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Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis

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Abstract

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increasedBcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.

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Abbreviations

3-MA:

3-Methyl adenine

Ab:

Antibody

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

H&E:

Hematoxylin and eosin

MDC:

Monodansylcadaverine

MTT:

3-[4,5-Dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide

NSE:

Neuron specific enolase

PI:

Propidium iodide

PI3K:

Phosphatidylinositol-3-kinase

PVDF:

Polyvinylidinedifluoride

SCI:

Spinal cord injury

SD:

Standard deviation

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TEM:

Transmission electron microscopy

TNF:

Tumor necrosis factor

References

  1. Kwon BK, Tetzlaff W, Grauer JN, Beiner J, Vaccaro AR (2004) Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J 4:451–464

    Article  PubMed  Google Scholar 

  2. Furlan JC, Sakakibara BM, Miller WC, Krassioukov AV (2013) Global incidence and prevalence of traumatic spinal cord injury. Can J Neurol Sci 40:456–464

    PubMed  Google Scholar 

  3. Varma AK, Das A, Wallace Gt, Barry J, Vertegel AA, Ray SK, Banik NL (2013) Spinal cord injury: a review of current therapy, future treatments, and basic science frontiers. Neurochem Res 38:895–905

    Article  PubMed  CAS  Google Scholar 

  4. Rabchevsky AG, Patel SP, Springer JE (2011) Pharmacological interventions for spinal cord injury: where do we stand? How might we step forward? Pharmacol Ther 132:15–29

    Article  PubMed  CAS  Google Scholar 

  5. Springer JE (2002) Apoptotic cell death following traumatic injury to the central nervous system. J Biochem Mol Biol 35:94–105

    Article  PubMed  CAS  Google Scholar 

  6. Beattie MS (2004) Inflammation and apoptosis: linked therapeutic targets in spinal cord injury. Trends Mol Med 10:580–583

    Article  PubMed  CAS  Google Scholar 

  7. Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Bao JK (2012) Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif 45:487–498

    Article  PubMed  CAS  Google Scholar 

  8. Kanno H, Ozawa H, Sekiguchi A, Itoi E (2009) The role of autophagy in spinal cord injury. Autophagy 5:390–392

    Article  PubMed  CAS  Google Scholar 

  9. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein DC (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383–1435

    Article  PubMed  CAS  Google Scholar 

  10. Todde V, Veenhuis M, van der Klei IJ (2009) Autophagy: principles and significance in health and disease. Biochim Biophys Acta 1792:3–13

    Article  PubMed  CAS  Google Scholar 

  11. Springer JE, Azbill RD, Knapp PE (1999) Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat Med 5:943–946

    Article  PubMed  CAS  Google Scholar 

  12. Yakovlev AG, Faden AI (2001) Caspase-dependent apoptotic pathways in CNS injury. Mol Neurobiol 24:131–144

    Article  PubMed  CAS  Google Scholar 

  13. Wu KL, Chan SH, Chao YM, Chan JY (2003) Expression of pro-inflammatory cytokine and caspase genes promotes neuronal apoptosis in pontine reticular formation after spinal cord transection. Neurobiol Dis 14:19–31

    Article  PubMed  CAS  Google Scholar 

  14. Yu Y, Matsuyama Y, Yanase M, Ito S, Adachi K, Satake K, Ishiguro N, Kiuchi K (2004) Effects of hyperbaric oxygen on GDNF expression and apoptosis in spinal cord injury. NeuroReport 15:2369–2373

    Article  PubMed  Google Scholar 

  15. Casha S, Yu WR, Fehlings MG (2005) FAS deficiency reduces apoptosis, spares axons and improves function after spinal cord injury. Exp Neurol 196:390–400

    Article  PubMed  CAS  Google Scholar 

  16. Inukai T, Uchida K, Nakajima H, Yayama T, Kobayashi S, Mwaka ES, Guerrero AR, Baba H (2009) Tumor necrosis factor-alpha and its receptors contribute to apoptosis of oligodendrocytes in the spinal cord of spinal hyperostotic mouse (twy/twy) sustaining chronic mechanical compression. Spine 34:2848–2857

    Article  PubMed  Google Scholar 

  17. Cavallucci V, D’Amelio M (2011) Matter of life and death: the pharmacological approaches targeting apoptosis in brain diseases. Curr Pharm Des 17:215–229

    Article  PubMed  CAS  Google Scholar 

  18. Diskin T, Tal-Or P, Erlich S, Mizrachy L, Alexandrovich A, Shohami E, Pinkas-Kramarski R (2005) Closed head injury induces upregulation of Beclin 1 at the cortical site of injury. J Neurotrauma 22:750–762

    Article  PubMed  Google Scholar 

  19. Erlich S, Alexandrovich A, Shohami E, Pinkas-Kramarski R (2007) Rapamycin is a neuroprotective treatment for traumatic brain injury. Neurobiol Dis 26:86–93

