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Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans

Nature volume 521, pages 525–528 (2015)Cite this article

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Abstract

Impaired mitochondrial maintenance in disparate cell types is a shared hallmark of many human pathologies and ageing1,2,3,4,5,6,7,8. How mitochondrial biogenesis coordinates with the removal of damaged or superfluous mitochondria to maintain cellular homeostasis is not well understood. Here we show that mitophagy, a selective type of autophagy targeting mitochondria for degradation, interfaces with mitochondrial biogenesis to regulate mitochondrial content and longevity in Caenorhabditis elegans. We find that DCT-1 is a key mediator of mitophagy and longevity assurance under conditions of stress in C. elegans. Impairment of mitophagy compromises stress resistance and triggers mitochondrial retrograde signalling through the SKN-1 transcription factor that regulates both mitochondrial biogenesis genes and mitophagy by enhancing DCT-1 expression. Our findings reveal a homeostatic feedback loop that integrates metabolic signals to coordinate the biogenesis and turnover of mitochondria. Uncoupling of these two processes during ageing contributes to overproliferation of damaged mitochondria and decline of cellular function.

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Figure 1: Selective mitochondrial autophagy regulates mitochondrial content and morphology.
Figure 2: Mitophagy is required for longevity under conditions of low insulin/IGF-1 signalling or impaired mitochondrial function.
Figure 3: Mitophagy deficiency compromises stress resistance and impairs mitochondrial function.
Figure 4: Mitophagy deficiency engages the mitochondrial retrograde signalling pathway through activation of SKN-1.

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Acknowledgements

We thank A. Pasparaki for technical support with experiments, N. Kourtis for the GCaMP2.0-expressing C. elegans strain, N. Charmpilas for the neuronal CTS-1::mCherry-expressing C. elegans strain and the unc-43 RNA interference (RNAi) plasmid, and K. Kounakis for the rpn-6 RNAi plasmid. We thank B. P. Braeckman for the Hyper-expressing C. elegans strain and D. Sieburth for the INVOM::RFP-expressing C. elegans strain. We thank R. Devenish for providing the pAS1NB-CS-Rosella plasmid. Mass spectrometry analysis was performed at the Institute of Molecular Biology and Biotechnology Proteomics Facility. We are grateful to M. Aivaliotis for the characterization and relative quantification of DCT-1 tryptic peptides by nanoflow liquid chromatography with tandem mass spectrometry. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources of the National Institutes of Health, and S. Mitani (National Bioresource Project) in Japan. We thank A. Fire for plasmid vectors. This work was funded by grants from the European Research Council, the European Commission 7th Framework Programme and the Greek General Secretariat for Research and Technology.

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Authors and Affiliations

  1. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Nikolaou Plastira 100, Heraklion, 70013, Crete, Greece

    Konstantinos Palikaras, Eirini Lionaki & Nektarios Tavernarakis

  2. Department of Biology, University of Crete, Heraklion, 70013, Crete, Greece

    Konstantinos Palikaras

  3. Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, 71110, Crete, Greece

    Nektarios Tavernarakis

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  1. Konstantinos Palikaras
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Contributions

K.P., E.L. and N.T. designed and performed experiments. K.P. and N.T. analysed data and wrote the manuscript.

Corresponding author

Correspondence to Nektarios Tavernarakis.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to N.T. (tavernarakis@imbb.forth.gr).

Extended data figures and tables

Extended Data Figure 1 Mitochondrial accumulation during ageing and upon mitophagy depletion.

