Neurotoxicity Research

, Volume 32, Issue 2, pp 204–217 | Cite as

Clathrin-Dependent Uptake of Paraquat into SH-SY5Y Cells and Its Internalization into Different Subcellular Compartments

  • Fengrui Li
  • Xiaofei Tian
  • Xiaoni Zhan
  • Baojie Wang
  • Mei Ding
  • Hao Pang


The herbicide paraquat (PQ) is an exogenous toxin that allows the selective activation of dopaminergic neurons in the mesencephalon to induce injury and also causes its apoptosis in vitro. However, uptake mechanisms between PQ and neurons remain elusive. To address this issue, we undertook a study of PQ endocytosis in a dopaminergic SH-SY5Y cell line as well as explored the subsequent subcellular location and potential functional analysis of PQ. The PQ was found to bind the SH-SY5Y cell membrane and then became internalized via a clathrin-dependent pathway. PQ was internalized by many subcellular organelles in a time- and dose-dependent manner. Interestingly, the taken up PQ and secretogranin III (SCG3), which became dysregulated with PQ treatment that induced SH-SY5Y apoptosis in our previous study, colocalized in cytoplasmic vesicles. Taken together, our findings indicate that PQ is endocytosed by SH-SY5Y cells and that its multiple, subcellular localizations indicate PQ may potentially be involved in subcellular-level functions. More importantly, PQ distributing preferentially into SCG3-positive vesicles demonstrates its selective targeting which may affect SCG3 and cargoes carried by SCG3-positive vesicles. Therefore, it is reasonable to infer that PQ toxic insults may potentially interfere with neurotransmitter storage and transport associated with secretory granules.


Paraquat SH-SY5Y Endocytosis Internalization SCG3 



This research was supported by grants from the National Natural Science Foundation of China (81172713 and 81471826). We appreciate BioMed Proofreading, LLC for manuscript language editing.

Compliance with Ethical Standards

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12640_2017_9722_MOESM1_ESM.doc (32 kb)
ESM 1 (DOC 32 kb)


