Address correspondence and reprint requests to Li-Fang Hu, Ph.D., Institute of Neuroscience, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou 215123, P.R. China. E-mail: email@example.com Wei-Feng Luo, MD, Ph.D., Department of Neurology, Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, China. E-mail: firstname.lastname@example.org
Higher plasma urate level is reported to be associated with a reduced risk and slower progression of Parkinson's disease (PD). In this study, we explored the effects of urate on dopaminergic neurons in nigrostriatal pathway in the 6-hydroxydopamine (6-OHDA) unilaterally lesioned rats. Uric acid (UA), when given twice daily at 200 mg/kg intraperitoneally for 10 consecutive days, elevated urate (the anionic form of UA) in plasma and striatum by 55% and 36.8%, respectively, as compared with vehicle group. This regimen of UA was found to ameliorate the behavioral deficits, dopaminergic neuron loss as well as dopamine depletion in the nigrostriatal system. Moreover, UA administration was capable of increasing glutathione level and superoxide dismutase activity while decreasing malondialdehyde accumulation in striatum. In addition, the phosphorylation of both protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) in the lesioned striata of 6-OHDA-lesioned rats was dramatically reduced as compared with sham-operated rats. This reduction was attenuated in the Parkinsonian rats receiving UA treatment. Similarly, in vitro findings showed that UA alleviated the decrease in Akt activation and the increase in GSK3β activity caused by 6-OHDA. Furthermore, neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9 was found to be abolished in the presence of PI3K inhibitor. Therefore, our findings demonstrated that urate was able to protect dopaminergic neurons in rat nigrostriatal pathway against the neurotoxicity of 6-OHDA, and showed that its beneficial effects may be related to its regulation on Akt/GSK3β signaling.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in substantia nigra (SN) and depletion of the neurotransmitter dopamine (DA) in striatum. Although it has been extensively investigated for many decades, the exact pathogenic factors responsible for dopaminergic neuron degeneration remain to be illuminated. Accumulating evidence suggests that oxidative stress and mitochondrial dysfunction are major contributing factors in the pathogenesis of PD (Jenner and Olanow 1998; Mizuno et al. 1998). In fact, these two pathogenic factors interact with each other. An increase in oxidative stress causes damage to mitochondria, resulting in moreaccumulation of reactive oxygen species (ROS), mitochondrial membrane permeabilization, energy depletion, and eventually cell death. Hence, strategies that aim to limit or clear reactive oxygen/nitrogen species have been proposed to relieve dopaminergic neuron degeneration in PD.
Urate is the anionic form of uric acid (UA) and an endogenous antioxidant in humans. Its antioxidant property is mainly attributed to its capacity to react with ROS, such as peroxynitrite, peroxides, and hypochlorous acid, and to chelate transition metal ions (Davies et al. 1986; Becker 1993). On the other hand, urate acts as a pro-oxidant under some circumstances. For instance, urate may enhance the copper-induced low-density lipoprotein oxidation (Patterson et al. 2003). In fact, altered serum urate concentrations have been linked to a number of disease conditions. An abnormally high urate level has been associated with gout, hypertension, cardiovascular disease, and renal disease (Johnson et al. 2003); whereas reduced urate concentration has been linked to PD, Alzheimer's disease, multiple sclerosis, and optic neuritis (Kutzing and Firestein 2008). In 1994, Church WH and Ward VL reported that urate was significantly lower by 54% in the SN of PD patient compared with age-matched controls (Church and Ward 1994). Consistently, other groups further demonstrated that besides SN, plasma urate levels were also lower in PD patients (Fitzmaurice et al. 2003; Annanmaki et al. 2007). In the meantime, several prospective and cohort studies have shown that higher plasma urate levels are associated with a lower risk of PD (de Lau et al. 2005; Weisskopf et al. 2007). The clinical findings further raised the possibility that the higher the urate level in serum, the slower the PD clinical progression (Schwarzschild et al. 2008). Thus, urate was postulated to be a neuroprotective agent. However, this assumption has not yet been verified in animal model and clinical trials, although several in vitro studies including our previous work have shown that urate could protect against 6-hydroxydopamine (6-OHDA)-induced cell injury via antioxidant mechanisms in PC12 cells (Zhu et al. 2012). Here, we further tested this assumption in 6-OHDA unilaterally lesioned rat model of PD with histochemical, behavioral, neurochemical, and also molecular techniques, by intraperitoneal (i.p.) injection of UA for 10 consecutive days to enhance plasma urate levels.
