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The memory of neuronal mitochondrial stress is inherited transgenerationally via elevated mitochondrial DNA levels

Abstract

The memory of stresses experienced by parents can be passed on to descendants as a forecast of the challenges to come. Here, we discovered that the neuronal mitochondrial perturbation-induced systemic mitochondrial unfolded protein response (UPRmt) in Caenorhabditis elegans can be transmitted to offspring over multiple generations. The transgenerational activation of UPRmt is mediated by maternal inheritance of elevated levels of mitochondrial DNA (mtDNA), which causes the proteostasis stress within mitochondria. Furthermore, results from intercrossing studies using wild C. elegans strains further support that maternal inheritance of higher levels of mtDNA can induce the UPRmt in descendants. The mitokine Wnt signalling pathway is required for the transmission of elevated mtDNA levels across generations, thereby conferring lifespan extension and stress resistance to offspring. Collectively, our results reveal that the nervous system can transmit stress signals across generations by increasing mtDNA in the germline, enabling descendants to better cope with anticipated challenges.

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Fig. 1: Neuronal mitochondrial perturbations transmit the systemic UPRmt across multiple generations.
Fig. 2: Descendants with transgenerational UPRmt carry elevated levels of mtDNA.
Fig. 3: Wnt signalling is required for the transmission of elevated mtDNA levels across generations.
Fig. 4: Mitochondrial fusion and mitochondrial DNA polymerase are required for the maintenance of elevated mtDNA levels.
Fig. 5: Higher levels of mtDNA inherited from wild C. elegans strains cause the induction of UPRmt in descendants.
Fig. 6: The transgenerational effects of elevated mtDNA levels.

Data availability

The accession numbers for the raw sequencing files and the processed data reported in this paper are NCBI GEO: GSE157031, and NCBI BioProject: PRJNA607689 and PRJNA727630. Previously published DNA-sequencing data that were used here are available under accession code PRJNA13758. The KEGG pathway database used in this study is available online (http://www.genome.jp/kegg/). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the members of the Tian laboratory, D. Chen and X. Zhan for discussion and technical assistance; Y. Qi for the polg-1 mutant strain; S. Zuryn for the germline mitochondrial marker strain; and S. Cai for the wild C. elegans strains. Several C. elegans strains used in this work were provided by CGC (supported by the NIH-Officer of Research Infrastructure Programs (P40 OD010440) and the Japanese National BioResource Project. This work was supported by the National Key R&D Program of China (2017YFA0506400, to Y.T.; and 2019YFA0508700, to W.Q.), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB39000000, to Y.T.) and the National Natural Science Foundation of China (nos 31930023 and 31771333, to Y.T.; and no. 31922014, to W.Q.).

Author information

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Authors

Contributions

Y.T. and Q.Z. conceived the study and designed the experiments. Q.Z., Q.W., Z.W. and J.Z. performed the C. elegans crosses and western blotting. Q.Z., W.Z. and Q.W. performed the mtDNA measurement, qPCR experiments, lifespan experiments and stress-resistance assay. Q.Z. performed BN-PAGE experiments, OCR measurement and data analysis. W.Z. performed the mtDNA measurement, qPCR experiments, western blotting in cell lines. X.L. performed the RNAi experiments and drug treatment. X.W. performed the ATP-level measurement. Y.G. and Y.L. performed plasmid and strain constructions. C.W. and W.Q. performed the RNA-seq analyses. Q.Z. prepared the original figures. Y.T. and Q.Z. wrote the manuscript.

Corresponding author

Correspondence to Ye Tian.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks Doris Germain, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Representative fluorescence visualization of the transgenerational induction of the UPRmt across multiple generations.

a, Fluorescence visualization of the hsp-6p::gfp reporter in descendant animals generated from crosses between animals with neuronal mitochondrial stresses and wild-type animals. The schematic design of the experiment is presented in Fig. 1a. Worms with neuronal expression of Q40 or Wnt were crossed with WT males to generate the P0 animals with the expression of neuronal Q40 or Wnt. Then, P0 worms were allowed to produce F1 offspring through self-fertilization. 20%–30% of the F1 animals without expression of neuronal Q40 or Wnt still exhibited the strong induction of hsp-6p::gfp (indicated with white stars). We followed three independent lines with strong hsp-6p::gfp expression and one line that was not selected for the hsp-6p::gfp expression (randomly maintained) from each cross for over 50 generations. Worms with hsp-6p::gfp fluorescence over one-third of the intestine were considered to show the strong induction of the UPRmt. Scale bar, 250 μm. b, Percentage of worms showing the strong UPRmt induction in each generation as shown in a.

