Implicación de la oxido nitrico sintasa (nos) en la enfermedad de parkinson. Estrategias neuroprotectoras con antagonistas sintéticos de la nos

Zusammenfassung

SUMMARY Participation of nitric oxide synthase (NOS) in Parkinson Disease. Neuroprotective strategies with synthetic antagonists of NOS. Group of Investigation CTS-101: Intercellular Comunication Institute of Biotecnology Center of Biomedical Research Technologyc Park os Health Department of Physiology Medicine Faculty UNIVERSIDAD DE GRANADA Ana López Ramírez 2011 Introduction The participation of factors such as oxidative/nitrosative stress, excitotoxicity, inflammation, and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson's disease (PD) may be studied in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. The active glial metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+), is taken up into the dopaminergic neurons through the dopamine transporter, and it accumulates in the mitochondria of the substantia nigra pars compacta (SNpc) (Przedborski et al., 2004). Here, MPP+ binds to and inhibits complex I of the electron transport chain (ETC) (Greenamyre et al., 2001), which in turn increases the production of reactive oxygen species (ROS), leading to a sustained oxidative damage to the ETC, ATP reduction, and nigral cell death (Przedborski et al., 2004;Tretter et al., 2004). MPP+ also induces microglia activation and iNOS expression in the SN of mice (Liberatore et al., 1999), producing large amounts of NO¿ and neuronal death (Brown et al., 2006). Here, NO¿ rapidly reacts with O2¿- generating the highly toxic peroxynitrite (ONOO), which impairs the mitochondria, resulting in the irreversible inhibition of all ETC complexes (Brown & Borutaite, 2002), and neuronal death (Zhang et al., 2006). Although it is yet unclear why the inflammatory reaction occurs in PD, recent data support that the Ros produced in the mitochondria may induce this response (Zhou et al., 2011) The presence of both constitutive (c-mtNOS) and inducible (i-mtNOS) NOS isoforms in the mitochondria of several tissues has been recently showed (Escames et al., 2003). A correlation between i-mtNOS expression and mitochondrial failure during inflammation has been also reported (López et al., 2006;Escames et al., 2003). In turn, iNOS mutant mice, which also lack i-mtNOS, are protected against MPTP toxicity (Dehmer et al., 2000). These data further support the existence of an inflammatory process in the pathogenesis of PD, and suggest a role for i-mtNOS in the mitochondrial dysfunction. By contrast, it has been suggested that the neuronal damage in the ST during PD mainly depends on the excitotoxicity derived from the nNOS (Liberatore et al., 1999). Melatonin (aMT) exerts important antioxidant and antiinflammatory actions. The antioxidant actions involve direct scavenging of both reactive oxygen (ROS) nitrogen (RNS) species (Tan et al., 2000;Acuna-Castroviejo et al., 2001) and indirect up-regulation of antioxidant enzymes (Antolin et al., 1996). The antiinflammatory actions of aMT depend on the inhibition of the expression of iNOS and, here, i-mtNOS (Crespo et al., 1999). It was reported that the brain aMT metabolite N-acetyl-5-methoxykynuramine (AMK) (Hirata et al., 1974) is a better antioxidant than its precursor aMT (Reiter et al., 2007), and it is a highly efficient NO scavenger, forming a stable product that does not easily redonate NO (Hardeland & Pandi-Perumal, 2005). Both compounds, aMT and AMK, easily cross the brain blood barrier after their systemic administration, reaching neuronal and glial cells (Borlongan et al., 2000;Leon et al., 2006). Previous reports showed that aMT prevents dopamine autooxidation (Khaldy et al., 2000) and exerts neuroprotective properties in different types of neurodegeneration, including the MPTP model of PD (Acuna-Castroviejo et al., 1997;Thomas & Mohanakumar, 2004;Khaldy et al., 2003). Although iNOS participates in the inflammatory reaction, the role of i-mtNOS in mitochondrial dysfunction and nigrostriatal degeneration during PD remains unclear. Moreover, increasing evidence suggest that the pharmacological inhibition of iNOS/i-mtNOS could be a promising therapetical approach to the treatment of PD. Hypothesis and Aims A direct relationship