The Roles of Hydrogen Peroxide Exposure in the Toxic Aggregation of Alpha-synuclein and Translocation of DNA Methyltransferase-1 in Human Neuroblastoma Cell Model of Parkinson's Disease

Main Article Content

O. A. Olorunyomi
S. G. Mafulul
K. M. Jiyil
D. P. Smith


Background: Oxidative stress has been implicated in neuronal damage in Parkinson’s disease (PD). However, the specific roles of reactive oxygen species such as Hydrogen peroxide (H2O2) and Iron in the pathogenesis of PD especially, alpha-synuclein (α-Syn) aggregation and translocation of nuclear DNA Methyltransferase-1(Dnmt1), are yet to be fully understood.

Aims: This study investigated and compared the effects of H2O2 and ferrous iron (Fe2+) on α-Syn aggregation and localization of Dnmt1 in human neuroblastoma cells (SH-SY5Y), using a Parkinson’s disease model expressing A53T mutation and wild typed (WT) α-Syn respectively.

Materials and Methods: The study was done using CellTox™ assay, Immunocytochemical and Enzyme-linked immunosorbent Assay (ELISA) methods. Statistical analysis of triplicate data were analysed on Microsoft Excel 2010 and Stats Direct© using one-way analysis of variance (ANOVA) and Dunnet comparison tests.

Results: Specifically, 100 µM of H2O2 caused significant reduction of cell viability, translocation of Dnmt1 from nucleus into the cytoplasm and expression of relatively higher amount of α-Syn proteins, compared to 500 µM iron after 24 hours treatment. H2O2 elicited the highest expression of both WT α-Syn (13.7 ± 0.5) ng/ml and (16.0 ± 0.2) ng/ml A53T α-Syn proteins respectively. While Iron caused the expression of (9.1± 1.1) ng/ml and (14.8 ± 1.1) ng/ml of WT and A53T α-Syn proteins respectively. The untreated controls expressed (3.2 ± 0.1) ng/ml and (7.5 ± 0.0) ng/ml of WT and A53T α-Syn proteins respectively. Furthermore, the A53T mutation also promoted the expression and aggregation of α-Syn, as evidenced with the relatively higher amount of A53T α-Syn protein compared to WT α-Syn expressed in control, H2O2 and Iron treated cells.

Conclusion: This study demonstrated that H2O2 and Fe2+ induced α-Syn aggregation and Dnmt-1 translocation, which promotes the pathogenesis of Parkinson's disease. Likewise, the A53T genetic alterations increased the overexpression and aggregation of α-Syn proteins. Hence, novel therapies targeting reactive oxygen species, oxidative stress and mutations may be beneficial for long term treatment of Parkinson’s disease.

Alpha-synuclein (α-Syn), DNA methyltransferase (Dnmt1), parkinson’s disease, hydrogen perioxide (H2O2), A53T mutation, wild type (WT), neurotoxicity

Article Details

How to Cite
Olorunyomi, O. A., Mafulul, S. G., Jiyil, K. M., & Smith, D. P. (2020). The Roles of Hydrogen Peroxide Exposure in the Toxic Aggregation of Alpha-synuclein and Translocation of DNA Methyltransferase-1 in Human Neuroblastoma Cell Model of Parkinson’s Disease. Journal of Applied Life Sciences International, 23(4), 28-44.
Original Research Article


Rokad D, Ghaisas S, Harischandra DS, Jin H, Anantharam V, Kanthasamy A, et al. Role of neurotoxicants and traumatic brain injury in α-synuclein protein misfolding and aggregation. Brain Research Bulletin. 2017;133:60-70.

Kim WS, Kågedal K, Halliday GM. Alpha-synuclein biology in Lewy body diseases. Alzheimer's Research & Therapy. 2014;6(5):73.

Gajula MB, Griesinger C, Herzig A, Zweckstetter M, Jäckle H. Pre-fibrillar α-synuclein mutants cause Parkinson's disease-like non-motor symptoms in Drosophila. PLoS One. 2011;6(9):e24701.

Giráldez-Pérez RM, Antolín-Vallespín M, Muñoz MD, Sánchez-Capelo A. Models of α-synuclein aggregation in Parkinson’s disease. Acta Neuropathologica Communications. 2014;2(1):176.

