Skip to main content

Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement

Journal of Huazhong University of Science and Technology [Medical Sciences] volume 30, pages 1–7 (2010)Cite this article

Summary

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury. Although melatonin is known as an effective antioxidant, the mechanism of the protection cannot be explained merely by antioxidation. This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy, since autophagy has been frequently suggested to play a crucial role in neuron survival. To find it out, an ischemia/reperfusion model in N2a cells was established for examinations. The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reperfusion onset following ischemia and greatly promoted cell survival, while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT, transmission electron microscopy, and laser confocal scanning microscopy. Besides, the protein levels of LC3II and Beclin1 were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin, whereas the expression of p-PKB, key kinase in PI3K/PKB signaling pathway, showed a decrease when compared with untreated subjects as accessed by immunoblotting. Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  1. Olivieri G, Brack C, Muller-Spahn, et al. Mercury induces cell cytotoxicity and oxidative stress and increases β-amyloid secretion and Tau phosphorylation in SHSY’5Y neuroblastoma cells. J Neurochem, 2000,74(1):231–236

    Article  CAS  PubMed  Google Scholar 

  2. Cheung RT. Cerebrovascular disease—advances in management. Hong Kong Med J, 2001,7(1):58–66

    CAS  PubMed  Google Scholar 

  3. Chan PH. Role of oxidants in ischemic brain damage. Stroke, 1996,27(6):1124–1129

    CAS  PubMed  Google Scholar 

  4. Margaill I, Plotkine M, Lerouet D. Antioxidant strategies in the treatment of stroke. Free Rad Biol Med, 2005,39(4):429–443

    Article  CAS  PubMed  Google Scholar 

  5. Lipton P. Ischemic cell death in brain neurons. Physiol Rev, 1999,79(4):1431–1568

    CAS  PubMed  Google Scholar 

  6. Warner DS, Sheng H, Batinie-Haberle I. Oxidants, antioxidants and the ischemic brain. J Exp Biol, 2004,207(18): 3221–3231

    Article  CAS  PubMed  Google Scholar 

  7. Letechipía-Vallejo G, López-Loeza E, Espinoza-González V, et al. Long-term morphological and functional evaluation of the neuroprotective effects of post-ischemic treatment with melatonin in rats. J Pineal Res, 2007,42(2):138–146

    Article  PubMed  Google Scholar 

  8. Pei Z, Cheung RT. Pretreatment with melatonin exerts anti-inflammatory effects against ischemia/reperfusion injury in a rat middle cerebral artery occlusion stroke model. J Pineal Res, 2004,37(2):85–91

    Article  PubMed  Google Scholar 

  9. Watanabe K, Wakatsuki A, Shinohara K, et al. Maternally administered melatonin protects against ischemia and reperfusion-induced oxidative mitochondrial damage in premature fetal rat brain. J Pineal Res, 2004,37(4):276–280

    Article  CAS  PubMed  Google Scholar 

  10. Lipartiti M, Franceschini D, Zanoni R, et al. Neuroprotective effects of melatonin. Adv Exp Med Biol, 1996,398:315–321

    CAS  PubMed  Google Scholar 

  11. Kilic E, Ozdemir YG, Bolay H, et al. Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia. J Cereb Blood Flow Metab, 1999,19(5):511–516

    Article  CAS  PubMed  Google Scholar 

  12. Sarrafzadeh AS, Thomale UW, Kroppenstedt SN, et al. Neuroprotective effect of melatonin on cortical impact injury in the rat. Acta Neurochir (Wien), 2000,142(11):1293–1299

    Article  CAS  Google Scholar 

  13. Duan QH, Wang ZQ, Lu T, et al. Comparison of 6-hydroxylmelatonin or melatonin in protecting neurons against ischemia/reperfusion-mediated injury. J Pineal Res, 2006,41(4):351–357

    Article  CAS  PubMed  Google Scholar 

  14. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science, 2000,290(5497):1717–1721

    Article  CAS  PubMed  Google Scholar 

  15. Pan T, Kondo S, Zhu W, et al. Neuroprotection of rapamycin in lactacystin-induced neurodegeneration via autophagy enhancement. Neurobiol Dis, 2008,32(1):16–25

    Article  CAS  PubMed  Google Scholar 

  16. Carloni S, Buonocore G, Balduini W. Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury. Neurobiol Dis, 2008,32(3):329–339

    Article  CAS  PubMed  Google Scholar 

  17. Blomgren K, Zhu CL, Hallin U, et al. Mitochondria and ischemic reperfusion damage in the adult and in the developing brain. Biochem Biophys Res Commun, 2003,304(3):551–559