between inflammation, iNOS expression and mitochondrial damage exist in PD, and these events occur before dopaminergic degeneration is observable in this disease. After MPTP administration, the induction of iNOS in SN increases i-mtNOS and NO production in mitochondria from SN, which in turn potentiates the MPP+-dependent complex I inhibition. The pathogenic events in the ST after MPTP administration, however, seems to depend more directly from the increase in nNOS and the subsequent NO-dependent excitotoxicity. Accordingly, the SN of iNOS deficient mice and the ST of nNOS deficient mice, respectively, should be more resistent to the damage induced by MPTP administration. Melatonin, which inhibits the expression and activity of iNOS and nNOS, should display neuroprotective features against MPTP administration to mice. The development of new neuroprotective drugs is being braking by our relative lack of knowledge regarding the mechanisms involved in neuronal cell death in PD. Because previous data showed that melatonin reduces iNOS activity and improves the mitochondrial function, the indoleamine may be used as template for the design and synthesis of new active molecules against iNOS/i-mtNOS, which may yield new therapeutical drugs for PD treatment. Thus, we considered to examine, in the MPTP model of PD in mice 1.- The capacity and selectivity of a series of synthetic compounds, structurally related to melatonin and its brain metabolites, on the activity of NOS isoforms "in vitro". 2.- The effect of some of these synthetic compounds on NOS activity, complex I activity and lipid peroxidation in the SN and ST of mice treated with MPTP. 3.- The effects of melatonin on the mitochondria (complex I activity, respiratory complexes expression), NOS activity and mitocondrial DNA damage, in wild type mice, iNOS, and nNOS deficient mice treated with MPTP. Materials and methods Animals and Treatments C57/Bl6 mice weighing 28-30 g (3-4 months old) were obtained from the University of Granada's facility and housed in clear plastic cages maintained at 22°C - 28°C and 40% humidity, with a 12-hr light/dark cycle. Mice were feed with tap water and a standard diet ad libitum. All experiments were performed according to the Spanish Government guide and the European Community guide for animal care. Mice were divided into the following groups: 1) control group, injected with vehicle (ethanol/saline); 2) MPTP group; 3) MPTP + aMT, and 4) MPTP + synthetic compound to be asssayed. Four doses of 15 mg/kg MPTP dissolved in 30 µl saline were injected (s.c.) to mice at the the first day, with 2-hr intervals between them, following 24 hr later by three additional injections of the same MPTP dose and with the same time interval. Melatonin and synthetic compounds (10 mg/kg dissolved in 30 ¿l of 2.5% ethanol/saline (v/v) solution) were injected (s.c.) 1 hr before the dose of MPTP. Thirty-two hours after starting treatments, the animals were sacrificed by cervical dislocation, their brains were removed, and fresh SN and ST were dissected for subsequent mitochondria preparation. Mitochondrial pellets were stored at -80°C until assays. Isolation of Cytosol and Pure Mitochondria Mitochondrial and cytosolic fractions of ST and SN were prepared as described elsewhere (Rice J.C. & Lindsay J.G., 1997), with minor modifications. All procedures were carried out at 0-4 °C. Briefly, ST and SN were dissected, weighed, washed with saline, placed into ice-cold buffer (25 mM Tris, 0.5 mM DTT, 10 ¿g/ml aprotinin, 10 ¿g/ml leupeptin, 10 ¿g/ml pepstatin, 1 mM PMSF, pH 7.6) and homogenized (10%, w/v) at 700 rpm in a Teflon pestle. The homogenates were centrifuged twice at 1,300 g for 3 min, and the supernatants were mixed and centrifuged at 21,200 g for 10 min, yielding the crude mitochondrial (pellet) and cytosol fractions. The supernatants of this second centrifugation, which correspond to the crude cytosolic fraction, were frozen at at -80°C. The crude mitochondrial pellets were suspended in 15% v/v Percoll prepared in isolation buffer (0.32 M sucrose, 1 mM EDTA-K1, 10 mM Tris-HCl, pH 7.4), in a proportion of 1 g original brain homogenate/10 ml Percoll. Then, this mixture were carefully layered onto a discontinuous Percoll density gradient consisting in 1 