Journal of Molecular Neuroscience

, Volume 30, Issue 3, pp 297–310

Oxidative stress and 17-α- and 17-β-estradiol modulate neurofilaments differently

  • Keith Chiasson
  • Vicky Lahaie-Collins
  • Julie Bournival
  • Benoit Delapierre
  • Sylvie Gélinas
  • Maria-Grazia Martinoli
Original Article


Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). Neuronal death in the substantia nigra of PD patients is partly caused by exacerbated oxidative damage. Our previous studies demonstrated that oxidative stress can alter the structure and stability of neuro-filament (NF) proteins and that 17-α- and 17-β-estradiol are potent neuroprotective agents. The aim of this study was to investigate the cytoskeletal target of neuroprotection by estrogens in neuronal PC12 cells. We induced oxidative stress by MPP+ administration for 24 h, and 17-α- and 17-β-estradiol were used as neuroprotective drugs. We measured gene expression and protein expression of each NF subunit, NFL, NFM, and NFH, by semiquantitative RT-PCR, Western blot, and immunofluorescence. Our results demonstrate that NFL mRNA and protein levels are not modulated by MPP+ or estradiol isomers, whereas NFM gene expression, as well as protein expression, are strongly influenced by MPP+, 17-α-, and 17-β-estradiol after a 24-h treatment. Finally, mRNA levels of the most phosphorylated subunits, NFH, are not changed by MPP+ or treatment with both estradiol isomers, whereas NFH protein expression is decreased by the same treatments. These results suggest that oxidative stress affects neuronal cytoskeleton, maybe though proteolysis and/or abnormal structural changes in NFs. Then, 17-α- and 17-β-estradiol might help the neuronal cell in recovering after oxidative stress by inducing protein expression of NFM and NFH subunits.

Index Entries

Neuroprotection MPP+ estradiol isomers neurofilaments expression 


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  1. Al-Chalabi A. and Miller C. C. (2003) Neurofilaments and neurological disease. Bioessays 25(4), 346–355.PubMedCrossRefGoogle Scholar
  2. Amantea D., Russo R., Bagetta G., and Corasaniti M. T. (2005) From clinicalevidence to molecular mechanisms underlying neuroprotection afforded by estrogens. Pharmacol. Res. 52(2), 119–132.PubMedCrossRefGoogle Scholar
  3. Anneser J. (2002) Molecular basis of treatment in motor neurone disease. Neurol. Sci. 21(Suppl. 5), S913-S918.Google Scholar
  4. Bains J. S. and Shaw C. A. (1997) Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res. Brain Res. Rev. 25(3), 335–358.PubMedCrossRefGoogle Scholar
  5. Barp J., Araujo A. S., Fernandes T. R., Rigatto K. V., Llesuy S., Bello-Klein A. and Singal P. (2002) Myocardial antioxidant and oxidative stress changes due to sex hormones. Braz. J. Med. Biol. Res 35(9), 1075–1081.PubMedCrossRefGoogle Scholar
  6. Berlett B. S. and Stadtman E. R. (1997) Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272(33) 20,313–20,316.CrossRefGoogle Scholar
  7. Bjorkdahl C., Sjogren M. J., Winblad B. and Pei J. J. (2005) Zinc induces neurofilament phosphorylation independent of p70S6 kinase in N2a cells. Neuroreport 16(6), 591–595.PubMedCrossRefGoogle Scholar
