Neurochem Res (2013) 38:2631–2639 DOI 10.1007/s11064-013-1181-2
ORIGINAL PAPER
Protective Effects of Zonisamide Against Rotenone-Induced Neurotoxicity Salvatore Condello • Monica Curro` • Nadia Ferlazzo • Gregorio Costa • Giuseppa Visalli • Daniela Caccamo • Laura Rosa Pisani • Cinzia Costa Paolo Calabresi • Riccardo Ientile • Francesco Pisani
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Received: 12 August 2013 / Revised: 3 October 2013 / Accepted: 14 October 2013 / Published online: 20 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Zonisamide (ZNS), an antiepileptic drug having beneficial effects also against Parkinson’s disease symptoms, has proven to display an antioxidant effects in different experimental models. In the present study, the effects of ZNS on rotenone-induced cell injury were investigated in human neuroblastoma SH-SY5Y cells differentiated towards a neuronal phenotype. Cell cultures were exposed for 24 h to 500 nM rotenone with or without pre-treatment with 10–100 lM ZNS. Then, the following parameters were analyzed: (a) cell viability; (b) intracellular reactive oxygen species production; (c) mitochondrial Salvatore Condello and Monica Curro` have contributed equally to this study. S. Condello M. Curro` N. Ferlazzo G. Visalli D. Caccamo R. Ientile (&) Department of Biomedical Sciences and Morphological and Functional Imaging, University of Messina, AOU Policlinico ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy e-mail: [email protected] G. Costa Department of Human Pathology, Laboratory of Immunology and Biotherapy, University of Messina, AOU ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy L. R. Pisani F. Pisani Department of Neurosciences, Neurology Clinic, University of Messina, AOU ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy C. Costa P. Calabresi Neurology Clinic, Ospedale Santa Maria Della Misericordia, University of Perugia, Via Santa Andrea Delle Fratte, San Sisto, 06158 Perugia, Italy C. Costa P. Calabresi IRCCS Fondazione S. Lucia, Via Ardeatina 306, 00179 Rome, Italy
transmembrane potential; (d) cell necrosis and apoptosis; (e) caspase-3 activity. ZNS dose-dependently suppressed rotenone-induced cell damage through a decrease in intracellular ROS production, and restoring mitochondrial membrane potential. Similarly to ZNS effects, the treatment with N-acetyl-cysteine (100 lM) displayed significant protective effects against rotenone-induced ROS production and Dwm at 4 and 12 h respectively, reaching the maximal extent at 24 h. Additionally, ZNS displayed antiapoptotic effects, as demonstrated by flow cytometric analysis of annexin V/propidium iodide double staining, and significant attenuated rotenone-increased caspase 3 activity. On the whole, these findings suggest that ZNS preserves mitochondrial functions and counteracts apoptotic signalling mechanisms mainly by an antioxidant action. Thus, ZNS might have beneficial effect against neuronal cell degeneration in different experimental models involving mitochondrial dysfunction. Keywords Zonisamide Neuroprotection Oxidative stress Mitochondrial impairment Apoptosis
Introduction Glutamate-mediated excitotoxicity and related oxidative stress have been reported to be among the main triggers of neuronal cell death observed in neurological disorders, i.e. epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease (PD) [1]. In particular, a major role for oxidative stress has been postulated in PD, based on the evidence of iron accumulation in the substantia nigra as well as increased superoxide dismutase activity, glutathione depletion, and
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oxidative damage of lipids and DNA in dopaminergic neurons [2–5]. The oxidative stress, likely resulting from impaired mitochondrial functions, has been involved in the pathogenesis of neurodegenerative diseases. The complex I is the first protein component of the mitochondrial respiratory chain and plays a crucial role in ATP production and mitochondrial function in general. Mitochondrial toxins selectively targeting complex I, such as 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine, or rotenone, are capable of producing neuronal cell death and have been widely used to produce in vitro and in vivo models of PD [6]. In particular, due to its ability to freely cross biological membranes, independently from transporters, rotenone produces a strong inhibition of mitochondrial complex I [7] which increases the rate of mitochondrial reactive oxygen species (ROS) release, leading to cell apoptosis [8, 9]. These observations strongly indicate that mitochondrial dysfunction underlying the massive cell death could be related to deficiency of mitochondrial complex I. Advances in understanding the pathogenesis of mitochondrial complex I dysfunction likely involved in neurodegeneration may allow the development of new therapeutic approaches to management of neurological dysfunctions. Interestingly, previous experimental studies have shown that some antiepileptic drugs, such as carbamazepine or the newly designed zonisamide (ZNS), could act as neuroprotective agents. The carbamazepine and ZNS neuroprotective potential has been tested in an in vitro model of neurotoxicity triggered by acute administration of rotenone to corticostriatal brain slices. In this regard, carbamazepine strongly prevented rotenone-induced striatal neuronal dysfunction by reducing membrane depolarization [10], while ZNS effects could also be mediated by inhibition of voltage-dependent Na? and T-type Ca(2?) channels, reduction of depolarizationinduced glutamate release, enhanced GABAergic and monoaminergic transmission [11–13]. Indeed, ZNS at micromolar concentrations was able to protect striatum against mitochondrial impairment suggesting its possible use in the therapy of basal ganglia neurodegenerative diseases [14]. Moreover, in PD mice model, ZNS provided an effective inhibition of monoamine oxidase B activity, which may contribute to the drug ability of improving clinical symptoms in PD patients [15]. The present study has been aimed at specifically investigating in differentiated SH-SY5Y neuronal like cells underlying mechanisms of ZNS effects against cell damage caused by prolonged cell exposure to rotenone.
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Experimental Procedures Materials The human neuroblastoma SH-SY5Y cell line (CRL-2266) was purchased from American Type Culture Collections (ATCC) (Rockville, Maryland, USA). Eagle’s Minimum Essential Medium (MEM), Ham’s F-12 Nutrient Mixture (F12), foetal bovine serum (FBS), penicillin/streptomycin mixture, all-trans retinoic acid (RA), sodium dodecyl sulphate (SDS), 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), 20 ,70 -dichlorofluorescein diacetate (H2DCFDA), dimethylsulfoxide (DMSO), glutamine, sodium pyruvate, phosphate buffered saline solution (PBS), the caspase-3 substrate Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-LeuLeu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-p-nitroaniline (DEVDpNa), propidium-iodide, and other chemicals of analytical grade were from Sigma (Milano, Italy). Zonisamide was from EISAI (London, UK). Rhodamine-123, fluorescein isothiocyanate-conjugated anti-Annexin V antibody and ECL Chemiluminescence detection kit were from GE Healthcare (Milan, Italy). Developer, fixer, and Kodak X-ray film were from Kodak (Milan, Italy). Cell Culture and Treatment Human neuroblastoma SH-SY5Y cells were maintained at 37 °C in a humidified incubator with 5 % CO2 and 95 % air, and cultured in MEM/F12 (1:1) medium containing 10 % (v/v) FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), 10 units/ml penicillin, and 100 mg/ml streptomycin. Sub-confluent cells were washed twice with PBS, then incubated in MEM/F12 medium containing 10 lM RA (10 mM in DMSO stock solution), 1 % FBS, L-glutamine (2 mM), sodium pyruvate (1 mM). The medium was renewed every 2 days. After 5 days of RA exposure, differentiated SH-SY5Y cells were incubated for 24 h with rotenone at indicated concentration, in the presence or absence of ZNS (10–250 lM) that was added to the culture medium 30 min prior to rotenone treatment. In a subset of experiments, rotenone effects were tested also in the presence or absence of 100 lM N-acetyl-cysteine (NAC), the well known antioxidant compound. Cell Viability Assay To assess rotenone adverse effects on cell viability, we evaluated the mitochondrial activity of living cells by a MTT quantitative colorimetric assay.
