Molecular Neurobiology

, Volume 51, Issue 2, pp 558–570 | Cite as

1,25-dyhydroxyvitamin D3 Attenuates l-DOPA-Induced Neurotoxicity in Neural Stem Cells

  • Wooyoung Jang
  • Hyun-Hee Park
  • Kyu-Yong Lee
  • Young Joo Lee
  • Hee-Tae KimEmail author
  • Seong-Ho KohEmail author


The neurotoxicity of levodopa (l-DOPA) on neural stem cells (NSCs) and treatment strategies to protect NSCs from this neurotoxicity remain to be elucidated. Recently, an active form of vitamin D3 has been reported to display neuroprotective properties. Therefore, we investigated the protective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) on l-DOPA-induced NSC injury. We measured cell viability via the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays and Annexin V/PI staining followed by flow cytometry, cell proliferation using the BrdU and colony-forming unit (CFU) assays, cell differentiation via immunocytochemistry, the levels of free radicals via 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, apoptosis via DAPI and TUNEL staining, and intracellular signaling protein expression via Western blot. Antibody microarrays were also employed to detect changes in the expression of prosurvival- and death-related proteins. Treatment of NSCs with l-DOPA reduced their viability and proliferation. This treatment also increased the levels of free radicals and decreased the expression levels of intracellular signaling proteins that are associated with cell survival. However, simultaneous exposure to calcitriol significantly reduced these effects. The calcitriol-mediated protection against l-DOPA toxicity was blocked by the phosphoinositide 3-kinase (PI3K) inhibitor LY294004. l-DOPA also inhibited the expression of Nestin and Ki-67, and co-treatment with calcitriol alleviated these effects. The expression levels of GFAP, DCX, and Tuj1 were not significantly affected by treatment with l-DOPA or calcitriol. Calcitriol protects against l-DOPA-induced NSC injury by promoting prosurvival signaling, including activation of the PI3K pathway, and reducing oxidative stress.


l-DOPA Neural stem cells 1,25-dyhydroxyvitamin D3 Neuroprotection 



This work was supported by the NanoBio R&D Program of the Korea Science and Engineering Foundation, the Ministry of Education, Science, and Technology (2007–04717), and the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101712).

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12035_2014_8835_MOESM1_ESM.pdf (85 kb)
Supplementary Fig. 1 The effect of calcitriol on the proliferation of NSCs. The BrdU assays revealed that treatment with high-dose calcitriol (100 μM) reduces NSC proliferation. *p < 0.05. (PDF 85 kb)
12035_2014_8835_MOESM2_ESM.pdf (359 kb)
Supplementary Fig. 2 Immunostaining of NSC proliferation and the expression of neuronal markers in NSCs exposed to various concentrations of calcitriol. Treatment with calcitriol at concentrations greater than 10 μM decreased the expression of Nestin and Ki-67 but displayed no effect on that of DAPI or Tuj-1. *p < 0.05 and **p < 0.01 (compared to the control group). (PDF 359 kb)
12035_2014_8835_MOESM3_ESM.pdf (192 kb)
Supplementary Fig. 3 The levels of TH and AADC activity in NSCs after exposure to 200 μM l-DOPA for 48 hr and different concentrations of calcitriol. There was no significant difference in the TH and AADC activity levels between the control, l-DOPA alone, and l-DOPA and various concentrations of calcitriol groups. (PDF 191 kb)


