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LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria

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Abstract

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG–mtDNA binding, which is antagonized by LONP-1.

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Fig. 1: OXPHOS dysfunction increases mtDNA content through ATFS-1.
Fig. 2: Defective OXPHOS impedes the degradation of ATFS-1 and facilitates mitochondrial-localized ATFS-1 and POLG binding to mtDNAs.
Fig. 3: LONP-1 limits ATFS-1 binding to WT mtDNA and impairs replication.
Fig. 4: ATFS-1 and POLG primarily interact with ∆mtDNAs in heteroplasmic worms.
Fig. 5: LONP-1 is required to maintain heteroplasmy.
Fig. 6: LONP1 inhibition improves heteroplasmy and OXPHOS function in cybrid cells.
Fig. 7: Pharmacological inhibition of LONP1 improves heteroplasmy and OXPHOS function in cybrid cells.

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Data availability

The ChIP–seq data have been deposited to the Gene Expression Omnibus database under the BioProject accession code PRJNA590136. The next-generation sequencing data for mtDNA have been deposited in the NCBI Sequence Read Archive database under the BioProject accession code PRJNA780293. 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 Caenorhabditis Genetics Center for providing C. elegans strains (funded by the NIH Office of Research 362 Infrastructure Programs (P40 OD010440)). We thank C. Moraes for the KSS and G. Manfredi for the CoxI G6930A cybrid cell lines. This work was supported by the HHMI, the Mallinckrodt Foundation and National Institutes of Health grants (R01AG040061 and R01AG047182 to C.M.H., R01GM115911 and R01AI117839 to S.A.W., R01GM111706 and R35GM130320 to P.C., and F31HL147482 to K.L.).

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Authors and Affiliations

  1. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Qiyuan Yang, Pengpeng Liu, Nadine S. Anderson, Tomer Shpilka, YunGuang Du, Nandhitha Uma Naresh, Rui Li, Lihua Julie Zhu, Kevin Luk, Josh Lavelle, Scot A. Wolfe & Cole M. Haynes

  2. Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA, USA

    Rilee D. Zeinert & Peter Chien

  3. Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, MA, USA

    Rilee D. Zeinert & Peter Chien

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  1. Qiyuan Yang
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Contributions

Q.Y. and C.M.H. planned the experiments. Q.Y., Y.D., T.S., N.U.N., J.L., R.D.Z. and P.C. generated the worm strains. R.L. and L.J.Z. analysed ATFS-1::GFP and TMRE quantification. Q.Y. performed the C. elegans and cybrid mtDNA analysis including ChIP and respiratory function. P.L., K.L. and S.A.W. performed and analysed mtDNA sequencing. Q.Y., N.S.A. and C.M.H. wrote the manuscript.

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Correspondence to Cole M. Haynes.

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Extended data

Extended Data Fig. 1 OXPHOS dysfunction increases mtDNAs.

a, Quantification of total mtDNA in wildtype and nduf-7(et19) worms. n = 3, biologically independent samples (Each sample contains 40-60 animals; every dot stands for averaged value from 3 technical replicates; data shown represent mean ± S.E.M.). **P = 0.0015, Two-tailed Student’s t test. b, POLG immunoblot of wildtype worms following fractionation into total lysate (T), post-mitochondrial supernatant (S), and mitochondrial pellet (M). Tubulin (Tub) and the OXPHOS protein (NDUFS3) serve as loading controls. Representative immunoblots from four biological repeats. c, POLG immunoblot of lysates from wildtype worms raised on control(RNAi) or polg(RNAi). Tubulin (Tub) serves as a loading control. Representative immunoblots from seven biological repeats.

