The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans

Abstract

The increased longevity of the C. elegans electron transport chain mutants isp-1 and nuo-6 is mediated by mitochondrial ROS (mtROS) signaling. Here we show that the mtROS signal is relayed by the conserved, mitochondria-associated, intrinsic apoptosis signaling pathway (CED-9/Bcl2, CED-4/Apaf1, and CED-3/Casp9) triggered by CED-13, an alternative BH3-only protein. Activation of the pathway by an elevation of mtROS does not affect apoptosis but protects from the consequences of mitochondrial dysfunction by triggering a unique pattern of gene expression that modulates stress sensitivity and promotes survival. In vertebrates, mtROS induce apoptosis through the intrinsic pathway to protect from severely damaged cells. Our observations in nematodes demonstrate that sensing of mtROS by the apoptotic pathway can, independently of apoptosis, elicit protective mechanisms that keep the organism alive under stressful conditions. This results in extended longevity when mtROS generation is inappropriately elevated. These findings clarify the relationships between mitochondria, ROS, apoptosis, and aging.

Figure 1

Whole genome expression profiling of isp-1 and nuo-6 mutants, and the wild type treated with 0.1mM paraquat. A) Venn diagrams illustrating the number of significantly up-regulated or down-regulated transcripts found in each condition tested when compared to untreated wild type. Bolded numbers represent the actual number of probes whose expression was significantly changed relative to wild type expression, while numbers in brackets represent the maximum number of different transcripts that could be detected as a result of high homology. B) Lifespan changes resulting from treatment of wild type and isp-1 mutants with RNAi against genes that are up-regulated in all three conditions and whose activities are expected to be involved in genome stability. The majority of genes had large effects on the mutant but no, or very little, effect on the wild type. Bars represent the degree of lifespan shortening relative to control and error bars represent SEM. See also Figures S1, S2, S3; Tables S1, S2, S3, S4 and S6. Numerical values and statistical analyses for all lifespan experiments are presented in Table S5. The GEO ascension number for all gene array data in this paper is GSE54024.

Figure 2

Genetic interactions between longevity and cell death genes. A) and B): effect of ced-4(n1162) on the survival of isp-1(qm150) and nuo-6(qm200). C) and D): effect of egl-1(n1084n3082) on the survival of isp-1 and nuo-6. E) and F): effect of ced-13(sv32) on the survival of isp-1 and nuo-6. G) Effects of ced-4 and ced-13 on isp-1 survival in the triple mutant combination. H) Effects of ced-4 and egl-1 on isp-1 survival in the triple mutant combination. I) Effects of ced-4 and ced-13 on nuo-6 survival in the triple mutant combination. J) Effects of egl-1 and ced-4 on nuo-6 survival in the triple mutant combination. See also Table S7; Figures S4 and S5. Numerical values and statistical analyses for all lifespan experiments are presented in Table S5.

Figure 3

Lifespan extension by 0.1mM paraquat (PQ) requires the intrinsic apoptosis pathway. A) Effect of 0.1mM PQ treatment on the wild type. Effects of 0.1mM PQ treatment on: B) ced-4(n1162), C) ced-9(n1950gf), D) ced-3(n717), E) ced-13(sv32) and F) egl-1(n1084n3082). See also Table S5. Numerical values and statistical analyses for all lifespan experiments are presented in Table S5.

Figure 4

The behavioural and growth defects of isp-1(qm150) and nuo-6(qm200) mutants are partially suppressed by ced-4(n1162) and ced-13(sv32) but not egl-1(n1084n3082). A) Pharyngeal pumping rate. isp-1 and nuo-6 pump at a significantly slower rate than the wild type. Loss of ced-4 or ced-13 but not egl-1 partially rescues the slow pumping rates of isp-1 and nuo-6. None of the cell death genes affects the pumping rate of the wild type. B) Defecation cycle length. isp-1 and nuo-6 mutants have a significantly lengthened defecation cycle length. Loss of ced-4 or ced-13 but not egl-1 partially rescues the slow defecation phenotype of isp-1 and nuo-6. None of the cell death genes affect the defecation cycle length of the wild type. C) Thrashing rate. isp-1 and nuo-6 mutants have a significantly decreased rate of thrashing. Loss of ced-4 or ced-13 but not egl-1 partially rescues the slow thrashing phenotype of isp-1 and nuo-6. None of the cell death genes affect the thrashing rate of the wild type. D) Brood size (the number of progeny produce by self-fertilization of a single hermaphrodite). Both isp-1 and nuo-6 have significantly reduced brood sizes. The reduction in brood size was enhanced by loss of ced-4 but not ced-13 or egl-1. Loss of ced-4, and to a lesser degree egl-1, also significantly reduced brood size the wild-type background. E) Length of Embryonic Development. The time taken for a 2-cell stage embryo to reach hatching is significantly increased in isp-1 and nuo-6 mutants. Loss of ced-4 and ced-13 but not egl-1 partially rescues this phenotype of isp-1 and nuo-6. None of the cell death genes affect the rate of embryonic development of the wild type. F) Length of post-embryonic development. The time taken for freshly hatched L1-stage larva to reach the young adult stage is significantly increased in isp-1 and nuo-6 mutants. Loss of ced-4 and ced-13 but not egl-1 partially rescues this phenotype of isp-1 and nuo-6. None of the cell death genes affect the rate of post-embryonic development of the wild type. Bars represent the mean value of 25 animals. Error bars represent standard error of the mean. Significance was determined using a Student’s t-test (*** denotes P < 0.0001 as compared to the wild type; # denotes P < 0.0001 as compared to either isp-1 or nuo-6 single mutants). Complete numerical values and statistics are provided in Table S5.

