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2 activity in striatum of dopamine deficient “weaver” mouse
Neurochemistry International 56 (2010) 245–249
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Blockade of adenosine A2A receptors downregulates DARPP-32 but increases ERK1/2 activity in striatum of dopamine deficient ‘‘weaver’’ mouse Konstantinos Botsakis a,1, Ourania Pavlou b,1, Paraskevi D. Poulou c,1, Nikolaos Matsokis a, Fevronia Angelatou c,* a b c
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 26 500, Greece Department of Materials Science, School of Natural Sciences, University of Patras, Patras 26 500, Greece Department of Physiology, School of Medicine, University of Patras, Patras 26 500, Greece
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 October 2009 Accepted 13 October 2009 Available online 21 October 2009
In the present study we investigated the signal transduction cascade modulated by adenosine A2A receptors under chronic dopamine deficiency in the ‘‘weaver’’ mouse. We determined the phosphorylation state of cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34 and of Extracellular Signal-regulated Protein Kinases 1/2 (ERK1/2), under basal conditions and after in vivo stimulation of A2A receptors by administration of the agonist CGS21680. Our results revealed that the endogenous levels of phospho-DARPPP-32 and phospho-ERK1/2 are elevated in ‘‘weaver’’ striatum probably as an adaptation phenomenon to gradual dopaminergic neurodegeneration appearing in this animal model, characterized as phenocopy of Parkinson’s disease. Stimulation of A2A receptors by CGS21680 further increases phospho-DARPP-32 but downregulates significantly the elevated phosphoERK1/2 levels bringing them close to those observed in wild type animals. Consistently, blockade of A2A receptors by MSX-3 (A2A receptor antagonist) downregulates phospho-DARPP-32 but significantly increases even more the phosphorylation/activation of ERK1/2. These results indicate that under chronic dopamine deficiency (a) the A2A/cAMP/PKA/DARPP-32 cascade is overactive due to the elevated endogenous phospho-DARPP-32 levels and (b) the A2A receptor modulatory effect on ERK1/2 signaling is dysregulated exerting opposing action compared to that observed in normal animals (Quiroz et al., 2006), i.e. in ‘‘weaver’’ animals A2A receptor blockade increases the activity of ERK1/2 cascade. This could be of clinical relevance since A2A antagonists are already used in clinical trials for ameliorating Parkinson’s disease (PD) symptoms. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: A2A receptors DARPP-32 ERK1/2 Dopamine deficiency ‘‘Weaver’’ mutant
1. Introduction Basal ganglia are involved in the integration of sensorimotor, associative and limbic information to produce motor behaviors and some forms of learning related plasticity (Bolam et al., 2000; Graybiel, 2005, 2008). The striatum is the main input and information processing structure of the basal ganglia. Corticolimbic-thalamic glutamatergic and mesencephalic dopaminergic systems converge in the GABAergic medium-sized spiny neurons of striatum (Gerfen, 2004). These are the striatal efferent neurons which can be classified into two neuronal pathways the ‘‘indirect’’, which projects via the globus pallidus and the subthalamic nucleus to output nuclei (substantia nigra and entopeduncular
* Corresponding author. Tel.: +30 2610 969159; fax: +30 2610 997215. E-mail address: [email protected] (F. Angelatou). 1 Equal contribution to this work. 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.10.007
nucleus) and the ‘‘direct’’, which sends axons directly to them (Gerfen, 2004). Both pathways are differentially regulated by dopaminergic nigrostriatal afferents via two types of receptors, D1-type which is confined to the ‘‘direct’’ pathway and D2-type confined to the ‘‘indirect’’ pathway (Gerfen, 2004). A balanced control of these two pathways is essential for the proper function of the extrapyramidal motor system (Graybiel, 2000). In Parkinson’s disease (PD), the gradual degeneration of the nigrostriatal dopaminergic system produces striatal dopamine depletion, which consequently impairs the function of the basal ganglia circuits resulting in akinesia, bradykinesia, tremor, and rigidity (Singh et al., 2007). Adenosine, a major neuromodulator of the synaptic function, exerts its effects in basal ganglia, by acting through its A1 and A2A adenosine receptors. The A2A receptors are co-localized and interact functionally with D2 receptors, on the medium-sized spiny neurons of the ‘‘indirect’’ pathway in striatum exerting opposing actions on neurotransmitter release, gene expression,
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and motor behavior (Ferre´ et al., 1997, 2008). As shown previously, overactivity of the ‘‘indirect’’ striatal pathway is a key component of the neural mechanisms responsible for generating parkinsonian symptoms (Graybiel, 2000). A previous study from our laboratory (Ekonomou et al., 2004) has shown that in vivo activation of adenosine A2A receptors by the agonist CGS21680, elicits a strong expression of the immediate early gene (IEG) zif/268 mRNA in striatum and motor cortex of ‘‘weaver’’ mice, but not in control animals. The ‘‘weaver’’ mutant represents the only genetic animal model of gradual nigrostriatal neurodegeneration, which can be characterized as a pathophysiological phenocopy of PD (Roffler-Tarlov and Graybiel, 1984; Triarhou, 2002). In the present study we investigated the signal transduction pathway that modulates IEG expression via A2A receptor stimulation under dopamine deficiency. We examined the in vivo induction of phosphorylation of DARPP-32 in striatum of ‘‘weaver’’ and control mice after A2A receptor activation. DARPP-32 is a central molecule in the signal transduction pathway of D1 and A2A receptors, which are both Gs/olf-coupled receptors and their activation stimulates the cAMP/PKA/DARPP-32 signaling cascade (Greengard et al., 1999). Furthermore we investigated whether, under dopamine deficiency, besides the A2A/cAMP/PKA/DARPP-32 established pathway an alternative pathway is also activated by the A2A receptors. Towards this end we examined the possible activation of the Mitogen-activated Protein Kinase (MAPK) pathway by determining ERK1/2 phosphorylation. ERK activation is involved in physiological processes and long-term changes in synaptic plasticity (Sweatt, 2004; Thomas and Huganir, 2004; Yoon and Seger, 2006). Moreover, ERK activation has been implicated in dopamine D1 receptor agonist supersensitivity in PD models (Gerfen et al., 2002, 2008). Before exploring either pathway we first established the endogenous levels of phosphorylated DARPP-32 and ERK1/2 in ‘‘weaver’’ animals. 