Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila

Nature Aging volume 2, pages 494–507 (2022)Cite this article

Subjects

Abstract

The effects of aging on the brain are widespread and can have dramatic implications on the overall health of an organism. Mitochondrial dysfunction is a hallmark of brain aging, but the interplay among mitochondrial quality control, neuronal aging and organismal health is not well understood. Here, we show that aging leads to a decline in mitochondrial autophagy (mitophagy) in the Drosophila brain with a concomitant increase in mitochondrial content. We find that induction of BCL2-interacting protein 3 (BNIP3), a mitochondrial outer membrane protein, in the adult nervous system induces mitophagy and prevents the accumulation of dysfunctional mitochondria in the aged brain. Importantly, neuronal induction of BNIP3-mediated mitophagy increases organismal longevity and healthspan. Furthermore, BNIP3-mediated mitophagy in the nervous system improves muscle and intestinal homeostasis in aged flies, indicating cell nonautonomous effects. Our findings identify BNIP3 as a therapeutic target to counteract brain aging and prolong overall organismal health with age.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Neuronal BNIP3 induction prevents accumulation of dysfunctional mitochondria in the aged brain.
Fig. 2: Neuronal BNIP3 induction induces mitophagy to improve mitochondrial homeostasis.
Fig. 3: Neuronal BNIP3 induction extends lifespan and improves healthspan.
Fig. 4: Neuronal BNIP3 induction improves mitochondrial homeostasis and proteostasis in aged muscle.
Fig. 5: Neuronal BNIP3 induction improves intestinal homeostasis during aging.
Fig. 6: Neuronal BNIP3 induction requires autophagy to delay systemic aging.

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in the figures and text, with representative images accompanying quantified results where applicable, unless otherwise noted. Further information is available from the corresponding author upon reasonable request.

References

  1. Alexander, G. E. et al. Characterizing cognitive aging in humans with links to animal models. Front. Aging Neurosci. 4, 21 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Dykiert, D., Der, G., Starr, J. M. & Deary, I. J. Age differences in intra-individual variability in simple and choice reaction time: systematic review and meta-analysis. PLoS ONE 7, e45759 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Levin, O., Fujiyama, H., Boisgontier, M. P., Swinnen, S. P. & Summers, J. J. Aging and motor inhibition: a converging perspective provided by brain stimulation and imaging approaches. Neurosci. Biobehav. Rev. 43, 100–117 (2014).

    Article  PubMed  Google Scholar 

  4. Prince, M. J. et al. The burden of disease in older people and implications for health policy and practice. Lancet 385, 549–562 (2015).

    Article  PubMed  Google Scholar 

  5. Mattson, M. P. & Arumugam, T. V. Hallmarks of brain aging: adaptive and pathological modification by metabolic states. Cell Metab. 27, 1176–1199 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Grimm, A. & Eckert, A. Brain aging and neurodegeneration: from a mitochondrial point of view. J. Neurochem. 143, 418–431 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sun, N., Youle, R. J. & Finkel, T. The mitochondrial basis of aging. Mol. Cell 61, 654–666 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hansen, M., Rubinsztein, D. C. & Walker, D. W. Autophagy as a promoter of longevity: insights from model organisms. Nat. Rev. Mol. Cell Biol. 19, 579–593 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Feng, Y., He, D., Yao, Z. & Klionsky, D. J. The machinery of macroautophagy. Cell Res. 24, 24–41 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Pickles, S., Vigie, P. & Youle, R. J. Mitophagy and quality control mechanisms in mitochondrial maintenance. Curr. Biol. 28, R170–R185 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Drummond, M. J. et al. Downregulation of E3 ubiquitin ligases and mitophagy-related genes in skeletal muscle of physically inactive, frail older women: a cross-sectional comparison. J. Gerontol. A Biol. Sci. Med. Sci. 69, 1040–1048 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sun, N. et al. Measuring in vivo mitophagy. Mol. Cell 60, 685–696 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rana, A. et al. Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster. Nat. Commun. 8, 448 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. D’Amico, D. et al. The RNA-binding protein PUM2 impairs mitochondrial dynamics and mitophagy during aging. Mol. Cell 73, 775–787 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Aparicio, R., Rana, A. & Walker, D. W. Upregulation of the autophagy adaptor p62/SQSTM1 prolongs health and lifespan in middle-aged Drosophila. Cell Rep. 28, 1029–1040 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rana, A., Rera, M. & Walker, D. W. Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan. Proc. Natl Acad. Sci. USA 110, 8638–8643 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ryu, D. et al. Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents. Nat. Med. 22, 879–888 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Martinez-Vicente, M. Neuronal mitophagy in neurodegenerative diseases. Front. Mol. Neurosci. 10, 64 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Palikaras, K., Lionaki, E. & Tavernarakis, N. Coupling mitogenesis and mitophagy for longevity. Autophagy 11, 1428–1430 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Palikaras, K., Lionaki, E. & Tavernarakis, N. Mitophagy: in sickness and in health. Mol. Cell. Oncol. 3, e1056332 (2016).