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For these assays, RA differentiated SH-SY5Y cells were cultured in 96-well culture plates. After treatments, cells were washed and incubated with fresh medium containing MTT (0.5 mg/ml) at 37 °C for 4 h. Then, insoluble formazan crystals were dissolved in 100 ll of a 10 % (w/v) SDS solution in HCl 0.01 M for 10 min. The optical density in each well was evaluated by spectrophotometrical measurement of absorbance at 570 nm using a microplate reader (Tecan Italia, Cologno Monzese, Italy). All experiments were performed in triplicate. Measurement of Intracellular ROS The amount of intracellular ROS was quantified by fluorescence with H2DCF-DA. At the end of each treatment, the cells, seeded at 2.5 9 105 cells/ml in 6-well plates, were incubated with 5 lM H2DCF-DA (dissolved in DMSO) for 30 min at 37 °C. For assay, cells were washed twice with PBS (pH 7.4), harvested with non-enzymatic cell dissociation solution, and resuspended in 500 ll of PBS supplemented with 0.1 M KH2PO4 and 0.5 % peroxide-free Triton X-100. After centrifugation at 13,000 rpm for 10 min, supernatants were analyzed under fluorescein optics, as previously described [16]. Measurement of Mitochondrial Transmembrane Potential (Dwm)
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of stained cells of each treatment were immediately analyzed by flow cytometry (FACSCanto II, Becton–Dickinson, San Jose, CA, USA). The difference between apoptosis and necrosis was detected by histogram analysis of the cell populations positive or negative for Annexin V-FITC only and from PI only. In particular, the viable cells are highlighted in the lower left quadrants of the cytograms, which exclude PI and are negative for Annexin V-FITC binding. The lower right-hand quadrants show the early apoptotic cells with bind Annexin V-FITC but exclude PI. Upper right-hand quadrant contain late apoptotic and necrotic cells, both Annexin V-FITC and PI positive. Necrotic cells, damaged during the cytotoxic treatments, are located in the up lefthand quadrants [18]. Caspase-3 Activity Assay The activity of caspase-3 was measured by a colorimetric assay using DEVD-pNa substrate, as previously described [16]. Briefly, aliquots from cell lysates were incubated at 37 °C for 60 min, and then DEVD-pNa cleavage was evaluated by measuring sample absorbance at 405 nm, using DU 800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA), and measurements were normalized by protein content. Statistical Analysis
Changes in mitochondrial transmembrane potential (Dwm) were assayed by the incorporation of a cationic fluorescent dye rhodamine 123 [17]. After treatments as previously described, the cells (2.5 9 105 cells/ml in 6-well plates) were changed to fresh medium containing 10 lM rhodamine 123 and incubated for 30 min at 37 °C in the dark. The cells were then collected, washed twice with PBS (pH 7.4), and the fluorescence intensity was analyzed at wavelength of 488-nm excitation and 525-nm emission by a microplate plate fluorescence reader Tecan Infinite F200 (Tecan Italia, Cologno Monzese, Italy). Apoptosis Assay Necrotic and apoptotic death in SH-SY5Y cells was detected with the use of double staining with Annexin V-FITC/propidium iodide (PI) according to the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, USA). After treatments as above described, cells were harvested with non-enzymatic cell dissociation solution and washed twice with cold PBS. The cell pellets were re-suspended in binding buffer (10 mM HEPES/NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 9 106 cells/ml. Then, cells were incubated with AnnexinV- FITC and PI for 15 min at room temperature in the dark. Ten thousand
All values are expressed as mean ± SEM. Statistical analysis was carried out by one-way ANOVA, followed by Newman-Keuls post hoc test. p values less than 0.05 were considered statistically significant.
