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NR4A1 Promotes Cerebral Ischemia Reperfusion Injury by Repressing Mfn2-Mediated Mitophagy and Inactivating the MAPK–ERK–CREB Signaling Pathway

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Abstract

Mitochondrial dysfunction has been acknowledged as the key pathogenic mechanism in cerebral ischemia–reperfusion (IR) injury. Mitophagy is the protective system used to sustain mitochondrial homeostasis. However, the upstream regulator of mitophagy in response to brain IR injury is not completely understood. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been found to be associated with mitochondrial protection in a number of diseases. The aim of our study is to explore the functional role of NR4A1 in cerebral IR injury, with a particular focus on its influence on mitophagy. Wild-type mice and NR4A1-knockout mice were used to generate cerebral IR injury in vivo. Mitochondrial function and mitophagy were detected via immunofluorescence assays and western blotting. Cellular apoptosis was determined via MTT assays, caspase-3 activity and western blotting. Our data revealed that NR4A1 was significantly increased in the reperfused brain tissues. Genetic ablation of NR4A1 reduced the cerebral infarction area and repressed neuronal apoptosis. The functional study demonstrated that NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, evoked cellular oxidative stress, interrupted ATP generation, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-mediated mitophagy. Knockdown of NR4A1 elevated Mfn2 expression and therefore reversed mitophagic activity, sending a prosurvival signal for mitochondria in the setting of cerebral IR injury. Further, we demonstrated that NR4A1 modulated Mfn2 expression via the MAPK–ERK–CREB signaling pathway. Blockade of the ERK pathway could abrogate the permissive effect of NR4A1 deletion on mitophagic activation, contributing to neuronal mitochondrial apoptosis. Overall, our results demonstrate that the pathogenesis of cerebral IR injury is closely associated with a drop in protective mitophagy due to increased NR4A1 through the MAPK–ERK–CREB signaling pathway.

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References

  1. Kim T, Koo J, Kim SH, Song IU, Chung SW, Lee KS (2018) Blood-brain barrier permeability assessed by perfusion computed tomography predicts hemorrhagic transformation in acute reperfusion therapy. Neurol Sci 39:1579–1584

    Article  Google Scholar 

  2. Luo Q, Fan Y, Lin L, Wei J, Li Z, Li Y, Nakae S, Lin W, Chen Q (2018) Interleukin-33 protects ischemic brain injury by regulating specific microglial activities. Neuroscience 385:75–89

    Article  CAS  Google Scholar 

  3. Asadi Y, Gorjipour F, Behrouzifar S, Vakili A (2018) Irisin peptide protects brain against ischemic injury through reducing apoptosis and enhancing BDNF in a rodent model of stroke. Neurochem Res 43:1549–1560

    Article  CAS  Google Scholar 

  4. Zhou H, Ma Q, Zhu P, Ren J, Reiter RJ, Chen Y (2018) Protective role of melatonin in cardiac ischemia-reperfusion injury: From pathogenesis to targeted therapy. J Pineal Res 64:e12471

    Article  CAS  Google Scholar 

  5. Zhu P, Hu S, Jin Q, Li D, Tian F, Toan S, Li Y, Zhou H, Chen Y (2018) Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: a mechanism involving calcium overload/XO/ROS/mPTP pathway. Redox Biol 16:157–168

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ligeza J, Marona P, Gach N, Lipert B, Miekus K, Wilk W, Jaszczynski J, Stelmach A, Loboda A, Dulak J, Branicki W, Rys J, Jura J (2017) MCPIP1 contributes to clear cell renal cell carcinomas development. Angiogenesis 20:325–340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Griffiths HR, Gao D, Pararasa C (2017) Redox regulation in metabolic programming and inflammation. Redox Biol 12:50–57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu D, Zeng X, Li X, Mehta JL, Wang X (2017) Role of NLRP3 inflammasome in the pathogenesis of cardiovascular diseases. Basic Res Cardiol 113:5

    Article  CAS  PubMed  Google Scholar 

  9. Zhou H, Zhu P, Wang J, Zhu H, Ren J, Chen Y (2018) Pathogenesis of cardiac ischemia reperfusion injury is associated with CK2alpha-disturbed mitochondrial homeostasis via suppression of FUNDC1-related mitophagy. Cell Death Differ 25:1080–1093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li R, Xin T, Li D, Wang C, Zhu H, Zhou H (2018) 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rossello X, Riquelme JA, He Z, Taferner S, Vanhaesebroeck B, Davidson SM, Yellon DM (2017) The role of PI3Kalpha isoform in cardioprotection. Basic Res Cardiol 112:66

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Han L, Wang H, Li L, Li X, Ge J, Reiter RJ, Wang Q (2017) Melatonin protects against maternal obesity-associated oxidative stress and meiotic defects in oocytes via the SIRT3-SOD2-dependent pathway. J Pineal Res 63:e12431

