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Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia.

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Increased D1 Dopamine Receptor Signaling
in Levodopa-Induced Dyskinesia
Incarnation Aubert, PhD,1 Céline Guigoni, PhD,2 Kerstin Håkansson, PhD,3 Qin Li, BsC,4 Sandra Dovero,2
Nicole Barthe, MD, PhD,5 Bernard H. Bioulac, MD, PhD,2 Christian E. Gross, PhD,2 Gilberto Fisone, PhD,3
Bertrand Bloch, MD, PhD,1 and Erwan Bezard, PhD2
Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa therapy for Parkinson’s disease.
Although changes affecting D1 and D2 dopamine receptors have been studied in association with this condition, no
causal relationship has yet been established. Taking advantage of a monkey brain bank constituted to study levodopainduced dyskinesia, we report changes affecting D1 and D2 dopamine receptors within the striatum of normal, parkinsonian, nondyskinetic levodopa-treated parkinsonian, and dyskinetic levodopa-treated parkinsonian animals. Whereas D1
receptor expression itself is not related to dyskinesia, D1 sensitivity per D1 receptor measured by D1 agonist-induced
[35S]GTP␥S binding is linearly related to dyskinesia. Moreover, the striata of dyskinetic animals show higher levels of
cyclin-dependent kinase 5 (Cdk5) and of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32).
Our data suggest that levodopa-induced dyskinesia results from increased dopamine D1 receptor–mediated transmission
at the level of the direct pathway.
Ann Neurol 2005;57:17–26
Long-term L-3,4-dihydroxyphenylalanine (L-dopa) treatment of Parkinson’s disease (PD)1–3 induces adverse
fluctuations in motor response and involuntary movements, known as L-dopa–induced dyskinesia (LID)
(for a review, see Bezard and colleagues4).
Denervation-induced supersensitivity of dopamine
(DA) receptors (D1-like and D2-like) has been widely
suggested as the most plausible mechanism of LID.
Indeed, striatal D2 receptor–binding sites are increased in postmortem tissue of untreated parkinsonian patients and in animal models.5,6 Although supersensitivity of D2 receptors is expected when
parkinsonism is first apparent, the first L-dopa dose
administered does not generally induce dyskinesia,
but dyskinesia develops gradually over time.7 Accordingly, the D2/D3 receptor agonists exert an antiparkinsonian effect with a reduced propensity to elicit
dyskinesia when administered de novo in PD patients.8 There is some evidence that D1 messenger
RNA (mRNA) levels are increased after dopaminergic
treatment of the DA-depleted striatum in animal
models of LID9; that downstream signal transduction
cascades is abnormal in LID,10,11 including increased
phosphorylation of cAMP-regulated phosphoprotein
of 32kDa (DARPP-32)12; and that an altered subcellular localization of D1 receptors is involved in
LID.13 Moreover, a DA D1 receptor agonist with
proven antiparkinsonian action14 induced LID similar
to that induced by L-dopa in PD patients,15 further
suggesting that D1 supersensitivity plays a key role in
LID occurrence. Together, these observations call for
a reassessment of the changes affecting D1 and D2
DA receptors in LID.
In this study, taking advantage of a nonhuman primate (NHP) brain bank constituted to study the
pathophysiology of LID,16 we determined changes affecting D1 and D2 DA receptors within the striatum of
four experimental groups: normal, parkinsonian, parkinsonian chronically treated with L-dopa without exhibiting dyskinesia, and parkinsonian chronically
treated with L-dopa that shows overt dyskinesia. We
show that LIDs are linked to a modification of both
D1 receptor expression and sensitivity of the D1signaling cascade, reinforcing the hypothesis of the piv-
From the 1Centre National de la Recherche Scientifique Unite
Mixte de Recherche 5541 and 2Basal Gang Centre National de la
Recherche Scientifique UMR 5543, Bordeaux Cedex, France;
3
Karolinska Institutet, Department of Neuroscience, Stockholm,
Sweden; 4Lab Animal Research Center, China Agricultural University, Beijing, China; and 5Institut National de la Sante et de la Recherche Médicale U443, Université Victor Segalen-Bordeaux 2, Bordeaux Cedex, France.
Received Jun 3, 2004, and in revised form Aug 19. Accepted for
publication Aug 24, 2004.
