Chronic A2A antagonist treatment alleviates parkinsonian locomotor deficiency in MitoPark mice

Neurobiology of Disease 40 (2010) 460–466

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i

Chronic A2A antagonist treatment alleviates parkinsonian locomotor deficiency in MitoPark mice Daniel Marcellino a, Eva Lindqvist a, Marion Schneider b, Christa E. Müller b, Kjell Fuxe a, Lars Olson a, Dagmar Galter a,⁎ a b

Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, D-53121 Bonn, Germany

a r t i c l e

i n f o

Article history: Received 5 May 2010 Revised 12 July 2010 Accepted 15 July 2010 Available online 23 July 2010 Keywords: Striatum Parkinson's disease L-DOPA Dopamine Spontaneous locomotion Drug-induced locomotor activity HPLC Western blot Mass spectrometry

a b s t r a c t Adenosine A2A receptor (A2AR) antagonists are being investigated as promising treatment strategy for Parkinson's disease (PD). To test whether A2AR antagonists are beneficial in early PD stages we used MitoPark mice, a genetic model with gradual degeneration of DA cells. Daily treatment of young MitoPark mice for eight weeks with the A2AR antagonist MSX-3 prevented the reduction of spontaneous locomotor activity observed in saline or L-DOPA treated animals. Chronic A2AR antagonist treatment neither induced desensitization of receptors nor accumulation of the drug in brain tissue. Despite beneficial effects on behavior, which are not improved upon addition of a low dose of L-DOPA, the characteristic decline of dopamine levels was not changed. Our results indicate that effective dosing with A2AR antagonists should be tested as monotherapy in early PD, and serves to remind us that positive behavioral effects of such treatment need not be reflected in rescue of striatal dopamine levels. © 2010 Elsevier Inc. All rights reserved.

Introduction Adenosine receptor 2A (A2AR) is highly expressed in a subpopulation of basal ganglia neurons where they structurally and functionally interact with dopamine D2 receptors by forming receptor heteromers and by targeting common intracellular signaling cascades. Neurochemical evidence from cell culture and animal models show that A2AR oppose the action of D2 receptors (Ferre et al., 1991), indicating that A2AR antagonists might reduce effects of dopamine (DA) depletion and improve motor symptoms in Parkinson's disease (PD) (Fuxe et al., 2007; Schwarzschild et al., 2006). In several rodent and primate PD models induced by infusion of the neurotoxins 6-OHDA or MPTP, treatment with A2AR antagonists such as KW-6002, MSX-3 or caffeine counteracted hypokinesia. When administered shortly after the DA lesion caffeine and other A2AR antagonists have been shown to be neuroprotective (Chen et al., 2001). Blockade of A2AR may therefore be responsible for the reduced risk for PD reported for caffeine users (Ross et al., 2000).

⁎ Corresponding author. Department of Neuroscience, Retzius väg 8, B2:4, Karolinska Institutet, 171 77 Stockholm, Sweden. Fax: + 46 8 323 742. E-mail address: [email protected] (D. Galter). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2010.07.008

Initial clinical trials with the A2AR antagonist istradefylline (KW6002) indicated that it prolonged the L-DOPA action without worsening of dyskinesia in advanced PD (Bara-Jimenez et al., 2003; Chase et al., 2003). Istradefylline treatment in addition to an optimal L-DOPA therapy ameliorated dyskinesia and reduced “off” periods in late PD (Jankovic, 2008; Jenner et al., 2009). Recently, istradefylline was also tested as monotherapy in early PD in a random double-blind trial that failed to demonstrate efficacy in improving motor symptoms (Fernandez et al., 2010). To address the discrepancy between the encouraging results from A2AR antagonist treatments in toxin-induced PD models and the disappointing results from a clinical trial of istradefylline as monotherapy in early PD, we used a genetic PD model, the MitoPark mouse, in which DA neurons undergo a slow and progressive degeneration due to the cell-type specific induction of mitochondrial dysfunction in midbrain DA neurons (Ekstrand et al., 2007). These mice display gradual development of motor dysfunction, reproducing progressive stages of PD without the potential side effects of toxins used in other animal models (Galter et al., 2009). As A2AR antagonist we used MSX-3, a prodrug of the water-soluble, highly specific A2AR antagonist MSX-2, which is hydrolyzed by cellular phosphatases and exhibits more than a 100-fold higher affinity to A2AR than A1R and is almost completely inactive at A2BR and A3R (Sauer et al., 2000). Young adult MitoPark mice were analyzed to ask (i) if chronic treatment with the most selective