Material and methods
Drugs and chemicals
UA, 6-OHDA, mouse anti-tyrosine hydroxylase (TH) monoclonal antibody were purchased from Sigma (St Louis, MO, USA). Antibodies against Akt, phospho-Akt (Ser473), GSK3β, and phospho-GSK3β (Ser9) were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody against phospho-GSK3β (Tyr216) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Experimental procedure and animal surgery
Sprague–Dawley (SD) male rats (180–220 g), 130 in total, were purchased from the Center for Experimental Animals, Soochow University (certificate No 20020008, Grade II). Animals were housed at a 12-h light/dark cycle with free access to food and water. NIH Guidelines for the Care and Use of Laboratory Animals were followed in all animal procedures.
The overall experimental procedure was illustrated as below (Fig. 1). Our study comprised two parts of work. In the first part, the effects of UA injection via i.p. on both plasma and brain urate levels in rats were assessed. In brief, 70 male SD rats were used and randomly divided into five treatment groups, and subject to injection for five and 10 consecutive days, respectively. Rats were injected (i.p.) with UA at doses of 50, 100, 200, 400 mg/kg, or its vehicle twice daily 2 h apart (n = 7 for each treatment at one time point). On the 5th and 10th day, 1 h after last injection, blood was sampled via caudal vein and put into an anticoagulant tube. After centrifugation at 835 g for 10 min, plasma was transferred into Eppendorf tubes and stored at −80°C for urate measurement later. Rats were killed and brain tissues (striatum) were then harvested.
In the second part, the potential protection by urate at an appropriate dose, which was chosen in accordance with the results obtained from part one, in 6-OHDA-induced Parkinsonian rats were further investigated. In total, 60 rats were randomly divided into four groups (n = 15 for each group. Among these, 10 rats were used for behavioral test, histology study, and DA assay. The others were used for measuring oxidative parameters and western blot analysis of protein expression in striatum): sham-operated group, UA treatment plus sham-operated group, 6-OHDA-injected group, and UA treatment plus 6-OHDA-injected group. Briefly, rats were injected (i.p.) with UA or its vehicle twice daily for five consecutive days before and after stereotaxic surgery. On the 5th day between the two injections, all rats received a unilateral stereotaxic injection of 6-OHDA or saline into the right striatum. The injection was performed on anesthetized rats (3.6% chloral hydrate, 0.18 g/kg, i.p.) via a 10-μL Hamilton syringe using stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) according to rat brain atlas. The lesions were made by injection of 6-OHDA into the right striatum at two sites (10 μg 6-OHDA hydrochloride in 5 μL of 0.02% ascorbic acid saline solution, 5 μg 6-OHDA each site) with the coordinates: (i) AP, −0.7; L, −3.0; DV, −4.5 mm; (ii) AP, −0.2; L, −2.6; DV, −6.0 mm from bregma at the rate of 0.5 μL/min. At the end of injection, the needle was left in place for an additional 5 min and then withdrawn at a rate of 1 mm/min. Sham-lesioned rats were infused with 5 μL saline containing 0.02% ascorbic acid. After surgery, the rats were kept in cages with constant temperature (25°C) and humidity, and were exposed to a 12:12-h light/dark cycle with unrestricted access to tap water and food.
