Edinburgh Research Explorer Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator 3H-1,2-dithiole-3-thione Citation for published version: Soriano, FX, Leveille, F, Papadia, S, Higgins, LG, Varley, J, Baxter, P, Hayes, JD & Hardingham, GE 2008, 'Induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective Nrf2 activator 3H-1,2-dithiole-3-thione', Journal of Neurochemistry, vol. 107, no. 2, pp. 533-543. https://doi.org/10.1111/j.1471-4159.2008.05648.x Digital Object Identifier (DOI): 10.1111/j.1471-4159.2008.05648.x Link: Link to publication record in Edinburgh Research Explorer Document Version: Peer reviewed version Published In: Journal of Neurochemistry Publisher Rights Statement: Published in final edited form as: J Neurochem. 2008 October ; 107(2): 533–543. doi:10.1111/j.1471-4159.2008.05648.x. 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Sep. 2021 JOURNAL OF NEUROCHEMISTRY | 2008 | 107 | 533–543 doi: 10.1111/j.1471-4159.2008.05648.x *Centre for Neuroscience Research, University of Edinburgh, Edinburgh, UK Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Abstract responsible for reducing hyperoxidized peroxiredoxins. Peroxiredoxins are an important family of cysteine-based Induction of sulfiredoxin expression is mediated by Nrf2, act- antioxidant enzymes that exert a neuroprotective effect in ing via a cis-acting antioxidant response element (ARE) in its several models of neurodegeneration. However, under oxi- promoter. The ARE element in Srxn1 contains an embedded dative stress they are vulnerable to inactivation through activator protein-1 (AP-1) site which directs induction of Srxn1 hyperoxidation of their active site cysteine residues. We show by synaptic activity. Thus, raising Nrf2 activity in neurons that in cortical neurons, the chemopreventive inducer 3H-1,2- prevents peroxiredoxin hyperoxidation and induces a new dithiole-3-thione (D3T), that activates the transcription factor member of the ARE-gene family, whose enzymatic function of Nuclear factor erythroid 2-related factor (Nrf2), inhibits the reducing hyperoxidized peroxiredoxins may contribute to the formation of inactivated, hyperoxidized peroxiredoxins fol- neuroprotective effects of Nrf2 activators. lowing oxidative trauma, and protects neurons against oxi- Keywords: chemoprevention, neurodegeneration, neuropro- dative stress. In both neurons and glia, Nrf2 expression and tection, oxidative stress, phase II enzymes, thioredoxin. treatment with chemopreventive Nrf2 activators, including J. Neurochem. (2008) 107, 533–543. D3T and sulforaphane, up-regulates sulfiredoxin, an enzyme Oxidative stress occurs because of an imbalance between These Prxs contain a peroxidatic cysteine residue, oxidized production of reactive oxygen species and the cell’s capacity by peroxides to cysteine sulfenic acid, which then forms a to neutralize them through its intrinsic antioxidant defences. disulfide bond with the resolving cysteine, which is in turn Key among these is the thioredoxin-peroxiredoxin system reduced by thioredoxin (Wood et al. 2003). Sometimes, Prx- which is an important reducer of oxidative stressors such as sulfenic acid is further oxidized by peroxide to sulfinic peroxides (Winyard et al. 2005). The thioredoxin system (-SO2H) or sulfonic (-SO3H) acid, causing inactivation of protects against H2O2-induced apoptosis, and its inhibition peroxidase activity (Rhee et al. 2007). Prx-SO2/3H is not a promotes oxidative stress and cell death (Yoshida et al. substrate for the resolving cysteine and cannot be reduced by 2005). The thioredoxin-peroxiredoxin system detoxifies peroxides by transferring reducing equivalents from NADPH to peroxides via thioredoxin reductase, thioredoxin and Received July 23, 2008; revised manuscript received August 8, 2008; finally peroxiredoxins (Prxs). Prxs are a ubiquitous family of accepted August 12, 2008. Address correspondence and reprint requests to Giles E. Hardingham, peroxidases with cytoprotective and antioxidative effects Centre for Neuroscience Research, University of Edinburgh, Edinburgh (Immenschuh and Baumgart-Vogt 2005). The 2-Cys Prxs is EH8 9XD, UK. E-mail: [email protected] the predominant Prx subfamily, comprising Prx I-IV (Wood Abbreviations used: ARE, antioxidant response element; AP-1, acti- et al. 2003) and are implicated in protecting neuronal cells vator protein-1; D3T, 3H-1,2-dithiole-3-thione; GAPDH, Glycer- from