Source data

Extended Data Fig. 2 atfs-1-dependent transcription is impaired in atfs-1nuc(−) worms.

a, Schematic highlighting the R (Arginine) to A (Alanine) substitution to impair the nuclear localization sequence (NLS) within ATFS-1 yielding ATFS-1nuc(−) confirmed by Sanger sequencing. b, UPRmt signaling schematic highlighting the ATFS-1nuc(−) with an impaired NLS. c, Expression level of hsp-6 mRNA in wildtype and atfs-1nuc(−) worms raised on control(RNAi) or spg-7(RNAi) examined by qRT-PCR. n = 3, biologically independent samples. **P = 0.0028, One-way ANOVA. d–e, Photomicrographs of wildtype, atfs-1(et18) and atfs-1(et18)nuc(-);hsp-6pr::gfp worms (Scale bar 0.1 mm) (d); Quantification of fluorescence pixel intensity in wildtype (n = 117; Max: 12.51; Min: 1.87; Median: 5.12), atfs-1(et18) (n = 74; Max: 75.251; Min: 15.790; Median: 32.021) and atfs-1(et18)nuc(−) strains (n = 121; Max: 15.100; Min: 1.55; Median: 5.43). Box & whiskers plots Min to Max. ****P < 0.0001, One-way ANOVA. n means the number of sampling areas. Average pixel intensity signals were calculated from sampling areas at each condition in biological triplicates (e). f, hsp-6 mRNA expression in wildtype, atfs-1(et18) or atfs-1(et18)nuc(−) worms examined by qRT-PCR. n = 4 (wildtype), n = 5 (atfs-1(et18)), n = 3 (atfs-1(et18)nuc(−)), biologically independent samples. *P = 0.0114 (wildtype vs. atfs-1(et18)), *P = 0.0407 (atfs-1(et18) vs. atfs-1(et18)nuc(-)), one-way ANOVA. g, hsp-6 mRNA expression in wildtype and atfs-1nuc(−) worms raised on control(RNAi) or cco-1(RNAi) examined by qRT-PCR. n = 3, biologically independent samples. ***P = 0.0004, one-way ANOVA. h, polg mRNA expression in atfs-1nuc(−) worms raised on control(RNAi) or cco-1(RNAi) examined by qRT-PCR. n = 3, biologically independent samples. Two-tailed Student’s t test. i, POLG immunoblots of lysates from wildtype, atfs-1nuc(−) and atfs-1(null) worms raised on control or cco-1(RNAi). Representative immunoblots from four biological repeats. j, Immunoblots of lysates from wildtype and atfs-1nuc(−) worms raised on control or lonp-1(RNAi). ATFS-1 or ATFS-1nuc(−) are indicated with an arrowhead. Representative immunoblots from four biological repeats. In c, f–h, each dot represents the average from 3 technical replicates; data shown represent mean ± S.E.M.

Source data

Extended Data Fig. 3 LONP-1 inhibition promotes mtDNA content via ATFS-1.

a, FLAG immunoblots of lysates from wildtype and LONP-1FLAG wildtype worms. Tubulin (Tub) serves as a loading control. Representative immunoblots from four biological repeats. b, Images of wildtype or LONP-1FLAG worms 48 hours after synchronization indicating worms expressing LONP-1FLAG at the endogenous locus develop normally (Scale bar 1 mm). Representative images from four biological repeats. c, Fluorescent photomicrographs of wildtype hsp-6pr::gfp or lonp-1FLAG;hsp-6pr::gfp worms 48 hours after synchronization indicating worms expressing LONP-1FLAG do not cause UPRmt activation (Scale bar 0.05 mm). Representative images from four biological repeats. d, Schematic of the putative ATFS-1 and LONP-1 binding sites within the mtDNA non-coding region (NCR) highlighting the proximity of both sites (~200 base pairs). e, POLG Immunoblots of lysates from wildtype worms raised on control or lonp-1(RNAi). Representative images from four biological repeats. f, Total mtDNA quantification in wildtype homoplasmic atfs-1nuc(−) worms raised on control(RNAi) or lonp-1(RNAi). n = 5, biologically independent samples. ***P = 0.0004, Two-tailed Student’s t test). g, Total mtDNA quantification in wildtype homoplasmic atfs-1mts(−);nuc(−) worms raised on control(RNAi) or lonp-1(RNAi). n = 3, biologically independent samples. Two-tailed Student’s t test. In f and g, each biologically independent sample contained 40-60 animals; every dot stands for averaged value from 3 technical replicates; data shown represent mean ± S.E.M. *p < 0.05, **p < 0.01, ****p < 0.0001.