Results ZNS Attenuates the Cytotoxic Effects of Rotenone According to previous studies [16], cell cultures were treated for 24 h with rotenone concentrations ranging between 50 and 500nM. The evaluation of cell viability by MTT assay showed that rotenone toxic effect was dosedependent, with the maximal toxicity (40 % of cell death compared with controls) displayed by a dose of 500 nM rotenone. Rotenone cytotoxicity was dose-dependently attenuated by ZNS (10–100 lM) that was added 30 min prior to rotenone treatment (F = 17.36; p \ 0.0001) (Fig. 1). In particular, the greatest protective effect was displayed by 100 lM ZNS that produced a cell viability recovery up to 80 %, as compared to the control (rotenone free) group, and the extent of beneficial effects was similar at higher
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Fig. 1 Effect of zonisamide against rotenone-induced reduction in cell viability. Cell viability was assayed by MTT assay. SH-SY5Y neuroblastoma cells were differentiated in presence of retinoic acid (10 lM) for 5 days. Differentiated cells were exposed to zonisamide only or to rotenone (500 nM) for 24 h, in presence or absence of zonisamide at indicated concentrations. Zonisamide was added 30 min before rotenone incubation. Data are the mean ± SEM from four independent experiments. Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to control; §§p \ 0.01 significant differences in comparison to rotenone-treated cultures
dose (i.e. 250 lM) (data not shown). At the employed concentrations, ZNS alone did not produce significant differences in comparison to controls (Fig. 1). ZNS Reduces ROS Generation and Mitochondrial Dysfunction Induced by Rotenone Treatment Rotenone treatment also increased ROS production by 70 % as compared to untreated control cells (p \ 0.001). When cell cultures were pre-incubated with ZNS, a dosedependent reduction of rotenone-induced ROS elevation was achieved in the presence of ZNS concentrations ranging from 50 to 100 lM (F = 34.33; p \ 0.0001, df = 5), while the lowest ZNS dose (10 lM) was not effective (Fig. 2a). The incubation with ZNS (100 lM) alone did not cause any significant changes in ROS production when compared to controls (data not reported). Given that alteration of the mitochondrial membrane potential (Dwm) is one of the earliest intracellular events following induction of cell damage [19], we also evaluated the extent of rotenone-induced impairment of mitochondrial functions in RA-differentiated SH-SY5Y cells. Cell treatment with rotenone (500 nM) produced a 35 % reduction in Dwm, evaluated by fluorimetric microplate assay (p \ 0.001). The pre-incubation of cell cultures with ZNS (10–100 lM) counteracted rotenone-induced Dwm
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Fig. 2 Effects of zonisamide on rotenone-induced ROS production (a) and mitochondrial membrane potential (b). ROS levels in differentiated SH-SY5Y cells treated with rotenone (500 nM) for 24 h in the presence or absence of zonisamide at indicated concentrations were determined by dichlorodihydrofluorescein diacetate reduction assay. Changes in Dwm were assessed by using cationic dye rhodamine-123. Cells were collected from culture plates and rhodamine-123 fluorescence was measured by using a fluorimetric plate reader. Data are the mean ± SEM from four separate experiments. Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to control; §p \ 0.05; §§p \ 0.01 and §§§p \ 0.001 significant differences in comparison to rotenone-treated cell cultures
decrease in a dose-dependent way, the almost complete recovery to basal Dwm being observed at the highest dose of ZNS (100 lM) (F = 15.43; p \ 0.0001; df = 5) (Fig. 2b). To further characterize the mechanism of ZNS protective effects on RA-differentiated SH-SY5Y cells, we evaluated the time-course of rotenone-induced changes in both ROS levels and Dwm, in the presence or absence of ZNS. Similarly, we tested the changes occurring in the presence or absence of NAC. As shown in Fig. 3a, both ZNS and NAC significantly reduced pro-oxidant effects of rotenone at 4 h from rotenone addition; moreover, both ZNS and NAC were able to reduce the alteration of Dwm at the same extent and protective effects against rotenone damage were significant after 12 h of incubation (Fig. 3b).
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cells; moreover, 4.8 ± 0.4 % of cells were in both early and late apoptosis, whereas 3.8 ± 0.3 % of cells were necrotic (Fig. 4a). Cell culture exposure to rotenone for 24 h increased up to 22.6 ± 2.1 % the number of early and lately apoptotic cells, and to 14.8 ± 1.3 % the number of necrotic ones (Fig. 4b). Notably, cell pre-incubation with 100 lM ZNS reduced by near 58 % the rotenone-increased total number of both early and lately apoptotic cells (22.6 ± 2.1 vs. 10.8 ± 1.1 %) (F = 50.08; p \ 0.0001,df = 2), while rotenone effect on necrotic cell number was reduced at a lower extent (14.8 ± 1.3 vs. 9.8 ± 0.8 %) (F = 44.81; p \ 0.0001,df = 2) (Fig. 4c). In Fig. 3d we show the number of cells undergoing apoptosis and necrosis after different treatments, expressed as percentage of whole cell number. To further investigate cell pathways involved in rotenone-induced apoptosis via mitochondrial dysfunction in RA-differentiated SH-SY5Y cells, we analyzed whether caspase-3 was acting as down-stream effector in our experimental conditions. As reported in Table 1, caspase-3 activity was increased by 230 % after 24 h treatment with 500 nM rotenone as compared with the control cells (p \ 0.001). The rotenone-induced increase in caspase-3 activity was reduced by 36 % in cells pre-treated with ZNS (100 lM) (F = 53.83; p \ 0.0001; df = 2).
Discussion
Fig. 3 Time course of ZNS (100 lM) and NAC (100 lM) effects on rotenone-induced ROS production (a) and Dwm (b) in RA-differentiated SH-SY5Y cells. For ROS production evaluation, dichlorofluorescein (DCF) signal was reported as percent of control values as described in ‘‘Materials and Methods’’. The changes in Dwm were assessed by using cationic dye rhodamine-123 and values were reported as percent of controls. The data are the mean from four experiments for SH-SY5Y cells treated with rotenone (500nM). Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to controls
Protective Effect of ZNS Against Rotenone-Induced Apoptosis We also performed a flow-cytometric counting of RAdifferentiated SH-SY5Y cells differentially stained with Annexin V-FITC and propidium iodide to evaluate the occurrence of apoptotic or necrotic features in both rotenone-exposed and rotenone-free cell cultures. Under our experimental conditions, RA-differentiated SH-SY5Y control cultures showed 91.4 ± 4.4 % of living
The inhibition of mitochondrial complex I causes bioenergetic defects and mitochondrial dysfunction, which is associated with the generation of oxidative stress. Indeed, much research efforts provided evidence that mitochondrial complex I activity is strongly reduced in the substantia nigra of PD brains, and that complex I deficiency persisting over the lifespan of a human being causally contributes to PD [20], although the molecular mechanisms are not clearly understood. Recent epidemiological studies have linked exposure to environmental agents, including pesticides, with an increased risk of developing PD [21]. As a result, over the last two decades the ‘‘environmental hypothesis’’ of PD has gained considerable interest. This speculates that agricultural chemicals in the environment, by producing selective dopaminergic cell death, can contribute to the development of the disease. However, a causal role for pesticides in the etiology of PD has yet to be definitively established. Importantly, most insights into PD pathogenesis came from investigations performed in experimental models of PD, especially those produced by neurotoxins [22]. The two most popular parkinsonian pesticide neurotoxins are namely paraquat and rotenone. Rotenone induces cell degeneration by inhibiting mitochondrial respiration at the level of complex I. The selective toxicity of rotenone is
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Fig. 4 Quantification of apoptosis after 24 h exposure to rotenone (500 nM) in the absence or presence of 100 lM zonisamide. Annexin V/propidium iodide staining was carried out on RAdifferentiated SH-SY5Y cells. Quantification (%) of cells undergoing apoptosis/necrosis was performed by flow cytometric analysis. Control cultures (a), rotenone-treated cells (b), rotenone-treated cells in presence of zonisamide (100 lM) (c). The percent of positive cells in the individual quadrants are reported (Q1, upper left panel, necrotic cells; Q2, upper right panel, late stage apoptosis; Q3, lower left, viable cells; Q4, lower right panel, early to mid state of apoptosis). d The histogram represents mean values (± SEM) of events for stained cells obtained from three independent experiments. *p \ 0.05 and ***p \ 0.001 significant differences in comparison to control; §§ p \ 0.01 and §§§p \ 0.001 significant differences in comparison to rotenone-treated cell cultures
especially relevant because, other than being a herbicide, it is also an insecticide widely used in several powders for delousing humans or animals. Notably, experimental evidence coming from in vitro and in vivo treatments with rotenone demonstrates that this toxin can be useful to mimic the biochemical mechanisms of neurodegeneration associated with mitochondrial impairment [21, 22]. In fact, rotenone other than targeting the dopaminergic system has also deleterious effects on other neuronal populations. Likewise, in PD in which neurodegeneration extends beyond the dopaminergic system, rotenone is associated
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with 35 % reduction in serotonin, 26 % in noradrenergic, and 29 % in cholinergic neurons [22]. In the present study, the effects of cell exposure to rotenone were tested using RA-differentiated SH-SY5Y neuroblastoma cells since this is a largely accepted in vitro model to investigate neuronal cell death [23–25]. The SHSY5Y is a human neuroblastoma cell line widely used as neuronal-like cells which express dopaminergic markers so that they are widely used in PD research [26–28]. In our experimental approach, the differentiation of SHSY5Y cells was carried out by incubation with RA alone.
