Functional characterization of a Xenopus tropicalis knockout and a human cellular model of RCBTB1-associated inherited retinal disease shows involvement of RCBTB1 in the cellular response to oxidative stress

Abstract

Purpose : The function of RCBTB1, a gene that has recently been implicated in syndromic and non-syndromic inherited retinal disease (IRD), remains unknown so far. Patients with biallelic missense variants in RCBTB1 display diverse IRD phenotypes varying from retinitis pigmentosa and reticular dystrophy to chorioretinal atrophy. Here, we tested the hypothesis that RCBTB1 is involved in NRF2-regulated protection against reactive oxygen species (ROS) in the eye, and more specifically in the retinal pigment epithelium (RPE).

Methods : A Xenopus tropicalis knockout (KO) animal model rcbtb1^-/- was generated using CRISPR/Cas9 genome editing. Histological examination and additional three-dimensional electron microscopy was performed on retinas of the rcbtb1^-/- frogs. RNA-seq analysis was performed on RCBTB1-mutated patients' lymphocytes, treated with H₂O₂, as well as on embryos from the Xenopus tropicalis knockout treated with CdCl₂. An RCBTB1 knockdown (KD) cell line was generated in ARPE-19 cells and a variety of functional assays (e.g. flow cytometry, MTT-assay, cell death kinetics) was used to assess the consequences of RCBTB1 loss-of-function.

Results : Rcbtb1^-/- animals showed dystrophic changes in the RPE, similar to observations in human cases, including loss of apical-basal cell polarity, cuboidal cell morphology, spreading of the pigment granules and vacuolisation. NRF2 downstream targets and several metallothioneins were differentially expressed in RNA-seq experiments, both in the KO animal and in human cellular models. The functional assays in ARPE-19 cells revealed that RCBTB1 depletion affects cellular responses to external insults of oxidative stress.

Conclusions : We showed that the Xenopus tropicalis rcbtb1^-/- KO recapitulates the human IRD phenotype, making it an excellent model to study RCBTB1-disease. Both in vivo results together with in vitro functional data generated on a human cellular model show involvement of RCBTB1 in the cellular response to oxidative stress. This provides insight into the mechanism underlying RCBTB1-associated IRD and uncovers potential therapeutic opportunities.

This is a 2020 ARVO Annual Meeting abstract.