    Article  PubMed  CAS  Google Scholar 

  24. Sliter, D. A. et al. Parkin and PINK1 mitigate STING-induced inflammation. Nature 561, 258–262 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Fang, E. F. et al. Mitophagy inhibits amyloid-beta and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease. Nat. Neurosci. 22, 401–412 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Onishi, M., Yamano, K., Sato, M., Matsuda, N. & Okamoto, K. Molecular mechanisms and physiological functions of mitophagy. EMBO J. 40, e104705 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pickrell, A. M. & Youle, R. J. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson’s disease. Neuron 85, 257–273 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dorn, G. W. 2nd Mitochondrial pruning by Nix and BNip3: an essential function for cardiac-expressed death factors. J. Cardiovasc. Transl. Res. 3, 374–383 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Burton, T. R. & Gibson, S. B. The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death. Cell Death Differ. 16, 515–523 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Kale, J., Osterlund, E. J. & Andrews, D. W. BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ. 25, 65–80 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Kubli, D. A., Ycaza, J. E. & Gustafsson, A. B. Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak. Biochem. J 405, 407–415 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Regula, K. M., Ens, K. & Kirshenbaum, L. A. Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes. Circ. Res. 91, 226–231 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Rikka, S. et al. Bnip3 impairs mitochondrial bioenergetics and stimulates mitochondrial turnover. Cell Death Differ. 18, 721–731 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Bellot, G. et al. Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol. Cell. Biol. 29, 2570–2581 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang, T. et al. BNIP3 protein suppresses PINK1 kinase proteolytic cleavage to promote mitophagy. J. Biol. Chem. 291, 21616–21629 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hanna, R. A. et al. Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy. J. Biol. Chem. 287, 19094–19104 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zhu, Y. et al. Modulation of serines 17 and 24 in the LC3-interacting region of Bnip3 determines pro-survival mitophagy versus apoptosis. J. Biol. Chem. 288, 1099–1113 (2013).

    Article  CAS  PubMed  Google Scholar 

  38. O’Sullivan, T. E., Johnson, L. R., Kang, H. H. & Sun, J. C. BNIP3- and BNIP3L-mediated mitophagy promotes the generation of natural killer cell memory. Immunity 43, 331–342 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Glick, D. et al. BNip3 regulates mitochondrial function and lipid metabolism in the liver. Mol. Cell. Biol. 32, 2570–2584 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li, R. et al. Therapeutic effect of Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: the role of the ERK-CREB pathway and Bnip3-mediated mitophagy. Redox Biol. 18, 229–243 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tang, C. et al. Activation of BNIP3-mediated mitophagy protects against renal ischemia-reperfusion injury. Cell Death Dis. 10, 677 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Cho, J., Hur, J. H. & Walker, D. W. The role of mitochondria in Drosophila aging. Exp. Gerontol. 46, 331–334 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Osterwalder, T., Yoon, K. S., White, B. H. & Keshishian, H. A conditional tissue-specific transgene expression system using inducible GAL4. Proc. Natl Acad. Sci. USA 98, 12596–12601 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Poirier, L., Shane, A., Zheng, J. & Seroude, L. Characterization of the Drosophila gene-switch system in aging studies: a cautionary tale. Aging Cell 7, 758–770 (2008).

