Genetic hyperactivation of Nrf2 causes larval lethality in Keap1a and Keap1b-double-knockout zebrafish

• Published in: Redox biology Vol. 62; p. 102673
• Main Authors: Bian, Lixuan, Nguyen, Vu Thanh, Tamaoki, Junya, Endo, Yuka, Dong, Guilin, Sato, Ayaka, Kobayashi, Makoto
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2023; Elsevier
• Subjects: Animals; Animals, Genetically Modified; Carrier Proteins - metabolism; Eating defects; Gene Knockout Techniques; keap1a;keap1b-double knockout zebrafish; Keap1–Nrf2 pathway; Kelch-Like ECH-Associated Protein 1 - genetics; Kelch-Like ECH-Associated Protein 1 - metabolism; Larva - genetics; Mammals - metabolism; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Nrf2-inhibiting compounds; Oxidative Stress; Short Communication; Visual cycle genes; Zebrafish - genetics; Zebrafish - metabolism; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism; Nrf2-inhibiting compounds; Keap1–Nrf2 pathway; keap1a;keap1b-double knockout zebrafish; Visual cycle genes; Eating defects
• ISSN: 2213-2317; 2213-2317
• DOI: 10.1016/j.redox.2023.102673

The Keap1–Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds. [Display omitted] •keap1a;keap1b-double-knockout in zebrafish caused larval lethality and eating defects.•Double-knockout larvae showed lethality even under non-feeding conditions.•Nrf2 mutation rescued all the phenotypes in keap1a;keap1b-double-knockout zebrafish.•Nrf2 inhibitors partially rescued the lethality of keap1a;keap1b-double-knockout larvae.