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Tetracyclines promote survival and fitness in mitochondrial disease models

Nature Metabolism volume 3, pages 33–42 (2021)Cite this article

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Abstract

Mitochondrial diseases (MDs) are a heterogeneous group of disorders resulting from mutations in nuclear or mitochondrial DNA genes encoding mitochondrial proteins1,2. MDs cause pathologies with severe tissue damage and ultimately death3,4. There are no cures for MDs and current treatments are only palliative5,6,7. Here we show that tetracyclines improve fitness of cultured MD cells and ameliorate disease in a mouse model of Leigh syndrome. To identify small molecules that prevent cellular damage and death under nutrient stress conditions, we conduct a chemical high-throughput screen with cells carrying human MD mutations and discover a series of antibiotics that maintain survival of various MD cells. We subsequently show that a sub-library of tetracycline analogues, including doxycycline, rescues cell death and inflammatory signatures in mutant cells through partial and selective inhibition of mitochondrial translation, resulting in an ATF4-independent mitohormetic response. Doxycycline treatment strongly promotes fitness and survival of Ndufs4−/− mice, a preclinical Leigh syndrome mouse model8. A proteomic analysis of brain tissue reveals that doxycycline treatment largely prevents neuronal death and the accumulation of neuroimmune and inflammatory proteins in Ndufs4−/− mice, indicating a potential causal role for these proteins in the brain pathology. Our findings suggest that tetracyclines deserve further evaluation as potential drugs for the treatment of MDs.

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Fig. 1: Small-molecule screen identifies antibiotics as suppressors of cell death in cellular models of human mitochondrial disease.
Fig. 2: Antibiotic-mediated mitochondrial translation attenuation is required to promote cell survival in human mitochondrial disease mutant cells.
Fig. 3: Doxycycline significantly increases the lifespan and fitness of a mouse model of complex I deficiency.
Fig. 4: Doxycycline corrects neuroimmune and inflammatory proteins and increases metabolites that suppress oxidative stress in models of complex I deficiency.

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Data availability

Datasets generated during this study are included as Supplementary Information or are publicly available. Chemical screen data have been deposited in PubChem under accession 1508586 and proteomic data in PRIDE under accession PXD022860. Source data are provided with this paper. Any additional data not included in this manuscript are available from the corresponding author upon request.

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Acknowledgements

We thank members of the Puigserver Laboratory for helpful discussions regarding this project. We also thank C. Moraes (University of Miami Medical School) and J. Smeitink and R. Vogel (Radboud University Medical Centre) for providing the cell lines used in this study. We acknowledge the Nikon Imaging Center at Harvard Medical School for assistance with brightfield microscopy; the ICCB-Longwood Screening Facility at Harvard Medical School for facilitating our screening efforts; J.M. Asara and M. Yuan at the Beth Israel Deaconess Medical Center Mass Spectrometry Core for providing metabolomics profiling data, R. Bronson and the Rodent Histopathology Core at Harvard Medical School for careful sectioning and analysis of mouse sections; and the Specialized Histopathology Core at Brigham and Women’s Hospital for immunohistochemistry. We thank C. Vidoudez of the Harvard Center for Mass Spectrometry for analysis of doxycycline tissue concentrations. This work was supported by National Institutes of Health (NIH) grants RO1 DK089883-07 NIDDK and RO1 GM121452 NIGMS (to P.P.), F30 DE028206-01A1 NIDCR (to E.A.P.) and F32 GM125243-01A1 NIGMS (to C.F.B.), an EMBO postdoctoral fellowship and MDA Development Grant (to E.B.), and the Human Frontier Science Program (LT-000033/2019-L) to P.L.M.

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Author notes

  1. These authors contributed equally: Elizabeth A. Perry, Christopher F. Bennett.

Authors and Affiliations

  1. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

    Elizabeth A. Perry, Christopher F. Bennett, Chi Luo, Eduardo Balsa, Mark Jedrychowski, Katherine E. O’Malley, Pedro Latorre-Muro & Pere Puigserver

  2. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Elizabeth A. Perry, Christopher F. Bennett, Chi Luo, Eduardo Balsa, Mark Jedrychowski, Katherine E. O’Malley, Pedro Latorre-Muro, Steven P. Gygi & Pere Puigserver

