Journal of Molecular Neuroscience

, Volume 66, Issue 2, pp 229–237 | Cite as

Nrf2 Signaling in Sodium Azide-Treated Oligodendrocytes Restores Mitochondrial Functions

  • Annette Liessem-Schmitz
  • Nico Teske
  • Miriam Scheld
  • Stella Nyamoya
  • Adib Zendedel
  • Cordian Beyer
  • Tim Clarner
  • Athanassios FragoulisEmail author


Mitochondrial dysfunctions mark a critical step in many central nervous system (CNS) pathologies, including multiple sclerosis (MS). Such dysfunctions lead to depolarization of mitochondrial membranes and imbalanced redox homeostasis. In this context, reactive oxygen species (ROS) are potentially deleterious but can also act as an important signaling step for cellular maintenance. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the key regulator in the cellular oxidative stress-response, induces a battery of genes involved in repair and regeneration. Here, we investigated the relevance of Nrf2 signaling for the prevention of cellular damage caused by dysfunctional mitochondria. We employed sodium azide (SA) as mitochondrial inhibitor on oligodendroglial OliNeu cells in vitro, and the cuprizone model with wild type and GFAP-Cre+::Keap1loxP/loxP mice to induce mitochondrial defects. The importance of Nrf2 for cellular functions and survival after SA treatment was elucidated by in vitro knockdown experiments with shRNA directed against Nrf2 and its inhibitor Keap1 as well as by methysticin treatment. Metabolic activity, cytotoxicity, and depolarization of the mitochondrial membrane were analyzed after SA treatment. The expression of Nrf2 target genes as well as endoplasmic reticulum stress response genes was additionally measured by real-time PCR (in vitro) and PCR gene arrays (in vivo). Treatment of OliNeu cells with SA resulted in significant depolarization of the mitochondrial membrane, decreased metabolic activity, and increased cytotoxicity. This was partly counteracted in Nrf2-hyperactivated cells and intensified in Nrf2-knockdown cells. Our studies demonstrate a key role of Nrf2 in maintaining cellular functions and survival in the context of mitochondrial dysfunction.


Nrf2 Oligodendrocytes Mitochondrial dysfunction Complex IV Sodium azide Depolarization 



We thank Helga Helten, Petra Ibold, and Uta Zahn for their excellent technical assistance. We thank Sandra Amor for her helpful input.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12031_2018_1159_MOESM1_ESM.pdf (3.1 mb)
Fig. S1 The Nrf2/Keap1 axis and applied shRNA strategies for KD experiments. (left side) Under steady-state conditions, Nrf2 is scavenged by its intracellular inhibitor Keap1. This interaction leads directly to 26S proteasome-mediated degradation of Nrf2. In the presence of Nrf2 activating stimuli, Keap1 dissociates from Nrf2 and enables its nuclear translocation. There, Nrf2 binds to the anti-oxidant response elements (ARE) within the promoter region of its target genes and thereby induces or enhances their gene expression. (right side) In our experiments, we applied shRNA against Keap1 to decrease Keap1 protein content and thereby boost the Nrf2 activation in OliNeu cells even under steady-state conditions. The use of shRNA directed against Nrf2 was chosen to decrease Nrf2 activity in OliNeu cells. (PDF 3138 kb)
12031_2018_1159_MOESM2_ESM.xlsx (33 kb)
ESM 1 (XLSX 32 kb)


