Probucol Affords Neuroprotection in a 6-OHDA Mouse Model of Parkinson’s Disease
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- Ribeiro, R.P., Moreira, E.L.G., Santos, D.B. et al. Neurochem Res (2013) 38: 660. doi:10.1007/s11064-012-0965-0
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons. Although the etiology of the majority of human PD cases is unknown, experimental evidence points to oxidative stress as an early and causal event. Probucol is a lipid-lowering phenolic compound with anti-inflammatory and antioxidant properties that has been recently reported as protective in neurotoxicity and neurodegeneration models. This study was designed to investigate the effects of probucol on the vulnerability of striatal dopaminergic neurons to oxidative stress in a PD in vivo model. Swiss mice were treated with probucol during 21 days (11.8 mg/kg; oral route). Two weeks after the beginning of treatment, mice received a single intracerebroventricular (i.c.v.) infusion of 6-hydroxydopamine (6-OHDA). On the 21st day, locomotor performance, striatal oxidative stress-related parameters, and striatal tyrosine hydroxylase and synaptophysin levels, were measured as outcomes of toxicity. 6-OHDA-infused mice showed hyperlocomotion and a significant decrease in striatal tyrosine hydroxylase (TH) and synaptophysin levels. In addition, 6-OHDA-infused mice showed reduced superoxide dismutase activity and increased lipid peroxidation and catalase activity in the striatum. Notably, probucol protected against 6-OHDA-induced hyperlocomotion and striatal lipid peroxidation, catalase upregulation and decrease of TH levels. Overall, the present results show that probucol protects against 6-OHDA-induced toxicity in mice. These findings may render probucol as a promising molecule for further pharmacological studies on the search for disease-modifying treatment in PD.
KeywordsProbucolOxidative stressParkinson’s disease6-HydroxydopamineTherapeutic strategies
Parkinson’s disease (PD) is a progressive, neurodegenerative disorder that is characterized by severe motor symptoms, including resting tremor, rigidity, bradykinesia and postural instability , as well as non-motor symptoms such as olfactory disturbances, rapid-eye-movement sleep behavior disorder, depression, anxiety, and urinary and sexual disturbances [2, 3]. The primary pathological hallmark of PD is a profound loss of dopamine-producing neurons in the substantianigrapars compacta (SNpc), resulting in a drastic depletion of dopamine in the striatum [4, 5]. Whereas some forms of PD are genetic, most cases are idiopathic and likely results from the combined effects of multiple factors including aging, genetic predisposition, and environmental exposures [6, 7].
Increasing evidence points to oxidative stress as an early and causal event of PD [8, 9]. Indeed, nigral dopaminergic neurons are particularly exposed to oxidative stress because the metabolism of dopamine gives rise to various molecules that can act as endogenous toxins if not handled properly . Specifically, dopamine can auto-oxidize at normal pH into toxic dopamine-quinone species, superoxide radicals (·O2−) and hydrogen peroxide (H2O2) . Moreover, a state of oxidative imbalance is also triggered by one or more factors, among which are brain aging, genetic predisposition, mitochondrial dysfunction, free radical production and environmental toxins [9, 12–14]. Accordingly, one of the earliest biochemical changes seen in PD is a reduction in the levels of glutathione (GSH) in SNpc, resulting in a selective decrease in mitochondrial complex I activity and a marked reduction in overall mitochondrial function [15, 16]. Further evidence that oxidative stress participates in the loss of nigral neurons in PD comes from studies using the parkinsonism-inducing toxin 6-hydroxydopamine (6-OHDA). 6-OHDA is a hydroxylated analogue of dopamine which uses the same transport system to produce specific degeneration of dopaminergic neurons . Particularly, the toxic mechanism of this compound is dependent on its oxidation with concomitant production of 6-OHDA quinine and H2O2, ·O2−, and hydroxyl radical (·OH) [18–20]. Taken together, the above mentioned evidence has led to the belief that antioxidants could be useful as disease-modifying treatments in PD. However, studies with PD patients on the potential beneficial effects of antioxidants, such as tocopherol and CoQ10, showed not promising results [21, 22].