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A2AR antagonist MSX-3 is neuroprotective; (ii) if co-administration with a low dose of L-DOPA is more effective; and (iii) how A2A antagonist treatment compare to L-DOPA treatment. Materials and methods Behavior experiments Drugs were injected intra-peritoneally in a volume of 10 ml/kg of body weight. MSX-3 was synthesized at the University of Bonn (Hockemeyer et al., 2004) and L-DOPA was prepared as described in Galter et al. (2010), using Madopark quick® (Roche AB, Sweden), a combination of 100 mg L-DOPA and 25 mg benserazide per 0.5 g tablet. Adult littermates of MitoPark mice of both sexes were used and locomotion tests were performed at the beginning of the animal's active period at 6:00 pm using an automated registration system (AccuScan Instruments, Ohio, USA). For acute drug-induced locomotion 4 to 6 pairs of MitoPark and control littermates of 12, 22 or 32 weeks of age were used. For chronic treatment 5–6 pairs of 12-week old MitoPark and control littermates were injected for 56 consecutive days with either saline; 5 mg/kg MSX-3; the combination of 2 mg/kg L-DOPA + 5 mg/kg MSX-3; 2 mg/kg (low) L-DOPA or 16 mg/kg (high) L-DOPA as detailed in Supplementary Fig. 1A. On the first treatment day and subsequently every other week the spontaneous and drug-induced locomotor activity of all animals was assessed and analyzed using appropriate software (Prism GraphPad5) and method (two-way ANOVA repeated measure with Bonferroni post hoc test). On day 57, 23 h after the last treatment, mice were sacrificed, brain tissues were dissected, frozen on dry ice and kept at −70° C until analysis. The Stockholm Animal Ethics committee had approved all experiments. Membrane preparation and competition binding analyses Frozen tissue samples from 32-week old mice were homogenized in ice-cold preparation buffer [20 mM Tris-HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7mM) and EDTA (1 mM)] by sonication. The membranes were precipitated by centrifugation at 47,000 × g for 20 min and washed through rehomogenzation in 10 ml of preparation buffer three sequential times. The final pellet was rehomogenized and sample protein concentration was determined using the BCA Protein Assay (Pierce, Rockford, IL, USA). Membranes were resuspened in incubation buffer (IB) [20 mM Tris-HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM) and EDTA (1 mM)] and the suspension of membranes were subsequently used in the radioligand binding experiments. Competition experiments of MSX-2, MSX-3 or CGS 21680 versus the A2A receptor antagonist [3H]-ZM 241385 (27.4 Ci/ mmol, PerkinElmer Life Sciences, USA) were carried out by membrane (20 μg protein/tube) incubation for 90 min at 30° C in incubation buffer [20 mM Tris-HCl pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM) and EDTA (1 mM)] and the incubation was terminated by rapid filtration through GF/C filters using a Brandel cell harvester.