Extended Data Fig. 2 Disrupting mitochondrial function by RNAi or drugs did not trigger the transgenerational induction of UPRmt in their progeny.

a, qRT-PCR analysis of exogenous expression of neuronal egl-20 in F1 animals with or without neuronal EGL-20 expression as described in Fig. 1d. n = 4 independent experiments. b, Fluorescence visualization of the hsp-6p::gfp reporter in F1 animals with or without neuronal cco-1 KD expression. Animals exhibiting the transgenerational induction of UPRmt generated from animals with neuronal cco-1 KD were referred to as ‘TGCK’. Data shown represent five independent experiments with similar results. c, The schematic diagram of the strategy used to induce the UPRmt in parental animals using RNAi or drug treatments and subsequently bleached these animals and transferred the progeny to the untreated plates. d-g, Fluorescence visualization of the hsp-6p::gfp reporter animals treated with different drugs and RNAi to perturb mitochondrial function, and their progeny recovered on OP50 plates as described. Data shown represent five independent experiments with similar results. h, Fluorescence visualization of the hsp-4p::gfp reporter in F1 animals with or without neuronal spliced xbp-1 generated from animals expressing neuronal xbp-1s. No transgenerational induction of UPRER was observed. Data shown represent five independent experiments with similar results. Scale bars, 250 μm. Data are the mean ± s.e.m.. Statistics source data are provided.

Source data

Extended Data Fig. 3 A schematic design of experiment to generate animals with the transgenerational induction of the UPRmt for mtDNA sequencing.

Worms used for the mtDNA sequencing are P0 animals with expression of neuronal Q40 or neuronal Wnt, and their F5 progeny with or without UPRmt induction. Data shown represent five independent experiments with similar results. Scale bar, 250 μm.

Extended Data Fig. 4 Animals with elevated levels of mtDNA causes mitochondrial proteostasis stress.

a, qRT-PCR analysis of nDNA-encoded and mtDNA-encoded OXPHOS genes in TGQ and WT animals. n = 3 independent experiments. b, qRT-PCR analysis of UPRmt target genes and polg-1, hmg-5, rpom-1, tfbm-1 in TGQ and WT animals. n = 3 independent experiments. c, Relative mtDNA levels of WT worms at different developmental stages. n = 7 independent experiments. d, e, mtDNA/nDNA ratios of TGW and WT animals. WT n = 102 worms, TGW n = 89 worms, WT D1 n = 22 worms, TGW D1 n = 22 worms, WT D5 n = 30 worms, TGW D5 n = 31 worms. f, Fold change in mtDNA/nDNA ratios of animals exhibited transgenerational UPRmt generated from animals with neuronal Q40 expression, or neuronal cco-1 KD. n = 4 independent experiments. g, Actual nDNA copies per worm of TGW, TGQ and WT animals. n = 5 independent experiments. Related to Fig. 2f. h, Fold change in germline mtDNA/nDNA ratios of animals expressing neuronal EGL-20 compared to WT. n = 5 independent experiments. i, Actual mtDNA copies per oocyte (position at ‘−1’, the one near to the spermatheca) of animals expressing neuronal EGL-20 compared to WT. n = 33 oocytes. j, qRT-PCR analysis of hsp-6 transcripts in the germline of TGW and WT animals. n = 3 independent experiments. k, Fluorescence visualization of the hsp-6p::gfp reporter in two groups of TGW animals according to their hsp-6p::gfp fluorescent intensities. Data shown represent three independent experiments with similar results. Scale bar, 250 μm. l, m, Representative immunoblots (l) and quantifications (m) of antibody sensitivity by western blot analyses of WT worms. n-p, Representative immunoblots (n) and quantifications (o, p) of mitochondrial OXPHOS proteins in TGQ and WT animals. n = 3 independent experiments. q, Western blot of purified mitochondria from TGQ and WT worms separated by Blue-native page gel using antibody against ATP5A (complex V). r, The oxygen consumption rates (r, n = 8 independent experiments) and the relative ATP levels (s, n = 3 independent experiments) of TGQ and WT animals. t-v, The mitochondria morphology in muscle (t), intestine (u), and oocytes (v) in TGW and WT worms. Scale bars, 10 μm. Data shown represent three independent experiments with similar results. Data are the mean ± s.e.m., ***P < 0.001, **P < 0.01, *P < 0.05, ns denotes P > 0.05, P values were determined using two-way ANOVA (a, b), one-way ANOVA (f, g) and two-sided Student’s t-test (d, e, h, i, j, o, p, r, s). The exact P values are provided in Source Data Extended Data Fig. 4. Statistics source data and unprocessed western blots are provided.