Probucol is a phenolic lipid-lowering prototype agent with anti-inflammatory and antioxidant properties that has a long history of clinical application for the treatment and prevention of cardiovascular diseases . Moreover, probucol has been reported to play protective effects in experimental models of neurotoxicity/neuropathology [24, 25]. Specifically, previous experimental studies from our research group showed (1) neuroprotective effects against Aβ1–40-induced synaptic loss and cognitive impairments in mice ; (2) beneficial effects against oxidative stress and excitotoxicity in an in vitro model of Huntington’s disease , and (3) protective effects against the neurotoxicity elicited by methylmercury, which were significantly higher compared with classical antioxidants, such as ascorbic acid and trolox .
Based on the mentioned evidence, herein we sought to investigate the effects of probucol in an in vivo model of PD induced by intracerebroventricular (i.c.v.) injection of 6-OHDA in mice. Behavioral analyses (mainly based on the motor performance) and biochemical alterations (oxidative stress-related parameters) were evaluated in the striatum of mice treated with 6-OHDA as outcomes of toxicity.
Materials and Methods
Probucol, 6-hydroxydopamine hydrochloride, desipramine, ascorbic acid, β-Nicotinamide adenine dinucleotide phosphate sodium salt reduced form, 5-5′-dithio-bis (2-nitrobenzoic) acid, glutathione reductase from baker’s yeast, reduced glutathione and dimethyl sulfoxide were obtained from Sigma (St. Louis, MO, USA). All other chemicals were of the highest grade available commercially.
Male adult Swiss Albino mice (3 months old and 40–50 g weight) were provided by the animal facility of the Universidade Federal de Santa Catarina (UFSC, Florianópolis, Brazil). Animals were maintained under controlled temperature (22 ± 2 °C), on a 12-h light/dark cycle (lights on at 7:00 a.m.), with free access to food and water. All experiments were conducted in compliance with the guidelines on the animal care of the UFSC Ethics Committee on the Use of Animals (protocol number PP00546; 23080.037849/2010-71), which follows the NIH “Principles of Laboratory Animal Care”.
Thirty mice were divided into 4 groups (6–8 animals each), as follows: (1) Control, (2) 6-OHDA, (3) Probucol and (4) Probucol + 6-OHDA. Animals from groups 3 and 4 received probucol (57 mg/L) diluted in a 1 % DMSO solution ad libitum as sole source of liquid, during 21 days. Groups 1 and 2 were treated with vehicle (1 % DMSO, ad libitum) during 21 days. For probucol-exposed mice (groups 3 and 4), a daily probucol dose of 11.8 mg/kg of body weight was calculated based on their daily liquid ingestion (9.5 ± 0.9 ml/animal). Liquid ingestion of these animals (groups 1–4) was not different from that of a parallel group of animals that received just tap water (data not shown). Two weeks after the beginning of probucol treatment, mice received a single intracerebroventricular (i.c.v.) infusion of 6-OHDA (see “6-Hydroxydopamine Lesions” section), and probucol treatment lasted for one more week. Probucol dose was based on a previous study from our group , which showed a protective effect of probucol in an experimental model of Alzheimer’s disease.
6-OHDA administrations were performed 2 weeks after the beginning of probucol treatment. Thirty minutes prior to 6-OHDA injection, all animals were treated with desipramine (25 mg/kg, i.p.) to protect noradrenaline-containing neurons. Under anesthesia with isoflurane (1 mL/mL; using a vaporizer system), mice from groups 2 and 4 were injected with 60 μg 6-OHDA (dissolved in 3 μL physiological saline containing 0.05 % ascorbic acid) into the right cerebral ventricle via a 5-μL Hamilton microsyringe . Control animals (groups 1 and 3) received the same volume of the vehicle (physiological saline containing 0.05 % ascorbic acid). The sterilization of the injection site was carried out using gauze embedded in 70 % ethanol. Under light anesthesia (i.e. just that necessary for loss of the postural reflex), the needle was inserted unilaterally 1 mm to the right of the midline point equidistant from each eye and 1 mm posterior to a line drawn through the anterior base of the eyes (used as external reference). The solutions was injected into the lateral ventricle, at the following coordinates from bregma: anteroposterior (AP) = −0.1 mm, mediolateral (ML) = 1 mm, and dorsoventral (DV) = −2.4 mm [30, 31]. This model causes a mortality rate of 50 %, which is observed within 1 h after 6-OHDA administration. Animals that survived the first hour did not lose weight and did not present detectable signs of systemic toxicity during the experimental period. Mortality rate within the first hour after 6-OHDA administration decreased to 25 % when probucol was administered simultaneously. Additionally, one control animal died after i.c.v. injection. The number of animals at the end of treatments was twenty-three, as follows: (1) Control (n = 7), (2) 6-OHDA (n = 4), (3) Probucol (n = 6) and (4) Probucol + 6-OHDA (n = 6).
