Light-independent photoreceptor degeneration and ciliary dysregulation in a novel Q344X rhodopsin knock-in mouse model

Abstract

Purpose : Rhodopsin mutation can cause blindness. The objective of this study was to create a mouse model that replicates the genetic condition of human autosomal dominant retinitis pigmentosa (adRP) resulting from the class I Q344X rhodopsin mutation. Additionally, the study aimed to elucidate the biological alterations triggered by the mislocalization of rhodopsin, the primary consequence of the class I mutation.

Methods : The Q344X mutation was introduced into the endogenous rhodopsin (Rho) gene using the CRISPR/Cas9 system. Outer nuclear layer (ONL) thicknesses were measured using optical coherence tomography (OCT) and light microscopy. The ratio of wild-type and Q344X mutant Rho mRNA was compared in Rho^Q344X/+ retinas by RT-PCR. Rhodopsin protein expression was assessed by immunoblotting. Rhodopsin mislocalization was examined through immunohistochemistry (IHC). Global protein expression changes in the retina were analyzed using quantitative proteomics. The effect of light on ONL thickness was investigated by OCT after raring mice under dark, 200 lux, and 2500 lux light conditions.

Results : The Q344X mutation led to adRP rod degeneration in mice. In Rho^Q344X/+ knock-in mice, mRNA transcripts from the wild-type and Q344X mutant Rho alleles were expressed at equivalent levels. IHC indicated that RHO^Q344X is mislocalized outside the outer segments (OSs) of rods both in Rho^Q344X/+ and Rho^Q344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting rhodopsin delivery to the OS. The mislocalization of RHO^Q344X was associated with the concurrent mislocalization of wild-type rhodopsin in Rho^Q344X/+ mice. Quantitative proteomics analysis showed attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Rods degenerated similarly among different light conditions in Rho^Q344X/+ mice.

Conclusions : These studies unveiled a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a reduction in the various ciliary proteins. Notably, in contrast to prior reports, our findings demonstrate that light exposure does not exacerbate photoreceptor degeneration in Rho^Q344X/+ mice.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.