    Article  CAS  PubMed  Google Scholar 

  45. Walker, D. W. & Benzer, S. Mitochondrial “swirls” induced by oxygen stress and in the Drosophila mutant hyperswirl. Proc. Natl Acad. Sci. USA 101, 10290–10295 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Brandt, T. et al. Changes of mitochondrial ultrastructure and function during ageing in mice and Drosophila. eLife https://doi.org/10.7554/eLife.24662 (2017).

  47. Ferguson, M., Mockett, R. J., Shen, Y., Orr, W. C. & Sohal, R. S. Age-associated decline in mitochondrial respiration and electron transport in Drosophila melanogaster. Biochem. J. 390, 501–511 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 17, 1–382 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Chang, J. T., Kumsta, C., Hellman, A. B., Adams, L. M. & Hansen, M. Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging. eLife https://doi.org/10.7554/eLife.18459 (2017).

  50. Lee, T. V., Kamber Kaya, H. E., Simin, R., Baehrecke, E. H. & Bergmann, A. The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila. Cell Death Differ. 23, 1555–1564 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lee, J. J. et al. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J. Cell Biol. 217, 1613–1622 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. McWilliams, T. G. et al. mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J. Cell Biol. 214, 333–345 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nakatogawa, H., Suzuki, K., Kamada, Y. & Ohsumi, Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell Biol. 10, 458–467 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. Shell, B. C. et al. Measurement of solid food intake in Drosophila via consumption-excretion of a dye tracer. Sci. Rep. 8, 11536 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Partridge, L., Gems, D. & Withers, D. J. Sex and death: what is the connection? Cell 120, 461–472 (2005).

    Article  CAS  PubMed  Google Scholar 

  56. Miller, H. A., Dean, E. S., Pletcher, S. D. & Leiser, S. F. Cell non-autonomous regulation of health and longevity. eLife https://doi.org/10.7554/eLife.62659 (2020).

  57. Weir, H. J. & Mair, W. B. SnapShot: neuronal regulation of aging. Cell 166, 784–784 (2016).

    Article  CAS  PubMed  Google Scholar 

  58. Twig, G. & Shirihai, O. S. The interplay between mitochondrial dynamics and mitophagy. Antioxid. Redox Signal. 14, 1939–1951 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Cho, H. M. et al. Drp1-Zip1 interaction regulates mitochondrial quality surveillance system. Mol. Cell 73, 364–376 (2019).

    Article  CAS  PubMed  Google Scholar 

  60. Demontis, F. & Perrimon, N. FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging. Cell 143, 813–825 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Schinaman, J. M., Rana, A., Ja, W. W., Clark, R. I. & Walker, D. W. Rapamycin modulates tissue aging and lifespan independently of the gut microbiota in Drosophila. Sci. Rep. 9, 7824 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Ulgherait, M., Rana, A., Rera, M., Graniel, J. & Walker, D. W. AMPK modulates tissue and organismal aging in a non-cell-autonomous manner. Cell Rep. 8, 1767–1780 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Rera, M., Azizi, M. J. & Walker, D. W. Organ-specific mediation of lifespan extension: more than a gut feeling? Ageing Res. Rev. 12, 436–444 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Jasper, H. Exploring the physiology and pathology of aging in the intestine of Drosophila melanogaster. Invertebr. Reprod. Dev. 59, 51–58 (2015).