  1. Acs P, Selak MA, Komoly S, Kalman B (2013) Distribution of oligodendrocyte loss and mitochondrial toxicity in the cuprizone-induced experimental demyelination model. J Neuroimmunol 262:128–131. CrossRefPubMedGoogle Scholar
  2. Al-Sawaf O, Clarner T, Fragoulis A, Kan YW, Pufe T, Streetz K, Wruck CJ (2015) Nrf2 in health and disease: current and future clinical implications. Clin Sci (Lond) 129:989–999. CrossRefGoogle Scholar
  3. Baird L, Lleres D, Swift S, Dinkova-Kostova AT (2013) Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex. Proc Natl Acad Sci U S A 110:15259–15264. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bellezza I, Giambanco I, Minelli A, Donato R (2018) Nrf2-Keap1 signaling in oxidative and reductive stress. Biochim Biophys Acta 1865:721–733. CrossRefGoogle Scholar
  5. Bennett MC, Mlady GW, Kwon YH, Rose GM (1996) Chronic in vivo sodium azide infusion induces selective and stable inhibition of cytochrome c oxidase. J Neurochem 66:2606–2611CrossRefGoogle Scholar
  6. Bomprezzi R (2015) Dimethyl fumarate in the treatment of relapsing-remitting multiple sclerosis: an overview. Ther Adv Neurol Disord 8:20–30. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bradl M, Lassmann H (2010) Oligodendrocytes: biology and pathology. Acta Neuropathol 119:37–53. CrossRefPubMedGoogle Scholar
  8. Campbell G, Mahad D (2018) Mitochondrial dysfunction and axon degeneration in progressive multiple sclerosis. FEBS Lett. CrossRefPubMedGoogle Scholar
  9. Campbell GR, Worrall JT, Mahad DJ (2014) The central role of mitochondria in axonal degeneration in multiple sclerosis. Mult Scler 20:1806–1813. CrossRefPubMedGoogle Scholar
  10. Carvalho KS (2013) Mitochondrial dysfunction in demyelinating diseases. Semin Pediatr Neurol 20:194–201. CrossRefPubMedGoogle Scholar
  11. Cassina P et al (2008) Mitochondrial dysfunction in SOD1G93A-bearing astrocytes promotes motor neuron degeneration: prevention by mitochondrial-targeted antioxidants. J Neurosci 28:4115–4122. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Clarner T, Parabucki A, Beyer C, Kipp M (2011) Corticosteroids impair remyelination in the corpus callosum of cuprizone-treated mice. J Neuroendocrinol 23:601–611. CrossRefPubMedGoogle Scholar
  13. Cozzolino M, Carri MT (2012) Mitochondrial dysfunction in ALS. Prog Neurobiol 97:54–66. CrossRefPubMedGoogle Scholar
  14. Cross AH, Manning PT, Keeling RM, Schmidt RE, Misko TP (1998) Peroxynitrite formation within the central nervous system in active multiple sclerosis. J Neuroimmunol 88:45–56CrossRefPubMedGoogle Scholar
  15. Das NR, Sharma SS (2016) Cognitive impairment associated with Parkinson’s disease: role of mitochondria. Curr Neuropharmacol 14:584–592CrossRefPubMedPubMedCentralGoogle Scholar
  16. De Riccardis L et al (2018) Copper and ceruloplasmin dyshomeostasis in serum and cerebrospinal fluid of multiple sclerosis subjects. Biochim Biophys Acta 1864:1828–1838. CrossRefGoogle Scholar
  17. Draheim T et al (2016) Activation of the astrocytic Nrf2/ARE system ameliorates the formation of demyelinating lesions in a multiple sclerosis animal model. Glia 64:2219–2230. CrossRefPubMedGoogle Scholar
  18. Faes L, Callewaert G (2011) Mitochondrial dysfunction in familial amyotrophic lateral sclerosis. J Bioenergetics Biomembranes 43:587–592. CrossRefGoogle Scholar
  19. Fragoulis A et al (2017) Oral administration of methysticin improves cognitive deficits in a mouse model of Alzheimer’s disease. Oxid Med Cell Longev 12:843–853. CrossRefGoogle Scholar
  20. Gao C et al (2018) Neuroprotective effects of hydrogen sulfide on sodium azide-induced oxidative stress in PC12 cells. Int J Mol Med 41:242–250. CrossRefPubMedGoogle Scholar
  21. Guzman-Villanueva D, Weissig V (2017) Mitochondria-targeted agents: mitochondriotropics, mitochondriotoxics, and mitocans. Handb Exp Pharmacol 240:423–438. CrossRefPubMedGoogle Scholar
  22. Haider L et al (2011) Oxidative damage in multiple sclerosis lesions brain. J Neurol 134:1914–1924. CrossRefGoogle Scholar
  23. Harvey J, Hardy SC, Ashford ML (1999) Dual actions of the metabolic inhibitor, sodium azide on K (ATP) channel currents in the rat CRI-G1 insulinoma cell line. Br J Pharmacol 126:51–60. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hayashi G et al (2017) Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans. Hum Mol Genet 26:2864–2873. CrossRefPubMedGoogle Scholar
