The loss of efficiency of lysosomal degradation may be due to inactivation of lysosomal enzymes by components of lipofuscin or generation of products no longer susceptible for lysosomal degradation, such as oxidatively modified and cross-linked proteins, lipids, nucleic acids, and carbohydrates. Inhibition of autophagy by 3-methyladenine or inhibition of lysosomal enzymes by leupeptin leads to rapid intracellular accumulation of autofluorescent material in confluent fibroblasts and astrocytes and eventually results in their apoptotic death [6]. Also, inhibition of proteasomal pathways of protein degradation leads to increased accumulation of lipofuscin in neurons [7]. Accumulation of lipofuscin may consecutively induce proteasome inhibition, which eventually leads to cell death [8, 9].

Fluorescence is a characteristic feature of all lipofuscins, which emit yellow-orange light on photoexcitation with ultraviolet (UV) or blue light (330–490 nm). The spectral characteristics of lipofuscin fluorescence vary depending on the type of tissue and type of lipofuscin.

To identify the components of lipofuscin responsible for its fluorescence, several different products with fluorescent properties have been synthesized by incubation of products of lipid peroxidation, such as 4-hydroxynonenal or malondialdehyde, with proteins or amino acids [10]. Also, polymerized products of lipid oxidation and modified proteins due to nonenzymatic glycation exhibit autofluorescence.

It has been demonstrated that there are some similarities in fluorescence features between those synthetic products and autofluorescence of oxidized subcellular components. Oxidative stress seems to play a key role in lipofuscin accumulation. For example, the accumulation of lipofuscin in heart muscle cells in vitro is strongly increased in the presence of iron ions, at increased oxygen tension, and at decreased levels of reduced glutathione, a peptide that plays a major role in antioxidant defenses. Antioxidants, such as vitamin E and chelators of metal ions, inhibit accumulation of lipofuscin.

To sum, accumulation of lipofuscin is a characteristic feature of postmitotic cells exhibiting high metabolism or under oxidative stress conditions. There is a growing body of evidence that age-related increase of lipofuscin in metabolically active postmitotic cells may, on exceeding a certain threshold, adversely affect cell function and viability and contribute to numerous age-related pathologies.

LIPOFUSCIN OF THE RETINAL PIGMENT EPITHELIUM

In the retina, the greatest accumulation of lipofuscin occurs in the retinal pigment epithelium (RPE) and is strongly dependent on age [11, 12]. The RPE separates the retina from the choroidal blood supply and plays multiple roles essential for survival and function of photoreceptors (Fig. 1) [13].

With age, there is a linear increase in lipofuscin as well as in complex granules, containing both melanin and lipofuscin, called melanolipofuscin. Accumulation of lipofuscin exhibits strong racial differences and occurs faster in white people with lighter pigmentation of the iris and choroid than in black people with darker pigmentation [14]. In white people, lipofuscin fluorescence in the RPE cells increases linearly up to the age of 70, after which it exhibits a gradual decline [12]. Morphometric data indicate that lipofuscin occupies almost 20% of RPE cytoplasmic volume in people above 80 years old [11].

Fig. 1. Schematic diagrams of the retina (A) and shedding, phagocytosis, and lysosomal degradation of the outer segment distal tips (B). The diagram of the retina depicts only cells involved in primary phototransduction (photoreceptors: rods and cones) and regeneration of visual pigment chromophore (retinal pigment epithelium [RPE] and Müller cells). All second-order neurons are omitted for clarity. The photoreceptor cells are roughly cylindrical in shape and are organized into specific specialized regions: synaptic terminal, cell body with nucleus (N), mitochondria (Mt)-rich inner segment (IS), and outer segment (OS) containing visual pigments. The outer retina is exposed to high oxygen tension provided by the choroidal blood supply. Light passes through most of the retina before being absorbed in the OSs. In rods, the outer segments consist of stacks of flattened membraneous disks discontinuous from the plasma membrane. In cones, the disks are continuous with the plasma membrane and are open to the extracellular space. The OSs undergo a continual process of renewal. The OSs grow outward from their bases adjacent to the IS. The tips of OSs are shed daily from the photoreceptors and are phagocytosed by the RPE. Phagosomes (Ph) fuse with lysosomes (Ls) to form phagolysosome (PhL), where the material undergoes degradation. The incomplete degradation results in accumulation of autofluorescent bodies, lipofuscin (LF). Apart from typical cellular organelles, RPE cells contain melanosomes (Ms), which absorb light passing through OSs, and retinosomes (Rs), which store retinyl esters. The drawings are not to scale.