  8. Bomont P., Cavalier L., Blondeau F., Ben Hamida C., Belal S., Tazir M., et al. (2000), The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy. Nat. Genet 26(3), 370–374.PubMedCrossRefGoogle Scholar
  9. Booth, E. A., Marchesi M., Kilbourne E. J. and Lucchesi B. R. (2003) 17β-estradiol as a receptor-mediated cardioprotective agent. J. Pharmacol. Exp. Ther. 307(1), 395–401.PubMedCrossRefGoogle Scholar
  10. Callier S., Morissette M., Grandbois M. and Di Paolo T. (2000) Stereospecific prevention by 17β-estradiol of MPTP-induced dopamine depletion in mice. Synapse 37(4), 245–251.PubMedCrossRefGoogle Scholar
  11. Carter J., Gragerov A., Konvicka K., Elder G., Weinstein H., and Lazzarini R. A. (1998) Neurofilament (NF) assembly: divergent characteristics of human and rodent NF-L subunits. J. Biol. Chem. 273(9), 5101–5108.PubMedCrossRefGoogle Scholar
  12. Dave K. R., Bradley W. G. and Perez-Pinzon M. A. (2003) Early mitochondrial dysfunction occurs in motor cortex and spinal cord at the onset of disease in the Wobbler mouse. Exp. Neurol. 182(2), 412–420.PubMedCrossRefGoogle Scholar
  13. De Girolamo L. A., Hargreaves A. J. and Billett E. E. (2001) Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells. J. Neurochem. 76(3), 650–660.PubMedCrossRefGoogle Scholar
  14. Dluzen D. E. (2000) Neuroprotective effects of estrogen upon the nigrostriatal dopaminergic system. J. Neurocytol. 29(5–6), 387–399.PubMedCrossRefGoogle Scholar
  15. Egana J. T., Zambrano C., Nunez M. T., Gonzalez-Billault C., and Maccioni R. B. (2003). Iron-induced oxidative stress modify tau phosphorylation patterns in hippocampal cell cultures. Biometals 16(1), 215–223.PubMedCrossRefGoogle Scholar
  16. Fall C. P. and Bennett J. P. Jr. (1999) Characterization and time course of MPP+-induced apoptosis in human SH-SY5Y neuroblastoma cells. J. Neurosci. Res. 55(5), 620–628.PubMedCrossRefGoogle Scholar
  17. Farah C. A., Nguyen M. D., Julien J. P. and Leclerc N. (2003) Altered levels and distribution of microtubule-associated proteins before disease onset in a mouse model of amyotrophic lateral sclerosis. J. Neurochem. 84(1), 77–86.PubMedCrossRefGoogle Scholar
  18. Gagne B., Gelinas S., Bureau G., Lagace B., Ramassamy C., Chiasson K., et al. (2003). Effects of estradiol, phyto-estrogens, and Ginkgo biloba extracts against 1-methyl-4-phenyl-pyridine-induced oxidative stress. Endocrine 21(1), 89–95.PubMedCrossRefGoogle Scholar
  19. Garcia-Segura L. M., Azcoitia I., and DonCarlos L. L. (2001) Neuroprotection by estradiol. Prog. Neurobiol. 63(1), 29–60.PubMedCrossRefGoogle Scholar
  20. Gelinas S. and Martinoli M. G. (2002) Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells. J. Neurosci. Res. 70(1), 90–96.PubMedCrossRefGoogle Scholar
  21. Gelinas S., Bureau G., Valastro B., Massicotte G., Cicchetti F., Chiasson K., et al. (2004) Alpha and beta estradiol protect neuronal but not native PC12 cells from paraquat-induced oxidative stress. Neurotox. Res. 6(2), 141–148.PubMedCrossRefGoogle Scholar
  22. Gelinas S., Chapados C., Beauregard M., Gosselin I., and Martinoli M. G. (2000) Effect of oxidative stress on stability and structure of neurofilament proteins. Biochem. Cell Biol. 78(6), 667–674.PubMedCrossRefGoogle Scholar