Neurochem Res (2013) 38:2631–2639 Table 1 Effects of zonisamide (100 lM) on rotenone-induced caspase-3 activity in RA-differentiated SH-SY5Y neuroblastoma cells Treatment
Caspase-3 activity (pmol pNA released/min/mg)
None
15.11 ± 0.9
Rotenone (500 nM)
34.95 ± 1.86***
Rotenone (500 nM)? ZNS (100 lM)
22.38 ± 1.23**,§§§
The evaluation of caspase-3 activity was carried out based on measurements of specific cleavage of DEVD-pNa as described in Materials and methods. Results are mean ± SEM of four independent experiments. ** p \ 0.01 and *** p \ 0.001 significant differences in comparison to control; §§§ p \ 0.0001 significant differences in comparison to rotenone-treated cultures
However, it has been shown that the bioenergetic profile of RA-differentiated SH-SY5Y cells possesses most of the hallmarks of neurons, including more antioxidant defenses in comparison to undifferentiated ones [25]. Previous observations suggest that apoptosis induced by mitochondrial toxins is an active process that requires a trigger for initiation and then other biochemical events take place to stop it [29]. NAD(P)H oxidase serves as a source of O2- generation [30, 31]. Rotenone blocks NADH dehydrogenase of complex I [32], an effect followed by an increase in NADH. Noteworthy, in mitochondrial preparations supplemented with NADH rotenone was able to stimulate O2production by the NADH dehydrogenase [33]. Indeed, rotenone has been shown to induce apoptosis through a dose-dependent ROS generation, change in Dwm, caspase-3 activation and DNA ladder formation [34, 35]. Consequently, one may expect that free radical scavengers inhibit rotenone-induced apoptosis. In the present study, we observed a scavenger effect of ZNS against rotenone-evoked ROS level increase. Our data support the possibility that ZNS also exerts an antioxidant activity, at micromolar concentrations. Additionally, ZNS has been suggested to prevent dopamine quinone formation induced by excessive amount of cytosolic dopamine outside the synaptic vesicles [36], and reduce neuronal cell death induced by MPP(?) via an anti-apoptotic effect and by up-regulating manganese superoxide dismutase levels [37]. More recently, it has been shown that ZNS reduces the lipid peroxide and cytosolic free Ca(2?) levels increased by MPP(?) group, and counteracts the MPP(?)induced decrease of reduced glutathione and glutathione peroxidase levels [38]. Given these reported observations, stimulating effects of ZNS on the expression and activity of either cellular antioxidant enzymes or molecules in our experimental setting cannot be excluded. In cell models of PD in which inhibition of complex I of electron transport chain occurs, ROS production and
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mitochondrial potential have been considered temporally correlated with irreversibility of the apoptotic machinery [29]. In particular, it is thought that ROS generation precedes and is causal to changes in Dwm in rotenone-induced cell damage. Several findings demonstrate the loss of mitochondrial Dw is an early event in cell committed to die by apoptosis [39, 40]. Under our experimental conditions, it was shown that increases in ROS production is associated with changes in Dwm producing both apoptotic and necrotic cell damage. Our data give evidence for an early antioxidant effect produced by ZNS, similar to that displayed by a conventional antioxidant such as NAC. Therefore, the findings described in this study suggest that the mechanism of rotenone neurotoxicity may involve oxidative stress, and ZNS may act on reactive oxygen species to inhibit apoptosis by their antioxidant properties. The observations on Dwm changes further extend the antioxidant mechanism of ZNS. Remarkably, ZNS treatment reduced the number of apoptotic cells and caspase 3 activity, the well known executor of apoptosis. Therefore, ZNS might scavenge oxygen radicals produced by rotenone-induced complex I inhibition [41, 42] leading to oxidative damage of membrane lipids and proteins. On the other hand, ZNS might stabilize Dwm and this could reduce alterations occurring in the mitochondrial membrane. Stabilization of Dwm could play a crucial role in counteracting seizure activity. In this context, it has been shown that ZNS is able to block both Na(?) channels and Ca(2?) channels, thus resulting more effective in attenuating seizure-induced hippocampal neurodegeneration [43]. Noteworthy, the Ca(2?) channel blockers have been clinically used and ZNS has been shown as an emerging therapeutic tool for tremor disorders [44]. The results of the present study may be considered as suggestive of neuroprotective effects of ZNS on neuronal damage associated with oxidative stress. ZNS has been shown to exert beneficial effects in a variety of experimental models [45–47]. Particularly, the major evidence in favour of ZNS neuroprotective effects come from PD experimental models [37, 48, 49]. Previous studies demonstrated that ZNS significantly reduces MPTP-induced neurotoxicity in mice by enhancing antioxidant effects [38, 50]. Additionally, it has been shown that high ZNS doses reduced striatal neuronal excitability while lower concentrations protected the striatum against mitochondrial impairment [14]. In the present study, we have demonstrated a direct pharmacological effects on neuron-like cells. In line with other results [14, 37], we have also shown that ZNS reduces apoptosis triggered by mitochondrial damage. Interestingly, ZNS has shown a neuroprotective action in PD patients [51, 52]. Moreover, an adjunctive treatment with low dose of ZNS to levodopa has induced
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improvement of all the cardinal symptoms of PD [52]. ZNS has shown to exert beneficial effects in patients with Parkinson’s disease, especially against motor function and impulse control [46, 53]. In conclusion, further characterization of ZNS properties could reveal a neuroprotective and hence disease-slowing agent in the neurodegenerative disorders. Acknowledgments This research received no Grant from any funding agency in the public, commercial or not-for-profit sectors. Conflict of interest interest.
