Studying melanin and lipofuscin in RPE cell culture models.

The retinal pigment epithelium contains three major types of pigment granules; melanosomes, lipofuscin and melanolipofuscin. Melanosomes in the retinal pigment epithelium (RPE) are formed during embryogenesis and mature during early postnatal life while lipofuscin and melanolipofuscin granules accumulate as a function of age. The difficulty in studying the formation and consequences of melanosomes and lipofuscin granules in RPE cell culture is compounded by the fact that these pigment granules do not normally occur in established RPE cell lines and pigment granules are rapidly lost in adult human primary culture. This review will consider options available for overcoming these limitations and permitting the study of melanosomes and lipofuscin in cell culture and will briefly evaluate the advantages and disadvantages of the different protocols.

Synthesis of antioxidants for prevention of age-related macular degeneration.

Photooxidation of A2E may be involved in diseases of the macula, and antioxidants could serve as therapeutic agents for these diseases. Inhibitors of A2E photooxidation were prepared by Mannich reaction of the antioxidant quercetin. These compounds contain water-solubilizing amine groups, and several were more potent inhibitors of A2E photooxidation than quercetin.

Indirect antioxidant protection against photooxidative processes initiated in retinal pigment epithelial cells by a lipofuscin pigment.

Oxidative mechanisms are considered to contribute to the aging changes in retinal pigment epithelial (RPE) cells that underlie the pathogenesis of age-related macular degeneration. An important source of oxidative damage is likely to be the photoreactive pigments that progressively accumulate and constitute the lipofuscin of retinal pigment epithelial cells. Evidence for a link between RPE lipofuscin and cellular dysfunction is also provided by the understanding of disease progression in Stargardt disease. Using a culture model previously used to demonstrate photooxidative damage to retinal pigment epithelial cells that have accumulated the lipofuscin fluorophore A2E, it was shown that the propensity for cell death is increased under conditions that deplete cellular levels of glutathione. Additionally, sulforaphane, a phytochemical and inducer of phase 2 enzymes, protected RPE cells that accumulated A2E and were irradiated at 430 nm. The protection afforded by sulforaphane was paralleled by elevated levels of glutathione and increases in the activities of the phase 2 enzymes NAD(P)H:quinone reductase and glutathione-S-transferases. Moreover, transcriptional induction of NAD(P)H:quinone reductase was indicated by the increases in mRNA determined by real time RT-PCR. There has been considerable interest in the intake of carotenoids and antioxidant vitamins and the related incidence of age-related macular degeneration. The present results indicate that the indirect antioxidant activity of plant-derived phase 2 inducers also may be potentially important.

Aggregates of oxidized proteins (lipofuscin) induce apoptosis through proteasome inhibition and dysregulation of proapoptotic proteins.

Cellular senescence may be accompanied by accumulation of large aggregates of oxidized proteins, also known as lipofuscin. The hypothesis that cellular accumulation of lipofuscin-like materials (LIP) results in cell death as a result of proteasome inhibition was examined. Rat neonatal cardiomyocytes were incubated with synthetic LIP for up to 48 h. This was accompanied by increases in cellular autofluorescence (207% by 48 h; p < 0.05) and electron microscopic evidence of internalization of LIP particles. LIP incubation resulted in loss of viability (-46% by 48 h; p < 0.05) through apoptotic cell death. Although 20S-proteasome activity was increased by 74% after 6 h, both 20S- and 26S-proteasome activities were decreased after 48 h of incubation (-54% (p < 0.05) and -50%, respectively), accompanied by large increases in ubiquitinated proteins. Several proteasome-regulated proapoptotic proteins, including c-Jun (2.9-fold; p < 0.05), Bax (1.8-fold; p < 0.05), and p27(kip1) (3.2-fold; p < 0.05), were observed to be increased by 48 h. Observation of ubiquitinated homologues of Bax and p27(kip1) suggested that part of the increase was due to decreased proteasomal degradation of these proteins. The results of this study are consistent with the conclusion that accumulation of LIP results in inhibition of the proteasome, which initiates an apoptotic cascade as a result of dysregulation of several proapoptotic proteins.

Melano-macrophage centres and their role in fish pathology.

