Neurotoxicity Research

, Volume 32, Issue 3, pp 351–361 | Cite as

Autophagy Activation Alleviates Amyloid-β-Induced Oxidative Stress, Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells

  • Abhishek Kumar SinghEmail author
  • Akalabya Bissoyi
  • Mahendra Pratap Kashyap
  • Pradeep Kumar Patra
  • Syed Ibrahim Rizvi


Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-β (Aβ) production and deposition, its role in pathogenesis of Alzheimer’s disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aβ-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aβ1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aβ1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca2+ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aβ1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aβ1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aβ1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aβ-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.


Amyloid-beta Autophagy Neuroblastoma SH-SY5Y cells Neuroprotection Oxidative stress Rapamycin 3-Methyladenine 



Dr. D. S. Kotahri Post Doctoral Fellowship scheme of University Grants Commission, New Delhi, India, is acknowledged for providing financial support (F.4-2/2006(BSR)/BL/14-15/0326) and fellowship to A. K. Singh.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Anantharaman M, Tangpong J, Keller JN, Murphy MP, Markesbery WR, Kiningham KK, St Clair DK (2006) Beta-amyloid mediated nitration of manganese superoxide dismutase: implication for oxidative stress in a APPNLH/NLH X PS-1P264L/P264L double knock-in mouse model of Alzheimer’s disease. Am J Pathol 168:1608–1618CrossRefPubMedPubMedCentralGoogle Scholar
  2. Arrázola MS, Ramos-Fernández E, Cisternas P, Ordenes D, Inestrosa NC (2017) Wnt signaling prevents the Aβ oligomer-induced mitochondrial permeability transition pore opening preserving mitochondrial structure in hippocampal neurons. PLoS One 12:e0168840. doi: 10.1371/journal.pone.0168840 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Behera SS, Das U, Kumar A, Bissoyi A, Singh AK (2017) Chitosan/TiO2 composite membrane improves proliferation and survival of L929 fibroblast cells: application in wound dressing and skin regeneration. Int J Biol Macromol 98:329–340. doi: 10.1016/j.ijbiomac.2017.02.017 CrossRefPubMedGoogle Scholar
  4. Berger Z, Ravikumar B, Menzies FM, Oroz LG, Underwood BR, Pangalos MN, Schmitt I, Wullner U, Evert BO, O’Kane CJ, Rubinsztein DC (2006) Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum Mol Genet 15:433–442. doi: 10.1093/hmg/ddi458 CrossRefPubMedGoogle Scholar
  5. Brouillette J, Caillierez R, Zommer N, Alves-Pires C, Benilova I, Blum D, De Strooper B, Buée L (2012) Neurotoxicity and memory deficits induced by soluble low-molecular-weight amyloid-β1-42 oligomers are revealed in vivo by using a novel animal model. J Neurosci 32:7852–7861. doi: 10.1523/JNEUROSCI.5901-11.2012 CrossRefPubMedGoogle Scholar
  6. Carloni S, Girelli S, Scopa C, Buonocore G, Longini M, Balduini W (2010) Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia. Autophagy 6:366–377CrossRefPubMedGoogle Scholar
  7. Collingridge GL, Isaac JTR, Wang YT (2004) Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 5:952–962. doi: 10.1038/nrn1556 CrossRefPubMedGoogle Scholar
  8. Cuervo AM, Bergamini E, Brunk UT, Dröge W, Ffrench M, Terman A (2005) Autophagy and aging: the importance of maintaining “clean” cells. Autophagy 1:131–140CrossRefPubMedGoogle Scholar
  9. Deng M, Huang L, Ning B, Wang N, Zhang Q, Zhu C, Fang Y (2016) β-asarone improves learning and memory and reduces acetyl cholinesterase and Beta-amyloid 42 levels in APP/PS1 transgenic mice by regulating Beclin-1-dependent autophagy. Brain Res 1652:188–194. doi: 10.1016/j.brainres.2016.10.008 CrossRefPubMedGoogle Scholar
  10. Esterbauer H, Cheeseman KH (1990) Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 186:407–421CrossRefPubMedGoogle Scholar
