MicroRNA-210 Downregulates ISCU and Induces Mitochondrial Dysfunction and Neuronal Death in Neonatal Hypoxic-Ischemic Brain Injury

  • Qingyi MaEmail author
  • Chiranjib Dasgupta
  • Yong Li
  • Lei Huang
  • Lubo ZhangEmail author


Neonatal hypoxic-ischemic (HI) brain injury causes significant mortality and long-term neurologic sequelae. We previously demonstrated that HI significantly increased microRNA-210 (miR-210) in the neonatal rat brain and inhibition of brain endogenous miR-210 was neuroprotective in HI brain injury. However, the molecular mechanisms underpinning this neuroprotection remain unclear. Using both in vivo and in vitro models, herein we uncover a novel mechanism mediating oxidative brain injury after neonatal HI, in which miR-210 induces mitochondrial dysfunction via downregulation of iron-sulfur cluster assembly protein (ISCU). Inhibition of miR-210 significantly ameliorates mitochondrial dysfunction, oxidative stress, and neuronal loss in the neonatal brain subjected to HI, as well as in primary cortical neurons exposed to oxygen-glucose deprivation (OGD). These effects are mediated through ISCU, in that miR-210 mimic decreases ISCU abundance in the brains of rat pups and primary cortical neurons, and inhibition of miR-210 protects ISCU against HI in vivo or OGD in vitro. Deletion of miR-210 binding sequences at the 3′UTR of ISCU transcript ablates miR-210-induced downregulation of ISCU protein abundance in PC12 cells. In primary cortical neurons, miR-210 mimic or silencing ISCU results in mitochondrial dysfunction, reactive oxygen species production, and activation of caspase-dependent death pathways. Of importance, knockdown of ISCU increases HI-induced injury in the neonatal rat brain and counteracts the neuroprotection of miR-210 inhibition. Therefore, miR-210 by downregulating ISCU and inducing mitochondrial dysfunction in neurons is a potent contributor of oxidative brain injury after neonatal HI.


Neonatal hypoxia-ischemia MicroRNA-210 Mitochondrial dysfunction Oxidative stress Neuronal death 


Author Contributions

Q.M. and L.Z. contributed to the study design, manuscript preparation, and drafting the manuscript. Q.M., C.D., Y.L., and L.H. contributed to experiment conducting, and data collection and analysis. All authors read and approved the final manuscript.

