Sparrow Lab

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 susceptibility 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 susceptibility 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.

Vitamin A aldehyde-conjugates accumulate as lipofuscin in retinal pigment epithelial (RPE) cells and have been linked to disease processes in some inherited forms of macular degeneration as well as age-related macular degeneration. These bisretinoid constituents of lipofuscin are unique to RPE and in addition to A2E include all-trans-retinal dimer and its conjugates, phosphatidyl-dihydropyridine bisretinoid (A2-DHP-PE) and a conjugate of all-trans-retinal and glycerophosphoethanolamine (A2-GPE). The excitation and emission spectra of these compounds can also account for the inherent autofluorescence of the retina (fundus autofluorescence).

Dr. Sparrow’s laboratory has shown that the adverse effects of RPE lipofuscin pigments are attributable, at least in part, to their detergent-like structure and their photo-sensitive properties. In the latter case, bisretinoids can generate reactive forms of oxygen; they also undergo photooxidation and photodegradation. The photo-cleavage products of A2E consists of a complex mixture of aldehyde-bearing fragments that includes the small dicarbonyls methylglyoxal and glyoxal that are responsible for damaging modifications of proteins – advanced glycation end-products (AGEs). AGE-modified proteins are present in drusen. We are applying our understanding of RPE bisretinoids to clinical interpretations and measurements of fundus autofluorescence. Taken together work in the laboratory contributes to the elucidation of pathology in several retinal disorders including recessive Stargardt disease, retinitis pigmentosa, pattern dystrophies and age-related macular degeneration.

Therapeutic strategies her laboratory investigates to target bisretinoids include antioxidants, inhibitors of complement activation, small molecules that inhibit their formation and gene-based therapy.