The broad, long-term objectives of the proposed research are to elucidate the function of the RP1 protein in vision, and identify the links between mutations in RP1 and photoreceptor cell death so that rational strategies to prevent vision loss can be devised. Research by the applicant, and other investigators, in the past 5 years has shown that mutations in RP1 are a common cause of retinitis pigmentosa (RP). The RP1 protein is part of the axoneme of rod and cone photoreceptor cells. Data from mice with targeted disruption of the Rp1 gene show that RP1 is required for correct stacking of outer segment discs. The interaction between RP1 and microtubules of the axoneme is mediated by the doublecortin domains in the N-terminus of RP1. The N-terminal portion of RP1 can stimulate tubulin polymerization into microtubules and stabilize microtubules, indicating that RP1 is a microtubule-associated protein (MAP). ? ? Specific Aim 1 of the proposed research is to investigate the MAP function of the RP1 protein, and test the hypothesis that RP1 serves to link newly formed outer segment discs to the axoneme, thus aligning discs into organized outer segments. This will be investigated by using yeast two-hybrid analysis and coimmunoprecipitation assays to identify the proteins that interact with RP1.
Specific Aim 2 will investigate the mechanism of RP1 disease, and test the hypothesis that lack of functional RP1 protein leads to photoreceptor dysfunction and death. This will be studied by producing Rp1-null and Rp1-knockin mice, and then characterizing the phenotype of the mice by electroretinography (ERG), histologic and ultrastructural analyses. Three-dimensional reconstruction of serial electron microscopic images will be used to provide more detailed insight into the defects in outer segment structure caused by mutations in RP1. The goal of Specific Aim 3 is to test the hypothesis that gene replacement therapy can be used to treat RP1 disease. This will be accomplished by preparing trans-splicing adeno-associated viruses to deliver wild-type Rp1 to the retina, and testing these in mice with mutant Rp1 alleles. It is hoped that the proposed investigations will lead to an improved understanding of how photoreceptor outer segments are formed, and ultimately to the development of rational therapies to prevent vision loss from RP1, and perhaps other forms of inherited blindness. ? ?
Showing the most recent 10 out of 41 publications