Seven days after the i.c.v. infusion, mice were submitted to activity cages for the evaluation of locomotor activity. Thereafter, animals’ striatum was dissected to determine the activities of the antioxidant enzymes activities glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), as well as thiobarbituric acid reactive substances (TBARS) levels, and the level of tyrosine hydroxylase (TH) and synaptophysin (SYP) (“Activity Cage” to “Western blot analysis” section).
The activity cage (70 × 30 × 22 cm) had a steel grid floor and was equipped with three parallel horizontal infrared beams positioned 3 cm above the floor and spaced evenly along the longitudinal axis. There was a digital counter that recorded photocell beam interruptions. Total locomotor activity was monitored every during a 30-min period .
Twenty four hours after the behavioral analyses, the animals were euthanized by decapitation and the striatum was removed and homogenized (1:10 w/v) in HEPES buffer (20 mM, pH 7.0). The tissue homogenates were centrifuged at 16,000×g, at 4 °C for 20 min and the supernatants obtained were used for the determination of enzymatic activities (GPx, GR, CAT and SOD) and thiobarbituric acid reactive substances (TBARS).
Antioxidant Enzymes Activities
Glutathione reductase (GR) activity was determined based on the protocol developed by Carlberg and Mannervik . Briefly, GR reduces GSSG to GSH at the expense of NADPH, the disappearance of which can be followed at 340 nm. Glutathione peroxidase (GPx) activity was determined based on the protocol developed by Wendel  by indirectly measuring the consumption of NADPH at 340 nm. The GPx uses GSH to reduce the tert-butyl hydroperoxide, producing GSSG, which is readily reduced to GSH by GR (purified glutathione reductase) using NADPH as a reducing equivalent donor. Data are expressed as nmol of NADPH oxidized/min/mg protein.
Catalase activity was measured by the rate of decrease in hydrogen peroxide (H2O2; Vetec, Rio de Janeiro, Brazil) absorbance at 240 nm . Enzyme activity is expressed as μmol H2O2 consumed/min/mg protein.
Superoxide dismutase (SOD) activity was determined in striatal supernatant according to the methodology of Misra and Fridowich . The addition of tissue supernatants containing SOD inhibits the auto-oxidation of epinephrine, which is monitored at 480 nm. The amount of enzyme required to produce 50 % inhibition of epinephrine auto-oxidation was defined as one unit of enzyme activity. The SOD enzymatic activity was expressed as units (U)/mg protein.
Lipid Peroxidation Assay
Lipid peroxidation was assessed in striatal homogenates by the measurement of thiobarbituric acid-reactive substances (TBARS) formation, according to previous protocol described by Ohkawa et al. , in which malondialdehyde (MDA), an end-product of lipid peroxidation, reacts with thiobarbituric acid to form a colored complex. The samples were incubated at 100 °C for 60 min in acid medium containing 0.45 % sodium dodecyl sulphate and 0.67 % thiobarbituric acid. After centrifugation, the reaction product was determined at 532 nm using MDA as standard.
Western Blot Analysis
Tissues were homogenized (1:10 w/v) in ice-cold lysis buffer (50 mM Tris–HCl pH 7.5, 1 % Triton X-100, 100 mM NaCl, 5 mM EDTA pH 8.0, 40 mM β-glycerolphosphate, 50 mM NaF, 200 μM orthovanadate, 5 % glycerol and protease inhibitors). The homogenates were centrifuged at 13,000×g, at 4 °C for 45 min. Prior to western blot analysis, equivalent amounts of proteins were mixed in buffer (Tris 200 mM, glycerol 10 %, SDS 2 %, β-mercaptoethanol 2.75 mM and bromophenol blue 0.04 %), boiled for 5 min and kept at −20 °C until western blot analyses.
