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Lipofuscin and Melanin in Aging Cells
Posted on: March 21, 2006

Early histologists observed that human retinal pigment epithelium (RPE) contained two kinds of pigment granules, melanin and lipoidal granules. However, it was not until 1961 that a histochemical study by Streeten established that the lipoidal granules were similar to lipofuscin, the "age pigment" or "wear-and-tear pigment" of brain and other organs. She also noted that certain staining features of these RPE lipoidal granules were common to rod outer segments (ROS).

Lipofuscin granules of the brain were found by Essner and Novikoff to be part of the lysosomal system, specifically, that they were the residual bodies of lysosomes. In 1965 the infrastructure of lipofuscin granules in the RPE as well as other bodies resembling portions of ROS in the apical cytoplasm of the RPE was described. Using the knowledge accrued to that time, it was postulated that broken or detached portions of rod and cone outer segments were engulfed by the pigment epithelial cell and that during the life of the retina, which equals the life of the individual, fragments of outer segment were partially broken down by enzymatic digestion, leaving residues to accumulate as lipofuscin granules. Subsequently the elegant autoradiographic studies of Young demonstrated that the photoreceptor outer segments are continually renewed; later, Young and Bok showed that detached ROS terminal fragments were phagocytized by the RPE. Enzyme cytochemical studies by Ishikawa and Yamada demonstrated that the phagosomes, composed of stacks of outer segment disks, contained acid phosphatase, indicating that they had been converted to phagolysosomes. Moreover, they showed that some lipofuscin granules in human RPE also had acid phosphatase activity. These findings established a biological paradigm which has guided research on the RPE during this decade: outer segment disks that are detached from the photoreceptor are phagocytized by the RPE and digested via the lysosomal system; for some unknown reason this process gives rise to lipofuscin granules in human RPE cells. Lipofuscin pigment has been described as intracellular yellow-brown refractile granules exhibiting sudanophilic, osmiophilic, argyrophilic, and periodic acid-Schiff-positive and acid-fast staining characteristics. Enzyme cytochemistry has revealed the presence of hydrolytic (i.e., lysosomal) enzymes, and the reduction of diaminobenzidine by lipofuscin granules has been interpreted as evidence of peroxidase activity. Ultrastructurally, the granules are membrane bound, but their internal structure as well as their chemical composition vary with cell type and from one granule to another in a given cell. Such heterogeneity is not surprising, since the granules represent terminal stages of lysosomal digestion of various products the cell phagocytizes or autophagocytizes. Complex lipids, however, are the most characteristic components of lipofuscin. Extracts of lipofuscin granules fluoresce at 430 to 470 nm when irradiated at 360 to 365 nm, reportedly due to the presence of Schiff-base fluorophore formed during peroxidation and polymerization of unsaturated lipids. Melanin, an insoluble high-molecular weight polymer derived from the enzymatic oxidation of tyrosine and dihydroxyphenylalanine, is contained in membrane-limited granules in the RPE. RPE melanin differs from that in melanocytes in its origin from neural ectoderm rather than neural crest and in its apparent static condition during adult life. Although the melanosomes of uveal and dermal melanocytes show ultrastructural and enzymatic properties suggestive of continual synthesis during life. RPE melanin granules are synthesized in utero, achieve maturity before the first decade of life and thereafter are thought to be unchanging structures in the cytoplasm of these essentially nondividing cells. The RPE of old human eyes usually contains complex granules composed of both melanin and lipofuscin, suggesting that these two components of RPE cytoplasm may have interrelated life histories. The present study was undertaken to examine the life history and composition of lipofuscin and melanin in human RPE cells. The characteristic fluorescence of lipofuscin was used to demonstrate these granules in wet preparations of unfixed RPE cells. The identical cells were subsequently fixed and examined ultrastructurally, before and after extraction of lipids, for the precise identification of the fluorescent components. These studies, carried out on eyes spanning nine decades of life, reveal differences in the structure and content of melanin and lipofuscin between childhood and old age. Enzyme cytochemical techniques for demonstration of acid hydrolases reveal a number of complex interrelationships among the lysosomes and lipofuscin and melanin granules of the RPE cytoplasm. These findings indicate that these subcellular organelles are in a dynamic state and capable of considerable rearrangement and alteration during the life of the RPE cell.

Fig. 1. Formation of melanin (above) and the postulated dissolution of melanin (below). Primary lysosomes fuse with fully melanized granule, forming a melanolysosome. As dissolution begins, lipofuscin granules also fuse, forming a melanolipofuscin granule with a fluorescent rim.

Fig. 2. Sequence of formation of fluorescent granules in human RPE.
1, Phagocytized outer segment disks; fluorescent properties unknown.
2, Phagolysosome or large secondary lysosome; fluorescence seems to begin here.
3a to 3c, Sequential diminution in size of fluorescent secondary lysosomes in young eyes.
4, Sequence in old eyes (all fluorescent): 4a, fusion of phagolysosomes with pre-existing lipofuscin; 4b, fusion of primary lysosomes with lipofuscin; 4c, fusion of multiple lipofuscin granules and continued fusion of primary lysosomes.

Source: Lynette Feeney; Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies; Investigative Ophthalmology & Visual Science A Journal of Clinical and Basic Research July 1978 Vol. 17/7.
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