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Western blot Protein levels of TH and A2AR were analyzed in striatal samples from all animals treated with saline or MSX-3 using appropriate antibodies (Ab, rabbit anti-TH Ab, Pel-Freeze Biologicals, Arkansas, USA, mouse anti-A2AR Ab, UpState, Charlottesville, VA, and mouse anti-α-tubulin Ab, Sigma-Aldrich Sweden, Stockholm). Analysis was performed on infrared imaging system (Odyssey LI-COR, Biosciences, NE, USA). MSX-2 detection with mass spectroscopy The MSX-2 content was analyzed 23 h after the last injection in cerebellum of 6 groups: MitoPark or control mice treated with saline, MSX-3 alone and MSX-3 in combination with L-DOPA. 4 brain samples (2 female and 2 male) from each group were combined, lyophilized and extracted. Samples were analyzed by HPLC (Agilent 1100, Böblingen, Germany) coupled to electrospray ionization mass spectroscopy (API 2000, Applied Biosystems, Darmstadt, Germany) equipped with a turbo ion spray ion source. The compounds were detected using Multi Reaction Monitoring with positive ionization using 3 pairs of MS/MS for each sample (395–268 u, 395–337 u and 395–377 u). Results Acute drug-induced motor activity during different stages of disease progression Effects of MSX-3 on locomotion and rearing of MitoPark mice were determined at three different ages reflecting different disease stages and compared with control littermates at corresponding ages (Fig. 1). At 12 weeks, MitoPark mice displayed a significant increase of locomotion in response to 5 mg/kg MSX-3. At later stages of disease the enhanced locomotion produced by MSX-3 was not significant (Bonferroni post hoc test following two-way ANOVA). Control littermates demonstrated a significant increase of locomotion in response to 5 mg/kg MSX-3 at all ages. A2AR radioligand competition experiments similar between genotypes To determine whether the pharmacology of A2AR in striatum of MitoPark mice differed from control littermates, A2AR radioligand competition experiments were performed using the antagonist [3H] ZM 241385 in striatal membrane preparations. MSX-2 bound to murine A2AR with a Ki of 45.3 ± 2.22 nM, around one logarithm more affine compared to its prodrug MSX-3 (567.6 ± 53.7 nM), (Fig. 2A). In addition, the affinity of MSX-3 to A2AR did not differ between MitoPark and controls (Fig. 2A). Similarly, the affinity for the A2AR agonist CGS 21680 did not differ between MitoPark mice and controls (Ki = 120nM, Fig. 2B). Chronic treatment with MSX-3 rescues spontaneous locomotor activity; daily MSX-3 effects are maintained and high dose L-DOPA effects increase with time

Determination of monoamine levels Monoamine and metabolite levels were determined using highperformance liquid chromatography (HPLC) with electrochemical detection (ESA Coulochem III, Dalco Chromtech AB, Sweden). Separations were performed on a reverse-phase column (ReprosilPur, C18-AQ) and norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels are expressed as ng/g wet weight of tissue. Groups were compared by one-way ANOVA using appropriate software (GraphPad Prism 5).

Young MitoPark and littermate control mice (12-week old) were treated daily with MSX-3, L-DOPA, or a combination of these drugs for 8 continuous weeks. Daily administration of MSX-3 prevented the expected decrease in spontaneous activity of MitoPark mice observed in saline treated animals (Fig. 3, upper panel). Already after 2 weeks, MSX-3 treated mice displayed around 30% more spontaneous locomotion and they maintained similar activity levels throughout the study. In contrast, saline treated mice displayed a steady decline of spontaneous activity, which had dropped to roughly half of their initial activity by the end of the study. Combining MSX-3 and a low,

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Fig. 1. Acute effects of two doses of MSX-3 on locomotion and rearing of pre-symptomatic (12-week), middle aged (22-week) and old (32-week) MitoPark and control mice. MSX-3 treatment groups were compared to saline treated animals using two-way ANOVA followed by Bonferroni post hoc test. Locomotion of MitoPark and control mice is significantly increased by MSX-3. A significant interaction of treatment and age was found in MitoPark mice (F(4, 37) = 3.08, P = 0.0275) whereas the treatment effect is highly significant (F(2,37) = 10.50, P = 0.0002) and the age effect is very significant (F(2,37) = 6.00, P = 0.0055). Locomotion of control mice showed a highly significant effect of treatment at all ages (F(2, 36) = 63.53, P b 0.0001). MSX-3 induced rearing of MitoPark mice significantly decreased with age (F(2,39) = 8.15, P = 0.0011). In contrast, MSX-3 induced rearing of control mice increased significantly in an age- (F(2,35) = 5.10, P = 0.0114) and dose-dependent manner (F(2, 35) = 11.5, P = 0.0002). Data are presented as mean ± SEM, N = 6–9. *, P b 0.05; ***, P b 0.001.