Published online Oct 27, 2004, in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.20296
Address correspondence to Dr Bezard, Laboratoire de Physiologie et
Physiopathologie de la Signalisation Cellulaire, Centre National de
la Recherche Scientifique Unite Mixte de Recherche 5543, Université Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex,
France. E-mail: erwan.bezard@umr5543.u-bordeaux2.fr
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
17
otal role played by the so-called direct pathway in LID
genesis.
Materials and Methods
Animals
We used 17 female cynomolgus monkeys (Macaca fascicularis; Shared Animal Health, Beijing, China) for this study
(Table). Animals were housed in individual primate cages
under controlled conditions of humidity (50% ⫾ 5%), temperature (24°C ⫾ 1°C) and light (12-hour light/12-hour
dark cycles, time lights on 8:00 AM), food and water were
available ad libitum, and animal care was supervised by veterinarians skilled in the health care and maintenance of
NHPs. Experiments were carried out in accordance with European Communities Council Directive of 24 November
1986 (86/609/EEC) for care of laboratory animals.
Experimental Protocol
Four monkeys were kept normal (control group), and 13
were intoxicated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) hydrochloride. Once a bilateral parkinsonian syndrome had stabilized (ie, unchanged disability score
over several weeks), four monkeys were kept without any dopaminergic supplementation (MPTP group), and the nine
were treated chronically with twice daily administration of
L-dopa (Modopar; L-dopa/carbidopa, ratio 4:1) for 6 to 8
months at a tailored dose designed to produce a full reversal
of parkinsonian condition (see Table). Five monkeys developed severe and reproducible dyskinesia (MPTP-intoxicated,
dyskinetic monkey group), whereas four did not (MPTPintoxicated, nondyskinetic monkey group). Animals were
killed by sodium pentobarbital overdose (150mg/kg of body
weight, intravenously). Brains were removed quickly after
death. Each brain was bisected along the midline, the most
rostral part of the striatum was removed for western blotting
experiments, and the two hemispheres were immediately frozen by immersion in isopentane (⫺45°C) and then stored at
⫺80°C. Tissue was sectioned coronally at 20␮m in a cryostat at ⫺17°C, thaw-mounted onto gelatine-subbed slides,
dried on a slide warmer, and stored at ⫺80°C.
Behavioral Assessment
Parkinsonian condition (and reversal) was assessed on a parkinsonian monkey rating scale using videotape recordings of
monkeys as previously described.16,17 A score of 0 corresponds to a normal animal, and a score above 6 corresponds
to a parkinsonian animal. The severity of dyskinesia was
rated using the Dyskinesia Disability Scale: 0, dyskinesia absent; 1, mild, fleeting, and rare dyskinetic postures and
movements; 2, moderate, more prominent abnormal movements, but not interfering significantly with normal behavior; 3, marked, frequent and, at times, continuous dyskinesia
intruding on the normal repertoire of activity; or, 4, severe,
virtually continuous dyskinetic activity, disabling to the animal and replacing normal behavior.
Assessment of Lesion
DA transporter binding using [125I](E)-N-(3-iodoprop-2enyl)-2␤-carboxymethyl-3␤-(4⬘-methylphenyl)-nortropane
(PE2I; Chelatec, Nantes, France) was measured as previously
described.18 Processing of mesencephalic sections for tyrosine
hydroxylase (TH) immunohistochemistry, counterstaining
with cresyl violet (Nissl staining), and cell counts (Visioscan
version 4.12; Biocom, Les Ulis, France) were performed as
Table. Characteristics of the Monkey Subgroups
L-Dopa
0 min
Group
Control
C1
C2
C3
C4
M1
M2
M3
M4
L1
L2
L3
L4
D1
D2
D3
D4
D5
MPTP
MPTP/L-dopa nondyskinetic
MPTP/L-dopa dyskinetic
90 min
Age (yr)
Weight (kg)
mg
P
D
P
D
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
3
3
3.0
2.7
3.0
3.1
3.4
2.9
3.0
3.1
2.7
2.5
2.7
3.3
3.2
3.3
4.5
2.8
3.0
—
—
—
—
—
—
—
—
54
59
56
50
47
60
66
53
50
0.0
0.0
0.0
0.0
9.0
9.2
8.2
8.8
9.4
10.6
9.2
8.4
10.0
8.8
7.5
7.2
7.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
2.2
0.7
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.4
1.9
3.7
2.8
3.7
3.3
L-Dopa dose (mg) is given for each individual as well as their parkinsonian (P) and dyskinetic (D) scores before administration (0 min) and 90
min after administration. L-Dopa dose was tailored to produce a full reversal of parkinsonian motor abnormalities as shown by the drastic
decrease in parkinsonian score after 90 min. Despite comparable levels of lesion and duration of treatment, only the “dyskinetic” animals
displayed severe LID 90 min after L-dopa administration.