  23. Gomez-Mancilla B. and Bedard P. J. (1992) Effect of estrogen and progesterone on L-dopa induced dyskinesia in MPTP-treated monkeys. Neurosci. Lett. 135(1), 129–132.PubMedCrossRefGoogle Scholar
  24. Gong C. X., Wang J. Z., Iqbal K. and Grundke-Iqbal I. (2003) Inhibition of protein phosphatase 2A induces phosphorylation and accumulation of neurofilaments in metabolically active rat brain slices. Neurosci. Lett. 340(2), 107–110.PubMedCrossRefGoogle Scholar
  25. Green P. S. and Simpkins J. W. (2000) Neuroprotective effects of estrogens: potential mechanisms of action. Int. J. Dev. Neurosci. 18(4–5), 347–358.PubMedCrossRefGoogle Scholar
  26. Hale S. L., Birnbaum Y. and Kloner R. A. (1997) Estradiol, administered acutely, protects ischemic myocardium in both female and male rabbits. J. Cardiovasc. Pharmacol. Ther. 2(1), 47–52.PubMedCrossRefGoogle Scholar
  27. Han H. J., Park S. H., Park H. J., Park K. M., Kang J. W., Lee J. H., et al. (2002) Effect of various oestrogens on cell injury and alteration of apical transporters induced by tert-butyl hydroperoxide in renal proximal tubule cells. Clin. Exp. Pharmacol. Physiol. 29(1–2), 60–67.PubMedCrossRefGoogle Scholar
  28. Hand C. K. and Rouleau G. A. (2002) Familiar amyotrophic lateral sclerosis. Muscle Nerve 25(2) 135–139.PubMedCrossRefGoogle Scholar
  29. Hirano A. (1994) Hirano bodies and related neuronal inclusions. Neuropathol. Appl. Neurobiol. 20(1), 3–11.PubMedCrossRefGoogle Scholar
  30. Itano Y., Kitamura Y. and Nomura Y. (1994) 1-Methyl-4-phenylpyridinium (MPP+)-induced cell death in PC12 cells: inhibitory effects of several drugs. Neurochem. Int. 25(5) 419–424.PubMedCrossRefGoogle Scholar
  31. Jayaraman D., Giasson B. I. and Mushynski W. E. (1995) Increased phosphorylation of neurofila ment subunits in PC12 cells and rat dorsal root ganglion neurons treated with N-Acetyl-Leu-Leu-norleucinal. Int J. Dev. Neurosci. 13(7), 753–758.PubMedCrossRefGoogle Scholar
  32. Jenner P. (2003), Oxidative stress in Parkinson's disease. Ann. Neurol. 53(Suppl. 3), S26-S36.PubMedCrossRefGoogle Scholar
  33. Kajta M. and Beyer C. (2003) Cellular strategies of estrogen-mediated neuroprotection during brain development. Endocrine 21(1), 3–9.PubMedCrossRefGoogle Scholar
  34. Kam K. W., Qi J. S., Chen M. and Wong, T. M. (2004) Estrogen reduces cardiac injury and expression of beta 1-adrenoceptor upon ischemic insult in the rat heart. J. Pharmacol. Exp. Ther. 309(1), 8–15.PubMedCrossRefGoogle Scholar
  35. Kocaturk P. A., Akbostanci M. C., Tan F. and Kavas G. O. (2000) Superoxide dismutase activity and zinc and copper concentrations in Parkinson's disease. Pathophysiology 7(1), 63–67.PubMedCrossRefGoogle Scholar
  36. Kosta, P., Argyropoulou M. I., Markoula S. and Konitsiotis S. (2005) MRI evaluation of the basal ganglia size and iron content in patients with Parkinson's disease. J. Neurol 253, 26–32.PubMedCrossRefGoogle Scholar
  37. Lee V., Trojanowski J. Q., and Schlaepfer W. W. (1982) Induction of neurofilament trip let proteins in PC12 cells by nerve growth factor. Brain Res. 238(1), 169–180.PubMedCrossRefGoogle Scholar