Plasma and striatum urate level determination in normal rats
Striatum tissues were homogenized and sonicated in normal saline (1 : 5, mg/uL) for 1 min and then centrifuged at 20 000 g for 15 min at 4°C. Protein concentrations were determined using the BCA kit (Pierce Chemical, Rockford, IL, USA). The urate levels in plasma and striatum were assessed using the Uric Acid Assay Kit from Bio Vision, according to the manufacturer's instructions. The urate levels in plasma and striatum were expressed as μmol/L and nmol per mg wet tissue, respectively.
Deficits in forepaw adjusting steps in PD rat model provide a simple and consistent behavioral phenomenon similar to akinesia in PD. Forepaw adjusting step was measured once a week after 6-OHDA lesion as previously described (Chang et al. 1999). The rat was held by the rear part of torso and placed on treadmill surface so that its weight was on one forepaw. The treadmill was set to move at a rate of 90 cm/5 s in the direction opposite to the weight-bearing forepaw, resulting in the outward lateral shifting of the torso relative to the weight-bearing forepaw. The number of forepaw adjusting steps, defined as the movement of weight-bearing forepaw toward torso to compensate for the outward lateral movement of body, was counted. Each stepping test consisted of five trials for each forepaw, alternating between forepaws, and each trial lasted 5 s. The average of five trials for each forepaw was used for analysis.
Formalin-fixed and paraffin-embedded sections (5 μm in thickness) received deparaffinization and rehydration treatments. Endogenous peroxidase activity was inactivated with 0.3% H2O2 for 30 min. The sections were then blocked with 5% normal goat serum in 1% bovine serum albumin for 30 min and incubated with a mouse monoclonal TH antibody (1 : 5000) at 4°C overnight. The slides were incubated with biotin-conjugated anti-mouse IgG and thereafter, streptavidin-horseradish peroxidase (1 : 1000) for another 1 h. Immunoreactivities were finally developed with diaminobenzidine solution and sections were counter-stained with Mayer's Hematoxylin, dehydrated, and mounted. The sections were observed and photographed using a Zeiss microscope (AXIO SCOPE A1; Zeiss Corp, Goettingen, Germany). The number of TH+ neurons in SN was manually counted using a superimposed grid to facilitate the procedure. At least five sections for each rat were examined and counting was performed by researchers blind to the treatments received.
Determination of DA and its metabolites in striatum
The levels of DA and its metabolites 3,4-dihydroxyphenyl-acetic acid (DOPAC), and homovanilic acid (HVA) in striatum were determined by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). HPLC was carried out on an Agilent 1100 system (Agilent Technologies, Palo Alto, CA, USA) consisting of a vacuum degasser, a quaternary pump, and an autosampler. An API 4000 triple-quadrupole mass spectrometer (Applied Biosystems Sciex, Ontario, Canada) equipped with a Turbo IonSpray ionization (ESI) source was used for mass analysis and detection. Data acquisition and integration were controlled using Applied Biosystems Analyst Software (Applied Biosystems/MDS SCIEX, version 1.4.2). The tissue was homogenized and sonicated in a mixture of normal saline and methanol (1 : 1, v/v, 10 uL mixture per mg brain tissue) for 1 min and then centrifuged at 18 000 g for 5 min at 4°C. After that, the supernatant was mixed with internal standard at the same volume, and was filtered (0.2 μm, Millipore, Billerica, MA, USA). A volume of 20 μL final solution was injected into HPLC–MS/MS system for analysis. The mobile phase was a mixture of water containing 0.015% formic acid and methanol (92 : 8, v/v), and was pumped at a flow rate of 0.3 mL/min under gradient elution. The precursor to product–ion transitions m/z 152.1→122.1, m/z 138.1→122.0, and m/z 180.9→137.0 were used to quantify DA, DOPAC, and HVA, respectively. The concentrations were expressed as ng/mg tissue.
Malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activity measurement
The striatal contents of MDA—a compound produced during lipid peroxidation, and SOD—an important antioxidant enzyme that plays a pivotal role in clearing ROS, as well as GSH were measured using commercially available detection kits (Nanjing Jiancheng Biochemical Reagent Co. Nanjing, China) according to the manufacturers' instructions.
