The Photoreactivity of the Retinal Age Pigment Lipofuscin

Abstract

The presence of the age pigment lipofuscin is associated with numerous age-related diseases. In the retina lipofuscin is located within the pigment epithelium where it is exposed to high oxygen and visible light, a prime environment for the generation of reactive oxygen species. Although we, and others, have demonstrated that retinal lipofuscin is a photoinducible generator of reactive oxygen species it is unclear how this may translate into cell damage. The position of lipofuscin within the lysosome infers that irradiated lipofuscin is liable to cause oxidative damage to either the lysosomal membrane or the lysosomal enzymes. We have found that illumination of lipofuscin with visible light is capable of extragranular lipid peroxidation, enzyme inactivation, and protein oxidation. These effects, which were pH-dependent, were significantly reduced by the addition of the antioxidants, superoxide dismutase and 1,4-diazabicyclo(2,2,2)-octane, confirming a role for both the superoxide anion and singlet oxygen. We postulate that lipofuscin may compromise retinal cell function by causing loss of lysosomal integrity and that this may be a major contributory factor to the pathology associated with retinal light damage and diseases such as age-related macular degeneration.The age pigment lipofuscin accumulates within the lysosomal system of a variety of postmitotic cells throughout life and is considered to be a biomarker of cell aging. Evidence has shown that the rate of lipofuscin accumulation corresponds to the aging rates in different species, being influenced by both metabolic activity and extent of oxidative stress (1). However, a causal role for lipofuscin in the aging process or development of age-related diseases such as neuronal ceroid lipofuscinosis, age-related macular degeneration, Ménière's disease, and cardiac hypertrophy has yet to be established (see Refs. 2 and 3). Unlike other cells in the body, in which lipofuscin occurs through the autophagic breakdown of intracellular organelles (2), the major substrate for lipofuscin in the retinal pigment epithelium (RPE) 1 of the eye is the undegradable end product resulting from the phagocytosis of photoreceptor outer segments (4, 5) that are rich in polyunsaturated fatty acids and vitamin A.Ocular lipofuscin may have a unique role to play in aging of the RPE, a tissue that is continually exposed to visible light (400 -700 nm) and high oxygen tensions (ϳ70 mm Hg). Studies have shown this type of lipofuscin to be a photoinducible generator of superoxide ions, singlet oxygen, hydrogen peroxide, and lipid peroxides (6 -9), all of which are reactive oxygen species implicated in general aging processes. These species can adversely affect cell function by damaging proteins, carbohydrates, DNA, and lipids (10). The position of lipofuscin within the lysosome infers that the first site of oxidative damage will be either the lysosomal membrane or the lysosomal enzymes. To test this hypothesis we have assessed the effect of photoactivated lipofuscin on (i) lipid peroxidat...

Retinal Pigment Epithelium Lipofuscin Proteomics

“…Purification of Lipofuscin Granules-RPE lipofuscin granules are usually isolated by sucrose density gradient centrifugation (26) and typically contain significant amounts of extragranular debris as shown (Fig. 1, A, C, E, F, I, and K) by light, confocal, and transmission electron microscopy.…”

“…RPE cells were isolated from human eyecups by gentle brushing in PBS and stored at Ϫ80°C. Lipofuscin was isolated from RPE lysates using high speed ultracentrifugation in a discontinuous sucrose density gradient (2.0 -0.3 M) as described previously (26) and designated "crude" lipofuscin. Three crude lipofuscin preparations were analyzed: preparation 1, from 65 donors (130 eyes), mean age 70 years; preparation 2, from 30 donors (60 eyes), mean age 65 years; and preparation 3, from 41 donors (82 eyes), mean age 76 years.…”

Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells

“…RPE cells may therefore be an important source of ROS in the eye. Indeed, focused light (Dorey et al, 1990), high oxygen tension in the macula (Alder and Cringle, 1985), and photoreceptor outer segment phagocytosis and degradation (Tate et al, 1995b;Higgins et al, 2003) all promote RPE oxidative stress (Tso, 1987;Gottsch et al, 1990;Cai et al, 1999;Wassell et al, 1999;Higgins et al, 2003Kindzelskii et al, 2004. Godley et al (2005) showed that ROS levels were strikingly higher in macular RPE compared to peripheral RPE.…”

The Role of Oxidative Stress in the Pathogenesis of Age-Related Macular Degeneration

“…180 Further study has revealed that the lipofuscinmediated generation of ROIs in response to irradiation is wavelength-dependent, being greatest for the blue region of the visible spectrum. 181 This photoinducible free radical generation by lipofuscin has been shown to result in lipid peroxidation and enzyme inactivation, 216 as well as RPE cellular dysfunction. 47 A2-E is a lysosomotropic agent that has the capacity to compromise both lysosomal function and integrity.…”