Proteins (40 μg) were separated by SDS-PAGE in 10 % gels. Proteins were detected immunologically following electrotransfer onto nitrocellulose membranes (Amersham-Pharmacia Biotechnology, USA). Protein and molecular weight markers (Bio-Rad, Mississauga, Canada) were revealed by Ponceau Red staining. Membranes were blocked in PBS containing 5 % powdered milk and 0.05 % Tween-20 for 1 h at 25 ± C. Membranes were then incubated overnight at 4 °C with anti-TH antibody (sc-7847; Santa Cruz Biotechnology, USA), anti-synaptophysin antibody (sc-9116; Santa Cruz Biotechnology, USA) or anti-β-actin (sc-4778; Santa Cruz Biotechnology, USA) in blocking solution and after that with horseradish peroxidase-conjugated anti-rabbit (SYP), anti-goat (TH) or anti-mouse (β-actin) IgG antibody for 1 h. Blots were visualized using the PerkinElmer ECL system. Optical density (O.D.) of the western blotting bands was quantified using the Scion Image® Software (Scion Corporation, Frederick, MD, USA). Values from TH and SYP bands were normalized with respect to β-actin bands.
The protein content was quantified by the method of Bradford , using bovine serum as a standard.
Data are presented as mean ± SEM. After Grubbs test (to discriminate potential outliers), Kolmogorov–Smirnov test was performed to detect normal (Gaussian) distribution. Most of the data was analyzed by two-way analysis of variance (ANOVA). Following significant ANOVAs, multiple post hoc comparisons were performed using the Fisher LSD post hoc test. Western blot data, which did not present normal distribution, were analyzed by the nonparametric Kruskal–Wallis test. Differences were considered significant when P ≤ 0.05. Statistical analysis of the data was performed using the STATISTICA software system, version 8.0. (StatSoft, Inc., 2008). All graphics were made using the GraphPad Prism (GraphPad Software, San Diego, CA, USA).
Probucol Prevents Hyperlocomotion Induced by 6-OHDA
Probucol Prevents Striatal Oxidative Stress Induced by 6-OHDA
Cellular antioxidant enzymes such as superoxide dismutases (SODs), catalase, GPx, and GR play important roles in cellular defense against oxidative damage by ROS . Specifically, SOD coverts superoxide radicals to form H2O2, and catalase and GPx are involved in the detoxification of H2O2. GPx oxidizes GSH in this process, which in turn is converted back to its reduced form in a reaction catalyzed by GR . Thus, in another set of experiments, we investigated whether 6-OHDA modulates antioxidant enzymes in the striatum of mice. Firstly, we observed main effects of 6-OHDA factor (F(1,19) = 13.45; P < 0.01) on catalase activity. Specifically, 6-OHDA induced a significantly increase in striatal catalase activity, which was remarkably attenuated by probucol treatment (Fig. 2b). Moreover, we observed a significant main effect of 6-OHDA factor (F(1,16) = 20.80; P < 0.001) on SOD activity. Specifically, 6-OHDA induced a significantly decrease in striatal SOD activity, but this effect was not attenuated by probucol treatment (Fig. 2c).
Probucol Prevents Striatal Toxicity Signs Induced by 6-OHDA
Increasing evidence points to oxidative stress as an early and causal event of PD [8, 9]. In fact, analyses of postmortem brain tissue from PD patients and of brain tissues from animal models have shown that lipid peroxidation , oxidative DNA damage , and protein carbonylation [47, 48] induced by oxidative stress might represent causative factors in PD. This hypothesis has led researchers to hypothesize that the therapeutic use of antioxidants may be beneficial in aging and neurodegenerative disorders, such as PD [9, 49]. In this study, we have examined the effects of probucol, a non-statin lipid lowering drug with anti-inflammatory and antioxidant properties, on the subsequent vulnerability of dopaminergic neurons to oxidative stress induced by 6-OHDA. We found that probucol decreases the oxidative stress and signs of neurotoxicity (such as hyperlocomotion and decreased levels of TH and synaptophysin in the striatum) observed in 6-OHDA-exposed mice.