a, Stained wild-type animals with MitoTracker Red FM dye were monitored throughout adulthood for mitochondrial content. Indicative images are shown on the right for days 1, 3, 5 and 10 (n = 90; ***P < 0.001; one-way ANOVA). b, Total worm lysates from transgenic animals expressing a mitochondria-tagged GFP in the intestine on day 1, 3, 5 and 10 of adulthood were loaded on Tricine-SDS–PAGE and immunoblotted against the indicated proteins. c, Transgenic animals expressing mitochondria-targeted GFP in the intestine were monitored throughout adulthood for mitochondrial content. (n = 100; ***P < 0.001; one-way ANOVA). d, Transgenic animals expressing mitochondria-targeted mCherry in neurons were monitored throughout adulthood for mitochondrial content. Indicative images are shown on the right for days 1, 3, 5 and 10 (n = 70; ***P < 0.001; one-way ANOVA). e, mRNA levels of dct-1 in wild-type animals, dct-1(tm376) mutants, dct-1(RNAi) animals, daf-2(e1370) and daf-16(mu86) mutants. Expression of dct-1 is transcriptionally regulated by DAF-16 (***P < 0.001; one-way ANOVA). f, Efficacy of dct-1 silencing by RNAi. Transgenic animals expressing a full-length pdct-1DCT-1::GFP reporter fusion subjected to dct-1 RNAi. g, mRNA levels in ges-1 and myo-3 in wild-type animals, dct-1(RNAi) and pink-1(RNAi) animals. Expression of ges-1 and myo-3 is not changed upon mitophagy inhibition (NS, P > 0.05; one-way ANOVA). h, Mutant dct-1(tm376) animals expressing mitochondria-targeted GFP in body wall muscle cells display increased mitochondrial mass (n = 100; ***P < 0.0001; unpaired t-test). Scale bars, 100 μm. Images were acquired using a ×5 objective lens. i, Knockdown of either DCT-1 or PINK-1 increases mitochondrial mass in worms that express a mitochondria-targeted GFP in body wall muscles (n = 120; ***P < 0.001; one-way ANOVA). Anterior, left; posterior, right. j, Stained wild-type animals and dct-1(tm376), pink-1(tm1779) and pdr-1(gk448) mutants with MitoTracker Red FM dye were monitored for mitochondrial content (n = 120; ***P < 0.001; one-way ANOVA). Scale bars, 20 μm. Images were acquired using a ×10 objective lens. k, Mitochondrial network morphology is altered in dct-1(tm376) mutants. The mitochondrial network in wild-type animals is well-organized and runs parallel with the myofilament lattice. By contrast, dct-1(tm376) mutant animals display fragmented and disorganized mitochondrial network morphology. Scale bar, 20 μm. Images were acquired using a ×40 objective lens. Error bars, s.e.m.

Extended Data Figure 2 DCT-1 expression and sub-cellular localization.

a, DCT-1 is the homologue of the mammalian BNIP3 and NIX/BNIP3L in C. elegans. Alignment of C. elegans DCT-1, M. musculus BNIP3 and M. musculus NIX/BNIP3L proteins. The predicted transmembrane domain of DCT-1, BNIP3 and NIX/BNIP3L is depicted in green. The conserved WXXL motif that is essential for interaction with the autophagosomal protein LC3/Atg8 is shown in red. The conserved MER and BH3 domains are depicted in blue and yellow, respectively. b, Spatiotemporal expression and sub-cellular localization of DCT-1. Images of transgenic animals expressing a full-length pdct-1DCT-1::GFP reporter fusion. Expression of dct-1 occurs in all somatic tissues of adult animals, including neurons, the pharynx, the intestine, body wall muscles and vulva muscles (indicated by the arrows). Scale bars, 50 μm. c, Expression is detectable in embryos and remains high during all postembryonic developmental stages, throughout adulthood. Scale bars, 50 μm. Images were acquired using × 10 and × 40 objective lenses. d, e, DCT-1::GFP co-localizes with mitochondria in different tissues. Transgenic animal expressing a full-length pdct-1DCT-1::GFP reporter fusion localizing in mitochondria. Animals were stained with Mitotracker Red FM (Invitrogen, Molecular Probes). Scale bars, 20 μm. Images were acquired using a ×100 objective lens. f, Transgenic animal co-expressing a full-length pdct-1DCT-1::GFP reporter fusion together with pmyo-3INVOM::RFP outer mitochondrial membrane reporter. DCT-1::GFP co-localizes with INVOM::RFP, demonstrating that DCT-1 is an outer mitochondrial membrane protein. Scale bars, 10 μm and 5 μm. Images were acquired using a ×100 objective lens. g, Isolated mitochondria from transgenic animals expressing a full-length pdct-1DCT-1::GFP reporter fusion were untreated (lane 1, M), treated with proteinase K (lane 2, MPK), or treated with sodium carbonate (lanes 3 and 4) to separate soluble from membrane material. DCT-1::GFP is accessible and degraded by proteinase K while HSP-60, a mitochondrial matrix protein, is protected. Soluble (lane 3, S) and membrane (lane 4, P) fractions after carbonate extraction are separated by ultracentrifugation. DCT-1::GFP is detected in the membrane fraction (P) while soluble HSP-60 protein is found in the soluble material, indicating that DCT-1 is an integral membrane protein.