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previously described.18 The boundaries of the substantia
nigra pars compacta (SNc) were chosen on three consecutive
sections corresponding to a representative median plane of
the SNc by examining the size and shape of the different
tyrosine hydroxylase–immunoreactive (TH-IR) neuronal
groups, cellular relationships to axonal projections, and
nearby fiber bundles. The number of both TH–IR and
Nissl-stained neurons per SNc representative plane was
counted three times by one examiner blind about the experimental condition. Split cell counting error was corrected by
using the formula of Abercrombie.19 Mean cell number per
plane and standard error of the mean were then calculated
for each group of monkeys.
Receptor Binding
Both the D1 and D2 receptors were labeled using ligands
specific for D1-like sites ([3H]SCH 23390, 75Ci/mmol; New
England Nuclear, Paris, France) or D2-like sites ([3H]YM09151-2, 85Ci/mmol, New England Nuclear) as previously
described18: tissue sections were incubated for 1 hour at
room temperature in a buffer solution (50mM Tris-HCl,
120mM NaCl, 5mM KCl, 2mM CaCl2, 1mM MgCl2, pH
7.4) containing either 2nM [3H]SCH 23390 or 0.3nM
[3H]YM-09151-2. Nonspecific binding was defined in the
presence of 10␮M of (⫹)butaclamol for both subtypes of
DA receptor. Sections were exposed to a ␤-imager (Biospace,
Paris, France) to assess directly the radioactivity bound to
regions of interest.20,21
GTP␥S Binding
Labeling of monkey brain sections with [35S]GTP␥S (Amersham, Uppsala, Sweden) was carried out essentially as described by Sim et al. (1995) with minor modifications. The
slides were incubated for 10 minutes at 25°C in assay buffer
(50mM Tris-HCl, 3mM MgCl2, 0.2mM EGTA, 100mM
NaCl, pH 7.7). Slides were then incubated with 2mM GDP
in assay buffer for 15 minutes at 25°C. Agonist-stimulated
activity was determined by incubation in [35S]GTP␥S
(0.01nM) with 2mM GDP and D1 agonist (SKF38393) in
assay buffer for 2 hours at 25°C. In each experiment, basal
activity was assessed with GDP in absence of agonist, and
nonspecific binding was assessed in presence of 10␮M unlabelled GTP␥S. Slides were exposed to ␤-imager (Biospace)
to assess directly the radioactivity bound to regions of interest.20,21
Image Analysis
Densitometric analysis of autoradiographs (in situ hybridization) and direct measurement of radioactivity of ␤-imager
images (binding) was performed using, respectively, an image
analysis system (Visioscan version 4.12; Biocom) and
␤-vision (version 4.2; Biospace), at three rostrocaudal levels
in accordance with the functional organization of the striatum as previously described18,25: a rostral level including the
caudate, putamen, and nucleus accumbens (A21.0); a
midlevel including the caudate, putamen, and globus pallidus pars externalis (A17.2); and a caudal level including the
body of the caudate, the putamen, and both parts of the
globus pallidus (ie, pars externalis and pars internalis)
(A14.6). Where appropriate, both caudate and putamen were
divided into dorsolateral, dorsomedial, ventrolateral, and
ventromedial quadrants for analysis. Three sections per animal per striatal level were analyzed by an examiner blind
about the experimental condition. For autoradiographs, optical densities were averaged for each region in each animal,
converted to amount of radioactivity bound by comparison
to the standards, and expressed in femtomoles per milligram
of tissue equivalent (mean ⫾ standard error of the mean).
Since ␤-imager images allow direct measurements of the radioactivity, data are expressed in counts per minute per
square micrometer.
