This is our first year at NIH and we estimate that 2/3 of our time has been spent physically moving and setting up our laboratory. Our main research interst is genetic retinal cell degeneration which is a major cause of blindness. We are studying animal models with inherited and induced retinal photoreceptor degeneration to learn about the causes and ways to prevent retinal cell death in these diseases. One of our goals during our first year at NIH was to establish certain critical animal models of retinal degeneration: RCS rat, P23H rhodopsin transgenic rat, light damaged rat, rhodopsin knockout mouse, G90D rhodopsin transgenic mouse. Breeding pairs of RCS rat and P23H rats were shipped to NIH from our laboratory at the University of Michigan and bred in quarantine. Litters were transferred to dim light rooms for housing since light can affect the time course and severity of retinal degeneration. We previously created a transgenic mouse with the G90D rhodopsin mutation which causes night blindness without degeneration in patients. Like patients this mouse shows suppression of signal transduction consistent with constitutive activity of rhodopsin. We completed an analysis of wild type and mutant rhodopsin RNA levels in these mice and are now looking at protein expression to find out whether rhodopsin activity and/or mutant protein levels affect wild type expression. We have discovered that intraocular injection of the small heat shock protein HSP25 protects photoreceptors in the RCS rat model of inherited retinal degeneration and also from the damaging effects of constant light exposure. We are currently investigation the mechanism of this protection by two approaches: One is to localize the site of action in the retina and the other is to identify the subcellular and molecular effects of drug application in rodent models of retinal degeneration. We are anatomically tracking the movement of biotinylated protein through the retina and are quantifying the RNA levels by RT-PCR of certain neurotrophic and cell cycle proteins with and without exogenous HSP25. In one animal model we have correlated GFAP immunoreactivity in retinal glial cells using confocal microscopy with electrophysiological response changes in progressive retinal degeneration. The retina pigment epithelium (RPE) is main cellular compartment in which the metabolism and recycling of the vitamin A and related retinoids occurs which is necessary for the production of the visual pigment rhodopsin. This metabolically demanding process, called the visual cycle, involves the production of reactive compounds which could potentially produce retinal damage. We have discovered that a commonly used clinical compound, 13-cis-retinoic acid, slows this recycling by competitive inhibition with the one or more of the RPE enzymes, and protects the retina from damage by light. It also offers us a convenient tool for examining the production of various retinoids under normal conditions of rhodopsin bleach in wild type mice and mice with mutations that affect the visual cycle. Thus far, we have quantified the effect of various doses of this compound on electroretinographic responses in mice subjected to bleach produced by strong illumination. We are engaged in a colloaboritive study exploring the therapeutic potential of this compound in the ABCA4 knockout mouse model of human Stargardt juvenile macular degeneration. Juvenile X-linked retinoschisis is a Mendelian genetic retinal degeneration that causes vision loss from formation of peri-macular cysts and, in the peripheral retina, splitting through the nerve fiber layer. The RS1 gene was identified in 1998 and elaborates a 224 amino acid protein that is expressed in the photoreceptor cells; the protein appears to be secreted and localizes in several cell types of the inner retina. We created a knockout construct by deleting exon 5 and substituting a neomycin cassette. Three ES cell clones were isolated and chimeras have now been generated. We currently are breeding the F1 founder mice these to determine whether the construct is incorporated in the germ line. The resulting progeny will then be characterized, toward our next goal of exploring therapy by protein replacement.
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