  38. Leranth C., Roth R. H., Elsworth J. D., Naftolin F., Horvath T. L., and Redmond D. E. Jr. (2000) Estrogen is essential for maintaining nigrostriatal dopamine neurons in primates: implications for Parkinson's disease and memory. J. Neurosci. 20(23), 8604–8609.PubMedGoogle Scholar
  39. Leranth C., Shanabrough M., and Redmond D. E. Jr. (2002) Gonadal hormones are responsible for maintaining the integrity of spine synapses in the CA1 hippocampal subfield of female nonhuman primates. J. Comp. Neurol. 447(1), 34–42.PubMedCrossRefGoogle Scholar
  40. Leterrier J. F. and Eyer J. (1992) Age-dependent changes in the ultrastructure and in the molecular composition of rat brain microtubules. J. Neurochem. 59(3), 1126–1137.PubMedCrossRefGoogle Scholar
  41. Lindenbaum M. H., Carbonetto S., and Mushynski W. E. (1987) Nerve growth factor enhances the synthesis, phosphorylation, and metabolic stability of neurofilament proteins in PC12 cells. J. Biol. Chem. 262(2), 605–610.PubMedGoogle Scholar
  42. Liu Q., Raina A. K., Smith M. A., Sayre L. M., and Perry G. (2003) hydroxynonenal, tocxic carbonyl, and Alzheimer disease. Mol. Aspects Med. 24, 305–313.PubMedCrossRefGoogle Scholar
  43. Liu S. J., Fang Z. Y., Yang Y., Deng H. M., and Wang J. Z. (2003) Alzheimer-like phosphorylation of tau and neurofilament induced by cocaine in vivo. Acta Pharmacol. Sin. 24(6), 512–518.PubMedGoogle Scholar
  44. Ma Z. Q., Spreafico E., Pollio G., Santagati S., Conti, E., Cattaneo E., and Maggi A. (1993) Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line. Proc. Natl. Acad. Sci. U. S. A. 90(8), 3740–3744.PubMedCrossRefGoogle Scholar
  45. MacLusky N. J., Chalmers-Redman R., Kay G., Ju W., Nethrapalli I. S., and Tatton W. G. (2003) Ovarian steroids reduce apoptosis induced by trophic insufficiency in nerve growth factor-differentiated PC12 cells and axotomized rat facial motoneurons. Neuroscience 118(3), 741–754.PubMedCrossRefGoogle Scholar
  46. Miller D. B., Ali S. F., O'Callaghan J. P., and Laws S. C. (1998) The impact of gender and estrogen on striatal dopaminergic neurotoxicity. Ann. N. Y. Acad. Sci. 844, 153–165.PubMedCrossRefGoogle Scholar
  47. Mitsumoto H., and Gambetti P. (1986) Impaired slow axonal transport in wobbler mouse motor neuron disease. Ann. Neurol. 19(1), 36–43.PubMedCrossRefGoogle Scholar
  48. Mori H., Oda M., and Mizuno Y. (1996) Cortical ballooned neurons in progressive supranuclear palsy. Neurosci. Lett. 209(2), 109–112.PubMedCrossRefGoogle Scholar
  49. Nilsen J., Mor G., and Naftolin F. (1998) Raloxifene induces neurite outgrowth in estrogen receptor positive PC12 cells. Menopause 5(4), 211–216.PubMedCrossRefGoogle Scholar
  50. Perez-Olle R., Lopez-Toledano M. A., Goryunov D., Cabrera-Poch N., Stefanis L., Brown K., and Liem R. K. (2005) Mutations in the neurofilament light gene linked to Charcot-Marie-Tooth disease cause defects in transport. J. Neurochem. 93(4), 861–874.PubMedCrossRefGoogle Scholar
  51. Pernas-Alonso R., Perrone-Capano C., Volpicelli F., and di Porzio U. (2001) Regionalized neurofilament accumulation and motoneuron degeneration are linked phenotypes in Wobbler neurom uscular disease. Neuro biol. Dis. 8(4), 581–589.CrossRefGoogle Scholar