  3. Biological Sciences in Dental Medicine Program, Harvard School of Dental Medicine, Boston, MA, USA

    Elizabeth A. Perry

  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Richard Porter Ladley, Kamar Reda, Peter M. Wright & Andrew G. Myers

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Contributions

Conceptualization: E.A.P., C.F.B. and P.P.; methodology: E.A.P. and C.F.B.; formal analysis: E.A.P. and C.F.B.; investigation: E.A.P., C.F.B., C.L., K.E.O., E.B., P.L.-M., M.J., R.P.L., K.R. and P.M.W.; resources: M.J., S.P.G., A.G.M. and P.P.; writing (original draft): E.A.P., C.F.B. and P.P.; writing (review and editing): E.A.P., C.F.B., P.P., K.E.O., E.A.P., E.B., P.L.-M., C.L., M.J., R.P.L., K.R., P.M.W., A.G.M. and P.P.; visualization: E.A.P. and C.F.B.; supervision: E.A.P., C.F.B. and P.P.; funding acquisition: E.A.P., C.F.B. and P.P.

Corresponding author

Correspondence to Pere Puigserver.

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The authors declare no competing interests.

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Peer review information Primary Handling Editors: Christoph Schmitt, Pooja Jha. Nature Metabolism thanks Navdeep Chandel, Riekelt Houtkooper and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 MELAS cybrid cells exhibit deficient mitochondrial respiration and undergo apoptosis from nutrient stress.

a, Oxygen Consumption Rates (OCR) measured in control and MELAS cybrid cells. Measurements 4–6 follow the injection of 4 μM oligomycin, measurements 7–9 follow the injection of 4 μM FCCP, and measurements 10–12 follow the injection of 1.5 μM rotenone/4 μM antimycin (n = 5 biologically independent samples). b, 48 hour low-glucose survival assay of MELAS cybrid cells treated with the pan-caspase inhibitor Z-VAD-FMK or doxycycline (Dox) (n = 2 biologically independent samples). c, RFLP mapping of ND1 and MELAS mtDNA mutations in cybrids after 24 hour galactose or low-glucose conditions with 1 μM doxycycline. Expected band sizes are 193/159 bp for ND1 and 117/213 bp for MELAS mutations. d-f, BN-PAGE of isolated mitochondria from MELAS cybrids, ND1 cybrids, and Rieske KO fibroblasts treated with 1 μM doxycycline. MELAS cybrids were propagated 24 hours in low-glucose media, ND1 cybrids were propagated for 48 hours in galactose media, and Rieske KO fibroblasts were propagated for 48 hours in high-glucose media. Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

Source data

Extended Data Fig. 2 Doxycycline promotes cell survival through attenuation of mitochondrial translation.

a, Mitochondrial protein synthesis (%DMSO) versus antibiotic concentration based on band quantification relative to DMSO from Fig. 2b (n = 2 experiments). b, 35S-labelled cysteine and methionine pulse in cybrid cells treated with doxycycline at 1 μM or 10 μM with a 48 hour pre-treatment and 1 hour pulse (n = 2 experiments). c, Western blot of MRPL4 in sgMRPL4 ND1 cybrid cells. d, Western blot of GFM2 in sgGFM2 ND1 cybrid cells.

Source data

Extended Data Fig. 3 Doxycycline does not promote cell survival in MELAS or ND1 cybrid cells through the integrated protein response (ATF4) or reported protein targets.

a, Western blot of integrated response proteins in MELAS cybrid cells treated with doxycycline (Dox) for 24 hours (n = 2 experiments). b, Western blot of ATF4 in siATF4 MELAS cybrid cells (n = 2 experiments). c, 48 hour low-glucose survival of siATF4 MELAS cybrid cells treated with doxycycline (n = 3 biologically independent samples). d, 48 hour low-glucose survival of MELAS cybrid cells treated with doxycycline with or without ISRIB (Integrated Stress Response Inhibitor) (n = 3 biologically independent samples over n = 2 independent experiments) e, 48 hour low-glucose survival of MELAS cybrid cells treated with pan matrix-metalloprotease (MMP) inhibitor BB-94 (n = 2 biologically independent samples). f, 4 day galactose survival assay of ND1 cybrid cells treated with BB-94 (n = 2 biologically independent samples). g, 48 hour low-glucose survival of MELAS cybrid cells treated with the PARP inhibitor Olaparib (n = 2 biologically independent samples). h, 4 day galactose survival of ND1 cybrid cells treated with Olaparib (n = 2 biologically independent samples). i, 48 hour low-glucose survival of MELAS cybrid cells treated with the PAR1 inhibitor Vorapaxar (n = 2 biologically independent samples). j, 8 day galactose survival of ND1 cybrid cells treated with Vorapaxar (n = 2 biologically independent samples). k, Western blot of PAR1 in siF2R MELAS cybrid cells (n = 2 experiments). l, 48 hour low-glucose survival of MELAS cybrid cells depleted of PAR1 (siF2R) (n = 3 biologically independent samples). Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

