Janet R. Sparrow, PhD

Work in the laboratory is aimed at examining a causal link between the intracellular accumulation of lipofuscin fluorophores and retinal pigmented epithelial (RPE) cell death. We have shown that a major fluorophore of RPE lipofuscin, A2E, confers a susceptibilty to blue light-mediated cell death and can lead to a detergent-like perturbation of membranes. We are also examining mechanisms involved in A2E biosynthesis and conditions under which the formation of A2E may be accelerated. The death of RPE cells in several retinal disorders including Stargardt's disease and atrophic age-related macular degeneration (AMD), precedes the degeneration of photoreceptor cells and the impairment of vision. A major focus of work in the laboratory involves studies of a causal link between the intracellular accumulation of aging pigments (lipofuscin) in the RPE and the death of those cells. A major hydrophobic constituent of RPE lipofuscin is the fluorophore A2E, a bis-retinoid whose biosynthesis begins randomly in photoreceptor cell outer segments. A2E is generated from the precursor A2-PE after phosphate hydrolysis, it is deposited in RPE cells secondary to phagocytosis of shed outer segment membrane and it accumulates as lipofuscin because it cannot be degraded by RPE lysosomal enzymes. Light is also involved in mechanisms that lead to the atrophy of A2E-laden RPE. Thus, accumulation of A2E in the lysosomal compartment of the cultured RPE confers a susceptibilty to blue light-mediated cell death; the wavelength dependency of this effect is consistent with the excitation spectra of A2E and with the known susceptibility of RPE cells to blue light damage in animal models. The cell death program which is initiated by blue light illumination of A2E involves the activation of caspase-3 a downstream cysteine-dependent protease and is regulated by Bcl-2 an anti-apoptotic protein situated in the outer mitochondrial membrane. Evidence indicates that the generation of singlet oxygen upon photoexcitation of A2E is integral to the death of the cells. In particular singlet oxygen which is generated by photosensitization of A2E becomes inserted into carbon-carbon double bonds of the retinoid-side arms of A2E to generate highly reactive epoxides. Since the considerable ring strain and electrophilicity of these three-membered oxygen and carbon containing rings makes them susceptible to reaction with nucleophilic macromolecules such as DNA and protein, A2E-epoxides may be agents that ravage the cell. Indeed, we have shown that at least one of the subcellular structures damaged by A2E-epoxides, is DNA with guanine bases of DNA being oxidatively modified to generate 8-oxo-dG and perhaps other structurally related lesions. In other experiments, A2E-epoxides have been shown to generate products of lipid peroxidation in RPE cells. The mediation of light damage may not be the only mechanism by which A2E induces cell injury, since A2E can also mediate a detergent-like perturbation of cell membranes. The structure of A2E, both its possession of hydrophobic and hydrophilic domains and its wedge-shaped configuration, is central to this property. These studies are relevant to the non-neovascular, atrophic form of age-related macular degeneration that accounts for up to 21% of the visual loss associated with AMD and that is characterized by a massive accumulation of lipofuscin preceding RPE cell death. These observations also revive the unresolved issue of whether lifelong exposure to bright light contributes to AMD.

Email: jrs88@cumc.columbia.edu