Defective responses to oxidative stress in protein l-isoaspartyl repair-deficient Caenorhabditis elegans

Abstract

We have shown that Caenorhabditis elegans lacking the PCM-1 protein repair l-isoaspartyl methyltransferase are more sensitive to oxidative stress than wild-type nematodes. Exposure to the redox-cycling quinone juglone upon exit from dauer diapause results in defective egg-laying (Egl phenotype) in the pcm-1 mutants only. Treatment with paraquat, a redox-cycling dipyridyl, causes a more severe developmental delay at the second larval stage in pcm-1 mutants than in wild-type nematodes. Finally, exposure to homocysteine and homocysteine thiolactone, molecules that can induce oxidative stress via distinct mechanisms, results in a more pronounced delay in development at the first larval stage in pcm-1 mutants than in wild-type animals. Homocysteine treatment also induced the Egl phenotype in mutant but not wild-type nematodes. All of the effects of these agents were reversed upon addition of vitamin C, indicating that the developmental delay and egg-laying defects result from oxidative stress. Furthermore, we have demonstrated that a mutation in the gene encoding the insulin-like receptor DAF-2 suppresses the Egl phenotype in pcm-1 mutants treated with juglone. Our results support a role of PCM-1 in the cellular responses mediated by the DAF-2 insulin-like signaling pathway in C. elegans for optimal protection against oxidative stress.

Fig. 1. Genetic organization of the C. elegans PCM-1 region

Gene map for C. elegans pcm-1 and the partially overlapping C10F3.4 gene. Exons are shown by boxes; introns by lines. The regions deleted in each mutant are indicated; the direction of transcription is shown with an arrow at the end of the transcript.

Fig. 2

Juglone-treated pcm-1(qa201) mutant dauer larvae develop a defect in egg-laying as gravid adults. Pcm-1(qa201) mutant dauer larvae (total of 300 animals) were incubated in liquid droplets containing either S-media (control) or S-media with 236 µM juglone for 90 min and were transferred NGM + OP50 plates. The animals were monitored for development and viability every 24 hr for 3 days. Most of the 150 control qa201 mutant dauers developed into healthy gravid adults as shown by Nomarski differential interference contrast microscopy at 200-fold magnification (upper panel), with some animals only progressing to L3 or L4 larvae. On the other hand, about one fifth of the 150 juglone-treated mutant dauer larvae developed into adults with a defect in egg-laying shown in the lower panel, with the remainder progressing apparently normally to L3, L4, and gravid adults. Scale bar = 50 µm.

Fig. 3

(A) Paraquat delays development in wild-type (N2) C. elegans. Eggs (about 100) were transferred to each of two NGM+OP50 plates containing 0, 0.1 mM and 0.2 mM paraquat at 25 °C. The developmental stage of each worm was determined after 48 h as L1, L2, L3, and L4 larvae and as young adult/egg-laying adult (YA/ELA) nematodes. Data from four replicate experiments (2 plates each) are averaged. Asterisks indicate a statistically significant difference in developmental delay between control and treated animals (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). (B) Paraquat treatment results in more severe developmental delays in pcm-1 mutant nematodes. Eggs from qa201 (pcm-1 mutant) and tm363 (pcm-1 mutant) were incubated on paraquat-containing plates and the developmental stage was identified as above. Data from each mutant set were combined. (C) Vitamin C reverses the delay in development induced by paraquat in C. elegans. Eggs from three C. elegans strains (N2 (wild-type), qa201 (pcm-1 mutant), and tm363 (pcm-1 mutant)) were transferred to NGM+OP50 plates containing 0, 0.1 mM and 0.2 mM paraquat in the presence and absence of 1 mM vitamin C and were then scored for larval development after 48 h as above. Data from four replicate experiments are shown; the data from the two mutant strains were pooled. Asterisks indicate a statistically significant difference in developmental delay between wild-type and pcm-1 mutant animals treated under the paraquat versus paraquat plus vitamin C concentrations (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). The numbers of animals scored in each of the groups in this figure are given in Supplemental Table S7 along with the standard deviation values for the percentage of each larval stage under each condition.