Extended Data Figure 3 Mitophagy inhibition does not alter intestinal or muscle cell mitochondrial network morphology of FZO-1- and DRP-1-depleted animals.

a, Mitophagy inhibition by RNAi against dct-1, pink-1 and pdr-1 does not affect mitochondrial network morphology in FZO-1- and DRP-1-depleted animals that express a mitochondria-targeted GFP in the intestine. The mitochondrial network in wild-type animals is well-organized and highly connected. By contrast, animals subjected to RNAi against dct-1, pink-1, pdr-1 and fzo-1 display fragmented and disorganized mitochondrial network morphology. Simultaneous knocking down of fzo-1/dct-1, pink-1/fzo-1 and fzo-1/pdr-1 does not alter the already fragmented mitochondrial network of FZO-1-deficient worms. DRP-1-depleted animals display disorganized aggregated and globular mitochondria compared with wild-type worms. Simultaneous knocking down of drp-1/dct-1, pink-1/drp-1 and pdr-1/drp-1 does not change the impaired mitochondrial network of DRP-1-depleted animals. Scale bar, 20 μm. Images were acquired using a ×40 objective lens. b, Transgenic animals expressing a mitochondria-targeted GFP in muscle cells subjected to RNAi against dct-1, pink-1, pdr-1 and fzo-1. The mitochondrial network in wild-type animals is well organized and runs parallel with the myofilament lattice. By contrast, DCT-1, PINK-1, PDR-1 and FZO-1 depleted animals display fragmented and disorganized mitochondrial network morphology. Simultaneous knocking down of fzo-1/dct-1, pink-1/fzo-1 and fzo-1/pdr-1 does not alter the already fragmented mitochondrial network of FZO-1-deficient worms (n = 120; ***P < 0.001 and NS, P > 0.05; one-way ANOVA). c, DRP-1-deficient animals present disorganized and aggregated mitochondrial morphology. Simultaneous knocking down of drp-1/dct-1, pink-1/drp-1 and pdr-1/drp-1 does not change the impaired mitochondrial network of DRP-1-depleted animals (n = 100; ***P < 0.001 and NS, P > 0.05; one-way ANOVA). Error bars, s.e.m.

Extended Data Figure 4 Μitophagy is induced under several stress conditions.

