International Journal of Neuropsychopharmacology (2013), 16, 365–376. f CINP 2012 doi:10.1017/S1461145712000132

ARTICLE

Neuroprotective effects of chronic exposure of SH-SY5Y to low lithium concentration involve glycolysis stimulation, extracellular pyruvate accumulation and resistance to oxidative stress Riadh Nciri1,2, Frank Desmoulin3, Mohamed Saleh Allagui1,2, Jean-Claude Murat1, Abdelfattah El Feki2, Christian Vincent1 and Franc¸oise Croute1 1 Laboratoire de Biologie Cellulaire, Faculte´ de Me´decine Purpan, Universite´ Paul Sabatier Toulouse III, 37 Alle´es Jules Guesde, Toulouse, France 2 Laboratoire d ’e´cophysiologie, Faculte´ des Sciences, Sfax, Tunisie 3 INSERM U1048, Universite´ Paul Sabatier Toulouse III, 1 avenue Jean Poulhe´s, Toulouse, Cedex 4, France

Abstract

Received 26 June 2011 ; Reviewed 3 August 2011 ; Revised 14 January 2012 ; Accepted 19 January 2012 ; First published online 22 March 2012 Key words : Glutathione, glycolysis, lithium, pyruvate, SH-SY5Y.

Introduction Multiple functional, structural, cellular and molecular changes occur in the brain during ageing, including cognitive decline and reduced brain weight and volume, probably caused by the progressive degeneration and loss of neurons (Shankar, 2010 ; West et al. 1994). These disorders involve an impaired glucose uptake/ metabolism (Cunnane et al. 2011), mitochondrial dysfunctions (Beal, 2005 ; Mu¨ller et al. 2010), increased Address for correspondence : Dr R. Nciri, Laboratoire d’Ecophysiologie Animale, Faculte´ des Sciences de Sfax, PB 802, 3018 Sfax, Tunisie. Tel. : (216) (74) 276 400 Fax : (216) (74) 274 437 Email : [email protected]

oxidative stress (Dro¨ge & Schipper, 2007 ; Muller et al. 2007) and accumulation of abnormal proteins (Calabrese et al. 2006 ; Trojanowski & Mattson, 2003). During the past century, life expectancy has significantly increased. Unfortunately, prevalence of neurodegenerative diseases (e.g. Alzheimer’s, Parkinson’s, and Huntington’s diseases) has also progressively increased, with the need to find means to prevent or cure these age-related disorders (Mattson, 2003 ; Mu¨ller et al. 2010). Recent in vitro and in vivo studies suggest that lithium, which has been used for >50 yr for treatment of bipolar disorders (MachadoVieira et al. 2009 ; Manji et al. 1999), could be used to prevent or treat age-related neurodegenerative diseases (Wada et al. 2005).

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Recent studies suggest that lithium protects neurons from death induced by a wide array of neurotoxic insults, stimulates neurogenesis and could be used to prevent age-related neurodegenerative diseases. In this study, SH-SY5Y human neuronal cells were cultured in the absence (Con) or in the presence (Li+) of a low lithium concentration (0.5 mM Li2CO3, i.e. 1 mM lithium ion) for 25–50 wk. In the course of treatment, growth rate of Con and Li+ cells was regularly analysed using Alamar Blue dye. Resistance to oxidative stress was investigated by evaluating : (1) the adverse effects of high concentrations of lithium (4–8 mM) or glutamate (20–90 mM) on cell growth rate ; (2) the levels of lipid peroxidation (TBARS) and total glutathione ; (3) the expression levels of the anti-apoptotic Bcl-2 protein. In addition, glucose metabolism was investigated by analysing selected metabolites in culture media and cell extracts by 1H-NMR spectroscopy. As compared to Con, Li+ cells multiplied faster and were more resistant to stress, as evidenced by a lower dose-dependent decrease of Alamar Blue reduction and dose-dependent increase of TBARS levels induced by toxic doses of lithium and glutamate. Total glutathione content and Bcl-2 level were increased in Li+ cells. Glucose consumption and glycolytic activity were enhanced in Li+ cells and an important release of pyruvate was observed. We conclude that chronic exposure to lithium induces adaptive changes in metabolism of SH-SY5Y cells involving a higher cell growth rate and a better resistance to oxidative stress.

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R. Nciri et al. some metabolites in culture media and cell extracts by H-NMR spectroscopy.

