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Contents lists available at ScienceDirect
NeuroToxicology
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The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2
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Aguirre-Vidal a, Sergio Montes b, Luis Tristan-Lo´pez b, Laura Anaya-Ramos a, John Teiber , Camilo Rı´os b,d, Vero´nica Baron-Flores d, Antonio Monroy-Noyola a,*
Q1 Yoshajandith c a
Laboratorio de Neuroproteccio´n, Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Mexico Departamento de Neuroquı´mica, Instituto Nacional de Neurologı´a y Neurocirugı´a, M.V.S., D.F., Mexico Division of Epidemiology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA d Q2 Laboratorio de Neurofarmacologia Molecular, Departamento de Sistemas Biolo´gicos, Universidad Auto´noma Metropolitana-Xochimilco, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 December 2014 Accepted 24 March 2015 Available online xxx
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta with subsequent striatal dopamine (DA) deficiency and increased lipid peroxidation. The etiology of the disease is still unclear and it is thought that PD may be caused by a combination of genetic and environmental factors. In the search of new pharmacological options, statins have been recognized for their potential application to treat PD, due to their antioxidant effect. The aim of this work is to contribute in the characterization of the neuroprotective effect of lovastatin in a model of PD induced by 1-methyl-4-phenylpyridinium (MPP+). Male Wistar rats (200–250 g) were randomly allocated into 4 groups and administered for 7 days with different pharmacological treatments. Lovastatin administration (5 mg/kg) diminished 40% of the amphetamine-induced circling behavior, prevented the striatal DA depletion and lipid peroxides formation by MPP+ intrastriatal injection, as compared to the group of animals treated only with MPP+. Lovastatin produced no change in paraoxonase-2 (PON2) activity. It is evident that lovastatin conferred neuroprotection against MPP+-induced protection but this effect was not associated with the induction of PON2 in the rat striatum. ß 2015 Published by Elsevier Inc.
Keywords: Parkinson Statin Lipoperoxidation Dopamine Lovastatin Q4 Paraoxonase-2
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1. Introduction Q5
Parkinson disease (PD) is an important neurodegenerative disorder; affecting between 0.5 and 1% of the population aged 65– 69 years of age, and increasing to 1–3% of the population over 80 years of age (Toulouse and Sullivan, 2008). PD is biochemically characterized by a decrease of dopamine (DA) levels, caused by the death of dopaminergic neurons in the substantia nigra pars
Abbreviations: MPP+, 1-methyl-4-phenylpyridinium; PD, Parkinson’s disease; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine; MAO-B, monoamine oxidase B; DA, dopamine; ROS, reactive oxidant species; PON2, paraoxonase-2; HVA, homovanillic acid; DOPAC, 3,4-dihydroxyphenylacetic acid; DMSO, dimethylsulfoxide; HPLC, high performance liquid chromatography; PL, lipidperoxides; CNS, central nervous system; FU, fluorescence units; DNA, deoxyribonucleic acid. * Corresponding author at: Laboratorio de Neuroproteccio´n, Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Av. Universidad 1001 Col. Q3 Chamilpa, CP 62209 Cuernavaca, Morelos, Mexico. Tel.: +52 7773297089. E-mail address: [email protected] (A. Monroy-Noyola).
compacta (SNpc). Dopamine is the neurotransmitter involved in the control of extrapyramidal motor function of the nigrostriatal pathway. As this pathway is injured in PD, dopamine depletion is the cause of the remarkable motor symptoms of the disease; including tremor, rigidity, bradykinesia and abnormal posture (Toulouse and Sullivan, 2008). The neurodegenerative process of the nigrostriatal pathway in PD has been related to oxidative stress and mitochondrial dysfunction. Oxidative stress initiates chain reactions leading to lipid peroxidation, therefore decreases unsaturated fatty acids content in cell membranes. This change affects membrane properties and enzyme activities, receptor functions and DA oxidation, increasing apoptosis and other cell mechanisms of death (Eunsung and Mouradian, 2001). Nowadays, there are several treatments for the symptoms, but the beneficial effects are reduced in the long-term and many of the alternative therapies (surgery, electric stimulation) have severe side effects (Toulouse and Sullivan, 2008). Recent studies have suggested that statins, drugs used as cholesterol-lowering agents, as potential treatments in
http://dx.doi.org/10.1016/j.neuro.2015.03.012 0161-813X/ß 2015 Published by Elsevier Inc.
Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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coordinates 0.5 mm posterior, 3.0 mm lateral and 4.5 mm ventral to the bregma, according to (Paxinos and Watson, 1998), as described by (Rubio-Osornio et al., 2009). Groups C and L only were injected with saline solution.
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2.3. Circling behavior evaluation
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The number of apomorphine-induced turns was recorded six days after MPP+ administration. Rats received a subcutaneous apomorphine administration (1 mg/kg) dissolved in ascorbic acid/ saline (1 mg/mL) as an antioxidant solution. Five minutes later, the total number of complete rotations (3608) was recorded, during one hour.