sub-threshold dose of L-DOPA (2 mg/kg) did not improve spontaneous activity over levels reached by MSX-3 alone. In contrast, L-DOPA alone, at a low or high dose (16 mg/kg) did not prevent the progressive decrease of spontaneous activity over 8 weeks of treatment. With the high dose of L-DOPA, there was a tendency for increased motor activity during the last month of the experiment. Similar changes were observed in rearing activity of MitoPark mice

Fig. 2. Binding characteristics of A2A receptor antagonists in striatal membrane preparations from MitoPark and control mice. (A) MSX-2 binds with more than one-log higher affinity than MSX-3 to murine A2AR [45.3 ± 2.22 versus 567.6 ± 53.7 nM, respectively (mean ± SEM, N = 3)] and MSX-3 binds with equal affinity to striatal A2AR from control and MitoPark mice. (B) Likewise, the agonist CGS 21680 binds with equal affinity to striatal A2AR from control and MitoPark mice.

(Supplementary Fig. 2); MSX-3 or combined treatment increased spontaneous rearing at all time points analyzed, in which MSX-3 treatment alone induced a significant increase after 6 and 8 weeks of treatment while in combination with L-DOPA a significant increase only after 8 weeks of treatment. No significant changes were detected in the spontaneous locomotor activity of control littermates with any treatment (Supplementary Fig. 3). Immediately after treatment, mice were returned to the activity boxes and their behavior was recorded for an additional hour. Saline treated MitoPark mice displayed low horizontal activity, with negligible changes during the 8 weeks of study, although there was a tendency towards a decrease with time (Fig. 3, upper panel). MSX-3 treatment alone induced a two- to three-fold increase in locomotion in both MitoPark (Fig. 3, upper panel) and their control littermates (Supplementary Fig. 3). Remarkably, the degree of MSX-3-induced locomotor activity did not change over time, thus indicating a lack of A2AR desensitization opposite to what has been described for caffeine (Karcz-Kubicha et al., 2003). Combining MSX-3 and a sub-threshold dose of L-DOPA, a very similar enhancement of activity was observed as with MSX-3 alone, and the effect was constant during the two months of treatment for both MitoPark and control mice. The subthreshold dose of L-DOPA did not produce any increase in locomotor activity over saline at any age of either genotype. However, treatment with the high L-DOPA dose induced a strong increase of locomotion in MitoPark mice that also increased considerably with the duration of treatment. As early as 2 weeks into the study, MitoPark mice displayed twice the level of drug-induced horizontal activity than saline treated animals and reached up to a 5-fold increase in locomotion after 6 or 8 weeks of treatment. These large increases in locomotion and rearing were present although the spontaneous activity of these animals was similar to saline treated mice before the administration of L-DOPA (Fig. 3 and Supplementary Fig. 2). In control littermates, chronic sub-threshold L-DOPA treatment had no effect while the high dose induced a small but distinct decrease in locomotion (Supplementary Fig. 3), similar to the behavior described in control mice following acute L-DOPA treatment (Galter et al., 2010).

