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A drug-like molecule engages nuclear hormone receptor DAF-12/FXR to regulate mitophagy and extend lifespan

Nature Aging volume 3, pages 1529–1543 (2023)Cite this article

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Abstract

Autophagy–lysosomal function is crucial for maintaining healthy lifespan and preventing age-related diseases. The transcription factor TFEB plays a key role in regulating this pathway. Decreased TFEB expression is associated with various age-related disorders, making it a promising therapeutic target. In this study, we screened a natural product library and discovered mitophagy-inducing coumarin (MIC), a benzocoumarin compound that enhances TFEB expression and lysosomal function. MIC robustly increases the lifespan of Caenorhabditis elegans in an HLH-30/TFEB-dependent and mitophagy-dependent manner involving DCT-1/BNIP3 while also preventing mitochondrial dysfunction in mammalian cells. Mechanistically, MIC acts by inhibiting ligand-induced activation of the nuclear hormone receptor DAF-12/FXR, which, in turn, induces mitophagy and extends lifespan. In conclusion, our study uncovers MIC as a promising drug-like molecule that enhances mitochondrial function and extends lifespan by targeting DAF-12/FXR. Furthermore, we discovered DAF-12/FXR as a previously unknown upstream regulator of HLH-30/TFEB and mitophagy.

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Fig. 1: MIC enhances TFEB/HLH-30 expression and lysosomal activity.
Fig. 2: MIC modulates activity of the NHR DAF-12/FXR.
Fig. 3: MIC-induced mitophagy enhances mitochondrial health.
Fig. 4: MIC extends lifespan and reduces neuropathologies in C. elegans.
Fig. 5: Conserved therapeutic potential of MIC in mammalian cells.

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Acknowledgements

The authors thank past and present members of the Lithgow and Andersen laboratories and Q. T. Le (Stanford University), D. Bhaumik, D. Tailor and S. Rajagopalan for help with laboratory resources. The authors also thank N. Tavernarakis (University of Crete Medical School) and C. E. de Carvalho (University of Saskatchewan) for the C. elegans reporter strains. C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center (CGC), funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40OD010440), and the Japanese National BioResource Project. This work was supported by NIH RF1 AG057358 to J.K.A. and R01 AG067325 to G.J.L. M.C. was supported by a postdoctoral fellowship from the Larry L. Hillblom Foundation. M.H. was supported by NIH AG 038664. The Lithgow and Andersen laboratories are also supported by the Larry L. Hillblom Foundation Center Grant. Figures were created using BioRender. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Buck Institute for Research on Aging, Novato, CA, USA

    Manish Chamoli, Anand Rane, Anna Foulger, Shankar J. Chinta, Azar Asadi Shahmirzadi, David Hall, Angelina Holcom, Suzanne Angeli, Minna Schmidt, Malene Hansen, Gordon J. Lithgow & Julie K. Andersen

  2. Touro University California, Vallejo, CA, USA

    Shankar J. Chinta

  3. University of Southern California, Los Angeles, CA, USA

    Azar Asadi Shahmirzadi, Angelina Holcom & Minna Schmidt

  4. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

    Caroline Kumsta & Malene Hansen

  5. Stanford University, Stanford, CA, USA

    Dhanya K. Nambiar & Sharon Pitteri

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  1. Manish Chamoli
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Contributions

Conceptualization: M.C., S.J.C., G.J.L. and J.K.A. Investigation: M.C., A.R., A.F., S.J.C., A.A.S., C.K., D.K.N., A.H., S.A., D.H. and M.S. Methodology: M.C., A.R., A.F., S.J.C., A.A.S., C.K., D.K.N., A.H. and S.A. Validation: M.C., A.R., A.F., A.A.S., C.K., D.K.N., S.A. and D.H. Visualization: M.C., A.R., C.K. and S.A. Resources: D.K.N. and S.P. Writing—original draft: MC. Writing—review and editing: M.C., M.H., G.J.L. and J.K.A. Supervision: M.C., G.J.L. and J.K.A. Funding acquisition: G.J.L. and J.K.A.