2. Experimental procedures 2.1. Animals Control (+/+) and ‘‘weaver’’ male (wv/wv) mice, 2 months old, were used. The animals were bred in our laboratory from a breeding stock of adult heterozygous animals purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in groups of adult controls (+/+) or adult heterozygous (wv/+) of five to six per cage in a room with controlled light/dark cycle (12-h light/dark) and free access to food and water. In the present work, all efforts were made to minimize animal suffering, according to the European Communities Council Directive Guidelines (86/609/EEC) and to the Greek National Laws (Animal Acts, PD 160/91) for the care and use of laboratory animals. Identification of mice as wv/wv or wv/+ was performed by virtue of behavior. The abnormal behavior of wv/wv mice was characterized by weakness, hypotonia, resting and intention tremor, poor limb coordination and instability of gait (Simon and Ghetti, 1994). At the age of treatment (2 months old wv/wv mice) the neurodegeneration of midbrain dopaminergic neurons reaches about 60% and consequently the content of dopamine is reduced about 70% in dorsal striatum (Triarhou et al., 1988; Smith et al., 1990; Verney et al., 1995; Roffler-Tarlov and Graybiel, 1984, 1986, 1987). For the study the following animal groups were used: (i) control mice receiving saline, (ii) control mice receiving the A2A receptor agonist, CGS21680 (20 mg/kg body weight), (iii) ‘‘weaver’’ mice receiving saline, (iv) ‘‘weaver’’ mice receiving CGS21680 (20 mg/kg body weight), (v) ‘‘weaver’’ mice receiving the A2A receptor antagonist, MSX-3 (10 mg/kg body weight), (vi) ‘‘weaver’’ mice receiving the D2 receptor agonist, quinpirole (0.5 mg/kg body weight), (vii) ‘‘weaver’’ mice receiving the D1 receptor agonist, SKF38393 (20 mg/kg body weight), and (viii) ‘‘weaver’’ mice receiving the serotonin receptor antagonists ketanserin (H-T2) (25 mg/kg body weight) and NAN-190 (H-T1A) (5 mg/kg body weight) injected 5 min after the ketanserin. 2.2. Materials Drugs were purchased from Sigma (Germany) and Tocris (UK). All drugs were dissolved in physiological saline (with a few drops of 0.1N NaOH for MSX-3, final pH 7.4) immediately before use and were injected intraperitoneally.
2.3. Tissue preparation Animals were decapitated 20 min after the last injection, the brains were rapidly removed and flash-frozen in liquid nitrogen. Both sides of the striatum were then dissected out in a cryostat at 22 8C and stored at 80 8C (Picconi et al., 2003). Frozen tissue samples were sonicated in boiling 1% SDS (400 ml) with 4 ml Sigma Phosphatase-Inhibitor-Cocktail-I, 4 ml Sigma Phosphatase-Inhibitor-Cocktail-II, 4 ml Sigma Protease-Inhibitor-Cocktail and boiled for 5 min. Protein concentration was determined for each sample using the BSA protein kit (Pierce, Rockford, IL) and spectrophotometry. 2.4. Western blotting Equal amounts of protein (50 mg) from each sample were loaded onto 12% polyacrylamide gels, separated by SDS-PAGE and then transferred electrophoretically to nitrocellulose membranes (Schleicher and Schuell). The membranes were immunoblotted using rabbit polyclonal antibodies, anti-phospho-Thr202/ Tyr204-ERK1/2 (Cell Signaling Technology), and anti-phospho-Thr34–DARPP-32 (Novus-Biologicals). Mouse monoclonal antibodies against ERK1/2 (Cell Signaling Technology), DARPP-32 (BD Transduction-Laboratories) and Tubuline (SIGMA) were used to estimate the total amount of proteins. Antibody binding was revealed using goat anti-rabbit horseradish peroxidase-linked IgG (for the polyclonal primary antibodies) or goat anti-mouse horseradish peroxidase-linked IgG (for the monoclonal antibodies) followed by the Enhanced Chemiluminescence Detection System (Amersham). Luminescence from the blots was detected by exposing the membranes to Fuji-Hyperfilm for 30 s to 5 min, to ensure that we were operating within the linear range of the film, followed by digital scanning of the developed film in transparency mode. The scanned image of the membranes and bandintensities were calibrated and quantified using NIH-ImageJ software (version 1.34). For each animal, the values obtained from the experiments with phosphorylated and total ERK1/2 corresponded to the total of the bands intensities for both ERK1 and ERK2 (also called p44 and p42 MAP kinases, in relation to their molecular weights, 44 and 42 kDa, respectively). For each animal, the values of phosphorylated DARPP-32 Thr34 and phosphorylated ERK1/2 were normalized to total DARPP-32 and total ERK1/2 respectively. The amount of total DARPP-32 and total ERK1/2 did not differ between control and ‘‘weaver’’ animals as shown using tubuline as reference (data not shown). In each Western blot, all values were normalized to the average of the values obtained from the weaver saline animals included in the same blot. Each experiment was performed two to four times. 2.5. Statistical analysis Data concerning wild type mice and weaver mice treated with saline or CGS21680 were analyzed using two-way ANOVA, in which genotype and drug treatment were the independent variables. When comparing the groups of ‘‘weaver’’ mice receiving saline with all the other ‘‘weaver’’ groups the data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test for specific comparisons.
3. Results 3.1. Phosphorylation state of DARPP-32 at Thr34 in control and ‘‘weaver’’ mice The amount of endogenous phospho-Thr-34-DARPP-32 in striata homogenates of ‘‘weaver’’ compared to wild type animals was significantly elevated by 49% (Fig. 1). Upon stimulation of A2A receptors by in vivo administration of the agonist CGS21680 we observed a significant increase of phospho-Thr-34-DARPP-32, which in control animals is 21%, compared to saline-injected ones, while in ‘‘weaver’’ animals is 17%, compared to saline-injected ‘‘weaver’’ (Fig. 1). When comparing the amount of phosphoDARPP-32 between ‘‘weaver’’ and control animals after A2A stimulation we found that they differed significantly, with phospho-DARPP-32 in ‘‘weaver’’ animals being elevated by about 50% compared to controls (Fig. 1). Administration of the A2A antagonist MSX-3 in ‘‘weaver’’ animals decreased the levels of phospho-DARPP-32 by 28% when compared to saline-injected ‘‘weaver’’ mice (Fig. 1). Administration of the serotonin receptor antagonists, ketanserin and NAN-190, had no significant effect on the levels of phospho-DARPP-32 in ‘‘weaver’’ animals compared to saline-injected ones (Fig. 1). Activation of the D1 receptors by SKF38393 led to a moderate increase (12%) of basal phosphoDARPP-32 in ‘‘weaver’’ animals, which however was significant
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Fig. 1. Striatal DARPP-32 phosphorylation of control (+/+) animals treated with saline (SAL) and CGS21680 (CGS) and ‘‘weaver’’ (wv/wv) animals treated with saline (SAL), CGS21680 (CGS), MSX-3 (MSX), KETANSERIN and NAN-190 (KE/NA), SKF38893 (SKF) and Quinpirole (QUIN). Data were normalized to the average of the values obtained from weaver saline animals. Data represent means SEM (n = 6–20 per group) of representative Western blots. #p < 0.05 significantly different compared to the control saline treated group. *p < 0.05 and **p < 0.01 significantly different compared with the ‘‘weaver’’ saline treated group. yp < 0.05 ‘‘weaver’’ CGS compared to the control CGS treated group. Statistical analysis concerning wild type and weaver mice treated with saline or CGS21680 used two-way ANOVA (genotype drug treatment), while statistical analysis concerning ‘‘weaver’’ groups used one-way ANOVA followed by Dunnett’s post hoc test.
