Establishment of a novel nrf2 mutant zebrafish model to study oxidative stress in retinal disease pathology.

Abstract

Purpose : Establish and validate a novel nrf2a/nrf2b double homozygous mutant zebrafish to investigate oxidative stress-provoked photoreceptor degeneration in zebrafish models of retinopathies.

Methods : The nrf2a/nrf2b double homozygous mutants were generated with an established nrf2a^fh318 mutant allele (Moens Lab) and a nrf2b CRISPR large deletion mutant. A light damage assay was used to induce retinal cell death in five days post-fertilization (dpf) nrf2a/nrf2b double mutants, nrf2a and nrf2b single mutants and wildtype zebrafish (n=15). Larvae were exposed to either normal (150 lux) or elevated (3500 lux) light conditions for three consecutive days. To evaluate photoreceptor death, pyknotic nuclei and TUNEL-positive cells in the outer nuclear layer (ONL) were counted from retinal cryosections from each experimental group (8dpf). With the same experimental groups, gene expression of three Nrf2 target genes – glutamate-cysteine ligase (gclc), a NAD(P)H dehydrogenase (nqo1) and heme oxygenase-1 (hmox1a) – were quantified using qPCR (n=3 pooled sets of eyes, 5 biological replicates). Fold change of gene expression was calculated using ΔCt analysis and differences in cell death count were tested for statistical significance using two-way ANOVA (p<0.05).

Results : After light damage, nrf2a/nrf2b double mutants had significantly higher pyknotic and TUNEL-positive cells in the ONL compared to wildtype fish. Light-damaged nrf2a single mutants did not show an increase in cell death in the ONL, but light-damaged nrf2b single mutants had significantly higher TUNEL-positive cells. Transcript levels of the Nrf2 targets gclc, nqo1, and hmox1a were significantly elevated in light-damaged wildtype fish, while in light-damaged nrf2a/nrf2b double and single mutants, gene expression remained at basal levels.

Conclusions : In light damage conditions, nrf2a/nrf2b double mutants failed to regulate Nrf2 target genes and showed increased photoreceptor death. While nrf2a and nrf2b single mutants lost regulation of target genes, only nrf2b mutants had a significant increase in light-induced photoreceptor death. To confirm heightened oxidative stress in nrf2 mutants after a light damage assay, antioxidant pathway activity will be assessed using photoreceptor redox-sensitive GFP reporter lines. In future studies, the nrf2 mutants and redox-reporters will be applied to models of human photoreceptor degenerations.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.