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Materials and method Chemicals and reagents Lithium carbonate (Li2CO3), pro analysis grade, was from Prolabo/Rhone-Poulenc (France). AB assay was from Biosource (USA). Rabbit monoclonal antihuman/mouse Bcl-2 antibody (clone : E17) against Bcl-2 from Epitomics (USA) was purchased from Cliniscience (France). Super-Signal1 West Pico chemiluminescent substrate was from Pierce biotechnology (USA). Foetal calf serum (FCS) and other cell culture reagents were from Institute Jacques Boy (France). Proteases inhibitors, i.e. phenylmethanesulfonyl fluoride (PMSF), N-ethylmaleimide (NEM) and aprotinin and anti-rabbit IgG peroxidase antibody produced in goat were purchased from Sigma-Aldrich (France). Long-term treatment of SH-SY5Y cells by Li2CO3 SH-SY5Y human neuroblastoma cells were chosen because they have been widely used to investigate functions related to neurodegenerative and neuroadaptive processes, neurotoxicity and neuroprotection (Xie et al. 2010). The SH-SY5Y cell line is a thricecloned sub-line of SK-N-SH cells (ECACC #86012802), which were originally established from a bone marrow biopsy. SH-SY5Y cells were derived from immature neoplastic neural crest cells. They exhibit properties of stem cells and possess the capability of proliferating in culture for long periods. SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % (v/v) FCS and 20 mg/ml gentamycin at 37 xC in humidified 5 % CO2 atmosphere. In order to avoid the rapid medium exhaustion (due to the quick proliferation of cells) and to reduce the stress phases (due to the number of re-seeding occurring during extended lithium exposures), DMEM containing a high-glucose level (25 mM) was used. Under such culture conditions, cells could be left in the same medium for 1 wk. The experiments of chronic treatment by lithium were initiated by seeding cells in T25 flasks containing 8 ml culture medium at a density 20 000/cm2 (i.e. 500 000/flask) and allowed to attach overnight. Then, Li2CO3 (150 mM stock solution adjusted to pH 7.0) was added in half of the cultures in order to obtain a final concentration of 0.5 mM. For 1 yr, once per week, cells from Con and Li+ treated cultures were dissociated by

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Indeed, therapeutic relevant concentrations of lithium (0.5–1 mM) were found to stimulate neurogenesis (Chen et al. 2000 ; Chuang, 2004), to increase neuronal differentiation of progenitor cells (Kim et al. 2004) , to exert neuroprotective effects by increasing resistance to oxidative damages (Cui et al. 2007 ; Kim et al. 2011 ; Shao et al. 2008) and to glutamate-induced excitotoxicity (Chen et al. 2003 ; Chuang et al. 2002), to decrease the production of amyloid-(Ab) peptides (Phiel et al. 2003) and to prevent t phosphorylation (Engel et al. 2006). Moreover, lithium was shown to promote neuronal survival by increasing the action of anti-apoptotic proteins and inhibiting pro-apoptotic signal transduction (Bielecka & Obuchowicz, 2008 ; Machado-Vieira et al. 2009 ; Manji et al. 2000 ; Marmol, 2008). SH-SY5Y cells, derived from a human neuroblastoma, are used in our laboratory to investigate neuroprotective effects of lithium (Allagui et al. 2009). SHSY5Y cells were chosen because they have been widely used as an in vitro model for the study of neurodegenerative disorders (Alzheimer’s or Parkinson’s diseases) and to evaluate the protective effects of antiapoptotic substances (Pasquariello et al. 2009 ; Peng et al. 2008 ; Xie et al. 2010). We previously showed that long-term lithium treatment at therapeutic concentration (0.5 mM Li2CO3 for 25 wk) stimulated SH-SY5Y proliferation, modified expression level of HSP27 stress protein and enhanced resistance to oxidative stress (Allagui et al. 2009). It was suggested that chronic treatment (over several months) by lithium could favour neurogenesis, decrease the vulnerability of neuronal cells to oxidative stress and induce post-translational changes of molecular chaperones. The aim of the present study was to improve our knowledge on neuroprotective effects of lithium in SH-SY5Y cells cultured in the presence of 0.5 mM Li2CO3 (Li+ ; i.e. 1 mM lithium ion) for 25–50 wk. Alamar Blue (AB) assay was used to compare the growth rate of control (Con) and Li+ treated cells. The resistance of Con and Li+ cells to oxidative stress was evaluated by : (1) measuring the effect of increasing doses of lithium carbonate (4–8 mM) or glutamate (20–90 mM) on cell growth ; (2) analysing the changes of lipid peroxidation level and total glutathione (tGSH) content ; (3) examining the expression level of the anti-apoptotic Bcl-2 protein. In light of some recent works, which showed that lithium activated cerebral glucose metabolism in healthy men (Fan et al. 2010 ; Kohno et al. 2007), we have studied lithium effects on glucose metabolism by measuring the pH of culture media and analysing