Western blot analysis
Striatal tissues were homogenized in lysis buffer and centrifuged at 12 000 g for 30 min. Protein samples were heated at 97°C for 5 min before loading. Equal amounts of protein were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (PALL, East Hills, NY, USA). The resulting blots were blocked with 5% milk in Tris Buffer Saline Tween20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween-20, pH 8.0) for 1 h and then incubated at 4°C overnight with primary antibodies that recognize proteins of interest. Bound antibodies were detected by a secondary antibody conjugated to horseradish peroxidase and visualized by ECL chemiluminescence (GE healthcare, Buckinghamshire, UK).
Cell viability determination
Cell viability was measured using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. In brief, regular medium was replaced, and MTT at the final concentration of 0.5 mg/mL was added at the end of treatment. Cells were further incubated at 37°C for 4 h. After that the insoluble formazan was dissolved with dimethyl sulphoxide. The absorbance was finally measured at 570 nm with a reference wavelength at 630 nm using a microplate reader (Tecan M200; TECAN, GmbH, Austria).
All data were presented as mean ± SEM. Statistical significance was assessed with one-way analysis of variance followed by Dunnett's test for multiple group comparison. In addition, we further analyzed the time- and dose-dependent effects of the behavioral test results jointly by using a two-way anova followed by a several post hoc paired t-tests. Differences with p-values of less than 0.05 were considered statistically significant.
Intraperitoneal injection of UA raises urate levels in plasma and striatum
The temporal changes of plasma and striatal urate levels were determined and monitored before exploring the potential of urate neuroprotection. UA, at various doses of 0, 50, 100, 200, and 400 mg/kg was administered i.p. twice daily at 2-h intervals for five and 10 consecutive days, respectively. On the 5th and 10th day, the plasma and striatum urate levels were determined at 1 h after last injection. It was found that at doses above 200 mg/kg, UA administration was able to enhance the urate level both in plasma and striatum in a dose-dependent manner, as shown in Fig. 2a and b. Specifically, on the 5th and 10th day after injection with UA at 200 mg/kg twice daily, the plasma urate level increased by 52.9% (from 130.1 ± 0.46 μmol/L to 198.9 ± 0.71 μmol/L) and 55% (from 142.5 ± 0.49 μmol/L to 220.8 ± 0.58 μmol/L), whereas the urate level in striatum increased by 32.3% (from 6.8 ± 0.17 nmol/mg to 9.0 ± 0.18 nmol/mg) and 36.8% (from 6.9 ± 0.13 nmol/mg to 9.5 ± 0.12 nmol/mg), respectively.
UA treatment improves behavioral deficits in 6-OHDA-lesioned rats
To evaluate the effects of UA treatment on motor performance, the rats that received different treatments were subjected to forepaw adjusting step test for five times at 1 week interval after surgery. Sham-operated rats (S) served as controls and data were shown in Fig. 3. As anticipated, unilateral 6-OHDA injection caused a significant reduction in forepaw adjusting steps at the 4th and 5th week after surgery compared with controls. Post hoc analysis revealed that UA treatment (200 mg/kg twice daily for five consecutive days before and after surgery) to 6-OHDA-lesioned rats significantly improved stepping in the forepaws when compared with saline-treated rats. Of note, UA treatment at the same regimen to the sham-operated rats did not affect the forepaw adjusting steps.