    Article  PubMed  Google Scholar 

  65. Clark, R. I. et al. Distinct shifts in microbiota composition during Drosophila aging impair intestinal function and drive mortality. Cell Rep. 12, 1656–1667 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Rera, M., Clark, R. I. & Walker, D. W. Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila. Proc. Natl Acad. Sci. USA 109, 21528–21533 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Li, H. & Jasper, H. Gastrointestinal stem cells in health and disease: from flies to humans. Dis. Model. Mech. 9, 487–499 (2016).

    PubMed  PubMed Central  Google Scholar 

  68. Biteau, B., Hochmuth, C. E. & Jasper, H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell 3, 442–455 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Hu, D. J. & Jasper, H. Epithelia: understanding the cell biology of intestinal barrier dysfunction. Curr. Biol. 27, R185–R187 (2017).

    Article  CAS  PubMed  Google Scholar 

  70. Dambroise, E. et al. Two phases of aging separated by the Smurf transition as a public path to death. Sci. Rep. 6, 23523 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Kavanagh, K. et al. Microbial translocation and skeletal muscle in young and old vervet monkeys. Age (Dordr.) 38, 58 (2016).

    Article  CAS  Google Scholar 

  72. Rera, M. et al. Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog. Cell Metab. 14, 623–634 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Thevaranjan, N. et al. Age-associated microbial dysbiosis promotes intestinal permeability, systemic inflammation, and macrophage dysfunction. Cell Host Microbe 21, 455–466 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Palikaras, K., Lionaki, E. & Tavernarakis, N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat. Cell Biol. 20, 1013–1022 (2018).

    Article  CAS  PubMed  Google Scholar 

  75. Lou, G. et al. Mitophagy and neuroprotection. Trends Mol. Med. 26, 8–20 (2020).

    Article  CAS  PubMed  Google Scholar 

  76. Zhang, J. & Ney, P. A. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 16, 939–946 (2009).

    Article  CAS  PubMed  Google Scholar 

  77. Gao, A., Jiang, J., Xie, F. & Chen, L. Bnip3 in mitophagy: novel insights and potential therapeutic target for diseases of secondary mitochondrial dysfunction. Clin. Chim. Acta 506, 72–83 (2020).

    Article  CAS  PubMed  Google Scholar 

  78. Ney, P. A. Mitochondrial autophagy: origins, significance, and role of BNIP3 and NIX. Biochim. Biophys. Acta 1853, 2775–2783 (2015).

    Article  CAS  PubMed  Google Scholar 

  79. Jung, J. et al. Mitochondrial NIX promotes tumor survival in the hypoxic niche of glioblastoma. Cancer Res. 79, 5218–5232 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Raji, J. I. & Potter, C. J. The number of neurons in Drosophila and mosquito brains. PLoS ONE 16, e0250381 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Herculano-Houzel, S. & Lent, R. Isotropic fractionator: a simple, rapid method for the quantification of total cell and neuron numbers in the brain. J. Neurosci. 25, 2518–2521 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Z. Zhang (Xiangya Medical School), E. Baehrecke (UMass Medical School), the Vienna Drosophila RNAi Center and the Bloomington Drosophila Stock Center (NIH no. P40OD018537) for fly stocks; J. Guerrero and V. Patel for help with fly work; and L. Palacios Castillo and R. Aparicio for technical assistance. We thank N. Prunet and the MCDB/BSCRC Microscopy Core for training and microscope facilities. This work was supported by NIH grant nos. R01AG037514 and RF1AG049157 to D.W.W. This research was conducted while D.W.W. was a Julie Martin Mid-Career Awardee in Aging Research supported by The Ellison Medical Foundation and AFAR.