Melano-macrophage centres, also known as macrophage aggregates, are distinctive groupings of pigment-containing cells within the tissues of heterothermic vertebrates. In fish they are normally located in the stroma of the haemopoietic tissue of the spleen and the kidney, although in amphibians and reptiles, and some fish, they are also found in the liver. They may also develop in association with chronic inflammatory lesions elsewhere in the body and during ovarian atresia. In higher teleosts, they often exist as complex discrete centres, containing lymphocytes and macrophages, and may be primitive analogues of the germinal centres of lymph nodes. Melano-macrophage centres usually contain a variety of pigments, including melanins, and these increase in range and volume in older fish or in the presence of cachectic disease. Melano-macrophage centres act as focal depositories for resistant intracellular bacteria, from which chronic infections may develop. Iron capture and storage in haemolytic diseases appears to be a primary function, but antigen trapping and presentation to lymphocytes, sequestration of products of cellular degradation and potentially toxic tissue materials, such as melanins, free radicals and catabolic breakdown products are among other functions that have been ascribed. Recent work suggests that they are a site of primary melanogenesis rather than mere storage. Melano-macrophage centres increase in size or frequency in conditions of environmental stress and have been suggested as reliable biomarkers for water quality in terms of both deoxygenation and iatragenic chemical pollution.

No reduction of energy metabolism in Clk mutants.

Mutation in any of the four clock genes (clk-1, clk-2, clk-3, gro-1) causes an average slowing down of many temporal processes, and an increase of mean life span. The latter effect has been linked to the slow phenotype, and it has been reasoned that any reduction of the rate of living would reduce the load of oxidative damage, which is thought to drive the ageing process. To test this model we measured several parameters describing metabolic output in wild type worms and all four Clk mutants. We found no gross changes in metabolic output, as assessed from oxygen consumption and heat production rates, lucigenin-mediated light production capacity, ATP content, and lipofuscin autofluorescence. Catalase and superoxide dismutase (SOD) were variably altered, but not cooperatively, as would be expected to enhance reactive oxygen species (ROS) scavenging activity. Thus we conclude that the prolonged life span of Clk mutants cannot be attributed to reduced metabolic rate or an increased activity of the major antioxidant enzymes catalase and SOD.

Oxidative damage and age-related macular degeneration.

This article provides current information on the potential role of oxidation in relation to age-related macular degeneration (AMD). The emphasis is placed on the generation of oxidants and free radicals and the protective effects of antioxidants in the outer retina, with specific emphasis on the photoreceptor cells, the retinal pigment epithelium and the choriocapillaris. The starting points include a discussion and a definition of what radicals are, their endogenous sources, how they react, and what damage they may cause. The photoreceptor/pigment epithelium complex is exposed to sunlight, is bathed in a near-arterial level of oxygen, and membranes in this complex contain high concentrations of polyunsaturated fatty acids, all considered to be potential factors leading to oxidative damage. Actions of antioxidants such as glutathione, vitamin C, superoxide dismutase, catalase, vitamin E and the carotenoids are discussed in terms of their mechanisms of preventing oxidative damage. The phototoxicity of lipofuscin, a group of complex autofluorescent lipid/protein aggregates that accumulate in the retinal pigment epithelium, is described and evidence is presented suggesting that intracellular lipofuscin is toxic to these cells, thus supporting a role for lipofuscin in aging and AMD. The theory that AMD is primarily due to a photosensitizing injury to the choriocapillaris is evaluated. Results are presented showing that when protoporphyric mice are exposed to blue light there is an induction in the synthesis of Type IV collagen synthesis by the choriocapillary endothelium, which leads to a thickened Bruch's membrane and to the appearance of sub-retinal pigment epithelial fibrillogranular deposits, which are similar to basal laminar deposits. The hypothesis that AMD may result from oxidative injury to the retinal pigment epithelium is further evaluated in experiments designed to test the protective effects of glutathione in preventing damage to cultured human pigment epithelial cells exposed to an oxidant. Experiments designed to increase the concentration of glutathione in pigment epithelial cells using dimethylfumarate, a monofunctional inducer, are described in relation to the ability of these cells to survive an oxidative challenge. While all these models provide undisputed evidence of oxidative damage to the retinal pigment epithelium and the choriocapillaris that is both light- and oxygen-dependent, it nevertheless is still unclear at this time what the precise linkage is between oxidation-induced events and the onset and progression of AMD.

Extra-cutaneous melanin.

Extra-cutaneous melanocytes derive from either the neural crest, the outer wall of the optic cup, or the cranial neural tube. Those of neural crest origin reach most bodily regions, and may give rise to primary melanoma in various tissues. The Kupffer cell produces a form of melanin, but is hardly a melanocyte. Melanocytes of the internal ear may be concerned with secretion of endolymph, trans-epithelial ion transport, and with protection against ototoxic drugs and high-intensity noise damage. There is evidence from albino animals that retinal pigment epithelium determines co-ordinates of the neural retina, and its decussation pattern during development. Neuromelanin derives from Dopamine, and is found in dopaminergic neurons widely distributed throughout the brain-stem and hypothalamus, and which project to the striatum and limbic system. Parkinsonism is due to degeneration of melanin-containing dopaminergic neurons of locus coeruleus and substantia nigra, and MPTP provides an investigative probe for studying the causes of Parkinsonism. Neuromelanin should not be regarded as a waste-product, but as something which can affect the firing properties of neurons with specific functional effect.