6-OHDA is a selective catecholaminergic neurotoxin that has been widely used to produce in vivo PD models [17, 50]. The toxic mechanism of this compound is dependent on its oxidation with concomitant production of ROS and para- and semiquinone products that are able to induce oxidative stress [51, 52]. The selective loss of central dopamine neurons induced by 6-OHDA administration produces neuroplasticity in the striatum, thus altering the regulation and function of dopaminergic system [39, 40]. For instance, several compensatory mechanisms, such as the increase of dopamine D2 postsynaptic receptor density, are activated in response to the lesion to compensate for the decrease of dopamine . In the present study, we showed that after 7 days of i.c.v. infusion of 6-OHDA, mice developed hyperlocomotion in the activity cages. Although this result was unexpected, one might posit that compensatory mechanisms may initially mask underlying motor deficiencies that arise as a result of altered striatal output. Nonetheless, the hyperlocomotion induced by 6-OHDA administration was temporally correlated with a significant reduction in the levels of the enzyme TH in the striatum of mice. Notably, the present findings demonstrated that probucol administration was able to prevent both hyperlocomotion and the decrease in TH levels in the striatum of 6-OHDA-infused mice, suggesting a potential link between both events.
As discussed above, the nigrostriatal damage caused by 6-OHDA is dependent on its oxidation with concomitant production of 6-OHDA quinine and H2O2, ·O2− and ·OH [18–20]. Accordingly, in the present study we showed that the i.c.v. 6-OHDA infusion increased lipid peroxidation as well as catalase activity in striatum of mice as compared to control group. Indeed, brain cells are vulnerable to oxidative stress and then excess generation of ROS may alter the antioxidant defense mechanism in the cells and increase the lipid peroxidation [54–56]. Although catalase has a low activity in the brain, it could became more important in removing H2O2 than GPx, mainly when there is a high concentration of H2O2, since the kM of catalase for H2O2 is much higher than the GPx one [57, 58]. Of particular importance, a previous study from our group showed that catalase activity was increased in the brain of animals exposed to methylmercury, as a compensatory response to the pro-oxidative properties of this toxicant . Thus, catalase seems to be highly responsive to an increased ROS burst. Interestingly, in that study, methylmercury-induced catalase up-regulation was abolished by the co-treatment with a thiol-peroxidase mimetic (Ebselen), reinforcing the potential cooperative actions of GPx and catalase .
On the other hand, 6-OHDA caused a significant decrease in striatal SOD activity. This enzyme has an important role in catalyzing the dismutation of superoxide anion (O2·−) to H2O2, which can be detoxified by catalase . In our study, catalase activity was increased in response to 6-OHDA treatment. Accordingly, experimental evidence indicates that events leading to oxidative stress and increased H2O2 levels can lead to increased catalase activity [59, 61], probably as a compensatory response to counteract H2O2-mediated oxidative damage. Although striatal SOD activity was decreased in our protocol, this decreased was likely not enough to avoid increased H2O2 levels, which can be resultant from either disrupted mitochondrial function or dopamine oxidation . Moreover, here we showed that probucol, which blunted 6-OHDA-induced catalase up-regulation, also displayed a main effect in increasing striatal GPx and GR activity. Although we observed a positive modulatory effect of probucol toward GPx-1 activity, corroborating previous findings , the direct capability of probucol in scavenging ROS  cannot be ruled out as a potential mechanism by which it prevents 6-OHDA-induced striatal lipid peroxidation and, consequently, signs of striatal neurotoxicity (i.e., hyperlocomotion and decreased TH and synaptophysin levels).
Overall, our results disclose novel molecular mechanisms associated with the pharmacological properties of probucol , thus demonstrating the potential therapeutic properties of probucol for pathological conditions related to increased peroxides levels, such as PD [65, 66]. Moreover, our data are of particular relevance taking into account the actual scenario of the pharmacological studies with probucol, in which clinical studies have shown its promising antiatherogenic and anti-diabetic  properties.
The financial supports by (1) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), (2) IBN.NET/CNPq, (3) PRONEX-CNPq/FAPESC (NENASC project), (4) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and (5) INCT-CNPq-Excitotoxicity and Neuroprotection are gratefully acknowledged. M.F. and C.P.F. are recipients of CNPq fellowships.
Conflict of interest
The authors have no financial or personal conflicts of interest related to this work.