UA treatment alleviates dopaminergic neuron loss in the nigrostriatal pathway in 6-OHDA-lesioned rats
We continued to examine the effect of UA on dopaminergic neuron degeneration in the nigrostriatal pathway in intrastriatal 6-OHDA unilaterally lesioned rats with immunohistochemistry study and western blot analysis as well. As shown in the representative pictures in Fig. 4a and c, unilateral 6-OHDA lesion induced a dramatic loss of TH+ neurons in both SN pars compacta (SNc, Fig. 4a) and striatum (Fig. 4c) in the lesioned side. However, there was no significant reduction of TH+ staining in the intact hemisphere and in sham-lesioned rats. UA, when given twice daily at 200 mg/kg intraperitoneally, markedly attenuated the loss of TH+ neurons in SN and fibers in striatum caused by 6-OHDA injection. The observation was verified by counting the number of TH+ neurons in SN (Fig. 4b), and by western blot analysis of protein lysates from striatum (Fig. 4d). As can be seen from Fig. 4b, the number of TH+ neurons in the 6-OHDA-lesioned side was reduced by 45.4% as compared with sham-lesioned side, while it merely reduced by 28.2% in the 6-OHDA-lesioned side of rats receiving UA treatment. Western blot analysis also showed that TH expression in the lesioned striata of 6-OHDA-treated group decreased by 72.8%, as compared with sham-lesioned side. This decrease was obviously attenuated in the UA-treated Parkinsonian rats, which reduced only by 46.2%. The findings indicate that UA treatment, by enhancing plasma urate level, was able to protect against dopaminergic neuron loss in the nigrostriatal pathway of 6-OHDA unilaterally lesioned rats.
UA attenuates dopamine reduction in striatum
Next, the contents of DA and its metabolites in striatum were determined by HPLC to confirm the neuroprotection on dopaminergic neurons by urate. The effects of both saline and UA treatment on the levels of DA, DOPAC, and HVA in the intact (left) versus lesioned (right) striata of 6-OHDA unilaterally lesioned rats were shown in Fig. 5. It was observed that in the saline-treated Parkinsonian rats, the DA content in the lesioned striata was significantly reduced by 64.9% compared with intact side. Likewise, the DA metabolites, DOPAC and HVA levels also dramatically dropped in the lesioned side. These observations indicate that 6-OHDA administration induces a significant DA depletion in striatum, consistent with our findings on dopaminergic neuron loss in SN as shown in Fig. 4a and b. These data also suggest the successful establishment of Parkinsonian rat model in this study. The levels of DA, HVA, as well as DOPAC in the lesioned side of UA-treated group were about 69.1%, 70.2%, and 69.0% of those in intact striatum, significantly higher than those in saline-treated group, as shown in Fig. 5.
UA treatment ameliorates oxidative damage in striatum
As urate is a natural antioxidant, the effect of UA treatment on oxidative damage in the striata of 6-OHDA-lesioned rats was also determined by measuring the levels of MDA, total GSH, and the activities of SOD as well. As shown in Fig. 6, there was no significant difference of the striatal MDA levels in the intact side between saline- and UA-treated Parkinsonian rats, Similar trends were observed for SOD activity and GSH content between the two groups. Of note, the MDA level in the injured side of saline-treated group was significantly higher than that of the intact side and also higher than the injured side of UA-treated rats lesioned by 6-OHDA intrastriatal injection (Fig. 6a), implying that 6-OHDA lesion caused the increase of lipid peroxidation, which was relieved by UA administration. Furthermore, it was observed that in the saline-treated group, the GSH level and SOD activity in the injured side were markedly lower than those in intact side, indicating GSH depletion and SOD activity reduction as a result of 6-OHDA lesion. UA treatment significantly enhanced the GSH contents and SOD activity in the injured striata compared to those of saline-treated group (Fig. 6b and c). Interestingly, it was found that in the UA-treated group, the SOD activity in the injured side was remarkably higher than that of the intact side.
AKT/GSK3β signaling contributes to the neuroprotection by urate
To explore the possible signaling pathways mediating the neuroprotection by urate, the phosphorylation levels of Akt at Ser473 and GSK3β at Ser9 and Tyr216 sites in the striatum were examined by western blot analysis as the changes in Akt and GSK3β activity have been reported in in vivo and in vitro PD models (Chen et al. 2004; Chung et al. 2007). As shown in Fig. 7a and b, the phosphorylation levels of Akt (Ser473) and GSK3β (Ser9) were obviously reduced in the injured side (right) of 6-OHDA-injected rats, as compared with either the intact side (left) of 6-OHDA-injected rats or the lesioned side of sham-operated rats. This reduction was markedly alleviated in the 6-OHDA-injected rats receiving UA treatment (200 mg/kg, i.p. twice daily). However, no significant change of phosphorylated GSK3β at Tyr216 was observed in the injured striata of 6-OHDA-injected rats compared with sham-operated group (data not shown). UA administration did not affect the phosphorylation of Akt or GSK3β in sham-operated rats.
