Abstract
Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns–Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells1 and dopaminergic neurons2 during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPRmt), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria3,4. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes5. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPRmt activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPRmt activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.
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Acknowledgements
We thank B. Lemire, the National Bioresource Project and the Caenorhabditis Genetics Center for providing C. elegans strains (funded by NIH Office of Research 362 Infrastructure Programs (P40 OD010440), and the Genomics and Bioinformatics Facilities at Memorial Sloan Kettering Cancer Center). This work was supported by National Institutes of Health grants (R01AG040061 and R01AG047182) to C.M.H. and (R01HD078703 and R01NS081490) to S.S., and a Parkinson’s Disease Foundation grant (PDF-FBS-1314) to Y.-F.L. and Deutsche Forschungsgemeinschaft (DFG, SCHU 3023/1-1) to A.M.S.
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Y.-F.L. and C.M.H. planned the experiments. Y.-F.L. generated the worm strains and performed the mtDNA quantification and obtained the images. A.M.S. and Y.-F.L. performed the oxygen consumption analysis and M.W.P. performed the microarray experiments. Electron microscopy was performed by Y.Lu under the supervision of S.S. Y.-F.L. and C.M.H. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 RNAi of nuclear encoded OXPHOS components activates the UPRmt, and UPRmt signalling components are required for ∆mtDNA propagation.
a, Images of hsp-6pr::gfp worms raised on nuo-2 (complex I), cyc-1 (cytochrome c) or atp-2 (complex V) RNAi. Scale bar, 0.1 mm. b, Relative ∆mtDNA quantification as determined by qPCR in ∆mtDNA or atfs-1(tm4525);∆mtDNA worms. n = 3; error bars, mean ± s.d.; *P < 0.03 (Student’s t-test). c, Relative ∆mtDNA quantification as determined by qPCR in ∆mtDNA worms raised on control, dve-1, or ubl-5 RNAi. n = 3; error bars, mean ± s.d.; *P < 0.03 (Student’s t-test). d, Images of dve-1pr::dve-1::gfp in wild-type or ∆mtDNA worms. Scale bar, 0.1 mm. e, Ratios of ∆mtDNA and nuclear genomic DNA as determined by qPCR in individual ∆mtDNA and atfs-1(tm4919);∆mtDNA worms. Black bars represent the mean. n = 16; mean P < 0.0001 (Student’s t-test).
Extended Data Figure 2 The reduction in ∆mtDNA caused by atfs-1(RNAi) is largely independent of autophagy, and ATFS-1 activation in the presence of a deleterious mtDNA is harmful.
a, Relative ∆mtDNA quantification as determined by qPCR in ∆mtDNA and atg-18(gk378);∆mtDNA worms raised on control or atfs-1 RNAi. n = 3; error bars, mean ± s.d.; *P < 0.03 (Student’s t-test). b, Images of hsp-6pr::gfp worms raised on control or spg-7 RNAi. Scale bar, 0.1 mm. c, Images of TMRE-stained wild-type, ∆mtDNA, atfs-1(et18) and atfs-1(et18);∆mtDNA worms. Scale bar, 0.1 mm. d, Images of synchronized atfs-1(et18) and atfs-1(et18);∆mtDNA worms raised on 2.5 μM rotenone 4 days after hatching. Scale bar, 1 mm.
Extended Data Figure 3 polg-1, hmg-5 and drp-1 mRNAs are induced in atfs-1(et18) worms, and mitochondrial fusion is required for the development of atfs-1(et18) worms harbouring ∆mtDNA.
a, polg-1, hmg-5 and drp-1 transcripts as determined by qRT–PCR in wild-type and atfs-1(et18) worms. n = 3; error bars, mean ± s.d.; *P < 0.03 (Student’s t-test). RFU, random fluorescent units. b, Images of synchronized ∆mtDNA, atfs-1(et18), and atfs-1(et18);∆mtDNA worms raised on control or fzo-1 RNAi. Scale bar, 1 mm. c, ∆mtDNA quantification as determined by qPCR in ∆mtDNA worms raised on control or hmg-5 RNAi. n = 3; error bars, mean ± s.d.; *P < 0.03 (Student’s t-test).
Supplementary information
Supplementary Tables
This file contains Supplementary Tables 1-2 comprising: (1) the relative fold induction and P values for each mRNA and (2) list of Primers that specifically amplify wild-type or ΔmtDNA . (PDF 242 kb)
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Lin, YF., Schulz, A., Pellegrino, M. et al. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response. Nature 533, 416–419 (2016). https://doi.org/10.1038/nature17989
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DOI: https://doi.org/10.1038/nature17989
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