Author information

Authors and Affiliations

  1. Department of Integrative Biology and Physiology, University of California, Los Angeles, CA, USA

    Edward T. Schmid, Jung-Hoon Pyo & David W. Walker

  2. Molecular Biology Institute, University of California, Los Angeles, CA, USA

    David W. Walker

Authors
  1. Edward T. Schmid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jung-Hoon Pyo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David W. Walker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.T.S. and D.W.W conceived the project. E.T.S. and J.-H.P. performed experiments. E.T.S. and J.-H.P. analyzed the data and prepared figures. E.T.S., J.-H.P. and D.W.W. wrote the manuscript.

Corresponding author

Correspondence to David W. Walker.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Aging thanks Evandro Fang and Edward Owusu-Ansah for their contribution to the peer review of this work.

Additional information

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

Extended data

Extended Data Fig. 1 RU486 induces BNIP3 expression in the brain of elavGS>UAS-BNIP3 flies.

(a) Immunostaining of brains from 10-day-old elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing BNIP3 expression level (green channel, anti-HA) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 50 µm. (b) Quantification of BNIP3 expression level in brain as shown in (a). n = 10 biologically independent animals per condition. ***p<0.0001; unpaired t test. Data are presented as scatter plots overlaying mean values +/− SEM. (c) Western blot detection of BNIP3 transgene induction in the heads of day 14 elavGS>UAS-BNIP3 flies with or without 9 days of RU486 treatment. n = 5 biological replicates per condition with 10 flies pooled per replicate. (d) Immunostaining of brains from young (10-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondrial morphology (green channel, anti-ATP5a) and BNIP3 (red channel). Arrows and outlines indicate sites of colocalization. Scale bar is 3 µm. RU486 was provided in the media at a concentration of 5 µg ml–1. Images are representative of 5 samples treated with RU and 6 samples provided vehicle.

Extended Data Fig. 2 Neuronal BNIP3 induction prevents loss of neurons in aged brains.

(a) TOPRO staining of nuclei in brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. Scale bar is 5 µm. (b) Quantification of nuclei per 350 µm2 of optic lobe as shown in (a). n = 12 young, 12 aged RU- and 15 aged RU- biologically independent animals per condition, as indicated. *p=0.0219, **p=0.0097; one-way ANOVA/Tukey’s multiple comparisons test. (c) Immunostaining of brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing cleaved (activated) caspase-3 (green channel) and nuclear DNA (blue channel, stained with To-Pro-3). Scale bar is 5 µm. (d) Quantification of cleaved caspase-3 in one optic lobe per fly as shown in (a). n = 8 young, 9 aged RU- and 10 aged RU+ biologically independent animals per condition, as indicated. *p=0.0140 (young vs. aged RU-), *p=0.0119 (aged RU- vs. aged RU+); one-way ANOVA/Tukey’s multiple comparisons test. (e) Immunostaining of nuclei isolated via isotropic fractionation from brains of young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing ELAV (green channel) and DNA (blue channel, stained with To-Pro-3). Scale bar is 50 µm. (f) Quantification of neuronal (elav+) nuclei isolated from brains via isotropic fractionation as shown in (e). n = 6 biological replicates per condition with 3 pooled brains per replicate. *p=0.0263, **p=0.0098; one-way ANOVA/Tukey’s multiple comparisons test. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 3 RU486 treatment in control flies has no effect on mitochondria homeostasis in aged brains.

(a) Immunostaining of brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondria morphology (red channel, anti-ATP5a) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 5 µm. (b) Quantification of mitochondria area in brain as shown in (a). n = 8 biologically independent animals per condition. *p=0.0327 (both), non-significant (n.s.); Kruskal-Wallis test/Dunn’s multiple comparisons test. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 4 Neuronal specific BNIP3 induction reduces ATG8 levels in aged brains.