Lu Y, Prudent M, Fauvet B, Lashuel HA, Girault HH. Phosphorylation of α-synuclein at Y125 and S129 alters its metal binding properties: Implications for understanding the role of α-synuclein in the pathogenesis of Parkinson’s disease and related disorders. ACS Chemical Neuroscience. 2011;2(11):667-75.

Desplats P, Spencer B, Coffee E, Patel P, Michael S, Patrick C, et al. α-Synuclein sequesters Dnmt1 from the nucleus a novel mechanism for epigenetic alterations in lewy body diseases. Journal of Biological Chemistry. 2011;286(11):9031-7.

Wales P, Pinho R, Lázaro DF, Outeiro TF. Limelight on alpha-synuclein: Pathological and mechanistic implications in neurodegeneration. Journal of Parkinson's Disease. 2013;3(4):415-59.

Oueslati A, Fournier M, Lashuel HA. Role of post-translational modifications in modulating the structure, function and toxicity of α-synuclein: Implications for Parkinson’s disease pathogenesis and therapies. In Progress in Brain Research. Elsevier. 2010;183:115-145.

Uversky VN, Li J, Fink AL. Metal-triggered structural transformations, aggregation, and fibrillation of human α-synuclein a possible molecular link between Parkinson′s disease and heavy metal exposure. Journal of Biological Chemistry. 2001;276(47):44284-96.

He Q, Song N, Xu H, Wang R, Xie J, Jiang H. Alpha-synuclein aggregation is involved in the toxicity induced by ferric iron to SK-N-SH neuroblastoma cells. Journal of Neural Transmission. 2011;118(3):397-406.

Danielson SR, Andersen JK. Oxidative and nitrative protein modifications in Parkinson's disease. Free Radical Biology and Medicine. 2008;44(10):1787-94.

Waldron RT, Rozengurt E. Oxidative stress induces protein kinase D activation in intact cells involvement of Src and dependence on protein kinase C. Journal of Biological Chemistry. 2000;275(22): 17114-21.

Garcimartín A, Merino JJ, González MP, Sánchez-Reus MI, Sánchez-Muniz FJ, Bastida S, et al. Organic silicon protects human neuroblastoma SH-SY5Y cells against hydrogen peroxide effects. BMC Complementary and Alternative Medicine. 2014;14(1):384.

Barnham KJ, Masters CL, Bush AI. Neurodegenerative diseases and oxidative stress. Nature Reviews Drug Discovery. 2004;3(3):205.

Li W, Jiang H, Song N, Xie J. Oxidative stress partially contributes to iron-induced alpha-synuclein aggregation in SK-N-SH cells. Neurotoxicity Research. 2011;19(3): 435-42.

Ostrerova-Golts N, et al. The A53T alpha- synuclein mutation increases iron- dependent aggregation and toxicity. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2000;20(16):6048-54.

Nita M, Grzybowski A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxidative Medicine and Cellular Longevity; 2016. Article ID: 3164734.


PHE Centre for Radiation, Chemical and Environmental Hazards. Hydrogen Peroxide – Toxicological Overview. 2009; Version 1.
(Accessed 12/2019)

International Programme on Chemical Safety (IPCS), Hydrogen Peroxide (>60% solution in water). International Chemical Safety Card: 0164. 2000, WHO: Geneva.

Xu Y, Li K, Qin W, Zhu B, Zhou Z, Shi J, Wang K, Hu J, Fan C, Li D. Unraveling the role of hydrogen peroxide in α-synuclein aggregation using an ultrasensitive nano-plasmonic probe. Analytical Chemistry. 2015;87(3):1968-73.

Kim YJ, Kim JY, Kang SW, Chun GS, Ban JY. Protective effect of geranylgeranylacetone against hydrogen peroxide-induced oxidative stress in human neuroblastoma cells. Life Sciences. 2015;131:51-6.

Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: Production, metabolism and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity. 2014;10(1155):360438.

Krishnan CV, Garnett M, Chu B. Spatiotemporal oscillations in biological molecules: Hydrogen peroxide and Parkinson’s disease. Int. J. Electrochem. Sci. 2008;3:1364-85.

Bienert GP, Schjoerring JK, Jahn TP. Membrane transport of hydrogen peroxide. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2006;1758(8):994-1003.