Ab toxicity (Yao et al. 2007), excitotoxicity (Hattori aldehyde-3-phosphate dehydrogenase; GFAP, Glial fibrillary acidic protein; DAPI, 4,6-diamino-2-phenylindole; eGFP, enhanced green et al. 2003), oxygen-glucose deprivation (Boulos et al. fluorescent protein; NeuN, Neuronal nuclear antigen; Nrf2, Nuclear 2007), peroxide (Sanchez-Font et al. 2003; Fang et al. factor erythroid 2-related factor; Prx, Peroxiredoxin; tBHQ, tert- 2007), and MPP+ toxicity (Qu et al. 2007). butylhydroquinone. Ó 2008 The Authors Journal Compilation Ó 2008 International Society for Neurochemistry, J. Neurochem. (2008) 107, 533–543 533 534 | F. X. Soriano et al. thioredoxin. Hyperoxidation takes place when there is not firedoxin. We find that this induction is mediated at the enough reduced Prx to deal with peroxides present, and is transcriptional level and that sulfiredoxin is a new member of associated with oxidative neuronal death in vitro, and also the Nrf2/ARE regulated gene family. Given its function, it ischemic brain damage in vivo (Papadia et al. 2008). may form part of the cytoprotective gene battery, the Previously, hyperoxidation of Prx was thought to be transcription of which is promoted by chemopreventive irreversible. However, more recently it has been found that agents. Prx-SO2H can be reduced back to the catalytically active thiol form in eukaryotic cells by the ATP-dependent reductase, sulfiredoxin (Biteau et al. 2003; Rhee et al. Materials and methods 2007; Jonsson et al. 2008). The activity of sulfiredoxin restores inactive Prxs back to the thioredoxin cycle and Tissue culture and the induction of oxidative stress prevents permanent oxidative inactivation of Prxs by strong Cortical rat neurons were cultured as described (Hardingham et al. oxidative insults. Over-expression of sulfiredoxin has been 2002) from E21 rats except that growth medium contained B27 (Invitrogen, Carlsbad, CA, USA). A single dose of anti-mitotic shown to prevent Prx hyperoxidation in response to an agent (AraC, 4.8 lM) was added to the cultures at days in vitro oxidative insult (Woo et al. 2005). Conversely, knockdown (DIV) 4 to minimize glial numbers. Experiments were carried out of sulfiredoxin prevents reduction of Prx-SO2H following a after being cultured for 8–10 days during which cortical neurons transient oxidative insult (Chang et al. 2004). develop a network of processes, express functional NMDA-type and One known defence against oxidative insults is the a-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate-type induction of a group of genes encoding antioxidative and glutamate receptors, and form synaptic contacts. Experiments were drug-metabolizing enzymes (also known as Phase II performed after transferring neurons at DIV8 into defined medium enzymes). These genes are induced by a variety of small lacking trophic support ‘TMo’ (Papadia et al. 2005): this is thiol-active molecules including the potent chemopreventive composed of 10% MEM (Invitrogen) and 90% Salt-Glucose- agent 3H-1,2-dithiole-3-thione (D3T), as well as dietary Glycine medium (SGG: 114 mM NaCl, 0.219% NaHCO3, phytochemicals such as Sulforaphane (Nguyen et al. 2004). 5.292 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM Glycine, 30 mM Glucose, 0.5 mM sodium pyruvate, 0.1% Mild oxidative stress also induces these genes (Giudice and Phenol Red; osmolarity 325 mosm/L, hereafter TMo). D3T (10– Montella 2006). Transcriptional regulation of this group of 25 lM) was applied to neurons 16 h prior to the application of an genes is mediated by a cis-acting promoter element termed oxidative insult in the form of H2O2 (100 lM, stabilized solution: the antioxidant response element (ARE), which recruits the Sigma, St Louis, MO, USA). Neurons were fixed after a further 24 h transcription factor Nuclear factor erythroid 2-related factor and subjected to 4,6-diamino-2-phenylindole (DAPI) staining and (Nrf2) as a heterodimer with small Maf proteins (Zhang cell death quantified by counting (blind) the number of apoptotic 2006). Nrf2 levels are constitutively low because of being nuclei as a percentage of the total. Approximately 1500 cells were targeted for degradation by Keap1. Under conditions of counted per treatment, across four independent experiments. oxidative stress, Nrf2 degradation is slowed and Nrf2 Morphologically, peroxide-treated neurons show typical signs of accumulates in