ml of 23% (v/v) Percoll layered onto 1 ml of 40% (v/v) Percoll. The samples were centrifuged at 28,200 g for 12 min in an angle-flexed rotor. The bands in the interface between 15% and 23% Percoll layers were aspirated with a syringe, carefully diluted 1:1 with isolation medium, and centrifuged at 16,800 g for 10 min. The pellet was washed in 1 ml isolation buffer to yield a highly pure mitochondrial preparation without contaminating organelles and broken mitochondria (Lopez et al., 2006). Assay of Cytosolic and Mitochondrial NOS Activity The samples from cytosol and mitochondria were either stored at -80 °C for total protein determination (Bradford, 1976) or used immediately for NOS activity assays, monitoring the conversion of L-[3H]arginine to L-[3H]citrulline (Bredt & Snyder, 1989). The final incubation volume was 100 ¿l and comprised 10 ¿l cytosol or mitochondrial samples added to a 25 mM Tris buffer containing 1 mM DTT, 30 ¿M H 4-biopterin, 10 ¿M FAD, 50 nM L-[3H]arginine, 10 ¿M L-arginine, 0.5 mg/ml BSA, 0.5 mM inosine, and 0.1 mM CaCl2 (final concentrations), pH 7.6. The reaction was started by the addition of 10 ¿l NADPH (0.75 mM final concentration) and continued for 30 min at 37°C. Control incubations were performed in the absence of NADPH. To determine the Ca2+-independent, iNOS activity, 10 mM EDTA was added to the reaction medium. The reaction was stopped by the addition of 400 ¿l cold 0.1 M Hepes containing 10 mM EGTA, 1 mM L-citrulline, pH 5.5. The reaction mixture was decanted into a 2 ml column packed with Dowex-50W ion exchange resin (Na+ form) and eluted with 1.2 ml distilled water. L-[3H]citrulline was quantified by liquid scintillation spectroscopy. The retention of L-[3H]arginine by the column was greater than 98%. Specific enzyme activity determined by subtracting the control value, which usually amounted less than 1% of the radioactivity added. NOS activity was expressed as pmol of L-[3H]citrulline/min/mg protein. Lipid Peroxidation Assay For lipid peroxidation (LPO) measurements, cytosolic aliquots were thawed and treated with ice-cold 10 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA-K, whereas mitochondrial fractions were thawed, suspended in ice-cold 10 mM Tris-HCl buffer, pH 7.4, containing 0.32 M sucrose and 1 mM EDTA-K 1, and sonicated to break mitochondrial membranes. Aliquots of the samples were either stored at -80°C for total protein determination (Bradford, 1976) or used for LPO assay. For this purpose, a commercial LPO assay kit able to determine both malondialdehide (MDA) and 4-hydroxyalkenals (4HDA) was used (Bioxytech LPO-568 assay kit; OxisResearch, Portland, OR; Esterbauer and Cheeseman, 1990). LPO concentration is expressed in nmol/mg protein. Complex I (NADH:Ubiquinone Oxidoreductase) Assay To prepare submitochondrial particles, mitochondrial pellets were frozen and thawed twice, suspended in 160 ¿l of the incubation medium, and sonicated. Complex I activity (NADH CoQ oxidoreductase) was measured in the presence of decylubiquinone and succinate as the rotenone-sensitive decrease in NADH. Brielyy, an aliquot of the submitochondrial suspension (0.5 mg protein/ml, final concentration) was added to the reaction mixture containing 0.25 M sucrose, 50 mM KH2PO4, pH 7.4, 1 mM KCN, 10 ¿g/ml antimycin A, and 50 ¿M decylubiquinone. After preincubation for 3 min at 25 °C, the reaction was initiated by the addition of NADH (100 ¿M, final concentration), and the rate of decrease in the absorbance was monitored at 340 nm for 1.5 min. The NADH oxidase activity measured with this method corresponds to complex I was further confirmed insofar as incubation in the presence of 1 ¿M rotenone completely abolished the enzyme activity. The complex I activity was expressed as nmol of NADH oxidized/min/mgprot. mtDNA quantification Mouse mtDNA was quantitated by real-time PCR using using primers and probes for murine COX I gene (mtDNA) and mouse glyceraldehyde-3-phosphate dehydrogenase (nDNA) (Spinazzola et al., 2006). The values mtDNA levels were normalized by nDNA, and the data were expressed in terms of percent relative to wild-type mice. Western blot analyses Mice SN and ST were dissected and homogenized in 0.5 mM PMSF, 50 mM Tris/HCl (pH 7.6), 2 mM DTT, 5 mM benzamidin, 5% NP-40 