Includes bibliographical references (pages [167]-189).The retinal pigment epithelium (RPE) is interposed between the photoreceptor cells of the neural retina and the choriocapillaris, and lies on a bed of extracellular matrix called Bruch’s membrane. Dysfunction in one of these components can cause a cascade of events that results in RPE and photoreceptor cell death, which causes permanent vision loss. The most debilitating vision loss occurs in the macular region due to its high concentration of photoreceptor and RPE cells and its role in central vision. Mutations in the gene for the ATP-dependent binding cassette transport protein, ABCR, have been shown to result in a high accumulation of the autofluorescent pigment mixture, lipofuscin, within the RPE cells. The ABCR protein is located in the outer segments of the photoreceptor cells. Stargardt’s macular dystrophy (STDG), retinitis pigmentosa (RP) and cone/rod dystrophy (CRD) are associated with mutations in this gene. Additionally, mutations are implicated in some forms of age-related macular degeneration (AMD). Although AMD is a multi-factorial disease, the prolific accumulation of autofluorescent compounds in the RPE or in Bruch’s membrane is a positive indicator for the disease. Clearly, understanding the mechanisms of the ABCR protein function and lipofuscin-mediated damage to RPE cells are the foundation of vision loss by retinal maculopathies. The first part of the research presented in this dissertation describes the cloning, transfection and expression of the ABCR transporter protein that is often found defective in STGD patients and other retinal diseases. ABCR dysfunction leads to the prolific accumulation of lipofuscin in the RPE, which may in turn be related to the accumulation of age-related debris on Bruch’s membrane. Therefore, a RPE cell line that mimics the in vivo state was isolated and characterized, including melanin pigmentation, ECM and differentiation. These cells were then used to determine the phototoxicity of lipofuscin and the lipofuscin precursor, A2PE. Photooxidative damage to RPE-challenged A2PE was performed and the lipophilic extracts analyzed. From these studies, the mechanisms that damage RPE cells are addressed, which facilitate the progression of AMD, STGD and other retinal dystrophies.Ph.D. (Doctor of Philosophy