  1. Aakalu G, Smith WB, Nguyen N, Jiang C, Schuman EM (2001) Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 30:489–502CrossRefPubMedGoogle Scholar
  2. Andersen JK (2003) Paraquat and iron exposure as possible synergistic environmental risk factors in Parkinson’s disease. Neurotox Res 5:307–313CrossRefPubMedGoogle Scholar
  3. Augustine GJ, Morgan JR, Villalba-Galea CA, Jin S, Prasad K, Lafer EM (2006) Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse. Biochem Soc Trans 34:68–72CrossRefPubMedPubMedCentralGoogle Scholar
  4. Baltazar T, Dinis-Oliveira RJ, Duarte JA, de Lourdes BM, Carvalho F (2013) Paraquat research: do recent advances in limiting its toxicity make its use safer? Br J Pharmacol 168:44–45CrossRefPubMedPubMedCentralGoogle Scholar
  5. Berry C, La Vecchia C, Nicotera P (2010) Paraquat and Parkinson’s disease. Cell Death Differ 17:1115–1125CrossRefPubMedGoogle Scholar
  6. Breckenridge CB, Sturgess NC, Butt M, Wolf JC, Zadory D, Beck M, Mathews JM, Tisdel MO, Minnema D, Travis KZ, Cook AR, Botham PA, Smith LL (2013) Pharmacokinetic, neurochemical, stereological and neuropathological studies on the potential effects of paraquat in the substantia nigra pars compacta and striatum of male C57BL/6J mice. Neurotoxicology 37:1–14CrossRefPubMedGoogle Scholar
  7. Castello PR, Drechsel DA, Patel M (2007) Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain. J Biol Chem 282:14186–14193CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chanyachukul T, Yoovathaworn K, Thongsaard W, Chongthammakun S, Navasumrit P, Satayavivad J (2004) Attenuation of paraquat-induced motor behavior and neurochemical disturbances by L-valine in vivo. Toxicol Lett 150:259–269CrossRefPubMedGoogle Scholar
  9. Chen Y, Zhang S, Sorani M, Giacomini KM (2007) Transport of paraquat by human organic cation transporters and multidrug and toxic compound extrusion family. J Pharmacol Exp Ther 322:695–700CrossRefPubMedGoogle Scholar
  10. Choi WS, Abel G, Klintworth H, Flavell RA, Xia Z (2010) JNK3 mediates paraquat- and rotenone-induced dopaminergic neuron death. J Neuropathol Exp Neurol 69:511–520CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cochemé HM, Murphy MP (2008) Complex I is the major site of mitochondrial superoxide production by paraquat. J Biol Chem 283:1786–1798CrossRefPubMedGoogle Scholar
  12. Colosio C, Birindelli S, Corsini E, Galli CL, Maroni M (2005) Low level exposure to chemicals and immune system. Toxicol Appl Pharmacol 207:320–328CrossRefPubMedGoogle Scholar
  13. Damm EM, Pelkmans L, Kartenbeck J, Mezzacasa A, Kurzchalia T, Helenius A (2005) Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae. J Cell Biol 168:477–488CrossRefPubMedPubMedCentralGoogle Scholar
  14. Del Zompo M, Piccardi MP, Ruiu S, Corsini GU, Vaccari A (1992) Characterization of a putatively vesicular binding site for [3H]MPP+ in mouse striatal membranes. Brain Res 571:354–357CrossRefPubMedGoogle Scholar
  15. Dinis-Oliveira RJ, Remião F, Carmo H, Duarte JA, Navarro AS, Bastos ML, Carvalho F (2006) Paraquat exposure as an etiological factor of Parkinson’s disease. Neurotoxicology 27:1110–1122CrossRefPubMedGoogle Scholar
  16. Dinis-Oliveira RJ, Duarte JA, Sánchez-Navarro A, Remião F, Bastos ML, Carvalho F (2008) Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 38:13–71CrossRefPubMedGoogle Scholar
  17. Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902CrossRefPubMedGoogle Scholar
  18. Ebrahimi-Fakhari D, Wahlster L, McLean PJ (2012) Protein degradation pathways in Parkinson’s disease: curse or blessing. Acta Neuropathol 124:153–172CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fei Q, McCormack AL, Di Monte DA, Ethell DW (2008) Paraquat neurotoxicity is mediated by a Bak-dependent mechanism. J Biol Chem 283:3357–3364CrossRefPubMedGoogle Scholar
  20. Franco R, Li S, Rodriguez-Rocha H, Burns M, Panayiotidis MI (2010) Molecular mechanisms of pesticide-induced neurotoxicity: relevance to Parkinson’s disease. Chem Biol Interact 188:289–300CrossRefPubMedPubMedCentralGoogle Scholar
  21. Jiao Y, Lu L, Williams RW, Smeyne RJ (2012) Genetic dissection of strain dependent paraquat-induced neurodegeneration in the substantia nigra pars compacta. PLoS One 7:e29447CrossRefPubMedPubMedCentralGoogle Scholar
  22. Krueger MJ, Singer TP, Casida JE, Ramsay RR (1990) Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone. Biochem Biophys Res Commun 169:123–128CrossRefPubMedGoogle Scholar
  23. Le PU, Benlimame N, Lagana A, Raz A, Nabi IR (2000) Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility. J Cell Sci 113:3227–3240PubMedGoogle Scholar