and 2.5% glycerol. Thirty micrograms of the extracts were electrophorezed in an SDS 12% PAGE gel, transferred onto Immun-Blot PVDF membranes and probed with Rodent Total OXPHOS Complexes Detection Kit cocktail of antibodies (MitoSciences, Eugene, OR, USA). Protein antibody interactions were detected with peroxidase-conjugated mouse anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA), using SuperSignal chemi-luminiscence detection kit (Thermo Fisher, Waltham, MA, USA). Quantication of proteins was carried out using a Kodak 2000R image station. Statistical Analysis Eight SN and ST (corresponding to four mice) were mixed and processed together as one sample to obtain a minimal amount of mitochondria for analytical purposes. Thus, the data represent the mean of six experiments (corresponding to 24 mice) performed in triplicate. One-way ANOVA I followed by Student-Newman-Keuls multiple-comparisons test was used for statistical purposes. P < 0.05 was considered statistically signicant. Disscusion Neurotoxicity of MPTP In the present study the neurotoxin MPTP has been used to induce parkinsonism in mice. This model reproduces most of the biochemical and pathophysiological features found in patients of Parkinson's disease, allowing us to study the mitochondrial dysfunction that occurs in this disease. Physiologicaly, an equilibrium exists between the production and elimination of free radicals. But in conditions such as the neuronal dopaminergic damage induced by MPTP administration, the formation of free radicals surpasses the endogenous antioxidative capacity and, consequently, a hyperoxidative status is originated (Jenner, 2003). Increasing evidences suggest that oxidative stress is directly implied in neurodegenerative events. Mitochondrial oxidative damage is a typical feature of PD, and it constitutes the common final pathway of the multiple pathophysiological events in this disease (Atlante et al., 2001). The complexes I and III of the ETC are the main locus of ROS production (Rock ET to., 2001; Liu ET to., 2002). These ROS, which include O2¿, OH¿ and H2O2, are sequently produced by the partial reduction of oxygen by one, two and three electrons, respectively (Vera Adam-Vizi & Christos Chinopoulos, 2006). High levels of O2¿ favor their reaction with the NO yielding ONOO, a highly toxic nitrogen radical that irreversibly damages the four complexes of the ETC (Boczkowski et al., 1999;Germaine Escames et al., 2003). The MPP+, the active form of the neurotoxin MPTP, selectively inhibits to the complex I, contributing to the ROS generation by this respiratory complex. Moreover, the SN contains high levels of iron that may lead to the OH¿ formation through the Fenton's reaction, increasing the OH¿ production in dopaminergic neurons (Speciale, 2002). Thus, basal ROS production by the mitochondria is amplified by MPP+ and the Fenton's reaction. In addition, the ATP reduction caused by the inhibition of complex I is related to an increase of the toxicity by glutamate, releasing the blockade of the NMDA by Mg2+. Consequently, the Ca2+ flux increases within the cell activating nNOS and NO¿ production, reducing even more the activity of the complex I (Brown et al., 2006; Escames et al., 2004). In our study, we verified that the MPTP administration increased the total activity in SN by 256%, and of 192% in ST. In both cases, the total NOS activity increased due to the inducible component, iNOS. These data further confirm the existence of an inflammatory response in SN of mice after MPTP administration elsewhere reported (Liberatore et al., 1999). Of note, is the increased activity of i-mtNOS in these conditions, which was responsible for the high levels of mitochondrial NO¿ found in this model of parkinsonism. The presence of high levels of NO¿ in the mitochondria are involved in the death of the dopaminergic neurons by nitrosative/oxidating damage (Zhang et al., 2006). In our study we observed that after MPTP administration, the activity of the complex I decreases in SN (66%) and, in a lesser extent, in ST (26%) of wilt-type mice. Interestingly, mice lacking iNOS or nNOS showed a comparable complex I inhibition after MPTP administration. Even more, the inhibition of the complex I by MPTP was similar in wild-type and