How lithium protects SH-SY5Y cells trypsin-EDTA treatment, counted and then re-seeded at low density, as above (20 000/cm2), lithium being added 12 h later in Li+ cultures. Con and Li+ treated cells were periodically collected for analysis.

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100 ml thiobarbituric acid and heated at 90 xC for 30 min. After cooling, absorbance of the mixture was read at 535 nm. TBARS values were calculated by using a molar absorption coefficient of 1.56r 105 Mx1 cmx1.

AB assay for cell quantification

Determination of lipid peroxidation Lipid peroxidation was estimated by measuring the thiobarbituric acid reactive substances (TBARS) amount after addition of butylated hydroxytoluene (BHT) as antioxidant and alkaline hydrolysis to release protein-bound malondialdehyde (Carbonneau et al. 1991 ; Hong et al. 2000). Briefly, the mixture including 500 ml cell extract, BHT (0.02 % final concentration) and NaOH (0.5 N final concentration) was incubated at 60 xC for 30 min. Proteins were precipitated by adding 500 ml trichloroacetic acid (15 %) and the mixture was placed in ice for 10 min and centrifuged (1000 g for 10 min). Then, 800 ml supernatant was mixed with

Colorimetric microplate assay for tGSH The assay utilizes an enzymatic recycling method, based on the reaction of the thiol group of glutathione (GSH) with 5k-thio-2-nitrobenzoic acid after the action of GSH reductase in the presence of NADPH2 (Coutelle et al. 1992). As recommended by this author, proteins from samples were eliminated by precipitation with metaphosphoric acid 10 % (v/v) at 4 xC for 10 min. After centrifugation (10 000 g for 10 min), supernatants (containing GSH) were diluted (1 : 5) in phosphate buffer saline (PBS ; 100 mM) with EDTA (5 mM), pH 7.8. The determination of tGSH in sample extracts was carried out in parallel with GSH standards (1, 2, 5, 10, 15, 20 mM) using 96 well plates. Absorbance was measured at 405 nm using a kinetic program, which could monitor the reaction at 1 min intervals for 4 min. Samples were analysed in triplicate. Analysis of Bcl-2 protein expression on Western blot Cell layers were rinsed with ice-cold PBS, collected into a lysis buffer (PBS containing 0.5 % NP-40 and protease inhibitors : 10 mM EDTA, 2 mM PMSF, 5 mM NEM, 1 mg/ml aprotinin, pH 7.4) and stored at x20 xC until use. In total, 100 ml lysis buffer was used for about 106 cells. Cell lysates were sonicated for 10 s and protein concentration was determined using the Lowry’s method (Lowry et al. 1951). Equal amounts of protein (20 mg) from each homogenate were separated by SDS–PAGE according to Laemmli’s (1970) method and electroblotted (100 V, 75 min) on to 0.45 mm pore size nitrocellulose membranes. In order to saturate unspecific sites, membranes were incubated for 1 h at 37 xC in a Tris-buffered saline (TBS : 10 mM Tris, 140 mM NaCl, pH 7.4) containing 0.1 % Tween 20 (TBST). Membranes were then incubated for 1 h at room temperature under continuous shaking in the presence of Bcl-2 rabbit monoclonal antibody diluted (1 : 1000) in TBST and allowed to stand overnight at 4 xC. After three washing steps, (1) TBS for 5 min, (2) TBS containing 0.1 % Nonidet-P40 for 15 min and (3) TBS for 5 min (twice), the blots were incubated at room temperature, in darkness, for 2 h under continuous shaking in the presence of a peroxidase-conjugated secondary antibody diluted (1 : 10 000) in TBS containing 3 % dried skimmed milk.

