Neurotoxicity Research

, Volume 33, Issue 2, pp 377–387 | Cite as

Cypermethrin Activates Autophagosome Formation Albeit Inhibits Autophagy Owing to Poor Lysosome Quality: Relevance to Parkinson’s Disease

  • Abhishek Kumar Mishra
  • Saumya Mishra
  • Charul Rajput
  • Mohd Sami ur Rasheed
  • Devendra Kumar Patel
  • Mahendra Pratap SinghEmail author


Parkinson’s disease (PD) is the second most familiar, progressive and movement-related neurodegenerative disorder after Alzheimer disease. This study aimed to decipher the role of autophagy in cypermethrin-induced Parkinsonism, an animal model of PD. Indicators of autophagy [expression of beclin 1, autophagy-related protein 12 (Atg 12), unc-51 like autophagy activating kinase 1 (Ulk 1), p62 and lysosome-associated membrane protein 2 (LAMP 2) and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3) I to II], signalling cascade [phosphorylated (p) 5′ adenosine monophosphate-activated protein kinase (p-AMPK), sirtuin 1 (Sirt 1), phosphorylated-mammalian target of rapamycin (p-mTOR), tuberous sclerosis complex 2 (TSC 2), p317Ulk 1 and p757Ulk 1 levels] and lysosome morphology were assessed in control and cypermethrin-treated rat model of PD. Autophagy markers were also measured in cypermethrin-treated neuroblastoma cells in the presence of 3-methyl adenine, a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) class III inhibitor; vinblastine, an autophagosome elongation inhibitor; bafilomycin A1, an autophagolysosome and lysosome fusion/abnormal acidification inhibitor or torin 1, a mechanistic target of rapamycin inhibitor. Cypermethrin reduced LAMP 2 and increased p-AMPK and Sirt 1 without causing any change in other signalling proteins. 3-Methyl adenine did not change LC3 conversion; vinblastine and bafilomycin A1 decreased LAMP 2 expression in controls. While cypermethrin increased LC3 conversion in the presence of 3-methyl adenine, LAMP 2 reduction was more pronounced in vinblastine and bafilomycin A1-treated cells. Torin 1 normalized the expression of LAMP 2 without any change in other autophagy markers. Results demonstrate that albeit cypermethrin activates autophagosome formation, it reduces LAMP 2 expression and lysosome quality leading to autophagy inhibition.


Cypermethrin Autophagy Pyrethroid Parkinson’s disease Parkinsonism 



The authors sincerely acknowledge the Council of Scientific and Industrial Research (CSIR), University Grants Commission and Department of Science and Technology India, respectively, for extending fellowship to Abhishek Kumar Mishra and Charul Rajput; Saumya Mishra and Mohd Sami ur Rasheed. The Science and Engineering Research Board (SERB), India (Project Reference No.: EMR/2016/005041), is gratefully appreciated for approving the study for financial support. CSIR-IITR communication number of this article is 3446.