Age-related accumulation of lipofuscin is greatest in the parafoveal area corresponding to the greatest density of rods [11, 12, 14–17]. Interestingly, in the center of the fovea, corresponding to the greatest density of cones, lipofuscin concentration is almost twice as small than elsewhere in the macula [14].

Lipofuscin accumulation in the RPE is strongly accelerated in patients with Stargardt’s disease [18, 19], Best’s disease [20], and some cases of retinitis pigmentosa [21, 22]. Retinas with different features of age-related macular degeneration (AMD) exhibit characteristic patterns of autofluorescence depending on the AMD status [23–26].

Composition of RPE Lipofuscin

RPE lipofuscin is an amorphous pigment with heterogeneous chemical composition, including mainly lipids and proteins, which account for at least 93% of the dry mass

of lipofuscin [27–30], and a number of fluorophores [31, 32]. Chloroform/methanol/ phosphate-buffered saline extraction yields chloroform-soluble components and insoluble interphase material accounting for about 0.08–0.10 and 0.08–0.14 pg of dry mass per lipofuscin granule, respectively [33]. Lipids of lipofuscin consist of free fatty acids ( 40%), phosphatidylcholine ( 30%), phosphatidylethanolamine (PE; 13%), phosphatidylinositol ( 7%), phosphatidylserine ( 4%), and diacylglycerols ( 3%). Free fatty acids and acyl chains of phospholipids contain a large proportion of polyunsaturated chains, including docosahexaenoate (DHA) and arachidoneate [27].

Aging is accompanied by a decrease of lipid-to-protein ratio in lipofuscin granules. It has been estimated that RPE lipofuscin isolated from donors below 40 years old contains 0.77 nmol of lipids per 1 mg of protein, while lipofuscin isolated from donors above 47 years old has 0.41 mmol/mg protein [27]. The content of insoluble components of lipofuscin granule increases from 0.08 pg in donors below 40 years old to 0.14 pg in donors above 80 years old, while the content of soluble components does not show any significant difference with age [33].

Proteomic analyses of lipofuscin identified up to 160 proteins, including several lysosomal enzymes; proteins of photoreceptor outer segment (POS), mitochondrial, and endoplasmic reticulum origin; cytoskeletal proteins; and retinoid chaperones but also proteins originating from blood plasma and erythrocytes [28–30]. The purification of lipofuscin granules before proteomic analysis remains a challenge. Currently employed protocols include homogenization of RPE cells followed by a series of centrifugations and ultracentrifugations, imposing a risk that the lipofuscin fraction may become contaminated by other organelles during the isolation procedure, while individual lipofuscin granules may be contaminated by adhering molecules. To remove superficial molecules from lipofuscin, Gugiu et al. employed incubations with sodium dodecyl sulfate (SDS) and proteinase K [30]. Interestingly, this treatment resulted in complete removal of peptides from the lipofuscin granule while preserving its granular appearance under transmission electron microscopy [30].

Proteins present in lipofuscin exhibit many oxidative modifications, such as oxidation of methionine residues to methionine sulfoxides and sulfones or adducts with products of lipid peroxidation (malondialdehyde, 4-hydroxynonenal, carboxyethylpyrrole [CEP]) or with advanced glycation end products [29, 30, 34]. In particular, lipofuscin proteins exhibit extensive damage as detected by the presence of abundant carbonyl groups, which may impede protein identification [29].