  25. Hroudova J, Singh N, Fisar Z (2014) Mitochondrial dysfunctions in neurodegenerative diseases: relevance to Alzheimer’s disease. BioMed Res Int 2014:175062. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Irvin CW, Kim RB, Mitchell CS (2015) Seeking homeostasis: temporal trends in respiration, oxidation, and calcium in SOD1 G93A Amyotrophic Lateral Sclerosis mice. Front Cell Neurosci 9:248. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Juurlink BHJ, Thorburne SK, Hertz L (1998) Peroxide-scavenging deficit underlies oligodendrocyte susceptibility to oxidative stress. Glia 22:371–378.<371::AID-GLIA6>3.0.CO;2-6 CrossRefPubMedGoogle Scholar
  28. Kaasik A, Safiulina D, Choubey V, Kuum M, Zharkovsky A, Veksler V (2007) Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons. J Biol Chem 282:32821–32826. CrossRefPubMedGoogle Scholar
  29. Karlik M, Valkovic P, Hancinova V, Krizova L, Tothova L, Celec P (2015) Markers of oxidative stress in plasma and saliva in patients with multiple sclerosis. Clin Biochem 48:24–28. CrossRefPubMedGoogle Scholar
  30. Lan M, Tang X, Zhang J, Yao Z (2018) Insights in pathogenesis of multiple sclerosis: nitric oxide may induce mitochondrial dysfunction of oligodendrocytes. Rev Neurosci 29:39–53. CrossRefPubMedGoogle Scholar
  31. Leoni V et al (2016) Mitochondrial dysfunctions in 7-ketocholesterol-treated 158N oligodendrocytes without or with alpha-tocopherol: impacts on the cellular profil of tricarboxylic cycle-associated organic acids, long chain saturated and unsaturated fatty acids, oxysterols, cholesterol and cholesterol precursors. J Steroid Biochem Mol Biol. CrossRefPubMedGoogle Scholar
  32. Licht-Mayer S et al (2015) Cell type-specific Nrf2 expression in multiple sclerosis lesions. Acta Neuropathol 130:263–277. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795. CrossRefPubMedGoogle Scholar
  34. Linnane AW, Marzuki S, Ozawa T, Tanaka M (1989) Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1:642–645CrossRefPubMedGoogle Scholar
  35. Liu JS, Zhao ML, Brosnan CF, Lee SC (2001) Expression of inducible nitric oxide synthase and nitrotyrosine in multiple sclerosis lesions. Am J Pathol 158:2057–2066. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lu MC, Ji JA, Jiang ZY, You QD (2016) The Keap1-Nrf2-ARE pathway as a potential preventive and therapeutic target: an update. Med Res Rev 36:924–963. CrossRefPubMedGoogle Scholar
  37. Mao P, Reddy PH (2010) Is multiple sclerosis a mitochondrial disease? Biochim Biophys Acta 1802:66–79. CrossRefPubMedGoogle Scholar
  38. Morel A, Bijak M, Niwald M, Miller E, Saluk J (2017) Markers of oxidative/nitrative damage of plasma proteins correlated with EDSS and BDI scores in patients with secondary progressive multiple sclerosis. Redox Rep 22:547–555. CrossRefPubMedGoogle Scholar
  39. Patergnani S et al (2017) Mitochondria in multiple sclerosis: molecular mechanisms of pathogenesis. Int Rev Cell Mol Biol 328:49–103. CrossRefPubMedGoogle Scholar
  40. Qin J, Goswami R, Balabanov R, Dawson G (2007) Oxidized phosphatidylcholine is a marker for neuroinflammation in multiple sclerosis brain. J Neurosci Res 85:977–984. CrossRefPubMedGoogle Scholar
  41. Safiulina D, Veksler V, Zharkovsky A, Kaasik A (2006) Loss of mitochondrial membrane potential is associated with increase in mitochondrial volume: physiological role in neurones. J Cell Physiol 206:347–353. CrossRefPubMedGoogle Scholar
  42. Shu Y et al (2017) Association of serum gamma-glutamyltransferase and C-reactive proteins with neuromyelitis optica and multiple sclerosis. Mult Scler Relat Disord 18:65–70. CrossRefPubMedGoogle Scholar
  43. Teske N, Liessem A, Fischbach F, Clarner T, Beyer C (2018) Chemical hypoxia-induced integrated stress response activation in oligodendrocytes is mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2). J Neurochem 144:285–301. CrossRefPubMedGoogle Scholar
  44. van Horssen J, Schreibelt G, Drexhage J, Hazes T, Dijkstra CD, van der Valk P, de Vries HE (2008) Severe oxidative damage in multiple sclerosis lesions coincides with enhanced antioxidant enzyme expression. Free Radic Biol Med 45:1729–1737. CrossRefPubMedGoogle Scholar
  45. Witte ME, Mahad DJ, Lassmann H, van Horssen J (2014) Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis. Trends Mol Med 20:179–187. CrossRefPubMedGoogle Scholar
  46. Wruck CJ, Gotz ME, Herdegen T, Varoga D, Brandenburg LO, Pufe T (2008) Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation. Mol Pharmacol 73:1785–1795. CrossRefPubMedGoogle Scholar
  47. Ziabreva I et al (2010) Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes. Glia 58:1827–1837. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of NeuroanatomyUniklinik RWTH Aachen UniversityAachenGermany
  2. 2.Department of Anatomy IILudwig-Maximilians-University of MunichMunichGermany
  3. 3.Department of Anatomy and Cell Biology, Medical FacultyUniklinik RWTH Aachen UniversityAachenGermany

Personalised recommendations