    Article  CAS  Google Scholar 

  13. Zhou H, Du W, Li Y, Shi C, Hu N, Ma S, Wang W, Ren J (2018) Effects of melatonin on fatty liver disease: the role of NR4A1/DNA-PKcs/p53 pathway, mitochondrial fission, and mitophagy. J Pineal Res 64:e12450

    Article  CAS  Google Scholar 

  14. Zhou H, Zhang Y, Hu S, Shi C, Zhu P, Ma Q, Jin Q, Cao F, Tian F, Chen Y (2017) Melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis. J Pineal Res 63:e12413

    Article  CAS  PubMed Central  Google Scholar 

  15. Jin Q, Li R, Hu N, Xin T, Zhu P, Hu S, Ma S, Zhu H, Ren J, Zhou H (2018) DUSP1 alleviates cardiac ischemia/reperfusion injury by suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK pathways. Redox Biol 14:576–587

    Article  CAS  PubMed  Google Scholar 

  16. Zhou H, Yue Y, Wang J, Ma Q, Chen Y (2018) Melatonin therapy for diabetic cardiomyopathy: a mechanism involving Syk-mitochondrial complex I-SERCA pathway. Cell Signal 47:88–100

    Article  CAS  PubMed  Google Scholar 

  17. Zhou H, Wang S, Hu S, Chen Y, Ren J (2018) ER-mitochondria microdomains in cardiac ischemia-reperfusion injury: a fresh perspective. Front Physiol 9:755

    Article  PubMed  PubMed Central  Google Scholar 

  18. Zhou H, Shi C, Hu S, Zhu H, Ren J, Chen Y (2018) BI1 is associated with microvascular protection in cardiac ischemia reperfusion injury via repressing Syk-Nox2-Drp1-mitochondrial fission pathways. Angiogenesis 21:599–615

    Article  CAS  PubMed  Google Scholar 

  19. Nunez-Gomez E, Pericacho M, Ollauri-Ibanez C, Bernabeu C, Lopez-Novoa JM (2017) The role of endoglin in post-ischemic revascularization. Angiogenesis 20:1–24

    Article  CAS  PubMed  Google Scholar 

  20. Hong H, Tao T, Chen S, Liang C, Qiu Y, Zhou Y, Zhang R (2017) MicroRNA-143 promotes cardiac ischemia-mediated mitochondrial impairment by the inhibition of protein kinase Cepsilon. Basic Res Cardiol 112:60

    Article  CAS  PubMed  Google Scholar 

  21. Rodriguez-Garcia A, Hevia D, Mayo JC, Gonzalez-Menendez P, Coppo L, Lu J, Holmgren A, Sainz RM (2017) Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells. Redox Biol 12:634–647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang G, Zhang X, Weng X, Liang P, Dai X, Zeng S, Xu H, Huan H, Fang M, Li Y, Xu D, Xu Y (2017) SUV39H1 mediated SIRT1 trans-repression contributes to cardiac ischemia-reperfusion injury. Basic Res Cardiol 112:22

    Article  CAS  PubMed  Google Scholar 

  23. Lee HJ, Jung YH, Choi GE, Ko SH, Lee SJ, Lee SH, Han HJ (2017) BNIP3 induction by hypoxia stimulates FASN-dependent free fatty acid production enhancing therapeutic potential of umbilical cord blood-derived human mesenchymal stem cells. Redox Biol 13:426–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hu Z, Cheng J, Xu J, Ruf W, Lockwood CJ (2017) Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy. Angiogenesis 20:85–96

    Article  CAS  PubMed  Google Scholar 

  25. Zhang W, Tao A, Lan T, Cepinskas G, Kao R, Martin CM, Rui T (2017) Carbon monoxide releasing molecule-3 improves myocardial function in mice with sepsis by inhibiting NLRP3 inflammasome activation in cardiac fibroblasts. Basic Res Cardiol 112:16

    Article  CAS  PubMed  Google Scholar 

  26. Liu Z, Gan L, Luo D, Sun C (2017) Melatonin promotes circadian rhythm-induced proliferation through Clock/histone deacetylase 3/c-Myc interaction in mouse adipose tissue. J Pineal Res 62:e12413

    Article  CAS  Google Scholar 

  27. Zhou H, Wang J, Zhu P, Zhu H, Toan S, Hu S, Ren J, Chen Y (2018) NR4A1 aggravates the cardiac microvascular ischemia reperfusion injury through suppressing FUNDC1-mediated mitophagy and promoting Mff-required mitochondrial fission by CK2alpha. Basic Res Cardiol 113:23