Corresponding authors

Correspondence to Manish Chamoli, Gordon J. Lithgow or Julie K. Andersen.

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Competing interests

The Buck Institute has filed a provisional patent application on which M.C., S.J.C., G.J.L. and J.K.A. are listed as an inventor. G.J.L. is a co-founder of Gerostate Alpha and declares no financial interests related to this work. M.C., G.J.L. and J.K.A. are co-founders of Symbiont Bio and declare no financial interests related to this work. The remaining authors declare no competing interests.

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Nature Aging thanks Javier Irazoqui and the other, anonymous, reviewer(s) for their contribtiuon to the peer review of this work.

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Extended data

Extended Data Fig. 1 MIC enhances TFEB/HLH-30 expression and lysosomal activity.

a, Day-2 HLH-30::GFP (MAH235) images 24 h post-treatment (20 °C) with DMSO or MIC. On 5 min starvation, animals show 100% nuclear localization (arrowheads). Assay was independently repeated three times (n ≥ 30 animals). Scale: 10 µm. b, Punctae count (muscles) of day-2 lgg-1p::gfp::lgg-1 (DA2123) post 24 h DMSO/MIC treatment (20 °C). Image highlighting LGG-1 punctae (arrow). Assay was independently repeated three times (n = 48 and 32 animals). Graph show mean ± s.d. (p-values via two-tailed unpaired t-test). Scale: 10 µm. c, Punctae count (pharynx) of day-2 lgg-1p::gfp::lgg-1 (DA2123) post 24 h DMSO/MIC treatment (20 °C). Image highlighting LGG-1 punctae (arrow). Assay was independently repeated three times (n = 28 and 32 animals). Graph show mean ± s.d. ns ≥ 0.05 (p-values via two-tailed unpaired t-test). Scale: 10 µm. d, Punctae count (neurons) in day-2 rgef-1p::gfp::lgg-1 (MAH242) post 24 h DMSO/MIC treatment (20 °C). Image highlighting LGG-1 punctae (arrow). Assay was independently repeated three times (n = 30 and 31 animals). Graph show mean ± s.d. (p-values via two-tailed unpaired t-test). Scale: 10 µm. e, Lysosomal area changes in day-2 WT(N2) 24 h post-treatment (20 °C) via lysotracker red. Assay was independently repeated three times with ≥15 animals/repeat (n = 311 and 571 lysosomes). The box plot shows the 25th–75th percentiles, median line, and min–max whiskers. p = 0.0004, (p-values via two-tailed unpaired t-test). f, Day-2 plmp1::lmp-1::GFP (RT258) images 24 h post-treatment (20 °C). Assay was independently repeated three times (n = 19 and 22 animals). The box plot shows the 25th–75th percentiles, median line, and min–max whiskers. p < 0.0001, (p-values via two-tailed unpaired t-test). Scale: 10 µm. g, Day-2 unc-54p: SEP::mCherry::laat-1 (arrow) images 24 h post-treatment (20 °C). Graph show mean ± s.d. Assay was independently repeated three times (n = 9 and 7 animals). p = 0.0066, (p-values via two-tailed unpaired t-test). Scale: 10 µm.

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Extended Data Fig. 2 MIC modulates activity of a nuclear hormone receptor DAF-12/FXR.

