Synchronized photoactivation of T4K rhodopsin causes a chromophore-dependent retinal degeneration that is moderated by interaction with phototransduction cascade components.

Multiple mutations in the Rhodopsin gene cause sector retinitis pigmentosa in humans and a corresponding light-exacerbated retinal degeneration (RD) in animal models. Previously we have shown that T4K rhodopsin requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in mixed male and female Xenopus laevis In this model, RD was prevented by rearing animals in constant darkness but surprisingly also in constant light. RD was maximized by light cycles containing at least one hour of darkness and 20 minutes of light exposure, light intensities > 750 lux, and by sudden light onset. Under conditions of frequent light cycling, RD occured rapidly and synchronously, with massive shedding of ROS fragments into the RPE initiated within hours, and subsequent death and phagocytosis of rod cell bodies. RD was minimized by reduced light levels, pre-treatment with constant light, and gradual light onset. RD was prevented by genetic ablation of the retinal isomerohydrolase RPE65, and exacerbated by ablation of phototransduction components GNAT1, SAG, and GRK1. Our results indicate that photoactivated T4K rhodopsin is toxic, that cell death requires synchronized photoactivation of T4K rhodopsin, and that toxicity is mitigated by interaction with other rod outer segment proteins regardless of whether they participate in activation or shutoff of phototransduction. In contrast, RD caused by P23H rhodopsin does not require photoactivation of the mutant protein, as it was exacerbated by RPE65 ablation, suggesting that these phenotypically similar disorders may require different treatment strategies.Significance Statement Many different rhodopsin mutations are linked to the inherited degenerative disease retinitis pigmentosa. Although the end result in each case is the loss of photoreceptor cells and blindness, not all mutations cause cell death via the same mechanism. In order to design and test treatment therapies that target the disease at points as upstream as possible in the process, we require detailed understanding of the range and nature of these disease mechanisms. This study using a transgenic Xenopus laevis model has extended our understanding of how T4K rhodopsin and related mutations cause rod cell photoreceptor death via a phototoxic product, and how this mechanism differs from the more extensively researched protein misfolding mechanism underlying cell death caused by P23H rhodopsin.

Electrophysiological Changes During Early Steps of Retinitis Pigmentosa.

Purpose: The rhodopsin mutation P23H is responsible for a significant portion of autosomal-dominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration. Methods: We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure. Results: Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rod-saturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations. Conclusions: Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9.

Xenopus laevis have proven to be a useful system for rapid generation and analysis of transgenic models of human retinal disease. However, experimental approaches in this system were limited by lack of a robust knockdown or knockout technology. Here we describe a protocol for generation of Cas9-edited X. laevis embryos. The technique introduces point mutations into the genome of X. laevis resulting in in-frame and out-of-frame insertions and deletions that allow modeling of both dominant and recessive human diseases and efficiently generates gene knockdown and knockout. Our techniques can produce high-frequency gene editing in X. laevis, permitting analysis in the F0 generation.

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Opposing Effects of Valproic Acid Treatment Mediated by Histone Deacetylase Inhibitor Activity in Four Transgenic X. laevis Models of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in Rhodopsin; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA on Xenopus laevis models of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases.SIGNIFICANCE STATEMENT Retinitis pigmentosa (RP) is an inherited, degenerative retinal disease that leads to blindness for which no therapy is available. We determined that valproic acid (VPA), currently undergoing a phase II trial for RP, has both beneficial and detrimental effects in animal models of RP depending on the underlying disease mechanism and that both effects are due to histone deacetylase (HDAC) inhibition possibly linked to autophagy regulation. Off-label use of VPA and other HDAC inhibitors for the treatment of RP should be limited to the research setting until this effect is understood and can be predicted. Our study suggests that, unless genotype is accounted for, clinical trials for RP treatments may give negative results due to multiple disease mechanisms with differential responses to therapeutic interventions.