The glutathione reductase GSR-1 determines stress tolerance and longevity in Caenorhabditis elegans

Abstract

Glutathione (GSH) and GSH-dependent enzymes play a key role in cellular detoxification processes that enable organism to cope with various internal and environmental stressors. However, it is often not clear, which components of the complex GSH-metabolism are required for tolerance towards a certain stressor. To address this question, a small scale RNAi-screen was carried out in Caenorhabditis elegans where GSH-related genes were systematically knocked down and worms were subsequently analysed for their survival rate under sub-lethal concentrations of arsenite and the redox cycler juglone. While the knockdown of γ-glutamylcysteine synthetase led to a diminished survival rate under arsenite stress conditions, GSR-1 (glutathione reductase) was shown to be essential for survival under juglone stress conditions. gsr-1 is the sole GSR encoding gene found in C. elegans. Knockdown of GSR-1 hardly affected total glutathione levels nor reduced glutathione/glutathione disulphide (GSH/GSSG) ratio under normal laboratory conditions. Nevertheless, when GSSG recycling was impaired by gsr-1(RNAi), GSH synthesis was induced, but not vice versa. Moreover, the impact of GSSG recycling was potentiated under oxidative stress conditions, explaining the enormous effect gsr-1(RNAi) knockdown had on juglone tolerance. Accordingly, overexpression of GSR-1 was capable of increasing stress tolerance. Furthermore, expression levels of SKN-1-regulated GSR-1 also affected life span of C. elegans, emphasising the crucial role the GSH redox state plays in both processes.

Figure 1. Analyses of GSR-1 and GCS-1 dependent stress tolerance.

Wild-type eggs/L1 were placed on RNAi plates (A) gsr-1(RNAi) or (B) gcs-1(RNAi) and cultured until they have reached the L4/young adult stage, before being transferred to corresponding RNAi plates containing the indicated stressors. As control, plates with empty control vector pL4440 were used. Following 16 h incubation at 20°C, the survival rate was determined. Data represent means of at least three independent triplicate determinations (n≥120 animals, p values from Fisher test).

Figure 2. Determination of γ-glutamylcysteine, GSH and GSSG levels in lysate of synchronised 3-days-old worms.

(A) Worms were cultured on RNAi-plates containing (i) HT115 bacteria carrying empty pL4440 vector or (ii) HT115 bacteria carrying empty pL4440 vector plus 150 µM juglone or (iii) gsr-1(RNAi) feeding bacteria or (iv) gsr-1(RNAi) feeding bacteria plus 150 µM juglone. (B) Analysis of the respective GSH/GSSG ratios.

Figure 3. GSR-1 expression pattern under standard and stress conditions.

(A) The GSR-1 expression pattern of all stages was examined under standard culture conditions using the Pgsr-1::GFP worms. L4/young adults were transferred to (B) NGM plates containing 0.15 mM juglone. (C) Effect of dietary deprivation on gsr-1 promoter activity. Pgsr-1::GFP worms were grown under standard culture conditions until they reach the L4/young adult stage. Animals were then transferred to NGM agar plates without E. coli food for 16 h, before being analysed by fluorescence microscopy (quantified by ImageJ). Note the different scale bars for L1–L3 (50 µm), for L4 and adult (100 µm).

Figure 4. Northern blot analyses.

C. elegans N2 wild-type worms were cultured on NGM agar under standard conditions (lane 1) or exposed to 150 µM (lane 2) and 300 µM juglone for 4 h, before total RNA was isolated and applied to Northern blot analyses using a radiolabeled gsr-1 probe. Since there is only a 40 bp difference in transcript size (C46F11.2a, C46F11.2b1-3), only one band can be observed.

Figure 5. Juglone-induced GSR-1 expression is not regulated by DAF-16.

Worms carrying the Pgsr-1::GFP reporter construct were grown on HT115 bacteria carrying empty pL4440 vector (A, B) or subjected to daf-16(RNAi) (C, D). The respective effects on GFP-expression were monitored under standard culture conditions (A, C) or after induction by juglone (B, D) (quantified by ImageJ, Scale bars = 100 µm).

Figure 6. Regulation of gsr-1 promoter activity by SKN-1.

Worms carrying the Pgsr-1::GFP reporter construct were grown on HT115 bacteria carrying empty pL4440 vector (A, C) or subjected to skn-1(RNAi) (B, D). The respective effects on GFP-expression were monitored under standard culture conditions (A, B) or after induction by juglone (C, D) (quantified by ImageJ, Scale bars = 100 µm).

Figure 7. Starvation and regulation of gsr-1 promoter activity.

Worms carrying the Pgsr-1::GFP reporter construct were grown on HT115 bacteria carrying empty pL4440 vector (A and B) or subjected to skn-1(RNAi) (C and D) or daf-16(RNAi) (E and F). The respective effects on GFP-expression were monitored under standard culture conditions (A, C, E) or after hunger (B, D, F) (quantified by ImageJ, Scale bars = 100 µm).

Figure 8. Analyses of the interrelation between GSH synthesis and GSH redox cycling.

Applying RNAi, GCS-1 expression was suppressed in the C. elegans Pgsr-1::GFP reporter strain (left) or GSR-1 expression in the Pgcs-1::GFP (middle) or Pgst-4::GFP reporter strain (right). RNAi-treated worms were analysed by fluorescence microscopy and GFP-expression was compared with control worms grown on HT115 bacteria carrying empty pL4440 vector. Fluorescence intensities for Pgsr-1::GFP reporter strain were scored as low (A), medium (B) and high (C) according to . Fluorescence intensity scoring for Pgcs-1::GFP and Pgst-4::GFP reporter strains is given in Figure S4. Significant alterations in GFP expression was detected only in Pgcs-1::GFP (p<0.001) and Pgst-4::GFP worms (p<0.001) exposed to GSR-1(RNAi). Results are the means of at least three independent assays (n = 100 worms; p values from Fisher test).

Figure 9. Enhanced stress tolerance by GSR-1 overexpression.

L4/young adult of C. elegans overexpressing GSR-1::GFP fusion protein or of control worms expressing a GFP protein only were exposed to 0.25 mM juglone. Survival rate was determined after 16 h incubation. Owing to the pha-1(e2123) genetic background, assays were performed at 25°C. Results are means of three independent triplicate assays (n = 120 animals, Fisher test).

Figure 10. GSR-1 dependent life span.

(A) gsr-1(RNAi) resulted in a reduced life span (log-rank test p<0.001, results are cumulative from three independent experiments with 50 worms per trial), whereas (B) overexpression of GSR-1 led to longevity (p<0.001, results are cumulative from three independent experiments with 100 worms per trial). As control, pha-1 transgenic worms were used that express GFP in a similar expression pattern (see Material and Methods).