A Dominant Mutation in Rpe65, D477G, Delays Dark Adaptation and Disturbs the Visual Cycle in the Mutant Knock-In Mice

Abstract

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.

Aberrant RNA splicing is the major pathogenic effect in a knock‐in mouse model of the dominantly inherited c.1430A>G humanRPE65mutation

“…The great reduction in Rpe65 mRNA level is only revealed when PCR analysis is done over the region containing the mutation. The importance of the location of the quantitation assays may explain why previous studies have reported comparable levels of mRNA generated in the c.1430G KI mice as that in WT controls (Choi et al., ; Shin et al., ). Similar novel splicing events are also found when the human RPE65 genomic sequence carrying the c.1430G mutation is transcribed in cultured cells, suggesting that the pathogenesis associated with the c.1430G mutation may also be involved in defects in splicing in human.…”

“…However, when forced‐expressed in cultured cells, RPE65/D477G behaves as a functional RPE65 (Bowne et al., ), with regular subcellular localization, and adequate isomerization activity (Choi et al., ). Moreover, heterozygous RPE65/D477G knock‐in (KI) mice produce sufficient chromophore and have relatively normal retinal structure and function (Choi et al., ; Shin, Moiseyev, Chakraborty, & Ma, ). Taken together, these data do not support the notion that RPE65/D477G acts as a gain‐of‐function missense mutant.…”

“…If these impinge on recognition sites for splicing or other elements, they can be deleterious to target gene expression (Meier et al, 2010;Turlo, Gallaher, Vora, Laski, & Iruela-Arispe, 2010). Moreover, the mixed genetic background of mouse stem cells can further complicate downstream analysis (Choi et al, 2018;Shin et al, 2017). CRISPR/Cas9 genome editing enables production of transgenic animals in a considerably shorter time frame (Hsu, Lander, & Zhang, 2014;Zhang, Wen, & Guo, 2014), and importantly, without the necessity of foreign sequence insertion (Hsu et al, 2014), or complications of varying genetic background.…”

Noninvasive Electroretinographic Procedures for the Study of the Mouse Retina

“…This recovery can be monitored using a two-flash protocol (Pepperberg et al, 1996), at the single-cell level and by ERG analysis. Alternate Protocol 1 can be used to monitor photoreceptor deactivation in mice (Chen et al, 1995; Kim et al, 2005; Budzynski et al, 2010; Shin et al, 2017). …”

“…Many proteins are involved, and mutations in the genes that encode these proteins have been linked to human retinal disease (Lamb and Pugh, 2004) and these discoveries have also been used to guide the development of relevant mouse models. In these models, dark adaptation recovery is monitored in terms of the growth of the amplitude of the ERG as a function of time following exposure to a bright light that causes a substantial fraction of the rhodopsin molecules to be bleached (e.g., Saari et al, 2001; Kim et al, 2005; Budzynski et al, 2010; Shin et al, 2017). Alternative Protocol 2 can be used to monitor the recovery of the ERG to a previously determined dark-adapted baseline level.…”