Why photoreceptors die when expressing mutant proteins such as rhodopsin, and why cones should die in such instances when they do not express the mutated gene is still unclear. X. laevis frogs have abundant rods and cones in their retinas and develop a functioning retina within 14 days post-fertilization. We developed models of retinal degeneration in transgenic X. laevis. Adult transgenic frogs transmit the transgenes to offspring. We have generated more than 100 transgenic lines and published studies of 56 transgenic animals in 6 years.
We aim 1) to evaluate the localization, function and impact of mutations of prominin-1 on rods. Prominin-1 is a pentaspan membrane protein localized exclusively to basal disks in rods and mutations of it cause retinal degeneration in humans. We have isolated the cDNA of Xenopus prominin-1 and made GFP fusion constructs that localize appropriately. We have also prepared antibodies to GST-fusion peptides of prominin and will use the antibodies and the fusion peptides in pull-down assays to isolate prominin's binding partners. The protein will be evaluated to determine which domains account for its unusual localization and it will be mutated to evaluate the impact by comparison with human mutations. 2) A new model of retinal degeneration has been created by expression of mutant rab8T22N which induces apoptosis early in the development of central rods, blocks docking of post-Golgi membranes to the base of the cilium and leads to formation of a nearly all-cone retina after 14 d. We will pursue the binding partners of rab8 in order to determine its role in vesicle docking. 3) We are initiating new studies of moesin in photoreceptors because perturbation of its localization has been shown in propranolol treated retinas similar to the effects of rab8T22N. We will use moesin PERM and actin binding domain fragments to block its function by expression of the fragments in transgenic frogs to determine the impact, in vivo, of the competition with endogenous moesin. 4) We will investigate the potential amelioration of cone death by antiapoptotic agents in our models of retinal degeneration in which the rods die early. Since the cones survive even beyond metamorphosis in the absence of rods, but eventually die, we will seek cone-preserving agents that may provide potential therapies in human retinal degenerations.
| Han, Zhou; Anderson, David W; Papermaster, David S (2012) Prominin-1 localizes to the open rims of outer segment lamellae in Xenopus laevis rod and cone photoreceptors. Invest Ophthalmol Vis Sci 53:361-73 |
| Han, Zhou; Papermaster, David S (2011) Identification of three prominin homologs and characterization of their messenger RNA expression in Xenopus laevis tissues. Mol Vis 17:1381-96 |
