Photoreceptor Degeneration Assay-Transgenic Xenopus(RMI)

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DESCRIPTION (provided by applicant): The long-term objective of these studies is to find therapies for the treatment of photoreceptor degenerations. Though most of our studies are aimed at understanding the mechanism of photoreceptor degeneration with the purpose of designing rational therapeutic interventions, in this study we propose to develop the technologies to directly screen for potential therapeutics using a frog animal model and a high throughput molecular screening (HTS) assay. We have developed lines of Xenopus laevis in which transgenes both cause rod photoreceptors to degenerate in a manner that resemble human retinitis pigmentosa as well as report the viability of photoreceptors externally and in live animals. In the line that will be used in this study, photoreceptors degenerate during the second week of life, during which period there is a change of fluorescence emitted through the lens of over an order of magnitude. Since adults of this line can produce over a thousand progeny in a single breeding, and the progeny can be raised and assayed in 96-well plates during the period of photoreceptor degeneration, this and similar lines offer great promise to serve as the substrates for HTS assays that aim to identify chemical compounds that prevent or slow photoreceptor degenerations. In this 1-year project, we propose to optimize the culturing of tadpoles in 96-well plates, optimize the imaging platform for fluorescence based imaging screens in cold-blooded vertebrate larvae, and perform a pilot screen. Besides having some potential to discover therapeutics and tools to study photoreceptor biology, this screen will serve as the prototype on which to build future HTS assays for neurodegenerative disorders based on fluorescent reporters using Xenopus or zebrafish embryos and larvae. These studies will take advantage of the chemical libraries and HTS infrastructure available at the Johns Hopkins ChemCORE facility. In return, we hope to be able to contribute tools and knowledge to advance future screens at the facility. The proposed studies have both direct and indirect relevance to promoting human health. The direct relevance is that we will be screening specifically for therapeutics that can be of benefit to individuals who suffer from retina degenerations. Though the frog animal model we will use most closely resembles retinitis pigmentosa, a disease which in of itself afflicts over 1 million people worldwide, we expect that both the chemicals and information that come out from our studies will have impact on a larger segment of those suffering from diseases of vision. The indirect relevance to human health is that we will be helping to bring HTS analyses to living vertebrate organisms, and any attempt to broaden the scope of HTS analyses will benefit human health in the long term.