(Fig. 1). Stimulation of the D2 receptors by the agonist quinpirole decreased significantly the levels of basal phospho-DARPP-32 in ‘‘weaver’’ mice by 21%. 3.2. Phosphorylation state of ERK1/2 in control and ‘‘weaver’’ mice The endogenous levels of phosphorylated ERK1/2 in ‘‘weaver’’ mice are significantly elevated by 48% when compared to wild type animals (Fig. 2). Administration of CGS21680, while it has no effect on ERK1/2 phosphorylation in control animals, it decreases significantly the high endogenous levels of phospho-ERK1/2 in ‘‘weaver’’ striata by 30% (Fig. 2). On the contrary administration of MSX-3 in ‘‘weaver’’ animals led to a further increase by 55% in phosphorylated ERK1/2 compared to the levels observed in salineinjected ‘‘weaver’’. Administration of the serotonin receptor antagonists, ketanserin and NAN-190, had no significant effect on the levels of phospho-ERK1/2 in ‘‘weaver’’ animals compared to saline-injected ones (Fig. 2). Use of a D1 receptor agonist (SKF38393) resulted in an increase of phospho-ERK1/2 levels by about 45% in ‘‘weaver’’ animals (Fig. 2). Stimulation of the D2 receptors by the agonist quinpirole decreased the levels of phospho-ERK1/2 in ‘‘weaver’’ mice by 34%. 4. Discussion The ‘‘weaver’’ mutation is autosomal recessive and the pathologic phenotype in the nervous system of the wv/wv mice
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Fig. 2. Striatal ERK1/2 phosphorylation of control (+/+) animals treated with saline (SAL) and CGS21680 (CGS) and ‘‘weaver’’ (wv/wv) animals treated with saline (SAL), CGS21680 (CGS), MSX-3 (MSX), KETANSERIN and NAN-190 (KE/NA), SKF38893 (SKF) and Quinpirole (QUIN). Data were normalized to the average of the values obtained from weaver saline animals. Data represent means SEM (n = 6–20 per group) of representative Western blots. ##p < 0.01 significantly different compared to the control saline treated group. **p < 0.01 significantly different compared with the ‘‘weaver’’ saline treated group. Statistical analysis concerning wild type and weaver mice treated with saline or CGS21680 used two-way ANOVA (genotype drug treatment). A significant interaction was found between genotype and treatment (F(1,177) = 16.28, p < 0.001). Statistical analysis concerning ‘‘weaver’’ groups used oneway ANOVA followed by Dunnett’s post hoc test.
includes loss of cerebellar granule cells and midbrain dopamine neurons. Thus the ‘‘weaver’’ mouse provides a unique animal model of nigrostriatal dopamine deficiency, which appears gradually during development, similar to that seen in several parkinsonian syndromes (Ghetti and Triarhou, 1992). More concrete the degeneration starts at day 7 after birth reaching about 45% at day 20 and about 60% at the age of 2 months (Triarhou et al., 1988; Smith et al., 1990; Verney et al., 1995). Thus, the ‘‘weaver’’ mice used in this study (at the age of 2 months) show a midbrain dopaminergic neuronal loss of approximately 60% and consistently they have a reduction of forebrain dopamine content as well as of tyrosine hydroxylase reactivity about 70% compared to wild type mice (Richter et al., 1992; Roffler-Tarlov and Graybiel, 1984, 1986, 1987). Moreover, the ‘‘weaver’’ mutant displays asynuclein aggregates in the degenerating dopaminergic neurons (Ebadi et al., 2005) and recent results also favor it as an appropriate model of neuroinflammation mediating dopaminergic neurodegeneration in PD (Peng et al., 2006). Finally the ‘‘weaver’’ mutant has been proposed not only as an appropriate neurological model but also as a behavioral model of PD, showing cognitive impairment (Derenne et al., 2007). According to our results, administration of the A2A receptor agonist CGS21680 in wild type animals leads to an increase of phospho-DARPP-32 levels. This is in agreement with previously published work for both in vitro (Sahin et al., 2007; Svenningsson et al., 1998) and in vivo systems (Svenningsson et al., 2000) establishing that stimulation of A2A receptors activates the cAMP/
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PKA/DARPP-32 pathway. However, this stimulation in wild type animals is not sufficient to induce zif/268 mRNA expression in striatum as shown in our previous work (Ekonomou et al., 2004). Under physiological conditions dopamine controls A2A receptor activation through D2 receptors in striatum, impairing the ability of A2A receptors to signal through the cAMP-PKA cascade (Ferre´ et al., 2004, 2005; Schiffmann et al., 2007). Under dopamine deficiency, which reaches about 70% in weaver mouse striatum at the age of 2 months, A2A receptors are presumably disinhibited, released from D2 counteracting control, thus eliciting increased endogenous levels of Thr34-phospho-DARPP-32 as we observed in this study. This hypothesis is further supported by the fact that MSX-3 downregulates significantly the elevated basal levels of phosphoDARPP-32 in ‘‘weaver’’ mice. Administration of CGS21680 in ‘‘weaver’’ mice induces a further increase of the already elevated endogenous levels of Thr34-phospho-DARPP-32. This could probably lead to the zif/268 mRNA expression in striatum seen in our previous work (Ekonomou et al., 2004). Thus, this hypersensitive response of the A2A receptors could rely, at least in our dopamine deficient model, on the upregulated endogenous state of Thr34-phospho-DARPP-32, which would enhance the activity of the A2A/cAMP/PKA/DARPP-32/IEG signaling in the ‘‘indirect’’ pathway by amplifying PKA function (Greengard et al., 1999; Hakansson et al., 2004). Furthermore a slight upregulation of A2A receptors’ density was detected in ‘‘weaver’’ mice which however was restricted in the lateral part of striatum (Ekonomou et al., 2004). The elevated endogenous levels of phospho-ERK1/2 in ‘‘weaver’’ mice compared to wild type ones might be of physiological importance since ERK1/2 are two mitogen-activated kinases involved in neuronal plasticity (Thomas and Huganir, 2004). Similarly, in the corresponding primate model in which the dopaminergic degeneration was achieved gradually, by chronic MPTP administration, the animals also exhibited elevated phospho-ERK levels (Bezard et al., 2005; Nicholas et al., 2008). However, acute dopaminergic lesion induced by 6-OHDA in adult rats did not lead to increased levels of phospho-ERK1/2 (Brown and Gerfen, 2006). There is no information on whether the levels of phosphoERK1/2 are elevated in human PD. However it seems that the gradual degeneration of neurons seen in ‘‘weaver’’ mouse as well as in the progressive MPTP-lesioned primate model (Bezard et al., 2005; Nicholas et al., 2008) allows