Funding Information

This work was supported by the National Institutes of Health grants HL118861 and NS103017 to L.Z., and American Heart Association Western States Affiliate winter 2015 Beginning Grant-in-Aid 15BGIA25750063 to Q.M.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Douglas-Escobar M, Weiss MD (2012) Biomarkers of hypoxic-ischemic encephalopathy in newborns. Front Neurol 3(144).
  2. 2.
    Graham EM, Ruis KA, Hartman AL, Northington FJ, Fox HE (2008) A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy. Am J Obstet Gynecol 199:587–595. CrossRefPubMedGoogle Scholar
  3. 3.
    Kurinczuk JJ, White-Koning M, Badawi N (2010) Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev 86:329–338. CrossRefPubMedGoogle Scholar
  4. 4.
    Koenigsberger MR (2000) Advances in neonatal neurology: 1950-2000. Rev Neurol 31:202–211PubMedGoogle Scholar
  5. 5.
    Zanelli G, Petrarca M, Cappa P, Castelli E, Berthoz A (2009) Reorientation ability of adults and healthy children submitted to whole body horizontal rotations. Cogn Process 10(Suppl 2):S346–S350. CrossRefPubMedGoogle Scholar
  6. 6.
    Johnston MV, Fatemi A, Wilson MA, Northington F (2011) Treatment advances in neonatal neuroprotection and neurointensive care. The Lancet Neurology 10:372–382. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Juul SE, Ferriero DM (2014) Pharmacologic neuroprotective strategies in neonatal brain injury. Clin Perinatol 41:119–131. CrossRefPubMedGoogle Scholar
  8. 8.
    Yin W, Signore AP, Iwai M, Cao G, Gao Y, Chen J (2008) Rapidly increased neuronal mitochondrial biogenesis after hypoxic-ischemic brain injury. Stroke 39:3057–3063. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Chavez-Valdez R, Martin LJ, Flock DL, Northington FJ (2012) Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia-ischemia. Neuroscience 219:192–203. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Lu Y, Tucker D, Dong Y, Zhao N, Zhuo X, Zhang Q (2015) Role of mitochondria in neonatal hypoxic-ischemic brain injury. J Neurosci Rehabil 2:1–14PubMedPubMedCentralGoogle Scholar
  11. 11.
    Al-Hasan YM, Evans LC, Pinkas GA et al (2013) Chronic hypoxia impairs cytochrome oxidase activity via oxidative stress in selected fetal Guinea pig organs. Reprod Sci 20:299–307. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Grivennikova VG, Vinogradov AD (2006) Generation of superoxide by the mitochondrial complex I. Biochim Biophys Acta 1757:553–561. CrossRefPubMedGoogle Scholar
  13. 13.
    Kussmaul L, Hirst J (2006) The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci U S A 103:7607–7612. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Perier C, Tieu K, Guegan C, Caspersen C, Jackson-Lewis V, Carelli V, Martinuzzi A, Hirano M et al (2005) Complex I deficiency primes Bax-dependent neuronal apoptosis through mitochondrial oxidative damage. Proc Natl Acad Sci U S A 102:19126–19131. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lopez-Fabuel I, Le Douce J, Logan A et al (2016) Complex I assembly into supercomplexes determines differential mitochondrial ROS production in neurons and astrocytes. Proc Natl Acad Sci U S A 113:13063–13068. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Ma Q, Dasgupta C, Li Y, Bajwa NM, Xiong F, Harding B, Hartman R, Zhang L (2016) Inhibition of microRNA-210 provides neuroprotection in hypoxic-ischemic brain injury in neonatal rats. Neurobiol Dis 89:202–212. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Esteller M (2011) Non-coding RNAs in human disease. Nat Rev Genet 12:861–874. CrossRefPubMedGoogle Scholar
  18. 18.
    Pasquinelli AE (2012) MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 13:271–282. CrossRefPubMedGoogle Scholar
  19. 19.
    Nallamshetty S, Chan SY, Loscalzo J (2013) Hypoxia: a master regulator of microRNA biogenesis and activity. Free Radic Biol Med 64:20–30. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chan SY, Zhang YY, Hemann C, Mahoney CE, Zweier JL, Loscalzo J (2009) MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metab 10:273–284. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Tong WH, Rouault TA (2006) Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab 3:199–210. CrossRefPubMedGoogle Scholar
  22. 22.
    Lee DC, Romero R, Kim JS, Tarca AL, Montenegro D, Pineles BL, Kim E, Lee JH et al (2011) miR-210 targets iron-sulfur cluster scaffold homologue in human trophoblast cell lines: siderosis of interstitial trophoblasts as a novel pathology of preterm preeclampsia and small-for-gestational-age pregnancies. Am J Pathol 179:590–602. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rice JE, Vannucci RC, Brierley JB (1981) The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol 9:131–141. CrossRefPubMedGoogle Scholar
  24. 24.
    Ma Q, Dasgupta C, Li Y, Huang L, Zhang L (2017) MicroRNA-210 suppresses junction proteins and disrupts blood-brain barrier integrity in neonatal rat hypoxic-ischemic brain injury. Int J Mol Sci 18.
  25. 25.
    Li Y, Xiao D, Dasgupta C, Xiong F, Tong W, Yang S, Zhang L (2012) Perinatal nicotine exposure increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of angiotensin II receptors. Stroke 43:2483–2490. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Beaudoin GM 3rd, Lee SH, Singh D et al (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754. CrossRefPubMedGoogle Scholar
  27. 27.
    Ma Q, Zhang L (2018) C-type natriuretic peptide functions as an innate neuroprotectant in neonatal hypoxic-ischemic brain injury in mouse via natriuretic peptide receptor 2. Exp Neurol 304:58–66. CrossRefPubMedGoogle Scholar
  28. 28.
    Frantseva MV, Carlen PL, El-Beheiry H (1999) A submersion method to induce hypoxic damage in organotypic hippocampal cultures. J Neurosci Methods 89:25–31CrossRefGoogle Scholar
  29. 29.
    Newcomb-Fernandez JK, Zhao X, Pike BR, Wang KKW, Kampfl A, Beer R, DeFord SM, Hayes RL (2001) Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures. J Cereb Blood Flow Metab 21:1281–1294. CrossRefPubMedGoogle Scholar
  30. 30.