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Fig. 3. Locomotion of MitoPark mice during 8 weeks of daily treatment with saline, MSX-3, MSX-3 + L-DOPA or two doses of L-DOPA. In saline treated mice (white bars) spontaneous locomotion slowly declines (P b 0.0001; repeated measures one-way ANOVA, post hoc test for linear trend). The spontaneous activity of MSX-3 treated animals is significantly increased over saline after 6 and 8 weeks of treatment. Thus, by the end of the study, MSX-3 treated MitoPark mice display almost 3 times the locomotion of saline treated littermates, recorded 23 h after the last administration of MSX-3. Combining MSX-3 and L-DOPA caused a similar, although less pronounced, improvement of spontaneous locomotion at all time points which reached significance over saline after 8 weeks of treatment. Both low and high doses of L-DOPA had no significant effect. Note that data from the same saline group are included in each diagram, to ease the visual comparison with the different treatment groups. (two-way ANOVA repeated measures (F(4,54) = 5.414, P b 0.0048); Bonferroni post hoc test; *, P b 0.05, **, P b 0.01, ***, P b 0.001). Drug-induced locomotor behavior was recorded and analyzed similarly (lower panel). A significant effect of treatment was found in the MSX-3 group and in the MSX-3 + L-DOPA treatment group at all time points. In the group treated with the high dose of L-DOPA a significant effect was observed from 4 weeks of treatment onward. In the low dose of L-DOPA group no significant effect was detected. (two-way ANOVA repeated measures (F(4,54) = 20.96, P b 0.0001); Bonferroni post hoc test; *, P b 0.05, **, P b 0.01, ***, P b 0.001).

Fig. 4. Dopamine, dopamine metabolites, TH and A2AR levels in striatum of 20-week old MitoPark mice chronically treated with saline, MSX-3, MSX-3 + L-DOPA, or L-DOPA at two different doses do not change. (A) No significant differences of dopamine, dopamine metabolites or the (HVA + DOPAC)/DA ratio were detected in any MitoPark treatment groups (one-way ANOVA). Respective levels of control mice chronically treated with saline are also presented for comparison. (B) TH protein expression was expectedly reduced in MitoPark mice compared to control littermates. No significant differences were detected between any MitoPark treatment groups (one-way ANOVA). A2AR protein levels were slightly reduced in MitoPark mice treated with MSX-3 or the combination of MSX-3 and L-DOPA. However, there were no significant effects (one-way ANOVA, N = 4–6).

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Monoamine, TH and receptor protein levels do not change with chronic drug treatment Twenty-three hours after the last treatment, mice were sacrificed and brain tissues were collected. Striatum (Fig. 4A) and frontal cortex (Supplementary Fig. 4), were selected for comparison of monoamine levels between MitoPark and control mice. DA levels and its metabolites DOPAC and HVA were significantly lower in MitoPark, but no significant change was observed between treatment groups. A more than two-fold increased in the ratio between DA metabolites and DA was observed in MitoPark striatum, indicating an increased DA turnover compared to controls. The ratio was particularly high for mice treated with a high L-DOPA dose, but was not significantly different from saline treated MitoPark mice (one-way ANOVA). The levels of NE and 5HT were not significantly changed in striatum or cortex between the two genotypes, nor between the treatment groups (Supplementary Fig. 5). At 20 weeks of age, only 11.1 ± 2.2% of control TH levels remained in the striatum of saline treated MitoPark mice (Fig. 4B). Although TH levels of MSX-3 treated MitoPark mice were slightly higher (14.4 ± 2.8%) they were not significantly different from saline treated mice (one-way ANOVA). A2AR protein levels were reduced in MitoPark mice treated with MSX-3 or the combination of MSX-3 and L-DOPA compared to saline treated mice (91.5 ± 16.8% and 84.0 ± 13.9% respectively), but were not significantly different from saline treated mice. Determination of residual MSX-2 levels in the brain MSX-3, a polar, water-soluble phosphate prodrug is readily hydrolyzed to lipophilic MSX-2, which penetrates into the CNS and blocks central A2AR. To test if the drug accumulated in the brain after chronic treatment, the concentration of MSX-2 was determined in brain samples by HPLC coupled to mass spectrometry (LC-MS) with a detection limit of 1 ng/ml. As positive control, a brain sample was spiked (Fig. 5A) and compared to chromatograms from brain samples from MitoPark mice treated with MSX-3 (Fig. 5B). Using this highly sensitive detection method, MSX-2 was not detected in the brain. Discussion A2ARs are highly localized to the basal ganglia and specifically to the indirect pathway, which is important in the control of voluntary movements (Svenningsson et al., 1999). Evidence from a number of PD models indicates that A2AR antagonists consistently reverse parkinsonian deficits in non-human primates and rodents. In the 1970s, it was reported that caffeine enhanced L-DOPA and DA agonistinduced contralatercal turning in unilaterally 6-OHDA-lesioned rats (Fuxe and Ungerstedt, 1974) and that caffeine exerts its effects through blockade of A1R and A2AR (Fredholm et al., 1976). A2AR antagonists appeared more promising in PD therapy in view of the antagonistic A2A-D2 receptor–receptor interactions present in striatum (Fuxe et al., 2003). Specific A2AR antagonists, such as SCH 58261, were found to dose dependently increase locomotor activity in combination with sub-threshold doses of L-DOPA or D2R agonists in rodent PD models (Fenu et al., 1997; Tanganelli et al., 2004). These preclinical findings lead to a search for more selective drugs with high affinity for A2ARs, which were introduced into clinical trials. Istradefylline (KW-6002) caused symptomatic but rather modest improvement in relatively advanced PD patients with dyskinetic complications (Bara-Jimenez et al., 2003; Hauser et al., 2003). A recent double-blind clinical trial studied the effect and safety of istradefylline as monotherapy in early PD patients who had not received dopaminergic drugs for at least 30 days (Fernandez et al., 2010). Treatment with 40 mg/day caused greater improvements in the motor scores measured by the Unified Parkinson's Disease Rating

