We propose to test the hypothesis that dominant rhodopsin mutations cause photoreceptor cell death by disrupting the structural role of rhodopsin in the morphogenesis and maintenance of photoreceptor outer segments, rather than the catalytic role in phototransduction. We will organize our approach on the basis of in vitro biochemical studies of rhodopsin mutants, current hypotheses regarding their pathogenic mechanisms, and previous findings from transgenic animal studies by us and other investigators. We will test two specific subplots: l. Rhodopsin mutants with known defect in protein folding/stability in vitro localize to the outer segments in vivo where they cause failure of outer segment integrity or maintenance, a condition presumed to be incompatible with photoreceptor survival. 2. Mutants exhibiting wild type properties in a non-photoreceptor milieu are, in photoreceptors, defective in post-Golgi transport to the nascent outer segment discs. The latter defect causes failure in outer segment maintenance as well as spillage of cellular content to interphotoreceptor space; both conditions may underlie lethality by these mutants. To investigate these hypotheses, morphological, electrophysiological and biochemical analyses will he carried out. Immunocytochemical evidence will be sought that mutant opsins are localized in the outer segments. A combination of methods will be employed to ask if misfolded mutant rhodopsins are present in the outer segments and if their presence perturb phototransduction above and beyond what is expected for reduced quantum catch due to a reduction in functional rhodopsin. Selected representatives from each class of mutants will be examined so as to infer the general applicability of the hypotheses to dominant rhodopsin mutants (more than 70) at large. We seek to understand the pathogenic mechanism about a gene whose mutations account for more RP families than any other. The knowledge learned through such a study may provide insight into how rhodopsin functions in an in vivo physiological context, and may serve as the foundation for the design of mechanism-based therapeutic strategies.
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