Western Blotting
Pieces of monkey caudate–putamen were sonicated in 1ml of
1% sodium dodecyl sulfate and boiled for 10 minutes. Aliquots (5␮l) of the homogenate were used for protein determination using the BCA (bicinchoninic acid) assay kit
(Pierce, Oud Beijerland, The Netherlands). Equal amounts
of protein (30␮g) from each sample were loaded onto 10%
polyacrylamide gels, separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Amersham). The membranes were immunoblotted using affinity-purified polyclonal
antibodies against cyclin-dependent kinase 5 (Cdk5; Santa
Cruz Biotechnology, Santa Cruz, CA) and DARPP-32 (Cell
Signaling Technology, Beverly, MA). Antibody binding was
indicated by incubation with goat anti–rabbit horseradish
peroxidase–linked immunoglobulin G (diluted 1:10,000;
Pierce Europe, Oud Beijerland, The Netherlands) and the
enhanced chemiluminescence (ECL) immunoblotting detection system. Autoradiograms were quantified with NIH Image software (version 1.62).
In Situ Hybridization Histochemistry
The in situ hybridization procedure was performed as previously described22 with probes designed to recognize the human D1R23 or the human D2R24. 35S-labeled antisense and
sense complementary RNA probes were prepared by in vitro
transcription from 100ng of linearized plasmid using
[35S]UTP (⬎1,000Ci/mmol; New England Nuclear), and
SP6, T3, or T7 polymerases. After alkaline hydrolysis to obtain 0.25kb complementary RNA fragments, the probes were
purified on G50-Sephadex and precipitated in sodium acetate (0.1 vol)–absolute ethanol (2.5 vol). Sections were then
hybridized as described by Aubert et al.22 Slides were then
exposed to Biomax film (Kodak) with autoradiographic microscale standard (Amersham).
Statistical Analysis
For multiple comparisons of cell counting, DAT binding
and GTP␥S binding, one-way analysis of variance (ANOVA)
was used. For multiple comparisons of DA receptor binding
and DA receptor mRNA levels, a two-way ANOVA, with
group and striatal subregions as factors, was used. ANOVAs
were followed when allowed by post hoc t tests corrected for
multiple comparisons by the method of Bonferroni. For
multiple comparisons of behavioral assessments, the Kruskal–
Wallis nonparametric test was used to estimate overall significance followed by post hoc t tests corrected for multiple
comparisons by the method of Dunn. These analyses were
completed using the Stata program (Intercooled Stata 6.0;
Aubert et al: Increased D1 Signaling in Dyskinesia
19
Stata Corporation, College Station, TX). A probability level
of 5% ( p ⬍ 0.05) was considered statistically significant.
Results
Changes in Motor Behavior
Five monkeys developed severe and reproducible dyskinesia (MPTP-lesioned, dyskinetic monkey group,
score ⫽ 3.08 ⫾ 0.34, of a maximum of 4) (see Table),
whereas four did not (MPTP-lesioned, nondyskinetic
monkey group, score ⱕ 0.4) (see Table). Parkinsonism
was comparable between the different MPTP-lesioned
groups ( p ⬎ 0.5), and motor abnormalities were fully
reversed by the L-dopa treatment (see Table). Dyskinetic animals presented choreic–athetoid (characterized
by constant writhing and jerking motions), dystonic,
and sometimes ballistic movements (large-amplitude
flinging, flailing movements). At the peak of dose (80 –
150 minutes after injection), dystonic rolling and
writhing on cage floor were common. Dyskinesia developed by MPTP animals was similar to the LID observed in PD patients.
Extent of Lesion Is Homogeneous among the MPTPLesioned Groups
Since the extent of nigrostriatal degeneration is an obvious variable that may play a role in the susceptibility
to develop LID, we first assessed whether all MPTPlesioned monkeys had similar loss in the number of
TH-IR neurons in the SNc and in striatal DA nerve
endings by measuring DA transporter (DAT) binding.