Source data

Extended Data Fig. 4 Mitochondrial mutant cells have basally elevated cytokines under nutrient stress conditions which are suppressed by doxycycline.

a, Gene expression of inflammatory cytokine panel under high-glucose conditions for control (CON), ND1, and MELAS cybrid cells (n = 3 biologically independent samples). b, Gene expression of inflammatory cytokines in MELAS cybrid cells after 24 hour low-glucose (LG) conditions (n = 6 biologically independent samples). c, Gene expression of inflammatory cytokines in ND1 cybrid cells after 48 hours galactose (GAL) conditions (n = 3 biologically independent samples). Tetracycline analogues that rescue cell survival such as doxycycline, 7015, 7039, and 7066 suppress inflammatory gene expression. Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

Source data

Extended Data Fig. 5 Doxycycline suppresses inflammatory gene expression and metabolite levels in a time-dependent manner.

a, Heatmap of metabolites in ND1 cybrid cells that significantly change with doxycycline (Dox) and 7066 compared to DMSO, 7004, 7013, and CMT-3 (n = 3 biologically independent samples, p < 0.05, Student’s t-test with Bonferroni’s correction). b, Time-dependent gene expression of inflammatory markers in ND1 cybrid cells under galactose conditions (n = 3 biologically independent samples). c, Time-dependent metabolite changes in ND1 cybrid cells under galactose conditions (n = 3 biologically independent samples). Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

Source data

Extended Data Fig. 6 p38 inhibition partially mimics the doxycycline anti-inflammatory metabolomic signature.

a, Gene expression of inflammatory markers in ND1 cybrid cells treated with doxycycline (Dox) or p38i SB203580 after 48 hours galactose conditions (n = 4 biologically independent samples). b, Metabolite changes in doxycycline signature in ND1 cybrid cells treated with doxycycline or SB203580 after 48 hours galactose conditions (n = 4 biologically independent samples). c-d, Quantitation of GSSG/GSH and NADP+/NADPH ratio in ND1 cybrid cells with doxycycline or SB203580 treatment after 48 hours galactose conditions (n = 4 biologically independent samples). Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

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Extended Data Fig. 7 Doxycycline promotes metabolite redox homeostasis independent of complex I protein levels.

a, Quantitation of doxycycline (Dox) in brains of wild-type and Ndufs4−/− (KO) animals fed 5000 or 8000 ppm doxycycline diets (n = 3 mice per treatment group). b, Quantitation of doxycycline in livers of wild-type and Ndufs4−/− (KO) animals fed 5000 or 8000 ppm doxycycline diets (n = 3 mice per treatment group). c, Heatmap of mitochondrial complex I subunits expressed in the mouse brains quantified through proteomics analysis (n = 3 WT, n = 3 KO, and n = 5 KO Dox mice). d, Heatmap of metabolites altered in Ndufs4−/− (KO) brains (p < 0.15, Student’s t-test, two-sided, unpaired) that are modulated by doxycycline (p < 0.15, Student’s t-test, two-sided, unpaired) (n = 5 mice). e, Heatmap of metabolites altered in ND1 cybrid cells (p < 0.05, Student’s t-test, two-sided, unpaired) that significantly change with doxycycline (p < 0.05, Student’s t-test, two-sided, unpaired) (n = 4 biologically independent samples). f-g, Quantitation of NADP+/NADPH and GSSG/GSH ratios in ND1 cybrid cells or Ndufs4−/− (KO) brains with doxycycline treatment (cybrid cells, n = 4 biologically independent samples; mice, n = 5). Data are presented as mean values ± s.e.m. error bars, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * q < 0.05.

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Perry, E.A., Bennett, C.F., Luo, C. et al. Tetracyclines promote survival and fitness in mitochondrial disease models. Nat Metab 3, 33–42 (2021). https://doi.org/10.1038/s42255-020-00334-y

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