a compensatory mechanism to arise, something that could not be detected in the acute 6-OHDA model (Brown and Gerfen, 2006). A novel finding of this work is that stimulation of A2A receptors in ‘‘weaver’’ mice by CGS21680 induces a significant decrease of the endogenous phosphorylation state of ERK1/2 bringing it close to basal levels observed in the wild type animals. In line with the above, administration of the A2A antagonist MSX-3 to ‘‘weaver’’ mice activates the MAP-kinase signaling by increasing even more the elevated basal phospho-ERK1/2 levels. This is in contrast with the results of Quiroz et al. (2006) obtained from wild type animals, showing exactly the opposite, i.e. that in vivo administration of MSX-3 prevents the phosphorylation of ERK1/2 in striatum induced by electrical cortical stimulation. We do not know the mechanism through which blockade of A2A receptors by MSX-3 in the dopamine deficient ‘‘weaver’’ mice induces a significant increase of phosphorylated ERK1/2. However, our results have shown that in vivo stimulation of D1 receptors by SKF38393 also induces a further increase of the endogenous phosho-ERK1/2, demonstrating an upregulation of ERK1/2 phosphorylation in the spiny neurons of the ‘‘direct’’ striatal pathway. This is in line with the results of Gerfen et al. (2002) showing that IEG expression in the 6-OHDA lesioned mouse relies on a switch of D1 receptors to the ERK1/2 signaling pathway. Moreover, according to Gerfen et al. (2008) the D1 receptor-mediated ERK1/2 activation seen in the
dorsal striatum of the dopamine depleted animal is DARPP-32independent. In our study, ERK1/2 activation induced by blockade of A2A receptors seems to be also DARPP-32-independent, since MSX-3 increases the phosphorylation of ERK1/2 while it downregulates the phosphorylation of DARPP-32 in ‘‘weaver’’ mouse. Furthermore, under dopamine deficiency a negative interaction between adenosine A2A and dopamine D1 receptors exists (Jiang et al., 1993; Morelli et al., 1994), which could probably account for the elevated levels of phospho-ERK1/2 seen after D1 receptor agonist or A2A receptor antagonist treatment. This however has to be further investigated. Our results could be of clinical relevance since endogenous levels of adenosine can be easily upregulated (by metabolic or other stress conditions) eliciting overactivity of the ‘‘indirect’’ pathway via A2A receptor stimulation, thus worsening the symptoms in PD patients (Graybiel, 2000). Blockade of A2A receptors with antagonists has been suggested for ameliorating PD symptoms if co-administered with L-Dopa, as has been shown to be beneficial in animal models and clinical trials (Simola et al., 2008). However we do not know the physiological consequences of the phospho-ERK1/2 increase induced by A2A receptor antagonists seen in our study, on the parkinsonian symptoms. It is well known that activation of ERK1/2 signaling plays a critical role in dyskinesias induced by chronic L-Dopa treatment (Santini et al., 2007; Westin et al., 2007). Neuroinflammation has been shown to be a critical factor involved in the degeneration process of dopaminergic neurons in PD (Long-Smith et al., 2009; Hirsch and Hunot, 2009; Peng et al., 2006). Since the ‘‘weaver’’ mouse has been thought as a model of neuroinflammation in PD (Peng et al., 2006), it would be of interest to investigate the effect of A2A receptor ligands as antiinflammatory drugs, since there is evidence for their therapeutic effects on inflammatory diseases (Chen and Pedata, 2008; Csoka et al., 2008; Hasko et al., 2008; Hasko and Pacher, 2008; Trevethick et al., 2008). The mechanism responsible for the increased endogenous pERK1/2 levels in ‘‘weaver’’ does not seem to engage the serotonin pathway acting through HT1A and HT2A/C receptors, seen in the 6OHDA model (Brown and Gerfen, 2006), Here the blockade of these serotonin receptors has no effect on the levels of either phosphoDARPP-32 or phospho-ERK. Dopamine D2 receptors also do not appear to be involved in the induction of the elevated p-ERK levels (Gerfen et al., 2002), since stimulation of these receptors downregulates the phosphorylation state. In vivo administration of CGS21680 to the wild type animals, according to our results, does not change phospho-ERK1/2 state. This is in contrast with in vitro experiments showing that administration of CGS21680 to striatal slices elevates phosphoERK1/2 (Sahin et al., 2007). This discrepancy indicates once more, that the in vitro experiments are not always in line with the in vivo systems, where all the network connections are present. In conclusion, in the ‘‘weaver’’ mouse, a genetic model of dopamine deficiency, the endogenous levels of phospho-DARPP-32 at Thr34 and phospho-ERK1/2 have been found to be elevated probably as an adaptation phenomenon to the dopaminergic neurodegeneration, which proceeds gradually resembling the human PD. Stimulation of A2A receptors further increases phospho-DARPP-32 which probably leads to IEG expression in striatum of ‘‘weaver’’ mouse but downregulates the elevated phospho-ERK1/2 state to control levels. Consistently, blockade of A2A receptors significantly increases the phosphorylation state of ERK1/2. Our results indicate for the first time, that under dopamine deficiency the modulatory effect of A2A receptors on ERK1/2 signaling changes, exerting opposing action compared to that observed in normal animals (Quiroz et al., 2006). This information might be of clinical relevance since blockade of A2A receptors is
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Neurochemistry International journal homepage: www.elsevier.com/locate/neuint
Blockade of adenosine A2A receptors downregulates DARPP-32 but increases ERK1/2 activity in striatum of dopamine deficient ‘‘weaver’’ mouse Konstantinos Botsakis a,1, Ourania Pavlou b,1, Paraskevi D. Poulou c,1, Nikolaos Matsokis a, Fevronia Angelatou c,* a b c
Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 26 500, Greece Department of Materials Science, School of Natural Sciences, University of Patras, Patras 26 500, Greece Department of Physiology, School of Medicine, University of Patras, Patras 26 500, Greece
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 October 2009 Accepted 13 October 2009 Available online 21 October 2009