Othman SB, Yabe T. Use of hydrogen peroxide and peroxyl radicals to induce oxidative stress in neuronal cells. Reviews in Agricultural Science. 2015;3:40-5.

Chetsawang J, Govitrapong P, Chetsawang B. Hydrogen peroxide toxicity induces Ras signaling in human neuroblastoma SH- SY5Y cultured cells. Journal of Biomedicine and Biotechnology. 2010;3–7.

Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Kravchenko JE, Chumakov PM. The antioxidant function of the p53 tumor suppressor. Nature Medicine. 2005;11(12):1306.

Rhee SG. H2O2, a necessary evil for cell signaling. Science. 2006;312(5782):1882-3.

Hung MC, Link W. Protein localization in disease and therapy. J Cell Sci. 2011;124(20):3381-92.

Xie HR, Hu LS, Li GY. SH-SY5Y human neuroblastoma cell line: In vitro cell model of dopaminergic neurons in Parkinson's disease. Chinese Medical Journal. 2010;123(8):1086-92.

Santner A, Uversky VN. Metalloproteomics and metal toxicology of α-synuclein. Metallomics. 2010;2(6):378-92.

Xicoy H, Wieringa B, Martens GJ. The SH-SY5Y cell line in Parkinson’s disease research: A systematic review. Molecular Neurodegeneration. 2017;12(1):10.

Bittremieux M, Mikoshiba K, Bultynck G. Data on cytotoxicity in HeLa and SU-DHL-4 cells exposed to DPB162-AE compound. Data in Brief. 2017;12:91-6.

Kanaan NM, Manfredsson FP. Loss of functional alpha-synuclein: A toxic event in Parkinson's disease? Journal of Parkinson's Disease. 2012;2(4):249-67.

Leng Y, Chase TN, Bennett MC. Muscarinic receptor stimulation induces translocation of an α-synuclein oligomer from plasma membrane to a light vesicle fraction in cytoplasm. Journal of Biological Chemistry. 2001;276(30):28212-8.

Zhou M, Xu S, Mi J, Uéda K, Chan P. Nuclear translocation of alpha-synuclein increases susceptibility of MES23. 5 cells to oxidative stress. Brain Research. 2013;1500:19-27.

Cole NB, DiEuliis D, Leo P, Mitchell DC, Nussbaum RL. Mitochondrial translocation of α-synuclein is promoted by intracellular acidification. Experimental Cell Research. 2008;314(10):2076-89.

Miraglia F, Ricci A, Rota L, Colla E. Subcellular localization of alpha-synuclein aggregates and their interaction with membranes. Neural Regeneration Research. 2018;13(7):1136.

Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, et al. Synphilin-1 associates with α-synuclein and promotes the formation of cytosolic inclusions. Nature Genetics. 1999;22(1): 110.

Smith WW, Liu Z, Liang Y, Masuda N, Swing DA, Jenkins NA, et al. Synphilin-1 attenuates neuronal degeneration in the A53T α-synuclein transgenic mouse model. Human Molecular Genetics. 2010;19(11):2087-98.

Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow AR, Zhu L, et al. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Molecular Neuro-degeneration. 2012;7(1):42.

Quah BJ, O'Neill HC. The immunogenicity of dendritic cell-derived exosomes. Blood Cells, Molecules and Diseases. 2005;35(2):94-110.

Flower TR. Insights into the mechanism and the suppression of alpha-synuclein-induced toxicity in a yeast model of Parkinson's disease. ProQuest. 2006; 68(12):239.

Han SM, Kim JM, Park KK, Chang YC, Pak SC. Neuroprotective effects of melittin on hydrogen peroxide-induced apoptotic cell death in neuroblastoma SH-SY5Y cells. BMC Complementary and Alternative Medicine. 2014;14(1):286.

Zhong L, Zhou J, Chen X, Lou Y, Liu D, Zou X, et al. Quantitative proteomics study of the neuroprotective effects of B12 on hydrogen peroxide-induced apoptosis in SH-SY5Y cells. Scientific Reports. 2016;6: 22635.

Vilema-Enríquez G, Arroyo A, Grijalva M, Amador-Zafra RI, Camacho J. Molecular and cellular effects of hydrogen peroxide on human lung cancer cells: Potential therapeutic implications. Oxidative Medicine and Cellular Longevity. 2016;1908164.