Compliance with Ethical Standards

Animal study was performed as per the guidelines of CPCSEA, India.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Agrawal S, Dixit A, Singh A, Tripathi P, Singh D, Patel DK, Singh MP (2015a) Cyclosporine A and MnTMPyP alleviate alpha-synuclein expression and aggregation in cypermethrin-induced Parkinsonism. Mol Neurobiol 52:1619–1628CrossRefPubMedGoogle Scholar
  2. Agrawal S, Singh A, Tripathi P, Mishra M, Singh PK, Singh MP (2015b) Cypermethrin-induced nigrostriatal dopaminergic neurodegeneration alters the mitochondrial function: a proteomics study. Mol Neurobiol 51:448–465CrossRefPubMedGoogle Scholar
  3. Basu S, Rajakaruna S, Reyes B, Van Bockstaele E, Menko AS (2014) Suppression of MAPK/JNK-MTORC1 signaling leads to premature loss of organelles and nuclei by autophagy during terminal differentiation of lens fiber cells. Autophagy 10(7):1193–1211CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bove J, Martinez-Vicente M, Dehay B, Perier C, Recasens A, Bombrun A, Antonsson B, Vila M (2014) BAX channel activity mediates lysosomal disruption linked to Parkinson disease. Autophagy 10:889–900CrossRefPubMedPubMedCentralGoogle Scholar
  5. Burbulla LF, Krebiehl G, Kruger R (2010) Balance is the challenge-the impact of mitochondrial dynamics in Parkinson’s disease. Eur J Clin Investig 40:1048–1060CrossRefGoogle Scholar
  6. Chen L, Xu B, Liu L, Luo Y, Zhou H, Chen W, Shen T, Han X, Kontos CD, Huang S (2011) Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death. Free Radic Biol Med 50(5):624–632CrossRefPubMedGoogle Scholar
  7. Cramer SL, Saha A, Liu J, Tadi S, Tiziani S, Yan W, Triplett K, Lamb C, Alters SE, Rowlinson S, Zhang YJ, Keating MJ, Huang P, DiGiovanni J, Georgiou G, Stone E (2017) Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth. Nat Med 23(1):120–127CrossRefPubMedGoogle Scholar
  8. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D (2004) Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 305:1292–1295CrossRefPubMedGoogle Scholar
  9. Dixit A, Srivastava G, Verma D, Mishra M, Singh PK, Prakash O, Singh MP (2013) Minocycline, levodopa and MnTMPyP induced changes in the mitochondrial proteome profile of MPTP and maneb and paraquat mice models of Parkinson's disease. Biochim Biophys Acta 1832:1227–1240CrossRefPubMedGoogle Scholar
  10. Dworak M, McCarley RW, Kim T, Kalinchuk AV, Basheer R (2010) Sleep and brain energy levels: ATP changes during sleep. J Neurosci 30(26):9007–9016CrossRefPubMedPubMedCentralGoogle Scholar
  11. Ebrahimi-Fakhari D, McLean PJ, Unni VK (2012) Alpha-synuclein’s degradation in vivo: opening a new (cranial) window on the roles of degradation pathways in Parkinson disease. Autophagy 8:281–283CrossRefPubMedPubMedCentralGoogle Scholar
  12. Giordano S, Darley-Usmar V, Zhang J (2013) Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease. Redox Biol 2:82–90CrossRefPubMedPubMedCentralGoogle Scholar
  13. Harlan FK, Lusk JS, Mohr BM, Guzikowski AP, Batchelor RH, Jiang Y, Naleway JJ (2016) Fluorogenic substrates for visualizing acidic organelle enzyme activities. PLoS One 11:e0156312CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hou YS, Guan JJ, Xu HD, Wu F, Sheng R, Qin ZH (2015) Sestrin2 protects dopaminergic cells against rotenone toxicity through AMPK-dependent autophagy activation. Mol Cell Biol 35:2740–2751CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kakko I, Toimela T, Tahti H (2004) The toxicity of pyrethroid compounds in neural cell cultures studied with total ATP, mitochondrial enzyme activity and microscopic photographing. Environ Toxicol Pharmacol 15:95–102CrossRefPubMedGoogle Scholar
  16. Klodowska-Duda G, Jasinska-Myga B, Safranow K, Boczarska-Jedynak M, Opala G (2005) The role of environmental factors in Parkinson’s disease may depend on disease onset age. Neurol Neurochir Pol 39:445–450PubMedGoogle Scholar
  17. Lan DM, Liu FT, Zhao J, Chen Y, Wu JJ, Ding ZT, Yue ZY, Ren HM, Jiang YP, Wang J (2012) Effect of trehalose on PC12 cells overexpressing wild-type or A53T mutant alpha-synuclein. Neurochem Res 37:2025–2032CrossRefPubMedGoogle Scholar
  18. Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 441:523–540CrossRefPubMedGoogle Scholar
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  20. Ma B, Cao W, Li W, Gao C, Qi Z, Zhao Y, Du J, Xue H, Peng J, Wen J, Chen H, Ning Y, Huang L, Zhang H, Gao X, Yu L, Chen YG (2014) Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation. Cell Res 24(8):912–924CrossRefPubMedPubMedCentralGoogle Scholar
  21. Mak SK, McCormack AL, Manning-Bog AB, Cuervo AM, Di Monte DA (2010) Lysosomal degradation of alpha-synuclein in vivo. J Biol Chem 285:13621–13629CrossRefPubMedPubMedCentralGoogle Scholar
  22. Mishra AK, ur Rasheed MS, Shukla S, Tripathi MK, Dixit A, Singh MP (2015) Aberrant autophagy and Parkinsonism: does correction rescue from disease progression? Mol Neurobiol 51:893–908CrossRefPubMedGoogle Scholar
