early experiments com­ Several antioxidant systems exist in the outer retina pared the fluorescence spectra of lipofuscin with and retinal pigment epithelium to deal with the free those of lipid peroxidation products created by the radicals that confront these cells. They include the incubation of subcellular organelles with oxygen. cellular enzymes superoxide dismutase, glutathione REVIEW: RPE CELL LIPOFUSCIN 767 peroxidase, peroxidase, and catalase, and the in the aqueous humour of the diurnal species was vitamins E (a-tocopherol) and C (ascorbate). found to be 35 times higher than that of the nocturnal Superoxide dismutase, which catalyses the conver­ species. It was suggested that this represented an sion of superoxide to hydrogen peroxide, has been adaptive response of the diurnal mouse in order for it found to be concentrated in ROS.29 RPE cells also to better withstand the free radicals generated by contain two distinct isoenzymes of superoxide solar radiation.48 dismutase: a copper-zinc and a manganous form, Nutrition may influencelipofuscin accumulation in with the copper-zinc form of superoxide dismutase several ways. In addition to the effects of dietary being distributed diffusely throughout the cytoplasm content of vitamin A, C and E discussed above, of the RPE cell and the manganous form being reduction in either total food intake or in caloric primarily localised within the mitochondria.42 Glu­ intake has been shown to slow lipofuscin accumula­ tathione peroxidase, which detoxifies hydrogen tion in rats.49 peroxide and fatty acid peroxides by converting them to water and hydroxy fatty acids, has been ROLE OF RPE CELL LYSOSOMAL identifiedin both neurosensory retina and RPE.5,29,35 PROTEASES Hydrogen peroxide can also be removed by catalase Incomplete proteolytic digestion qf ROS within the and peroxidase.5 Catalase is present in the retinal lysosomes of RPE cells may also play an important pigment epithelium at a concentration 6 times that in role in lipofuscin accumulation. Indeed, it has been any other ocular tissue, and phagocytosis of ROS has proposed that the protein components of ROS are been shown to cause an increase in catalase activity the major precursors of RPE lipofuscin.4,34,5o in cultured human RPE cells.43 Intravitreal injection of the specific cysteine pro­ Acting as a free radical scavenger, vitamin E can tease inhibitor leupeptin has been shown to impair absorb unpaired free electrons to terminate auto­ the digestion of phagocytosed ROS and result in oxidation chain reactions.5,35 Vitamin E is known to rapid accumulation of lipofuscin-like material within be concentrated in the lipid phase of ROS and in the RPE cells?4,50 In vitro experiments have vitro experiments have shown that vitamin E can reproduced these findings and confirmed that reduce lipid hydroperoxide formation in ROS specific protease inhibitors can impair ROS degrada­ exposed to light.29 In rats deficient in vitamin E, tion by RPE cells without diminishing the rate of but receiving an adequate vitamin A intake, there is ROS phagocytosis.25,51 It has been hypothesised that a marked increase in lipofuscin-like granules within either primary failure of the lysosomal proteases (as the RPE cells.15 Diets deficient in vitamin E have might occur with increasing age) or inhibition of the been shown to result in an enhanced rate of proteases by other factors (namely oxidatively lipofuscin-like granule accumulation in RPE cells of damaged ROS components) could allow non-oxida­ monkeys and rats, especially when the diet is high in tive amino acid modifications to occur and hence polyunsaturated fatty acids.5,15,44 Also, it has been lipofuscin to accumulate.4,34 The effect that antiox­ reported that the severity of oxygen-induced retino­ idant deficiency has on promoting lipofuscin forma­ pathy in rats may be diminished by supplementing tion may then be viewed as an indirect result of RPE the animals' diets with vitamin E.45 Although vitamin protease inhibition by the oxidised ROS and not a E deficiency causes an accumulation of autofluor­ result of the direct transformation of oxidised ROS escent lipofuscin-like granules in RPE which seem to components into lipofuscin.4 This would also explain in vitro be derived from ROS,32 there is some evidence to why lipofuscin cannot be produced by suggest that these pigments are in fact dissimilar to oxidising tissues.9,39 lipofuscin due to their different organ distribution, extractability and chromatographic characteris­ EFFECTS OF AGE ticsy,46 The progressive increase in cellular lipofuscin is one Like vitamin E, vitamin C can also terminate of the most characteristic features of ageing seen in oxidative chain reactions and quench singlet oxy­ RPE cells? The area of each macular RPE cell gen?9 It has been demonstrated that exposure of profile occupied by lipofuscin has been found to baboon and guinea pig neural retina to light