Sullivan, Alanna1; Nonarath, Hannah1; Clark, Eric1; Link, Brian1
1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Purpose: Oxidative stress-provoked photoreceptor degeneration is a critical factor in retinal disease pathologies such as retinitis pigmentosa. NF-E2-related factor 2 (NRF2; NFE2L2) is an important transcriptional regulator for redox homeostasis. In stressed conditions, Nrf2 translocates to the nucleus to bind and activate antioxidant response elements, which upregulate target antioxidant genes. The increased production of antioxidants break-down reactive oxygen species, allowing cells to return to redox homeostasis. Thus, nrf2 is an attractive target for treating retinopathies like retinitis pigmentosa. Danio rerio have two orthologs of the human NRF2 gene, nrf2a and nrf2b, but only nrf2a has been knocked out. A complete nrf2 mutant will provide a platform to investigate the role of oxidative stress in retinal diseases and inform therapeutic strategies. The purpose of this study is to establish and validate a novel nrf2 mutant zebrafish model where both nrf2a and nrf2b are knocked out.
Methods: The CRISPR-Cas9 system was used to produce a large deletion in nrf2b. The nrf2b mutants generated were bred with an established nrf2a mutant line, nrf2afh318 (Goessling lab). The double homozygous mutant offspring (nrf2) were confirmed with sequencing. A light damage assay (LDA) was used to induce retinal cell death by having five days post fertilization (dpf) fish exposed to normal or excessive light conditions for three days. To evaluate the susceptibility of photoreceptors to light damage, pyknotic nuclei and TUNEL positive cells in the outer nuclear layer (ONL) were quantified in wildtype and nrf2 mutants (8dpf, n=15). A qPCR assay was used to determine if nrf2 mutants have reduced activation of antioxidant pathways in LDA conditions compared to wildtype fish (n=3, 5 biological replicates). Three nrf2 target genes were measured, a glutamate-cysteine ligase (gclc), a NAD(P)H dehydrogenase (nqo1), and heme oxygenase-1 (hmox1a).
Results: A two-way ANOVA showed that after light damage, nrf2 mutants have significantly higher pyknotic (21.3 ± 1.37, p = 0.001) and TUNEL positive cell count (1.5±0.55, p = 0.018) in the ONL compared to wildtype fish (10.2±1.18; 0.73±0.17). Furthermore, the expected upregulation of nrf2 target genes in light damage conditions was not induced in nrf2 mutants. qPCR results indicated a significant fold change of gclc, nqo1, and hmox1a expression in light damaged wildtype fish (48.2±10.5; 91.9±41.3; 7.68±1.22, p = 0.03) while gene expression remained at basal levels in light damaged nrf2 mutants (1.27±0.17; 0.43±0.07; 0.40±0.07).
Conclusions: The data suggests nrf2 mutants fail to regulate downstream target genes in LDA conditions and are sensitive to light damage-induced photoreceptor death. To validate heightened oxidative stress in nrf2 mutants after LDA conditions, retinal antioxidant activity will be assessed using redox-sensitive GFP reporter lines. In future studies, nrf2 mutants will be applied to a model of Usher Syndrome subtype 2A (USH2A; Usherin), a rare retinopathy that causes progressive photoreceptor death. Oxidative stress may be a factor involved in the pathogenesis, thus, the loss of Nrf2 activity may increase susceptibly for further cell death in the USH2A mutant zebrafish model.
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