Figure 5

Effects of isp-1, nuo-6 and cell death genes on ATP levels and survival under heat stress. A) isp-1 and nuo-6 mutants, but not ced-4, ced-13 or egl-1 mutants exhibit reduced ATP levels when grown under standard conditions (20°C). Acute exposure (1.5h) to heat (37°C) reduces the ATP levels of all genotypes. B) Loss of ced-4, ced-13 or egl-1 does not affect ATP levels in isp-1 mutants at 20°C. However, the reduction in ATP levels after heat stress is significantly reduced in ced-4;isp-1 and isp-1;ced-13 but not egl-1;isp-1 double mutants compared to isp-1(qm150). C) Mutations in ced-4, ced-13 and egl-1 do not affect ATP levels in nuo-6 mutants at 20°C. However, the reduction in ATP levels after heat stress is significantly less in ced-4;nuo-6 and nuo-6;ced-13 but not egl-1;isp-1 double mutants compared to nuo-6(qm200). D) Exposure to heat stress for 4h significantly decreases the survival of all genotypes, but much more severely for isp-1(qm150) and nuo-6(qm200) mutants. However, loss of ced-4 or ced-13 but not egl-1 strongly rescues the survival of isp-1 and nuo-6 mutants. Significance was determined using a Student’s t-test (a) * denotes P < 0.05, *** denotes P < 0.0001 as compared to the wild type. # denotes P < 0.05 compared to the control at 20°C, ## denotes P < 0.05 compared to the wild type at 37°C. ### denotes P < 0.005 compared to the wild type at 37°C. (b) *** denotes P < 0.0005 relative to the wild type control, ## denotes P < 0.05 relative to isp-1(qm150) at 37°C. (c) * denotes P < 0.05 as compared to the wild type control at 20°C, *** denotes P < 0.001 as compared to the wild type control at 37°C, ## denotes P < 0.005 as compared to the nuo-6(qm200) at 37°C. Complete numerical values and statistics are provided in Table S5.

Figure 6

Epistatic relationships between genotypes and treatments. A) Treatment of isp-1;ced-13 with 0.1mM PQ rescues lifespan to the isp-1 level (n>50, P < 0.0001 for the difference between treated and untreated double mutants). B) Treatment with 0.1mM PQ does not affect the defecation of isp-1 ced-4 but partially restores the defecation of isp-1;ced-13 toward the isp-1 level (n=25). C) Treatment with 0.1mM PQ does not affect the pumping rate of isp-1 ced-4 but partially restores isp-1;ced-13 pumping toward the isp-1 level (n=10). D) Treatment with 0.1mM PQ does not affect the trashing rate of isp-1 ced-4 but partially restores isp-1;ced-13 thrashing toward the isp-1 level (n =15). F) Treatment with 0.1mM and 0.15mM PQ decreases the acute survival of isp-1;ced-13 worms but not of isp-1 ced-4 at 37°C (for 4 hours). Significance for all experiments was determined using the Student’s t-test (* denotes P < 0.05, ** denotes P < 0.01, *** denotes P < 0.001. G) Treatment with 0.1mM PQ increases wild type lifespan but not sod-3(tm783) lifespan (n =150, P <0.0001 for the difference between the wild type and sod-3 treated with PQ). Error bars represent mean + SEM. See also Figure S6. Complete numerical values and statistics are provided in Table S5.

Figure 7

A model for the regulation of lifespan by mtROS signaling through the intrinsic apoptosis pathway. The intrinsic apoptosis pathway (composed of CED-9, CED-4 and CED-3) is sensitive to mtROS from the ETC when it is activated by the alternative BH3-only protein CED-13. Mitochondrial dysfunction leads to an increase in mtROS which activates the CED signalling pathway to reduce ATP usage and redistribute it to protective rather than active functions. We propose that the mitochondrial dysfunction in isp-1(qm150) and nuo-6(qm200) mutants induces the mutant phenotypes, including longevity, both by directly lowering ATP generation and by stimulating mtROS signaling to alter ATP usage. In the wild type this mechanism could provide a protective role in case of transient mitochondrial dysfunction or nutrient shortage. In the mutants its continuous action leads to the mutant phenotypes, including longevity.