a, mRNA of lysosomal and mitophagy genes, day-1 WT(N2) and daf-12(rh61rh411) (20 °C). Assay was independently repeated four times. Graph show mean ± s.d. (p-values via two-tailed unpaired t-test). b, WT(N2) and daf-12(rh61rh411), day-1 (20 °C); Left: lysotracker intensity, (n = 311 and n = 325 lysosomes), p < 0.0001. Right: lysosomal area, (n = 311 and n = 321 lysosomes), p = 0.0013. Assay was independently repeated three times with ≥15 animals/repeat. (P-value via two-tailed unpaired t-test). Scale: 10 µm. c, WT(N2) and daf-12(rh61rh411) 24 h post-DMSO/MIC treatment, day-2 (20 °C); lysotracker intensity, Left: (n = 272 and n = 617 lysosomes), p < 0.0001; Right: (n = 325 and n = 477 lysosomes), p < 0.0001. Assay was independently repeated three times with ≥15 animals/repeat. (p-value via two-tailed unpaired t-test). d. mRNA of lysosomal genes, day-2 WT(N2) and hlh-30(tm1978) (20 °C). Assay was independently repeated five times. Graph show mean ± s.e.m. (p-values via two-tailed unpaired t-test). Right: lysotracker intensity, hlh-30(tm1978) 24 h post-DMSO/MIC treatment, day-2 (20 °C), (n = 447 and n = 156 lysosomes), p < 0.0001. Assay was independently repeated three times with ≥15 animals/repeat. (P-value via two-tailed unpaired t-test). The box plots (b, c and d) represent the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. e, DAF-12 activity post-dafachronic acid (ligand) doses quantified by one-hybrid reporter assay. Graph show mean ± s.d. Assay was independently repeated four times, except for ligand 50 nM that is, two, Ligand200 (p = 0.0045) and Ligand500 (p = 0.0003), (P-value via one-way ANOVA with Tukey’s multiple comparisons test). f, Schematic depicts dafadine A (DFA) action. DFA induces HLH-30 expression. HLH-30::GFP (MAH235), day-2 animals post-DFA/DMSO treatment (20 °C) probed with GFP antibody, Actin (ACT) as loading control. Assay was independently repeated two times. g, mRNA of lysosomal genes, day-2 WT(N2) and daf-12(rh61rh411) (20 °C). Assay was independently repeated two times. (p-values via two-tailed unpaired t-test).

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Extended Data Fig. 3 MIC-induced mitophagy enhances mitochondrial health.

a. Image depicts co-localization (arrowheads) in day-2 reporter animals treated with DMSO, MIC, or FCCP at 20 °C, expressing mCherry::LGG-1 and mitochondrial GFP. FCCP is used as positive control. Right: Spearman’s co-localization coefficient. Assay was independently repeated four times. Graph show mean ± s.d. MIC (p = 0.0464) and FCCP (p = 0.0003), (p-value via two-tailed unpaired t-test). Scale: 10 µm. b. mRNA of mitophagy genes in day-2 WT(N2) and daf-12(rh61rh411) mutants 24 h post DMSO or MIC (20 °C). Assay was independently repeated four times. Graph show mean ± s.d. daf-12 vs WT, for dct-1 (p = 0.0059) and drp-1, (p = 0.0476), WT (DMSO vs MIC), for dct-1 (p = 0.0071) and for drp-1 (p = 0.0326), (p-value by unpaired t-test). c. mRNA of mitophagy genes in day-2 WT(N2) and hlh-30(tm1978) mutants 24 h post DMSO or MIC (20 °C). hlh-30 vs WT, for dct-1 (p = 0.0099), pink-1 (p = 0.0277). WT (DMSO vs MIC), for dct-1 (p = 0.0004) and hlh-30 (DMSO vs MIC) for pink-1 (p = 0.0163). Assay was independently repeated three times. Graph show mean ± s.d., (p-value by unpaired t-test). d. Quantification of mitophagy in muscle-specific mito-rosella reporter upon RNAi treatment, performed at day-2, 24 h post DMSO/MIC (20 °C). Assay was independently repeated three times (n = 45, 44, 34 and 26 animals). Graph show mean ± s.d. control RNAi, MIC vs DMSO (p = 0.0020), (p-value via one-way ANOVA with Tukey’s multiple comparison test). e. Survival of DMSO/MIC treated day-5 pdr-1(gk448) mutants 5 h (20 °C) post-rotenone. Assay was independently repeated three times. Graph show mean ± s.d., p = 0.0226 (p-value via two-tailed unpaired t-test). f. Oxygen consumption rate, basal (B) and max (M) of day-2 WT(N2) and hlh-30(tm1978) mutants 24 h post DMSO/MIC (20 °C). Assay was independently repeated four times. Graph show mean ± s.e.m. (B vs M) for; WT-DMSO (p = 0.0169), WT-MIC (p < 0.0001), hlh-30-DMSO (p = 0.0269), hlh-30-MIC (p = 0.0005); (max-WT-DMSO vs max-WT-MIC, p < 0.0001), (max-WT-MIC vs max-hlh-30-MIC, p < 0.0001), (p-value via two-way ANOVA with Sidak’s multiple comparison test).