The Authors declare that there is no conflict of
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ORIGINAL PAPER
Protective Effects of Zonisamide Against Rotenone-Induced Neurotoxicity Salvatore Condello • Monica Curro` • Nadia Ferlazzo • Gregorio Costa • Giuseppa Visalli • Daniela Caccamo • Laura Rosa Pisani • Cinzia Costa Paolo Calabresi • Riccardo Ientile • Francesco Pisani
•
Received: 12 August 2013 / Revised: 3 October 2013 / Accepted: 14 October 2013 / Published online: 20 October 2013 Ó Springer Science+Business Media New York 2013
Abstract Zonisamide (ZNS), an antiepileptic drug having beneficial effects also against Parkinson’s disease symptoms, has proven to display an antioxidant effects in different experimental models. In the present study, the effects of ZNS on rotenone-induced cell injury were investigated in human neuroblastoma SH-SY5Y cells differentiated towards a neuronal phenotype. Cell cultures were exposed for 24 h to 500 nM rotenone with or without pre-treatment with 10–100 lM ZNS. Then, the following parameters were analyzed: (a) cell viability; (b) intracellular reactive oxygen species production; (c) mitochondrial Salvatore Condello and Monica Curro` have contributed equally to this study. S. Condello M. Curro` N. Ferlazzo G. Visalli D. Caccamo R. Ientile (&) Department of Biomedical Sciences and Morphological and Functional Imaging, University of Messina, AOU Policlinico ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy e-mail: [email protected] G. Costa Department of Human Pathology, Laboratory of Immunology and Biotherapy, University of Messina, AOU ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy L. R. Pisani F. Pisani Department of Neurosciences, Neurology Clinic, University of Messina, AOU ‘‘G. Martino’’, Via C. Valeria, 98125 Messina, Italy C. Costa P. Calabresi Neurology Clinic, Ospedale Santa Maria Della Misericordia, University of Perugia, Via Santa Andrea Delle Fratte, San Sisto, 06158 Perugia, Italy C. Costa P. Calabresi IRCCS Fondazione S. Lucia, Via Ardeatina 306, 00179 Rome, Italy
transmembrane potential; (d) cell necrosis and apoptosis; (e) caspase-3 activity. ZNS dose-dependently suppressed rotenone-induced cell damage through a decrease in intracellular ROS production, and restoring mitochondrial membrane potential. Similarly to ZNS effects, the treatment with N-acetyl-cysteine (100 lM) displayed significant protective effects against rotenone-induced ROS production and Dwm at 4 and 12 h respectively, reaching the maximal extent at 24 h. Additionally, ZNS displayed antiapoptotic effects, as demonstrated by flow cytometric analysis of annexin V/propidium iodide double staining, and significant attenuated rotenone-increased caspase 3 activity. On the whole, these findings suggest that ZNS preserves mitochondrial functions and counteracts apoptotic signalling mechanisms mainly by an antioxidant action. Thus, ZNS might have beneficial effect against neuronal cell degeneration in different experimental models involving mitochondrial dysfunction. Keywords Zonisamide Neuroprotection Oxidative stress Mitochondrial impairment Apoptosis
Introduction Glutamate-mediated excitotoxicity and related oxidative stress have been reported to be among the main triggers of neuronal cell death observed in neurological disorders, i.e. epilepsy, amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease (PD) [1]. In particular, a major role for oxidative stress has been postulated in PD, based on the evidence of iron accumulation in the substantia nigra as well as increased superoxide dismutase activity, glutathione depletion, and
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oxidative damage of lipids and DNA in dopaminergic neurons [2–5]. The oxidative stress, likely resulting from impaired mitochondrial functions, has been involved in the pathogenesis of neurodegenerative diseases. The complex I is the first protein component of the mitochondrial respiratory chain and plays a crucial role in ATP production and mitochondrial function in general. Mitochondrial toxins selectively targeting complex I, such as 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine, or rotenone, are capable of producing neuronal cell death and have been widely used to produce in vitro and in vivo models of PD [6]. In particular, due to its ability to freely cross biological membranes, independently from transporters, rotenone produces a strong inhibition of mitochondrial complex I [7] which increases the rate of mitochondrial reactive oxygen species (ROS) release, leading to cell apoptosis [8, 9]. These observations strongly indicate that mitochondrial dysfunction underlying the massive cell death could be related to deficiency of mitochondrial complex I. Advances in understanding the pathogenesis of mitochondrial complex I dysfunction likely involved in neurodegeneration may allow the development of new therapeutic approaches to management of neurological dysfunctions. Interestingly, previous experimental studies have shown that some antiepileptic drugs, such as carbamazepine or the newly designed zonisamide (ZNS), could act as neuroprotective agents. The carbamazepine and ZNS neuroprotective potential has been tested in an in vitro model of neurotoxicity triggered by acute administration of rotenone to corticostriatal brain slices. In this regard, carbamazepine strongly prevented rotenone-induced striatal neuronal dysfunction by reducing membrane depolarization [10], while ZNS effects could also be mediated by inhibition of voltage-dependent Na? and T-type Ca(2?) channels, reduction of depolarizationinduced glutamate release, enhanced GABAergic and monoaminergic transmission [11–13]. Indeed, ZNS at micromolar concentrations was able to protect striatum against mitochondrial impairment suggesting its possible use in the therapy of basal ganglia neurodegenerative diseases [14]. Moreover, in PD mice model, ZNS provided an effective inhibition of monoamine oxidase B activity, which may contribute to the drug ability of improving clinical symptoms in PD patients [15]. The present study has been aimed at specifically investigating in differentiated SH-SY5Y neuronal like cells underlying mechanisms of ZNS effects against cell damage caused by prolonged cell exposure to rotenone.
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Experimental Procedures Materials The human neuroblastoma SH-SY5Y cell line (CRL-2266) was purchased from American Type Culture Collections (ATCC) (Rockville, Maryland, USA). Eagle’s Minimum Essential Medium (MEM), Ham’s F-12 Nutrient Mixture (F12), foetal bovine serum (FBS), penicillin/streptomycin mixture, all-trans retinoic acid (RA), sodium dodecyl sulphate (SDS), 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), 20 ,70 -dichlorofluorescein diacetate (H2DCFDA), dimethylsulfoxide (DMSO), glutamine, sodium pyruvate, phosphate buffered saline solution (PBS), the caspase-3 substrate Ac-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-LeuLeu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-p-nitroaniline (DEVDpNa), propidium-iodide, and other chemicals of analytical grade were from Sigma (Milano, Italy). Zonisamide was from EISAI (London, UK). Rhodamine-123, fluorescein isothiocyanate-conjugated anti-Annexin V antibody and ECL Chemiluminescence detection kit were from GE Healthcare (Milan, Italy). Developer, fixer, and Kodak X-ray film were from Kodak (Milan, Italy). Cell Culture and Treatment Human neuroblastoma SH-SY5Y cells were maintained at 37 °C in a humidified incubator with 5 % CO2 and 95 % air, and cultured in MEM/F12 (1:1) medium containing 10 % (v/v) FBS, L-glutamine (2 mM), sodium pyruvate (1 mM), 10 units/ml penicillin, and 100 mg/ml streptomycin. Sub-confluent cells were washed twice with PBS, then incubated in MEM/F12 medium containing 10 lM RA (10 mM in DMSO stock solution), 1 % FBS, L-glutamine (2 mM), sodium pyruvate (1 mM). The medium was renewed every 2 days. After 5 days of RA exposure, differentiated SH-SY5Y cells were incubated for 24 h with rotenone at indicated concentration, in the presence or absence of ZNS (10–250 lM) that was added to the culture medium 30 min prior to rotenone treatment. In a subset of experiments, rotenone effects were tested also in the presence or absence of 100 lM N-acetyl-cysteine (NAC), the well known antioxidant compound. Cell Viability Assay To assess rotenone adverse effects on cell viability, we evaluated the mitochondrial activity of living cells by a MTT quantitative colorimetric assay.
