Role of Unfolded Protein Response and Endoplasmic Reticulum-Associated Degradation by Repeated Exposure to Inhalation Anesthetics in Caenorhabditis elegans

Abstract

Background: When an imbalance occurs between the demand and capacity for protein folding, unfolded proteins accumulate in the endoplasmic reticulum (ER) lumen and activate the unfolded protein response (UPR). In addition, unfolded proteins are cleared from the ER lumen for ubiquitination and subsequent cytosolic proteasomal degradation, which is termed as the ER-associated degradation (ERAD) pathway. This study focused on changes in the UPR and ERAD pathways induced by the repeated inhalation anesthetic exposure in Caenorhabditis elegans. Methods: Depending on repeated isoflurane exposure, C. elegans was classified into the control or isoflurane group. To evaluate the expression of a specific gene, RNA was extracted from adult worms in each group and real-time polymerase chain reaction was performed. Ubiquitinated protein levels were measured using western blotting, and behavioral changes were evaluated by chemotaxis assay using various mutant strains. Results: Isoflurane upregulated the expression of ire-1 and pek-1 whereas the expression of atf-6 was unaffected. The expression of both sel-1 and sel-11 was decreased by isoflurane exposure, possibly indicating the inhibition of retro-translocation. The expression of cdc-48.1 and cdc-48.2 was decreased and higher ubiquitinated protein levels were observed in the isoflurane group than in the control, suggesting that deubiquitination and degradation of misfolded proteins were interrupted. The chemotaxis indices of ire-1, pek-1, sel-1, and sel-11 mutants decreased significantly compared to N2, and they were not suppressed further even after the repeated isoflurane exposure. Conclusion: Repeated isoflurane exposure caused significant ER stress in C. elegans. Following the increase in UPR, the ERAD pathway was disrupted by repeated isoflurane exposure and ubiquitinated proteins was accumulated subsequently. UPR and ERAD pathways are potential modifiable neuroprotection targets against anesthesia-induced neurotoxicity.

Keywords: Caenorhabditis elegans; endoplasmic reticulum-associated degradation pathway; inhalation anesthetics; unfolded protein response.

Figure 1

Chemotaxis assay plate. A 9-cm petri plate covered with nutrient growth medium was used for chemotaxis assay. OP50 was used for attractant, and control site was blank. Before placing worm pellet in the center, 1 μl of 1 M sodium azide was dropped in both sites to immobilize worms when they reached there. Number of worms in a circle within 1.5-cm radius at each point and in other zone was counted and used for chemotaxis index calculation.

Figure 2

Expression of genes related to the unfolded protein response and endoplasmic reticulum associated degradation. (A) Isoflurane upregulated the expression of ire-1 and pek-1; however, the expression of atf-6 remained unaffected. (B) The expression of both sel-1 and sel-11 was decreased by isoflurane exposure. (C) The expression of cdc-48.1 and cdc-48.2 was decreased by isoflurane exposure. All batches included 3 plates in each group and same assay was performed three times. Error bar, standard deviation; *P<0.001.

Figure 3

Western blotting for ubiquitinated proteins. (A, B) Higher levels of ubiquitinated protein were observed in the isoflurane group. All batches included 3 plates in each group and same assay was performed three times. Error bar, standard deviation; *P<0.001.

Figure 4

Chemotaxis index. In several mutant strains related unfolded protein response and endoplasmic reticulum-associated degradation, the chemotaxis indices were measured (A) and the difference between the control and isoflurane group was calculated in each strains (B). All batches included 3 plates in each group and same assay was performed three times. Error bar, standard deviation; *P <0.05 vs. control in each strain; †P = 0.001 vs. N2 control group; ‡P <0.001 vs. N2.