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Extended Data Fig. 4 Lifespan extending effect of MIC.

a, Survival curve of WT(N2) animals, treatment (median days); N2-DMSO (22) and N2-MIC (29), ****p < 0.0001 (p-value via Mantel-Cox long-rank test). MIC exposure from days 1–12 (15 °C) of adulthood. Detailed statistical information for all lifespans is presented in Supplementary Table 1. b, Survival curve of WT(N2) animals, treatment (median days); N2-DMSO (17) and N2-MIC (17), MIC exposure (15 °C) during development only (egg to young adult). c, Left: Survival curve of WT(N2) and hlh-30(tm1978) animals on dafadine A (DFA) post-development, N2-DMSO (17) and N2-DFA (19); p = 0.0021, hlh-30-DMSO (15) and hlh-30-DFA (12); p < 0.0001, (p-value via Mantel-Cox long-rank test). Assay was independently repeated three times. Right: Survival of day-2 WT(N2) and hlh-30(tm1978), 3 h post-FCCP (20 °C). Assay was independently repeated two times, ≥30 animals/repeat. WT-DMSO vs WT-DFA 20 μM; (p = 0.0113), hlh-30-DMSO vs WT-DFA 40 μM; (p = 0.0116), (p-values via two-way ANOVA with Sidak’s multiple comparisons test). d, Survival curve of pdr-1(gk448) animals, pdr-1-DMSO (15) and pdr-1-MIC (17), (p = 0.0009) (p-value via Mantel-Cox long-rank test). Assay was independently repeated two times. MIC exposure from days 1–7 (20 °C) of adulthood. e, Median lifespan shown for conditions/genotypes post treatment. N2 (D-20 °C) indicates MIC treatment only during development. ‘Life-long’ means exposure throughout the life starting from day-of adulthood. Each dot depicts median lifespan from a plate of ~50 animals. Lifespan change is averaged across trials. DMSO vs MIC for N2(15 °C)-life-long; (p = 0.0149), N2(20 °C)-life-long; (p = 0.0400), N2(15 °C)-day1–15; (p < 0.0001), N2(20 °C)-day1–7; (<0.0001) and mev-1(20 °C)-day1–7; (p = 0.0020), (p-value via two-tailed unpaired t-test). Detailed statistical information for all lifespans is presented in Supplementary Table 1.

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Extended Data Fig. 5 Mitophagy inducer urolithin A modulates DAF-12/FXR activity.

a, Human FXR activity was measured post-treatment with GW4064 and MIC. Assay was independently repeated four times. Graph show mean ± s.d. GW4064 (p < 0.0001), (P-value via one-way ANOVA with Tukey’s multiple comparisons test). b, left: UA and MIC structure is shown with benzocoumarin rings depicted in grey. Right: Human FXR activity was measured post-treatment with GW4064 and (+/− UA). Assay was independently repeated four times. Graph show mean ± s.d. GW4064 vs ctrl (p < 0.0001) and GW4064-UA50 (p = 0.0012). (p-value via two-way ANOVA with Tukey’s multiple comparisons test). c, DAF-12 activity post-dafachronic acid treatment, alone or with UA, was quantified by one-hybrid reporter assay. Graph show mean ± s.d. Assay was independently repeated three times. DMSO + Ligand (p = 0.0005), Ligand+ UA30 (p = 0.0076) Ligand +UA50 (p = 0.0069), (P-value via one-way ANOVA with Tukey’s multiple comparisons test).

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Supplementary information

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Supplementary Figs. 1 and 2 and Supplementary Tables 1–3

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Chamoli, M., Rane, A., Foulger, A. et al. A drug-like molecule engages nuclear hormone receptor DAF-12/FXR to regulate mitophagy and extend lifespan. Nat Aging 3, 1529–1543 (2023). https://doi.org/10.1038/s43587-023-00524-9

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