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AB is a non-toxic, water-soluble dye that was designed to measure quantitatively cell proliferation. When added to cell cultures, the oxidized form of AB is converted to the reduced form due to chemical reduction of medium resulting from metabolic activity of cells. The redox reaction is accompanied by a shift in colour that can be easily measured by spectrophotometry. Magnitude of dye reduction correlates linearly with the number of cells (Al-Nasiry et al. 2007), but can be affected by glycolytic and oxidative metabolism of glucose (Abe et al. 2002). Preliminary experiments were carried out to determine the optimal density of seeding. In order to compare proliferation rate of Con and Li+ cells, they were seeded in 24-well culture plates at a concentration of 20 000 cells/well/ml. For 6 d, the medium of one culture plate was replaced daily by fresh media containing 10 % v/v AB and cultures were put back into the CO2 incubator for 6 h. The absorbance at 570 nm (oxidized form) and 600 nm (reduced form) was recorded with an automated spectrophotometric microplate reader and percentage of AB reduction was calculated as recommended by the manufacturer (Biosource). To analyse the relative resistance of Con cells and Li+ cells to toxic doses of glutamate or lithium, these chemicals were added to the culture medium to obtain final concentrations ranging from 4 to 8 mM for Li2CO3 and from 20 to 90 mM for glutamate. AB assay was performed after 6 d of growth. Percentage of AB reduction in Li+ culture media was normalized to the percentage of AB reduction measured in Con.

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After final washing, membranes were incubated with the chemoluminescent substrate for 2 min at room temperature and then exposed to Bio Max light-1 film (Kodak, USA). Protein levels (arbitrary units) were determined by computer-assisted densitometric analysis (Photo-Capt software ; 123 Multimedia, France) of immunoreactive bands visible on exposed film. Values were expressed as percent of controls (normalized to 100). Analysis of Con and Li+ cell metabolism Cellular metabolism was evaluated by monitoring pH decrease and by metabolomic analysis (1H-NMR spectroscopy). Con and Li+ cells were seeded at high density : i.e. 200 000 cells/cm2, in order to inhibit cell proliferation by contact inhibition and to limit cell density differences between Con and Li+ cultures (which could result from their growth rate differences). Forty-eight hours later, culture supernatants were rapidly collected for pH measurement and frozen at x80 xC. The cells were washed with ice-cold PBS and immediately frozen at x80 xC until extraction procedure. Cellular metabolites were extracted at 4 xC as previously described (Tyagi et al. 1996). Briefly, cells were thawed in 1 ml methanol : water (2 : 1, v/v) mixture at 4 xC and disrupted with an ultraturax (30 s in ice). Then, 1 ml

chloroform was added and the mixture was vortexed for 2 min. Finally, 1 ml methanol : water (2 : 1, v/v) and 1 ml chloroform were added and vortexed for 2 min. The mixture was kept on ice for 30 min, centrifuged (4000 g, 10 min, 4 xC) and the upper fraction (containing the hydrosoluble metabolites) was collected and lyophilized. Cells extracts were reconstituted in 650 ml D2O with 10 ml 10 mM 3-(trimethylsilyl)-1-propanesulfonate sodium salt (TMPS) solution and 200 ml D2O with 6 ml 60 mM TMPS solution was added to 400 ml culture medium prior to NMR analysis. 1 H-NMR spectra were obtained at 300 K on a Bruker Avance DRX-600 spectrometer (Bruker, France) operating at 600.13 MHz and equipped with a 5 mm TXI cryoprobe. Fully relaxed 1H spectra resulting from 60x excitation pulses and 5.5 s repetition time (SW of 9.4 kHz, 64 K data points) were obtained with presaturation pulse sequence to suppress residual intensity of 1H water resonance peak by accumulating 256 free induction decays and 32 for cell extract and culture medium, respectively. Line broadening of 1 Hz was applied prior to Fourier transform. Characteristic metabolites (Desmoulin et al. 1990 ; Govindaraju et al. 2000) have been assigned by reference to literature data and on the basis of 2D homonuclear correlation spectroscopy experiments performed on the extracts (data not shown). The signal of TMPS (d=0 ppm)

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Fig. 1. Time-related changes in % Alamar Blue (AB) reduction in sub-cultures of SH-SY5Y previously cultured in the absence (Con) or presence of 0.5 mM Li2CO3 (Li+) for 36 (a) or 47 wk (b). Data are presented as mean¡S.D. (n=4). (a) Percentages of AB reduction (i.e. growth rate) were significantly higher in Li+ vs. Con cells [two-way analysis of variance (ANOVA) ; F6,28=967.0, p