mutant mice. Thus, although the inflammatory process in PD can be involved in the mitochondrial dysfunction and neuronal death (Barcia & Herrero Ezquerro, 2004;Hunot & Hirsch, 2003), the absence of iNOS/i-mtNOS does not influence the activity of the complex I in mice with MPTP treatment, probably because the inhibition is maintained by the presence of MPP+. In this regard, data from septic mice showed that complex I activity is inhibited due to the inflammatory process, and this inhibition disappeared in iNOS-deficient mice (Escames et al., 2006), recovering the ATP production (Lopez et al., 2006). The inflammation-dependent complex I inhibition was related to the NO produced by the i-mtNOS, which was also induced during the inflammatory status (Lopez et al., 2006). Consequently, although we prevented the inflammatory response caused by MPTP in mice lacking iNOS, whereas we do no remove the MPP+, the complex I remains inhibited. The reduced activity of the complex I by MPP+ increases ROS production, an event reflected in our data by the high LPO levels found in these conditions. LPO levels were more elevated in the SN than in ST. The higher induction of iNOS/i-mtNOS in SN, together with a lower complex I activity in this nucleus compared with ST, explain the higher LPO and oxidative stress in the former. Again, iNOS/i-mtNOS-dependent NO production constitutes a pathogenic event of major importance in PD. The mitochondrial hyperoxidative status originated by MPTP administration causes oxidative injury to macromolecules. Besides LPO, another important target for oxidative damage is the mtDNA. Our data show that SN of wild-type animals resulted in a significant reduction in the amount of mtDNA after MPTP administration, and this reduction was comparable to that occurred in nNOS-deficient mice. Mice lacking iNOS, however, did not show changes in mtDNA integrity. In this case, we can observed a tendency to increase mtDNA in iNOS mutant mice. These data suggest that the iNOS/i-mtNOS induction after MPTP could participate in the mtDNA damage. How the iNOS induction and the inflammatory response occurs in PD is unclear, but based on recent data, we can suggest that the cause of iNOS induction probably depend on the ROS generated by the impaired mitochondria due to MPP+-inhibition of complex I (Zhou et al., 2011). Based on these results, three main targets could be proposed for pharmacological intervention in PD: 1) recovering the activity of complex I; 2) inhibiting the formation of ROS in mitochondria, and 3) reducing the production of NO¿. Effects of melatonin treatment against MPTP neurotoxicity Previous data reported that melatonin increases the efficiency of the mitochondrial ETC, harnessing the activity of the four respiratory complexes in a dose-dependent manner (Lopez et al., 2009). Melatonin administration also counteracts the inhibition of complex I induced by MPTP (Khaldy et al., 2003; Srinivasan et al., 2005), recovering the production of ATP and the cellular survival. Here, we found that melatonin administration recovered the complex I activity reduced by MPTP, and this effect was independent of the mice strain used; i.e., the presence or absence of iNOS/nNOS was unrelated to the melatonin action. Nevertheless, melatonin counteracted the expression of iNOS/i-mtNOS induced by MPTP, reducing the inflammatory response to the neurotoxin. Thus, the effect of melatonin concerning mitochondrial complex I seems to involve a direct mechanism and not depending on its anti-inflammatory properties. It is probably that the first event in melatonin action was the recovery of complex I activity; then, mitochondria reduced ROS generation, counteracting the inflammation (Zhou et al., 2011). Regarding to the expression of the respiratory complexes, the changes detected in the protein amount of each complex were small and similar in SN and ST. Melatonin administration tends to increase the expression of these complexes, a finding that could be partially related to its ability to prevent the mtDNA depletion induced by MPTP. Of note, the expression of complex I and III was reduced in iNOS deficient mice and increased in nNOS deficient mice. Therefore, it seems that the absence of NO¿ derived from iNOS inhibits the expression