In the present study we investigated the signal transduction cascade modulated by adenosine A2A receptors under chronic dopamine deficiency in the ‘‘weaver’’ mouse. We determined the phosphorylation state of cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34 and of Extracellular Signal-regulated Protein Kinases 1/2 (ERK1/2), under basal conditions and after in vivo stimulation of A2A receptors by administration of the agonist CGS21680. Our results revealed that the endogenous levels of phospho-DARPPP-32 and phospho-ERK1/2 are elevated in ‘‘weaver’’ striatum probably as an adaptation phenomenon to gradual dopaminergic neurodegeneration appearing in this animal model, characterized as phenocopy of Parkinson’s disease. Stimulation of A2A receptors by CGS21680 further increases phospho-DARPP-32 but downregulates significantly the elevated phosphoERK1/2 levels bringing them close to those observed in wild type animals. Consistently, blockade of A2A receptors by MSX-3 (A2A receptor antagonist) downregulates phospho-DARPP-32 but significantly increases even more the phosphorylation/activation of ERK1/2. These results indicate that under chronic dopamine deficiency (a) the A2A/cAMP/PKA/DARPP-32 cascade is overactive due to the elevated endogenous phospho-DARPP-32 levels and (b) the A2A receptor modulatory effect on ERK1/2 signaling is dysregulated exerting opposing action compared to that observed in normal animals (Quiroz et al., 2006), i.e. in ‘‘weaver’’ animals A2A receptor blockade increases the activity of ERK1/2 cascade. This could be of clinical relevance since A2A antagonists are already used in clinical trials for ameliorating Parkinson’s disease (PD) symptoms. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: A2A receptors DARPP-32 ERK1/2 Dopamine deficiency ‘‘Weaver’’ mutant
1. Introduction Basal ganglia are involved in the integration of sensorimotor, associative and limbic information to produce motor behaviors and some forms of learning related plasticity (Bolam et al., 2000; Graybiel, 2005, 2008). The striatum is the main input and information processing structure of the basal ganglia. Corticolimbic-thalamic glutamatergic and mesencephalic dopaminergic systems converge in the GABAergic medium-sized spiny neurons of striatum (Gerfen, 2004). These are the striatal efferent neurons which can be classified into two neuronal pathways the ‘‘indirect’’, which projects via the globus pallidus and the subthalamic nucleus to output nuclei (substantia nigra and entopeduncular
* Corresponding author. Tel.: +30 2610 969159; fax: +30 2610 997215. E-mail address: [email protected] (F. Angelatou). 1 Equal contribution to this work. 0197-0186/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2009.10.007
nucleus) and the ‘‘direct’’, which sends axons directly to them (Gerfen, 2004). Both pathways are differentially regulated by dopaminergic nigrostriatal afferents via two types of receptors, D1-type which is confined to the ‘‘direct’’ pathway and D2-type confined to the ‘‘indirect’’ pathway (Gerfen, 2004). A balanced control of these two pathways is essential for the proper function of the extrapyramidal motor system (Graybiel, 2000). In Parkinson’s disease (PD), the gradual degeneration of the nigrostriatal dopaminergic system produces striatal dopamine depletion, which consequently impairs the function of the basal ganglia circuits resulting in akinesia, bradykinesia, tremor, and rigidity (Singh et al., 2007). Adenosine, a major neuromodulator of the synaptic function, exerts its effects in basal ganglia, by acting through its A1 and A2A adenosine receptors. The A2A receptors are co-localized and interact functionally with D2 receptors, on the medium-sized spiny neurons of the ‘‘indirect’’ pathway in striatum exerting opposing actions on neurotransmitter release, gene expression,
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and motor behavior (Ferre´ et al., 1997, 2008). As shown previously, overactivity of the ‘‘indirect’’ striatal pathway is a key component of the neural mechanisms responsible for generating parkinsonian symptoms (Graybiel, 2000). A previous study from our laboratory (Ekonomou et al., 2004) has shown that in vivo activation of adenosine A2A receptors by the agonist CGS21680, elicits a strong expression of the immediate early gene (IEG) zif/268 mRNA in striatum and motor cortex of ‘‘weaver’’ mice, but not in control animals. The ‘‘weaver’’ mutant represents the only genetic animal model of gradual nigrostriatal neurodegeneration, which can be characterized as a pathophysiological phenocopy of PD (Roffler-Tarlov and Graybiel, 1984; Triarhou, 2002). In the present study we investigated the signal transduction pathway that modulates IEG expression via A2A receptor stimulation under dopamine deficiency. We examined the in vivo induction of phosphorylation of DARPP-32 in striatum of ‘‘weaver’’ and control mice after A2A receptor activation. DARPP-32 is a central molecule in the signal transduction pathway of D1 and A2A receptors, which are both Gs/olf-coupled receptors and their activation stimulates the cAMP/PKA/DARPP-32 signaling cascade (Greengard et al., 1999). Furthermore we investigated whether, under dopamine deficiency, besides the A2A/cAMP/PKA/DARPP-32 established pathway an alternative pathway is also activated by the A2A receptors. Towards this end we examined the possible activation of the Mitogen-activated Protein Kinase (MAPK) pathway by determining ERK1/2 phosphorylation. ERK activation is involved in physiological processes and long-term changes in synaptic plasticity (Sweatt, 2004; Thomas and Huganir, 2004; Yoon and Seger, 2006). Moreover, ERK activation has been implicated in dopamine D1 receptor agonist supersensitivity in PD models (Gerfen et al., 2002, 2008). Before exploring either pathway we first established the endogenous levels of phosphorylated DARPP-32 and ERK1/2 in ‘‘weaver’’ animals. 2. Experimental procedures 2.1. Animals Control (+/+) and ‘‘weaver’’ male (wv/wv) mice, 2 months old, were used. The animals were bred in our laboratory from a breeding stock of adult heterozygous animals purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in groups of adult controls (+/+) or adult heterozygous (wv/+) of five to six per cage in a room with controlled light/dark cycle (12-h light/dark) and free access to food and water. In the present work, all efforts were made to minimize animal suffering, according to the European Communities Council Directive Guidelines (86/609/EEC) and to the Greek National Laws (Animal Acts, PD 160/91) for the care and use of laboratory animals. Identification of mice as wv/wv or wv/+ was performed by virtue of behavior. The abnormal behavior of wv/wv mice was characterized by weakness, hypotonia, resting and intention tremor, poor limb coordination and instability of gait (Simon and Ghetti, 1994). At the age of treatment (2 months old wv/wv mice) the neurodegeneration of midbrain dopaminergic neurons reaches about 60% and consequently the content of dopamine is reduced about 70% in dorsal striatum (Triarhou et al., 1988; Smith et al., 1990; Verney et al., 1995; Roffler-Tarlov and Graybiel, 1984, 1986, 1987). For the study the following animal groups were used: (i) control mice receiving saline, (ii) control mice receiving the A2A receptor agonist, CGS21680 (20 mg/kg body weight), (iii) ‘‘weaver’’ mice receiving saline, (iv) ‘‘weaver’’ mice receiving CGS21680 (20 mg/kg body weight), (v) ‘‘weaver’’ mice receiving the A2A receptor antagonist, MSX-3 (10 mg/kg body weight), (vi) ‘‘weaver’’ mice receiving the D2 receptor agonist, quinpirole (0.5 mg/kg body weight), (vii) ‘‘weaver’’ mice receiving the D1 receptor agonist, SKF38393 (20 mg/kg body weight), and (viii) ‘‘weaver’’ mice receiving the serotonin receptor antagonists ketanserin (H-T2) (25 mg/kg body weight) and NAN-190 (H-T1A) (5 mg/kg body weight) injected 5 min after the ketanserin. 2.2. Materials Drugs were purchased from Sigma (Germany) and Tocris (UK). All drugs were dissolved in physiological saline (with a few drops of 0.1N NaOH for MSX-3, final pH 7.4) immediately before use and were injected intraperitoneally.
2.3. Tissue preparation Animals were decapitated 20 min after the last injection, the brains were rapidly removed and flash-frozen in liquid nitrogen. Both sides of the striatum were then dissected out in a cryostat at 22 8C and stored at 80 8C (Picconi et al., 2003). Frozen tissue samples were sonicated in boiling 1% SDS (400 ml) with 4 ml Sigma Phosphatase-Inhibitor-Cocktail-I, 4 ml Sigma Phosphatase-Inhibitor-Cocktail-II, 4 ml Sigma Protease-Inhibitor-Cocktail and boiled for 5 min. Protein concentration was determined for each sample using the BSA protein kit (Pierce, Rockford, IL) and spectrophotometry. 2.4. Western blotting Equal amounts of protein (50 mg) from each sample were loaded onto 12% polyacrylamide gels, separated by SDS-PAGE and then transferred electrophoretically to nitrocellulose membranes (Schleicher and Schuell). The membranes were immunoblotted using rabbit polyclonal antibodies, anti-phospho-Thr202/ Tyr204-ERK1/2 (Cell Signaling Technology), and anti-phospho-Thr34–DARPP-32 (Novus-Biologicals). Mouse monoclonal antibodies against ERK1/2 (Cell Signaling Technology), DARPP-32 (BD Transduction-Laboratories) and Tubuline (SIGMA) were used to estimate the total amount of proteins. Antibody binding was revealed using goat anti-rabbit horseradish peroxidase-linked IgG (for the polyclonal primary antibodies) or goat anti-mouse horseradish peroxidase-linked IgG (for the monoclonal antibodies) followed by the Enhanced Chemiluminescence Detection System (Amersham). Luminescence from the blots was detected by exposing the membranes to Fuji-Hyperfilm for 30 s to 5 min, to ensure that we were operating within the linear range of the film, followed by digital scanning of the developed film in transparency mode. The scanned image of the membranes and bandintensities were calibrated and quantified using NIH-ImageJ software (version 1.34). For each animal, the values obtained from the experiments with phosphorylated and total ERK1/2 corresponded to the total of the bands intensities for both ERK1 and ERK2 (also called p44 and p42 MAP kinases, in relation to their molecular weights, 44 and 42 kDa, respectively). For each animal, the values of phosphorylated DARPP-32 Thr34 and phosphorylated ERK1/2 were normalized to total DARPP-32 and total ERK1/2 respectively. The amount of total DARPP-32 and total ERK1/2 did not differ between control and ‘‘weaver’’ animals as shown using tubuline as reference (data not shown). In each Western blot, all values were normalized to the average of the values obtained from the weaver saline animals included in the same blot. Each experiment was performed two to four times. 2.5. Statistical analysis Data concerning wild type mice and weaver mice treated with saline or CGS21680 were analyzed using two-way ANOVA, in which genotype and drug treatment were the independent variables. When comparing the groups of ‘‘weaver’’ mice receiving saline with all the other ‘‘weaver’’ groups the data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test for specific comparisons.
3. Results 3.1. Phosphorylation state of DARPP-32 at Thr34 in control and ‘‘weaver’’ mice The amount of endogenous phospho-Thr-34-DARPP-32 in striata homogenates of ‘‘weaver’’ compared to wild type animals was significantly elevated by 49% (Fig. 1). Upon stimulation of A2A receptors by in vivo administration of the agonist CGS21680 we observed a significant increase of phospho-Thr-34-DARPP-32, which in control animals is 21%, compared to saline-injected ones, while in ‘‘weaver’’ animals is 17%, compared to saline-injected ‘‘weaver’’ (Fig. 1). When comparing the amount of phosphoDARPP-32 between ‘‘weaver’’ and control animals after A2A stimulation we found that they differed significantly, with phospho-DARPP-32 in ‘‘weaver’’ animals being elevated by about 50% compared to controls (Fig. 1). Administration of the A2A antagonist MSX-3 in ‘‘weaver’’ animals decreased the levels of phospho-DARPP-32 by 28% when compared to saline-injected ‘‘weaver’’ mice (Fig. 1). Administration of the serotonin receptor antagonists, ketanserin and NAN-190, had no significant effect on the levels of phospho-DARPP-32 in ‘‘weaver’’ animals compared to saline-injected ones (Fig. 1). Activation of the D1 receptors by SKF38393 led to a moderate increase (12%) of basal phosphoDARPP-32 in ‘‘weaver’’ animals, which however was significant
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Fig. 1. Striatal DARPP-32 phosphorylation of control (+/+) animals treated with saline (SAL) and CGS21680 (CGS) and ‘‘weaver’’ (wv/wv) animals treated with saline (SAL), CGS21680 (CGS), MSX-3 (MSX), KETANSERIN and NAN-190 (KE/NA), SKF38893 (SKF) and Quinpirole (QUIN). Data were normalized to the average of the values obtained from weaver saline animals. Data represent means SEM (n = 6–20 per group) of representative Western blots. #p < 0.05 significantly different compared to the control saline treated group. *p < 0.05 and **p < 0.01 significantly different compared with the ‘‘weaver’’ saline treated group. yp < 0.05 ‘‘weaver’’ CGS compared to the control CGS treated group. Statistical analysis concerning wild type and weaver mice treated with saline or CGS21680 used two-way ANOVA (genotype drug treatment), while statistical analysis concerning ‘‘weaver’’ groups used one-way ANOVA followed by Dunnett’s post hoc test.
(Fig. 1). Stimulation of the D2 receptors by the agonist quinpirole decreased significantly the levels of basal phospho-DARPP-32 in ‘‘weaver’’ mice by 21%. 3.2. Phosphorylation state of ERK1/2 in control and ‘‘weaver’’ mice The endogenous levels of phosphorylated ERK1/2 in ‘‘weaver’’ mice are significantly elevated by 48% when compared to wild type animals (Fig. 2). Administration of CGS21680, while it has no effect on ERK1/2 phosphorylation in control animals, it decreases significantly the high endogenous levels of phospho-ERK1/2 in ‘‘weaver’’ striata by 30% (Fig. 2). On the contrary administration of MSX-3 in ‘‘weaver’’ animals led to a further increase by 55% in phosphorylated ERK1/2 compared to the levels observed in salineinjected ‘‘weaver’’. Administration of the serotonin receptor antagonists, ketanserin and NAN-190, had no significant effect on the levels of phospho-ERK1/2 in ‘‘weaver’’ animals compared to saline-injected ones (Fig. 2). Use of a D1 receptor agonist (SKF38393) resulted in an increase of phospho-ERK1/2 levels by about 45% in ‘‘weaver’’ animals (Fig. 2). Stimulation of the D2 receptors by the agonist quinpirole decreased the levels of phospho-ERK1/2 in ‘‘weaver’’ mice by 34%. 4. Discussion The ‘‘weaver’’ mutation is autosomal recessive and the pathologic phenotype in the nervous system of the wv/wv mice
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Fig. 2. Striatal ERK1/2 phosphorylation of control (+/+) animals treated with saline (SAL) and CGS21680 (CGS) and ‘‘weaver’’ (wv/wv) animals treated with saline (SAL), CGS21680 (CGS), MSX-3 (MSX), KETANSERIN and NAN-190 (KE/NA), SKF38893 (SKF) and Quinpirole (QUIN). Data were normalized to the average of the values obtained from weaver saline animals. Data represent means SEM (n = 6–20 per group) of representative Western blots. ##p < 0.01 significantly different compared to the control saline treated group. **p < 0.01 significantly different compared with the ‘‘weaver’’ saline treated group. Statistical analysis concerning wild type and weaver mice treated with saline or CGS21680 used two-way ANOVA (genotype drug treatment). A significant interaction was found between genotype and treatment (F(1,177) = 16.28, p < 0.001). Statistical analysis concerning ‘‘weaver’’ groups used oneway ANOVA followed by Dunnett’s post hoc test.
includes loss of cerebellar granule cells and midbrain dopamine neurons. Thus the ‘‘weaver’’ mouse provides a unique animal model of nigrostriatal dopamine deficiency, which appears gradually during development, similar to that seen in several parkinsonian syndromes (Ghetti and Triarhou, 1992). More concrete the degeneration starts at day 7 after birth reaching about 45% at day 20 and about 60% at the age of 2 months (Triarhou et al., 1988; Smith et al., 1990; Verney et al., 1995). Thus, the ‘‘weaver’’ mice used in this study (at the age of 2 months) show a midbrain dopaminergic neuronal loss of approximately 60% and consistently they have a reduction of forebrain dopamine content as well as of tyrosine hydroxylase reactivity about 70% compared to wild type mice (Richter et al., 1992; Roffler-Tarlov and Graybiel, 1984, 1986, 1987). Moreover, the ‘‘weaver’’ mutant displays asynuclein aggregates in the degenerating dopaminergic neurons (Ebadi et al., 2005) and recent results also favor it as an appropriate model of neuroinflammation mediating dopaminergic neurodegeneration in PD (Peng et al., 2006). Finally the ‘‘weaver’’ mutant has been proposed not only as an appropriate neurological model but also as a behavioral model of PD, showing cognitive impairment (Derenne et al., 2007). According to our results, administration of the A2A receptor agonist CGS21680 in wild type animals leads to an increase of phospho-DARPP-32 levels. This is in agreement with previously published work for both in vitro (Sahin et al., 2007; Svenningsson et al., 1998) and in vivo systems (Svenningsson et al., 2000) establishing that stimulation of A2A receptors activates the cAMP/
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PKA/DARPP-32 pathway. However, this stimulation in wild type animals is not sufficient to induce zif/268 mRNA expression in striatum as shown in our previous work (Ekonomou et al., 2004). Under physiological conditions dopamine controls A2A receptor activation through D2 receptors in striatum, impairing the ability of A2A receptors to signal through the cAMP-PKA cascade (Ferre´ et al., 2004, 2005; Schiffmann et al., 2007). Under dopamine deficiency, which reaches about 70% in weaver mouse striatum at the age of 2 months, A2A receptors are presumably disinhibited, released from D2 counteracting control, thus eliciting increased endogenous levels of Thr34-phospho-DARPP-32 as we observed in this study. This hypothesis is further supported by the fact that MSX-3 downregulates significantly the elevated basal levels of phosphoDARPP-32 in ‘‘weaver’’ mice. Administration of CGS21680 in ‘‘weaver’’ mice induces a further increase of the already elevated endogenous levels of Thr34-phospho-DARPP-32. This could probably lead to the zif/268 mRNA expression in striatum seen in our previous work (Ekonomou et al., 2004). Thus, this hypersensitive response of the A2A receptors could rely, at least in our dopamine deficient model, on the upregulated endogenous state of Thr34-phospho-DARPP-32, which would enhance the activity of the A2A/cAMP/PKA/DARPP-32/IEG signaling in the ‘‘indirect’’ pathway by amplifying PKA function (Greengard et al., 1999; Hakansson et al., 2004). Furthermore a slight upregulation of A2A receptors’ density was detected in ‘‘weaver’’ mice which however was restricted in the lateral part of striatum (Ekonomou et al., 2004). The elevated endogenous levels of phospho-ERK1/2 in ‘‘weaver’’ mice compared to wild type ones might be of physiological importance since ERK1/2 are two mitogen-activated kinases involved in neuronal plasticity (Thomas and Huganir, 2004). Similarly, in the corresponding primate model in which the dopaminergic degeneration was achieved gradually, by chronic MPTP administration, the animals also exhibited elevated phospho-ERK levels (Bezard et al., 2005; Nicholas et al., 2008). However, acute dopaminergic lesion induced by 6-OHDA in adult rats did not lead to increased levels of phospho-ERK1/2 (Brown and Gerfen, 2006). There is no information on whether the levels of phosphoERK1/2 are elevated in human PD. However it seems that the gradual degeneration of neurons seen in ‘‘weaver’’ mouse as well as in the progressive MPTP-lesioned primate model (Bezard et al., 2005; Nicholas et al., 2008) allows a compensatory mechanism to arise, something that could not be detected in the acute 6-OHDA model (Brown and Gerfen, 2006). A novel finding of this work is that stimulation of A2A receptors in ‘‘weaver’’ mice by CGS21680 induces a significant decrease of the endogenous phosphorylation state of ERK1/2 bringing it close to basal levels observed in the wild type animals. In line with the above, administration of the A2A antagonist MSX-3 to ‘‘weaver’’ mice activates the MAP-kinase signaling by increasing even more the elevated basal phospho-ERK1/2 levels. This is in contrast with the results of Quiroz et al. (2006) obtained from wild type animals, showing exactly the opposite, i.e. that in vivo administration of MSX-3 prevents the phosphorylation of ERK1/2 in striatum induced by electrical cortical stimulation. We do not know the mechanism through which blockade of A2A receptors by MSX-3 in the dopamine deficient ‘‘weaver’’ mice induces a significant increase of phosphorylated ERK1/2. However, our results have shown that in vivo stimulation of D1 receptors by SKF38393 also induces a further increase of the endogenous phosho-ERK1/2, demonstrating an upregulation of ERK1/2 phosphorylation in the spiny neurons of the ‘‘direct’’ striatal pathway. This is in line with the results of Gerfen et al. (2002) showing that IEG expression in the 6-OHDA lesioned mouse relies on a switch of D1 receptors to the ERK1/2 signaling pathway. Moreover, according to Gerfen et al. (2008) the D1 receptor-mediated ERK1/2 activation seen in the
dorsal striatum of the dopamine depleted animal is DARPP-32independent. In our study, ERK1/2 activation induced by blockade of A2A receptors seems to be also DARPP-32-independent, since MSX-3 increases the phosphorylation of ERK1/2 while it downregulates the phosphorylation of DARPP-32 in ‘‘weaver’’ mouse. Furthermore, under dopamine deficiency a negative interaction between adenosine A2A and dopamine D1 receptors exists (Jiang et al., 1993; Morelli et al., 1994), which could probably account for the elevated levels of phospho-ERK1/2 seen after D1 receptor agonist or A2A receptor antagonist treatment. This however has to be further investigated. Our results could be of clinical relevance since endogenous levels of adenosine can be easily upregulated (by metabolic or other stress conditions) eliciting overactivity of the ‘‘indirect’’ pathway via A2A receptor stimulation, thus worsening the symptoms in PD patients (Graybiel, 2000). Blockade of A2A receptors with antagonists has been suggested for ameliorating PD symptoms if co-administered with L-Dopa, as has been shown to be beneficial in animal models and clinical trials (Simola et al., 2008). However we do not know the physiological consequences of the phospho-ERK1/2 increase induced by A2A receptor antagonists seen in our study, on the parkinsonian symptoms. It is well known that activation of ERK1/2 signaling plays a critical role in dyskinesias induced by chronic L-Dopa treatment (Santini et al., 2007; Westin et al., 2007). Neuroinflammation has been shown to be a critical factor involved in the degeneration process of dopaminergic neurons in PD (Long-Smith et al., 2009; Hirsch and Hunot, 2009; Peng et al., 2006). Since the ‘‘weaver’’ mouse has been thought as a model of neuroinflammation in PD (Peng et al., 2006), it would be of interest to investigate the effect of A2A receptor ligands as antiinflammatory drugs, since there is evidence for their therapeutic effects on inflammatory diseases (Chen and Pedata, 2008; Csoka et al., 2008; Hasko et al., 2008; Hasko and Pacher, 2008; Trevethick et al., 2008). The mechanism responsible for the increased endogenous pERK1/2 levels in ‘‘weaver’’ does not seem to engage the serotonin pathway acting through HT1A and HT2A/C receptors, seen in the 6OHDA model (Brown and Gerfen, 2006), Here the blockade of these serotonin receptors has no effect on the levels of either phosphoDARPP-32 or phospho-ERK. Dopamine D2 receptors also do not appear to be involved in the induction of the elevated p-ERK levels (Gerfen et al., 2002), since stimulation of these receptors downregulates the phosphorylation state. In vivo administration of CGS21680 to the wild type animals, according to our results, does not change phospho-ERK1/2 state. This is in contrast with in vitro experiments showing that administration of CGS21680 to striatal slices elevates phosphoERK1/2 (Sahin et al., 2007). This discrepancy indicates once more, that the in vitro experiments are not always in line with the in vivo systems, where all the network connections are present. In conclusion, in the ‘‘weaver’’ mouse, a genetic model of dopamine deficiency, the endogenous levels of phospho-DARPP-32 at Thr34 and phospho-ERK1/2 have been found to be elevated probably as an adaptation phenomenon to the dopaminergic neurodegeneration, which proceeds gradually resembling the human PD. Stimulation of A2A receptors further increases phospho-DARPP-32 which probably leads to IEG expression in striatum of ‘‘weaver’’ mouse but downregulates the elevated phospho-ERK1/2 state to control levels. Consistently, blockade of A2A receptors significantly increases the phosphorylation state of ERK1/2. Our results indicate for the first time, that under dopamine deficiency the modulatory effect of A2A receptors on ERK1/2 signaling changes, exerting opposing action compared to that observed in normal animals (Quiroz et al., 2006). This information might be of clinical relevance since blockade of A2A receptors is
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