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  21. Zhang YB, Li SX, Chen XP, Yang L, Zhang YG, Liu R, Tao LY (2008) Autophagy is activated and might protect neurons from degeneration after traumatic brain injury. Neurosci Bull 24:143–149

    Article  PubMed  Google Scholar 

  22. Jing CH, Wang L, Liu PP, Wu C, Ruan D, Chen G (2012) Autophagy activation is associated with neuroprotection against apoptosis via a mitochondrial pathway in a rat model of subarachnoid hemorrhage. Neuroscience 213:144–153

    Article  PubMed  CAS  Google Scholar 

  23. Sekiguchi A, Kanno H, Ozawa H, Yamaya S, Itoi E (2012) Rapamycin promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury in mice. J Neurotrauma 29:946–956

    Article  PubMed  Google Scholar 

  24. Kanno H, Ozawa H, Sekiguchi A, Itoi E (2009) Spinal cord injury induces upregulation of Beclin 1 and promotes autophagic cell death. Neurobiol Dis 33:143–148

    Article  PubMed  Google Scholar 

  25. Puyal J, Vaslin A, Mottier V, Clarke PG (2009) Postischemic treatment of neonatal cerebral ischemia should target autophagy. Ann Neurol 66:378–389

    Article  PubMed  CAS  Google Scholar 

  26. Kanno H, Ozawa H, Sekiguchi A, Yamaya S, Itoi E (2011) Induction of autophagy and autophagic cell death in damaged neural tissue after acute spinal cord injury in mice. Spine 36:E1427–E1434

    Article  PubMed  Google Scholar 

  27. Hao HH, Wang L, Guo ZJ, Bai L, Zhang RP, Shuang WB, Jia YJ, Wang J, Li XY, Liu Q (2013) Valproic acid reduces autophagy and promotes functional recovery after spinal cord injury in rats. Neurosci Bull 29:484–492

    Article  PubMed  CAS  Google Scholar 

  28. Smith CM, Chen Y, Sullivan ML, Kochanek PM, Clark RS (2011) Autophagy in acute brain injury: feast, famine, or folly? Neurobiol Dis 43:52–59

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  29. Kang R, Zeh HJ, Lotze MT, Tang D (2011) The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18:571–580

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  30. Zhou F, Yang Y, Xing D (2011) Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis. FEBS J 278:403–413

    Article  PubMed  CAS  Google Scholar 

  31. Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N (2013) Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 1833:3448–3459

    Article  PubMed  CAS  Google Scholar 

  32. Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, Brunner T, Simon HU (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8:1124–1132

    Article  PubMed  CAS  Google Scholar 

  33. Cho DH, Jo YK, Hwang JJ, Lee YM, Roh SA, Kim JC (2009) Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett 274:95–100

    Article  PubMed  CAS  Google Scholar 

  34. Djavaheri-Mergny M, Maiuri MC, Kroemer G (2010) Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1. Oncogene 29:1717–1719

    Article  PubMed  CAS  Google Scholar 

  35. Li H, Wang P, Yu J, Zhang L (2011) Cleaving Beclin 1 to suppress autophagy in chemotherapy-induced apoptosis. Autophagy 7:1239–1241

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  36. Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q (2011) Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell 1:468–477

    Article  CAS  Google Scholar 

  37. Grishchuk Y, Ginet V, Truttmann AC, Clarke PG, Puyal J (2011) Beclin 1-independent autophagy contributes to apoptosis in cortical neurons. Autophagy 7:1115–1131

    Article  PubMed  CAS  Google Scholar 

  38. Tang P, Hou H, Zhang L, Lan X, Mao Z, Liu D, He C, Du H (2013) Autophagy reduces neuronal damage and promotes locomotor recovery via inhibition of apoptosis after spinal cord injury in rats. Mol Neurobiol 49:276–287

    Article  PubMed  CAS  Google Scholar 

  39. Que H, Liu Y, Jia Y, Liu S (2011) Establishment and assessment of a simple and easily reproducible incision model of spinal cord neuron cells in vitro. In Vitro Cell Dev Biol Anim 47:558–564

    Article  PubMed  Google Scholar 

  40. Morrison B 3rd, Saatman KE, Meaney DF, McIntosh TK (1998) In vitro central nervous system models of mechanically induced trauma: a review. J Neurotrauma 15:911–928

    Article  PubMed  Google Scholar 

  41. LaPlaca MC, Simon CM, Prado GR, Cullen DK (2007) CNS injury biomechanics and experimental models. Prog Brain Res 161:13–26

    Article  PubMed  CAS  Google Scholar 

  42. Akhtar AZ, Pippin JJ, Sandusky CB (2008) Animal models in spinal cord injury: a review. Rev Neurosci 19:47–60

    Article  PubMed  Google Scholar 

  43. Ma YH, Zeng X, Zhang K, Zeng YS (2012) A new in vitro injury model of mouse neurons induced by mechanical scratching. Neurosci Lett 510:14–19

