Molecular Neurobiology

, Volume 55, Issue 8, pp 6369–6386 | Cite as

Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury

  • Jinghan Feng
  • Xingmiao Chen
  • Binghe Guan
  • Caiming Li
  • Jinhua Qiu
  • Jiangang Shen


Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO-induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.


Cerebral ischemia-reperfusion injury Autophagy Mitochondria Mitophagy Peroxynitrite Nitrative stress 



We thank the Faculty Core Facility, Li Ka Shing Faculty of Medicine, The University of Hong Kong, for providing the Carl Zeiss LSM 780 used in capturing confocal fluorescent images. We also appreciate the Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, for supplying the MALDI-TOF/TOF tandem mass spectrometry analysis service. Alternatively, we greatly appreciate Lingen Kong, Chunsheng Cai, and Qianwu Zhou in the Department of Neurology and the Clinical Laboratory of Huizhou First People’s Hospital, Huizhou.

Author Contributions

J.-H.F. designed and performed the experiments, analyzed the data, and wrote the manuscript. X.-M.C. and B.-H.G. carried out the clinical trial, detected the ELISA analysis, and analyzed the data. C.-M.L. and J.-H.Q. supervised the clinical trial. J.-G.S conceived of and supervised this research, designed the experiments, and co-wrote the manuscript.

Funding information

This work was supported by the National Natural Science Foundation of China (No. 31570855) and the Research Grants Council, University Grants Committee (No. 776512M) and RGC Area of Excellence Sheme (AoE/P-705/16).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12035_2017_859_MOESM1_ESM.doc (6 mb)
ESM 1 (DOC 6162 kb)