Fig. 5. MSX-2 is not detected in brain tissue after 8 weeks of daily MSX-3 injections. Representative MS chromatograms from saline treated control mice spiked with 5 ng/ ml MSX-2 in an injection volume of 10 μl (A) and from MitoPark mice treated with MSX-3 in an injection volume of 50 μl (B). Different colors represent three different mass transitions used for detection.

Scale UPDRS (Subscale III) at each of the 4 rating time points compared to the placebo group. However, the change from baseline to endpoint in the UPDRS score was not significantly different between the treatment groups. However, the change from baseline to endpoint was not significantly different between the groups. The authors discuss that the istradefylline dose was too low as monotherapy; a dose that had been recommended for advanced PD patients to supplement L-DOPA treatment. The results from the MitoPark model of PD fully support this view. Instead of istradefylline we used MSX-3, a water-soluble prodrug of the highly specific A2AR antagonist MSX-2, which exhibits greater potency for A2AR than other antagonists including istradefylline (Yang et al., 2007). We first determined the effective dose at different PD stages modeled by different ages of MitoPark mice (Fig. 1). As expected, acute treatment of 12-week old MitoPark mice with MSX-3 increased locomotion and rearing at doses similar to those used in rats (Karcz-Kubicha et al., 2003; Nagel et al., 2003). Pharmacological analysis confirmed receptor binding and specificity properties comparable to those for rats (Fig. 2). The effect MSX-3 had on locomotion was reduced in older MitoPark mice, possibly due to insufficient DA levels in striatum. In control mice, acute MSX-3 increased locomotion and rearing similar to the response described in rats (Karcz-Kubicha et al., 2003). To model long-term treatment of early PD with an effective dose of an A2AR antagonist, we treated young MitoPark mice with 5 mg/kg MSX-3 daily for 8 weeks. One potentially harmful effect of chronic administration of MSX-3 is delayed and reduced food intake, as described in rats after its micro-infusion into nucleus accumbens (Nagel et al., 2003). Throughout the treatment, weight development of all mice was therefore monitored and no significant effects were found (Supplementary Fig. 1B). Thus, there might be differences