results in increase from 1 % in firstdecade of life to as much as an increase in oxidised ascorbate and a concomitant 19% in the 81- to 90-year-old age group.l Somewhat decrease in reduced ascorbate.47 Whilst dietary surprisingly, the fastest rate of lipofuscin accumula­ deficiency of vitamin C has been shown to increase tion occurs in the first two decades of life.1,2,7 With light-induced ROS damage in guinea pigs, dietary increasing age there is an increase in the lipid content vitamin C supplementation has been shown to of the lipofuscin granules and a reduction in the protect against such light-induced damage in amount of polyunsaturated fatty acyl chainsp,52 rats?9,47 In a study comparing two closely related Histopathological studies have shown that fusion of species of spiny mice, the concentration of vitamin C primary lysosomes with lipofuscin granules is 768 C. J. KENNEDY ET AL. common in older eyes, suggesting that repeated Histopathological case studies probably provide unsuccessful attempts at digesting the material are the strongest available evidence linking lipofuscin to occurring.52 As illustrated in Fig. 1, there is also a RPE cell dysfunction, although they are unable to higher rate of lipofuscin granule fusion with melano­ prove a direct causal relationship. An abnormal somes to form complex granules with advancing accumulation of lipofuscin in the RPE cells has been age.1,5 noted to be associated with degeneration of RPE For reasons detailed above, there has been cells and adjacent photoreceptors in a dominantly increasing interest in the possible influence of age­ inherited pigment epithelial dystrophy affecting dogs related changes in RPE enzyme activity. The main and in chronic lead subacetate poisoning in rabbits.57 lysosomal protease of RPE cells responsible for ROS Lipofuscin accumulation in RPE cells has also been digestion is thought to be cathepsin D.l Ageing rat correlated with photoreceptor death in histopatho­ organs have been shown to accumulate immunolo­ logical studies on humans. Photoreceptor loss in gically active, but enzymatically inactive, cathepsin Caucasians has been reported to be very significantly In vitro D.53 experiments on cultured human RPE and directly correlated with the lipofuscin concentra­ cells have revealed that there is a decline in tion of the apposing RPE cells?6 . extracellular cathepsin D release from the basal A particularly controversial topic is the association surface of the cells with increasing donor age.54 A between lipofuscin and age-related macular degen­ separate study on cultured human RPE cells has eration, the commonest cause of blindness in shown a reduction in the activity of another Western society and a disease considered by many lysosomal enzyme, a-mannosidase, with increasing to be principally caused by gradual RPE cell donor age. Whilst age-dependent reductions in failure.58--62 As suggested by its title, the strongest lysosomal enzyme activity could possibly increase risk factor identified for development of age-related the rate of lipofuscin accumulation, an age-depen­ macular degeneration is chronological age.47,63 As dent decline in retinal antioxidant enzyme activity noted above, RPE cell lipofuscin content is also could also theoretically promote lipofuscin accumu­ highly correlated with age,l,7 One histopathological lation. Indeed, a study on post-mortem ocular tissue study noted that the ratio of photoreceptors to RPE has reported that RPE cell catalase activity declines cells was higher in the macula than in the paramacula significantlywith advancing age.43 or equatorial region and that the number of photoreceptors per RPE cell profile increased with RELATION TO RETINAL DISEASE age. The age-related increase in the ROS burden on individual RPE cells was found to be particularly The issue of whether lipofuscin accumulation has pronounced at the macula, the favoured anatomical significant deleterious effects on RPE cell and, site of disease in age-related macular degeneration?6 consequently, overall retinal function continues to be of great interest. Several mechanisms have been In the 'dry', atrophic form of age-related macular suggested by which lipofuscin may disrupt normal degeneration which culminates in geographic atro­ RPE cellular function. Lipofuscin may be a basically phy, the RPE cells show massive lipofuscin accumu­ inert substance which leads to cellular dysfunction lation, loss of cellular organelles, nuclear only by means of engorging the cells with non­ displacement, and loss of apical microvilli prior to functional waste, causing a reduction in functional cell death and loss.62 cytoplasmic space and distortion of cellular architec­ Drusen are extracellular deposits that form ture which result in progressive failure of metabolic beneath the RPE cells in age-related macular processes?9,55,56 On the other hand, lipofuscin degeneration. Although lipofuscin