Fig. 3

(A) Paraquat delays development in wild-type (N2) C. elegans. Eggs (about 100) were transferred to each of two NGM+OP50 plates containing 0, 0.1 mM and 0.2 mM paraquat at 25 °C. The developmental stage of each worm was determined after 48 h as L1, L2, L3, and L4 larvae and as young adult/egg-laying adult (YA/ELA) nematodes. Data from four replicate experiments (2 plates each) are averaged. Asterisks indicate a statistically significant difference in developmental delay between control and treated animals (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). (B) Paraquat treatment results in more severe developmental delays in pcm-1 mutant nematodes. Eggs from qa201 (pcm-1 mutant) and tm363 (pcm-1 mutant) were incubated on paraquat-containing plates and the developmental stage was identified as above. Data from each mutant set were combined. (C) Vitamin C reverses the delay in development induced by paraquat in C. elegans. Eggs from three C. elegans strains (N2 (wild-type), qa201 (pcm-1 mutant), and tm363 (pcm-1 mutant)) were transferred to NGM+OP50 plates containing 0, 0.1 mM and 0.2 mM paraquat in the presence and absence of 1 mM vitamin C and were then scored for larval development after 48 h as above. Data from four replicate experiments are shown; the data from the two mutant strains were pooled. Asterisks indicate a statistically significant difference in developmental delay between wild-type and pcm-1 mutant animals treated under the paraquat versus paraquat plus vitamin C concentrations (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). The numbers of animals scored in each of the groups in this figure are given in Supplemental Table S7 along with the standard deviation values for the percentage of each larval stage under each condition.

Fig. 4

(A) Homocysteine and homocysteine thiolactone delay development in wild-type (N2) C. elegans. Eggs (about 100) were transferred to NGM+OP50 plates containing 0, 10 mM and 20 mM homocysteine (Hcy), homocysteine thiolactone (HCTL), and homocystine (Hcy-Hcy) and the developmental stage of each worm was determined after 48 h at 25 °C as L1, L2, L3, and L4 larvae and as young adult/egg-laying adult (YA/ELA) nematodes. Data from a total of five experiments each with duplicate plates are shown. Asterisks indicate a statistically significant difference in developmental delay between control and treated animals (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). (B) Homocysteine treatment results in more severe developmental delays in pcm-1 mutant nematodes. Eggs from qa201 and tm363 pcm-1 mutants were exposed to homocysteine, homocysteine thiolactone, and homocystine and development was scored as above, again with five duplicate experiments for each mutant. Asterisks indicate a statistically significant difference in developmental delay between wild-type and pcm-1 mutant animals treated under the specified homocysteine compound concentrations (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). The numbers of animals scored in each of the groups in this figure are given in Supplemental Table S7 along with the standard deviation values for the percentage of each larval stage under each condition.

Fig. 5

Vitamin C reverses the delay in development induced by homocysteine thiolactone (HCTL) in C. elegans. Eggs (about 100) from strains N2 (wild-type), qa201 (pcm-1 mutant), and tm363 (pcm-1 mutant) were transferred to duplicate NGM+OP50 plates containing 0 and 10 mM homocysteine thiolactone (panel A) or 0 and 20 mM homocysteine thiolactone (panel B) in the presence and absence of 1 mM vitamin C at 25 °C and scored for larval development at 48 h as in Fig. 4. Data are shown for four replicate experiments. Data for the two pcm-1 mutants are combined. Asterisks indicate a statistically significant difference in developmental delay between wild-type and pcm-1 mutant animals treated under the specified homocysteine compound versus homocysteine plus vitamin C concentrations (two-tailed Student’s t-test of unequal variance: *p < 0.05, **p <0.005). The numbers of animals scored in each of the groups in this figure are given in Supplemental Table S7 along with the standard deviation values for the percentage of each larval stage under each condition.