To establish the role of Akt/GSK3β signaling pathway in the neuroprotection by urate against 6-OHDA-induced neurotoxicity, we further assessed its beneficial effects in the presence and absence of a phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with in vitro study using human neuroblastoma cell line SH-SY5Y. Western blot analysis displayed that consistent with our in vivo results, 6-OHDA markedly inhibited the phosphorylation of Akt at Ser473, implying an inhibitory effect on Akt activity. Pre-treatment with UA for 30 min, at concentrations from 25 μM to 400 μM, appeared to alleviate the inhibition on Akt activation caused by 6-OHDA in a concentration-dependent manner, as shown in the upper two panels in Fig. 7c. The cell viability measurement showed that UA (200 μM) pre-treatment was able to protect against 6-OHDA (50 μM, 14 h)-induced toxicity to SH-SY5Y cells. But, this beneficial effect was abolished in the presence of LY294002 (5 μM), which inhibits Akt activation, as shown in Fig. 7d. Furthermore, western blot analyses demonstrated that 6-OHDA markedly dephosphorylated GSK3β at Ser9 (Fig. 7c), but hyperphosphorylated that at Tyr216 (Fig. 7e), both of which indicated a stimulatory effect by 6-OHDA on GSK3β activity. LY294002 was observed to enhance the inhibition by 6-OHDA on GSK3β phosphorylation at Ser9, whereas it failed to affect that at Tyr216, implying that GSK3β activation was secondary to Akt inhibition by LY294002. Of interest, UA was able to attenuate the alterations in GSK3β phosphorylation at both Ser9 and Tyr216 caused by 6-OHDA, which indicates a dual inhibition by urate on GSK3β activity. Similarly, LY294002 was found to abolish the effect by urate on GSK3β phosphorylation at Ser9, but not that at Tyr216. These results clearly suggest that the neuroprotective effects by urate on 6-OHDA-induced Parkinsonian rats may be mediated, at least in part, by Akt/GSK3β signaling.
Urate has emerged as a promising candidate therapeutic target for neuroprotection in PD based on a remarkable convergence of biological, epidemiological, and clinical data (Church and Ward 1994; Fitzmaurice et al. 2003). However, this still lacks consolidated experimental evidence both in vivo and in vitro. Our previous study demonstrated that UA pre-treatment was capable of protecting PC12 cells against 6-OHDA-induced injury in vitro (Zhu et al. 2012). In this study, intraperitoneal injection of UA was found to elevate plasma and brain urate levels. This elevation was particularly obvious when the dose of UA was above 200 mg/kg. Therefore, treatment with UA, at a dose of 200 mg/kg twice daily was chosen to explore the effects and underlying mechanisms of UA administration on 6-OHDA-induced Parkinsonian rats. Our data showed that UA treatment alleviated the impairment of motor performance, loss of dopaminergic neurons in SN, reduction of DA and its metabolites in striatum, accumulation of lipid oxidation, as well as depletion of GSH and SOD activity in striatum caused by 6-OHDA unilaterally injected into striatum (right side). These findings clearly demonstrate that UA treatment at this regimen could protect dopaminergic neurons in SN against 6-OHDA-induced degeneration, in agreement with our previous findings (Wang et al. 2010). In addition, western blot analysis showed that 6-OHDA lesion remarkably reduced the phosphorylation of Akt (activation) and induced the dephosphorylation of GSK3β (activation) in the lesioned striatum, which was significantly alleviated in the UA-treated Parkinsonian rats. This indicates that Akt activation and GSK3β inactivation may, at least in part, be involved in the beneficial effects of UA treatment.