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2.4. Measurement of dopamine and metabolites
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Striatal dopamine and their metabolites concentrations were measured by HPLC/ED. Rat striatum was homogenized in 10 volumes of perchloric acid/sodium metabisulfite solution (1 M, 0.1% w/v) and centrifuged at 10,000 g for 10 min at 4 8C. The concentration of DA, DOPAC and HVA were measured in the supernatants by the use of a high performance liquid chromatography (HPLC) system (LC 250 Perkin-Elmer) with electrochemical detection (Metrohm 641-electrochemical), using a catecholamine analytical column (100 mm 4.8 mm with 3 mm of particle size). The mobile phase was a phosphate buffer (pH 3.2) prepared with 0.2 mM sodium octyl sulfate, 0.1 mM EDTA and 15% (v/v) methanol. Concentrations were calculated using a linear calibration curve, constructed with catecholamine standards.
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2.5. Striatal lipid peroxidation
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The striatum from rats were homogenized in 2.5 mL of deionized water. One-milliliter homogenate aliquots were mixed with 4 mL of a chloroform-methanol mixture (2:1 v/v). The mix was covered from light, shaken for 2 min and placed on ice for 30 min in the dark to allow phase separation. To detect the lipid fluorescence products, 1 mL chloroform phase aliquots were placed in a quartz cuvette with 0.1 mL of methanol and were read at 370 nm excitation and 430 nm of emission in a PerkinElmer LS50B luminescence spectrophotometer. The sensitivity was adjusted to 140 fluorescence units with a (0.1 mg/L) quinine standard. The levels of lipid fluorescent products were expressed as fluorescence units per mg of protein content (Lowry et al., 1951).
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2.6. PON2 activity
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The PON2 activity was determined in the right striatum, using the technique described by Teiber and Draganov (Teiber and Draganov, 2011). Briefly, the striatal tissue was homogenized in Tris–HCl buffer 25 mM, pH 7.4 containing 0.5% n-dodecyl b-Dmaltoside (Dojindo), 1 mM CaCl2, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (Sigma) and protease inhibitors as described (Teiber and Draganov, 2011). N-3-oxododecanoyl-Lhomoserine lactone (3OC12; Sigma) was used as a specific substrate. The detection was determined by HPLC with UV detection (205 nm), Restek Pinnacle II C-18 column (250 mm 4.6 mm, 5 mm particles) equipped with a pre-column Restek cap frit (4 mm, 0.5 mm) and mobile phase: 75% acetonitrile, 0.2% acetic acid.
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2.2. MPP+ PD model
2.7. Statistical analysis
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Twenty-four hours after of the last lovastatin dose, the animals were anesthetized with pentobarbital sodium (40 mg/kg i.p., Sigma). Groups M and LM were intrastriatally injected with 15 mg of MPP+ in 8 mL of saline solution in the right striatum at
The circling behavior, striatal catecholamine concentrations, lipid peroxidation and PON2 results are expressed as mean one SEM. Statistical significance between the different assays was assessed by using one-way analysis of variance (ANOVA), followed
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neurodegenerative diseases, like PD. Statins possess an antioxidant effect that decreases oxidative stress (Dolga et al., 2011) and prevents striatal DA, HVA (homovanillic acid) and DOPAC (dihydroxyphenylacetic acid) depletion (Selley, 2005). Specifically, lovastatin reduced the oxidized metabolites of cholesterol in mouse brain and prevented a-synuclein aggregates and neurodegeneration (Bosco et al., 2006). Xu et al., 2013 had demonstrated that simvastatin inhibited the decrease of cell viability induced by dopaminergic toxin MPP+ in PC12 cells by inhibition of MPP+induced intracellular ROS production. However, Santiago et al., 2009 reported that this statin has no antioxidant effect against the neurotoxicity of MPP+ in vivo. On the other hand, C57BL/6 mice treated with statins for 21 days showed that simvastatin increased the expression of 38 genes in cerebral cortex, particularly genes associated with cell growth and signaling and trafficking that could be fundamental contributors to neuroprotection. In the case of lovastatin, at least 15 genes were up-regulated in this mice brain region (Johnson-Anuna et al., 2005). Another clinical study showed that simvastatin administration in patients increases concentrations and activities of antioxidant proteins such as plasma paraoxonase-1 (PON1) increasing PON1 promoter activity in a dose-dependent manner (Deakin et al., 2003). The paraoxonases are a family of 3 enzymes (PON1, PON2 and PON3) widely distributed in the body. Their main cellular function is not clear; however, they have been associated with antioxidant effects, including the diminution of lipid hydroperoxides (Aviram et al., 1998; Marathe et al., 2003). The concentration of mice brain PON2 protein was measured, showing its highest concentration in the substantia nigra, nucleus accumbens and striatum (Giordano et al., 2011). All these brain regions are involved in PD neurodegeneration, suggesting a possible participation of PON2 in the antioxidant and neuroprotective effects. Recently, in vitro experiments showed that neurons deficient in PON2 are hypersensitive to oxidative stress induced by MPP+ (Parsanejad et al., 2014). The aims of this work were to (i) determine if lovastatin could protect against MPP induced behavioral changes, increasing levels of catecholamine including dopamine, decreasing levels of lipid peroxidation and (ii) determine whether or not the effects of lovastatin are mediated by an increase in PON2 activity in the model of PD induced by MPP+ in rats.