Source data

Extended Data Fig. 4 Mitochondrial ATFS-1 is required to maintain ∆mtDNA in heteroplasmic worms.

a, Crossing strategy of atfs-1(null);pdr-1(tm598);uaDf5 strain. b. TMRE quantification of heteroplasmic (∆mtDNA) worms raised on control(RNAi) (n = 475; Max: 1.052; Min: 0.21; Median: 0.618), or wildtype worms raised on control (n = 232; Max: 1.318; Min: 0.725; Median: 0.995) or spg-7(RNAi) (n = 114; Max: 0.798; Min: 0.134; Median: 0.402). Box & whiskers plots Min to Max. n means the number of sampling areas. Average pixel intensity signals were calculated from sampling areas at each condition in biological triplicates. c,d, Photomicrographs of uaDf5 and atfs-1nuc(−);uaDf5;hsp-6pr::gfp worms (Scale bar 0.1 mm) (c); Quantification of fluorescence pixel intensity in uaDf5 (n = 199; Max: 30.89; Min: 3.430; Median:11.590) and atfs-1nuc(−);uaDf5;hsp-6pr::gfp (n = 234; Max: 15.640; Min: 2.540; Median: 6.915). Box & whiskers plots Min to Max. n means the number of sampling areas. Average pixel intensity signals were calculated from sampling areas at each condition in biological triplicates (d). e, ∆mtDNA quantification as determined by qPCR in heteroplasmic uaDf5 worms, atfs-1(null);uaDf5 worms and atfs-1nuc(−);uaDf5 worms. n = 3, biologically independent samples. f, ∆mtDNA quantification as determined by qPCR in heteroplasmic atfs-1nuc(−);uaDf5 worms and atfs-1mts(−);nuc(−);uaDf5. n = 3 (atfs-1nuc(−);uaDf5) and n = 4 (atfs-1mts(−);nuc(−);uaDf5), biologically independent samples. ***P = 0.0007. g, Quantification of total mtDNA following POLG ChIP-mtDNA in homoplasmic wildtype or uaDf5 worms. n = 4 (wildtype) and n = 3 (uaDf5), biologically independent samples. *P = 0.0229. In e and f, each biologically independent sample contained 40-60 animals; in g, each biologically independent sample contained about 150,000 animals; each dot stands for averaged value from 3 technical replicates in f,g; Two-tailed Student’s t test was used in d, f and g, One-way ANOVA was used in b; data shown represent mean ± S.E.M. *p < 0.05, **p < 0.01, ****p < 0.0001.

Source data

Extended Data Fig. 5 ATFS-1 and POLG primarily interact with ∆mtDNAs in heteroplasmic worms.

a, Overview of the qPCR strategy to quantify the ∆mtDNA percentage in heteroplasmic worms or heteroplasmic cells. Plasmids containing a sequence specific to the ∆mtDNA or wildtype mtDNA were created7. Standard curves were generated using the indicated concentration of each plasmid harboring sequences specific to either wildtype or ∆mtDNAs. Both PCR reactions were carried out simultaneously in the same qPCR machine. b,c, Scatter plots (b) and results (c) of 3D digital PCR quantification of wildtype mtDNA and ∆mtDNA following ATFS-1 ChIP-mtDNA in heteroplasmic uaDf5 worms. n = 4, biologically independent samples. d-e, Scatter plots (d) and results (e) of 3D digital PCR quantification of wildtype mtDNA and ∆mtDNA following POLG ChIP-mtDNA in heteroplasmic uaDf5 worms. n = 4, biologically independent samples. f, HMG-5/TFAM immunoblot of wildtype worms following fractionation into total lysate (T), post-mitochondrial supernatant (S), and mitochondrial pellet (M). Tubulin (Tub) and the OXPHOS component (NDUFS3) serve as loading controls. Representative immunoblots from two biological repeats. g, HMG-5/TFAM immunoblots of lysates from wildtype worms raised on control or hmg-5/tfam(RNAi). Tubulin (Tub) serves as a loading control. Representative immunoblots from three biological repeats. Each biologically independent sample contained 150,000 animals in c,e; data shown represent mean ± S.E.M.