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between species or, more likely, the much higher acute dose infused in the rat affects food intake. One advantage of the MitoPark mouse model is the slow and progressive degeneration of striatal DA innervation, reflected by the gradual reduction of spontaneous activity observed in saline treated mice. The positive effect of daily MSX-3 treatment was most evident in preventing this expected decrease of spontaneous locomotion over time. During the length of chronic treatment, the spontaneous locomotor activity of MitoPark mice was substantially and significantly higher. This parameter has not been analyzed in any other model to date, but may more accurately reflect the disease progression in PD patients. A significant change appeared in the mouse model after 6 weeks of continuous treatment, indicating that recently reported studies in PD patients with istradefylline may have been too short (12 weeks long) to witness any significant improvement of motor symptoms (Fernandez et al., 2010). Our data on drug-induced locomotor activity indicate that chronic daily treatment of MitoPark mice produces no desensitization of the A2AR. The extent of drug-induced increase in locomotor behavior was similar throughout the duration of the 8-week study. These results are in line with observations in marmosets, primates and rats that were treated daily for up to 3 weeks with the A2AR specific antagonist KW6002 (Kanda et al., 1998) and are in contrast to the effects of the unspecific A2AR antagonist, caffeine (Karcz-Kubicha et al., 2003). Furthermore, A2AR protein levels were unchanged in striatum of MSX3 treated mice (Fig. 4B) in line with data from post mortem studies in PD patients without dyskinesia (Calon et al., 2004). The long chronic treatment did not provide any detectable neuroprotection in MitoPark mice, in contrast to results from other acute PD models: mice pretreated with caffeine or other A2AR antagonists including SCH 58261, KW-6002 or DMPX, 10 min before they were lesioned with MPTP had up to 25% higher levels of striatal DA seven days later (Chen et al., 2001). A further study strengthened these results by demonstrating that daily treatment with KW-6002 starting less than one hour after the first dose of MPTP prevented the decline by ~30% of both striatal DA and TH protein levels. In addition, the authors showed a similar effect using the unilateral 6-OHDA rat model, where daily oral treatment with KW-6002, starting 50 min before the stereotactic toxin injection prevented the reduction of DA cells in SN from 50% to around 10% after one week (Ikeda et al., 2002). Beside the much longer treatment period in our model (8 weeks of daily treatment compared to 1 or 7 days of treatment), a more important difference between the neurotoxic PD models and MitoPark mice is an acute DA neurodegeneration compared to the much slower and progressive degeneration in the genetic PD model. In addition, it seems that neuroprotection of DA neurons is possible only when A2AR inhibition occurs before or very soon after (around 1 h) the toxic injury. In MitoPark mice, we started the chronic treatment with A2AR antagonist at an age when striatal DA levels were already reduced to approximately 50% of control levels (Galter et al., 2010). We did not detect any significant change of DA levels compared to MitoPark mice treated with saline or L-DOPA in either striatum or frontal cortex (Fig. 4A and Supplementary Fig. 4). These data are consistent with results from the ELLDOPA study in which PD patients failed to show improvements in DA neuroimaging after 40 weeks of L-DOPA treatment, but showed clear positive effects in motor scores (Fahn, 2005; Hauser, 2009). In addition to MSX-3, we have compared the effects of long chronic treatment of MitoPark mice with both a low and a high dose of LDOPA. The low dose did not induce any locomotor activity in MitoPark mice even at 20 weeks of age, whereas the high dose induced strong horizontal and vertical locomotor activity already after 2 weeks of treatment (at 14 weeks of age) and after 4 weeks of treatment the effect was comparable to that of MSX-3 (Fig. 3 and Supplementary Fig. 2). Importantly, L-DOPA treatment did not lead to any improvement in the animals' spontaneous locomotor activity.

