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Calcium dysregulation mediates mitochondrial and neurite outgrowth abnormalities in SOD2 deficient embryonic cerebral cortical neurons

Cell Death & Differentiation (2018) | Download Citation

Abstract

Mitochondrial superoxide dismutase 2 (SOD2) is a major antioxidant defense enzyme. Here we provide evidence that SOD2 plays critical roles in maintaining calcium homeostasis in newly generated embryonic cerebral cortical neurons, which is essential for normal mitochondrial function and subcellular distribution, and neurite outgrowth. Primary cortical neurons in cultures established from embryonic day 15 SOD2+/+ and SOD2−/− mice appear similar during the first 24 h in culture. During the ensuing two days in culture, SOD2−/− neurons exhibit a profound reduction of neurite outgrowth and their mitochondria become fragmented and accumulate in the cell body. The structural abnormalities of the mitochondria are associated with reduced levels of phosphorylated (S637) dynamin related protein 1 (Drp1), a major mitochondrial fission-regulating protein, whereas mitochondrial fusion regulating proteins (OPA1 and MFN2) are relatively unaffected. Mitochondrial fission and Drp1 dephosphorylation coincide with impaired mitochondrial Ca2+ buffering capacity and an elevation of cytosolic Ca2+ levels. Treatment of SOD2−/− neurons with the Ca2+ chelator BAPTA-AM significantly increases levels of phosphorylated Drp1, reduces mitochondrial fragmentation and enables neurite outgrowth.

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References

  1. 1.

    Mattson MP, Gleichmann M, Cheng A. Mitochondria in neuroplasticity and neurological disorders. Neuron. 2008;60:748–66.

  2. 2.

    Cheng A, Wan R, Yang J, Kamimura N, Son TG, Ouyang X, et al. Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines. Nat Commun. 2012;3:1250.

  3. 3.

    Li Z, Okamoto K, Hayashi Y, Sheng M. The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell. 2004;119:873–87.

  4. 4.

    Vayssière JL, Cordeau-Lossouarn L, Larcher JC, Basseville M, Gros F, Croizat B. Participation of the mitochondrial genome in the differentiation of neuroblastoma cells. Vitr Cell Dev Biol. 1992;28A:763–72.

  5. 5.

    Mattson MP, Partin J. Evidence for mitochondrial control of neuronal polarity. J Neurosci Res. 1999;56:8–20.

  6. 6.

    Khacho M, Slack RS. Mitochondrial dynamics in the regulation of neurogenesis: from development to the adult brain. Dev Dyn. 2018;247:47–53.

  7. 7.

    Knapp LT, Klann E. Role of reactive oxygen species in hippocampal long-term potentiation: contributory or inhibitory? J Neurosci Res. 2002;70:1–7.

  8. 8.

    Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat Med. 2004;10:Suppl: S18–25.

  9. 9.

    Nicholls DG, Vesce S, Kirk L, Chalmers S. Interactions between mitochondrial bioenergetics and cytoplasmic calcium in cultured cerebellar granule cells. Cell Calcium. 2003;34:407–24.

  10. 10.

    Giacomello M, Drago I, Pizzo P, Pozzan T. Mitochondrial Ca2+ as a key regulator of cell life and death. Cell Death Differ. 2007;14:1267–74.

  11. 11.

    Yang JL, Tadokoro T, Keijzers G, Mattson MP, Bohr VA. Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1. J Biol Chem. 2010;285:28191–9.

  12. 12.

    Carlsson LM, Jonsson J, Edlund T, Marklund SL. Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia. Proc Natl Acad Sci USA. 1995;92:6264–8.

  13. 13.

    Sentman ML, Granström M, Jakobson H, Reaume A, Basu S, Marklund SL. Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase. J Biol Chem. 2006;281:6904–9.

  14. 14.

    Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, et al. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 1995;11:376–81.

  15. 15.

    Lebovitz RM, Zhang H, Vogel H, Cartwright J Jr, Dionne L, Lu N, et al. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci USA. 1996;93:9782–7.

  16. 16.

    Andreassen OA, Ferrante RJ, Dedeoglu A, Albers DW, Klivenyi P, Carlson EJ, et al. Mice with a partial deficiency of manganese superoxide dismutase show increased vulnerability to the mitochondrial toxins malonate, 3-nitropropionic acid, and MPTP. Exp Neurol. 2001;167:189–95.

  17. 17.

    Cheng A, Yang Y, Zhou Y, Maharana C, Lu D, Peng W, et al. Mitochondrial SIRT3 mediates adaptive responses of neurons toexercise and metabolic and excitatory challenges. Cell Metab. 2016;23:128–42.

  18. 18.

    Keller JN, Kindy MS, Holtsberg FW, St Clair DK, Yen HC, Germeyer A, et al. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J Neurosci. 1998;18:687–97.

  19. 19.

    Massaad CA, Washington TM, Pautler RG, Klann E. Overexpression of SOD-2 reduces hippocampal superoxide and prevents memory deficits in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA. 2009;106:13576–81.

  20. 20.

    Mattson MP, Dou P, Kater SB. Outgrowth-regulating actions of glutamate in isolated hippocampal pyramidal neurons. J Neurosci. 1988;8:2087–100.

  21. 21.

    Mattson MP, Murain M, Guthrie PB. Localized calcium influx orients axon formation in embryonic hippocampal pyramidal neurons. Brain Res Dev Brain Res. 1990;52:201–9.

  22. 22.

