Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu Melatonin prevents calcineurin-activated the nuclear translocation of nuclear factor of activated T-cells in human neuroblastoma SH-SY5Y cells undergoing hydrogen peroxide-induced cell death Asawin Premratanachai a , Wilasinee Suwanjang b , Piyarat Govitrapong a,c , Jirapa Chetsawang d , Banthit Chetsawang a, * a Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom, Thailand b Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand c Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok, Thailand d Department of Anatomy, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand ARTICLE INFO Keywords: Melatonin Cell death Hydrogen peroxide NFAT Calcineurin Neuroblastoma cells ABSTRACT The interaction between the activation of protein phosphatase, calcineurin (CaN), and the dephosphorylation and nuclear translocation of nuclear factor of activated T-cells (NFAT), a transcriptional factor in the immune system, has attracted interest as a key factor responsible for the cell death process. In this study, the effects of melatonin on the interaction between CaN and NFAT signaling during oxidative stress-induced cell death were investigated. Human neuroblastoma SH-SY5Y cells were treated with the non-radical reactive oxygen species hydrogen peroxide (H 2 O 2 ). Cells were treated with 200 μMH 2 O 2 for the indicated time. Some H 2 O 2 -treated cells were pretreated with melatonin for 1 h. Control cells were treated with the same concentration of ethanol used to dilute melatonin. H 2 O 2 -induced cell death promoted increases in reactive oxygen species (ROS) production and the nuclear translocation of NFAT, which were related to increased levels the active, cleaved form of CaN (32.5 kDa). In addition, pretreatment of H 2 O 2 -treated cells with melatonin decreased cell death, ROS production, the levels of the active-cleaved form of CaN and the nuclear translocation of NFAT. Based on these findings, melatonin may exert its neuroprotective effects on oxidative damage-induced cell death by inhibiting CaN- activated the nuclear translocation of NFAT. 1. Introduction Oxidative stress, an imbalance between the production of reactive oxygen species (ROS) and oxidant detoxification of a biological system, is involved in the development of various neurological diseases, in- cluding Alzheimer’s disease (AD), Parkinson’s disease (PD) and cere- brovascular accident (CVA) (Behl, 1999; Choi et al., 2006; Finkel and Holbrook, 2000; Olanow, 1993). A non-radical ROS, hydrogen peroxide (H 2 O 2 ), elevates intracellular Ca 2+ levels by either inducing en- doplasmic reticulum (ER) stress (Scorrano et al., 2003) or damage to Ca 2+ channels in the cell membrane (Mattson, 2004). The excessive Ca 2+ levels play a pivotal role in pathological events by triggering the apoptotic cell death pathway that includes calcineurin (CaN), a Ca 2+ / calmodulin (CaM)-dependent protein phosphatase that is thought to be a key factor responsible for inducing the cell death process (Hajimohammadreza et al., 1997; Lee et al., 1991; Liu et al., 2005; Nakagawa et al., 2000; Saito et al., 1993). CaN consists of two subunits: the 62 kDa subunit A (CaNA) and the 19 kDa subunit B (CaNB). The CaNA domains are divided into catalytic and regulatory domains. The regulatory domains contain a CaNB-binding site, a CaM-binding site and autoinhibitory domains. CaN is activated upon the binding of the Ca 2+ -CaNB complex or Ca 2+ -CaM complex to CaNA, leading to the release of the autoinhibitory domain from the catalytic domain (Aramburu et al., 2000; Klee et al., 1998; Shioda and Fukunaga, 2011; Wang et al., 1989). In addition to Ca 2+ /CaM-dependent activation of CaN, CaN is also activated by cleavage of the autoinhibitory domain that transforms it into a constitutively active form, which remains ac- tivated and cannot be reversed to the resting or inactive form. Three cleaved forms of CaN have been reported, including 48 kDa CaN mediated by calpain cleavage of the autoinhibitory domain (Shioda and Fukunaga, 2011; Croall and DeMartino, 1991; Pedrosa et al., 2010; Wu et al., 2004), 45 kDa CaN mediated by caspase-3 cleavage of both the https://doi.org/10.1016/j.jchemneu.2020.101793 Received 3 January 2020; Received in revised form 2 April 2020; Accepted 3 April 2020 ⁎ Corresponding author at: Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom 73170, Thailand. E-mail address: banthit.che@mahidol.ac.th (B. Chetsawang). Journal of Chemical Neuroanatomy 106 (2020) 101793 Available online 26 April 2020 0891-0618/ © 2020 Elsevier B.V. All rights reserved. T