Human blinding disorders are often initiated by hereditary mutations that impact rod and/or cone photoreceptors (PRs) and cause subsequent cellular death. Generally, the disease phenotype can be predicted from the specific mutation since many PR genes are specific to rods or cones; however certain genes, such as Retinal Degeneration Slow (Rds), are expressed in both cell types and cause different forms of retinal disease affecting rods, cones, or both. Rds is a member of the tetraspanin family known to interact with other proteins to build a membrane domain responsible for the morphogenesis of the rod and cone PR disc rim. Without this protein the outer segments (OSs) fail to develop, and over 80 different Rds mutations have been shown to cause several inherited retinal diseases. The goals of this program are: 1) to uncover the mechanisms underlying the differential behavior of Rds in rods and cones; 2) to identify Rds' interacting partners from both PRs types; and 3) to determine how Rds complexes traffic to OSs and assemble the disc rim. Our ultimate goal is to determine how the structure of Rds, Rds/Rom-1 complexes, and Rds interactions with secondary unknown binding partners affect the development of the OS and the formation of the rim microdomain. Using biochemical, structural and functional approaches along with different genetically modified mouse models, we have shown that Rds functions differently in rods vs. cones, Rds inter-molecular disulfide oligomerization occurs in the OS, and that Rds traffics in the inner segment as homo- and hetero-tetramers with its partner Rom-1. In this application, we will test the hypothesis that the differential roles of Rds in rods and cones are a result of the presence of distinct Rds-associated partners in the rod OS vs the cone OS. Four aims are proposed to test our hypotheses. Aim 1 will identify Rds/Rom-1 associated partners involved in Rds complex trafficking and OS assembly in rods & cones. In this aim, we will use proteomic studies on affinity purified Rds complexes from various retinal samples isolated from different genetically modified models to identify and study interacting proteins. Aim 2 will determine the functional role of the Rds C-terminal region in rod and cone OS morphogenesis. Transgenic mice expressing a chimeric protein made of the body of Rom-1 but with the Rds C-terminal region have been generated and will be evaluated structurally, functionally, and biochemically. Aim 3 will evaluate the role of Rds in cone OS lamellae formation. We have shown that in the absence of Rds, rods do not form OSs and enter apoptosis, whereas cone PRs develop viable but dysmorphic OS structures devoid of lamellae that are able to photo-transduce. Aim 4 will study the differential role of certain Rds residues in rod vs. cone OSs morphogenesis and maintenance. Experiments put forth in this application will provide biochemical and physiological evidence for the role of Rds and its associated proteins during genesis of rod and cone OSs. PUBLIC HEALTH RELEVANCE: This proposal is designed to further our understanding of the role of Rds in outer segment morphogenesis and maintenance. Mutations in the Rds gene lead to a variety of debilitating retinal disorders. Our studies will extend our fundamental knowledge of Rds and other molecules involved in normal disc membrane morphogenesis and turnover processes, both steps are essential for normal vision but poorly understood. Also, our work will provide a basis for understanding how different mutations in the Rds gene can result in diverse retinal diseases. We will employ different genetically modified mouse models to test our main hypothesis that in rods, Rds plays an important role in rod rim outer segment morphogenesis and closure of the disc rim while in cones, Rds is only necessary for the evagination step of the lamellae formation. We also hypothesize that the differential behavior of Rds in rods vs. cones is a function of differences in Rds-associated protein partners and in this application we propose to identify these partners and test their functional role in normal and diseased retinas.
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