    Yin HZ, Sensi SL, Ogoshi F, Weiss JH (2002) Blockade of Ca2+-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn2+ accumulation and neuronal loss in hippocampal pyramidal neurons. J Neurosci 22:1273–1279CrossRefGoogle Scholar
  31. 31.
    Zhao X, Strong R, Zhang J, Sun G, Tsien JZ, Cui Z, Grotta JC, Aronowski J (2009) Neuronal PPARgamma deficiency increases susceptibility to brain damage after cerebral ischemia. J Neurosci 29:6186–6195. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Gonzalez-Rodriguez PJ, Xiong F, Li Y, Zhou J, Zhang L (2014) Fetal hypoxia increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of glucocorticoid receptors. Neurobiol Dis 65:172–179. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ischiropoulos H (1998) Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 356:1–11. CrossRefPubMedGoogle Scholar
  34. 34.
    Dalleau S, Baradat M, Gueraud F, Huc L (2013) Cell death and diseases related to oxidative stress: 4-hydroxynonenal (HNE) in the balance. Cell Death Differ 20:1615–1630. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Aoyama K, Suh SW, Hamby AM, Liu J, Chan WY, Chen Y, Swanson RA (2006) Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci 9:119–126. CrossRefPubMedGoogle Scholar
  36. 36.
    Blanchard-Fillion B, Prou D, Polydoro M, Spielberg D, Tsika E, Wang Z, Hazen SL, Koval M et al (2006) Metabolism of 3-nitrotyrosine induces apoptotic death in dopaminergic cells. J Neurosci 26:6124–6130. CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang W, Liu J, Hu X, Li P, Leak RK, Gao Y, Chen J (2015) N-3 polyunsaturated fatty acids reduce neonatal hypoxic/ischemic brain injury by promoting phosphatidylserine formation and Akt signaling. Stroke 46:2943–2950. CrossRefPubMedGoogle Scholar
  38. 38.
    Zhao X, Wang H, Sun G, Zhang J, Edwards NJ, Aronowski J (2015) Neuronal interleukin-4 as a modulator of microglial pathways and ischemic brain damage. J Neurosci 35:11281–11291. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Chen Z, Li Y, Zhang H et al (2010) Hypoxia-regulated microRNA-210 modulates mitochondrial function and decreases ISCU and COX10 expression. Oncogene 29:4362–4368. CrossRefPubMedGoogle Scholar
  40. 40.
    Escobar-Khondiker M, Hollerhage M, Muriel MP, Champy P, Bach A, Depienne C, Respondek G, Yamada ES et al (2007) Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons. J Neurosci 27:7827–7837. CrossRefPubMedGoogle Scholar
  41. 41.
    Imaizumi N, Kwang Lee K, Zhang C, Boelsterli UA (2015) Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I. Redox Biol 4:279–288. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Buonocore G, Groenendaal F (2007) Anti-oxidant strategies. Semin Fetal Neonatal Med 12:287–295. CrossRefPubMedGoogle Scholar
  43. 43.
    Davidson JO, Wassink G, van den Heuij LG, Bennet L, Gunn AJ (2015) Therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy—where to from here? Front Neurol 6(198).
  44. 44.
    Wu Q, Chen W, Sinha B, Tu Y, Manning S, Thomas N, Zhou S, Jiang H et al (2015) Neuroprotective agents for neonatal hypoxic-ischemic brain injury. Drug Discov Today 20:1372–1381. CrossRefPubMedGoogle Scholar
  45. 45.
    Folkerth RD (2005) Neuropathologic substrate of cerebral palsy. J Child Neurol 20:940–949CrossRefGoogle Scholar
  46. 46.
    Semple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ (2013) Brain development in rodents and humans: identifying benchmarks of maturation and vulnerability to injury across species. Prog Neurobiol 106–107:1–16. CrossRefPubMedGoogle Scholar
  47. 47.
    Han BH, DeMattos RB, Dugan LL et al (2001) Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia. Nat Med 7:338–343. CrossRefPubMedGoogle Scholar
  48. 48.
    Perlman JM (2006) Summary proceedings from the neurology group on hypoxic-ischemic encephalopathy. Pediatrics 117:S28–S33. CrossRefPubMedGoogle Scholar
  49. 49.
    Northington FJ, Zelaya ME, O’Riordan DP, Blomgren K, Flock DL, Hagberg H, Ferriero DM, Martin LJ (2007) Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as “continuum” phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain. Neuroscience 149:822–833. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Hossain MA (2008) Hypoxic-ischemic injury in neonatal brain: involvement of a novel neuronal molecule in neuronal cell death and potential target for neuroprotection. Int J Dev Neurosci 26:93–101. CrossRefPubMedGoogle Scholar
  51. 51.
    Vasiljevic B, Maglajlic-Djukic S, Gojnic M, Stankovic S, Ignjatovic S, Lutovac D (2011) New insights into the pathogenesis of perinatal hypoxic-ischemic brain injury. Pediatr Int 53:454–462. CrossRefPubMedGoogle Scholar
  52. 52.
    Turrens JF, Alexandre A, Lehninger AL (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 237:408–414CrossRefGoogle Scholar
  53. 53.
    Turrens JF (1997) Superoxide production by the mitochondrial respiratory chain. Biosci Rep 17:3–8CrossRefGoogle Scholar
  54. 54.
    Patel RP, McAndrew J, Sellak H, White CR, Jo H, Freeman BA, Darley-Usmar VM (1999) Biological aspects of reactive nitrogen species. Biochim Biophys Acta 1411:385–400CrossRefGoogle Scholar
  55. 55.
    Voloboueva LA, Sun X, Xu L, Ouyang YB, Giffard RG (2017) Distinct effects of miR-210 reduction on neurogenesis: increased neuronal survival of inflammation but reduced proliferation associated with mitochondrial enhancement. J Neurosci 37:3072–3084. CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Fasanaro P, D’Alessandra Y, Di Stefano V, et al (2008) MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 283:15878–15883.
  57. 57.
    Ziu M, Fletcher L, Rana S, Jimenez DF, Digicaylioglu M (2011) Temporal differences in microRNA expression patterns in astrocytes and neurons after ischemic injury. PLoS One 6:e14724. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Rouault TA, Tong WH (2008) Iron-sulfur cluster biogenesis and human disease. Trends Genet 24:398–407. CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The Lawrence D. Longo Center for Perinatal Biology, Department of Basic SciencesLoma Linda University School of MedicineLoma LindaUSA

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