The Rpe65(rd12) Allele Exerts a Semidominant Negative Effect on Vision in Mice

• Type of Material: Article
• Authors: Charles B. Wright, Emory University; Micah A. Chrenek, Emory University; Wei Feng, Emory University; Shannon E. Getz, Emory University; Todd Duncan, National Eye Institute; Machelle Pardue, Emory University; Yue Feng, Emory University; T. Michael Redmond, National Eye Institute; Jeffrey Boatright, Emory University; John Nickerson, Emory University
• Language: English
• Date: 2014-04-01
• Publisher: The Association for Research in Vision and Ophthalmology
• Publication Version: Final Published Version
• Copyright Statement: © 2014 The Association for Research in Vision and Ophthalmology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1167/iovs.13-13574
• Title of Journal or Parent Work: Investigative Ophthalmology and Visual Science
• Volume: 55
• Issue: 4
• Start Page: 2500
• End Page: 2515
• Grant/Funding Information: Supported by National Institutes of Health Grants P30EY006360, R01EY016470, T32EY007092, R01EY014026, R01EY016435, R01EY021592, R01NS070526, and Z01EY000444; an unrestricted Departmental Award from Research to Prevent Blindness; the Intramural Research Program of the National Eye Institute; and a grant from the Abraham J. and Phyllis Katz Foundation.
• Keywords: GENE-THERAPY; LEBER CONGENITAL AMAUROSIS; RETINAL DYSTROPHY; visual cycle; ISOMEROHYDROLASE ACTIVITY; MOUSE MODEL; PERFORMANCE LIQUID-CHROMATOGRAPHY; Science & Technology; rd12; Ophthalmology; RETINITIS-PIGMENTOSA; RPE65 MUTATIONS; ADENOASSOCIATED VIRUS; MACULAR DEGENERATION; Life Sciences & Biomedicine; RPE65
• Research Categories: Health Sciences, Opthamology

Abstract

PURPOSE. The rd12 mouse was reported as a recessively inherited Rpe65 mutation. We asked if the rd12 mutation resides in Rpe65 and how the mutation manifests itself. METHODS. A complementation test was performed by mating Rpe65KO (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the Rpe65 gene. Visual function of wildtype (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRTPCR quantified Rpe65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. Rpe65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of Rpe65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. RESULTS. The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of Rpe65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wildtype mRNA was enriched on actively translating polyribosomes. CONCLUSIONS. The rd12 lesion is in Rpe65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.