a, DCT-1 specifically localizes on the outer mitochondrial membrane (shown for muscle cells), where it co-localizes with the autophagosomal protein LGG-1/Atg8. Confocal images of body wall muscle cells of wild-type animals expressing fluorescently tagged DCT-1::GFP along with the autophagosomal marker LGG-1/Atg8 (tagged with DsRed). b, DCT-1 co-localizes with the E3 ubiquitin ligase PDR-1/Parkin. Confocal images of body wall muscle cells in wild-type animals expressing fluorescently tagged DCT-1::GFP and PDR-1::DsRed. Scale bar, 20 μm. Images were acquired using a ×40 objective lens. c, C. elegans transgenic embryos expressing a full-length plgg-1GFP::LGG-1 fusion protein indicative of autophagic activity subjected to single daf-2, dct-1, pink-1 RNAi or subjected simultaneous daf-2/dct-1 and pink-1/daf-2 RNAi (n = 70; NS, P > 0.05, ***P < 0.001; one-way ANOVA). d, C. elegans transgenic animals expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity subjected to single daf-2, dct-1, pink-1, pdr-1 RNAi or subjected simultaneous daf-2/dct-1, pink-1/daf-2, daf-2/pdr-1 RNAi. Autophagosome number monitored in the pharynx of the animals (n = 100; NS, P > 0.05, ***P < 0.001; one-way ANOVA). e, The daf-2(e1370) mutant subjected to RNAi against dct-1, pink-1, pdr-1 and lgg-1 or subjected simultaneous against lgg-1/dct-1, pink-1/lgg-1, lgg-1/pdr-1 RNAi. Lifespan values are given in Supplementary Table 1; assays were performed at 20 °C. f, Transgenic animals expressing the mtRosella biosensor in body wall muscle cells, were subjected to daf-2(RNAi), treated with paraquat or CCCP and exposed to heat stress (37 °C). Mitophagy stimulation is signified by the reduction of the ratio between pH-sensitive GFP to pH-insensitive DsRed. DCT-1, PINK-1 and PDR-1 are required for mitophagy. Mitophagy is not activated under stress conditions when transgenic animals are subjected to RNAi against dct-1, pink-1 and pdr-1 (n = 120; NS, P > 0.05, ***P < 0.001; one-way ANOVA). Scale bars, 20 μm. Images were acquired using a ×10 objective lens. g, Mitophagy is not further reduced under oxidative stress conditions in transgenic animals subjected to RNAi against dct-1, pdr-1 or subjected to simultaneous pdr-1/dct-1 RNAi (NS, P > 0.05, ***P < 0.001; one-way ANOVA). h, Mitophagy inhibition under stress conditions in transgenic animals subjected to RNAi against lgg-1 (NS, P > 0.05, ***P < 0.001; one-way ANOVA). Error bars, s.e.m.

Extended Data Figure 5 Monitoring mitophagy in vivo.

Transgenic animals co-expressing a mitochondria-tagged GFP (mtGFP) in body wall muscle cells and the autophagosomal protein LGG-1 fused with DsRed, were exposed to heat stress (37 °C), treated with CCCP, paraquat or subjected to RNAi against daf-2. Mitophagy induction is signified by co-localization of GFP and DsRed signals (for each group of images mitochondria are shown in green on top, autophagosomes in red below, with a merged image at the bottom). a, DCT-1 and PINK-1 are required for mitophagy. Mitophagy inhibition under stress conditions when transgenic animals are subjected to RNAi against dct-1 and pink-1. Scale bars, 20 μm. Images were acquired using a ×40 objective lens. b, High-magnification images showing induction of mitophagy. Scale bars, 10 μm. Images were acquired using a ×40 objective lens.

Extended Data Figure 6 Mitochondrial content is not affected upon mitochondrial dysfunction.

a, Knockdown of either PHB-1, PHB-2, CLK-1 or ISP-1 does not affect the mitochondrial mass in transgenic worms that express a mitochondria-targeted GFP in the intestine (n = 100; NS, P > 0.05; one-way ANOVA). b, c, Simultaneous RNAi against phb-1/dct-1, phb-1/pink-1, phb-1/pdr-1, phb-2/dct-1, pink-1/ phb-2, phb-2/pdr-1, isp-1/dct-1, pink-1/isp-1, pdr-1/isp-1, clk-1/dct-1, pink-1/clk-1, clk-1/pdr-1 increases mitochondrial mass (n = 100; ***P < 0.001; one-way ANOVA). d, Knockdown of either DCT-1, PINK-1 or PDR-1 increases mitochondrial mass in transgenic worms that express a mitochondria-targeted GFP in the intestine. Transgenic worms expressing mitochondria-tagged GFP in the intestine were raised over one generation in the presence of EtBr to block mitochondrial biogenesis. Knockdown of either DCT-1, PINK-1 or PDR-1 increases mitochondrial content of animals exposed to EtBr (n = 100; ***P < 0.001; one-way ANOVA). e, Knockdown of either PBS-5 or RPN-6 promotes fragmentation of mitochondrial network and decreases mitochondrial content in worms that express a mitochondria-targeted GFP in the intestine (n = 100; ***P < 0.001; one-way ANOVA). Scale bar, 20 μm. Images were acquired using a ×40 objective lens. f–h, The expression levels of ges-1, myo-3 and unc-119 gene are relatively stable throughout ageing. Inhibition of autophagy or proteasome system does not alter the expression levels of ges-1, myo-3 and unc-119 genes during ageing (NS, P > 0.05, **P < 0.01; one-way ANOVA). Error bars, s.e.m.

