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

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.

Genistein protects against amyloid‐beta‐induced toxicity in SH‐SY5Y cells by regulation of Akt and Tau phosphorylation

“…SH-SY5Y are neuroblast-like cells that can be differentiated into a mature neuronal phenotype using several differentiating agents, including RA, phorbol esters, and neurotrophins (Kovalevich & Langford, 2013). Cells under RA treatment have shown to develop long and extensively branched neurites, express neuronal cell markers (NeuN, tyrosine hydroxylase, neuron specific enolase, synaptophysin, post-synaptic associated protein-97, and β-III tubulin) (Cheung et al, 2009;Kunzler et al, 2017;Lopes et al, 2010), and exhibit mature excitability (Toselli, Tosetti, & Taglietti, 1996;Tosetti, Taglietti, & Toselli, 1998). We observed an alteration in cell morphology, as well as an increase in NeuN levels (a marker for mature neurons) after RA treatment, as previously reported (Cheung et al, 2009;Lopes et al, 2010).…”

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells

“…There are many lines of evidence showing the effect of RA-differentiation in SH-SY5Y regarding the evaluation of proliferation rates (Ross 1996;Pezzini et al 2016;Kunzler et al 2016). Previous studies have demonstrated that RAinduced differentiation can cause cell cycle arrest either in G1/G0 phase or in G2/M phase and a decrease in proliferation rates, which leads to terminal differentiation of neuroblastoma cells (Qiao et al 2012;Hämmerle et al 2013).…”

Effects of silica nanoparticle exposure on mitochondrial function during neuronal differentiation

“…Recent data demonstrate that proliferative neuronal stem cells have high ROS levels, which is required for self-renewal and neurogenesis with underlying PI3-K/Akt signaling [ 45 ]. A significant increase in the differentiation markers (PI3-K/Akt and MAP-K/ERK) was found in differentiated SH-SY5Y compared to undifferentiated cells indicating that these kinases are involved in neuronal differentiation [ 46 ]. This finding is corroborating previously published data [ 5 ] and other studies also demonstrated that the PI3-K/Akt improved neurite elongation in primary hippocampal and cortical neurons [ 21 , 22 , 47 ].…”