  1. 1.
    Jankovic J, Poewe W (2012) Therapies in Parkinson’s disease. Curr Opin Neurol 25(4):433–447PubMedCrossRefGoogle Scholar
  2. 2.
    Katzenschlager R, Lees AJ (2002) Treatment of Parkinson’s disease: levodopa as the first choice. J Neurol 249(2):ii19–ii24PubMedGoogle Scholar
  3. 3.
    Schapira AHV, Olanow CW (2008) Drug selection and timing of initiation of treatment in early Parkinson’s disease. Ann Neurol 64(S2):S47–S55PubMedCrossRefGoogle Scholar
  4. 4.
    Fahn S (2005) Does levodopa slow or hasten the rate of progression of Parkinson’s disease? J Neurol 252(4):iv37–iv42PubMedGoogle Scholar
  5. 5.
    Lipski J, Nistico R, Berretta N, Guatteo E, Bernardi G, Mercuri NB (2011) l-DOPA: a scapegoat for accelerated neurodegeneration in Parkinson’s disease? Prog Neurobiol 94(4):389–407PubMedCrossRefGoogle Scholar
  6. 6.
    Müller T, Hefter H, Hueber R, Jost W, Leenders K, Odin P, Schwarz J (2004) Is levodopa toxic? J Neurol 251(6):vi44–vi46Google Scholar
  7. 7.
    Parkkinen L, O’Sullivan SS, Kuoppamäki M, Collins C, Kallis C, Holton JL, Williams DR, Revesz T, Lees AJ (2011) Does levodopa accelerate the pathologic process in Parkinson disease brain? Neurology 77(15):1420–1426PubMedCrossRefGoogle Scholar
  8. 8.
    Agil A, Durán R, Barrero F, Morales B, Araúzo M, Alba F, Miranda MT, Prieto I, Ramírez M, Vives F (2006) Plasma lipid peroxidation in sporadic Parkinson’s disease. Role of the l-dopa. J Neurol Sci 240(1–2):31–36PubMedCrossRefGoogle Scholar
  9. 9.
    Chen J, Wersinger C, Sidhu A (2003) Chronic stimulation of D1 dopamine receptors in human SK-N-MC neuroblastoma cells induces nitric-oxide synthase activation and cytotoxicity. J Biol Chem 278(30):28089–28100PubMedCrossRefGoogle Scholar
  10. 10.
    Maharaj H, Sukhdev Maharaj D, Scheepers M, Mokokong R, Daya S (2005) l-DOPA administration enhances 6-hydroxydopamine generation. Brain Res 1063(2):180–186PubMedCrossRefGoogle Scholar
  11. 11.
    Soliman MK, Mazzio E, Soliman KFA (2002) Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells. Life Sci 72(2):185–198PubMedCrossRefGoogle Scholar
  12. 12.
    Koshimura K, Tanaka J, Murakami Y, Kato Y (2000) Effects of dopamine and L-DOPA on survival of PC12 cells. J Neurosci Res 62(1):112–119PubMedCrossRefGoogle Scholar
  13. 13.
    Mytilineou C, Walker RH, JnoBaptiste R, Olanow CW (2003) Levodopa is toxic to dopamine neurons in an in vitro but not an in vivo model of oxidative stress. J Pharmacol Exp Ther 304(2):792–800PubMedCrossRefGoogle Scholar
  14. 14.
    Shin JY, Park H-J, Ahn YH, Lee PH (2009) Neuroprotective effect of l-dopa on dopaminergic neurons is comparable to pramipexol in MPTP-treated animal model of Parkinson’s disease: a direct comparison study. J Neurochem 111(4):1042–1050PubMedCrossRefGoogle Scholar
  15. 15.
    Olanow CW, Obeso JA (2011) Levodopa toxicity and Parkinson disease: still a need for equipoise. Neurology 77(15):1416–1417PubMedCrossRefGoogle Scholar
  16. 16.
    Arias-Carrión O, Yamada E, Freundlieb N, Djufri M, Maurer L, Hermanns G, Ipach B, Chiu W-H, Steiner C, Oertel W, Höglinger G (2009) Neurogenesis in substantia nigra of parkinsonian brains? In: Giovanni G, Di Matteo V, Esposito E (eds) Birth, life and death of dopaminergic neurons in the substantia nigra, vol 73. J Neural Transm. Supplementa. Springer Vienna, pp 279–285Google Scholar
  17. 17.
    Hoglinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH, Caille I, Hirsch EC (2004) Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci 7(7):726–735PubMedCrossRefGoogle Scholar