  1. 1.
    Ohsumi Y (2014) Historical landmarks of autophagy research. Cell Res 24(1):9–23. CrossRefPubMedGoogle Scholar
  2. 2.
    Boya P, Reggiori F, Codogno P (2013) Emerging regulation and functions of autophagy. Nat Cell Biol 15(7):713–720. CrossRefPubMedGoogle Scholar
  3. 3.
    Tian F, Deguchi K, Yamashita T, Ohta Y, Morimoto N, Shang J, Zhang X, Liu N et al (2010) In vivo imaging of autophagy in a mouse stroke model. Autophagy 6(8):1107–1114. CrossRefPubMedGoogle Scholar
  4. 4.
    Lu Q, Harris VA, Kumar S, Mansour HM, Black SM (2015) Autophagy in neonatal hypoxia ischemic brain is associated with oxidative stress. Redox Biol 6:516–523. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Li H, Qiu S, Li X, Li M, Peng Y (2015) Autophagy biomarkers in CSF correlates with infarct size, clinical severity and neurological outcome in AIS patients. J Transl Med 13(1):359. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Frugier T, Taylor JM, McLean C, Bye N, Beart PM, Devenish RJ, Crack PJ (2016) Evidence for the recruitment of autophagic vesicles in human brain after stroke. Neurochem Int 96:62–68. CrossRefPubMedGoogle Scholar
  7. 7.
    Chen W, Sun Y, Liu K, Sun X (2014) Autophagy: a double-edged sword for neuronal survival after cerebral ischemia. Neural Regen Res 9(12):1210–1216. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wei K, Wang P, Miao CY (2012) A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury. CNS Neurosci Ther 18(11):879–886. CrossRefPubMedGoogle Scholar
  9. 9.
    Yuan Y, Zhang X, Zheng Y, Chen Z (2015) Regulation of mitophagy in ischemic brain injury. Neurosci Bull 31(4):395–406. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Shi RY, Zhu SH, Li V, Gibson SB, XS X, Kong JM (2014) BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke. CNS Neurosci Ther 20(12):1045–1055. CrossRefPubMedGoogle Scholar
  11. 11.
    Dolman NJ, Chambers KM, Mandavilli B, Batchelor RH, Janes MS (2013) Tools and techniques to measure mitophagy using fluorescence microscopy. Autophagy 9(11):1653–1662. CrossRefPubMedGoogle Scholar
  12. 12.
    Liu K, Sun Y, Gu Z, Shi N, Zhang T, Sun X (2013) Mitophagy in ischaemia/reperfusion induced cerebral injury. Neurochem Res 38(7):1295–1300. CrossRefPubMedGoogle Scholar
  13. 13.
    Michalska B, Duszynski J, Szymanski J (2016) Mechanism of mitochondrial fission—structure and function of Drp1 protein. Postepy Biochem 62(2):127–137PubMedGoogle Scholar
  14. 14.
    Cho B, Choi SY, Cho HM, Kim HJ, Sun W (2013) Physiological and pathological significance of dynamin-related protein 1 (Drp1)-dependent mitochondrial fission in the nervous system. Exp Neurobiol 22(3):149–157. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zuo W, Yang PF, Chen J, Zhang Z, Chen NH (2016) Drp-1, a potential therapeutic target for brain ischaemic stroke. Br J Pharmacol 173(10):1665–1677. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zuo W, Zhang S, Xia CY, Guo XF, He WB, Chen NH (2014) Mitochondria autophagy is induced after hypoxic/ischemic stress in a Drp1 dependent manner: the role of inhibition of Drp1 in ischemic brain damage. Neuropharmacology 86:103–115. CrossRefPubMedGoogle Scholar
  17. 17.
    Buhlman L, Damiano M, Bertolin G, Ferrando-Miguel R, Lombes A, Brice A, Corti O (2014) Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance. Biochim Biophys Acta 1843(9):2012–2026. CrossRefPubMedGoogle Scholar
  18. 18.
    Tang YC, Tian HX, Yi T, Chen HB (2016) The critical roles of mitophagy in cerebral ischemia. Protein Cell 7(10):699–713. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Lin C, Chao H, Li Z, Xu X, Liu Y, Hou L, Liu N, Ji J (2016) Melatonin attenuates traumatic brain injury-induced inflammation: a possible role for mitophagy. J Pineal Res 61(2):177–186. CrossRefPubMedGoogle Scholar
  20. 20.
    Li Q, Zhang T, Wang J, Zhang Z, Zhai Y, Yang GY, Sun X (2014) Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke. Biochem Biophys Res Commun 444(2):182–188. CrossRefPubMedGoogle Scholar
  21. 21.
    Zhang XM, Zhang L, Wang G, Niu W, He Z, Ding L, Jia J (2015) Suppression of mitochondrial fission in experimental cerebral ischemia: the potential neuroprotective target of p38 MAPK inhibition. Neurochem Int 90:1–8. CrossRefPubMedGoogle Scholar
  22. 22.