6-OHDA is toxic in both central and peripheral nervous system. However, this neurotoxin is incapable of crossing the blood–brain barrier; thus, its toxicity to the central nervous system is achieved only when directly injected into brain by means of stereotaxic surgery (Simola et al. 2007). Therefore, it is generally accepted that the intact side (left in this study) could serve as a good internal control without the interference of individual variability. In addition, the infusion of 6-OHDA into striatum was reported to alter the redox homeostasis in rat brain. The chronic and persistent imbalance between ROS formation generation and antioxidant defenses characterizes many pathological processes and disease conditions including PD. Several studies demonstrated the oxidation products of lipid, protein, and nuclei acid were significantly enhanced in urine, serum, and substantia nigra of 6-OHDA-lesioned animal models of PD (Kikuchi et al. 2011). This implies that antioxidant agents are capable of relieving 6-OHDA-induced neurotoxicity.
Urate was found to reduce 6-OHDA-induced oxidative products such as MDA and 8-hydroxy-deoxyguanosine (8-OHdG), but increase the SOD activity and GSH levels in an in vitro model of PD (Zhu et al. 2012). Likewise, the increase in SOD activity and GSH level exerted by UA treatment was observed in this in vivo study. More importantly, we found that the SOD activity and GSH level in the injured striata of UA-treatment group were even higher than those in intact side. This may be explained by the mechanism that the locally accumulated urate in striatum resulted in an overshoot in enhancing SOD activity and GSH level to defend against the excessive ROS generation in striatum caused by 6-OHDA local injection. It is supported by our findings that i.p. injection of UA yielded an increase of urate in plasma and striatum by 52.9% and 32.3%, respectively, on the 5th day after injection. This also indicates a tight correlation between blood and brain urate, although detailed information on urate modulation in periphery and brain remains to be determined. In short, the data in this study provided evidence that UA treatment could elevate plasma urate level and enhance the resistance to oxidative injury in vivo.
However, it is still debatable whether urate is accumulated into neurons or not. Guerreiro et al. reported that urate is not significantly accumulated into neurons, which indicates that its antioxidant effect occurs extracellularly (Guerreiro et al. 2009). But, Cipriani et al. recently demonstrated that exogenous application of urate raises intracellular urate level about four folds 24 h later (Cipriani et al. 2012). This discrepancy was explained by the different sensitivity of the analytical methods applied. Although this discrepancy exists, researchers consistently agree that urate, at appropriate levels, may produce antioxidant action in cells, which could be mediated by some undefined receptor(s) even if urate does not enter cells. In fact, our recent findings showed that urate could produce antioxidant effect intracellularly by promoting nuclear factor erythroid-related factor (Nrf)-2 nuclear translocation and the up-regulation of antioxidant enzymes such as γ-glutamate cysteine ligase (unpublished data). Therefore, UA treatment, at the regimen employed in this work, could provide neuroprotection against oxidative stress and thus rescue the loss of dopaminergic neurons and terminals in the nigrostriatal pathway in 6-OHDA-lesioned rats. Because of the selective dopaminergic neuron degeneration induced by 6-OHDA, we believed that an appropriate elevation of plasma urate level could exert specific neuroprotective effects on dopaminergic neurons in PD models. Although the whole picture is yet to be uncovered, the dysregulation of urate homeostasis should play a crucial role in dopaminergic neuron degeneration in PD.