  11. Filomeni G, De Zio D, Cecconi F (2015) Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ 22:377–388. doi: 10.1038/cdd.2014.150 CrossRefPubMedGoogle Scholar
  12. Floyd RA, Hensley K (2002) Oxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseases. Neurobiol Aging 23:795–807CrossRefPubMedGoogle Scholar
  13. Flynn JM, Melov S (2013) SOD2 in mitochondrial dysfunction and neurodegeneration. Free Radic Biol Med 62:4–12. doi: 10.1016/j.freeradbiomed.2013.05.027 CrossRefPubMedGoogle Scholar
  14. Garg G, Singh S, Singh AK, Rizvi SI (2017) Antiaging effect of metformin on brain in naturally aged and accelerated senescence model of rat. Rejuvenation Res. doi: 10.1089/rej.2016.1883 Google Scholar
  15. Godoy JA, Lindsay CB, Quintanilla RA, Carvajal FJ, Cerpa W, Inestrosa NC (2016) Quercetin exerts differential neuroprotective effects against H2O2 and Aβ aggregates in hippocampal neurons: the role of mitochondria. Mol Neurobiol. doi: 10.1007/s12035-016-0203-x Google Scholar
  16. Harkany T, Abrahám I, Timmerman W, Laskay G, Tóth B, Sasvári M, Kónya C, Sebens JB, Korf J, Nyakas C, Zarándi M, Soós K, Penke B, Luiten PG (2000) Beta-amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis. Eur J Neurosci 12:2735–2745CrossRefPubMedGoogle Scholar
  17. Henry-Mowatt J, Dive C, Martinou JC, James D (2004) Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 23:2850–2860. doi: 10.1038/sj.onc.1207534 CrossRefPubMedGoogle Scholar
  18. Hong IS, Lee HY, Kim HP (2014) Anti-oxidative effects of rooibos tea (Aspalathus linearis) on immobilization-induced oxidative stress in rat brain. PLoS One 9:e87061. doi: 10.1371/journal.pone.0087061 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Huang HC, Jiang ZF (2009) Accumulated amyloid-beta peptide and hyperphosphorylated tau protein: relationship and links in Alzheimer’s disease. J Alzheimers Dis JAD 16:15–27. doi: 10.3233/JAD-2009-0960 CrossRefPubMedGoogle Scholar
  20. Jaeger PA, Pickford F, Sun CH, Lucin KM, Masliah E, Wyss-Coray T (2010) Regulation of amyloid precursor protein processing by the Beclin 1 complex. PLoS One 5:e11102. doi: 10.1371/journal.pone.0011102 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728. doi: 10.1093/emboj/19.21.5720 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kabuta T, Suzuki Y, Wada K (2006) Degradation of amyotrophic lateral sclerosis-linked mutant Cu, Zn-superoxide dismutase proteins by macroautophagy and the proteasome. J Biol Chem 281:30524–30533. doi: 10.1074/jbc.M603337200 CrossRefPubMedGoogle Scholar
  23. Kakkar P, Das B, Viswanathan PN (1984) A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21:130–132PubMedGoogle Scholar
  24. Karran E, Mercken M, Strooper BD (2011) The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal for the development of therapeutics. Nat Rev Drug Discov 10:698–712. doi: 10.1038/nrd3505 CrossRefPubMedGoogle Scholar
  25. Kashyap MP, Singh AK, Yadav DK, Siddiqui MA, Srivastava RK, Chaturvedi V, Rai N (2015) 4-Hydroxy-trans-2-nonenal (4-HNE) induces neuronal SH-SY5Y cell death via hampering ATP binding at kinase domain of Akt1. Arch Toxicol 89:243–258. doi: 10.1007/s00204-014-1260-4 CrossRefPubMedGoogle Scholar
  26. Kawahara M (2010) Neurotoxicity of β-amyloid protein: oligomerization, channel formation, and calcium dyshomeostasis. Curr Pharm Des 16:2779–2789CrossRefPubMedGoogle Scholar
  27. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175CrossRefPubMedGoogle Scholar
  28. Kim DI, Lee KH, Oh JY, Kim JS, Han HJ (2016) Relationship between β-amyloid and mitochondrial dynamics. Cell Mol Neurobiol. doi: 10.1007/s10571-016-0434-4 Google Scholar