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Extended Data Fig. 6 Inhibition of LONP-1 improves the deleterious heteroplasmy ratio.

a, LONP-1 immunoblots of lysates from wildtype worms raised on control(RNAi) or lonp-1(RNAi). Tubulin (Tub) serves as a loading control. Representative immunoblots from four biological repeats. b, ChIP-mtDNA using ATFS-1 or LONP-1 antibodies in wildtype worms followed by quantification of total mtDNA. n = 3, biologically independent samples. **P = 0.0042. c, ChIP-mtDNA using LONP-1 antibodies in wildtype or heteroplasmic worms followed by quantification of total mtDNA (both wildtype and ∆mtDNA). n = 3, biologically independent samples. d, ∆mtDNA quantification in atfs-1nuc(−);uaDf5 worms raised on control(RNAi) or lonp-1(RNAi). n = 3, biologically independent samples. *P = 0.0168. e, The brood size of heteroplasmic worms raised on control or lonp-1(RNAi). n = 9 worms. f, ∆mtDNA and wildtype mtDNA quantification following HMG-5/TFAM ChIP-mtDNA in uaDf5 heteroplasmic worms raised on lonp-1(RNAi) indicating that the binding of HMG-5/TFAM to wildtype mtDNAs or ∆mtDNAs is similar the input ratio. n = 4, biologically independent samples. g, wildtype mtDNA quantification in uaDf5 heteroplasmic worms raised on control(RNAi) or cco-1(RNAi). n = 3, biologically independent samples. **P = 0.0075. h, wildtype mtDNA quantification in uaDf5 or clk-1(qm30);uaDf5 heteroplasmic worms. n = 3, biologically independent samples. **P = 0.0029. In b,c and f, Each biologically independent sample contained 150,000 animals; in d,g,h each biologically independent sample contained 40-60 animals; every dot stands for averaged value from 3 technical replicates in b-d and f-h; Two-tailed Student’s t test was used; data shown represent mean ± S.E.M.

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Extended Data Fig. 7 Pharmacological inhibition of LONP1 improves heteroplasmy and OXPHOS function in heteroplasmic cybrid cells.

a, Mutant (G6930A) mtDNA ratio confirmation by sanger sequencing in CoxI G6930A cells treated by CDDO. b, Oxygen consumption rates (OCR) of 143B (wildtype) cells treated with DMSO (ctrl), 0.1 μM or 0.25 μM CDDO for 3 days. n = 22 (ctrl) and n = 24 (0.1 μM and 0.25 μM CDDO), biologically independent samples. c, Cell viability of 143b (WT) and KSS ∆mtDNA cells exposed to various concentrations of CDDO for 72 hours. n = 3, biologically independent samples. d, Basal respiration of KSS heteroplasmic cells treated with DMSO (ctrl), 0.1 μM or 0.25 μM CDDO for 4 or 13 weeks. n = 14 (ctrl) and n = 16 (0.1 μM and 0.25 μM CDDO), biologically independent samples. ****P < 0.0001, Two-tailed Student’s t test. Data shown represent mean ± S.E.M.

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Supplementary Tables

Supplementary Table 1: primers for qPCR, qRT–PCR, mtDNA quantification and guide RNAs for gene editing. Supplementary Table 2: antibodies used in this study.

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Yang, Q., Liu, P., Anderson, N.S. et al. LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria. Nat Cell Biol 24, 181–193 (2022). https://doi.org/10.1038/s41556-021-00840-5

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