  24. Li F, Tian X, Zhou Y, Zhu L, Wang B, Ding M, Pang H (2012) Dysregulated expression of secretogranin III is involved in neurotoxin-induced dopaminergic neuron apoptosis. J Neurosci Res 90:2237–2246CrossRefPubMedGoogle Scholar
  25. Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen RC (1997) Environmental risk factors and Parkinson’s disease: a case-control study in Taiwan. Neurology 48:1583–1588CrossRefPubMedGoogle Scholar
  26. Lotharius J, O’Malley KL (2000) The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity J Biol Chem 275:38581–38588PubMedGoogle Scholar
  27. Mahajan R, Blair A, Lynch CF, Schroeder P, Hoppin JA, Sandler DP, Alavanja MC (2006) Fonofos exposure and cancer incidence in the agricultural health study. Environ Health Perspect 114:1838–1842CrossRefPubMedPubMedCentralGoogle Scholar
  28. Manning-Bog AB, McCormack AL, Li J, Uversky VN, Fink AL, Di Monte DA (2002) The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice—paraquat and alpha-synuclein. J Biol Chem 277:1641–1644CrossRefPubMedGoogle Scholar
  29. Manning-Bog AB, McCormack AL, Purisai MG, Bolin LM, Di Monte DA (2003) Alpha-synuclein overexpression protects against paraquat-induced neurodegeneration. J Neurosci 23:3095–3099PubMedGoogle Scholar
  30. van der Mark M, Brouwer M, Kromhout H, Nijssen P, Huss A, Vermeulen R (2012) Is pesticide use related to Parkinson disease? Some clues to heterogeneity in study results. Environ Health Perspect 120:340–347CrossRefPubMedGoogle Scholar
  31. Mayor S, Viola A, Stan RV, del Pozo MA (2006) Flying kites on slippery slopes at keystone. Symposium on lipid rafts and cell function. EMBO Rep 7:1089–1093CrossRefPubMedPubMedCentralGoogle Scholar
  32. McCormack AL, Di Monte DA (2003) Effects of L-dopa and other amino acids against paraquat-induced nigrostriatal degeneration. J Neurochem 85:82–86CrossRefPubMedGoogle Scholar
  33. McCormack AL, Thiruchelvam M, Manning-Bog AB, Thiffault C, Langston JW, Cory-Slechta DA, Di Monte DA (2002) Environmental risk factors and Parkinson’s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol Dis 10:119–127CrossRefPubMedGoogle Scholar
  34. Mendola P, Selevan SG, Gutter S, Rice D (2002) Environmental factors associated with a spectrum of neurodevelopmental deficits. Ment Retard Dev Disabil Res Rev 8:188–197CrossRefPubMedGoogle Scholar
  35. Nuber S, Tadros D, Fields J, Overk CR, Ettle B, Kosberg K, Mante M, Rockenstein E, Trejo M, Masliah E (2014) Environmental neurotoxic challenge of conditional alpha-synuclein transgenic mice predicts a dopaminergic olfactory-striatal interplay in early PD. Acta Neuropathol 127:477–494CrossRefPubMedPubMedCentralGoogle Scholar
  36. Peter D, Jimenez J, Liu Y, Kim J, Edwards RH (1994) The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors. J Biol Chem 269:7231–7237PubMedGoogle Scholar
  37. Prasad K, Winnik B, Thiruchelvam MJ, Buckley B, Mirochnitchenko O, Richfield EK (2007) Prolonged toxicokinetics and toxicodynamics of paraquat in mouse brain. Environ Health Perspect 115:1448–1453PubMedPubMedCentralGoogle Scholar
  38. Purdue MP, Hoppin JA, Blair A, Dosemeci M, Alavanja MC (2007) Occupational exposure to organochlorine insecticides and cancer incidence in the agricultural health study. Int J Cancer 120:642–649CrossRefPubMedPubMedCentralGoogle Scholar
  39. Ramachandiran S, Hansen JM, Jones DP, Richardson JR, Miller GW (2007) Divergent mechanisms of paraquat, MPP+, and rotenone toxicity: oxidation of thioredoxin and caspase-3 activation. Toxicol Sci 95:163–171CrossRefPubMedGoogle Scholar
  40. Rappold PM, Cui M, Chesser AS, Tibbett J, Grima JC, Duan L, Sen N, Javitch JA, Tieu K (2011) Paraquat neurotoxicity is mediated by the dopamine transporter and organic cation transporter-3. Proc Natl Acad Sci U S A 108:20766–20771CrossRefPubMedPubMedCentralGoogle Scholar
  41. Reinhard JF Jr, Diliberto EJ Jr, Viveros OH, Daniels AJ (1987) Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells. Proc Natl Acad Sci U S A 84:8160–8164CrossRefPubMedPubMedCentralGoogle Scholar
  42. Richardson JR, Quan Y, Sherer TB, Greenamyre JT, Miller GW (2005) Paraquat neurotoxicity is distinct from that of MPTP and rotenone. Toxicol Sci 88:193–201CrossRefPubMedGoogle Scholar
  43. Ritz B, Yu F (2000) Parkinson’s disease mortality and pesticide exposure in California 198–1994. Int J Epidemiol 29:323–329CrossRefPubMedGoogle Scholar
  44. Rizzoli SO, Betz WJ (2005) Synaptic vesicle pools. Nat Rev Neurosci 6:57–69CrossRefPubMedGoogle Scholar