Extended Data Figure 7 Gene ced-9 acts in the same genetic pathway with dct-1, pink-1 and pdr-1 to mediate mitophagy.

a, CED-9 depletion does not affect the localization of DCT-1. Scale bars, 20 μm. Images were acquired using a ×63 objective lens. b, Knockdown of either CED-9, DCT-1, PINK-1 or PDR-1 increases mitochondrial mass in transgenic worms expressing a mitochondria-targeted GFP in the intestine. Simultaneous RNAi against ced-9/dct-1, pink-1/ced-9 and ced-9/pdr-1 does not increase further mitochondrial mass (n = 100; NS, P > 0.05, ***P < 0.001; one-way ANOVA). c, Transgenic animals expressing the mtRosella biosensor in body wall muscle cells, were treated with paraquat. Mitophagy induction is signified by the reduction of the ratio between pH-sensitive GFP to pH-insensitive DsRed. CED-9 is required for mitophagy. Mitophagy inhibition under stress conditions when transgenic animals are subjected to RNAi against ced-9 (n = 120; NS, P > 0.05, ***P < 0.001; one-way ANOVA). Error bars, s.e.m. d, Mitophagy inhibition under stress, in transgenic animals subjected to RNAi against ced-9. Scale bars, 20 μm. Images were acquired using a ×40 objective lens.

Extended Data Figure 8 Inhibition of mitophagy affects longevity in a genetic-background-specific manner.

a, Depletion of PDR-1, or PINK-1. b, c, Depletion of both DCT-1 and PINK-1. d, e, Depletion of both PDR-1 and PINK-1, f, Depletion of both DCT-1 and PDR-1, Knockdown of DCT-1 shortens the lifespan of long-lived clk-1(e2519) (g), and eat-2(ad465) (h) mutants. i, j, Mitochormesis is not engaged in mitophagy-deficient animals to influence lifespan. Depletion of either DCT-1, PINK-1 or PDR-1, with, or without NAC or BHA treatment. k, Depletion of DCT-1, PINK-1 or PDR-1, with, or without EGTA treatment. l, DCT-1 overexpression does not influence the lifespan of otherwise wild-type animals or pink-1(tm1779) mutants under normal conditions. DCT-1 overexpression extends only the maximum lifespan of DAF-2-depleted worms. However, PINK-1 deficiency abolishes the extended lifespan of both daf-2(RNAi) and daf-2(RNAi);Ex[pdct-1DCT-1::GFP] animals. Survival curves depict the percentage of animals remaining alive, plotted against animal age. Lifespan values are given in Supplementary Table 1; assays were performed at 20 °C.

Extended Data Figure 9 Mitophagy deficiency impairs mitochondrial homeostasis and stress resistance.