  52. Pernas-Alonso R., Schaffner A. E., Perrone-Capano C., Orlando A., Morelli F., Hansen C. T., et al. (1996), Early upregulation of medium neurofilament gene expression in developing spinal cord of the Wobbler mouse mutant. Brain Res. Mol. Brain Res. 38(2), 267–275.PubMedCrossRefGoogle Scholar
  53. Pong K. (2003) Oxidative stress in neurodegenerative diseases: therapeutic implications for superoxide dismutase mimetics. Expert Opin. Biol. Ther. 3(1), 127–139.PubMedCrossRefGoogle Scholar
  54. Radunovic A., Porto W. G., Zeman S., and Leigh P. N. (1997) Increased mitochondrial superoxide dismutase activity in Parkinson's disease but not amyotrophic lateral sclerosis motor cortex. Neurosci. Lett. 239(2–3), 105–108.PubMedCrossRefGoogle Scholar
  55. Rahaman S. O., Ghosh S., Mohanakumar K. P., Das S., and Sakar P. K. (2001) Hypothyroidism in the developing rat brain is associated with marked oxidative stress and aberrant intraneuronal accumulation of neurofilaments. Neurosci. Res. 40, 273–279.PubMedCrossRefGoogle Scholar
  56. Ramirez A. D., Liu X., and Menniti F. S. (2003) Repeated estradiol treatment prevents MPTP-induced dopamine depletion in male mice. Neuroendocrinology 77(4), 223–231.PubMedCrossRefGoogle Scholar
  57. Rau S. W., Dubal D. B., Bottner M., Gerhold L. M., and Wise P. M. (2003) Estradiolattenuates programmed cell death after stroke-like injury. J. Neurosci. 23(36), 11420–11426.PubMedGoogle Scholar
  58. Rousseau J., Cossette L., Grenier S., and Martinoli M.-G. (2002) Modulation of prolactin expression by xeno-estrogens. Gen. Comp. Endocrinol. 126, 175–182.PubMedCrossRefGoogle Scholar
  59. Ruiz-Larrea M. B., Garrido M. J., and Lacort M. (1993). Estradiol-induced effects on glutathione metabolism in rat hepatocytes. J. Biochem. (Tokyo) 113(5), 563–567.Google Scholar
  60. Schapira A. H., Cooper J. M., Dexter D., Clark J. B., Jenner P., and Marsden C. D. (1990a) Mitochondrial complex I deficiency in Parkinson's disease. J. Neurochem. 54(3), 823–827.PubMedCrossRefGoogle Scholar
  61. Schapira A. H., Holt I. J., Sweeney M., Harding A. E., Jenner P., and Marsden C. D. (1990b) Mitochondrial DNA analysis in Parkinson's disease. Mov. Disord. 5(4), 294–297.PubMedCrossRefGoogle Scholar
  62. Schimmelpfeng J., Weibezahn K. F., and Dertinger H. (2004) Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-LmRNA expression and neuronal outgrowth. J. Neurosci. Methods 139(2), 299–306.PubMedCrossRefGoogle Scholar
  63. Schmidt R. E., Beaudet L. N., Plurad S. B., and Dorsey D. A. (1997) A xonal cytoskeletal pathology in aged and diabetic human sympathetic autonomic ganglia. Brain Res. 769(2), 375–383.PubMedCrossRefGoogle Scholar
  64. Schmittgen T. D., and Zakrajsek B. A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46(1–2), 69–81.PubMedCrossRefGoogle Scholar
  65. Schober A. (2004) Classic toxin-induced animal models of Parkinson's disease: 6-OHDA and MPTP. Cell Tissue Res. 318(1), 215–224.PubMedCrossRefGoogle Scholar
  66. Schon E. A. and Manfredi G. (2003) Neuronal degeneration and mitochondrial dysfunction. J. Clin. Invest. 111(3), 303–312.PubMedGoogle Scholar