of the complex I, whereas the lack of NO¿ derived from nNOS increases its expression. These results open new pathways involved in the regulation of the respiratory complexes that should be further analyzed. The beneficial effects of melatonin against the mitochondrial dysfunction induced by MPTP here reported, support its utility in patients with PD. Moreover, melatonin itself may be used as template for the design and synthesis of new molecules with neuroprotective properties. Effects of synthetic molecules against MTPT neurotoxicity The following challenge was to find drugs targeting the mitochondria and/or reducing iNOS/i-mtNOS expression. For this purpose, iNOS/i-mtNOS and/or nNOS/c-mtNOS selective antagonists should be obtained. In this regard, we studied a series of synthetic compounds structurally related to melatonin and its brain metabolites, to assed their properties as selective iNOS and nNOS antagonists in vitro (Encarna Camacho et al., 2002;Camacho et al., 2004;Carrion et al., 2004;Entrena et al., 2005). From these studies, several interesting molecules were obtained. Some of them are being analyzing now for their neuroprotective properties. Among them, pyrazoline QFF-124 and pyrrols QFF-205 and QFF-212 were evaluated in vivo against mitochondrial dysfunction in the MPTP model of PD. The results obtained can be summarizes as follows: ¿ All compounds recovered the control level of the activity of complex I in SN and ST inhibited by MPTP, being especially important the effect of QFF-124. ¿ All compounds showed an inhibitory effect on iNOS activity induced by MPTP, mainly in SN. The greatest effect was found with the pyrrol compounds, followed by the pyrazoline. ¿ All compounds reduced the LPO levels induced by MPTP in cytosol and mitochondria of SN and ST, being pyrrols QFF-205 and QFF-212, the most effective. These results drive us to follow the search for neuroprotective compounds using this experimental approach. CONCLUSIONS 1. Besides complex I inhibition, MPTP administration increases cytosolic and mitochondrial iNOS, with a parallel increase in NO¿ and LPO levels. These changes reproduce some of the pathogenic features of PD, and confirm the existence of an inflammatory process in this neurologic illness. 2. The mitochondria from ST and SN possess a mtNOS with a constitutive (c-mtNOS) and inducible (i-mtNOS) components. The administration of MPTP increases mainly the inducible form. 3. The results obtained with iNOS and nNOS knockout mice showed that complex I activity remains low after the MPTP administration, independently of the presence or absence of these NOS isoforms. Thus, although the inflammatory process can be involved in the neuronal cell death and mitochondrial dysfunction in PD, we cannot detect any improvement in complex I activity in NOS deficient mice, probably due to the presence of MPP+, which maintains the inhibition of the complex I. 4. Melatonin administration counteracts the deleterious effects of MPTP, recovering the activity of complex I, inhibiting the activity of iNOS/i-mtNOS, and, therefore, the production of NO¿. Moreover, melatonin administration also normalizes the mitochondrial redox status, preventing the mitochondrial depletion of DNA. These results support the neuroprotective properties of melatonin. 5. The efefcts of melatonin were similar in iNOS/nNOS wild-type and mutant mice. Therefore, the effect of melatonin on complex I seems to be direct, independently of the antioxidant/anti-inflammatory properties of the indoleamine. 6. The changes found in the expression of the respiratory complexes indicate that NO¿ seems to control, at some extent, the expression of the complex I and V, without affecting the other respiratory complexes. These results further support that the modulation of the CTE by NO¿ goes beyond the simple direct regulatory paper of this gas on the activity of the complex IV. 7. The data obtained from the studies with the synthetic melatonin analogues have provided a series of compounds with selective activity against iNOS/nNOS in vitro. The results of in vivo studies show the neuroprotective capacity of some of them. These results open a new line in the search of new pharmacological agents with neuroprotective properties susceptible against PD.