a, b, Fifteen-day-old dct-1(tm376), pink-1(tm1779), pdr-1(gk448) and wild-type animals incubated (a) at 37 °C for 7 h (n = 150; ***P < 0.001; one-way ANOVA) and (b) treated with 8 mM paraquat (n = 150; ***P < 0.001; one-way ANOVA). c, Transgenic animals expressing the H2O2-biosensor HyPer subjected to either dct-1 or pink-1 RNAi. The graph depicts pixel intensity ratios of oxidized to reduced HyPer (n = 70; **P < 0.01; one-way ANOVA). d, Mitophagy-depleted animals display increased ratios of mitochondrial ROS formation (MitoTracker Red CM-H2X ROS) to total mitochondrial content (intestinal mtGFP) (n = 150; ***P < 0.001; one-way ANOVA). e, TMRE staining of mitophagy-depleted adults at 20 °C or 25 °C (n = 150; *P < 0.05, ***P < 0.001; one-way ANOVA). f, The percentage of mitochondrial DNA relative to wild type is shown. Quantitative PCR was performed in wild-type, dct-1(tm376), pink-1(tm1779) and pdr-1(gk448) mutant animals. The percentage of mtDNA is not affected in mitophagy-deficient animals (NS, P > 0.05; one-way ANOVA). g, The genetic locus of dct-1 gene is depicted; dct-1(tm376) is homozygous deletion. The dct-1(tm376) mutant carries a deletion of 900 base pairs at the promoter region of dct-1 gene. h, i, Transgenic animals were generated carrying extra copies of wild-type dct-1 gene and dct-1 gene carrying deletions for the corresponding sequences of WXXL motif, MER domain and BH3 domain, and were exposed to heat stress (37 °C) or treated with paraquat. WXXL motif of DCT-1 is required for mitophagy process since WXXL-deleted DCT-1 fails to rescue resistance of dct-1(tm376) animals under stress conditions (n = 150; NS, P > 0.05, ***P < 0.001; one-way ANOVA). j, k, Wild-type, pink-1(tm1779) and pdr-1(gk448) transgenic animals expressing a full-length pdct-1DCT-1::GFP reporter fusion were exposed to heat stress (37 °C) or treated with paraquat. DCT-1 overexpression is sufficient to promote stress resistance in wild-type worms. However, the stress resistance of DCT-1 overexpressing animals is abolished in pink-1 and pdr-1 mutant background (n = 150; ***P < 0.001; one-way ANOVA). Error bars, s.e.m. l–o, DCT-1 is ubiquitinated in a PINK-1-dependent manner upon oxidative stress. DTC-1::GFP was immunoprecipitated from total lysates of wild-type and pink-1(tm1779) transgenic animals expressing a full-length pdct-1DCT-1::GFP reporter fusion. The immunoprecipitated material was trypsinized and subjected to nLC-MS/MS analysis. l, DCT-1 was identified by mass spectrometry with high confidence and a sequence coverage of ∼60% (underlined on DCT-1 sequence). The two most prominent tryptic peptides of DCT-1 (highlighted in blue) were used for the relative quantification of the ubiquitination within the different samples. Ubiquitin-modified lysine is highlighted in red. m, Numbers represent the following samples: untreated wild-type worms (sample 1), untreated wild-type transgenic worms expressing a full-length pdct-1DCT-1::GFP reporter fusion (sample 2), paraquat-treated wild-type transgenic worms expressing a full-length pdct-1DCT-1::GFP (sample 3) and paraquat-treated pink-1(tm1779) mutants expressing a full-length pdct-1DCT-1::GFP (sample 4). The most prominently identified tryptic peptides 19–50 (peptide 1) and 101–121 (peptide 2) were used to determine the relative abundance of modified and unmodified DCT-1 in the four samples. The maximum intensities of the peptide ions of each peptide in the different samples were normalized and used for the comparative analysis and K26-GG abundance. The unmodified peptides showed similar pattern and abundance in the different samples (not identified in control sample 1, which did not express DCT-1::GFP, as expected), while the tryptic peptide with the K26-GG modification (19–50) was significantly enriched in sample 3 where mitophagy was induced in the presence of PINK-1. n, o, K26-GG modification on the tryptic peptide 19–50 of DCT-1 was manually validated on the MS/MS spectra of the unmodified (n) and modified (o) peptide. Both peptides were identified by high amino-acid coverage, as is shown on their corresponding assigned spectra. The intact peptide masses after the loss of H2O and NH3 molecules are shown in green, and the characteristic b and y fragment ions used for sequencing the amino-acid residues of the peptides are shown in red and blue, respectively. The characteristic fragments indicating the K26-GG modification are marked by blue circles and underlined fragment numbers (o).