  29. Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13:132–141. doi: 10.1038/ncb2152 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149:274–293. doi: 10.1016/j.cell.2012.03.017 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lee B, Cao R, Choi YS, Cho HY, Rhee AD, Hah CK, Hoyt KR, Obrietan K (2009) The CREB/CRE transcriptional pathway: protection against oxidative stress-mediated neuronal cell death. J Neurochem 108:1251–1265. doi: 10.1111/j.1471-4159.2008.05864.x CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lee JA (2012) Neuronal autophagy: a housekeeper or a fighter in neuronal cell survival? Exp Neurobiol 21:1. doi: 10.5607/en.2012.21.1.1 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li L, Zhang X, Le W (2010) Autophagy dysfunction in Alzheimer’s disease. Neurodegener Dis 7:265–271. doi: 10.1159/000276710 PubMedGoogle Scholar
  34. Liu J, Su H, Qu QM (2016) Carnosic acid prevents Beta-amyloid-induced injury in human neuroblastoma SH-SY5Y cells via the induction of autophagy. Neurochem Res 41:2311–2323. doi: 10.1007/s11064-016-1945-6 CrossRefPubMedGoogle Scholar
  35. Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1:120–129. doi: 10.1038/35040009 CrossRefPubMedGoogle Scholar
  36. Mattson MP, Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7:278–294. doi: 10.1038/nrn1886 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Meley D, Bauvy C, Houben-Weerts JHPM, Dubbelhuis PF, Helmond MTJ, Codogno P, Meijer AJ (2006) AMP-activated protein kinase and the regulation of autophagic proteolysis. J Biol Chem 281:34870–34879. doi: 10.1074/jbc.M605488200 CrossRefPubMedGoogle Scholar
  38. Milton NG (1999) Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown. Biochem J 344(Pt 2):293–296CrossRefPubMedPubMedCentralGoogle Scholar
  39. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147:728–741. doi: 10.1016/j.cell.2011.10.026 CrossRefPubMedGoogle Scholar
  40. Moreno-Ortega AJ, Buendia I, Mouhid L, Egea J, Lucea S, Ruiz-Nuño A, López MG, Cano-Abad MF (2015) CALHM1 and its polymorphism P86L differentially control Ca2+homeostasis, mitogen-activated protein kinase signaling, and cell vulnerability upon exposure to amyloid β. Aging Cell 14:1094–1102. doi: 10.1111/acel.12403 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Müller WE, Eckert A, Kurz C, Eckert GP, Leuner K (2010) Mitochondrial dysfunction: common final pathway in brain aging and Alzheimer’s disease—therapeutic aspects. Mol Neurobiol 41:159–171. doi: 10.1007/s12035-010-8141-5 CrossRefPubMedGoogle Scholar
  42. Onyango IG, Khan SM (2006) Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer’s disease. Curr Alzheimer Res 3:339–349CrossRefPubMedGoogle Scholar
  43. Pugazhenthi S, Wang M, Pham S, Sze CI, Eckman CB (2011) Downregulation of CREB expression in Alzheimer’s brain and in Aβ-treated rat hippocampal neurons. Mol Neurodegener 6:60. doi: 10.1186/1750-1326-6-60 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B (2003) Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 112:1809–1820. doi: 10.1172/JCI20039 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Salminen A, Kaarniranta K, Haapasalo A, Hiltunen M, Soininen H, Alafuzoff I (2012) Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer’s disease. Prog Neurobiol 96:87–95. doi: 10.1016/j.pneurobio.2011.11.005 CrossRefPubMedGoogle Scholar
  46. Sanchez PE, Zhu L, Verret L, Vossel KA, Orr AG, Cirrito JR, Devidze N, Ho K, Yu GQ, Palop JJ, Mucke L (2012) Levetiracetam suppresses neuronal network dysfunction and reverses synaptic and cognitive deficits in an Alzheimer’s disease model. Proc Natl Acad Sci 109:E2895–E2903. doi: 10.1073/pnas.1121081109 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Sarkar S (2013) Regulation of autophagy by mTOR-dependent and mTOR-independent pathways: autophagy dysfunction in neurodegenerative diseases and therapeutic application of autophagy enhancers. Biochem Soc Trans 41:1103–1130. doi: 10.1042/BST20130134 CrossRefPubMedGoogle Scholar