  45. Ruder AM (2006) Potential health effects of occupational chlorinated solvent exposure. Ann N Y Acad Sci 1076:207–227CrossRefPubMedGoogle Scholar
  46. Sakai Y, Hosaka M, Yoshinaga A, Hira Y, Harumi T, Watanabe T (2004) Immunocytochemical localization of secretogranin III in the endocrine pancreas of male rats. Arch Histol Cytol 67:57–64CrossRefPubMedGoogle Scholar
  47. Segura Aguilar J, Kostrzewa RM (2004) Neurotoxins and neurotoxic species implicated in neurodegeneration. Neurotox Res 6:615–630CrossRefPubMedGoogle Scholar
  48. Shimizu K, Ohtaki K, Matsubara K, Aoyama K, Uezono T, Saito O, Suno M, Ogawa K, Hayase N, Kimura K, Shiono H (2001) Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat. Brain Res 906:135–142CrossRefPubMedGoogle Scholar
  49. Shimizu K, Matsubara K, Ohtaki K, Fujimaru S, Saito O, Shiono H (2003a) Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats. Brain Res 976:243–252CrossRefPubMedGoogle Scholar
  50. Shimizu K, Matsubara K, Ohtaki K, Shiono H (2003b) Paraquat leads to dopaminergic neural vulnerability in organotypic midbrain culture. Neurosci Res 46:523–532CrossRefPubMedGoogle Scholar
  51. Shogomori H, Futerman AH (2001) Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism. J Biol Chem 276:9182–9188CrossRefPubMedGoogle Scholar
  52. Silva R, Carmo H, Vilas-Boas V, Barbosa DJ, Monteiro M, de Pinho PG, de Lourdes BM, Remião F (2015a) Several transport systems contribute to the intestinal uptake of paraquat, modulating its cytotoxic effects. Toxicol Lett 232:271–283CrossRefPubMedGoogle Scholar
  53. Silva R, Palmeira A, Carmo H, Barbosa DJ, Gameiro M, Gomes A, Paiva AM, Sousa E, Pinto M, Bastos Mde L, Remião F (2015b) P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity. Arch Toxicol 89:1783–1800CrossRefPubMedGoogle Scholar
  54. Stridsberg M, Eriksson B, Janson ET (2008) Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours. Regul Pept 148:95–98CrossRefPubMedGoogle Scholar
  55. Tanner CM, Ross GW, Jewell SA, Hauser RA, Jankovic J, Factor SA, Bressman S, Deligtisch A, Marras C, Lyons KE, Bhudhikanok GS, Roucoux DF, Meng C, Abbott RD, Langston JW (2009) Occupation and risk of parkinsonism: a multicenter case-control study. Arch Neurol 66:1106–1113CrossRefPubMedGoogle Scholar
  56. Traub LM, Bonifacino JS (2013) Cargo recognition in clathrin-mediated endocytosis. Cold Spring Harb Perspect Biol 5:a016790CrossRefPubMedPubMedCentralGoogle Scholar
  57. Van Maele-Fabry G, Hoet P, Vilain F, Lison D (2012) Occupational exposure to pesticides and Parkinson’s disease: a systematic review and meta-analysis of cohort studies. Environ Int 46:30–43CrossRefPubMedGoogle Scholar
  58. Vilas-Boas V, Silva R, Palmeira A, Sousa E, Ferreira LM, Branco PS, Carvalho F, Bastos Mde L, Remião F (2013) Development of novel rifampicin-derived P-glycoprotein activators/inducers. Synthesis, in silico analysis and application in the RBE4 cell model, using paraquat as substrate. PLoS One 8:e74425CrossRefPubMedPubMedCentralGoogle Scholar
  59. Vilas-Boas V, Silva R, Guedes-de-Pinho P, Carvalho F, Bastos ML, Remião F (2014) RBE4 cells are highly resistant to paraquat-induced cytotoxicity: studies on uptake and efflux mechanisms. J Appl Toxicol 34:1023–1030CrossRefPubMedGoogle Scholar
  60. Wang LH, Rothberg KG, Anderson RG (1993) Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 123:1107–1117CrossRefPubMedGoogle Scholar
  61. Yang W, Tiffany-Castiglioni E (2005) The bipyridyl herbicide paraquat produces oxidative stress-mediated toxicity in human neuroblastoma SH-SY5Y cells: relevance to the dopaminergic pathogenesis. J Toxicol Environ Health A 68:1939–1961CrossRefPubMedGoogle Scholar
  62. Yasuda T, Hayakawa H, Nihira T, Ren YR, Nakata Y, Nagai M, Hattori N, Miyake K, Takada M, Shimada T, Mizuno Y, Mochizuki H (2011) Parkin-mediated protection of dopaminergic neurons in a chronic MPTP-minipump mouse model of Parkinson disease. J Neuropathol Exp Neurol 70:686–697CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Fengrui Li
    • 1
    • 2
  • Xiaofei Tian
    • 1
    • 3
  • Xiaoni Zhan
    • 1
  • Baojie Wang
    • 1
  • Mei Ding
    • 1
  • Hao Pang
    • 1
  1. 1.School of Forensic MedicineChina Medical UniversityShenyangPeople’s Republic of China
  2. 2.Department of Forensic MedicineBaotou Medical UniversityBaotouPeople’s Republic of China
  3. 3.Department of Forensic MedicineHebei North UniversityZhangjiakouPeople’s Republic of China

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