(a) Immunostaining of brains from young (10-day-old) and aged (30-day-old) elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing ATG8a levels (red channel, anti-ATG8a). Scale bar is 5 µm. (b) Quantification of Atg8a levels in brain as shown in (a). n = 7 young, 6 aged RU- and 8 aged RU+ biologically independent animals, as indicated. *p=0.0446 (young vs. aged RU-), *p=0.0169 (aged RU- vs. aged RU+); Kruskal-Wallis test/Dunn’s multiple comparisons test. RU486 was provided in the media at a concentration of 5 µg/ml. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 5 Midlife neuronal induction of BNIP3 induces mitophagy and extends lifespan.

(a and c) mito-QC of brains from 37-day-old (a) and 44-day-old (c) flies. Genotypes analyzed were elavGS>UAS-mito-QC,UAS-lacZ, as a control, and elavGS>UAS-mito-QC,UAS-BNIP3. RU486-mediated transgenes were induced from day 30 to day 37 (a) or from day 30 to day 44 (c). Images shown of merged GFP and mCherry along with punctate mCherry-only foci (from merged images where GFP has been quenched; mitolysosomes). Scale bar is 5 µm. (b and d) Quantification of the number of mitolysosomes per 500 µm2 brain area and average size (µm2) as shown in (a) and (c) at day 37 (b) and day 44 (d). (b) n = 7 control and 11 BNIP3+ biologically independent animals. *p=0.0102, **p=0.0089; unpaired t tests. (d) n = 8 biologically independent animals per condition. *p=0.0115, **p=0.0053; Mann-Whitney test (#), unpaired t tests (size). (e) Survival curves of elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 30 onward. The shaded area indicates the duration of BNIP3 induction. ***p=0.0010; log-rank test; n = 150 RU- and 147 RU+ biologically independent animals. RU486 was provided in the media at a concentration of 25 µg/ml. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 6 RU486 induces BNIP3 expression in the brain of elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies.

(a) Immunostaining of brains from 10-day-old elavGS>UAS-Atg1RNAi,UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward, showing BNIP3 expression level (green channel, anti-HA) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 50 µm. (b) Quantification of BNIP3 expression level in brain as shown in (a). n = 20 RU- and 15 RU+ biologically independent animals. ***p=0.0008; unpaired t test. RU486 was provided in the media at a concentration of 5 µg/ml. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 7 RU486 treatment in control flies has no effect on lifespan and healthspan, and neuronal-specific BNIP3 induction does not alter food consumption or fecundity.

(a) Survival curve of elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward. non-significant (n.s.); log-rank test. n = 228 RU- and 218 RU+ biologically independent animals. (b) Con-Ex feeding assay of 10-day-old elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward. n = 6 vials of 10 flies per condition. non-significant (n.s.); unpaired t test. (c) Climbing index as a measure of endurance of 30-day-old elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward. n = 8 biological replicates with 100 flies per replicate. non-significant (n.s.); unpaired t test. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as mean values +/− SEM. (d) Con-Ex feeding assay of 10-day-old elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. n = 6 vials of 10 flies per condition. non-significant (n.s.); unpaired t test. (e) Fecundity of 37-day-old elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. n =6 vials of 30 RU- biologically independent animals and 5 vials of 30 RU+ biologically independent animals with values normalized per fly, as indicated. non-significant (n.s.); unpaired t test. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as scatter plots overlaying mean values +/− SEM unless otherwise indicated.

Extended Data Fig. 8 Ubiquitous, gut- or muscle-specific BNIP3 induction shortens lifespan.