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2. Materials and methods
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2.1. Animals and treatments
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Experiments were conducted on male Wistar rats, NIH strain weighing 250–280 g. Animals were maintained under standard conditions (12:12 light-dark cycles, 23 2 8C) and 40% of relative humidity. They were fed a standard chow diet (Purina chow) and allowed free access to water. All animals were treated humanely to minimize discomfort in accordance with the ethical principles and regulations specified by the Animal Care and Use Committee of the National Institute of Neurology and Neurosurgery and the standards of the National Institutes of Health of Mexico. Rats were distributed in four experimental groups (n = 6); control group (C), MPP+ group (M), lovastatin group (L) and lovastatin-MPP+ group (LM). The group L and LM were treated with lovastatin (5 mg/kg) intraperitoneally daily for 7 days, while C and M groups were administered with the vehicle (10% DMSO, 500 mL/kg i.p.).
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Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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(Figs. 3 and 4, respectively). Lovastatin treatment prevented this effect, because their DOPAC and HVA concentrations were similar as compared with the C group.
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3.3. Lipid peroxides in the striatum
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As shown in Fig. 5, treatment with lovastatin statistically decreased by 55% (p < 0.05) lipid peroxide formation (PL) generated by the MPP+ striatal administration. PL levels for control and lovastatin groups were about 4 UF/mg of protein, in contrast PL
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Fig. 1. Behavioral test. This graph shows the rotational effect induced by administration of apomorphine (1 mg/kg) s.c. six days after stereotactic surgery in which administered MPP+ (M and ML injured animals) or vehicle (C and L groups). Each bar represents the average number of turns in each group (n 7 per group) for 1 h. (* p < 0.05) Kruskal–Wallis test followed by Mann–Whitney.
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the Tukey post-test. For DA and its metabolites we applied a Kruskal– Wallis test, followed the multiple comparisons using the Mann– Whitney test. All statistical analyses were performed by using SPSS statistical software, version 17, p < 0.05 was considered statistically significant.
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3. Results
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3.1. Circling behavior induced by apomorphine
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Animals receiving saline intrastriatal administration showed no rotation behavior (Fig. 1) in response apomorphine administration (groups C and L). Rats injected with MPP+ (M group) displayed 170 26 turns/h. Lovastatin treatment to MPP+ injected animals (LM group) significantly reduced (p < 0.05) the number of turns by 58% (70 16 turns) as compared with turns displayed by animals without lovastatin treatment (M group).
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3.2. Striatal catecholamine concentrations
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Animals injected with MPP+ (M group) showed a statistically significant decrease (p < 0.05) in their striatal DA concentrations of 45%, as compared to the control group (C) (Fig. 2). Meanwhile, rats pretreated with lovastatin plus MPP+ (LM group) significantly preserved (p < 0.05) the DA levels (30%) as compared to the MPP+ group (M). On the other hand, striatal DOPAC and HVA levels were statistically decreased (p < 0.05) by MPP+ administration
Fig. 2. Effect of lovastatin on DA levels in the striatum rat. Each bar represents the mean SE of right striated DA levels in each treatment group, quantified by HPLC with electrochemical detector. The results show a significant change between M and LM group. Where C, Control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/ MPP+ group (* p < 0.05) ANOVA followed by Tukey test.
Fig. 3. Lovastatin pretreatment effect on DOPAC levels in the MPP+ model in rat. This chart illustrates DOPAC levels detected in the right striatum of each treatment group (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) by HPLC coupled to electrochemical detector (* p < 0.05) ANOVA followed by Tukey test.
Fig. 4. Lovastatin pretreatment effect on HVA levels in the MPP+ model in rat. This graph illustrates effect on HVA levels detected in the right striatum of each treatment group, identified by HPLC coupled to electrochemical detector, (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) (* p < 0.05) ANOVA followed by Tukey test.
Fig. 5. Lovastatin pretreatment effect on the levels of lipid peroxides in the MPP+ model in rat. Each bar represents the mean SE of the detected fluorescence units per gram of tissue (right striatum) of each group. These concentrations were obtained from a spectrophotometric method, adjusted to 140 UF with a quinine standard. (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) (* p < 0.05) by Kruskal–Wallis test followed by Mann–Whitney.
Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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level in the MPP+ neurotoxin (M group) were 4.5 times higher (18 UF/mg of protein) than in the control group (C group).
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3.4. PON2 lactonase activity
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Striatal PON2 lactonase activity values were similar in the four experimental groups, about 0.2 nmol/min/mg of tissue. The striatal PON2 enzymatic activity in the animals administrated only with MPP+ (M group) or lovastatin (L group) showed no significant difference among treatment groups (Fig. 6).
