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PubMed · 11222546

Mutant rhodopsin transgene expression on a null background.

J M Frederick·N V Krasnoperova·K Hoffmann·J Church-Kopish·K Rüther·K Howes·J Lem·W Baehr
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Abstract

PURPOSE: To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation. METHODS: Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG). RESULTS: Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS: GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.

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AnimalsDNA PrimersElectrophoresis, Polyacrylamide GelElectroretinographyFemaleFluorescent Antibody Technique, IndirectGene ExpressionGenotypeImmunoblottingMaleMiceMice, KnockoutMice, TransgenicMicroscopy, ImmunoelectronMutationPhotoreceptor Cells, VertebrateRetinal DegenerationRhodopsinTransgenesJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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J M Frederick, N V Krasnoperova, K Hoffmann, J Church-Kopish, K Rüther, K Howes, J Lem, W Baehr. 2001. Mutant rhodopsin transgene expression on a null background.. https://pubmed.ncbi.nlm.nih.gov/11222546/

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