Extended Data Figure 10 SKN-1 regulates the transcription of mitochondrial genes and modulates mitochondrial homeostasis and integrity.

a, mRNA level analysis of the SKN-1 target genes gst-4 and gst-10 in wild-type animals and mitophagy-deficient animals (**P < 0.01; one-way ANOVA). b, Expression of the DAF-16-regulated, psod-3GFP transgene in animals subjected to dct-1 or pink-1 RNAi (n = 70; NS, P > 0.05, ***P < 0.001; one-way ANOVA). c, Expression of the mitochondrial stress reporter phsp-60GFP, in animals subjected to RNAi against dct-1 or pink-1 (n = 70; NS, P > 0.05; one-way ANOVA). d, Expression of the ER stress reporter phsp-4GFP, in animals subjected to RNAi against dct-1 or pink-1 (n = 70; NS, P > 0.05; one-way ANOVA). e, mRNA levels of atp-5, gas-1, hmg-5, tim-17, W09C5.8 and dct-1 in wild-type and SKN-1-depleted animals (skn-1(zu67) and skn-1(zu135) animals) (*P < 0.01; one-way ANOVA). f, The percentage of mitochondrial DNA relative to wild type is shown. The percentage of mtDNA is reduced in SKN-1-depleted worms (**P < 0.01; one-way ANOVA). g, Knockdown of SKN-1 alters mitochondrial network morphology in the intestine of young adult animals. Scale bar, 20 μm. Images were acquired using a ×40 objective lens. h, mRNA levels of pink-1 and pdr-1 in wild-type animals, DAF-16- and SKN-1-depleted animals (NS, P > 0.05; one-way ANOVA). i, Mitophagy is not stimulated under oxidative stress, in transgenic animals expressing mtRosella subjected to RNAi against skn-1 (n = 120; NS, P > 0.05, ***P < 0.001; one-way ANOVA). j, Monitoring mitophagy under oxidative stress, in transgenic animals subjected to RNAi against skn-1, skn-1 and dct-1, daf-16, daf-16 and dct-1 (n = 120; NS, P > 0.05, ***P < 0.001; one-way ANOVA). k, SKN-1 compensates for inhibition of mitophagy in otherwise wild-type animals. Simultaneous depletion of DCT-1 and SKN-1, or PINK-1 and SKN-1. Survival curves depict the percentage of animals remaining alive, plotted against animal age. Lifespan values are given in Supplementary Table 1; assays were performed at 20 °C. l, Transgenic animals co-expressing a mitochondria-tagged GFP (mtGFP) in body wall muscle cells and the autophagosomal protein LGG-1 fused to DsRed were exposed to heat stress (37 °C) or treated with CCCP. Induction of mitophagy, as indicated by co-localization of GFP and DsRed (for each group of images, mitochondria are shown in green on top, autophagosomes in red below, with a merged image at the bottom). Mitophagy inhibition under stress, in transgenic animals subjected to RNAi against skn-1. Scale bars, 20 μm. Images were acquired using a ×40 objective lens. m, Cytoplasmic calcium elevation upon mitophagy depletion is abolished upon treatment with EGTA (n = 100; NS, P > 0.05, ***P < 0.001; one-way ANOVA). n, Transgenic animals expressing pgst-4GFP reporter subjected to RNAi against dct-1, pink-1 and pdr-1, with or without EGTA treatment (n = 120; NS, P > 0.05, ***P < 0.001; one-way ANOVA). o, UNC-43 regulates the transcriptional activity of SKN-1 upon mitophagy depletion (n = 90; ***P < 0.001; one-way ANOVA). Error bars, s.e.m.

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Palikaras, K., Lionaki, E. & Tavernarakis, N. Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature 521, 525–528 (2015). https://doi.org/10.1038/nature14300

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