Biology subjects

Chiu, C. N.

Publications and source records attributed to Chiu, C. N..

3 recordsLinked to original sources

bioRxiv2025

RPE65 knockout Xenopus laevis have a compromised but detectable electroretinogram and altered visual responses, without retinal degeneration or altered melanophore dispersion

The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the rpe65 gene cause a severe form of blindness called Lebers congenital amaurosis. Studies of RPE65-/- mice have shown dramatic depletion of 11- cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.

cell biology↗

bioRxiv2025

A survey of opsin localization, glycosylation, and light/chromophore influence on degeneration in 26 rhodopsin-associated retinitis pigmentosa models

PurposeMutations in rhodopsin (RHO) cause autosomal dominant retinitis pigmentosa (RP), which has multiple clinical subclasses, including class B1 ("sector") RP in which the asymmetric retinal degeneration (RD) suggests an environmental influence. The pathogenic mechanisms of most class B1 mutations are uncharacterized. We generated new animal models of RHO-associated RP to examine RHO expression, localization, glycosylation, and the influence of light and chromophore binding on RD. MethodsWe generated transgenic X. laevis expressing wildtype or mutant human RHO transgenes. Confocal images were used to evaluate RD and trafficking. Immunoassays were used to quantify RD and investigate RHO glycosylation. ResultsWe created X. laevis models of 26 different forms of RHO-associated RP. Most mutations caused RD, with the exception of those at residue R135 and G101. Many variants did not alter RHO localization. Multiple class B1-associated RHO mutants induced light-dependent RD, suggesting light is the environmental influence associated with the class B1 phenotype. However, the degeneration associated with two partially ER-retained class B1 mutants (S22R and D190G) was not mitigated by dark rearing. P23H and S176F constituted a distinct subclass associated with inner segment retention and proteolytic cleavage. ConclusionsMany RHO mutations do not substantially alter RHO localization or glycosylation. The exceptions we identified are P23H and S176F, which dramatically mislocalize, and constitute a distinct category of proteolytically-cleaved misfolding variants. L31Q and T58R induce RD by mechanisms similar to glycosylation-deficient variants, despite lack of glycosylation defects. Intermediate phenotypes indicate at least one previously undescribed mechanism for class B1 RP pathogenesis.

cell biology↗

bioRxiv2024

Synchronized photoactivation of T4K rhodopsin causes a chromophore-dependent retinal degeneration that 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 the rhodopsin mutation T4K requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in 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 of intensity 750 lux or greater, 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 benefit from different treatment strategies. SignificanceA large number of 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 of these 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.

neuroscience↗