    Baek SH, Noh AR, Kim KA, Akram M, Shin YJ, Kim ES, SW Y, Majid A et al (2014) Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage. Stroke 45(8):2438–2443. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Feng J, Chen X, Shen J (2017) Reactive nitrogen species as therapeutic targets for autophagy: implication for ischemic stroke. Expert Opin Ther Targets 21(3):305–317. CrossRefPubMedGoogle Scholar
  24. 24.
    Ansari S, Rahman M, Waters MF, Hoh BL, Mocco J (2011) Recanalization therapy for acute ischemic stroke, part 1: surgical embolectomy and chemical thrombolysis. Neurosurg Rev 34(1):1–9. CrossRefPubMedGoogle Scholar
  25. 25.
    Gomis M, Davalos A (2014) Recanalization and reperfusion therapies of acute ischemic stroke: what have we learned, what are the major research questions, and where are we headed? Front Neurol 5:226. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Shirley R, Ord EN, Work LM (2014) Oxidative stress and the use of antioxidants in stroke. Antioxidants (Basel) 3(3):472–501. CrossRefGoogle Scholar
  27. 27.
    Chen XM, Chen HS, MJ X, Shen JG (2013) Targeting reactive nitrogen species: a promising therapeutic strategy for cerebral ischemia-reperfusion injury. Acta Pharmacol Sin 34(1):67–77. CrossRefPubMedGoogle Scholar
  28. 28.
    Kuhn DM, Sakowski SA, Sadidi M, Geddes TJ (2004) Nitrotyrosine as a marker for peroxynitrite-induced neurotoxicity: the beginning or the end of the end of dopamine neurons? J Neurochem 89(3):529–536. CrossRefPubMedGoogle Scholar
  29. 29.
    Ding R, Chen Y, Yang S, Deng X, Fu Z, Feng L, Cai Y, Du M et al (2014) Blood-brain barrier disruption induced by hemoglobin in vivo: Involvement of up-regulation of nitric oxide synthase and peroxynitrite formation. Brain Res 1571:25–38. CrossRefPubMedGoogle Scholar
  30. 30.
    Nakamura T, Cieplak P, Cho DH, Godzik A, Lipton SA (2010) S-nitrosylation of Drp1 links excessive mitochondrial fission to neuronal injury in neurodegeneration. Mitochondrion 10(5):573–578. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Cho DH, Nakamura T, Fang J, Cieplak P, Godzik A, Gu Z, Lipton SA (2009) S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury. Science 324(5923):102–105. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Liu B, Tewari AK, Zhang L, Green-Church KB, Zweier JL, Chen YR, He G (2009) Proteomic analysis of protein tyrosine nitration after ischemia reperfusion injury: mitochondria as the major target. Biochim Biophys Acta 1794(3):476–485. CrossRefPubMedGoogle Scholar
  33. 33.
    Vattemi G, Mechref Y, Marini M, Tonin P, Minuz P, Grigoli L, Guglielmi V, Klouckova I et al (2011) Increased protein nitration in mitochondrial diseases: evidence for vessel wall involvement. Mol Cell Proteomics 10(4):M110 002964. CrossRefPubMedGoogle Scholar
  34. 34.
    Lozano-Juste J, Colom-Moreno R, Leon J (2011) In vivo protein tyrosine nitration in Arabidopsis thaliana. J Exp Bot 62(10):3501–3517. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Hatano S (1976) Experience from a multicentre stroke register: a preliminary report. Bull World Health Organ 54(5):541–553PubMedPubMedCentralGoogle Scholar
  36. 36.
    Brott T, Adams HP Jr, Olinger CP, Marler JR, Barsan WG, Biller J, Spilker J, Holleran R et al (1989) Measurements of acute cerebral infarction: a clinical examination scale. Stroke 20(7):864–870. CrossRefPubMedGoogle Scholar
  37. 37.
    Sebastian D, Palacin M, Zorzano A (2017) Mitochondrial dynamics: coupling mitochondrial fitness with healthy aging. Trends Mol Med 23(3):201–215. CrossRefPubMedGoogle Scholar
  38. 38.
    Ashrafi G, Schwarz TL (2013) The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ 20(1):31–42. CrossRefPubMedGoogle Scholar
  39. 39.
    Grivennikova VG, Kareyeva AV, Vinogradov AD (2010) What are the sources of hydrogen peroxide production by heart mitochondria? Biochim Biophys Acta 1797(6–7):939–944. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Kang J, Pervaiz S (2012) Mitochondria: redox metabolism and dysfunction. Biochem Res Int 2012:1–14. CrossRefGoogle Scholar
  41. 41.
    Horn T, Klein J (2013) Neuroprotective effects of lactate in brain ischemia: dependence on anesthetic drugs. Neurochem Int 62(3):251–257. CrossRefPubMedGoogle Scholar
  42. 42.