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4. Discussion
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In this study we show that lovastatin pretreatment was able to reduce the number of turns in the rats injured with MPP+, showing the neuroprotective effect of the statin in the model of Parkinson’s disease induced by this neurotoxin. Results of the present study are in agreement with previous reports of statins as protective agents against neurodegeneration in several CNS disorders (Selley, 2005; Stepien et al., 2005). To assess the damage to the nigrostriatal pathway, we compare the turns of the control group against those of MPP+ group, evidencing the motor asymmetry generated by the neurodegeneration after MPP+ administration. This resulting behavior 172 turns/h confirms the neurotoxic dopaminergic animal model of MPP+ as it has been reported in other neuroprotective studies (Hung et al., 2008). We found that lovastatin pretreatment was able to maintain the DA levels against the effect of the neurotoxin. This result is comparable with that reported for other statins, including simvastatin that has been administered at different doses (10, 20 and 40 mg/kg/day for 10 days) to show neuroprotection against MPTP model (Selley, 2005). Pretreatment with lovastatin produced no effect on striatal DA levels of uninjured rats, as was reported by Wang et al., 2006. However in the MPP+ treatment group, lovastatin preserved, DOPAC, HVA and dopamine concentrations, indicating a whole neuroprotective effect of the statin on the metabolic pathway of DA. Regarding the antioxidant effect of lovastatin observed in this study, there are reports showing that this group of drugs posses a clear effect against oxidative stress (Shishehbor et al., 2003). Recently, Xu et al. (2013) have shown that simvastatin inhibited the decrease of cell viability induced by MPP+ in PC12 cells by inhibiting the MPP+-induced intracellular ROS production. On the other hand, PON2 activity has been considered antioxidant, by its ability to limit peroxides formation (Ng et al., 2001). As PON1 has been reported to be induced by statins, in plasma of humans (Deakin et al., 2003), it was expected to see an up-regulation of the protein after lovastatin administration. In the case of PON2, there are reports showing an increased expression after statin treatment
Fig. 6. Effect of lovastatin on PON2 lactonase activity (3OC12) in the MPP+ model. This figure shows the mean S.E. lactone hydrolyzed by PON2 expressed as nmol hydrolyzed min/mg tissue (n = 3–5), (C, control group; L, Lovastatin group; M, MPP+ group and ML, Lovastatin/MPP+ group).
to mice (Johnson-Anuna et al., 2005). Therefore, it is also suggested that an up-regulation of PON2 would participate in the antioxidant mechanism of the statins. However, results of the present study indicate that lovastatin inhibition of MPP+-induced oxidative stress is produced by a mechanism independent of PON2 activity. The statins are considered antioxidant drugs by their molecular structure and their ability to induce the expression of endogenous antioxidant systems, among them, paraoxonases (PON1, PON2 and PON3). Specifically, PON2 reduces superoxide anion release from the inner mitochondrial membrane, resulting from complex I or complex III of the electron transport chain activity, presumably by acting on coenzyme Q10 (Altenhofer et al., 2010). As the expression of PON2 is higher in the brain as compared with the other paraoxonases (PON1 and PON3). A greater participation of PON2 in the regulation of oxidative stress associated to various pathologies would be expected (Giordano et al., 2011). However, the administration of MPP+ in this study did not affect the PON2 activity levels. Nevertheless, the levels of PON2 lactonase activity were the same among the treatment groups, suggesting the lack of participation of this enzyme in the mechanism of antioxidant neuroprotection induced by lovastatin. Assuming that the lactonase activity has a direct relationship with the concentration of PON2 protein, there is also a lack of effect on the expression of the enzyme by the statin. Thus, our results suggest a mechanism of antioxidant action of lovastatin unrelated to the induction of PON2 in the striatum. However, there are in vitro and in vivo reports that strongly support the differential PON2 cell expression in striatum brain region induced by these compounds (Giordano et al., 2013; Costa et al., 2014). It is possible that PON2 may have been induced by lovastatin in a smaller select group of cells. Thus it will be important in future work to evaluate in different specific cell types of rat brain nigrostriatal pathway if compounds known to induce this protein, including estrogens and polyphenols, increase PON2 levels. In conclusion, lovastatin has a neuroprotective effect against neurotoxicity of MPP+ by a mechanism that appears to be independent of PON2 activity. Conflict of interests
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The authors declare that there is no conflict of interests regarding the publication of this paper. Q7
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Acknowledgments
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This work was partially supported by CONACYT 106436. And Q8 Aguirre-Vidal receives a fellowship CONACYT 350320. We thank FERMIC Laboratories for the donation of the Lovastatin.