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Unexpectedly, the combined treatment with MSX-3 and a low dose of L-DOPA did not demonstrate any improvement over MSX-3 alone, either in spontaneous or drug-induced locomotor activity. Perhaps this is due to sufficient striatal DA levels in MitoPark mice at this age, allowing at least a 2-fold increase in locomotor activity. In conclusion chronic MSX-3 treatment alleviates the symptomatic parkinsonian locomotor deficiency in MitoPark mice. Based on our findings, A2AR antagonist monotherapy stands out as an attractive alternative in early PD when DA is still released from remaining DA terminals. According to our data, the dose required for an effective PD therapy in early stages of the disease is likely to be higher than the dose used in a previous study (40 mg/day of istradefylline). Chronic treatment with the A2AR antagonist as monotherapy was more effective than L-DOPA, used at either a high or a low dose, and more effective than a combination therapy using a low dose of L-DOPA and high dose of MSX-3. Our results suggest that chronic MSX-3 may confer a degree of disease modification by mechanisms that do not directly involve the nigrostriatal DA system. Acknowledgments This study was supported by: The Swedish Research Council, The Swedish Parkinson Foundation, Swedish Brain Power, The Michael J. Fox Foundation and Karolinska Institutet Funds. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nbd.2010.07.008. References Bara-Jimenez, W., et al., 2003. Adenosine A(2A) receptor antagonist treatment of Parkinson's disease. Neurology 61, 293–296. Calon, F., et al., 2004. Increased adenosine A2A receptors in the brain of Parkinson's disease patients with dyskinesias. Brain 127, 1075–1084. Chase, T.N., et al., 2003. Translating A2A antagonist KW6002 from animal models to parkinsonian patients. Neurology 61, S107–S111. Chen, J.F., et al., 2001. Neuroprotection by caffeine and A(2A) adenosine receptor inactivation in a model of Parkinson's disease. J. Neurosci. 21, RC143. Ekstrand, M.I., et al., 2007. Progressive parkinsonism in mice with respiratory-chaindeficient dopamine neurons. Proc. Natl Acad. Sci. USA 104, 1325–1330. Fahn, S., 2005. Does levodopa slow or hasten the rate of progression of Parkinson's disease. J. Neurol. 252 Suppl 4, IV37–IV42. Fenu, S., et al., 1997. Adenosine A2A receptor antagonism potentiates L-DOPA-induced turning behaviour and c-fos expression in 6-hydroxydopamine-lesioned rats. Eur. J. Pharmacol. 321, 143–147. Fernandez, H.H., et al., 2010. Istradefylline as monotherapy for Parkinson disease: results of the 6002-US-051 trial. Parkinsonism Relat. Disord. 16 (1), 16–20. Ferre, S., et al., 1991. Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes. Proc. Natl Acad. Sci. USA 88, 7238–7241. Fredholm, B.B., et al., 1976. Effect of some phosphodiesterase inhibitors on central dopamine mechanisms. Eur. J. Pharmacol. 38, 31–38. Fuxe, K., et al., 2003. Receptor heteromerization in adenosine A2A receptor signaling: relevance for striatal function and Parkinson's disease. Neurology 61, S19–S23. Fuxe, K., et al., 2007. Adenosine A(2A) receptors, dopamine D(2) receptors and their interactions in Parkinson's disease. Mov. Disord. 22, 1990–2017. Fuxe, K., Ungerstedt, U., 1974. Action of caffeine and theophyllamine on supersensitive dopamine receptors: considerable enhancement of receptor response to treatment with DOPA and dopamine receptor agonists. Med. Biol. 52, 48–54. Galter, D., et al., 2010. MitoPark mice mirror the slow progression of key symptoms and L-DOPA response in Parkinson's disease. Genes Brain Behav. 9 (2), 173–181. Hauser, R.A., 2009. New considerations in the medical management of early Parkinson's disease: impact of recent clinical trials on treatment strategy. Parkinsonism Relat. Disord. 15 (Suppl 3), S17–S21. Hauser, R.A., et al., 2003. Randomized trial of the adenosine A(2A) receptor antagonist istradefylline in advanced PD. Neurology 61, 297–303. Hockemeyer, J., et al., 2004. Multigram-scale syntheses, stability, and photoreactions of A2A adenosine receptor antagonists with 8-styrylxanthine structure: potential drugs for Parkinson's disease. J. Org. Chem. 69, 3308–3318. Ikeda, K., et al., 2002. Neuroprotection by adenosine A2A receptor blockade in experimental models of Parkinson's disease. J. Neurochem. 80, 262–270. Jankovic, J., 2008. Are adenosine antagonists, such as istradefylline, caffeine, and chocolate, useful in the treatment of Parkinson's disease? Ann. Neurol. 63, 267–269.

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