Molecular Neurobiology

, Volume 54, Issue 9, pp 6903–6916 | Cite as

Changes in Cell Cycle and Up-Regulation of Neuronal Markers During SH-SY5Y Neurodifferentiation by Retinoic Acid are Mediated by Reactive Species Production and Oxidative Stress

  • Alice KunzlerEmail author
  • Fares Zeidán-Chuliá
  • Juciano Gasparotto
  • Carolina Saibro Girardi
  • Karina Klafke
  • Lyvia Lintzmaier Petiz
  • Rafael Calixto Bortolin
  • Diana Carolina Rostirolla
  • Alfeu Zanotto-Filho
  • Matheus Augusto de Bittencourt Pasquali
  • Phillip Dickson
  • Peter Dunkley
  • José Cláudio Fonseca Moreira
  • Daniel Pens Gelain


Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson’s disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), β-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, β-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.


Retinoic acid Neuronal differentiation SH-SY5Y Oxidative stress Tyrosine hydroxylase 



This work was supported by the Brazilian funds CNPq (401260/2014-3, 400437/2013-9, 443514/2014-3, 401368/2012-2 and 303227/2015-0), CAPES, FAPERGS (2299-2551/14-6), and Propesq-UFRGS. The authors thank Mr. Henrique Biehl for their technical assistance at the CME.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Alice Kunzler
    • 1
    Email author
  • Fares Zeidán-Chuliá
    • 1
  • Juciano Gasparotto
    • 1
  • Carolina Saibro Girardi
    • 1
  • Karina Klafke
    • 1
  • Lyvia Lintzmaier Petiz
    • 1
  • Rafael Calixto Bortolin
    • 1
  • Diana Carolina Rostirolla
    • 1
  • Alfeu Zanotto-Filho
    • 1
  • Matheus Augusto de Bittencourt Pasquali
    • 1
  • Phillip Dickson
    • 2
  • Peter Dunkley
    • 2
  • José Cláudio Fonseca Moreira
    • 1
  • Daniel Pens Gelain
    • 1
  1. 1.Departamento de Bioquímica, Centro de Estudos em Estresse OxidativoUniversidade Federal do Rio Grande do Sul—UFRGSPorto AlegreBrazil
  2. 2.The School of Biomedical Sciences and The Hunter Medical Research Institute, Faculty of HealthThe University of NewcastleCallaghanAustralia

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