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For these assays, RA differentiated SH-SY5Y cells were cultured in 96-well culture plates. After treatments, cells were washed and incubated with fresh medium containing MTT (0.5 mg/ml) at 37 °C for 4 h. Then, insoluble formazan crystals were dissolved in 100 ll of a 10 % (w/v) SDS solution in HCl 0.01 M for 10 min. The optical density in each well was evaluated by spectrophotometrical measurement of absorbance at 570 nm using a microplate reader (Tecan Italia, Cologno Monzese, Italy). All experiments were performed in triplicate. Measurement of Intracellular ROS The amount of intracellular ROS was quantified by fluorescence with H2DCF-DA. At the end of each treatment, the cells, seeded at 2.5 9 105 cells/ml in 6-well plates, were incubated with 5 lM H2DCF-DA (dissolved in DMSO) for 30 min at 37 °C. For assay, cells were washed twice with PBS (pH 7.4), harvested with non-enzymatic cell dissociation solution, and resuspended in 500 ll of PBS supplemented with 0.1 M KH2PO4 and 0.5 % peroxide-free Triton X-100. After centrifugation at 13,000 rpm for 10 min, supernatants were analyzed under fluorescein optics, as previously described [16]. Measurement of Mitochondrial Transmembrane Potential (Dwm)
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of stained cells of each treatment were immediately analyzed by flow cytometry (FACSCanto II, Becton–Dickinson, San Jose, CA, USA). The difference between apoptosis and necrosis was detected by histogram analysis of the cell populations positive or negative for Annexin V-FITC only and from PI only. In particular, the viable cells are highlighted in the lower left quadrants of the cytograms, which exclude PI and are negative for Annexin V-FITC binding. The lower right-hand quadrants show the early apoptotic cells with bind Annexin V-FITC but exclude PI. Upper right-hand quadrant contain late apoptotic and necrotic cells, both Annexin V-FITC and PI positive. Necrotic cells, damaged during the cytotoxic treatments, are located in the up lefthand quadrants [18]. Caspase-3 Activity Assay The activity of caspase-3 was measured by a colorimetric assay using DEVD-pNa substrate, as previously described [16]. Briefly, aliquots from cell lysates were incubated at 37 °C for 60 min, and then DEVD-pNa cleavage was evaluated by measuring sample absorbance at 405 nm, using DU 800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA), and measurements were normalized by protein content. Statistical Analysis
Changes in mitochondrial transmembrane potential (Dwm) were assayed by the incorporation of a cationic fluorescent dye rhodamine 123 [17]. After treatments as previously described, the cells (2.5 9 105 cells/ml in 6-well plates) were changed to fresh medium containing 10 lM rhodamine 123 and incubated for 30 min at 37 °C in the dark. The cells were then collected, washed twice with PBS (pH 7.4), and the fluorescence intensity was analyzed at wavelength of 488-nm excitation and 525-nm emission by a microplate plate fluorescence reader Tecan Infinite F200 (Tecan Italia, Cologno Monzese, Italy). Apoptosis Assay Necrotic and apoptotic death in SH-SY5Y cells was detected with the use of double staining with Annexin V-FITC/propidium iodide (PI) according to the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, USA). After treatments as above described, cells were harvested with non-enzymatic cell dissociation solution and washed twice with cold PBS. The cell pellets were re-suspended in binding buffer (10 mM HEPES/NaOH, pH7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 9 106 cells/ml. Then, cells were incubated with AnnexinV- FITC and PI for 15 min at room temperature in the dark. Ten thousand
All values are expressed as mean ± SEM. Statistical analysis was carried out by one-way ANOVA, followed by Newman-Keuls post hoc test. p values less than 0.05 were considered statistically significant.