    Mattson MP, Lee RE, Adams ME, Guthrie PB, Kater SB. Interactions between entorhinal axons and target hippocampalneurons: a role for glutamate in the development of hippocampalcircuitry. Neuron. 1988;1:865–76.

  23. 23.

    Flippo KH, Strack S. Mitochondrial dynamics in neuronal injury, development and plasticity. J Cell Sci. 2017;130:671–81.

  24. 24.

    Friedman JS, Rebel VI, Derby R, Bell K, Huang TT, Kuypers FA, et al. Absence of mitochondrial superoxide dismutase results in a murine hemolytic anemia responsive to therapy with a catalytic antioxidant. J Exp Med. 2001;193:925–34.

  25. 25.

    Knott AB, Perkins G, Schwarzenbacher R, Bossy-Wetzel E. Mitochondrial fragmentation in neurodegeneration. Nat Rev Neurosci. 2008;9:505–18.

  26. 26.

    Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, et al. Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc Natl Acad Sci USA. 2008;105:15803–8.

  27. 27.

    Chang CR, Blackstone C. Cyclic AMP-dependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology. J Biol Chem. 2007;282:21583–7.

  28. 28.

    Floto RA, Mahaut-Smith MP, Somasundaram B, Allen JM. (1995) IgG-induced Ca2+oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Cell Calcium. 1995;18:377–89.

  29. 29.

    Schild D, Jung A, Schultens HA. Localization of calcium entry through calcium channels in olfactory receptor neurons using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red. Cell Calcium. 1994;15:341–8.

  30. 30.

    Lipp P, Niggli E. Ratiometric confocal Ca(2+)-measurements with visible wavelength indicators in isolated cardiac myocytes. Cell Calcium. 1993;14:359–72.

  31. 31.

    Harkins AB, Kurebayashi N, Baylor SM. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys J. 1993;65:865–81.

  32. 32.

    Kurebayashi N, Harkins AB, Baylor SM. Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophys J. 1993;64:1934–60.

  33. 33.

    Hou Y, Ouyang X, Wan R, Cheng H, Mattson MP, Cheng A. Mitochondrial superoxide production negatively regulates neural progenitor proliferation and cerebral cortical development. Stem Cells. 2012;30:2535–47.

  34. 34.

    Fukumitsu K, Fujishima K, Yoshimura A, Wu YK, Heuser J, Kengaku M. Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites. J Neurosci. 2015;35:5707–23.

  35. 35.

    Mattson MP. Mitochondrial regulation of neuronal plasticity. Neurochem Res. 2007;32:707–15.

  36. 36.

    Ligon LA, Steward O. Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons. J Comp Neurol. 2000;427:340–50.

  37. 37.

    Niescier RF, Chang KT, Min KT. Miro, MCU, and calcium: bridging our understanding of mitochondrial movement in axons. Front Cell Neurosci. 2013;7:148.

  38. 38.

    Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 2006;125:1241–52.

  39. 39.

    Elchuri S, Oberley TD, Qi W, Eisenstein RS, Jackson Roberts L, Van Remmen H, et al. CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. Oncogene. 2005;24:367–80.

  40. 40.

    Shefner JM, Reaume AG, Flood DG, Scott RW, Kowall NW, Ferrante RJ, et al. Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology. 1999;53:1239–46.

  41. 41.

    Fischer LR, Li Y, Asress SA, Jones DP, Glass JD. Absence of SOD1 leads to oxidative stress in peripheral nerve and causes a progressive distal motor axonopathy. Exp Neurol. 2012;233:163–71.

  42. 42.

    Kondo T, Reaume AG, Huang TT, Murakami K, Carlson E, Chen S, et al. Edema formation exacerbates neurological and histological outcomes after focal cerebral ischemia in CuZn-superoxide dismutase gene knockoutmutant mice. Acta Neurochir Suppl. 1997;70:62–64.

  43. 43.

    Abramov AY, Canevari L, Duchen MR. Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J Neurosci. 2004;24:565–75.

  44. 44.

    Pollock JD, Williams DA, Gifford MA, Li LL, Du X, Fisherman J, et al. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet. 1995;9:202–9.

  45. 45.

    Kishida KT, Hoeffer CA, Hu D, Pao M, Holland SM, Klann E. Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease. Mol Cell Biol. 2006;26:5908–20.

  46. 46.

    Berridge MV, Tan AS. Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys. 1993;303:474–82.

  47. 47.

    Berridge M, Tan A, McCoy K, Wang R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica. 1996;4:14–19.

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Acknowledgements

This research was supported by the Intramural Research Program of the National Institute on Aging and NSFC (Project 31521062).

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Author notes

  1. These authors contributed equally: Qijin Zhao, Daoyuan Lu.

Affiliations

  1. Laboratory of Neurosciences, Biomedical Research Center, National Institute on Aging Intramural Research Program, 251 Bayview Blvd, Baltimore, MD, 21224, USA

    • Qijin Zhao
    • , Daoyuan Lu
    • , Jing Wang
    • , Mark P. Mattson
    •  & Aiwu Cheng
  2. Laboratory of Calcium Signaling and Mitochondrial Biomedicine, Institute of Molecular Medicine, Peking University, 100871, Beijing, China

    • Qijin Zhao
    • , Beibei Liu
    •  & Heping Cheng
  3. Department of Neuroscience, Johns Hopkins University School Medicine, Baltimore, MD, 21205, USA

    • Mark P. Mattson

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The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Mark P. Mattson or Aiwu Cheng.

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DOI

https://doi.org/10.1038/s41418-018-0230-4