Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization
Ning Zhang, … , Vladimir J. Kefalov, Krzysztof Palczewski
Ning Zhang, … , Vladimir J. Kefalov, Krzysztof Palczewski
Published January 2, 2013; First published December 10, 2012
Citation Information: J Clin Invest. 2013;123(1):121-137. https://doi.org/10.1172/JCI66176.
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Research Article Ophthalmology

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

Abstract

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin’s stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.

Authors

Ning Zhang, Alexander V. Kolesnikov, Beata Jastrzebska, Debarshi Mustafi, Osamu Sawada, Tadao Maeda, Christel Genoud, Andreas Engel, Vladimir J. Kefalov, Krzysztof Palczewski

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Figure 1

Rhodopsin models and targeting strategy and molecular characterization of E150K knock-in mice.

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Rhodopsin models and targeting strategy and molecular characterization o...
(A) 2D model of rhodopsin indicate residue conservation and mutations that cause human adRP and arRP. Figure adapted with permission from Science (26). The E150K mutation is located at the interface of the cytoplasmic side with the disc membrane, close to the putative dimerization site indicated by arrows in the (B) 3D model of bovine rhodopsin. Figure adapted with permission from the Journal of Structural Biology (71). (C) E150K knock-in mice were generated by using a targeting vector containing a 2.11-kb 5′ short homology arm (SA) and a 5.36 kb long homology arm (LA). A loxP-flanked Neo cassette was upstream of exon 2. The target 284-bp sequence region included a G to A transversion at codon 150 in exon 2 (star). Codon 150 in exon 2 was changed to AAG by homologous recombination, and the flipase recognition target loxP-flanked Neo cassette was inserted before exon 2. The Neo cassette was removed by Cre-loxP recombination to generate E150K opsin knock-in mice. Whole-locus sequencing confirmed the knock-in (KI). This mouse retained a 44-bp loxP sequence in the intron between exons 1 and 2. (D) PCR primers were designed to amplify the intron where the 44-bp loxP sequence was inserted. The WT allele and E150K opsin allele produced 262- and 306-bp PCR products, respectively, whereas the negative control displayed no template contamination. (E) Chromatogram of opsin cDNA of 2 clones from one 15-day-old EK mouse retina confirmed the presence of both mutant and WT mRNAs.