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Contents lists available at ScienceDirect
NeuroToxicology
1
The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2
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Aguirre-Vidal a, Sergio Montes b, Luis Tristan-Lo´pez b, Laura Anaya-Ramos a, John Teiber , Camilo Rı´os b,d, Vero´nica Baron-Flores d, Antonio Monroy-Noyola a,*
Q1 Yoshajandith c a
Laboratorio de Neuroproteccio´n, Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Cuernavaca, Morelos, Mexico Departamento de Neuroquı´mica, Instituto Nacional de Neurologı´a y Neurocirugı´a, M.V.S., D.F., Mexico Division of Epidemiology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA d Q2 Laboratorio de Neurofarmacologia Molecular, Departamento de Sistemas Biolo´gicos, Universidad Auto´noma Metropolitana-Xochimilco, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 December 2014 Accepted 24 March 2015 Available online xxx
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of the pigmented dopaminergic neurons in the substantia nigra pars compacta with subsequent striatal dopamine (DA) deficiency and increased lipid peroxidation. The etiology of the disease is still unclear and it is thought that PD may be caused by a combination of genetic and environmental factors. In the search of new pharmacological options, statins have been recognized for their potential application to treat PD, due to their antioxidant effect. The aim of this work is to contribute in the characterization of the neuroprotective effect of lovastatin in a model of PD induced by 1-methyl-4-phenylpyridinium (MPP+). Male Wistar rats (200–250 g) were randomly allocated into 4 groups and administered for 7 days with different pharmacological treatments. Lovastatin administration (5 mg/kg) diminished 40% of the amphetamine-induced circling behavior, prevented the striatal DA depletion and lipid peroxides formation by MPP+ intrastriatal injection, as compared to the group of animals treated only with MPP+. Lovastatin produced no change in paraoxonase-2 (PON2) activity. It is evident that lovastatin conferred neuroprotection against MPP+-induced protection but this effect was not associated with the induction of PON2 in the rat striatum. ß 2015 Published by Elsevier Inc.
Keywords: Parkinson Statin Lipoperoxidation Dopamine Lovastatin Q4 Paraoxonase-2
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1. Introduction Q5
Parkinson disease (PD) is an important neurodegenerative disorder; affecting between 0.5 and 1% of the population aged 65– 69 years of age, and increasing to 1–3% of the population over 80 years of age (Toulouse and Sullivan, 2008). PD is biochemically characterized by a decrease of dopamine (DA) levels, caused by the death of dopaminergic neurons in the substantia nigra pars
Abbreviations: MPP+, 1-methyl-4-phenylpyridinium; PD, Parkinson’s disease; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine; MAO-B, monoamine oxidase B; DA, dopamine; ROS, reactive oxidant species; PON2, paraoxonase-2; HVA, homovanillic acid; DOPAC, 3,4-dihydroxyphenylacetic acid; DMSO, dimethylsulfoxide; HPLC, high performance liquid chromatography; PL, lipidperoxides; CNS, central nervous system; FU, fluorescence units; DNA, deoxyribonucleic acid. * Corresponding author at: Laboratorio de Neuroproteccio´n, Facultad de Farmacia, Universidad Auto´noma del Estado de Morelos, Av. Universidad 1001 Col. Q3 Chamilpa, CP 62209 Cuernavaca, Morelos, Mexico. Tel.: +52 7773297089. E-mail address: [email protected] (A. Monroy-Noyola).
compacta (SNpc). Dopamine is the neurotransmitter involved in the control of extrapyramidal motor function of the nigrostriatal pathway. As this pathway is injured in PD, dopamine depletion is the cause of the remarkable motor symptoms of the disease; including tremor, rigidity, bradykinesia and abnormal posture (Toulouse and Sullivan, 2008). The neurodegenerative process of the nigrostriatal pathway in PD has been related to oxidative stress and mitochondrial dysfunction. Oxidative stress initiates chain reactions leading to lipid peroxidation, therefore decreases unsaturated fatty acids content in cell membranes. This change affects membrane properties and enzyme activities, receptor functions and DA oxidation, increasing apoptosis and other cell mechanisms of death (Eunsung and Mouradian, 2001). Nowadays, there are several treatments for the symptoms, but the beneficial effects are reduced in the long-term and many of the alternative therapies (surgery, electric stimulation) have severe side effects (Toulouse and Sullivan, 2008). Recent studies have suggested that statins, drugs used as cholesterol-lowering agents, as potential treatments in
http://dx.doi.org/10.1016/j.neuro.2015.03.012 0161-813X/ß 2015 Published by Elsevier Inc.
Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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coordinates 0.5 mm posterior, 3.0 mm lateral and 4.5 mm ventral to the bregma, according to (Paxinos and Watson, 1998), as described by (Rubio-Osornio et al., 2009). Groups C and L only were injected with saline solution.
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2.3. Circling behavior evaluation
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The number of apomorphine-induced turns was recorded six days after MPP+ administration. Rats received a subcutaneous apomorphine administration (1 mg/kg) dissolved in ascorbic acid/ saline (1 mg/mL) as an antioxidant solution. Five minutes later, the total number of complete rotations (3608) was recorded, during one hour.