    Article  CAS  PubMed  Google Scholar 

  28. Merjaneh M, Langlois A, Larochelle S, Cloutier CB, Ricard-Blum S, Moulin VJ (2017) Pro-angiogenic capacities of microvesicles produced by skin wound myofibroblasts. Angiogenesis 20:385–398

    Article  CAS  PubMed  Google Scholar 

  29. Tobisawa T, Yano T, Tanno M, Miki T, Kuno A, Kimura Y, Ishikawa S, Kouzu H, Nishizawa K, Yoshida H, Miura T (2017) Insufficient activation of Akt upon reperfusion because of its novel modification by reduced PP2A-B55alpha contributes to enlargement of infarct size by chronic kidney disease. Basic Res Cardiol 112:31

    Article  CAS  PubMed  Google Scholar 

  30. Geng C, Wei J, Wu C (2018) Yap-Hippo pathway regulates cerebral hypoxia-reoxygenation injury in neuroblastoma N2a cells via inhibiting ROCK1/F-actin/mitochondrial fission pathways. Acta Neurol Belg. https://doi.org/10.1007/s13760-018-0944-6

    Article  PubMed  Google Scholar 

  31. Ackermann M, Kim YO, Wagner WL, Schuppan D, Valenzuela CD, Mentzer SJ, Kreuz S, Stiller D, Wollin L, Konerding MA (2017) Effects of nintedanib on the microvascular architecture in a lung fibrosis model. Angiogenesis 20:359–372

    Article  CAS  PubMed  Google Scholar 

  32. Blackburn NJR, Vulesevic B, McNeill B, Cimenci CE, Ahmadi A, Gonzalez-Gomez M, Ostojic A, Zhong Z, Brownlee M, Beisswenger PJ, Milne RW, Suuronen EJ (2017) Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction. Basic Res Cardiol 112:57

    Article  PubMed  Google Scholar 

  33. Brasacchio D, Alsop AE, Noori T, Lufti M, Iyer S, Simpson KJ, Bird PI, Kluck RM, Johnstone RW, Trapani JA (2017) Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization. Cell Death Differ 24:961–970

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhou H, Li D, Zhu P, Hu S, Hu N, Ma S, Zhang Y, Han T, Ren J, Cao F, Chen Y (2017) Melatonin suppresses platelet activation and function against cardiac ischemia/reperfusion injury via PPARgamma/FUNDC1/mitophagy pathways. J Pineal Res 63:e12438

    Article  CAS  Google Scholar 

  35. Das N, Mandala A, Naaz S, Giri S, Jain M, Bandyopadhyay D, Reiter RJ, Roy SS (2017) Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice. J Pineal Res 62:e12404

    Article  CAS  Google Scholar 

  36. Alghanem AF, Wilkinson EL, Emmett MS, Aljasir MA, Holmes K, Rothermel BA, Simms VA, Heath VL, Cross MJ (2017) RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells. Angiogenesis 20:341–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Du GQ, Shao ZB, Wu J, Yin WJ, Li SH, Wu J, Weisel RD, Tian JW, Li RK (2017) Targeted myocardial delivery of GDF11 gene rejuvenates the aged mouse heart and enhances myocardial regeneration after ischemia-reperfusion injury. Basic Res Cardiol 112:7

    Article  CAS  PubMed  Google Scholar 

  38. Dufour F, Rattier T, Shirley S, Picarda G, Constantinescu AA, Morle A, Zakaria AB, Marcion G, Causse S, Szegezdi E, Zajonc DM, Seigneuric R, Guichard G, Gharbi T, Picaud F, Herlem G, Garrido C, Schneider P, Benedict CA, Micheau O (2017) N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death. Cell Death Differ 24:500–510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Feng D, Wang B, Wang L, Abraham N, Tao K, Huang L, Shi W, Dong Y, Qu Y (2017) Pre-ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress-dependent autophagy via PERK and IRE1 signalings. J Pineal Res 62:e12395

    Article  CAS  Google Scholar 

  40. Couto JA, Ayturk UM, Konczyk DJ, Goss JA, Huang AY, Hann S, Reeve JL, Liang MG, Bischoff J, Warman ML, Greene AK (2017) A somatic GNA11 mutation is associated with extremity capillary malformation and overgrowth. Angiogenesis 20:303–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gadicherla AK, Wang N, Bulic M, Agullo-Pascual E, Lissoni A, De Smet M, Delmar M, Bultynck G, Krysko DV, Camara A, Schluter KD, Schulz R, Kwok WM, Leybaert L (2017) Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart. Basic Res Cardiol 112:27