  67. Scoville S. A., Bufton S. M., and Liuzzi F. J. (1997) Estrogen regulates neurofilament gene expression in adult female rat dorsal root ganglion neurons. Exp. Neurol. 146(2), 596–599.PubMedCrossRefGoogle Scholar
  68. Shepherd C. E., McCann H., Thiel E., and Halliday G. M. (2002) Neurofilament-immunoreactive neurons in Alzheimer's disease and dementia with Lewy bodies. Neurobiol Dis. 9(2), 249–257.PubMedCrossRefGoogle Scholar
  69. Smeyne R. J., and Jackson-Lewis V. (2005) The MPTP model of Parkinson's disease. Brain Res. Mol. Brain Res. 134(1), 57–66.PubMedCrossRefGoogle Scholar
  70. Smith D. S., and Tsai L. H. (2002) Cdk5 behind the wheel: a role in trafficking and transport? Trends Cell Biol. 12(1), 28–36.PubMedCrossRefGoogle Scholar
  71. Stadtman E. R., and Berlett B. S. (1997) Reactive oxygen-mediated protein oxidation in aging and disease. Chem. Res. Toxicol. 10(5), 485–494.PubMedCrossRefGoogle Scholar
  72. Topalli I., and Etgen A. M. (2004) Insulin-like growth factor-I receptor and estrogen receptor crosstalk mediates horm one-induced neurite outgrowth in PC12 cells. Brain Res. 1030(1), 116–124.PubMedCrossRefGoogle Scholar
  73. Toran-A'llerand C. D. (2004) Estrogen and the brain: beyond ER-alpha and ER-beta. Exp. Gerontol. 39(11–12), 1579–1586.CrossRefGoogle Scholar
  74. Toung T. J., Traystman R. J., and Hurn P. D. (1998) Estrogen-mediated neuroprotection after experimental stroke in male rats. Stroke 29(8), 1666–1670.PubMedGoogle Scholar
  75. Trimpin S., Mixon A. E., Stapels M. D., Kim M. Y., Spencer P. S., and Deinzer M. L. (2004) Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry. Biochemistry 43(7), 2091–2105.PubMedCrossRefGoogle Scholar
  76. Wise, P. M., Dubal D. B., Wilson M. E., Rau S. W., and Liu Y. (2001) Estrogens: trophic and protective factors in the adult brain. Front. Neuroendocrinol. 22(1), 33–66.PubMedCrossRefGoogle Scholar
  77. Wise P. M. (2002) Estrogens and neuroprotection. Trends Endocrinol. Metab. 13, 229–230.PubMedCrossRefGoogle Scholar
  78. Yang S. H., Liu R., Wu S. S., and Simpkins J. W. (2003) The use of estrogens and related compounds in the treatment of damage from cerebral ischemia. Ann. N. Y. Acad. Sci. 1007, 101–107.PubMedCrossRefGoogle Scholar
  79. Zecca L., Berg D., Arzberger T., Ruprecht P., Rausch W. D., Musicco M., et al. (2005) In vivo detection of iron and neuromelanin by transcranial sonography: A new approach for early detection of substantia nigra damage. Mov. Disord. 20, 1278–1285.PubMedCrossRefGoogle Scholar
  80. Zhang H., Joseph J., Feix J., Hogg N., and Kalyanaraman B. (2001) Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution. Biochemistry 40(25), 7675–7686.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Keith Chiasson
    • 1
    • 2
  • Vicky Lahaie-Collins
    • 1
  • Julie Bournival
    • 1
  • Benoit Delapierre
    • 1
  • Sylvie Gélinas
    • 1
  • Maria-Grazia Martinoli
    • 1
    • 2
  1. 1.Department of Biochemistry and Research Group in NeuroscienceUniversité du Québec à Trois-RivièresTrois-RivièresCanada
  2. 2.Neuroscience Research CenterCentre de Recherche de l'Université LavalSte-FoyCanada

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