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2.4. Measurement of dopamine and metabolites
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Striatal dopamine and their metabolites concentrations were measured by HPLC/ED. Rat striatum was homogenized in 10 volumes of perchloric acid/sodium metabisulfite solution (1 M, 0.1% w/v) and centrifuged at 10,000 g for 10 min at 4 8C. The concentration of DA, DOPAC and HVA were measured in the supernatants by the use of a high performance liquid chromatography (HPLC) system (LC 250 Perkin-Elmer) with electrochemical detection (Metrohm 641-electrochemical), using a catecholamine analytical column (100 mm 4.8 mm with 3 mm of particle size). The mobile phase was a phosphate buffer (pH 3.2) prepared with 0.2 mM sodium octyl sulfate, 0.1 mM EDTA and 15% (v/v) methanol. Concentrations were calculated using a linear calibration curve, constructed with catecholamine standards.
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2.5. Striatal lipid peroxidation
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The striatum from rats were homogenized in 2.5 mL of deionized water. One-milliliter homogenate aliquots were mixed with 4 mL of a chloroform-methanol mixture (2:1 v/v). The mix was covered from light, shaken for 2 min and placed on ice for 30 min in the dark to allow phase separation. To detect the lipid fluorescence products, 1 mL chloroform phase aliquots were placed in a quartz cuvette with 0.1 mL of methanol and were read at 370 nm excitation and 430 nm of emission in a PerkinElmer LS50B luminescence spectrophotometer. The sensitivity was adjusted to 140 fluorescence units with a (0.1 mg/L) quinine standard. The levels of lipid fluorescent products were expressed as fluorescence units per mg of protein content (Lowry et al., 1951).
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2.6. PON2 activity
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The PON2 activity was determined in the right striatum, using the technique described by Teiber and Draganov (Teiber and Draganov, 2011). Briefly, the striatal tissue was homogenized in Tris–HCl buffer 25 mM, pH 7.4 containing 0.5% n-dodecyl b-Dmaltoside (Dojindo), 1 mM CaCl2, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (Sigma) and protease inhibitors as described (Teiber and Draganov, 2011). N-3-oxododecanoyl-Lhomoserine lactone (3OC12; Sigma) was used as a specific substrate. The detection was determined by HPLC with UV detection (205 nm), Restek Pinnacle II C-18 column (250 mm 4.6 mm, 5 mm particles) equipped with a pre-column Restek cap frit (4 mm, 0.5 mm) and mobile phase: 75% acetonitrile, 0.2% acetic acid.
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2.2. MPP+ PD model
2.7. Statistical analysis
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Twenty-four hours after of the last lovastatin dose, the animals were anesthetized with pentobarbital sodium (40 mg/kg i.p., Sigma). Groups M and LM were intrastriatally injected with 15 mg of MPP+ in 8 mL of saline solution in the right striatum at
The circling behavior, striatal catecholamine concentrations, lipid peroxidation and PON2 results are expressed as mean one SEM. Statistical significance between the different assays was assessed by using one-way analysis of variance (ANOVA), followed
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neurodegenerative diseases, like PD. Statins possess an antioxidant effect that decreases oxidative stress (Dolga et al., 2011) and prevents striatal DA, HVA (homovanillic acid) and DOPAC (dihydroxyphenylacetic acid) depletion (Selley, 2005). Specifically, lovastatin reduced the oxidized metabolites of cholesterol in mouse brain and prevented a-synuclein aggregates and neurodegeneration (Bosco et al., 2006). Xu et al., 2013 had demonstrated that simvastatin inhibited the decrease of cell viability induced by dopaminergic toxin MPP+ in PC12 cells by inhibition of MPP+induced intracellular ROS production. However, Santiago et al., 2009 reported that this statin has no antioxidant effect against the neurotoxicity of MPP+ in vivo. On the other hand, C57BL/6 mice treated with statins for 21 days showed that simvastatin increased the expression of 38 genes in cerebral cortex, particularly genes associated with cell growth and signaling and trafficking that could be fundamental contributors to neuroprotection. In the case of lovastatin, at least 15 genes were up-regulated in this mice brain region (Johnson-Anuna et al., 2005). Another clinical study showed that simvastatin administration in patients increases concentrations and activities of antioxidant proteins such as plasma paraoxonase-1 (PON1) increasing PON1 promoter activity in a dose-dependent manner (Deakin et al., 2003). The paraoxonases are a family of 3 enzymes (PON1, PON2 and PON3) widely distributed in the body. Their main cellular function is not clear; however, they have been associated with antioxidant effects, including the diminution of lipid hydroperoxides (Aviram et al., 1998; Marathe et al., 2003). The concentration of mice brain PON2 protein was measured, showing its highest concentration in the substantia nigra, nucleus accumbens and striatum (Giordano et al., 2011). All these brain regions are involved in PD neurodegeneration, suggesting a possible participation of PON2 in the antioxidant and neuroprotective effects. Recently, in vitro experiments showed that neurons deficient in PON2 are hypersensitive to oxidative stress induced by MPP+ (Parsanejad et al., 2014). The aims of this work were to (i) determine if lovastatin could protect against MPP induced behavioral changes, increasing levels of catecholamine including dopamine, decreasing levels of lipid peroxidation and (ii) determine whether or not the effects of lovastatin are mediated by an increase in PON2 activity in the model of PD induced by MPP+ in rats.