  18. 18.
    Yoshimi K, Ren Y-R, Seki T, Yamada M, Ooizumi H, Onodera M, Saito Y, Murayama S, Okano H, Mizuno Y, Mochizuki H (2005) Possibility for neurogenesis in substantia nigra of parkinsonian brain. Ann Neurol 58(1):31–40PubMedCrossRefGoogle Scholar
  19. 19.
    Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen E-Y, Palfi S, Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling Z, Trono D, Hantraye P, Déglon N, Aebischer P (2000) Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 290(5492):767–773PubMedCrossRefGoogle Scholar
  20. 20.
    Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH (2002) The adult substantia nigra contains progenitor cells with neurogenic potential. The Journal of Neuroscience 22(15):6639–6649PubMedGoogle Scholar
  21. 21.
    Parain K, Murer MG, Yan Q, Faucheux B, Agid Y, Hirsch E, Raisman-Vozari R (1999) Reduced expression of brain-derived neurotrophic factor protein in Parkinson’s disease substantia nigra. NeuroReport 10(3):557–561PubMedCrossRefGoogle Scholar
  22. 22.
    Zhu Q, Ma J, Yu L, Yuan C (2009) Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats. Neurosci Lett 462(3):213–218PubMedCrossRefGoogle Scholar
  23. 23.
    Knekt P, Kilkkinen A, Rissanen H, Marniemi J, Saaksjarvi K, Heliovaara M (2010) Serum vitamin D and the risk of Parkinson disease. Arch Neurol 67(7):808–811PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Newmark HL, Newmark J (2007) Vitamin D and Parkinson’s disease—a hypothesis. Mov Disord 22(4):461–468PubMedCrossRefGoogle Scholar
  25. 25.
    Shinpo K, Kikuchi S, Sasaki H, Moriwaka F, Tashiro K (2000) Effect of 1,25-dihydroxyvitamin D3 on cultured mesencephalic dopaminergic neurons to the combined toxicity caused by L-buthionine sulfoximine and 1-methyl-4-phenylpyridine. J Neurosci Res 62(3):374–382PubMedCrossRefGoogle Scholar
  26. 26.
    Taniura H, Ito M, Sanada N, Kuramoto N, Ohno Y, Nakamichi N, Yoneda Y (2006) Chronic vitamin D3 treatment protects against neurotoxicity by glutamate in association with upregulation of vitamin D receptor mRNA expression in cultured rat cortical neurons. J Neurosci Res 83(7):1179–1189PubMedCrossRefGoogle Scholar
  27. 27.
    Wang J-Y, Wu J-N, Cherng T-L, Hoffer BJ, Chen H-H, Borlongan CV, Wang Y (2001) Vitamin D3 attenuates 6-hydroxydopamine-induced neurotoxicity in rats. Brain Res 904(1):67–75PubMedCrossRefGoogle Scholar
  28. 28.
    Bae EJ, Lee HS, Park CH, Lee SH (2009) Orphan nuclear receptor Nurr1 induces neuron differentiation from embryonic cortical precursor cells via an extrinsic paracrine mechanism. FEBS Lett 583(9):1505–1510PubMedCrossRefGoogle Scholar
  29. 29.
    Chang M-Y, Son H, Lee Y-S, Lee S-H (2003) Neurons and astrocytes secrete factors that cause stem cells to differentiate into neurons and astrocytes, respectively. Mol Cell Neurosci 23(3):414–426PubMedCrossRefGoogle Scholar
  30. 30.
    Kim J-Y, Koh HC, Lee J-Y, Chang M-Y, Kim Y-C, Chung H-Y, Son H, Lee Y-S, Studer L, McKay R, Lee S-H (2003) Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression. J Neurochem 85(6):1443–1454PubMedCrossRefGoogle Scholar
  31. 31.
    Jiang Q, Gu Z, Zhang G, Jing G (2000) Diphosphorylation and involvement of extracellular signal-regulated kinases (ERK1/2) in glutamate-induced apoptotic-like death in cultured rat cortical neurons. Brain Res 857(1–2):71–77PubMedCrossRefGoogle Scholar
  32. 32.
    Koh S-H, Song C, Noh MY, Kim HY, Lee K-Y, Lee YJ, Kim J, Kim SH, Kim H-T (2008) Inhibition of glycogen synthase kinase-3 reduces l-DOPA-induced neurotoxicity. Toxicology 247(2–3):112–118PubMedCrossRefGoogle Scholar
