Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans

Abstract

Aging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma.

Keywords: aging; eukaryotic initiation factor 4E; mRNA decapping; mRNA translation; processing bodies; protein synthesis; proteostasis; stress response.

Graphical abstract

Figure 1

IFE-2 Colocalizes with PBs under Stress Conditions and during Aging (A) Representative images of transgenic animals co-expressing p_ife-2IFE-2::GFP and the PB-specific marker p_dcap-1DCAP-1::DsRed before and after heat shock at 35°C for 1.5 hr. The anterior part of the animal is shown; inlays indicate higher magnification of the pharyngeal region and the hypodermal tissue. Scale bars, 25 μm. White arrows indicate possible docking between PBs and SGs. (B) Two-dimensional (2D) intensity histograms with regression lines and Pearson’s coefficient (ρ_x,y) as quantitative representation of IFE-2::GFP and DCAP-1::DsRed colocalization within regions of interest in the pharynx of the animals (compare A) before and after heat shock. (C–E) Quantification of DsRed-tagged PB-specific signal in the pharyngeal region of transgenic animals during aging for p_lsm-3LSM-3::DsRed (C), p_edc-3EDC-3::DsRed (D), and PB number (E) for animals expressing p_dcap-1DCAP-1::DsRed. Error bars denote SEM; n = 10 animals per time point and trial. ^∗p < 0.01, ^∗∗p < 0.001, and ^∗∗∗p < 0.0001 (unpaired t test). (F) Expression of the full-length p_ife-2IFE-2::GFP reporter decreases during aging. Error bars show SEM; n = 40 animals per experiment. ^∗p < 0.01, ^∗∗p < 0.001, and ^∗∗∗p < 0.0001 (unpaired t test). (G) Percentage of animals forming SGs in various tissues during aging at day 3, day 8, and day 15 of adulthood. Error bars represent SEM; n > 50 animals per experiment. ^∗p < 0.01, ^∗∗p < 0.001, and ^∗∗∗p < 0.0001 (unpaired t test to measure statistical significance between day 8 or 15 in reference to day 3, respectively). See also Figures S1 and S2 and Videos S1 and S2.

Figure 2

Downregulation of mRNA Degradation Components Extends the Lifespan of Otherwise Wild-Type Animals but Does Not Affect the Lifespan of IFE-2-Depleted Mutants (A) Scheme displaying highly conserved key factors of bulk mRNA degradation as studied in various species including yeast, mammals, and C. elegans. Edc3 is a central scaffolding protein, responsible for the recruitment of the Dcp2 decapping enzyme and its coactivator Dcp1, the formation of the mRNA decay complex and contributes to PB assembly. Schematic adapted from Decker and Parker (2012). (B) Adult animal expressing p_edc-3EDC-3::DsRed and the pan-neuronal reporter p_unc-119GFP. Inlays are also shown at higher magnification. Arrows indicate localization of p_edc-3EDC-3::DsRed in neurons. Asterisks indicate areas of increased zoom. Scale bars, 25 μm. (C) Animals overexpressing EDC-3 (p_edc-3EDC-3, p_edc-3EDC-3::DsRed) under its endogenous promoter are short lived compared with wild-type nematodes. RNAi-mediated knockdown of edc-3 extends lifespan, though to a lesser extent than the edc-3 (ok1427) deletion allele. (D) Loss of the PB-specific factor LSM-3 leads to a significant lifespan extension, while the overexpression of p_lsm-3LSM-3::DsRed significantly shortens lifespan compared with the wild-type. (E) Downregulation of edc-3 further prolongs the lifespan of daf-2(e1370), dietary-restricted eat-2(ad465), and clk-1(e2519) mutants, compared with genetically identical control animals fed with bacteria harboring the RNAi empty vector. (F) Knockdown of edc-3 does not further increase the lifespan of long-lived mutants harboring the ife-2(ok306) allele. ife-2(ok306);edc-3 (ok1427) double mutants do not show significant lifespan extension compared with ife-2(ok306) mutants. (G) Downregulation of ife-2 by RNAi partially suppresses the short-lived phenotype of EDC-3-overexpressing animals. See also Figure S4 and Table S1.

Figure 3

EDC-3 Deficiency Promotes IFE-2 Entrapment in P Bodies and Reduces Protein Synthesis in Somatic C. elegans Tissues during Aging (A) The number of DCAP-1::DsRed-labeled PBs increases in edc-3 mutants compared with wild-type at older age. Error bars denote SEM; n = 25 animals per trial. ^∗p < 0.01, ^∗∗p < 0.001, and ^∗∗∗p < 0.0001 (unpaired t test between the edc-3 mutant and the age-matched wild-type or wild-type day 10 and day 17 compared with wild-type at day 3). (B) IFE-2::GFP intensity decreases substantially with age in both wild-type and edc-3-mutant background but remains higher in animals carrying the edc-3 (ok1427) allele. Error bars denote SEM; n = 25 animals per trial. ^∗p < 0.01, ^∗∗p < 0.001, and ^∗∗∗p < 0.0001 (unpaired t test between the edc-3 mutant and the age-matched wild-type or wild-type at day 10 and day 17 compared with wild-type at day 3 of adulthood). (C–E) Fluorescence recovery after photobleaching (FRAP) studies to measure de novo protein synthesis at various time points. The percentage of fluorescence recovery is calculated on the basis of average pixel intensity measured before bleaching (100%) and directly after bleaching the signal to about 20%–40% of the original intensity. Error bars in all studies denote SD. ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.0001 (multiple t tests with the Holm-Sidak method, in reference to the control with alpha = 5.000%). (C) Fluorescence recovery is monitored in wild-type animals and edc-3 mutants expressing the pan-neuronal reporter p_unc-119GFP. Percentage of fluorescence recovery in the area of the nerve ring is plotted against time for 2-day-old and 7-day-old adult animals (n = 24 animals). (D) Recovery of the fluorescent signal in all somatic tissues of wild-type and edc-3-mutant animals expressing p_ife-2GFP, with or without cycloheximide treatment, at L4 stage and day 5 of adulthood (n = 10 animals). (E) FRAP study in animals fed with control (empty vector), edc-3, or ife-2 RNAi, at L4 stage and day 5 of adulthood (n = 10 animals). (F) IFE-2::GFP sequestration in PBs is increased in EDC-3-deficient animals compared with wild-type controls as quantified by mean voxel intensity over time in 2-day-old and 6-day-old nematodes. Animals are constantly exposed to 34°C. Error bars denote SEM; n = 5 animals. (G) IFE-2::GFP sequestration in PBs at 5.5 min after constant exposure to heat stress at 34°C is markedly decreased in animals subjected to hsf-1 RNAi. Error bars denote SEM; n = 5 animals. ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.0001 (unpaired t test to compare edc-3(ok1427) mutant background with the control). See also Figure S4.

