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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

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Abstract

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.

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Acknowledgments

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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Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Estudos em Estresse Oxidativo, Universidade Federal do Rio Grande do Sul—UFRGS, Rua Ramiro Barcelos, 2600 – Anexo, Porto Alegre, RS, CEP 90035-003, Brazil

    Alice Kunzler, 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, José Cláudio Fonseca Moreira & Daniel Pens Gelain

  2. The School of Biomedical Sciences and The Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, Australia

    Phillip Dickson & Peter Dunkley

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  1. Alice Kunzler
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  2. Fares Zeidán-Chuliá
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Correspondence to Alice Kunzler.

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Kunzler, A., Zeidán-Chuliá, F., Gasparotto, J. et al. 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. Mol Neurobiol 54, 6903–6916 (2017). https://doi.org/10.1007/s12035-016-0189-4

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