  33. 33.
    Lee YJ, Park KH, Park H-H, Kim YJ, Lee K-Y, Kim SH, Koh S-H (2009) Cilnidipine mediates a neuroprotective effect by scavenging free radicals and activating the phosphatidylinositol 3-kinase pathway. J Neurochem 111(1):90–100PubMedCrossRefGoogle Scholar
  34. 34.
    Fukuda S, Kato F, Tozuka Y, Yamaguchi M, Miyamoto Y, Hisatsune T (2003) Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus. J Neurosci 23(28):9357–9366PubMedGoogle Scholar
  35. 35.
    Kay JN, Blum M (2000) Differential response of ventral midbrain and striatal progenitor cells to lesions of the nigrostriatal dopaminergic projection. Dev Neurosci 22(1–2):56–67PubMedCrossRefGoogle Scholar
  36. 36.
    Lazarov O, Marr R (2010) Neurogenesis and Alzheimer’s disease: at the crossroads. Exp Neurol 223:267–281PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Steiner B, Wolf S, Kempermann G (2006) Adult neurogenesis and neurodegenerative disease. Regen Med 1(1):15–28PubMedCrossRefGoogle Scholar
  38. 38.
    Politis M, Lindvall O (2012) Clinical application of stem cell therapy in Parkinson’s disease. BMC Med 10(1):1PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Gerlach M, Braak H, Hartmann A, Jost WH, Odin P, Priller J, Schwarz J (2002) Current state of stem cell research for the treatment of Parkinson’s disease. J Neurol 249(3):iii33–ii35Google Scholar
  40. 40.
    Lindvall O, Björklund A (2011) Cell therapeutics in Parkinson’s disease. Neurotherapeutics 8(4):539–548PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Evatt ML, DeLong MR, Kumari M, Auinger P, McDermott MP, Tangpricha V (2011) High prevalence of hypovitaminosis D status in patients with early Parkinson disease. Arch Neurol 68(3):314–319PubMedCrossRefGoogle Scholar
  42. 42.
    Burne THJ, McGrath JJ, Eyles DW, Mackay-Sim A (2005) Behavioural characterization of vitamin D receptor knockout mice. Behav Brain Res 157(2):299–308PubMedCrossRefGoogle Scholar
  43. 43.
    Eyles DW, Smith S, Kinobe R, Hewison M, McGrath JJ (2005) Distribution of the vitamin D receptor and 1α-hydroxylase in human brain. J Chem Neuroanat 29(1):21–30PubMedCrossRefGoogle Scholar
  44. 44.
    Koh S-H, Park H-H, Choi N-Y, Lee K-Y, Kim S, Lee YJ, Kim H-T (2011) Protective effects of statins on l-DOPA neurotoxicity due to the activation of phosphatidylinositol 3-kinase and free radical scavenging in PC12 cell culture. Brain Res 1370:53–63PubMedCrossRefGoogle Scholar
  45. 45.
    Park H-H, Lee K-Y, Kim SH, Lee YJ, Koh S-H (2009) l-DOPA-induced neurotoxicity is reduced by the activation of the PI3K signaling pathway. Toxicology 265(3):80–86PubMedCrossRefGoogle Scholar
  46. 46.
    Nyholm D, Lennernäs H, Gomes-Trolin C, Aquilonius S-M (2002) Levodopa pharmacokinetics and motor performance during activities of daily living in patients with Parkinson’s disease on individual drug combinations. Clin Neuropharmacol 25(2):89–96PubMedCrossRefGoogle Scholar
  47. 47.
    Brewer LD, Thibault V, Chen K-C, Langub MC, Landfield PW, Porter NM (2001) Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons. J Neurosci 21(1):98–108PubMedGoogle Scholar
  48. 48.
    Garcion E, Wion-Barbot N, Montero-Menei CN, Berger F, Wion D (2002) New clues about vitamin D functions in the nervous system. Trends Endocrinol Metab TEM 13(3):100–105CrossRefGoogle Scholar
  49. 49.
    Liu W-G, Chen Y, Li B, Lu G-Q, Chen S-D (2004) Neuroprotection by pergolide against levodopa-induced cytotoxicity of neural stem cells. Neurochem Res 29(12):2207–2214PubMedCrossRefGoogle Scholar
