Biology subjects

Moritz, O. L.

Publications and source records attributed to Moritz, O. L..

6 recordsLinked to original sources

bioRxiv2026

Alterations in phototransduction timing, dark adaptation and GNAT1 trafficking in GNAT1-edited Xenopus laevis.

GNAT1 (also called -transducin) is the -subunit of a heterotrimeric G-protein present in rod photoreceptors that plays a critical role in phototransduction. Photoactivated rhodopsin binds GNAT1, which stimulates phosphodiesterase to hydrolyze cGMP, closing ion channels and hyperpolarizing the rod cell. To examine the effects of GNAT1 ablation on light-induced autophagy and light-exacerbated retinal degeneration, we previously generated genetically chimeric, GNAT1-edited Xenopus laevis using CRISPR technology. Here, we interbred these F0 animals to produce F1 animals with defined genotypes, including full knockouts, and characterized the F1 generation by Western blot, histology, and electroretinography (ERG). In X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles, the GNAT1 protein was absent, and the scotopic ERG was absent, indicating full knockout. In animals with reduced GNAT1 expression, we observed increased A- and B-wave implicit times. Greater GNAT1 protein reduction was associated with greater ERG delay, demonstrating a role for GNAT1 in the timing of phototransduction. We also identified two unexpected phenotypes linked to distinct gene edits. The Q184L-GNAT1.L mutation conferred an ERG phenotype similar to that of a full knockout, but with minimal reduction in GNAT1 protein levels, consistent with loss of function. This mutation is also present in the human population. A three-base-pair deletion (T183,Q184[->]K183) in GNAT1.S caused dominantly inherited diminished ERG response and defective dark adaptation, and was associated with photoreceptor structural abnormalities and GNAT1 accumulation at the connecting cilium, consistent with a dominant-negative effect on GNAT1 trafficking.

neuroscience↗

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

Prominin-1 null Xenopus laevis develop subretinal drusenoid-like deposits, cone-rod dystrophy, and dysmorphic retinal pigment epithelium.

Mutations in the PROMININ-1 (PROM1) gene are associated with inherited, non-syndromic vision loss. Here, we used CRISPR/Cas9 to induce truncating prom1-null mutations in Xenopus laevis to create a disease model. We then tracked progression of retinal degeneration in these animals from the ages of 6 weeks to 3 years old. We found that retinal degeneration caused by prom1-null is age-dependent and likely involves death or damage to the retinal pigment epithelium (RPE) that precedes photoreceptor degeneration. As prom1-null frogs age, they develop large cellular debris deposits in the subretinal space and outer segment layer which resemble subretinal drusenoid deposits (SDD) in their location, histology, and representation in color fundus photography and optical coherence tomography (OCT). In older frogs, these SDD-like deposits accumulate in size and number, and they are present before retinal degeneration occurs. Evidence for an RPE origin of these deposits includes infiltration of pigment granules into the deposits, thinning of RPE as measured by OCT, and RPE disorganization as measured by histology and OCT. The appearance and accumulation of SDD-like deposits and RPE thinning and disorganization in our animal model suggests an underlying disease mechanism for prom1-null mediated blindness of death and dysfunction of the RPE preceding photoreceptor degeneration, instead of direct effects upon photoreceptor outer segment morphogenesis, as was previously hypothesized.

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↗

bioRxiv2020

Genetically-modified X. laevisindicate distinct roles for prominin-1 and photoreceptor cadherin in outer segment morphogenesis and retinal dystrophy

Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders such as retinitis pigmentosa, cone-rod dystrophy, and juvenile macular dystrophy. The proteins encoded by these genes are hypothesized to regulate photoreceptor outer segment disc morphogenesis, but their functions remain unknown. We used CRISPR/Cas9 to generate prom1-, cdhr1-, and prom1 + cdhr1-null X. laevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in dysmorphic photoreceptors comprised of overgrown and disorganized disc membranes. Cones were more severely affected than rods; outer segments were elongated and fragmented, and ERG response was impaired. Autofluorescent deposits in the outer segment layer of aging prom1-null animals indicate that secondary toxic effects to the retina or RPE drive retinal degeneration for this mutation, instead of direct effects on outer segment disc morphogenesis. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or aligned vertically within the plasma membrane. Prom1 + cdhr1-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the membrane fusion event involved in disc sealing. Rather, they are necessary for higher-order organization of the nascent outer segment discs. Prom1 may align and reinforce interactions between the disc leading edges, a function more critical in cone photoreceptors for structural support. Cdhr1 may help to align nascent discs and maintain horizontal disc orientation prior to fusion.

cell biology↗