    Article  CAS  PubMed  Google Scholar 

  18. Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry, 2003,27(2):267–282

    Article  CAS  PubMed  Google Scholar 

  19. Kim I, Rodriguez-Enriquez S, Lemasters JJ. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys, 2007,462(2):245–253

    Article  CAS  PubMed  Google Scholar 

  20. Banasiak KJ, Xia Y, Haddad GG. Mechanisms underlying hypoxia-induced neuronal apoptosis. Prog Neurobil, 2000,62(3):215–249

    Article  CAS  Google Scholar 

  21. Poeggeler B, Reiter RJ, Tan DX, et al. Melatonin, hydroxyl radical-mediated oxidative damage, and aging: a hypothesis. J Pineal Res, 1993,14(4):151–168

    Article  CAS  PubMed  Google Scholar 

  22. Cervantes M, Morali G, Letechipia-Vallejo G. Melatonin and ischemia-reperfusion injury of the brain. J Pineal Res, 2008,45(1):1–7

    Article  CAS  PubMed  Google Scholar 

  23. Chan PH. Role of oxidants in ischemic brain damage. Stroke, 1996,27(6): 1124–1129

    CAS  PubMed  Google Scholar 

  24. Reiter RJ, Tan DX, Pappolla MA. Melatonin relieves the neural oxidative burden that contributes to dementias. Ann N Y Acad Sci, 2004,1035:179–196

    Article  CAS  PubMed  Google Scholar 

  25. Harms C, Lauternschlager M, Bergk A. Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures. FASEB J, 2000,14(12):1814–1824

    Article  CAS  PubMed  Google Scholar 

  26. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 2000,19(21):5720–5728

    Article  CAS  PubMed  Google Scholar 

  27. Mizushima, N. Methods for monitoring autophagy. Int J Biochem Cell Biol, 2004,36(12):2491–2502

    Article  CAS  PubMed  Google Scholar 

  28. Tassa A, Roux MP, Attaix D, et al. Class III Phosphoinositide 3-kinase-Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes. Biochem J, 2003,376(Pt 3):577–586

    Article  CAS  PubMed  Google Scholar 

  29. Li J, Ni M, Lee B, et al. The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells. Cell Death Differ, 2008,15(9):1460–1471

    Article  CAS  PubMed  Google Scholar 

  30. Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature, 1999,402(6762):672–676

    Article  CAS  PubMed  Google Scholar 

  31. Potter CJ, Padraza LG, Xu T. Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol, 2002,4(9):658–665

    Article  CAS  PubMed  Google Scholar 

  32. Manning BD, Tee AR, Logsdon MN, et al. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell, 2002,10(1):151–162

    Article  CAS  PubMed  Google Scholar 

  33. Inoki K, Li Y, Zhu T, et al. TSC is phosphorylated and inhibited by Akt and suppresses mTOR signaling. Nat Cell Biol, 2002,4(9):648–657

    Article  CAS  PubMed  Google Scholar 

  34. Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell, 2004,7(2):167–178

    Article  CAS  PubMed  Google Scholar 

  35. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell, 2006,124(3):471–484

    Article  CAS  PubMed  Google Scholar 

  36. Lum JJ, DeBerardinis RJ, Thompson CB. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol, 2005,6(6):439–448

    Article  CAS  PubMed  Google Scholar 

  37. Noda T, Ohsumi YJ. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem, 1998,273(7):3963–3966

    Article  CAS  PubMed  Google Scholar 

  38. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Atg8p, is localized in autophagosome membranes after processing. EMBO J, 2000,19(21):5720–5728

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

    Yanchun Guo, Jianfei Wang, Yi Yang, Ximing Wang & Qiuhong Duan

  2. Department of Emergency Medicine, Wuhan General Hospital of Guangzhou Command, Wuhan, 430070, China

    Zhongqiang Wang

Authors
  1. Yanchun Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianfei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhongqiang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yi Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ximing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qiuhong Duan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qiuhong Duan.

Additional information

The authors contributed equally to this wore.

This project was supported by a grant from the PhD Programs Foundation of Ministry of Education of China (No. 20070487101).

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Guo, Y., Wang, J., Wang, Z. et al. Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 30, 1–7 (2010). https://doi.org/10.1007/s11596-010-0101-9

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11596-010-0101-9

Key words

  • melatonin
  • autophagy
  • ischemia/reperfusion
  • rapamycin
  • 3-MA
  • LC3
  • Beclin1
  • PKB
  • N2a