Mutant rhodopsin transgene expression on a null background

PURPOSE. TO Study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rho dopsin P23H mutation. METHODS. Mice of a transgenic line expressing a rhodopsin triple mutant, V2 0G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Li ttermates of various ages and genotypes (GHL(+)rho(+/+), GHL(+)rho(+/+), an d GHL(+)rho(-/-)) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemist ry), and photoreceptor function (electroretinogram; ERG). RESULTS. Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsi n had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfa te-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin pre dominantly formed monomers. Expression level of mutant rhodopsin in predege nerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was obs erved at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalizat ion using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS. GHL-rhodopsin dimerizes, localizes to the ER, and fails to tra nsport and support outer segment formation. Additionally, the mutant protei n does not support a scotopic ERG a-wave and accelerates photoreceptor dege neration over that occurring with the rhodopsin knockout alone. These findi ngs indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.