Abstract
Purpose :
A wide range of blinding disorders have been associated with mutations in the rod visual pigment rhodopsin. Point mutations of Glycine 90 to Aspartate (G90D) or to Valine (G90V) have been reported to cause congenital stationary night blindness (CSNB) and retinitis pigmentosa (RP), respectively. In vitro studies have shown that the G90D/G90V mutations cause decreased stability of both chromophore-bound rhodopsin and chromophore-free opsin. This lower stability could produce persistent activation of rod photoreceptors even in darkness and interfere with normal light detection, causing CSNB or RP. However, the molecular mechanism by which these mutations cause abnormal photoreceptor function and degeneration remains controversial.
Methods :
Using CRISPR/Cas9 technology, we created homozygous mutant mice with either G90D or G90V rhodopsin and performed their initial characterization. Retinal morphology was analyzed by hematoxylin and eosin staining of retinal cross-sections. Scotopic function was assessed by in vivo electroretinography (ERG). Pigment content and stability of its 11-cis-retinal Schiff base in the dark were measured by spectrophotometry of dodecyl maltoside extracts from whole mouse eyes, in the absence or presence of hydroxylamine (HA), respectively.
Results :
The morphology of the retina in both G90D and G90V mutant mice was normal up to 4 months of age, with good preservation of both outer nuclear and photoreceptor layers. However, the rod maximal response (ERG a-wave) in G90V and G90D mice at 2 months was suppressed to ~70% and ~50%, respectively, compared to controls. The rod photosensitivity was also reduced to a greater degree in the G90D than in G90V mice. The amount of rhodopsin in eyes of 2-month-old G90V mice was reduced to 25% of wild type levels. However, its 11-cis-retinal chromophore was not readily accessible to HA in the dark. In contrast, the level of rhodopsin in the eyes of G90D eyes was 35% of that in controls and its chromophore was accessible to HA.
Conclusions :
Our preliminary data suggest that both G90D and G90V opsins in mouse rods have reduced chromophore binding. However, unlike in wild type and G90V rhodopsin, the covalent link between chromophore and opsin in G90D rhodopsin appears unstable and susceptible to hydrolysis. We conclude that G90V and G90D mutations in rhodopsin can cause night blindness by two different mechanisms that are being investigated further.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.