    Article  CAS  PubMed  Google Scholar 

  42. Glab JA, Doerflinger M, Nedeva C, Jose I, Mbogo GW, Paton JC, Paton AW, Kueh AJ, Herold MJ, Huang DC, Segal D, Brumatti G, Puthalakath H (2017) DR5 and caspase-8 are dispensable in ER stress-induced apoptosis. Cell Death Differ 24:944–950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dong X, Fu J, Yin X, Qu C, Yang C, He H, Ni J (2017) Induction of apoptosis in HepaRG cell line by Aloe-Emodin through generation of reactive oxygen species and the mitochondrial pathway. Cell Physiol Biochem 42:685–696

    Article  CAS  PubMed  Google Scholar 

  44. Zhou H, Wang J, Zhu P, Hu S, Ren J (2018) Ripk3 regulates cardiac microvascular reperfusion injury: the role of IP3R-dependent calcium overload, XO-mediated oxidative stress and F-action/filopodia-based cellular migration. Cell Signal 45:12–22

    Article  CAS  PubMed  Google Scholar 

  45. Fuhrmann DC, Brune B (2017) Mitochondrial composition and function under the control of hypoxia. Redox Biol 12:208–215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhang Y, Zhou H, Wu W, Shi C, Hu S, Yin T, Ma Q, Han T, Zhang Y, Tian F, Chen Y (2016) Liraglutide protects cardiac microvascular endothelial cells against hypoxia/reoxygenation injury through the suppression of the SR-Ca(2+)-XO-ROS axis via activation of the GLP-1R/PI3K/Akt/survivin pathways. Free Radic Biol Med 95:278–292

    Article  CAS  PubMed  Google Scholar 

  47. Zhu H, Jin Q, Li Y, Ma Q, Wang J, Li D, Zhou H, Chen Y (2018) Melatonin protected cardiac microvascular endothelial cells against oxidative stress injury via suppression of IP3R-[Ca(2+)]c/VDAC-[Ca(2+)]m axis by activation of MAPK/ERK signaling pathway. Cell Stress Chaperones 23:101–113

    Article  CAS  PubMed  Google Scholar 

  48. Randriamboavonjy V, Kyselova A, Elgheznawy A, Zukunft S, Wittig I, Fleming I (2017) Calpain 1 cleaves and inactivates prostacyclin synthase in mesenteric arteries from diabetic mice. Basic Res Cardiol 112:10

    Article  CAS  PubMed  Google Scholar 

  49. Thirusangu P, Vigneshwaran V, Prashanth T, Vijay Avin BR, Malojirao VH, Rakesh H, Khanum SA, Mahmood R, Prabhakar BT (2017) BP-1T, an antiangiogenic benzophenone-thiazole pharmacophore, counteracts HIF-1 signalling through p53/MDM2-mediated HIF-1alpha proteasomal degradation. Angiogenesis 20:55–71

    Article  CAS  PubMed  Google Scholar 

  50. Zhou H, Li D, Zhu P, Ma Q, Toan S, Wang J, Hu S, Chen Y, Zhang Y (2018) Inhibitory effect of melatonin on necroptosis via repressing the Ripk3-PGAM5-CypD-mPTP pathway attenuates cardiac microvascular ischemia-reperfusion injury. J Pineal Res. https://doi.org/10.1111/jpi.12503

    Article  PubMed  Google Scholar 

  51. Tomczyk M, Kraszewska I, Szade K, Bukowska-Strakova K, Meloni M, Jozkowicz A, Dulak J, Jazwa A (2017) Splenic Ly6C(hi) monocytes contribute to adverse late post-ischemic left ventricular remodeling in heme oxygenase-1 deficient mice. Basic Res Cardiol 112:39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhou H, Zhu P, Guo J, Hu N, Wang S, Li D, Hu S, Ren J, Cao F, Chen Y (2017) Ripk3 induces mitochondrial apoptosis via inhibition of FUNDC1 mitophagy in cardiac IR injury. Redox Biol 13:498–507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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  1. Department of Neurosurgery, First Affiliated Hospital, Hunan University of Chinese Medicine, Changsha, 410007, Hunan, China

    Zhanwei Zhang & Jianbai Yu

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  1. Zhanwei Zhang
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ZWZ and JBY conceived the research; ZWZ and JBY performed the experiments; all authors participated in discussing and revising the manuscript.

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Correspondence to Jianbai Yu.

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The animal study was performed in accordance with the Declaration of Helsinki. All experimental protocols were approved by the Ethics Committee of Hunan University of Chinese Medicine, Changsha, Hunan Province, China. The ethics reference number: SCSSJN20135.

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Zhang, Z., Yu, J. NR4A1 Promotes Cerebral Ischemia Reperfusion Injury by Repressing Mfn2-Mediated Mitophagy and Inactivating the MAPK–ERK–CREB Signaling Pathway. Neurochem Res 43, 1963–1977 (2018). https://doi.org/10.1007/s11064-018-2618-4

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