    Article  PubMed  CAS  Google Scholar 

  44. Mirzoeva OK, Hann B, Hom YK, Debnath J, Aftab D, Shokat K, Korn WM (2011) Autophagy suppression promotes apoptotic cell death in response to inhibition of the PI3K–mTOR pathway in pancreatic adenocarcinoma. J Mol Med (Berl) 89:877–889

    Article  CAS  Google Scholar 

  45. Sun H, Wang Z, Yakisich JS (2013) Natural products targeting autophagy via the PI3K/Akt/mTOR pathway as anticancer agents. Anticancer Agents Med Chem 13:1048–1056

    Article  PubMed  CAS  Google Scholar 

  46. Surviladze Z, Sterk RT, DeHaro SA, Ozbun MA (2013) Cellular entry of human papillomavirus type 16 involves activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway and inhibition of autophagy. J Virol 87:2508–2517

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  47. Dancey JE (2006) Therapeutic targets: MTOR and related pathways. Cancer Biol Ther 5:1065–1073

    Article  PubMed  CAS  Google Scholar 

  48. Alers S, Loffler AS, Wesselborg S, Stork B (2012) Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol 32:2–11

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  49. Kanno H, Ozawa H, Sekiguchi A, Yamaya S, Tateda S, Yahata K, Itoi E (2012) The role of mTOR signaling pathway in spinal cord injury. Cell Cycle 11:3175–3179

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  50. Park KK, Liu K, Hu Y, Smith PD, Wang C, Cai B, Xu B, Connolly L, Kramvis I, Sahin M, He Z (2008) Promoting axon regeneration in the adult CNS by modulation of the PTEN/mTOR pathway. Science 322:963–966

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  51. Codeluppi S, Svensson CI, Hefferan MP, Valencia F, Silldorff MD, Oshiro M, Marsala M, Pasquale EB (2009) The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord. J Neurosci 29:1093–1104

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  52. Don AS, Tsang CK, Kazdoba TM, D’Arcangelo G, Young W, Zheng XF (2012) Targeting mTOR as a novel therapeutic strategy for traumatic CNS injuries. Drug Discov Today 17:861–868

    Article  PubMed  CAS  Google Scholar 

  53. Cheever ML, Sato TK, de Beer T, Kutateladze TG, Emr SD, Overduin M (2001) Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat Cell Biol 3:613–618

    Article  PubMed  CAS  Google Scholar 

  54. Ellson CD, Gobert-Gosse S, Anderson KE, Davidson K, Erdjument-Bromage H, Tempst P, Thuring JW, Cooper MA, Lim ZY, Holmes AB, Gaffney PR, Coadwell J, Chilvers ER, Hawkins PT, Stephens LR (2001) PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox). Nat Cell Biol 3:679–682

    Article  PubMed  CAS  Google Scholar 

  55. Xu Y, Hortsman H, Seet L, Wong SH, Hong W (2001) SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P. Nat Cell Biol 3:658–666

    Article  PubMed  CAS  Google Scholar 

  56. Funderburk SF, Wang QJ, Yue Z (2010) The Beclin 1-VPS34 complex—at the crossroads of autophagy and beyond. Trends Cell Biol 20:355–362

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  57. He C, Levine B (2010) The Beclin 1 interactome. Curr Opin Cell Biol 22:140–149

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  58. Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, Herman B, Levine B (1998) Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein. J Virol 72:8586–8596

    PubMed Central  PubMed  CAS  Google Scholar 

  59. Yu D, Li M, Ni B, Kong J, Zhang Z (2013) Induction of neuronal mitophagy in acute spinal cord injury in rats. Neurotox Res 24:512–522

    Article  PubMed  Google Scholar 

  60. Saatman KE, Creed J, Raghupathi R (2010) Calpain as a therapeutic target in traumatic brain injury. Neurotherapeutics 7:31–42

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  61. Oberstein A, Jeffrey PD, Shi Y (2007) Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein. J Biol Chem 282:13123–13132

    Article  PubMed  CAS  Google Scholar 

  62. Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P (2008) Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie 90:313–323

    Article  PubMed  CAS  Google Scholar 

  63. Kubli DA, Gustafsson AB (2012) Mitochondria and mitophagy: the yin and yang of cell death control. Circ Res 111:1208–1221

    Article  PubMed Central  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (General Program, Grant No. 81371343), the Medical Innovation Project Foundation of Fujian Health Bureau (2012—CXB—020), the Scientific Research Foundation for Youth of Fujian Health Bureau (2011-2-7).

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Correspondence to Wen-Ge Liu.

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Wang, ZY., Lin, JH., Muharram, A. et al. Beclin-1-mediated autophagy protects spinal cord neurons against mechanical injury-induced apoptosis. Apoptosis 19, 933–945 (2014). https://doi.org/10.1007/s10495-014-0976-1

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