Research on Parkinson’s disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.
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Brazilian funds CNPq/MS/SCTIE/DECIT-Pesquisas Sobre Doenças Neurodegenerativas (no. 466989/2014-8), MCT/CNPq INCT-TM (no. 573671/2008-7), and Rapid Response Innovation Award/MJFF (no. 1326-2014) provided the financial support without interference in the ongoing work. FK received a fellowship from MCT/CNPq (no. 306439/2014-0). FML received a fellowship from Programa de Doutorado Sanduíche no Exterior (PDSE)/CAPES (no. 14581/2013-2). We thank Dr. Florencia M. Barbé-Tuana for technical assistance with flow cytometry and Dr. Tadeu Mello e Souza for kindly providing DATi.
F.M.L., L.L.M., L.F.S., D.M.V., P.S., G.F.L., and L.M. performed experiments. B.P. and B.W.A. performed the RNA extraction for the microarray analysis. G.Z. and V.N.F. performed the molecular docking. F.M.L., L.L.M., M.A.D.B., M.A.A.C., R.B.P., A.L.D., and F.K. analyzed and interpreted the data. F.M.L. and F.K. conceived and designed the experiments. F.M.L. and F.K. wrote the manuscript.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interests.
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