  50. 50.
    Kriebitzsch C, Verlinden L, Eelen G, van Schoor NM, Swart K, Lips P, Meyer MB, Pike JW, Boonen S, Carlberg C, Vitvitsky V, Bouillon R, Banerjee R, Verstuyf A (2011) 1,25-dihydroxyvitamin D3 influences cellular homocysteine levels in murine preosteoblastic MC3T3-E1 cells by direct regulation of cystathionine β-synthase. J Bone Miner Res 26(12):2991–3000PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Prufer K, Jirikowski GF (1997) 1.25-Dihydroxyvitamin D3 receptor is partly colocalized with oxytocin immunoreactivity in neurons of the male rat hypothalamus. Cell Mol Biol 43(4):543–548PubMedGoogle Scholar
  52. 52.
    Sutherland MK, Somerville MJ, Yoong LK, Bergeron C, Haussler MR, McLachlan DR (1992) Reduction of vitamin D hormone receptor mRNA levels in Alzheimer compared to Huntington hippocampus: correlation with calbindin-28k mRNA levels. Brain Res Mol Brain Res 13(3):239–250PubMedCrossRefGoogle Scholar
  53. 53.
    Hall A, Juckett M (2013) The role of vitamin D in hematologic disease and stem cell transplantation. Nutrients 5(6):2206–2221PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Samuel S, Sitrin MD (2008) Vitamin D’s role in cell proliferation and differentiation. Nutr Rev 66:S116–S124PubMedCrossRefGoogle Scholar
  55. 55.
    Klotz B, Mentrup B, Regensburger M, Zeck S, Schneidereit J, Schupp N, Linden C, Merz C, Ebert R, Jakob F (2012) 1,25-dihydroxyvitamin D3 treatment delays cellular aging in human mesenchymal stem cells while maintaining their multipotent capacity. PLoS One 7(1):e29959PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Höhler B, Lange B, Holzapfel B, Goldenberg A, Hänze J, Sell A, Testan H, Möller W, Kummer W (1999) Hypoxic upregulation of tyrosine hydroxylase gene expression is paralleled, but not induced, by increased generation of reactive oxygen species in PC12 cells. FEBS Lett 457(1):53–56PubMedCrossRefGoogle Scholar
  57. 57.
    Kastner A, Herrero MT, Hirsch EC, Guillen J, Luquin MR, Javoy-Agid F, Obeso JA, Agid Y (1994) Decreased tyrosine hydroxylase content in the dopaminergic neurons of MPTP-intoxicated monkeys: effect of levodopa and GM1 ganglioside therapy. Ann Neurol 36(2):206–214PubMedCrossRefGoogle Scholar
  58. 58.
    Walkinshaw G, Waters CM (1995) Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA. Implications for the treatment of Parkinson’s disease. J Clin Invest 95(6):2458–2464PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Nakao N, Nakai K, Itakura T (1997) Metabolic inhibition enhances selective toxicity of l-DOPA toward mesencephalic dopamine neurons in vitro. Brain Res 777(1–2):202–209PubMedCrossRefGoogle Scholar
  60. 60.
    de Groot MWGDM, Westerink RHS Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity. NeuroToxicology (0)Google Scholar
  61. 61.
    Peritore CS, Ho A, Yamamoto BK, Schaus SE (2012) Resveratrol attenuates L-DOPA-induced hydrogen peroxide toxicity in neuronal cells. NeuroReport 23(17):989–994PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of NeurologyHanyang University College of MedicineSeoulSouth Korea
  2. 2.Department of Neurology, Gangneung Asan Hospital, College of MedicineUniversity of UlsanGangneungSouth Korea
  3. 3.Department of Translational MedicineHanyang University Graduate School of Biomedical Science and EngineeringSeoulRepublic of Korea
  4. 4.Department of NeurologyHanyang University College of MedicineGuri-siSouth Korea
  5. 5.Department of NeurologyHanyang University College of MedicineSeoulSouth Korea

Personalised recommendations