Figure 4

Longevity and Oxidative Stress Resistance in EDC-3-Depleted Animals Depend on SKN-1, DAF-16, and HSF-1 Transcription Factors (A) Knockdown of daf-16 shortens the lifespan of both wild-type and edc-3-mutant animals. (B) Knockdown of hsf-1 shortens the lifespan of edc-3 mutants below that of wild-type animals. (C) Downregulation of hsf-1 reduces the lifespan of ife-2 mutants to levels of respective controls. (D) Knockdown of skn-1 reduces the lifespan of edc-3 mutants below wild-type levels. (E) N-acetylcysteine (NAC) diminishes longevity conferred by EDC-3 deficiency. (F) Survival curves of EDC-3-deficient animals subjected to skn-1 knockdown, compared with skn-1-deficient worms, with or without NAC treatment. The log rank (Mantel-Cox) test shows a significant difference between edc-3(ok1427);skn-1(RNAi)-NAC and edc-3(ok1427);skn-1(RNAi)+NAC (^∗∗∗p < 0.0001), while the lifespan of wild-type treated with skn-1(RNAi) with or without NAC is not significantly different (p = 0.6534). (G) Quantification of DCAP-1::DsRed foci in wild-type and edc-3-mutant animals at day 8 of adulthood. Error bars show SEM; n = 25 animals per experiment. ^∗p < 0.05 and ^∗∗∗p < 0.001 (one-way ANOVA). N-acetylcysteine (NAC) treatment suppresses the increased abundance of PBs caused by skn-1 knockdown. (H) Knockdown of edc-3 increases the lifespan of short-lived mev-1 mutants that are sensitized to oxidative stress. (I) Survival of 7-day-old, EDC-3-deficient nematodes exposed to the oxidative phosphorylation inhibitor sodium azide. Error bars denote SEM; n > 80 per trial; ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.0001 (unpaired t test). (J) Survival of 7-day-old, EDC-3-depleted nematodes under oxidative stress induced by the herbicide paraquat. Error bars represent the SEM; n > 80 per trial; ^∗p < 0.05, ^∗∗p < 0.005, and ^∗∗∗p < 0.0001 (unpaired t test). See also Figure S5 and Table S1.

Figure 5

EDC-3 Functions in the Nervous System to Modulate Aging (A) Pan-neuronal expression of p_unc-119EDC-3 abrogates longevity of the edc-3(ok1427)-mutant strain. (B) RNAi-mediated knockdown of ife-2 increases the lifespan of animals bearing the edc-3(ok1427) mutation. (C) Pan-neuronal expression of p_unc-119EDC-3 in edc-3 mutants abrogates lifespan extension caused by downregulation of ife-2. (D) Representative confocal images of 8- and 20-day-old wild-type and edc-3(ok1427)-mutant animals expressing the pan-neuronal p_unc-119GFP reporter. Scale bars, 100 μm. (E) Corresponding quantification of fluorescence intensity at day 8 of adulthood. Error bars represent the SEM; n = 40 animals per experiment. ^∗∗∗p < 0.0001 (unpaired t test). (F) Quantification of defects in the ventral nerve cord (VNC) of wild-type and edc-3 mutants bearing the p_unc-119GFP transgene during aging. Wild-type worms display a higher frequency of ventral nerve cord defects compared with edc-3 mutants at day 20 of adulthood. Error bars denote SEM; n = 25 animals per experiment; ^∗∗∗p < 0.0001 (unpaired t test between edc-3[ok1427] and the age-matched control). (G) Representative images of the VNC in wild-type and edc-3 mutant worms at day 3 and day 20 of adulthood. Scale bars, 10 μm. (H) Proposed model for PB-mediated control of protein synthesis in the soma and its causative role in the modulation of aging. The PB-specific component EDC-3 influences mRNA translation by modulating mRNA decapping. Properly functioning decapping limits eIF4E localization to PBs. By contrast, stalled translation initiation complexes accumulating in EDC-3-deficient PBs and SGs cause eIF4E sequestration, lowering its availability for translation initiation. In turn, protein synthesis reduction leads to an orchestrated activation of the transcription factors SKN-1, HSF-1, and DAF-16, possibly via ROS signaling, mediating gene expression changes that increase longevity and stress resistance. See also Figure S6 and Table S1.