DOI: 10.1155/2016/1908164

Gülden M, Jess A, Kammann J, Maser E, Seibert H. Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time. Free Radical Biology and Medicine. 2010;49(8):1298-305.

Ruffels J, Griffin M, Dickenson JM. Activation of ERK1/2, JNK and PKB by hydrogen peroxide in human SH-SY5Y neuroblastoma cells: Role of ERK1/2 in H2O2-induced cell death. European Journal of Pharmacology. 2004;483(2-3):163- 73.

Avery SV. Molecular targets of oxidative stress. Biochemical Journal. 2011;434(2): 201-10.

Imlay JA. Cellular defenses against superoxide and hydrogen peroxide. Annu. Rev. Biochem. 2008;77:755-76.

O'Hagan HM, Wang W, Sen S, Shields CD, Lee SS, Zhang YW, et al. Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Cancer Cell. 2011;20(5):606- 19.

Jowaed A, Schmitt I, Kaut O, Wüllner U. Methylation regulates alpha-synuclein expression and is decreased in Parkinson's disease patients' brains. Journal of Neuroscience. 2010;30(18): 6355-9.

Kodiha M, Stochaj U. Nuclear transport: A switch for the oxidative stress—signaling circuit? Journal of Signal Transduction. 2012;208650.

DOI: 10.1155/2012/208650

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology. 2007;39(1):44-84.

Emamzadeh FN. Alpha-synuclein structure, functions and interactions. Journal of Research in Medical Sciences: The Official Journal of Isfahan University of Medical Sciences. 2016;21.

Stadtman ER, Levine RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids. 2003;25(3-4):207-18.

Zhou C, Huang Y, Przedborski S. Oxidative stress in Parkinson's disease: A mechanism of pathogenic and therapeutic significance. Annals of the New York Academy of Sciences. 2008;1147:93.

Cole NB, Murphy DD, Lebowitz J, Di Noto L, Levine RL, Nussbaum RL. Metal-catalyzed oxidation of α-Synuclein helping to define the relationship between oligomers, protofibrils and filaments. Journal of Biological Chemistry. 2005;280(10):9678-90.

Min JY, Lim SO, Jung G. Down regulation of catalase by reactive oxygen species via hypermethylation of CpG island II on the catalase promoter. FEBS Letters. 2010;584(11):2427-32.

Emerit J, Edeas M, Bricaire F. Neurodegenerative diseases and oxidative stress. Biomedicine & Pharmacotherapy. 2004;58(1):39-46.

Giasson BI, Uryu K, Trojanowski JQ, Lee VM. Mutant and wild type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro. Journal of Biological Chemistry. 1999;274(12):7619-22.

Conway KA, Harper JD, Lansbury PT. Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid. Biochemistry. 2000;39(10):2552-63.

Markopoulou K, Wszolek ZK, Pfeiffer RF, Chase BA. Reduced expression of the G209A α‐synuclein allele in familial Parkinsonism. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society. 1999;46(3):374-81.

Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of α-synuclein: From structure and toxicity to therapeutic target. Nature Reviews Neuroscience. 2013;14(1):38.

Zhao H, Han Z, Ji X, Luo Y. Epigenetic regulation of oxidative stress in ischemic stroke. Aging and Disease. 2016;7(3):295.

Matsumoto L, Takuma H, Tamaoka A, Kurisaki H, Date H, Tsuji S, Iwata A. CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease. PloS One. 2010;5(11):e15522.

Grosso H, Woo JM, Lee KW, Im JY, Masliah E, Junn E, Mouradian MM. Transglutaminase 2 exacerbates α-synuclein toxicity in mice and yeast. The FASEB Journal. 2014;28(10):4280-91.

Olivares D, Huang X, Branden L, Greig N, Rogers J. Physiological and pathological role of alpha-synuclein in Parkinson’s disease through iron mediated oxidative stress; the role of a putative iron-responsive element. International Journal of Molecular Sciences. 2009;10(3):1226-60.

Lee HJ, Bae EJ, Lee SJ. Extracellular α-synuclein—a novel and crucial factor in Lewy body diseases. Nature Reviews Neurology. 2014;10(2):92