Source data

Extended Data Fig. 5 UPRmt machinery is not required for the transmission of increased mtDNA levels across generations.

a, b, Fluorescence visualization of the hsp-6p::gfp reporter (a) and fold change in relative mtDNA levels (b) in TGQ, TGQ;atfs-1(-/-) animals. n = 4 independent experiments. c-e, Representative immunoblots (c) and quantifications (d, e) of mitochondrial OXPHOS proteins in atfs-1 and TGQ; atfs-1 animals. n = 3 independent experiments. f, g, Fluorescence visualization (f) and quantifications (g) of the hsp-6p::gfp reporter in TGW, TGW;lin-65(-/-) and TGW;lin-65(+/+) animals. TGW n = 34 worms, TGW;lin-65(-/-) n = 41 worms, TGW;lin-65(+/+) n = 30 worms. h, Fold change in mtDNA/nDNA ratios of animals shown in (f). n = 3 independent experiments. i, Relative mtDNA levels in F1 animals generated from the cross between neuronal EGL-20; atfs-1(gk3094) hermaphrodites and atfs-1(gk3094) males. The cross strategy was described in Fig. 1a. n = 3 independent experiments. Scale bars, 250 μm. Data are the mean ± s.e.m., ***P < 0.001, **P < 0.01, *P < 0.05, ns denotes P > 0.05, P values were determined using one-way ANOVA (b, g, h, i) and two-sided Student’s t-test (d, e). The exact P values are provided in Source Data Extended Data Fig. 5. Statistics source data and unprocessed western blots are provided.

Source data

Extended Data Fig. 6 Wnt signaling is involved in the transmission of increased mtDNA levels across generations and is conserved to regulate mtDNA biogenesis.

a, qRT-PCR analysis of egl-20 in TGW, TGQ and WT animals. n = 8 independent experiments. b, c, Fluorescence visualization of the hsp-6p::gfp(b) and fold change in relative mtDNA levels (c) in TGQ, TGQ;egl-20(-/-) animals. n = 4 independent experiments. d-f, Representative immunoblots (d) and quantifications (e, f) of mitochondrial OXPHOS proteins in egl-20(-/-) and TGQ;egl-20(-/-) animals. n = 3 independent experiments. g, h, Fluorescence visualization (g) and quantifications (h) of the hsp-6p::gfp reporter in TGW (n = 32 worms), TGW;vps-35(-/-) (n = 30 worms) and TGW;vps-35(+/+) (n = 30 worms) animals. i, Fold change in mtDNA/nDNA ratios of animals shown in (g). n = 4 independent experiments. j, k, Fluorescence visualization (j) and quantifications (k) of the hsp-6p::gfp reporter in TGW (n = 31 worms), TGW;mig-14(-/-) (n = 30 worms) and TGW;mig-14(+/+) (n = 31 worms). l, Fold change in mtDNA/nDNA ratios of animals shown in (j). n = 4 independent experiments. m, n, Fluorescence visualization (m) and quantifications (n) of the hsp-6p::gfp reporter in TGW animals in different mutant background. WT n = 35 worms, cwn-1 n = 35 worms, cwn-2 n = 29 worms, lin-44 n = 34 worms, lin-17 n = 29 worms, mig-1 n = 30 worms. o, Fold change in mtDNA/nDNA ratios of animals shown in m. n = 3 independent experiments. p, Fluorescence visualization of the hsp-6p::gfp reporter in TGW animals treated with EV or bar-1 RNAi. Data shown represent three independent experiments with similar results. q, r, Fold change in mtDNA/nDNA ratios of TGW and WT animals treated with EV or bar-1 RNAi that since oogenesis (q, n = 6 independent experiments) and zygotically development (r, n = 5 independent experiments). s, t, Wnt target gene axin2 transcripts (s) and β-catenin protein level (t) in HEK293T cells treated with different concentrations of CHIR-99021 for 6 h. n = 3 independent experiments. u, v, Wnt target gene axin2 transcripts (u) and β-catenin protein level (v) in HEK293T cells treated with 10 µM CHIR-99021 at different time points. n = 4 independent experiments. w, x, qRT-PCR analysis of β-catenin or Myc in HEK293T cells treated with β-catenin or Myc siRNA. n = 4 independent experiments. Scale bars, 250 μm. Data are the mean ± s.e.m., ***P < 0.001, **P < 0.01, *P < 0.05, ns denotes P > 0.05, P values were determined using two-way ANOVA (q, r), one-way ANOVA (a, c, h, I, k, l, n, o, s, u) and two-sided Student’s t-test (e, f, w, x). The exact P values are provided in Source Data Extended Data Fig. 6. Statistics source data and unprocessed western blots are provided.