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2. Materials and methods
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2.1. Animals and treatments
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Experiments were conducted on male Wistar rats, NIH strain weighing 250–280 g. Animals were maintained under standard conditions (12:12 light-dark cycles, 23 2 8C) and 40% of relative humidity. They were fed a standard chow diet (Purina chow) and allowed free access to water. All animals were treated humanely to minimize discomfort in accordance with the ethical principles and regulations specified by the Animal Care and Use Committee of the National Institute of Neurology and Neurosurgery and the standards of the National Institutes of Health of Mexico. Rats were distributed in four experimental groups (n = 6); control group (C), MPP+ group (M), lovastatin group (L) and lovastatin-MPP+ group (LM). The group L and LM were treated with lovastatin (5 mg/kg) intraperitoneally daily for 7 days, while C and M groups were administered with the vehicle (10% DMSO, 500 mL/kg i.p.).
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Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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(Figs. 3 and 4, respectively). Lovastatin treatment prevented this effect, because their DOPAC and HVA concentrations were similar as compared with the C group.
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3.3. Lipid peroxides in the striatum
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As shown in Fig. 5, treatment with lovastatin statistically decreased by 55% (p < 0.05) lipid peroxide formation (PL) generated by the MPP+ striatal administration. PL levels for control and lovastatin groups were about 4 UF/mg of protein, in contrast PL
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Fig. 1. Behavioral test. This graph shows the rotational effect induced by administration of apomorphine (1 mg/kg) s.c. six days after stereotactic surgery in which administered MPP+ (M and ML injured animals) or vehicle (C and L groups). Each bar represents the average number of turns in each group (n 7 per group) for 1 h. (* p < 0.05) Kruskal–Wallis test followed by Mann–Whitney.
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the Tukey post-test. For DA and its metabolites we applied a Kruskal– Wallis test, followed the multiple comparisons using the Mann– Whitney test. All statistical analyses were performed by using SPSS statistical software, version 17, p < 0.05 was considered statistically significant.
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3. Results
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3.1. Circling behavior induced by apomorphine
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Animals receiving saline intrastriatal administration showed no rotation behavior (Fig. 1) in response apomorphine administration (groups C and L). Rats injected with MPP+ (M group) displayed 170 26 turns/h. Lovastatin treatment to MPP+ injected animals (LM group) significantly reduced (p < 0.05) the number of turns by 58% (70 16 turns) as compared with turns displayed by animals without lovastatin treatment (M group).
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3.2. Striatal catecholamine concentrations
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Animals injected with MPP+ (M group) showed a statistically significant decrease (p < 0.05) in their striatal DA concentrations of 45%, as compared to the control group (C) (Fig. 2). Meanwhile, rats pretreated with lovastatin plus MPP+ (LM group) significantly preserved (p < 0.05) the DA levels (30%) as compared to the MPP+ group (M). On the other hand, striatal DOPAC and HVA levels were statistically decreased (p < 0.05) by MPP+ administration
Fig. 2. Effect of lovastatin on DA levels in the striatum rat. Each bar represents the mean SE of right striated DA levels in each treatment group, quantified by HPLC with electrochemical detector. The results show a significant change between M and LM group. Where C, Control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/ MPP+ group (* p < 0.05) ANOVA followed by Tukey test.
Fig. 3. Lovastatin pretreatment effect on DOPAC levels in the MPP+ model in rat. This chart illustrates DOPAC levels detected in the right striatum of each treatment group (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) by HPLC coupled to electrochemical detector (* p < 0.05) ANOVA followed by Tukey test.
Fig. 4. Lovastatin pretreatment effect on HVA levels in the MPP+ model in rat. This graph illustrates effect on HVA levels detected in the right striatum of each treatment group, identified by HPLC coupled to electrochemical detector, (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) (* p < 0.05) ANOVA followed by Tukey test.
Fig. 5. Lovastatin pretreatment effect on the levels of lipid peroxides in the MPP+ model in rat. Each bar represents the mean SE of the detected fluorescence units per gram of tissue (right striatum) of each group. These concentrations were obtained from a spectrophotometric method, adjusted to 140 UF with a quinine standard. (C, control group; L, Lovastatin group; M, MPP+ group; ML, Lovastatin/MPP+ group) (* p < 0.05) by Kruskal–Wallis test followed by Mann–Whitney.
Please cite this article in press as: Aguirre-Vidal Y, et al. The neuroprotective effect of lovastatin on MPP+-induced neurotoxicity is not mediated by PON2. Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.03.012
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level in the MPP+ neurotoxin (M group) were 4.5 times higher (18 UF/mg of protein) than in the control group (C group).
