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. 2021 Mar 15;12(1):1678.
doi: 10.1038/s41467-021-21743-x.

Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation

Maxime J Derisbourg #  1 , Laura E Wester #  1 , Ruth Baddi  1 , Martin S Denzel  2   3   4
Affiliations

Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation

Maxime J Derisbourg et al. Nat Commun. .

Abstract

Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Unbiased forward longevity screen in C. elegans identifies mutations in ISR components.
a Mutagenesis screening strategy. b Mean lifespan extension (normalized to temperature-sensitive sterile CF512 control) visualized as the number of tested genomes in 3% bins. c Schematic representation of identified ISR genes and corresponding longevity alleles. d Cartoon depiction of mRNA translation initiation and the ISR. e Survival of outcrossed ppp-1(wrm10) and ppp-1(wrm15) mutants compared to WT controls (representative data from n = 3 independent experiments). f Survival of CRISPR/Cas9-generated ppp-1 alleles syb691 and syb728 compared to WT controls (representative data from n = 2 independent experiments). syb691 corresponds to wrm10 and syb728 to wrm15. g Survival of outcrossed gcn-2(wrm4), pek-1(wrm7), and gcn-2(wrm4); pek-1(wrm7) double mutants compared to WT controls (representative data from n = 2 independent experiments). h Survival of outcrossed iftb-1(wrm53) mutants compared to WT controls (representative data from n = 2 independent experiments). i Thermotolerance assays of day 1 ppp-1 mutant worms compared to WT controls (error bars represent means ± SD, two-way ANOVA Dunnett’s post hoc test with **p = 0.0032 ppp-1(wrm10) vs. WT at the 6 h time point; **p = 0.0039 ppp-1(wrm15) vs WT at the 6 h time point; *p = 0.0107 ppp-1(wrm10) vs. WT at the 8 h time point; *p = 0.0404 ppp-1(wrm15) vs. WT at the 8 h time point; n = 4 independent experiments with 50 animals each). j Motility assay using day 8 WT animals and ppp-1 mutants with an unc-54P-driven muscle-specific expression of polyQ35–YFP fusion protein (error bars represent means ± SD, one-way ANOVA Dunnett’s post hoc test with ***p < 0.001 vs. WT controls; n = 3 independent experiments with ≥12 animals each). k Motility assay using day 7 WT animals and ppp-1 mutants with an unc-54P-driven muscle-specific expression of α-synuclein (error bars represent means ± SD, one-way ANOVA Dunnett’s post hoc test with ***p < 0.001 vs. WT controls; n = 3 independent experiments with 15 worms each). See Supplementary Dataset 1 for survival statistics. See Supplementary Table 1 for statistics on thermotolerance assays. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Mutations in ppp-1 do not attenuate mRNA translation.
a Puromycin incorporation followed by Western blot analysis using antibodies detecting puromycin and α-tubulin in day 1 WT animals, iftb-1(wrm53) mutants, and control rsks-1(sv31) mutants. Images of the same membrane are shown with short and long exposures (error bars represent means + SEM, one-way ANOVA Dunnett’s post hoc test with **p = 0.0023 iftb-1(wrm53) vs. WT and ***p < 0.001 rsks-1(sv31) vs. WT; n = 3 independent experiments). b Puromycin incorporation assay in day 1 WT animals, ppp-1 mutants, and rsks-1(sv31) controls as described in (a). Images of the same membrane are shown with short and long exposures (error bars represent means + SEM, one-way ANOVA Dunnett’s post hoc test with ***p < 0.001 vs. WT; n = 6 independent experiments). c Quantification of methionine 35S labeling of day 1 WT worms, ppp-1 mutants, and control rsks-1(sv31) mutants (error bars represent means + SEM, one-way ANOVA Dunnett’s post hoc test with ***p < 0.001 vs. WT; the number of independent experiments (n) indicated in the figure). d, e Polysome profiling, and quantification of day 1 WT and ppp-1 animals. Quantification represents the relative abundance of ribosomal subunits (40S, 60S), monosomes (mono), and polysomes (poly; error bars represent means + SD, two-way ANOVA Dunnett’s post hoc test; n = 4 independent experiments). Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Translational efficiency is altered in ppp-1 mutants.
a Polysome sequencing strategy. b Volcano plot of polysome-associated mRNAs normalized to total mRNA levels between WT animals and ppp-1 mutants. All displayed mRNAs were found in both ppp-1 mutants. Mean p-values and mean log-2-fold changes of both ppp-1 mutants were used (Student’s two-sided t test, significance is reached for p < 0.05). FC fold change. The full dataset is in Supplementary Dataset 2. c DAVID gene ontology (GO) analysis of significantly changed mRNAs shown in (b). Processes involved in phosphorylation are highlighted in purple. d RNAi screen for suppressors of polyQ35; ppp-1(wrm10) motility. For reliability, assays of polyQ35 WT animals and polyQ35; ppp-1(wrm10) mutants on luciferase RNAi were performed four times (error bars represent means + SD). Bars highlighted in yellow indicate RNAi treatments with a reduction of motility of at least 50% compared to the luciferase control treatment of polyQ35; ppp-1(wrm10) worms. e Motility assays of day 8 polyQ35 transgenic animals, polyQ35; ppp-1(wrm10) and polyQ35; ppp-1(wrm15) mutants after indicated RNAi treatments (error bars represent