Results ZNS Attenuates the Cytotoxic Effects of Rotenone According to previous studies [16], cell cultures were treated for 24 h with rotenone concentrations ranging between 50 and 500nM. The evaluation of cell viability by MTT assay showed that rotenone toxic effect was dosedependent, with the maximal toxicity (40 % of cell death compared with controls) displayed by a dose of 500 nM rotenone. Rotenone cytotoxicity was dose-dependently attenuated by ZNS (10–100 lM) that was added 30 min prior to rotenone treatment (F = 17.36; p \ 0.0001) (Fig. 1). In particular, the greatest protective effect was displayed by 100 lM ZNS that produced a cell viability recovery up to 80 %, as compared to the control (rotenone free) group, and the extent of beneficial effects was similar at higher
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Fig. 1 Effect of zonisamide against rotenone-induced reduction in cell viability. Cell viability was assayed by MTT assay. SH-SY5Y neuroblastoma cells were differentiated in presence of retinoic acid (10 lM) for 5 days. Differentiated cells were exposed to zonisamide only or to rotenone (500 nM) for 24 h, in presence or absence of zonisamide at indicated concentrations. Zonisamide was added 30 min before rotenone incubation. Data are the mean ± SEM from four independent experiments. Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to control; §§p \ 0.01 significant differences in comparison to rotenone-treated cultures
dose (i.e. 250 lM) (data not shown). At the employed concentrations, ZNS alone did not produce significant differences in comparison to controls (Fig. 1). ZNS Reduces ROS Generation and Mitochondrial Dysfunction Induced by Rotenone Treatment Rotenone treatment also increased ROS production by 70 % as compared to untreated control cells (p \ 0.001). When cell cultures were pre-incubated with ZNS, a dosedependent reduction of rotenone-induced ROS elevation was achieved in the presence of ZNS concentrations ranging from 50 to 100 lM (F = 34.33; p \ 0.0001, df = 5), while the lowest ZNS dose (10 lM) was not effective (Fig. 2a). The incubation with ZNS (100 lM) alone did not cause any significant changes in ROS production when compared to controls (data not reported). Given that alteration of the mitochondrial membrane potential (Dwm) is one of the earliest intracellular events following induction of cell damage [19], we also evaluated the extent of rotenone-induced impairment of mitochondrial functions in RA-differentiated SH-SY5Y cells. Cell treatment with rotenone (500 nM) produced a 35 % reduction in Dwm, evaluated by fluorimetric microplate assay (p \ 0.001). The pre-incubation of cell cultures with ZNS (10–100 lM) counteracted rotenone-induced Dwm
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Fig. 2 Effects of zonisamide on rotenone-induced ROS production (a) and mitochondrial membrane potential (b). ROS levels in differentiated SH-SY5Y cells treated with rotenone (500 nM) for 24 h in the presence or absence of zonisamide at indicated concentrations were determined by dichlorodihydrofluorescein diacetate reduction assay. Changes in Dwm were assessed by using cationic dye rhodamine-123. Cells were collected from culture plates and rhodamine-123 fluorescence was measured by using a fluorimetric plate reader. Data are the mean ± SEM from four separate experiments. Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to control; §p \ 0.05; §§p \ 0.01 and §§§p \ 0.001 significant differences in comparison to rotenone-treated cell cultures
decrease in a dose-dependent way, the almost complete recovery to basal Dwm being observed at the highest dose of ZNS (100 lM) (F = 15.43; p \ 0.0001; df = 5) (Fig. 2b). To further characterize the mechanism of ZNS protective effects on RA-differentiated SH-SY5Y cells, we evaluated the time-course of rotenone-induced changes in both ROS levels and Dwm, in the presence or absence of ZNS. Similarly, we tested the changes occurring in the presence or absence of NAC. As shown in Fig. 3a, both ZNS and NAC significantly reduced pro-oxidant effects of rotenone at 4 h from rotenone addition; moreover, both ZNS and NAC were able to reduce the alteration of Dwm at the same extent and protective effects against rotenone damage were significant after 12 h of incubation (Fig. 3b).
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cells; moreover, 4.8 ± 0.4 % of cells were in both early and late apoptosis, whereas 3.8 ± 0.3 % of cells were necrotic (Fig. 4a). Cell culture exposure to rotenone for 24 h increased up to 22.6 ± 2.1 % the number of early and lately apoptotic cells, and to 14.8 ± 1.3 % the number of necrotic ones (Fig. 4b). Notably, cell pre-incubation with 100 lM ZNS reduced by near 58 % the rotenone-increased total number of both early and lately apoptotic cells (22.6 ± 2.1 vs. 10.8 ± 1.1 %) (F = 50.08; p \ 0.0001,df = 2), while rotenone effect on necrotic cell number was reduced at a lower extent (14.8 ± 1.3 vs. 9.8 ± 0.8 %) (F = 44.81; p \ 0.0001,df = 2) (Fig. 4c). In Fig. 3d we show the number of cells undergoing apoptosis and necrosis after different treatments, expressed as percentage of whole cell number. To further investigate cell pathways involved in rotenone-induced apoptosis via mitochondrial dysfunction in RA-differentiated SH-SY5Y cells, we analyzed whether caspase-3 was acting as down-stream effector in our experimental conditions. As reported in Table 1, caspase-3 activity was increased by 230 % after 24 h treatment with 500 nM rotenone as compared with the control cells (p \ 0.001). The rotenone-induced increase in caspase-3 activity was reduced by 36 % in cells pre-treated with ZNS (100 lM) (F = 53.83; p \ 0.0001; df = 2).
Discussion
Fig. 3 Time course of ZNS (100 lM) and NAC (100 lM) effects on rotenone-induced ROS production (a) and Dwm (b) in RA-differentiated SH-SY5Y cells. For ROS production evaluation, dichlorofluorescein (DCF) signal was reported as percent of control values as described in ‘‘Materials and Methods’’. The changes in Dwm were assessed by using cationic dye rhodamine-123 and values were reported as percent of controls. The data are the mean from four experiments for SH-SY5Y cells treated with rotenone (500nM). Statistically significant differences were evaluated by one-way ANOVA, followed by Newman-Keuls post hoc test. *p \ 0.05; **p \ 0.01 and ***p \ 0.001 significant differences in comparison to controls
Protective Effect of ZNS Against Rotenone-Induced Apoptosis We also performed a flow-cytometric counting of RAdifferentiated SH-SY5Y cells differentially stained with Annexin V-FITC and propidium iodide to evaluate the occurrence of apoptotic or necrotic features in both rotenone-exposed and rotenone-free cell cultures. Under our experimental conditions, RA-differentiated SH-SY5Y control cultures showed 91.4 ± 4.4 % of living
The inhibition of mitochondrial complex I causes bioenergetic defects and mitochondrial dysfunction, which is associated with the generation of oxidative stress. Indeed, much research efforts provided evidence that mitochondrial complex I activity is strongly reduced in the substantia nigra of PD brains, and that complex I deficiency persisting over the lifespan of a human being causally contributes to PD [20], although the molecular mechanisms are not clearly understood. Recent epidemiological studies have linked exposure to environmental agents, including pesticides, with an increased risk of developing PD [21]. As a result, over the last two decades the ‘‘environmental hypothesis’’ of PD has gained considerable interest. This speculates that agricultural chemicals in the environment, by producing selective dopaminergic cell death, can contribute to the development of the disease. However, a causal role for pesticides in the etiology of PD has yet to be definitively established. Importantly, most insights into PD pathogenesis came from investigations performed in experimental models of PD, especially those produced by neurotoxins [22]. The two most popular parkinsonian pesticide neurotoxins are namely paraquat and rotenone. Rotenone induces cell degeneration by inhibiting mitochondrial respiration at the level of complex I. The selective toxicity of rotenone is
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Fig. 4 Quantification of apoptosis after 24 h exposure to rotenone (500 nM) in the absence or presence of 100 lM zonisamide. Annexin V/propidium iodide staining was carried out on RAdifferentiated SH-SY5Y cells. Quantification (%) of cells undergoing apoptosis/necrosis was performed by flow cytometric analysis. Control cultures (a), rotenone-treated cells (b), rotenone-treated cells in presence of zonisamide (100 lM) (c). The percent of positive cells in the individual quadrants are reported (Q1, upper left panel, necrotic cells; Q2, upper right panel, late stage apoptosis; Q3, lower left, viable cells; Q4, lower right panel, early to mid state of apoptosis). d The histogram represents mean values (± SEM) of events for stained cells obtained from three independent experiments. *p \ 0.05 and ***p \ 0.001 significant differences in comparison to control; §§ p \ 0.01 and §§§p \ 0.001 significant differences in comparison to rotenone-treated cell cultures
especially relevant because, other than being a herbicide, it is also an insecticide widely used in several powders for delousing humans or animals. Notably, experimental evidence coming from in vitro and in vivo treatments with rotenone demonstrates that this toxin can be useful to mimic the biochemical mechanisms of neurodegeneration associated with mitochondrial impairment [21, 22]. In fact, rotenone other than targeting the dopaminergic system has also deleterious effects on other neuronal populations. Likewise, in PD in which neurodegeneration extends beyond the dopaminergic system, rotenone is associated
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with 35 % reduction in serotonin, 26 % in noradrenergic, and 29 % in cholinergic neurons [22]. In the present study, the effects of cell exposure to rotenone were tested using RA-differentiated SH-SY5Y neuroblastoma cells since this is a largely accepted in vitro model to investigate neuronal cell death [23–25]. The SHSY5Y is a human neuroblastoma cell line widely used as neuronal-like cells which express dopaminergic markers so that they are widely used in PD research [26–28]. In our experimental approach, the differentiation of SHSY5Y cells was carried out by incubation with RA alone.
