Using transgenic mouse models that have been developed for humans with mutations in the rds gene, the applicant and his collaborators will attempt to determine some of the molecular mechanisms underlying photoreceptor degeneration. These mice produce defective rds/peripherin, an outer segment intrinsic membrane protein, which alters photoreceptor morphology and function. The applicant will also test the efficacy of growth factor therapy in humans by applying it to these transgenic mice which, due to a null mutation in their rds gene, make no mutant rds/peripherin of their own. Finally, the applicant will attempt to rescue the normal photoreceptor phenotype in rds mice by adenoviral delivery via the subretinal space of a proven rds minigene that has produced rescue via the transgenic approach. The applicant will model and study molecular mechanisms underlying Sorsby's fundus dystrophy (SFD) by introducing a point-mutated minigene coding for tissue inhibitor of metalloproteinase-3 (TIMP-3) into cultured human RPE. The extracellular matrix phenotype and function of mutated TIMP-3 will be studied under these conditions. Additionally, the polarized expression of major histocompatibility class II (MHC II) antigens will be studied in cultured human RPE by metabolic labeling of MHC II and domain-selective biotinylation and recovery of apical and basolateral MHC II molecules. Finally, using a kidney cell line (MDCK), the applicant will study the polarized delivery of Na+/K+-ATPase subunit isoforms unique to the RPE to determine whether targeting signals are intrinsic to these subunits. These projects probe the cell and molecular biology of photoreceptors and RPE in health and disease. All are targeted toward an understanding of mechanisms that cause malfunction and blinding disease.
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