means ± SD, one-way ANOVA Kruskal Wallis test with ***p < 0.001 C01A2.5 vs. Luc, *p = 0.0282 D1014.3 vs. Luc, **p = 0.0074 M04F3.3/kin-35 vs. Luc, and *p = 0.0361 Y18D10a.10 vs. Luc in polyQ35; ppp-1(wrm10) animals (left panel) and *p = 0.0397 C01A2.5 vs. Luc, **p = 0.0036 M04F3.3/kin-35 vs. Luc, and ***p < 0.001 K04C2.3 vs. Luc in polyQ35; ppp-1(wrm15) animals (right panel); n = 1 with ≥14 animals per treatment). f Survival of WT animals and ppp-1 mutants upon M04F3.3/kin-35 or control luciferase RNAi knockdown (representative data from n = 2 independent experiments). g Survival of kin-35 over-expressing worms (line 1) compared to respective control animals without the extrachromosomal array (representative data from n = 2 independent experiments). h Thermotolerance assay of day 1 kin-35 over-expressing mutants (line 1) compared to respective controls as described in (g) (representative data from n = 2 independent experiments with ≥30 worms each). OE overexpressor. See Supplementary Dataset 1 for survival statistics. See Supplementary Table 1 for statistics on thermotolerance assays. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Mutations in ppp-1 reduce the ISR upon stress.
a Survival of WT animals and ppp-1 mutants upon RNAi treatment targeting ppp-1 or control luciferase (representative data from n = 2 independent experiments). b Survival of heterozygous ppp-1 mutants compared to WT control animals. c Fluorescence images of day 1 WT animals and ppp-1 mutants in the atf-4P::GFP::unc-54 3′UTR reporter background. Worms were treated with DMSO only (control) or with the indicated tunicamycin (TM) concentrations for 6 h each (scale bar 75 µm; representative data from n = 3 independent experiments with ≥7 animals each). d Quantification of GFP intensity in (c). Error bars represent means ± SD, two-way ANOVA Dunnett’s post hoc test with ***p < 0.001 vs. WT (n = 3 independent experiments with ≥7 animals each). See Supplementary Dataset 1 for survival statistics. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Estradiol valerate inhibits the ISR and extends lifespan.
a Fluorescence images of atf-4P::GFP::unc-54 3′UTR reporter animals grown on NGM plates supplemented with 10 µg/mL tunicamycin (TM) and with the indicated compounds (20 µM) or 1% DMSO vehicle control (Est Val = estradiol valerate, Propa = propafenone hydrochloride, Aza = azadirachtin). Scale bar is 75 µm, n = 1. b Survival of WT worms treated with 1% DMSO (control) or 20 µM estradiol valerate from day 1 (D1), day 5 (D5), or day 10 (D10) (representative data from n = 2 independent experiments). c Representative Western blot of day 1 worms treated with 1% DMSO (control) or 20 µM estradiol valerate. Worms were incubated without (−) or with 5 mM DTT (+) for 2 h. Levels of phospho-eIF2α (Ser51) were normalized to α-tubulin (error bars represent means +SEM, one-way ANOVA Dunnett’s post hoc test, **p = 0.0044 DTT vs. DMSO; ns = not significant vs. DMSO; n = 7 independent experiments). d Survival of WT worms treated with 1% DMSO (control) or 20 µM estradiol valerate from day 1 of adulthood upon RNAi treatment targeting kin-35 or control luciferase (representative data from n = 2 independent experiments). e Survival of ppp-1 mutants and WT controls treated with 1% DMSO (control) or 20 µM estradiol valerate from day 1 of adulthood (representative data from n = 2 independent experiments). See Supplementary Dataset 1 for survival statistics. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Direct ISR inhibition through phospho-defective eIF2αS51A mutations extends lifespan via eIF2B.
a Western blot of day 1 WT animals and eIF2αS51A mutants using anti-phospho-eIF2α (Ser51) and anti-α-tubulin antibodies. b Developmental tunicamycin (TM) resistance assay of WT animals and eIF2αS51A mutants treated with tunicamycin at the indicated concentrations (error bars represent means + SEM, two-way ANOVA Sidak’s post hoc test, ***p < 0.001 vs. WT at respective tunicamycin concentration; n = 4 independent experiments with ≥20 animals each). c Survival of eIF2αS51A mutants compared to WT control animals (representative data from n = 4 independent experiments). d Thermotolerance assays of eIF2αS51A mutants compared to WT animals (error bars represent means ± SD, two-way ANOVA Sidak’s post hoc test with ***p < 0.001 vs. WT controls; n = 4 independent experiments with 50 animals each). e, f Polysome profiling and quantification of day 1 WT worms and eIF2αS51A mutants (error bars represent means + SD, two-way ANOVA Dunnett’s post hoc test; n = 4 independent experiments). g Survival of WT worms and eIF2αS51A mutants upon RNAi treatment targeting kin-35 or control luciferase (representative data from n = 4 independent experiments). h Survival of WT animals and eIF2αS51A mutants upon RNAi treatment targeting ppp-1 or control luciferase (representative data from n = 2 independent experiments). i Survival of heterozygous phospho-mimic eIF2αS51D/+ mutants compared to WT control animals (representative data from n = 2 independent experiments). See Supplementary Dataset 1 for survival statistics. See Supplementary Table 1 for statistics on thermotolerance assays. Source data are provided as a Source Data file.
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