Neurochem Res (2013) 38:2631–2639 Table 1 Effects of zonisamide (100 lM) on rotenone-induced caspase-3 activity in RA-differentiated SH-SY5Y neuroblastoma cells Treatment
Caspase-3 activity (pmol pNA released/min/mg)
None
15.11 ± 0.9
Rotenone (500 nM)
34.95 ± 1.86***
Rotenone (500 nM)? ZNS (100 lM)
22.38 ± 1.23**,§§§
The evaluation of caspase-3 activity was carried out based on measurements of specific cleavage of DEVD-pNa as described in Materials and methods. Results are mean ± SEM of four independent experiments. ** p \ 0.01 and *** p \ 0.001 significant differences in comparison to control; §§§ p \ 0.0001 significant differences in comparison to rotenone-treated cultures
However, it has been shown that the bioenergetic profile of RA-differentiated SH-SY5Y cells possesses most of the hallmarks of neurons, including more antioxidant defenses in comparison to undifferentiated ones [25]. Previous observations suggest that apoptosis induced by mitochondrial toxins is an active process that requires a trigger for initiation and then other biochemical events take place to stop it [29]. NAD(P)H oxidase serves as a source of O2- generation [30, 31]. Rotenone blocks NADH dehydrogenase of complex I [32], an effect followed by an increase in NADH. Noteworthy, in mitochondrial preparations supplemented with NADH rotenone was able to stimulate O2production by the NADH dehydrogenase [33]. Indeed, rotenone has been shown to induce apoptosis through a dose-dependent ROS generation, change in Dwm, caspase-3 activation and DNA ladder formation [34, 35]. Consequently, one may expect that free radical scavengers inhibit rotenone-induced apoptosis. In the present study, we observed a scavenger effect of ZNS against rotenone-evoked ROS level increase. Our data support the possibility that ZNS also exerts an antioxidant activity, at micromolar concentrations. Additionally, ZNS has been suggested to prevent dopamine quinone formation induced by excessive amount of cytosolic dopamine outside the synaptic vesicles [36], and reduce neuronal cell death induced by MPP(?) via an anti-apoptotic effect and by up-regulating manganese superoxide dismutase levels [37]. More recently, it has been shown that ZNS reduces the lipid peroxide and cytosolic free Ca(2?) levels increased by MPP(?) group, and counteracts the MPP(?)induced decrease of reduced glutathione and glutathione peroxidase levels [38]. Given these reported observations, stimulating effects of ZNS on the expression and activity of either cellular antioxidant enzymes or molecules in our experimental setting cannot be excluded. In cell models of PD in which inhibition of complex I of electron transport chain occurs, ROS production and
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mitochondrial potential have been considered temporally correlated with irreversibility of the apoptotic machinery [29]. In particular, it is thought that ROS generation precedes and is causal to changes in Dwm in rotenone-induced cell damage. Several findings demonstrate the loss of mitochondrial Dw is an early event in cell committed to die by apoptosis [39, 40]. Under our experimental conditions, it was shown that increases in ROS production is associated with changes in Dwm producing both apoptotic and necrotic cell damage. Our data give evidence for an early antioxidant effect produced by ZNS, similar to that displayed by a conventional antioxidant such as NAC. Therefore, the findings described in this study suggest that the mechanism of rotenone neurotoxicity may involve oxidative stress, and ZNS may act on reactive oxygen species to inhibit apoptosis by their antioxidant properties. The observations on Dwm changes further extend the antioxidant mechanism of ZNS. Remarkably, ZNS treatment reduced the number of apoptotic cells and caspase 3 activity, the well known executor of apoptosis. Therefore, ZNS might scavenge oxygen radicals produced by rotenone-induced complex I inhibition [41, 42] leading to oxidative damage of membrane lipids and proteins. On the other hand, ZNS might stabilize Dwm and this could reduce alterations occurring in the mitochondrial membrane. Stabilization of Dwm could play a crucial role in counteracting seizure activity. In this context, it has been shown that ZNS is able to block both Na(?) channels and Ca(2?) channels, thus resulting more effective in attenuating seizure-induced hippocampal neurodegeneration [43]. Noteworthy, the Ca(2?) channel blockers have been clinically used and ZNS has been shown as an emerging therapeutic tool for tremor disorders [44]. The results of the present study may be considered as suggestive of neuroprotective effects of ZNS on neuronal damage associated with oxidative stress. ZNS has been shown to exert beneficial effects in a variety of experimental models [45–47]. Particularly, the major evidence in favour of ZNS neuroprotective effects come from PD experimental models [37, 48, 49]. Previous studies demonstrated that ZNS significantly reduces MPTP-induced neurotoxicity in mice by enhancing antioxidant effects [38, 50]. Additionally, it has been shown that high ZNS doses reduced striatal neuronal excitability while lower concentrations protected the striatum against mitochondrial impairment [14]. In the present study, we have demonstrated a direct pharmacological effects on neuron-like cells. In line with other results [14, 37], we have also shown that ZNS reduces apoptosis triggered by mitochondrial damage. Interestingly, ZNS has shown a neuroprotective action in PD patients [51, 52]. Moreover, an adjunctive treatment with low dose of ZNS to levodopa has induced
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improvement of all the cardinal symptoms of PD [52]. ZNS has shown to exert beneficial effects in patients with Parkinson’s disease, especially against motor function and impulse control [46, 53]. In conclusion, further characterization of ZNS properties could reveal a neuroprotective and hence disease-slowing agent in the neurodegenerative disorders. Acknowledgments This research received no Grant from any funding agency in the public, commercial or not-for-profit sectors. Conflict of interest interest.
The Authors declare that there is no conflict of
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