Growing evidence shows that Akt/GSK3β signaling takes a dominant role in preventing cellular degeneration (Franke et al. 1997). Decreased Akt signaling, associated with increase in GSK3β activity, has been reported in the in vitro and in vivo PD models (Chen et al. 2004; Chung et al. 2007). Akt is a serine/threonine protein kinase and its signaling depends on its phosphorylation by PI3K. GSK3β activity is differently regulated by the phosphorylation level of two critical sites Ser9 and Tyr216. Specifically, Ser9 phosphorylation reduces GSK3β activity, whereas Tyr216 phosphorylation increases its activity. Physiologically, Akt activation inhibits GSK3β activity by enhancing its phosphorylation at Ser9. Our in vivo and in vitro observations consistently showed that 6-OHDA treatment markedly dephosphorylated Akt at Ser473 and GSK3β at Ser9, implying the inactivation of Akt but activation of GSK3β by 6-OHDA. We found that 6-OHDA also enhanced the GSK3β activity by hyperphosphorylating it at Tyr216 in vitro, which was not observed in in vivo study. These led us to postulate that there are other mechanisms involved in modulating GSK3β phosphorylation at Tyr216 in vivo. Nevertheless, our present observations clearly show that UA treatment could attenuate the decrease in Akt phosphorylation at Ser473 and GSK3β phosphorylation at Ser9, as well as the increase in GSK3β phosphorylation at Tyr216 caused by 6-OHDA. The differentially regulatory effects by urate on GSK3β phosphorylation at Ser9 and Tyr216 consistently imply the inhibition on GSK3β activation exerted by urate. More importantly, our results showed that the PI3K inhibitor LY294002 was able to abolish the protective effects of urate on cell survival and its regulation on GSK3β phosphorylation at Ser9 in 6-OHDA-treated SH-SY5Y cells. Herein, we proposed that Akt/GSK3β signaling pathway may play a critical role in the neuroprotection by urate on dopaminergic neurons in SN of 6-OHDA-lesioned parkinsonian rats.
In addition to PD, the neuroprotection by urate have been reported in in vitro and in vivo models of other neurological disorders. In an experimental allergic encephalomyelitis model of multiple sclerosis, urate and its precursor inosine were found to delay the onset and improve the behavioral deficits in mice (Hooper et al. 1997; Scott et al. 2002). Urate has also been shown to protect embryonic rat spinal cord neuron cultures against glutamate toxicity (Du et al. 2007), and it protected against secondary damage and improved functional recovery after spinal cord injury in vivo (Scott et al. 2005). Studies also proved that in Alzheimer's disease, there is defective tubular urate transport and a plasma natriuretic factor (Maesaka et al. 1993). Of note, among these neurodegenerative diseases, PD is most closely related to urate. This may be because of a possible link between the antioxidant action of urate and the vulnerability of dopaminergic neurons to oxidative damage. To this end, further investigation is warranted to enhance our understanding of urate biology and to translate it into improved PD treatments.
In conclusion, our present data demonstrated a significantly neuroprotective effect by urate on dopaminergic neurons in SN, supported by the evidence that UA treatment enhanced DA and its metabolite DOPAC content in striatum and improved the behavioral activities. Apart from these, UA treatment alleviated oxidative stress and induced an increase in phosphorylation of Akt (Ser473) and GSK3β (Ser9), both in vivo and in vitro. The in vitro findings further show that PI3K inhibitor LY294002 abolished the neuroprotection by urate and its regulation on GSK3β phosphorylation at Ser9. Thus, the findings clearly demonstrate that the in vivo neuroprotection by urate was possibly mediated by Akt/GSK3β signaling. These findings offered an experimental basis for the clinical usage of urate, and also provided promising approaches or drug combinations for PD therapy.
We would like to thank Ms. Wang Fen for technical support. In our study, Dr Luo gave contributions to conception and design. Dr Hu made critical reading of the manuscript and gave helpful discussion and revision of our manuscript. Qi-Lin Zhang and Wen-Yan Hua focused on HPLC–MS/MS determination while Li Gong, Yi-Xian Huang, and Ping-Wei Di performed all other experiments in this study. This work was supported by grants from The Suzhou Foundation for Development of Science and Technology (200815404), Suzhou Technology Support Project (SS201112), Jiangsu Ordinary University Science Research Project (08KJB320012), and also Natural Science Foundation of Jiangsu Province, China (BK2010229). There is no conflict of interest to be disclosed.