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3.4. PON2 lactonase activity
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Striatal PON2 lactonase activity values were similar in the four experimental groups, about 0.2 nmol/min/mg of tissue. The striatal PON2 enzymatic activity in the animals administrated only with MPP+ (M group) or lovastatin (L group) showed no significant difference among treatment groups (Fig. 6).
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4. Discussion
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In this study we show that lovastatin pretreatment was able to reduce the number of turns in the rats injured with MPP+, showing the neuroprotective effect of the statin in the model of Parkinson’s disease induced by this neurotoxin. Results of the present study are in agreement with previous reports of statins as protective agents against neurodegeneration in several CNS disorders (Selley, 2005; Stepien et al., 2005). To assess the damage to the nigrostriatal pathway, we compare the turns of the control group against those of MPP+ group, evidencing the motor asymmetry generated by the neurodegeneration after MPP+ administration. This resulting behavior 172 turns/h confirms the neurotoxic dopaminergic animal model of MPP+ as it has been reported in other neuroprotective studies (Hung et al., 2008). We found that lovastatin pretreatment was able to maintain the DA levels against the effect of the neurotoxin. This result is comparable with that reported for other statins, including simvastatin that has been administered at different doses (10, 20 and 40 mg/kg/day for 10 days) to show neuroprotection against MPTP model (Selley, 2005). Pretreatment with lovastatin produced no effect on striatal DA levels of uninjured rats, as was reported by Wang et al., 2006. However in the MPP+ treatment group, lovastatin preserved, DOPAC, HVA and dopamine concentrations, indicating a whole neuroprotective effect of the statin on the metabolic pathway of DA. Regarding the antioxidant effect of lovastatin observed in this study, there are reports showing that this group of drugs posses a clear effect against oxidative stress (Shishehbor et al., 2003). Recently, Xu et al. (2013) have shown that simvastatin inhibited the decrease of cell viability induced by MPP+ in PC12 cells by inhibiting the MPP+-induced intracellular ROS production. On the other hand, PON2 activity has been considered antioxidant, by its ability to limit peroxides formation (Ng et al., 2001). As PON1 has been reported to be induced by statins, in plasma of humans (Deakin et al., 2003), it was expected to see an up-regulation of the protein after lovastatin administration. In the case of PON2, there are reports showing an increased expression after statin treatment
Fig. 6. Effect of lovastatin on PON2 lactonase activity (3OC12) in the MPP+ model. This figure shows the mean S.E. lactone hydrolyzed by PON2 expressed as nmol hydrolyzed min/mg tissue (n = 3–5), (C, control group; L, Lovastatin group; M, MPP+ group and ML, Lovastatin/MPP+ group).
to mice (Johnson-Anuna et al., 2005). Therefore, it is also suggested that an up-regulation of PON2 would participate in the antioxidant mechanism of the statins. However, results of the present study indicate that lovastatin inhibition of MPP+-induced oxidative stress is produced by a mechanism independent of PON2 activity. The statins are considered antioxidant drugs by their molecular structure and their ability to induce the expression of endogenous antioxidant systems, among them, paraoxonases (PON1, PON2 and PON3). Specifically, PON2 reduces superoxide anion release from the inner mitochondrial membrane, resulting from complex I or complex III of the electron transport chain activity, presumably by acting on coenzyme Q10 (Altenhofer et al., 2010). As the expression of PON2 is higher in the brain as compared with the other paraoxonases (PON1 and PON3). A greater participation of PON2 in the regulation of oxidative stress associated to various pathologies would be expected (Giordano et al., 2011). However, the administration of MPP+ in this study did not affect the PON2 activity levels. Nevertheless, the levels of PON2 lactonase activity were the same among the treatment groups, suggesting the lack of participation of this enzyme in the mechanism of antioxidant neuroprotection induced by lovastatin. Assuming that the lactonase activity has a direct relationship with the concentration of PON2 protein, there is also a lack of effect on the expression of the enzyme by the statin. Thus, our results suggest a mechanism of antioxidant action of lovastatin unrelated to the induction of PON2 in the striatum. However, there are in vitro and in vivo reports that strongly support the differential PON2 cell expression in striatum brain region induced by these compounds (Giordano et al., 2013; Costa et al., 2014). It is possible that PON2 may have been induced by lovastatin in a smaller select group of cells. Thus it will be important in future work to evaluate in different specific cell types of rat brain nigrostriatal pathway if compounds known to induce this protein, including estrogens and polyphenols, increase PON2 levels. In conclusion, lovastatin has a neuroprotective effect against neurotoxicity of MPP+ by a mechanism that appears to be independent of PON2 activity. Conflict of interests
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The authors declare that there is no conflict of interests regarding the publication of this paper. Q7
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Acknowledgments
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This work was partially supported by CONACYT 106436. And Q8 Aguirre-Vidal receives a fellowship CONACYT 350320. We thank FERMIC Laboratories for the donation of the Lovastatin.
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