and drusen are granules may slowly leach abnormal molecular both considered to be forms of cellular debris fragments which have some toxicity to normal produced by the RPE cells, they have distinctly cellular function.56 The only currently identified different histological features and their precise fluorophore of RPE lipofuscin (N-retinylidene-N­ relationship is not established.6l,64 However, the retinylethanolamine) is both a detegent and a weak RPE cell atrophy seen in both primary age-related base and when present in sufficient quantities could RPE atrophy and drusen-related atrophy precisely be expected to inactivate lysosomal enzymes and parallels the topographic distribution of lipofuscin eventually cause lysis of the lysosomal membrane across the fundus, increasing toward the posterior and cell death.l3 Some authors have also implicated pole, but tending relatively to spare the centre of the the autofluorescent properties of lipofuscin as a fovea.62 The process of RPE cell membrane blebbing possible contributing factor. It has been suggested and cytoplasm extrusion into Bruch's membrane may that following absorption of shorter-wavelength light contribute to drusen formation.56,65,66 This finding by lipofuscin, radiation re-emitted at other wave­ has been described in age-related macular degenera­ lengths inside the RPE cells could lead to further tion66 and in aged human eyes67 and is an expected photo�hemical damage?9 finding in cells which contain excessive amounts of REVIEW: RPE CELL LIPOFUSCIN 769 lysosomotropic detergent such as the recently of the human retinal pigment epithelium. Am J identified lipofuscin fluorophore N-retinylidene-N­ Ophthalmol 1980;90:783-9l. 3 6. Wing GL, Blanchard GC, Weiter n. The topography retinylethanolamine.1 and age relationship of lipofuscin concentration in the Fundus flavimaculatus (Stargardt's disease) and retinal pigment epithelium. Invest Ophthalmol Vis Sci vitelliform macular degeneration (Best's macular 1978;17:601-7. dystrophy) are two RPE diseases of unknown 7. Weiter n, Delori FC, Wing GL, Fitch KA. Retinal aetiology associated with abnormal accumulation of pigment epithelial lipofuscin and melanin and chor­ oidal melanin in human eyes. Invest Ophthalmol Vis lipofuscin in the RPE cells. Fundus flavimaculatus is Sci 1986;27:145-52. the collective name given to a group of inherited 8. Eldred GE, Katz ML. Fluorophores of the human macular dystrophies, the majority of which are retinal pigment epithelium: separation and spectral transmitted in autosomal recessive fashion.68 In characterisation. Exp Eye Res 1988;47:71-86. fundus flavimaculatus the RPE cells contain up to 7 9. Eldred GE, Katz ML. The lipid peroxidation theory of lipofuscinogenesis cannot yet be confirmed. Free Radic times more lipofuscin than normal and this is BioI Med 1991;10:445-7. associated with RPE cell degeneration and second­ 10. Eldred GE, Miller GE, Stark WS, Feeney-Burns L. ary atrophy of photoreceptors and choriocapil­ Lipofuscin: resolution of discrepant fluorescence data. laris.68.69 In vitelliform macular degeneration, a Science 1982;216:757-9. disease with autosomal dominant inheritance, there 11. Eldred GE. Vitamins A .and E in RPE lipofuscin is a marked accumulation of lipofuscin both within formation and implications for age-related macular degeneration. Prog Clin BioI Res 1989;314:113-29. and beneath the RPE cells. Even at an early stage in 12. Bazan HE, Bazan NG, Feeney-Burns L, Berman ER. the disease, the electro-oculogram is subnormal due Lipids in human lipofuscin-enriched subcellular frac­ to loss of the ability of the RPE cells to maintain a tions of two age popUlations: comparison with rod normal transepithelial potential. Later there is outer segments and neural retina. Invest Ophthalmol progression to frank dysfunction and degeneration Vis Sci 1990;31:1433-43. 13. Eldred GE, Lansky MR. Retinal age pigments of the macula?O Finally, several conditions which generated by self-assembling lysosomotropic deter­ secondarily cause RPE dysfunction are also asso­ gents. Nature 1993;361:724-6. ciated with excessive lipofuscin accumulation in the 14. Katz ML, Drea CM, Eldred GE, Hess HH, Robinson RPE cells. Such examples include the retinal pigment WG Jr. Influence of early photoreceptor degeneration epitheliopathy associated with choroidal mela­ on lipofuscin in the retinal pigment epithelium. Exp 70 7 7 Eye Res 1986;43:561-73. noma • and severe ocular trauma. 2 15. Robinson WG Jr, Kuwabara T, Bieri JG. Deficiencies Although there is an association between RPE of vitamins E and A in the rat: retinal damage and lipofuscin accumulation and disease of the retinal lipofuscin accumulation. Invest Ophthalmol Vis Sci pigment epithelium, firm evidence that lipofuscin is 1980;19:1030-7. directly responsible for RPE cell dysfunction is still 16. Katz ML, Norberg M, Stientjes HJ. 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