ER stress occurs in response to the accumulation of unfolded or misfolded proteins in the ER lumen, subsequently activating three signal transduction pathways collectively called the unfolded protein response (UPR). Although the goal of the UPR is to restore ER homeostasis, it can result in apoptosis when ER stress is too severe or prolonged. CHOP, which is induced by all three branches of the UPR, is central to inducing ER stress-induced apoptosis by regulating the expression of numerous pro-apoptotic genes. One of the downstream targets of CHOP is GADD34, which contributes to PP1-mediated dephosphorylation of eIF2α together with its homolog CReP to restore the attenuated global translation. A previous study reveals that morpholino knockdown of GADD34 rescued chronic ER stress-induced apoptosis in zebrafish caudal fin epidermal cells in vivo. However, due to the limitations of morpholino, any morpholino data about novel phenotypes should be verified with comparative mutant data. Therefore, we sought to investigate how GADD34 and CReP are involved in acute and chronic ER stress-induced apoptosis using GADD34 or CReP mutant zebrafish embryos. GADD34 heterozygous or CReP heterozygous zebrafish are crossed to produce the embryos of the following genotypes: wildtype, GADD34 heterozygous mutant, GADD34 homozygous mutant, CReP heterozygous mutant, and CReP homozygous mutant embryos. At 24hpf, embryos were treated with ER stress inducer Thapsigargin for either 4 hours or 24 hours to induce acute or chronic ER stress. After stained with acridine orange for an apoptosis assay, each embryo was imaged under a confocal microscope and subsequently genotyped. GADD34 or CReP mutation alone did not affect levels of apoptosis induced by acute ER stress. Similarly, CReP mutation alone did not affect levels of apoptosis induced by chronic ER stress. However, GADD34 mutation rescued chronic ER stress-induced apoptosis, and the differences between the apoptosis level in GADD34 homozygous mutants and those in wildtype and GADD34 heterozygous mutants were statistically significant. Our results indicate no obvious role of GADD34 and CReP in acute ER stress-induced apoptosis, which is consistent with the previous morpholino data. However, as GADD34 mutation rescued chronic ER stress-induced apoptosis, the CHOP- GADD34 downstream pathway is likely to be involved in chronic ER stress-induced apoptosis. In the future, GADD34 and CReP double mutant embryos should be used to analyze for the redundancy of GADD34 and CReP.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-5054 |
| Date | 01 January 2024 |
| Creators | Ohata, Ayano |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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