    Bleilevens C, Roehl AB, Goetzenich A, Zoremba N, Kipp M, Dang J, Tolba R, Rossaint R et al (2013) Effect of anesthesia and cerebral blood flow on neuronal injury in a rat middle cerebral artery occlusion (MCAO) model. Exp Brain Res 224(2):155–164. CrossRefPubMedGoogle Scholar
  43. 43.
    Zavodnik IB (2016) Mitochondria, calcium homeostasis and calcium signaling. Biomed Khim 62(3):311–317. CrossRefPubMedGoogle Scholar
  44. 44.
    Klingenberg M (2008) The ADP and ATP transport in mitochondria and its carrier. Biochim Biophys Acta 1778(10):1978–2021. CrossRefPubMedGoogle Scholar
  45. 45.
    Ferrer-Sueta G, Radi R (2009) Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem Biol 4(3):161–177. CrossRefPubMedGoogle Scholar
  46. 46.
    Gong J, Sun F, Li Y, Zhou X, Duan Z, Duan F, Zhao L, Chen H et al (2015) Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway. Neuropharmacology 91:123–134. CrossRefPubMedGoogle Scholar
  47. 47.
    Chen HS, Chen XM, Feng JH, Liu KJ, Qi SH, Shen JG (2015) Peroxynitrite decomposition catalyst reduces delayed thrombolysis-induced hemorrhagic transformation in ischemia-reperfused rat brains. CNS Neurosci Ther 21(7):585–590. CrossRefPubMedGoogle Scholar
  48. 48.
    Xu M, Chen X, Gu Y, Peng T, Yang D, Chang RC, So KF, Liu K et al (2013) Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury. J Ethnopharmacol 150(1):116–124. CrossRefPubMedGoogle Scholar
  49. 49.
    Bas DF, Topcuoglu MA, Gursoy-Ozdemir Y, Saatci I, Bodur E, Dalkara T (2012) Plasma 3-nitrotyrosine estimates the reperfusion-induced cerebrovascular stress, whereas matrix metalloproteinases mainly reflect plasma activity: a study in patients treated with thrombolysis or endovascular recanalization. J Neurochem 123 Suppl 2:138–147. CrossRefPubMedGoogle Scholar
  50. 50.
    Shin CM, Chung YH, Kim MJ, Lee EY, Kim EG, Cha CI (2002) Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats. Brain Res 931(2):194–199. CrossRefPubMedGoogle Scholar
  51. 51.
    Murdaugh LS, Wang Z, Del Priore LV, Dillon J, Gaillard ER (2010) Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch’s membrane. Exp Eye Res 90(5):564–571. CrossRefPubMedGoogle Scholar
  52. 52.
    Ahmad R, Rasheed Z, Ahsan H (2009) Biochemical and cellular toxicology of peroxynitrite: implications in cell death and autoimmune phenomenon. Immunopharmacol Immunotoxicol 31(3):388–396. CrossRefPubMedGoogle Scholar
  53. 53.
    Ramdial K, Franco MC, Estevez AG (2017) Cellular mechanisms of peroxynitrite-induced neuronal death. Brain Res Bull 133:4–11. CrossRefPubMedGoogle Scholar
  54. 54.
    Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N (2013) Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 1833(12):3448–3459. CrossRefPubMedGoogle Scholar
  55. 55.
    Booth LA, Tavallai S, Hamed HA, Cruickshanks N, Dent P (2014) The role of cell signalling in the crosstalk between autophagy and apoptosis. Cell Signal 26(3):549–555. CrossRefPubMedGoogle Scholar
  56. 56.
    Rubinstein AD, Eisenstein M, Ber Y, Bialik S, Kimchi A (2011) The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis. Mol Cell 44(5):698–709. CrossRefPubMedGoogle Scholar
  57. 57.
    Young MM, Takahashi Y, Khan O, Park S, Hori T, Yun J, Sharma AK, Amin S et al (2012) Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis. J Biol Chem 287(15):12455–12468. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Nagelkerke A, Bussink J, Geurts-Moespot A, Sweep FC, Span PN (2015) Therapeutic targeting of autophagy in cancer. Part II: pharmacological modulation of treatment-induced autophagy. Semin Cancer Biol 31:99–105. CrossRefPubMedGoogle Scholar
  59. 59.
    Seglen PO, Gordon PB (1982) 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A 79(6):1889–1892. CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Zheng XY, Li LJ, Li W, Jiang PF, Shen HQ, Chen YH, Chen X (2015) Low concentrations of chloroquine and 3-methyladenine suppress the viability of retinoblastoma cells synergistically with vincristine independent of autophagy inhibition. Graefes Arch Clin Exp Ophthalmol 253(12):2309–2315. CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.School of Chinese Medicine, Li Ka Shing Faculty of MedicineThe University of Hong KongHong KongChina
  2. 2.Department of NeurologyHuizhou First People’s HospitalHuizhouChina

Personalised recommendations