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 Kunzler 1 & 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 Received: 7 June 2016 /Accepted: 30 September 2016 # Springer Science+Business Media New York 2016 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 neuro- nal 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 prolif- erative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydrox- ylase (TH), β-III tubulin, and enolase-2. Evaluation of intra- cellular RS production by DCFH oxidation assay and quanti- fication 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 ca- pacity 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 ap- pearance of morphological characteristics such as neurite exten- sion 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. Keywords Retinoic acid . Neuronal differentiation . SH-SY5Y . Oxidative stress . Tyrosine hydroxylase Introduction Parkinson’ s disease (PD) is the most common movement dis- order, affecting approximately six million people worldwide [1] and is highly connected to oxidative stress, with increased lipid peroxidation, decreased glutathione, abnormalities in iron homeostasis, and protein aggregation [2]. Cellular models are often used to understand pathways and molecular events of pathological processes. In vitro research emerged as first choice for preliminary studies on the molecular action, cyto- toxic and genetic events of neurodegenerative diseases, in- cluding PD [1]. The human neuroblastoma SH-SY5Y cell line has been largely used in neuroscience studies, particularly to generate different PD cell models [3–6]. Originally derived as subclone of the neuroblastoma cell line SK-N-SH, SH-SY5Y * Alice Kunzler alice.bio@hotmail.com 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 2 The School of Biomedical Sciences and The Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, Australia Mol Neurobiol DOI 10.1007/s12035-016-0189-4