Lipofuscin contains a number of fluorophores, one of which was identified as 2-(2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,4E,7E-octatetrae- nyl)-1-(2-hydroxylethyl)-4-(4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E- hexatrienyl)-pyridinium [32, 35]. As the compound can be derived from two molecules of all-trans retinal (ATR; vitamin A aldehydes) and a molecule of ethanolamine, this pyridynium bisretinoid was called A2E. Other A2E isomers were also identified in the human RPE. A2E accounts for 7.8×10−20 mol per lipofuscin granule, which corresponds to 0.019–0.024% of dry mass of the lipofuscin [33, 36].

Another conjugate of two ATR molecules with PE, called ATR dimer–PE conjugate, has been detected in the human RPE extracts [37]. Chromophores of lipofuscin may potentially include ATR–lysine adducts, but their presence in the retina or lipofuscin has not been reported to date [38]. Such adducts with fluorescent properties were generated

in vitro as a result of 3-day incubation of bovine POSs in the presence of ATR in fourfold and greater molar excess over rhodopsin.

A2E and ATR dimer–PE conjugate are, like their precursor ATR, susceptible to oxidation and photodegradation [37, 39–42]. Several degradation products of A2E have been identified in the human RPE, including monoand polyperoxy-A2E, and furanA2E derivatives and many carbonyl derivatives of A2E [41–44].

Fluorescence Properties of RPE Lipofuscin

A characteristic feature of RPE lipofuscin is its golden-yellow fluorescence. Excitation of lipofuscin granules with 364 nm gives an emission with a broad maximum at 600 nm that exhibits multiexponential decay [45–47]. Different lifetimes of fluorescence indicate different environments of the fluorophore or several different fluorophores involved. Fluorescence properties of lipofuscin undergo age-related changes, suggesting that the composition of lipofuscin chromophores or the environment of fluorophores within lipofuscin granules changes with age.

Most studies of lipofuscin fluorophores were performed on chloroform-soluble extracts from lipofuscin or whole RPE [31, 32, 48]. At least ten fluorophores of different excitation and emission maxima were detected in these chloroform-soluble extracts from the RPE. Two were assigned as retinol and retinyl palmitate, emitting green light with a maximum at 520 nm on excitation with UV light centered at 330 nm. These two retinoids are normally present in the RPE cells independently of lipofuscin. Nevertheless, retinyl palmitate was identified as a component of lipofuscin [49]. Other fluorophores include a fluorophore emitting broadband yellow light (540–640 nm), at least three fluorophores emitting yellow-green light, and three fluorophores emitting orange-red light. One of the orange fluorophores was identified as A2E and was claimed to be “the major orangeemitting fluorophore” of lipofuscin [32, 35].

Interestingly, the quantum yield of lipofuscin fluorescence in solution is about 70 times greater than for A2E [48]. Fluorescence lifetimes of lipofuscin in solution exhibit at least four components of 60-ps, 320-ps, 1.2-ns, and 4.8-ns lifetimes [48], while the A2E fluorescence lifetime was determined to be only 12 ps [50]. These discrepancies were explained by Haralampus-Grynaviski and colleagues, who demonstrated that A2E can act as an energy acceptor from different blue-light-absorbing chromophores within lipofuscin granules. So indeed, the long-wavelength emission (>580nm) of the lipofuscin granule is due to A2E fluorescence, but it results mainly from energy transfer from other blue-light-absorbing chromophores of lipofuscin and not via direct photoexcitation of A2E [51]. Also, fundus fluorescence in vivo exhibits remarkable similarities to the fluorescence of A2E [52].

It needs to be stressed, however, that lipofuscins accumulated in vivo exhibit a substantial variability in fluorescence characteristics between different granules [51]. There is also a remarkable heterogeneity of fluorescence among different RPE cells within the retina [53].

Fluorescence of lipofuscin observed during fundoscopic examination serves as a diagnostic tool in several retinal degenerations as well as for quantification of blue- light-absorbing macular pigment in the neural part of the retina [52, 54]. Therefore, it is important to elucidate the fluorescence properties of lipofuscin in vivo, including their changes during normal aging and retinal degenerations.