  23. Moors T, Paciotti S, Chiasserini D, Calabresi P, Parnetti L, Beccari T, van de Berg WD (2016) Lysosomal dysfunction and alpha-synuclein aggregation in Parkinson’s disease: diagnostic links. Mov Disord 31:791–801CrossRefPubMedGoogle Scholar
  24. Musiwaro P, Smith M, Manifava M, Walker SA, Ktistakis NT (2013) Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy 9:1407–1417CrossRefPubMedGoogle Scholar
  25. Niso-Santano M, Bravo-San Pedro JM, Gomez-Sanchez R, Climent V, Soler G, Fuentes JM, Gonzalez-Polo RA (2011) ASK1 overexpression accelerates paraquat-induced autophagy via endoplasmic reticulum stress. Toxicol Sci 119:156–168CrossRefPubMedGoogle Scholar
  26. Ozeki N, Hase N, Hiyama T, Yamaguchi H, Kawai R, Kondo A, Matsumoto T, Nakata K, Mogi M (2015) Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells. PLoS One 10(4):e0124542CrossRefPubMedPubMedCentralGoogle Scholar
  27. Pan T, Kondo S, Le W, Jankovic J (2008) The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain 131:1969–1978CrossRefPubMedGoogle Scholar
  28. Plank TL, Yeung RS, Henske EP (1998) Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res 58(21):4766–4770PubMedGoogle Scholar
  29. Richter EA, Ruderman NB (2009) AMPK and the biochemistry of exercise: implications for human health and disease. Biochem J 418:261–275CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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 41:1103–1130CrossRefPubMedGoogle Scholar
  31. Sarkar S, Ravikumar B, Floto RA, Rubinsztein DC (2009) Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ 16:46–56CrossRefPubMedGoogle Scholar
  32. Singh AK, Tiwari MN, Dixit A, Upadhyay G, Patel DK, Singh D, Prakash O, Singh MP (2011) Nigrostriatal proteomics of cypermethrin-induced dopaminergic neurodegeneration: microglial activation-dependent and -independent regulations. Toxicol Sci 122:526–538CrossRefPubMedGoogle Scholar
  33. Singh AK, Tiwari MN, Upadhyay G, Patel DK, Singh D, Prakash O, Singh MP (2012) Long term exposure to cypermethrin induces nigrostriatal dopaminergic neurodegeneration in adult rats: postnatal exposure enhances the susceptibility during adulthood. Neurobiol Aging 33:404–415CrossRefPubMedGoogle Scholar
  34. Song L, Chen L, Zhang X, Li J, Le W (2014) Resveratrol ameliorates motor neuron degeneration and improves survival in SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Biomed Res 2014:483501Google Scholar
  35. Srivastava G, Dixit A, Yadav S, Patel DK, Prakash O, Singh MP (2012) Resveratrol potentiates cytochrome P450 2 d22-mediated neuroprotection in maneb and paraquat-induced Parkinsonism in the mouse. Free Radic Biol Med 52:1294–1306CrossRefPubMedGoogle Scholar
  36. Tripathi P, Singh A, Singh MP (2017) Ibuprofen protects from cypermethrin-induced changes in the striatal dendritic length and spine density. Mol Neurobiol.
  37. Ur Rasheed MS, Tripathi MK, Mishra AK, Shukla S, Singh MP (2016) Resveratrol protects from toxin-induced Parkinsonism: plethora of proofs hitherto petty translational value. Mol Neurobiol 53:2751–2760CrossRefPubMedGoogle Scholar
  38. Viollet B, Horman S, Leclerc J, Lantier L, Foretz M, Billaud M, Giri S, Andreelli F (2010) AMPK inhibition in health and disease. Crit Rev Biochem Mol Biol 45:276–295CrossRefPubMedPubMedCentralGoogle Scholar
  39. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC (2003) Alpha-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem 278:25009–25013CrossRefPubMedGoogle Scholar
  40. 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 285:10850–10861CrossRefPubMedPubMedCentralGoogle Scholar
  41. Xie X, Zhang D, Zhao B, Lu MK, You M, Condorelli G, Wang CY, Guan KL (2011) IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation. Proc Natl Acad Sci 108(16):6474–6479CrossRefPubMedPubMedCentralGoogle Scholar
  42. Xilouri M, Brekk OR, Stefanis L (2013) α-Synuclein and protein degradation systems: a reciprocal relationship. Mol Neurobiol 47:537–551CrossRefPubMedGoogle Scholar
  43. Zhang L, Fu L, Zhang S, Zhang J, Zhao Y, Zheng Y, He G, Yang S, Ouyang L, Liu B (2017) Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo. Chem Sci 8(4):2687–2701CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Abhishek Kumar Mishra
    • 1
    • 2
  • Saumya Mishra
    • 1
    • 2
  • Charul Rajput
    • 1
    • 2
  • Mohd Sami ur Rasheed
    • 1
    • 2
  • Devendra Kumar Patel
    • 2
    • 3
  • Mahendra Pratap Singh
    • 1
    • 2
    Email author
  1. 1.Toxicogenomics and Predictive Toxicology Laboratory, Systems Toxicology and Health Risk Assessment GroupCSIR-Indian Institute of Toxicology Research (CSIR-IITR)LucknowIndia
  2. 2.Academy of Scientific and Innovative ResearchLucknowIndia
  3. 3.Analytical Chemistry Laboratory, Regulatory Toxicology GroupCSIR-IITRLucknowIndia

Personalised recommendations