  48. Shen DN, Zhang LH, Wei EQ, Yang Y (2015) Autophagy in synaptic development, function, and pathology. Neurosci Bull 31:416–426. doi: 10.1007/s12264-015-1536-6 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Singh AK, Kashyap MP, Jahan S, Kumar V, Tripathi VK, Siddiqui MA, Yadav S, Khanna VK, Jain SK, Das V, Pant AB (2012) Expression and inducibility of cytochrome P450s (CYP1A1, 2B6, 2E1, 3A4) in human cord blood CD34(+) stem cell-derived differentiating neuronal cells. Toxicol Sci Off J Soc Toxicol 129:392–410. doi: 10.1093/toxsci/kfs213 CrossRefGoogle Scholar
  50. Singh AK, Kashyap MP, Kumar V, Tripathi VK, Yadav DK, Khan F, Jahan S, Khanna VK, Yadav S, Pant AB (2013) 3-methylcholanthrene induces neurotoxicity in developing neurons derived from human CD34+Thy1+ stem cells by activation of aryl hydrocarbon receptor. NeuroMolecular Med 15:570–592. doi: 10.1007/s12017-013-8243-0 CrossRefPubMedGoogle Scholar
  51. Singh AK, Singh S, Garg G, Rizvi SI (2016) Rapamycin alleviates oxidative stress-induced damage in rat erythrocytes. Biochem Cell Biol Biochim Biol Cell 94:471–479. doi: 10.1139/bcb-2016-0048 CrossRefGoogle Scholar
  52. Small SA, Kent K, Pierce A, Leung C, Kang MS, Okada H, Honig L, Vonsattel JP, Kim TW (2005) Model-guided microarray implicates the retromer complex in Alzheimer’s disease. Ann Neurol 58:909–919. doi: 10.1002/ana.20667 CrossRefPubMedGoogle Scholar
  53. Srivastava P, Dhuriya YK, Gupta R, Shukla RK, Yadav RS, Dwivedi HN, Pant AB, Khanna VK (2016) Protective effect of curcumin by modulating BDNF/DARPP32/CREB in arsenic-induced alterations in dopaminergic signaling in rat corpus striatum. Mol Neurobiol. doi: 10.1007/s12035-016-0288-2 Google Scholar
  54. Song G, Li Y, Lin L, Cao Y (2015) Anti-autophagic and anti-apoptotic effects of memantine in a SH-SY5Y cell model of Alzheimer’s disease via mammalian target of rapamycin-dependent and -independent pathways. Mol Med Rep 12:7615–7622. doi: 10.3892/mmr.2015.4382 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Tong L, Thornton PL, Balazs R, Cotman CW (2001) Beta-amyloid-(1-42) impairs activity-dependent cAMP-response element-binding protein signaling in neurons at concentrations in which cell survival is not compromised. J Biol Chem 276:17301–17306. doi: 10.1074/jbc.M010450200 CrossRefPubMedGoogle Scholar
  56. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ (2002) Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416:535–539. doi: 10.1038/416535a CrossRefPubMedGoogle Scholar
  57. Welberg L (2012) Neurotransmission: autophagy regulates transmission. Nat Rev Neurosci. doi: 10.1038/nrn3266 Google Scholar
  58. Wu YT, Tan HL, Shui G, Bauvy C, Huang Q, Wenk MR, Ong CN, Codogno P, Shen HM (2010) Dual role of 3-Methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem 285:10850–10861. doi: 10.1074/jbc.M109.080796 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Xue Z, Guo Y, Fang Y (2016) Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells. Neurosci Lett 618:50–57. doi: 10.1016/j.neulet.2016.02.044 CrossRefPubMedGoogle Scholar
  60. Yang DS, Stavrides P, Saito M, Kumar A, Rodriguez-Navarro JA, Pawlik M, Huo C, Walkley SU, Saito M, Cuervo AM, Nixon RA (2014) Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits. Brain J Neurol 137:3300–3318. doi: 10.1093/brain/awu278 CrossRefGoogle Scholar
  61. Yang Z, Klionsky DJ (2010) Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814–822. doi: 10.1038/ncb0910-814 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Abhishek Kumar Singh
    • 1
    Email author
  • Akalabya Bissoyi
    • 2
  • Mahendra Pratap Kashyap
    • 3
  • Pradeep Kumar Patra
    • 4
  • Syed Ibrahim Rizvi
    • 1
  1. 1.Department of BiochemistryUniversity of AllahabadAllahabadIndia
  2. 2.Department of Biomedical EngineeringNational Institute of TechnologyRaipurIndia
  3. 3.Department of UrologyUniversity of Pittsburgh School of MedicinePittsburghUSA
  4. 4.Department of BiochemistryPt. JNM Medical CollegeRaipurIndia

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