(a) Survival curves of daGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. ***p=0.0006 (RU0 vs. RU5) and ***p<0.0001 (RU0 vs. each other RU dose), (n.s.) non-significant; log-rank test; n = 208 RU-, 200 RU5, 204 RU10, 198 RU25 and 201 RU50 biologically independent animals. (b) Survival curves of 5966GS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. ***p<0.0001 (RU0 vs. each RU dose); log-rank test; n = 201 RU-, 194 RU5, 197 RU10, 199 RU25 and 204 RU50 biologically independent animals. (c) Survival curves of Act88FGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. ***p<0.0001 (RU0 vs. each RU dose); log-rank test; n = 239 RU-, 473 RU5, 233 RU10, 234 RU25 and 238 RU50 biologically independent animals. (d) Survival curves of daGS>UAS-BNIP3-RNAi flies with or without RU486-mediated transgene induction from day 5 onward. ***p=0.0006; log-rank test; n = 209 RU- and 233 RU+ biologically independent animals. (e) Survival curves of daGS>UAS-BNIP3-RNAi flies with or without RU486-mediated transgene induction from day 30 onward. ***p<0.001; log-rank test; n = 209 RU- and 202 RU+ biologically independent animals. (f) Survival curves of elavGS>UAS-BNIP3-RNAi flies with or without RU486-mediated transgene induction from day 5 onward. *p=0.0203; log-rank test; n = 165 RU- and 180 RU+ biologically independent animals.

Extended Data Fig. 9 RU486 treatment in control flies has no effect on mitochondria homeostasis in aged muscle and gut.

(a) Immunostaining of indirect flight muscles from young (10-day-old) and aged (30-day-old) elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondria morphology (red channel, anti-ATP5a), muscles (magenta channel, stained with phalloidin/F-Actin) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 10 µm. (b) Quantification of mitochondria area in muscles as shown in (a). n = 6 biologically independent animals per condition. **p=0.0020, ***p=0.0001, non-significant (n.s.); one-way ANOVA/Tukey’s multiple comparisons test. (c) Immunostaining of guts from young (10-day-old) and aged (30-day-old) elavGS>UAS-GFP flies with or without RU486-mediated transgene induction from day 5 onward, showing mitochondria morphology (red channel, anti-ATP5a) and nuclear DNA (blue channel, stained with DAPI). Scale bar is 5 µm. (d) Quantification of mitochondria area in guts as shown in (A). n = 21 biologically independent replicates per condition. *p=0.0131, **p=0.0017; one-way ANOVA/Tukey’s multiple comparisons test. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as scatter plots overlaying mean values +/− SEM.

Extended Data Fig. 10 Neuronal-specific BNIP3 upregulation induces midlife Drp1 expression in the thorax and gut but does not alter microbial dynamics in the gut.

(a) qPCR analyses of Drp1 mRNA levels relative to GAPDH in the thorax on days 10, 30 and 44 in elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. n = 5 biological replicates with 5 dissected thoraxes pooled per replicate. **p=0.0023; two-way ANOVA/Šídák’s multiple comparisons test. (b) qPCR analyses of Drp1 mRNA levels relative to GAPDH in dissected guts on days 10, 30 and 44 in elavGS>UAS-BNIP3 flies with or without RU486-mediated transgene induction from day 5 onward. n = 5 biological replicates with 5 dissected guts pooled per replicate. ***p<0.0001; two-way ANOVA/Šídák’s multiple comparisons test. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as scatter plots overlaying mean values +/− SEM. (c) Bacterial levels assayed by qPCR of 16S rRNA gene in surface sterilized, elavGS>UAS-BNIP3 flies with or without 5 μg ml–1 RU486 treatment from day 5 post-eclosion onwards. n.s.; not significant, two-way ANOVA/Šídák’s multiple comparisons test; n = 3 replicates of ten flies pooled per condition. RU486 was provided in the media at a concentration of 5 µg ml–1. Data are presented as scatter plots overlaying mean values +/− SEM.

Supplementary information

Source data

Source Data Fig. 1.

Unprocessed immunoblot.

Source Data Fig. 2.

Unprocessed immunoblot.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schmid, E.T., Pyo, JH. & Walker, D.W. Neuronal induction of BNIP3-mediated mitophagy slows systemic aging in Drosophila. Nat Aging 2, 494–507 (2022). https://doi.org/10.1038/s43587-022-00214-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s43587-022-00214-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing