Alterations of striatal NMDA receptor subunits ...

Neuropharmacology 48 (2005) 503–516 www.elsevier.com/locate/neuropharm

Alterations of striatal NMDA receptor subunits associated with the development of dyskinesia in the MPTP-lesioned primate model of Parkinson’s disease P.J. Halletta,*, A.W. Dunaha, P. Ravenscroftb, S. Zhoub, E. Bezardc, A.R. Crossmanb, J.M. Brotchied, D.G. Standaerta a

MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital and Harvard Medical School, 114 16th Street, Charlestown, MA 02129, USA b Division of Neuroscience, University of Manchester 1.124 Stopford Building, Oxford Road, Manchester, M13 9PT, UK c Basal Gang, Laboratoire de Neurophysiologie, CNRS UMR 5543, Universite Victor Segalen-Bordeaux 2, 146 rue Leo Saignat, BP 28, 33076 Bordeaux cedex, France d Toronto Western Research Institute, MC 11-419, Toronto Western Hospital, 399 Bathurst St, Toronto, Ontario, M5T 2S8, Canada Received 9 July 2004; received in revised form 16 October 2004; accepted 27 November 2004

Abstract The development of dyskinesias and other motor complications greatly limits the use of levodopa therapy in Parkinson’s disease (PD). Studies in rodent models of PD suggest that an important mechanism underlying the development of levodopa-related motor complications is alterations in striatal NMDA receptor function. We examined striatal NMDA receptors in the MPTP-lesioned primate model of PD. Quantitative immunoblotting was used to determine the subcellular abundance of NR1, NR2A and NR2B subunits in striata from unlesioned, MPTP-lesioned (parkinsonian) and MPTP-lesioned, levodopa-treated (dyskinetic) macaques. In parkinsonian macaques, NR1 and NR2B subunits in synaptosomal membranes were decreased to 66 G 11% and 51.2 G 5% of unlesioned levels respectively, while the abundance of NR2A was unaltered. Levodopa treatment eliciting dyskinesia normalized NR1 and NR2B and increased NR2A subunits to 150 G 12% of unlesioned levels. No alterations in receptor subunit tyrosine phosphorylation were detected. These results demonstrate that altered synaptic abundance of NMDA receptors with relative enhancement in the abundance of NR2A occurs in primate as well as rodent models of parkinsonism, and that in the macaque model, NR2A subunit abundance is further increased in dyskinesia. These data support the view that alterations in striatal NMDA receptor systems are responsible for adaptive and maladaptive responses to dopamine depletion and replacement in parkinsonism, and highlight the value of subtype selective NMDA antagonists as novel therapeutic approaches for PD. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Basal ganglia; Trafficking; Dopamine; Glutamate; Levodopa; Macaque

1. Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons of the substantia nigra pars compacta * Corresponding author. Tel.: C1 617 724 9147; fax: C1 617 724 1480. E-mail address: [email protected] (P.J. Hallett). 0028-3908/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.11.008

(SNc), which results in a deficiency of striatal dopamine. In the initial stages of disease, symptoms can be effectively controlled using the dopamine precursor L-3,4-dihydroxyphenylalanine (levodopa) (Cotzias et al., 1969). However, long-term levodopa treatment is limited by the development of ‘‘motor complications’’, including wearing off, on–off fluctuations, and abnormal movements termed levodopa-induced dyskinesia (LID) (Fahn, 1999). Approximately 40% of PD patients will

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develop motor fluctuations within 5 years of beginning levodopa therapy (Ahlskog and Muenter, 2001). These motor fluctuations can significantly impair quality of life and limit effective treatment of the disease. Abnormal function of striatal N-methyl-D-aspartate (NMDA) glutamate receptors has been implicated in the pathogenesis of parkinsonian symptoms as well as in the development of motor complications of levodopa therapy (Chase and Oh, 2000a; Hallett and Standaert, 2004). The striatum is the principal input structure of the basal ganglia, and the primary target of the dopamine depletion which occurs in PD. Striatal neurons receive extensive glutamatergic afferents from motor and non-motor regions of the cortex, which synapse on the spines of striatal output neurons. Dopaminergic synapses are found in close proximity on the shafts of these spines (Smith and Bolam, 1990) and are thought to exert a modulatory influence over the excitatory inputs (Bolam et al., 2000). NMDA receptors are essential for mediating excitatory transmission at corticostriatal synapses (Di Chiara et al., 1994) and for striatal long-term potentiation (Calabresi et al., 1992). In experimental models of PD, NMDA receptor antagonists are effective in alleviating parkinsonian symptoms (Nash et al., 1998, 2000; Steece-Collier et al., 2000; Loschmann et al., 2004). NMDA receptor antagonists can also block the development of motor complications of chronic dopaminergic therapy and attenuate motor complications after they have been induced (Papa and Chase, 1996; Blanchet et al., 1999; Hadj Tahar et al., 2004; Wessell et al., 2004), suggesting that a functional overactivity of NMDA receptors contributes to the development of wearing off and dyskinesia (Klockgether and Turski, 1993; Chase and Oh, 2000b; Hallett and Standaert, 2004). NMDA receptors are heteromeric assemblies of subunits (NRs). In the adult rat and human striatum, the most abundant subunit mRNAs are for the NR1 (NR1a isoform), NR2A and NR2B subunits (Standaert et al., 1994; Kosinski et al., 1998). In the unilateral 6-hydroxydopamine (6-OHDA) lesioned rat model of PD, dopamine depletion results in a reduction in the protein abundance of NR1 and NR2B subunits in striatal synaptosomal membrane fractions, reflecting a selective reduction of NMDA receptor complexes composed of NR1/NR2B (Dunah et al., 2000). NR2A abundance is not altered and therefore is enhanced relative to the other subunits. In these rodents, chronic levodopa treatment restores the abundance of NR1, NR2A and NR2B subunits to normal levels, but leads to hyperphosphorylation of both NR2A and NR2B subunits (Oh et al., 1998; Dunah et al., 2000). These changes in synaptic proteins in chronic lesion models appear to arise as a result of redistribution of receptor complexes between synaptic and intracellular sites. An underlying mechanism for this distribution may be

a rapid D1 dopamine receptor- and tyrosine kinasedependent trafficking system, which regulates delivery of NMDA receptors to synaptic sites in striatal neurons (Dunah and Standaert, 2001; Dunah et al., 2004). The rodent studies mentioned above have provided important insights into modifications of striatal NMDA receptors, which occur after dopamine depletion and behavioral sensitization following chronic dopamine treatment. An important limitation of these studies is that the rodent models do not reproduce all the features of human PD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned primate models of PD are a much more authentic model of human PD than rodent models, and they replicate many features of the human disorder including the development of LID which is unequivocally equivalent to that seen in man (Jenner, 2003). Assessment of the NMDA receptor hypothesis of Parkinson’s disease and LID in this model is, thus, a necessary step towards developing new strategies for the treatment of human disease. We have investigated the abundance, subcellular distribution and tyrosine phosphorylation of striatal NMDA receptor subunits in the MPTP-lesioned macaque model of PD and LID. Some of this work has been previously reported in abstract form (Hallett et al., 2003).

2. Methods 2.1. MPTP-lesioning of animals Eighteen female macaque monkeys (Macaca mulatta, Experimental Animal Lab Center, Fourth Military Medical University, Xi’an, PR, China) weighing 3–6 kg were used in this study. Animals were housed in individual primate cages (1.1 m high ! 0.9 m deep ! 0.8 m wide) under controlled conditions of humidity (50 G 5%), temperature (24 G 1%) and light (12 h light–dark cycles, lights on 08:00 h), food and water were available ad libitum. Animal care was supervised by veterinarians, skilled in the healthcare and maintenance of non-human primates. Experiments were carried out in accordance with the requirements of The European Union Council Directive 86/609/EEC as they apply to the care of laboratory animals. All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Five animals were treated solely with vehicle (water, orally, twice daily) for the whole experimental period (unlesioned group). Thirteen animals were rendered parkinsonian with 15 daily injections of MPTP hydrochloride (0.2 mg/kg i.v., given at 09:00 h, dissolved in ethanol; Sigma) according to a previously described protocol (Bezard et al., 2001b). Thirty days after commencing MPTP administration a stable parkinsonian

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condition was observed in all the treated animals. Nine MPTP-lesioned animals were given three daily doses of apomorphine (0.3 mg/kg s.c.,), followed by oral levodopa (25 mg/kg) and benserazide (0.5 mg/kg), as MadoparÒ, twice daily for 120 days (MPTP-lesioned, dyskinetic group). The remaining 4 MPTP-lesioned animals (MPTP-lesioned, non-dyskinetic group) were given vehicle treatment. On the day of the terminal procedure, the behavior of each animal was videotaped for subsequent blinded analysis by a neurologist with experience in movement disorders. Immediately following administration of vehicle or levodopa, animals were transferred to an observation cage (1.06 m high ! 1.0 m wide ! 0.8 m deep) and their behavior videotaped for 60 min. Dyskinesia was rated by post-hoc analysis of the videotapes and rated using a global dyskinesia rating scale where choreic and dystonic symptoms were graded from 0 to 4, where 0 Z absent, 1 Z mild, 2 Z moderate, but not interfering with normal activity, 3 Z marked, and at times interfering with normal activity, 4 Z severe, functionally disabling and replacing normal activity. Sixty minutes after the last treatment, a time at which dyskinesia was maximal in all animals in the MPTP-lesioned, dyskinetic group, all animals were killed by anesthetic overdose (sodium pentobarbital (150 mg/kg, i.v.)). Brains were removed rapidly and divided into the two hemispheres along the midline. Dissection was performed on ice with the brain immersed in cold saline water (0.9%). The striatum (combining caudate nucleus, putamen and nucleus accumbens, across the rostrocaudal extent of the structure) was dissected from each hemisphere and frozen at ÿ45 C in isopentane and then stored at ÿ80 C. The time taken to dissect the striata from each animal was approximately 10 min. In the following text, MPTP-lesioned animals treated with vehicle are termed ‘‘MPTP-lesioned, parkinsonian’’, and MPTP-lesioned rendered dyskinetic with repeated levodopa treatment are termed ‘‘MPTPlesioned, dyskinetic’’. 2.2. Subcellular fractionation of macaque striatal tissue Fractionation of tissue for immunoblotting was performed as described previously (Dunah and Standaert, 2001) with minor modifications to allow for differences in tissue. A schematic to illustrate the biochemical fractionation procedure is illustrated in Fig. 1. Aliquots of the pellet and supernatant fractions obtained from each centrifugation step were removed and kept on ice until the end of the fractionation procedure. Approximately 500 mg frozen weight striatal tissue was defrosted for 5 min in 10 ml ice-cold TEVP buffer, pH 7.4 (10 mM Tris–HCl, 5 mM NaF, 1 mM Na3VO4,

Striatal tissues homogenized in TEVP / 320mM sucrose buffer H (Total Homogenate) 800 x g P1 (Nuclei and large debris)

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P2 (Crude synaptosomal membranes) Lysis 25,000 x g LS1 LP1 (Synaptosomal membranes) LP2 (Synaptic vesicleenriched)

S2 165,000 x g P3 (Light membranes)

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Fig. 1. Characterization of biochemical fractionation procedure in macaque striatum. Schematic for the biochemical fractionation. The procedure for the subcellular separation of proteins as depicted in this schematic is described in Section 2.2.

1 mM EDTA, 1 mM EGTA), containing 320 mM sucrose. Tissue was homogenized in the TEVP buffer for 15 s using an Ultra-Turrax T25 homogenizer (JankeKunkel, IKA Labortechnik) at load setting 24,000 rpm. A 500 ml sample of homogenate (H) was removed and kept on ice and the remainder of the sample centrifuged at 800!g for 10 min at 4 C. Following centrifugation, the supernatant (S1) was carefully separated from the pellet and transferred into a clean centrifuge tube. The pellet (P1) was rinsed briefly with ice-cold TEVP buffer to avoid possible crossover contamination and was then resuspended into 2 ml TEVP buffer, pH 7.4. An aliquot of 500 ml was removed. The S1 supernatant was centrifuged at 9200 ! g for 15 min at 4 C. Following centrifugation, the supernatant (S2) was transferred to a clean centrifuge tube and placed on ice. A 500 ml sample of the supernatant was removed and placed on ice. The P2 pellet was rinsed briefly and resuspended in 2 ml TEVP buffer, pH 7.4 containing 35.6 mM sucrose, and hypo-osmotic lysis to release synaptic vesicles and other cytoplasmic organelles was performed by keeping on ice for 30 min. A 300 ml sample of the lysed P2 fraction was removed. The remaining P2 fraction was centrifuged at 25,000 ! g for 20 min at 4 C. Following centrifugation, the supernatant (LS1) was collected and a 300 ml sample removed and placed on ice. The LP1 pellet was rinsed briefly and resuspended in 1 ml TEVP buffer, pH 7.4 and kept on ice. The S2 and LS1 supernatant fractions were centrifuged at 165,000 ! g for 2 h at 4 C. The supernatants (S3 and LS2) obtained were removed and a 500 ml sample of each taken. The P3

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pellet obtained was rinsed briefly and resuspended in 200 ml TEVP buffer, pH 7.4, and was transferred to an Eppendorf tube. The LP2 pellet was rinsed briefly and resuspended in 50 ml TEVP buffer, pH 7.4, and was transferred to an Eppendorf tube. At the end of the centrifugation procedure all pellet fractions were sonicated for 10 s, using a probe sonicator. Pellet and supernatant fractions were then frozen and stored at ÿ80 C.

were sonicated for 5 s. Samples were centrifuged at 15,000! g and the supernatant transferred to a clean Eppendorf tube. Protein concentrations were determined using the BioRad DC protein assay kit (BioRad). The supernatants were then used for immunoblot and immunoprecipitation studies.

2.3. Assessment of lesions in MPTP-lesioned macaque striatum

The monoclonal anti-phosphotyrosine antibody (PY20) (Transduction Laboratories) was incubated with protein A-Sepharose beads (Sigma) at a concentration of 20 mg antibody per 50 ml of hydrated protein A-Sepharose overnight at 4 C in 100 mM sodium borate, pH 8.0, with gentle rotation. The beads were washed with 100 mM sodium borate, pH 8.0, and used for immunoprecipitation.

The extent of depletion of dopamine terminals in the striatum was assessed by determining binding of the dopamine transporter ligand (E )-N-(3-iodoprop-2-enyl)2-b-carbomethoxy-3-b-(4#-methylphenyl) nortropane (PE2I) in synaptosomal (LP1) membrane fractions using a modification of a method described previously (Guilloteau et al., 1998). Following fractionation as described above, 300 ml of the LP1 fraction was removed and diluted with 7 ml binding buffer (50 mM Tris–HCl, pH 7.4, containing 120 mM NaCl and 5 mM KCl). The sample was centrifuged at 50,000! g for 30 min at 4 C. The supernatant was removed and the pellet resuspended in 7 ml of binding buffer as above. Following centrifugation at 50,000! g for 20 min at 4 C, the resulting pellet was washed again before final resuspension in a minimal volume of assay buffer. Protein content was determined using the BioRad DC protein assay kit (BioRad) and the membrane sample was resuspended to a final protein concentration of 2 mg/ml. A sample of 50 mg protein (25 ml sample) was incubated with 4 nM [125I] PE2I, made up to a total of 200 ml volume with binding buffer. Non-specific binding was measured in the presence of 100 mM GBR-12909 (Sigma). For each animal, three samples were used for total binding, and one for non-specific binding. Samples were incubated for 90 min at 22 C. Incubation was terminated by vacuum filtration (pressure 600 mmHg) with ice-cold binding buffer using a 12-sample Brandel cell harvester for 30 s (flow rate 600 ml/min) through GF/B filters (Skatron) which had been previously soaked briefly in 0.1% polyethylenimine. Filters were transferred to scintillation vials and the radioactivity was measured in a gamma counter (Cobra 5010, Packard). Non-specific binding per animal was subtracted from corresponding total samples to obtain specific binding. The average specific binding per animal was then calculated and normalized to the mean binding observed in the unlesioned animals. 2.4. Denaturing conditions of protein solubilization Protein samples, 50–200 ml, were solubilized by the addition of one-twentieth volume of 20% SDS and

2.5. Precoupling phosphotyrosine antibody to protein A-Sepharose

2.6. Immunoprecipitation Solubilized protein samples were diluted 20-fold with immunoprecipitation buffer (150 mM NaCl, 50 mM Na3SO4, pH 7.2, 1% sodium deoxycholate, 2 mM EDTA, 1% Triton X-100). The diluted samples were incubated with 50 ml of protein A-Sepharose/antibodycoupled beads for each 200 mg of soluble protein for 3 h in a cold room with gentle rotation. The immunoprecipitated pellets were washed 3 times with ice-cold immunoprecipitation buffer after brief centrifugation. The pellets were resuspended in 50 ml loading buffer (125 Mm Tris–HCl, pH 6.8, 4% SDS, 100 mM DTT, 15% glycerol), boiled for 7 min with gentle agitation, and centrifuged at 15,000! g for 2 minutes. The supernatants were used for SDS-PAGE. 2.7. Quantitative immunoblotting The subunit specific monoclonal NR1 (Luo et al., 1997) (4 mg/ml working concentration) was developed in the laboratory of Dr. Barry B. Wolfe, Georgetown University, USA. The polyclonal NR2A (1 mg/ml working concentration) and NR2B antibodies (Sheng et al., 1994) (0.5 mg/ml working concentration) were developed in the laboratory of Dr. Morgan Sheng, Massachusetts Institute of Technology, USA. The following antibodies were obtained from commercial sources: polyclonal calnexin (1 mg/ml working concentration) (Stressgen) monoclonal syntaxin (0.02 mg/ml working concentration) (Sigma), polyclonal GluR2/3 (0.25 mg/ml working concentration) (Chemicon), monoclonal tubulin (1 mg/ml working concentration) (Promega) and monoclonal postsynaptic density (95 kDa) protein (PSD-95) (1 mg/ ml working concentration) (Sigma). Proteins were separated according to the method of Laemmli (1970). After determination of protein content,

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2.8. Statistical analysis A standard curve for each gel was generated using polynomial second-order non-linear regression of net intensities of the protein standards (cortical extracts from a normal control animal). This standard curve was then used to extrapolate representative protein values of the experimental bands from the same gel. Protein abundance in each fraction (H, LP1, P3, LP2) was expressed as a percentage of the average protein

abundance in the unlesioned group. Average values for each experimental group in the H, LP1, P3 and LP2 fractions were then calculated. Differences between group means (unlesioned, MPTP-lesioned parkinsonian, MPTP-lesioned dyskinetic) were tested using one-way ANOVA followed by Fisher’s PLSD test post-hoc. The level of significance was taken to be p % 0.05. For analysis of tyrosine phosphorylated NR2A and NR2B subunits the ratio of optical density of the pellet band to the input band was calculated and expressed as a percentage of the unlesioned group.

3. Results 3.1. MPTP-lesioning and chronic levodopa treatment of macaques MPTP induced a stable parkinsonian state in all 13 animals treated. Following levodopa therapy for 120 days, all 9 animals treated exhibited stable, treatmentrelated dyskinesia (most disabling chorea score 60 min post-levodopa Z 3 [range 0 – 4]; most disabling dystonia score 60 min post-levodopa Z 2 [range 0 – 4]). These dyskinesias were idiosyncratic in pattern but reproducible in each animal. LID was assessed using a global dyskinesia rating scale as described in the Section 2. Evaluation of PE2I binding revealed that all of the MPTP-lesioned animals had extensive depletion of dopamine transporter binding sites in the striatum (Fig. 2). In the MPTP-lesioned parkinsonian animals (n Z 4), PE2I binding was decreased to 11.5% G 0.7 of unlesioned values ( p ! 0.001). In the MPTP-lesioned dyskinetic animals (n Z 9), PE2I binding was reduced to 12.2% G 2.9 of unlesioned values ( p ! 0.001). There

Specific [125I]PE2I binding expressed as a percentage of average unlesioned animals

100 mg protein per fractionated sample was solubilized by boiling for 5 min in dithiothreitol (DTT) treatment buffer (0.625 M Tris–HCl, 2% sodium dodecyl sulfate [SDS], 50 mM DTT and 7.5% glycerol). For characterization of the fractionation procedure, 1–10 mg protein of each solubilized fraction was loaded into adjacent lanes of a 7.5% (for detection of NMDA receptor subunits, calnexin, PSD-95, GluR2/3 and bIII-tubulin) and 10% (for detection of syntaxin) SDS-polyacrylamide gel. For the quantification of NR2A and NR2B NMDA receptor subunits in experimental animals, equal amounts of the input proteins (2 mg [NR2B] and 10 mg [NR2A] for H, LP1, P3 fractions, and 10 mg [NR2B] and 20 mg [NR2A] for the LP2 fraction) were loaded adjacent to the respective immunoprecipitated pellet samples from the same animal (20 mg [NR2B] and 40 mg [NR2A]). The samples from one animal were loaded onto a single gel together with six protein standards prepared from increasing concentrations of homogenate prepared from cortex from a normal macaque. The same batch of standards was used for all experiments. Tyrosine phosphorylation analysis of NR1 in experimental animals was not carried out due to the absence of detectable tyrosine phosphorylation of this subunit in normal animals. Quantification of NR1 subunit abundance was carried out by loading input samples (2 mg [H, LP1, P3] and 10 mg [LP2]) onto a single gel with protein standards as above. The abundance of PSD-95 and GluR2/3 in LP1 fractions from experimental animals was also determined. In addition, in order to control for possible non-specific effects of MPTP-lesioning on the abundance of striatal proteins, the abundance of bIII-tubulin in LP1 fractions was determined. For the quantification of GluR2/3, PSD-95 and bIII-tubulin proteins LP1 samples from each animal were loaded in adjacent lanes together with protein standards as above. Following separation and transfer of proteins, blots were incubated in primary and secondary antibodies (anti-rabbit/anti-mouse horseradish peroxidase-conjugated secondary antibodies; BioRad; 0.1 mg/ml working concentration) and bands were visualized on film by enhanced chemiluminescence (BioRad). The net intensities of bands were quantified with computer-assisted densitometry (Kodak 1-D System; Kodak).

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Fig. 2. Effect of MPTP lesioning on dopamine transporter binding in striatal synaptosomal membranes. Data are presented as specific [125I] PE2I binding (nCi/mg protein) expressed as a percentage of average [125I] PE2I binding in unlesioned animals. Two groups of macaques were rendered parkinsonian with MPTP. One group was subsequently treated with levodopa to produce dyskinesia. The remaining group was treated with vehicle. The effect of treatment compared to unlesioned animals was assessed and tested using a one-way ANOVA followed by Fishers PLSD test post-hoc ( p ! 0.001), compared to unlesioned animals F2,17 Z 72.46).

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was no significant difference between the levels of PE2I loss in the parkinsonian compared to the dyskinetic group ( p O 0.05). 3.2. Characterization of the subcellular fractionation procedure The effectiveness of the subcellular fractionation procedure in macaque striatum was evaluated by the use of protein markers for subcellular compartments (Fig. 3, panel A). bIII-tubulin, which is a soluble microtubule-associated protein, was enriched in the cytosolic fractions (LS1, S3, LS2) but was detected in

all subcellular compartments including membrane-associated compartments (LP1, P3, LP2). Syntaxin, a protein that interacts with synaptotagmin and participates in the docking of synaptic vesicles (Bennett et al., 1992) was enriched in the light membrane (P3, lane 5), synaptosomal membrane (LP1, lane 7) and synaptic vesicleenriched (LP2, lane 9) fractions, but was absent from the purely cytosolic fractions S3 and LS2. The calciumbinding protein calnexin, which interacts with newlysynthesized glycoproteins in the endoplasmic reticulum (David et al., 1993) was enriched in the light membrane compartment (P3, lane 5), but was present only at much lower levels in the other compartments studied. PSD-95, a protein which interacts with NMDA receptors at the post-synaptic density (Kornau et al., 1995) was concentrated in the synaptosomal membrane fraction (LP1) but not detected in the cytosolic or vesicle-enriched fraction (S2, P3, S3, LP2, and LS2). The distributions of the proteins studied are comparable to those observed when the same separation technique is applied to rodent tissue (Dunah and Standaert, 2001). 3.3. Distributions of NMDA receptor subunit proteins in subcellular fractions of macaque striatum Each of the three NMDA receptor subunit proteins studied (NR1, NR2A, NR2B) exhibited a similar pattern of distribution in the various subcellular compartments (Fig. 3, panel B). All three proteins were highly concentrated in the synaptosomal membrane (LP1) fraction. They were also each detected in the two purified vesicular fractions, P3 (light membranes, lane 5), and LP2 (synaptic vesicle-enriched, lane 9). They were not detected in the purely cytosolic fractions (S3 and LS2, lanes 6 and 10). For comparison, we also examined the distribution of AMPA (a-amino-3hydroxy-5-methyl-4-isxazolepropionic acid) type glutamate receptors, using an antibody to GluR2/3. The Fig. 3. Characterization of biochemical fractionation procedure in macaque striatum. Characterization of subcellular compartments. The isolated biochemical fractions from striatal tissues were separated by SDS-PAGE and the blots were probed with antibodies against bIIItubulin, syntaxin, calnexin, PSD-95, GluR2/3 (Panel A) and NR1, NR2A and NR2B (Panel B). H, total homogenate; P1, nuclei and large debris; P2, crude synaptosomal membrane fraction; P3, light membrane fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction. S2, S3, LS1, and LS2 are supernatants from P2, P3, LP1 and LP2, respectively. bIII-tubulin was detected in membrane-associated and cytosolic subcellular fractions. Syntaxin is highly concentrated in the light membrane (P3), synaptosomal membrane (LP1), and synaptic vesicle-enriched (LP2) fractions. Calnexin is enriched in the light membrane (P3) fraction. PSD-95 is found in the synaptosomal membrane (LP1) fraction but not in the light membrane (P3) or synaptic vesicle-enriched (LP2) fractions. Glutamate receptor subunits (NR1, NR2A, NR2B, GluR2/ 3) are enriched in the synaptosomal membrane fraction (LP1) and are also found in the light membrane (P3) and synaptic vesicle-enriched (LP2) fractions.


distribution of immunoreactivity to GluR2/3 was identical to that of the NMDA receptor subunits. Based on these results, in subsequent studies to examine the effect of MPTP-lesioning and subsequent levodopa treatment of macaques on NMDA receptor subunit abundance, only the homogenate (H), light membrane (P3), synaptosomal membrane (LP1) and synaptic vesicle-enriched (LP2) fractions were used. 3.4. Differential subcellular localization of tyrosine phosphorylated NMDA receptor subunits in the macaque striatum Immunoprecipitation of fractionated striatal protein extracts with an anti-phosphotyrosine antibody and subsequent immunoblotting of the input and pellet samples with NMDA receptor antibodies was used to detect tyrosine phosphorylated NMDA receptor subunits in the different subcellular compartments (Fig. 4). Tyrosine phosphorylation of NR1 was not detected in any subcellular compartment, which is in accord with previous studies in the rat (Lau and Huganir, 1995; Dunah and Standaert, 2001). A moderate intensity of tyrosine phosphorylation of NR2B subunits was detected in the total homogenate (H, lane 2), nuclei and large debris (P1, lane 4), crude synaptosomal membrane (P2, lane 6) and synaptosomal membrane (LP1, lane 14) fractions. Tyrosine phosphorylated NR2A subunits were similarly distributed but phosphorylated to a much

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lower extent than NR2B subunits. Tyrosine phosphorylation of NR2 subunits in the light membrane (P3, lane 10) fraction was very low and was undetectable in the synaptic vesicle-enriched (LP2, lane 12) fraction, even with longer exposure periods.

3.5. Alterations in striatal NMDA receptor proteins following MPTP-lesioning and subsequent levodopa treatment In order to compare the NMDA receptor subunit abundance in the striatal extracts from the vehicletreated unlesioned macaques (n Z 5), MPTP-lesioned parkinsonian macaques (n Z 4) and MPTP-lesioned dyskinetic macaques (n Z 9), fractionated samples from each animal were separated on a single SDS-PAGE gel together with six standards of increasing protein concentrations prepared from cortical extracts from a normal control animal. The same standards were used on each gel and interpolation of these standards was used to normalize the band intensities among different gels as described in Section 2.8. Average protein abundance for each experimental group in each of the four subcellular compartments (H, LP1, P3, LP2) was calculated as a percentage of protein abundance in unlesioned animals and statistical analysis was carried out using one-way ANOVA followed by Fisher’s PLSD test post-hoc.

Fig. 4. Tyrosine phosphorylated NMDA receptors are enriched in the synaptosomal membrane fraction. Samples from macaque striatum were subjected to biochemical fractionation, solubilized and immunoprecipitated with anti-phosphotyrosine antibody. The inputs (I; lanes 1,3,5,7,9,11,13,15,17,19) (loaded with 2 mg protein for NR1 and NR2B subunits [10 mg in LP2 lanes] and 10 mg protein for NR2A subunits [20 mg in LP2 lane]), and pellets (P; lanes 2,4,6,8,10,12,14,16,18,20) (loaded with 20 mg protein for NR1 and NR2B subunits and 40 mg protein for NR2A subunits), were separated on SDS-PAGE gels, and the blots were probed with anti-NR1, anti-NR2A and anti-NR2B antibodies. Tyrosine phosphorylated NR2A and NR2B subunits were enriched in synaptosomal membranes (LP1, lane 13), with low levels in the light membrane fraction (P3, lane 10), and were not present in the synaptic vesicle-enriched fraction (LP2, lane 18).

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Fig. 5. Effect of MPTP-lesioning and subsequent levodopa treatment on striatal NMDA receptor subunit abundance. Striata from unlesioned (U) (n Z 5), MPTP-lesioned parkinsonian (P) (n Z 4) and MPTP-lesioned dyskinetic (D) (n Z 9) macaques were subjected to biochemical fractionation. 2 mg (NR1 and NR2B) and 10 mg (NR2A) samples of homogenate (H), synaptosomal membrane (LP1) and light membrane (P3), and 10 mg (NR1 and NR2B) and 20 mg (NR2A) samples of synaptic vesicle-enriched (LP2) fractions (data not illustrated), were separated by SDS-PAGE onto 7.5% polyacrylamide gels and probed with anti-NR1, anti-NR2A and anti-NR2B antibodies. Samples from a single animal were loaded onto a single gel together with protein standards prepared from macaque cortex homogenate. The optical density of bands was measured and the protein standards used to generate a standard curve, from which the relative protein abundance of the experimental bands were extrapolated. Data are expressed as mean per experimental group G SEM. Data were analyzed using one-way ANOVA and Fisher’s PLSD test post-hoc (*p % 0.05). NR1 (H [F2,17 Z 1.94], LP1 [F2,17 Z 4.85], P3 [F2,17 Z 4.93]); NR2A (H [F2,16 Z 3.40], LP1 [F2,16 Z 5.99], P3 [F2,14 Z 6.80]); NR2B (H [F2,16 Z 3.34], LP1 [F2,16 Z 5.35], P3 [F2,15 Z 2.20]). Insets show representative immunoblots from each experimental group demonstrating NMDA receptor subunit changes.

Analysis of NR1 subunit abundance in the fractionated samples revealed that MPTP-lesioning led to a reduction in the abundance of this subunit in the synaptosomal membrane (LP1) fraction (Fig. 5). Following levodopa treatment the abundance of NR1 subunit in

this compartment returned to unlesioned levels. A similar pattern of changes was observed in the whole homogenate samples, although in these the magnitude of change was smaller and did not reach statistical significance. In the light membrane compartment (P3), repeated levodopa

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3.6. Tyrosine phosphorylation of striatal NMDA receptors following MPTP-lesioning and subsequent levodopa treatment Immunoprecipitation of tyrosine phosphorylated NR2A and NR2B subunits was carried out in homogenate (H), synaptosomal membrane (LP1), light membrane (P3) and synaptic vesicle-enriched (LP2) fractions from the striatum of unlesioned, MPTP-lesioned and dyskinetic animals. The intensity of the signals from the phosphorylated proteins were adjusted for the abundance of the subunits in the input lanes, and the resulting measures of the extent of tyrosine phosphorylation were normalized to unlesioned vaules. In these experiments, we found substantially more variability in the extent of tyrosine phosphorylation in different animals than we had observed in rodent studies. In synaptosomal membrane (LP1) fractions, the mean extent of NR2A tyrosine phosphorylation was reduced in the MPTP-lesioned, parkinsonian animals (to 49.5 G 28.6% of that observed in the unlesioned animals), and in the MPTP-lesioned, dyskinetic animals (to 54.1 G 24.5% of the unlesioned value), but because of the large variance of these samples as well as that of

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treatment caused a significant increase in the abundance of NR1 subunits. LP2 (not illustrated) did not exhibit a statistically significant change in the comparisons of interest. NR2A protein abundance exhibited a different pattern of alterations than that observed in the analysis of NR1. MPTP lesioning alone did not alter NR2A abundance in any of the compartments studied, but treatment with levodopa produced a striking enhancement of this subunit LP1 synaptic fraction (150 G 12% in the compared to unlesioned values, p % 0.05). Levodopa treatment also enhanced the abundance of NR2A in the light membrane fraction (P3). Abundance in the LP2 fractions was not altered (not illustrated). The effect of MPTP lesioning and subsequent levodopa treatment on the abundance of NR2B was very similar to the effect of these treatments on NR1. MPTP-lesioning reduced the abundance of NR2B in the LP1 synaptic membrane fraction (51.2% of unlesioned, p % 0.05). Treatment with levodopa restored the abundance of NR2B in this compartment to unlesioned levels, but did not increase it above the level found in the unlesioned striatum. Neither P3 nor LP2 exhibited statistically significant alterations in NR2B. We also examined the abundance of three additional proteins, PSD-95, GluR2/3 and bIII-tubulin, in the LP1 synaptosomal membrane fractions from unlesioned, MPTP-lesioned parkinsonian, and MPTP-lesioned dyskinetic macaques. As illustrated in Fig. 6, there was no significant difference in the abundance of these proteins in any of the groups of animals studied.

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Fig. 6. Effect of MPTP-lesioning and subsequent levodopa treatment on the abundance of PSD-95, GluR2/3 and bIII-tubulin proteins. Striata from unlesioned (U) (n Z 5), MPTP-lesioned parkinsonian (P) (n Z 4) and MPTP-lesioned dyskinetic (D) (n Z 9) macaques were subjected to biochemical fractionation. PSD-95 and GluR2/3 (5 mg) and bIII-tubulin (1 mg) samples of synaptosomal membrane fractions (LP1) were separated by SDS-PAGE onto 7.5% polyacrylamide gels and probed with anti-PSD-95, anti-GluR2/3 and anti-bIII-tubulin antibodies. Samples were loaded in adjacent lanes on two separate gels so that each gel contained samples from each experimental group, and a set of protein standards prepared from macaque cortex homogenate were included on each gel. The optical density of bands was measured and the protein standards used to generate a standard curve, from which the relative protein abundance of the experimental bands was extrapolated. Data were expressed as mean per experimental group G SEM. Data were analyzed using one-way ANOVA. Treatment did not significantly alter PSD-95, GluR2/3 or bIII-tubulin protein abundance ( p O 0.05). PSD-95 (F2,16 Z 0.55); GluR2/3 (F2,17 Z 0.73); bIII-tubulin (F2,17 Z 0.96).

the unlesioned tissue (100 G 37.9%) neither change was statistically significant. Similar variability was observed in the measurements of NR2B phosphorylation, and none of the alterations observed in this receptor subunit were statistically significant either (100 G 5.6% [unlesioned]; 94.2 G 20.9% [MPTP-lesioned, parkinsonian]; 66.0 G 17.5% [MPTP-lesioned, dyskinetic]). There was also no significant difference among the experimental groups in the phosphorylation of NR2A and NR2B proteins in the whole homogenate or light membrane fraction (data not shown). No phosphorylation of the receptor subunits in the LP2 fraction was detected in any of the experimental groups.

4. Discussion In the striatum of MPTP-lesioned macaques we have found that dopamine depletion and subsequent pharmacological replacement with levodopa induces substantial alterations in the abundance of striatal NMDA receptor proteins. In MPTP-lesioned parkinsonian animals, there is a reduction in the abundance of NR1 and NR2B proteins in the synaptic membrane fractions, but the abundance of NR2A is not altered. Following

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repeated treatment with levodopa, there is normalization of NR1 and NR2B subunit abundance in the synaptic membrane fraction to unlesioned levels and in addition a striking increase in the abundance of NR2A subunits in the synaptic membrane fraction. In the present study we did not find any alteration in the tyrosine phosphorylation of striatal NR2A and NR2B subunits, but this may be related to constraints of the experimental method used in primate, as discussed below. 4.1. The MPTP-lesioned non-human primate as a model of PD The MPTP-lesioned macaque has important advantages over rodent models of PD and LID in which most previous studies of striatal NMDA receptor proteins have been studied. Rodents do not exhibit behaviors unequivocally comparable to LID although they can, depending on the dose employed, demonstrate sensitization of intensity of behavioral response, abnormal involuntary movements with some phonological simulation to LID, or a shortening of duration of behavioral response, which may be comparable to the wearing off phenomenon, and all of these may, to some extent, model the complications of long-term levodopa therapy. Using the MPTP treatment method described, we achieved extensive lesions of the nigrostriatal pathway. As assessed with the radioligand [125I] PE2I, a selective ligand for the dopamine transporter (Guilloteau et al., 1998), we observed substantial lesioning of all of the animals studied, with a mean loss of striatal dopamine transporter binding greater than 85% of unlesioned values. Repeated daily levodopa treatment reliably induces dyskinesia in MPTP-lesioned primates (Jenner, 2003), and in the current study all MPTP-lesioned animals chronically treated with levodopa exhibited dyskinesia. 4.2. Analysis of NMDA receptor subunit proteins in non-human primate brain Immunoblotting for NMDA NR1, NR2A and NR2B receptor subunits was carried out using antibodies raised against specific peptide sequences for the individual NMDA receptor subunits. The NMDA receptor antibodies used in this study have not been previously characterized in macaque tissue although they have been well-characterized in rodent brain (Sheng et al., 1994; Dunah et al., 2000). In all the fractions in which NR1 immunoreactivity was detected, a single band at approximately 115 kDa was identified, except in the P3 and LP2 fractions, in which a double band was observed. The monoclonal antibody for NR1 used in this study recognizes all splice variants of the NR1 subunit (Luo et al., 1997). It is probable that a doublet

occurs in all fractions where NR1 protein is detected, corresponding to NR1 splice variants, however, in the fractions where immunoreactivity is stronger the double bands appear as a single band due to a stronger intensity of signal. The polyclonal NR2A and NR2B antibodies identified a single band at approximately 180 kDa. In addition, the NR2B antibody also recognized two additional lower molecular weight bands and these bands may represent proteolytic breakdown of the NR2B subunit protein. NR1, NR2A and NR2B NMDA receptor subunits were confined to three membrane-associated compartments: the synaptosomal fraction (LP1), synaptic vesicle-enriched fraction (LP2) and the light membrane fraction (P3). NMDA receptor subunits were undetected in the purely cytosolic compartments (S3 and LS2). This localization of subunits is in agreement with previous descriptions of the subcellular distribution of NMDA receptor subunit proteins in the rat striatum (Dunah and Standaert, 2001). 4.3. Alterations in NMDA receptor subunit abundance in MPTP-lesioned parkinsonian macaques We found that in the macaque, MPTP-lesioning induced a substantial decrease in NR1 and NR2B subunits in the synaptosomal membrane fraction, with no alteration in NR2A subunits, implying relative enrichment of NR2A-containing receptor assemblies. This effect is identical to that previously described in the 6-OHDA-lesioned rat model of PD (Dunah et al., 2000). It is also compatible with a recent study using immunocytochemistry, which describes decreased NR1 immunoreactivity in the striatum of MPTP-lesioned macaques (Betarbet et al., 2004). It is more difficult to reconcile our results with studies employing indirect methods of detecting NMDA receptor subunits such as ligand binding. Calon et al. (2002) found no alterations in the binding of ligands with relative specificity for NR2A- and NR2B-containing receptor assemblies in the MPTP-lesioned macaque. Since ligand binding was not conducted in fractionated tissue samples, it may be more appropriate to compare these data to our observation in whole homogenate rather than the LP1 fraction. In the whole striatal homogenate, we did not observe any statistically significant differences in the abundance of the subunit proteins among the different experimental groups, although in some cases a trend was evident which mirrored the changes present in the LP1 fraction. We have previously demonstrated in the 6-OHDAlesioned rodent model, using co-immunoprecipitation, that the alterations in abundance of the individual subunits reflected a reduced number of receptors containing co-assembled NR1 and NR2B subunits, without a change in the number of NR1/NR2A


complexes (Dunah et al., 2000). We did not directly test this in the macaque model because of the limited amount of tissue available for study, but it seems likely that the underlying reorganization of receptor complexes is similar in the two species. This enhancement in the proportion of NR1/NR2A assemblies relative to NR1/NR2B assemblies may have an important effect on the function and pharmacological properties of striatal NMDA receptors. A shift to increased NR1/NR2A assemblies would be predicted to lead to NMDA receptors with fast deactivation times, a reduced affinity for glutamate, glycine and polyamines, and an increased sensitivity to Mg2C and Zn2C (Dingledine et al., 1999). NMDA receptor antagonists, and more recently antagonists with high affinity for NR2B-containing NMDA receptors, have been demonstrated to show antiparkinsonian actions in experimental models of PD (Nash et al., 2000; Steece-Collier et al., 2000; Loschmann et al., 2004). We show here in the present study that NR2B subunits are reduced in synaptosomal membranes, but clearly there are still substantial numbers of NR2B-containing receptors remaining in striatal synapses. Indeed, the efficacy of the NR2B selective antagonists in parkinsonism demonstrates that even though the number of NR2B receptors is reduced, they continue to have a crucial role in striatal signaling.

4.4. Alterations induced by repeated levodopa treatment in MPTP-lesioned macaques Repeated treatment of the animals with levodopa to induce dyskinesia led to further modulation of striatal NMDA receptor proteins. In dyskinetic animals, NR1 and NR2B subunit protein abundance in the LP1 fraction was restored to unlesioned levels. The same effect was observed after repeated levodopa treatment in 6-OHDA-lesioned rats (Dunah et al., 2000). Additionally, in MPTP-lesioned macaques the induction of dyskinesia is associated with an increase (150 G 12% of unlesioned levels) in the abundance of striatal NR2A subunits in synaptosomal membranes. Indeed, a trend to increased NR2A abundance was observed in all fractions, although it reached statistical significance in the synaptosomal membrane (LP1) and light membrane (P3) fractions only. Prior studies of NMDA receptor subunits in levodopatreated MPTP-lesioned primates or patients with PD have been few. The study by Calon et al. (2002) mentioned previously did examine binding of ligands for NR2A and NR2B, and found both to be increased in dyskinetic macaques. Two studies have reported NR2Asensitive binding in human post-mortem striatal tissue from parkinsonian patients with levodopa-induced motor complications, reporting it to be either increased (Lange et al., 1997) or unchanged (Calon et al., 2003).

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The finding of increased NR2A subunits in dyskinesia differs to the 6-OHDA-lesioned rat model of PD where no alteration in NR2A subunits was found in lesioned rats treated long-term with levodopa (Dunah et al., 2000). In addition to species differences in the molecular anatomy of the primate versus rodent basal ganglia, there are several differences between the application of rat and primate experimental models of PD that may contribute to this difference in NR2A subunit changes. The time between the lesion and the duration of dopaminergic treatment were both much longer in the primates (30 day post-lesion interval and 120 day levodopa treatment) than the 14 day post-lesion interval and 21 day levodopa treatment interval employed in the rodent study. In addition, the primates were sacrificed at the peak of the dyskinetic response, while the rodents were sacrificed 12 h after their last dose of levodopa. It would be of interest to determine whether rodents exhibit similar alterations in NR2A abundance if examined at shorter intervals after levodopa treatment and to examine in more detail the duration of the NMDA receptor alterations in both species after levodopa therapy is discontinued. In the primate model, MPTPlesioning results in a bilateral lesion, whereas in the rat studies, 6-OHDA was used to produce a unilateral lesion only. Another potential factor between primates and rodents is differences in the abundance of NR2A among different types of neurons present in the striatum. In rats, NR2A subunits are ubiquitously expressed on both indirect and direct pathway neurons and also in all interneuron types, except for cholinergic interneurons (Standaert et al., 1999). However, in the human, NR2A subunits are expressed predominantly on direct pathway neurons and in GABAergic interneurons (Ku¨ppenbender et al., 2000). Abnormal (hyperactive) transmission of the direct striatal output pathway has been implicated in the genesis of LID (Bezard et al., 2001a), and it is possible that the enhanced abundance of NR2A observed in the primate reflects the participation of the direct pathway in LID. The results from the present study in primates suggest that the upregulation of NR2A may be a potentially important phenomenon in LID. One previous study has investigated the effects of blocking NR2A-containing NMDA receptors on levodopa-induced response alterations in MPTP-lesioned primates using the competitive NR2A-selective antagonist MDL100,453 (Blanchet et al., 1999). MDL100,453 in fact worsened dyskinesia in MPTP-lesioned primates when administered together with levodopa. However, MDL100,453 shows only approximately 5-fold degree of selectivity for NR1/ NR2A subunit combinations over NR1/NR2B receptors, and may have other properties which have not been explored in detail. In light of the present results showing increased NR2A abundance in LID, studies of other agents targeted to NR2A may be warranted.

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4.5. Mechanisms of alterations in striatal NMDA receptor subunits Previous studies in rodents have implicated a D1 dopamine receptor- and tyrosine phosphorylation-dependent trafficking mechanism in dopamine-dependent redistribution of striatal NMDA receptors. When examined in vitro, activation of D1 dopamine receptors produces a rapid enhancement of NMDA receptor subunit abundance in the LP1 fraction of striatal tissue. This is associated with a reduction in the abundance of the subunits in P3 and LP2, and is blocked by tyrosine kinase inhibitors and by genetic deletion of Fyn, a tyrosine kinase (Dunah and Standaert, 2001; Dunah et al., 2004). The tyrosine phosphorylation of NMDA receptor subunits in primate brain has not been previously investigated. In the present study, NR2A and NR2B subunits in normal macaque striatum were found to be tyrosine phosphorylated, and in accordance with the rat, tyrosine phosphorylation of NR1 subunits was not detected. Tyrosine phosphorylated NR2 subunits were most abundant in the synaptosomal membrane fraction of macaque striatum, and this is in agreement with the rat (Dunah and Standaert, 2001). No tyrosine phosphorylation of NR2 subunits in the synaptic vesicleenriched fraction (LP2) was detected, even after increasing the amount of immunoprecipitated protein loaded (data not shown). However, a small proportion of NR2 subunits were tyrosine phosphorylated in the light membrane fraction (P3). This differs from studies in rat striatum (Dunah and Standaert, 2001) and may represent a species difference between rats and primates. Under the conditions used in the current study to assess tyrosine phosphorylated NMDA receptor subunits in unlesioned, MPTP-lesioned parkinsonian, and MPTP-lesioned dyskinetic macaque striatum, no alterations in the proportion of tyrosine phosphorylated NR2A and NR2B subunits were detected. This result was surprising given the body of literature reporting alterations of the phosphorylation of striatal NMDA receptors in 6-OHDA-lesioned rats (Menegoz et al., 1995; Oh et al., 1998; Dunah et al., 2000). The most striking effects on phosphorylation of NMDA receptor subunits are produced by chronic levodopa treatment of 6-OHDA-lesioned rats, which enhances tyrosine phosphorylation of striatal NR2A and NR2B subunits (Oh et al., 1998, 1999; Dunah et al., 2000). The lack of detectable changes in phosphorylation in the present study could present a genuine absence in altered phosphorylation of striatal NMDA receptors in parkinsonian and dyskinetic primates, and thereby imply that the mechanism of NMDA receptor trafficking differs in the two species. However, it is notable that in the present study the amount of tyrosine phosphorylation of NR2A and NR2B subunits expressed as a percentage of total input protein varied widely between animals.

Protein phosphorylation is very labile, and it is likely that there was degradation of tyrosine phosphorylation during the time taken to dissect the striatum out from the macaque brains. On average, the time taken from death of the animal to freezing of striatum was approximately 10 min and is undoubtedly substantially longer than the time taken for a similar dissection in rat brain (1–2 min). These considerations lead us to believe that the phosphorylation of NMDA subunits observed in our samples of macaque brain may not be a reliable measure of the state of striatal NMDA receptors in vivo. Since the direction and magnitudes of the receptor redistribution which occur in the macaque are similar to those seen in rodents, we think it is probable that the underlying mechanisms are indeed shared and involve changes in phosphorylation, but direct confirmation of this hypothesis awaits development of better methods for the study of protein phosphorylation in primates. We also did not observe any clear pattern of alterations in NMDA receptor subunits in the vesicular compartments examined (P3, LP2). These were of interest because the in vitro data in rodent suggested that these compartments can serve as a source for NMDA receptor subunits which enter LP1 after dopaminergic treatment. The lack of alteration in the macaque model may reflect the vastly different time scale of these studies: the in vitro measurements were conducted in tissue from normal rat striatum 20 min after pharmacological activation of D1 dopamine receptors, whilst in the macaque, the subunits were examined after 120 days of levodopa therapy. Depletion of subunits from P3 and LP2 may be an initial response to acute stimulation, while with chronic dopaminergic stimulation compensatory changes restore the abundance of these pools. No alterations were found in the abundance of either striatal GluR2/3 AMPA receptor subunits or PSD-95 in synaptosomal membrane fractions following MPTPlesioning and subsequent repeated levodopa treatment. The absence of changes in AMPA receptor subunit proteins is in accord with previous studies in 6-OHDAlesioned rats (Dunah et al., 2000; Picconi et al., 2004). Similarly, we have previously reported that PSD-95 abundance in the striatal LP1 fraction of 6-OHDAlesioned rats is also unchanged (Dunah et al., 2000), although it is worth noting that a recent report has described reduced PSD-95 in rodent striatal synaptosomes prepared using a different separation technique (Picconi et al., 2004), perhaps reflecting a change in the strength of interactions among components of the PSD. In conclusion, the current study has demonstrated that NMDA receptor subunit abundance is altered in both parkinsonian and dyskinetic MPTP-lesioned macaques. These changes are predicted to result in substantial changes in striatal NMDA receptor physiology and pharmacology. In the parkinsonian state in both rodents and macaques, there is a reduction in the


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Fig. 7. A hypothesis for the redistribution of striatal NMDA receptor subunits in synaptosomal membranes in the MPTP-lesioned primate model of PD and LID. In the ‘‘normal’’ state NMDA receptors localized in synaptosomal membranes are composed of heterodimeric NR1/NR2A and NR1/NR2B receptors, and heterotrimeric NR1/ NR2A/NR2B receptors (Dunah and Standaert, 2003). Following dopamine depletion in the ‘‘parkinsonian’’ state, there is a selective reduction of NR1/NR2B heterodimeric receptors. This results in a relative enrichment of NMDA receptors containing NR2A subunits. Following repeated levodopa treatment causing dyskinesia, there is a normalization of NMDA receptors composed of NR1 and NR2B subunits and an increase in NMDA receptors containing NR2A subunits. This increase in NR2A-containing NMDA receptors may bring about important changes to NMDA receptor-mediated signalling in dyskinesia.

abundance of NR1 and NR2B subunits, leading to relative enhancement of NR2A-containing receptor assemblies. In addition, the present study has identified increased NR2A subunit levels in dyskinetic macaques (Fig. 7). This result differs from rodent studies and highlights the importance of conducting research on non-human primates. Considering the (i) increased relative abundance of NR2A in a non-human primate in both the parkinsonian and dyskinetic state, and (ii) evidence for selective localization of NR2A in the striato-nigral direct pathway of humans, we propose that pharmacological or molecular manipulation of NR2A subunits in the direct pathway may represent an important target of therapies to prevent or reduce levodopa motor complications in human patients. Acknowledgements Parkinson’s Disease Foundation and National Parkinson Foundation, Francis and Ingeborg Heide Schumann Fellowship, and USPHS grant NS34361 are gratefully acknowledged. References Ahlskog, J.E., Muenter, M.D., 2001. Frequency of levodopa-related dyskinesias and motor fluctuations as estimated from the cumulative literature. Mov. Disord. 16, 448–458. Bennett, M.K., Calakos, N., Scheller, R.H., 1992. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255–259. Betarbet, R., Poisik, O., Sherer, T.B., Greenamyre, J.T., 2004. Differential expression and ser897 phosphorylation of striatal

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N-methyl-D-aspartate receptor subunit NR1 in animal models of Parkinson’s disease . Exp. Neurol. 187, 76–85. Bezard, E., Brotchie, J.M., Gross, C.E., 2001a. Pathophysiology of levodopa-induced dyskinesia: potential for new therapies. Nat. Rev. Neurosci. 2, 577–588. Bezard, E., Dovero, S., Prunier, C., Ravenscroft, P., Chalon, S., Guilloteau, D., Crossman, A.R., Bioulac, B., Brotchie, J.M., Gross, C.E., 2001b. Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J. Neurosci. 21, 6853–6861. Blanchet, P.J., Konitsiotis, S., Whittemore, E.R., Zhou, Z.L., Woodward, R.M., Chase, T.N., 1999. Differing effects of N-methyl-Daspartate receptor subtype selective antagonists on dyskinesias in levodopa-treated 1-methyl-4-phenyl-tetrahydropyridine monkeys. J. Pharmacol. Exp. Ther. 290, 1034–1040. Bolam, J.P., Hanley, J.J., Booth, P.A., Bevan, M.D., 2000. Synaptic organisation of the basal ganglia. J. Anat. 196 (Pt 4), 527–542. Calabresi, P., Pisani, A., Mercuri, N.B., Bernardi, G., 1992. Long-term potentiation in the striatum is unmasked by removing the voltagedependent magnesium block of NMDA receptor channels. Eur. J. Neurosci. 4, 929–935. Calon, F., Morissette, M., Ghribi, O., Goulet, M., Grondin, R., Blanchet, P.J., Bedard, P.J., Di, P.T., 2002. Alteration of glutamate receptors in the striatum of dyskinetic 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-treated monkeys following dopamine agonist treatment. Prog. Neuropsychopharmacol. Biol. Psychiatry 26, 127–138. Calon, F., Rajput, A.H., Hornykiewicz, O., Bedard, P.J., Di Paolo, T., 2003. Levodopa-induced motor complications are associated with alterations of glutamate receptors in Parkinson’s disease. Neurobiol. Dis. 14, 404–416. Chase, T.N., Oh, J.D., 2000a. Striatal dopamine- and glutamatemediated dysregulation in experimental parkinsonism. Trends Neurosci. 23, S86–S91. Chase, T.N., Oh, J.D., 2000b. Striatal mechanisms and pathogenesis of parkinsonian signs and motor complications. Ann. Neurol. 47, S122–S129. Cotzias, G.C., Papavasiliou, P.S., Gellene, R., 1969. Modification of parkinsonism-chronic treatment with L-DOPA. N. Engl. J. Med. 280, 337–345. David, V., Hochstenbach, F., Rajagopalan, S., Brenner, M.B., 1993. Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J. Biol. Chem. 268, 9585–9592. Di Chiara, G., Morelli, M., Consolo, S., 1994. Modulatory functions of neurotransmitters in the striatum - ACh/dopamine/NMDA interactions. Trends Neurosci. 17, 228–233. Dingledine, R., Borges, K., Bowie, D., Traynelis, S.F., 1999. The glutamate receptor ion channels. Pharmacol. Rev. 51, 7–61. Dunah, A.W., Sirianni, A.C., Fienberg, A.A., Bastia, E., Schwarzschild, M.A., Standaert, D.G., 2004. Dopamine D1-dependent trafficking of striatal N-methyl-D-aspartate glutamate receptors requires Fyn protein tyrosine kinase but not DARPP-32. Mol. Pharmacol. 65, 121–129. Dunah, A.W., Standaert, D.G., 2001. Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane. J. Neurosci. 21, 5546–5558. Dunah, A.W., Standaert, D.G., 2003. Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatum. J. Neurochem. 85, 935–943. Dunah, A.W., Wang, Y.H., Yasuda, R.P., Kameyama, K., Huganir, R.L., Wolfe, B.B., Standaert, D.G., 2000. Alterations in subunit expression, composition and phosphorylation of striatal NMDA glutamate receptors in the rat 6-OHDA models of Parkinson’s disease. Mol. Pharmacol. 57, 342–352.

516


Fahn, S., 1999. Parkinson disease, the effect of levodopa, and the ELLDOPA trial. earlier vs later L-DOPA. Arch. Neurol. 56, 529–535. Guilloteau, D., Emond, P., Baulieu, J.L., Garreau, L., Frangin, Y., Pourcelot, L., Mauclaire, L., Besnard, J.C., Chalon, S., 1998. Exploration of the dopamine transporter: in vitro and in vivo characterization of a high-affinity and high-specificity iodinated tropane derivative (E )-N-(3-iodoprop-2-enyl)-2-b-carbomethoxy3-b-(4#-methylphenyl) nortropane (PE2I). Nucl. Med. Biol. 25, 331–337. Hadj Tahar, A., Gregoire, L., Darre, A., Belanger, N., Meltzer, L., Bedard, P.J., 2004. Effect of a selective glutamate antagonist on L-dopa-induced dyskinesias in drug-naive parkinsonian monkeys. Neurobiol. Dis. 15, 171–176. Hallett, P.J., Dunah, A.W., Ravenscroft, P., Zhou, S., Bezard E., Crossman, A.R., Brotchie, J.M., Standaert, D.G., 2003. Striatal NMDA receptor subunit abundance and phosphorylation in the MPTP-lesioned macaque model of Parkinson’s disease and dyskinesia. 2003 Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DC (online program 946.7). Hallett, P.J., Standaert, D.G., 2004. Rationale for and use of NMDA receptor antagonists in Parkinson’s disease. Pharmacol. Ther. 102, 155–174. Jenner, P., 2003. The MPTP-treated primate as a model of motor complications in PD: primate model of motor complications. Neurology 61, S4–S11. Klockgether, T., Turski, L., 1993. Toward an understanding of the role of glutamate in experimental Parkinsonism: agonist-sensitive sites in the basal ganglia. Ann. Neurol. 34, 585–593. Kornau, H.C., Schenker, L.T., Kennedy, M.B., Seeburg, P.H., 1995. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737–1740. Kosinski, C.M., Standaert, D.G., Counihan, T.J., Scherzer, C.R., Kerner, J.A., Daggett, L.P., Velicelebi, G., Penney, J.B., Young, A.B., 1998. Expression of N-methyl-D-aspartate receptor subunit mRNA in the human neostriatum and globus pallidus. J. Comp. Neurol. 390, 63–74. Ku¨ppenbender, K.D., Standaert, D.G., Feuerstein, T.J., Penney Jr., J.B., Young, A.B., Landwehrmeyer, G.B., 2000. Expression of NMDA receptor subunit mRNAs in neurochemically identified projection and interneurons in the human striatum. J. Comp. Neurol. 419, 407–421. Lange, K.W., Kornhuber, J., Riederer, P., 1997. Dopamine/glutamate interactions in Parkinson’s disease. Neurosci. Biobehav. Rev. 21, 393–400. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lau, L.F., Huganir, R.L., 1995. Differential tyrosine phosphorylation of N-methyl-D-aspartate receptor subunits. J. Biol. Chem. 270, 20036–20041. Loschmann, P.A., De Groote, C., Smith, L., Wullner, U., Fischer, G., Kemp, J.A., Jenner, P., Klockgether, T., 2004. Antiparkinsonian activity of Ro 25-6981, a NR2B subunit specific NMDA receptor antagonist, in animal models of Parkinson’s disease. Exp. Neurol. 187, 86–93.

Luo, J., Wang, Y., Yasuda, R.P., Dunah, A.W., Wolfe, B.B., 1997. The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/ NR2A/NR2B). Mol. Pharmacol. 51, 79–86. Menegoz, M., Lau, L.F., Herve, D., Huganir, R.L., Girault, J.A., 1995. Tyrosine phosphorylation of NMDA receptor in rat striatum: effects of 6-OH-dopamine lesions. Neuroreport 7, 125–128. Nash, J.E., Fox, S.H., Henry, B., Hill, M.P., Peggs, D., McGuire, S., Maneuf, Y., Hille, C., Brotchie, J.M., Crossman, A.R., 2000. Antiparkinsonian actions of ifenprodil in the MPTP-lesioned marmoset model of Parkinson’s disease. Exp. Neurol. 165, 136–142. Nash, J.E., Hill, M.P., Brotchie, J.M., 1998. Antiparkinsonian actions of blockade of NR2B-containing NMDA receptors in the reserpine-treated rat. Exp. Neurol. 155, 42–48. Oh, J.D., Russell, D., Vaughan, C.L., Chase, T.N., 1998. Enhanced tyrosine phosphorylation of striatal NMDA receptor subunits: effect of dopaminergic denervation and L-DOPA administration. Brain Res. 813, 150–159. Oh, J.D., Vaughan, C.L., Chase, T.N., 1999. Effect of dopamine denervation and dopamine agonist administration on serine phosphorylation of striatal NMDA receptor subunits. Brain Res. 821, 433–442. Papa, S.M., Chase, T.N., 1996. Levodopa-induced dyskinesias improved by a glutamate antagonist in parkinsonian monkeys. Ann. Neurol. 39, 574–578. Picconi, B., Gardoni, F., Centonze, D., Mauceri, D., Cenci, M.A., Bernadi, G., Calabresi, P., Di Luca, M., 2004. Abnormal Ca2Ccalmodulin-dependent protein kinase II function mediates synaptic and motor deficits in experimental parkinsonism. J. Neurosci. 24, 5283–5291. Sheng, M., Cummings, J., Roldan, L.A., Jan, Y.N., Jan, L.Y., 1994. Changing subunit composition of heteromeric NMDA receptors during development. Nature 368, 144–147. Smith, A.D., Bolam, J.P., 1990. The neural network of the basal ganglia as revealed by the study of synaptic connections of identified neurones. Trends Neurosci. 13, 259–265. Standaert, D.G., Friberg, I.K., Landwehrmeyer, G.B., Young, A.B., Penney Jr., J.B., 1999. Expression of NMDA glutamate receptor subunit mRNAs in neurochemically identified projection and interneurons in the striatum of the rat. Brain. Res. Mol. Brain. Res. 64, 11–23. Standaert, D.G., Testa, C.M., Penney, J.B., Young, A.B., 1994. Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat. J. Comp. Neurol. 343, 1–16. Steece-Collier, K., Chambers, L.K., Jaw-Tsai, S.S., Menniti, F.S., Greenamyre, J.T., 2000. Antiparkinsonian actions of CP-101,606, an antagonist of NR2B subunit-containing N-methyl-D-aspartate receptors. Exp. Neurol. 163, 239–243. Wessell, R.H., Ahmed, S.M., Menniti, F.S., Dunbar, G.L., Chase, T.N., Oh, J.D., 2004. NR2B selective NMDA receptor antagonist CP-101,606 prevents levodopa-induced motor response alterations hemi-parkinsonian rats. Neuropharmacology 47, 184–194.