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Extended Data Fig. 7 Distribution of SNPs and indels of genome for ED3011 and KR314.

a, Circos-plot of mitochondrial genome features of ED3011 and KR314. The circles from the inside going in represent the C. elegans mitochondrial genome, nucleotide variations of KR314 and ED3011 (as the black line indicated) compared to N2. The circles on the outside represent the gene names on the mtDNA. b, Circos-plot of nuclear genome features of ED3011 and KR314. Segmented circles on the inside represent the six C. elegans chromosomes. Track 1, 2, 3, 4 are the histograms represent the numbers of variations per 10 kb (outward pointing bars = higher density), Track 1 shows the SNPs of ED3011. Track 2 shows the SNPs of KR314.Track 3 represents the indels of ED3011. Track 4 represents the indels of KR314. Track 5 is the heatmap depicts the density of CDS (dark red = higher density) on C. elegans chromosomes. Circos graph were generated with the basic information of C. elegans genome WBcel235 (BioProject PRJNA13758) using TBtools61. See Supplementary Table 5 for the list of genetic variations identified in wild C. elegans strain ED3011 and KR314.

Extended Data Fig. 8 The transgenerational induction of the UPRmt provides benefits to offspring under stress conditions.

a-d, Lifespan analysis of TGW and TGQ animals compared to WT animals. e-h, PA14 slow killing assay of TGW and TGQ animals compared to WT animals. i, Survival rates of day 1 adult WT and TGQ animals after paraquat treatment. n = 4 independent experiments. j, Survival rates of D1 adult WT and TGQ animals after heat shock treatment. n = 5 independent experiments. k, Lifespan analysis of N2 and other wild C. elegans strains ED3011 and KR314. l-o, Lifespan analysis of TGQ and TGW animals in polg-1(tm2685) (l), fzo-1(tm1133) (m), atfs-1(gk3094) (n) and egl-20(n585) (o) background. Data are the mean ± s.e.m., ***P < 0.001, **P < 0.01, P values were determined using two-sided Student’s t-test (i, j). The exact P values are provided in Source Data Extended Data Fig. 8. Statistics source data are provided.

Source data

Supplementary information

Reporting Summary

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Supplementary Table 1

Primer sequences used in this study.

Supplementary Table 2

Strain list used in this study.

Supplementary Table 3

Summary of mtDNA variations identified in TGQ and TGW animals.

Supplementary Table 4

Differentially expressed genes in TGQ worms as identified by RNA-seq analyses.

Supplementary Table 5

Summary of genetic variations identified in wild C. elegans strains ED3011, KR314 and CB4856.

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Unprocessed western blots.

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Unprocessed western blots.

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Unprocessed western blots.

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Statistical source data.

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Zhang, Q., Wang, Z., Zhang, W. et al. The memory of neuronal mitochondrial stress is inherited transgenerationally via elevated mitochondrial DNA levels. Nat Cell Biol 23, 870–880 (2021). https://doi.org/10.1038/s41556-021-00724-8

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