MPTP treatment induced a strong loss both in the
number of TH-IR cells and in the total number of
neurons, that is, Nissl-stained cells (Fig 1A). In addition, MPTP induced striatal dopaminergic denervation, as shown by a decrease in DAT binding, both in
the dorsolateral caudate nucleus and in the dorsolateral
putamen (see Figs 1B, C). There was no significant
difference in the decrease in the number of TH-IR
neurons and DAT binding between MPTP-lesioned
group, nondyskinetic L-dopa–treated MPTP-lesioned
group, and dyskinetic L-dopa–treated MPTP-lesioned
group. Thus, MPTP intoxication generated a similar
lesion in all MPTP-lesioned groups (see Fig 1C).
L-Dopa
Does Not Normalize D2 Levels
Previous studies have shown that DA denervation
causes an increase in striatal D2 DA receptor binding
sites in the postmortem tissue of untreated patients
with PD and animal models.5,6,18 Accordingly, we
found that MPTP alone produced a significant increase
in the levels of D2 mRNA in comparison with control
animals (Figs 2A, B). A similar effect was produced on
D2 DA receptor binding (see Figs 2C, D). When compared with the control group, the increase in D2 binding was particularly striking in the dorsal part of the
caudate nucleus and in the whole putamen (see Figs
2C, D).
L-Dopa treatment, in addition to MPTP intoxication, differentially affected D2 mRNA in nondyskinetic
and dyskinetic animals. In the nondyskinetic NHP,
L-dopa induced a normalization of D2 mRNA levels,
which were not significantly different from those determined in the control group (see Figs 2A, B). In the
dyskinetic animals, D2 mRNA levels remained signifi-
Fig 1. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and L-dopa treatment on (A) the estimated
number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons and Nissl-stained cells in the substantia nigra pars compacta
(SNc), and (B) on the striatal DA transporter (DAT) binding. MPTP treatment induced a strong loss in the estimated number of
TH-IR neurons (F(3,15) ⫽ 293.7; p ⬍ 0.0001) and Nissl-stained cells (F(3,15) ⫽ 180.9; p ⬍ 0.0001) and specific [125I]PE2I
binding (caudate: F(3,15) ⫽ 488.47; p ⬍ 0.0001; putamen: F(3,15) ⫽ 1911.82; p ⬍ 0.0001). Results represent the mean ⫾ standard error of the mean (* ⫽ statistically significant difference compared with control animals, p ⬍ 0.05). (C) Example of DAT
binding autoradiographs showing the striatal denervation at the caudal level.
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Fig 2. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and L-dopa treatment on (A, B) striatal D2
messenger RNA (mRNA) expression (group effect: F(3,95) ⫽ 55.38; p ⬍ 0.0001; interaction between group and striatal subregion: F(21,95) ⫽ 1.08) (DL ⫽ dorsolateral; DM ⫽ dorsomedian; VL ⫽ ventrolateral; VM ⫽ ventromedian) and (C, D) striatal D2 binding levels (group effect: F(3,135) ⫽ 104.9; p ⬍ 0.0001; interaction between group and striatal subregion:
F(21,135) ⫽ 0.61). Results are expressed as the percentage of control animals. For example, in the dorsolateral (DL) putamen of
control animals, mRNA level was 30.73 ⫾ 1.23fmol/mg of tissue equivalent, and binding level was of 2.07 ⫾ 0.17cpm/␮m2
(* ⫽ statistically significant difference compared with control animals, p ⬍ 0.05). (B) Examples of D2 mRNA expression autoradiographs and (D) of D2 binding ␤-imager images at the rostral level of striatum.
cantly higher than those of control monkeys, particularly in the ventromedian caudate nucleus and the putamen (see Figs 2A, B). Despite these differences in
mRNA, L-dopa therapy had no effect on D2 receptor
levels. Indeed, the D2 binding of the nondyskinetic
and dyskinetic groups remained comparable to that observed after MPTP treatment (see Figs 2C, D). Our
results are in agreement with previous studies that
showed a normalization of the D2 mRNA receptor after L-dopa treatment in patients with PD6,26,27 and in
MPTP-lesioned monkeys.28 They confirm the lack of
direct correlation between D2 regulation and the occurrence of LID and rule out the hypothesis of a predominant role for the indirect pathway in LID occurrence.
L-Dopa Increases D1 Levels
D1 receptor expression does not follow the same pattern. In MPTP-lesioned animals and in MPTPlesioned animals treated with L-dopa, D1 mRNA expression was similar to that of control group in all
striatal quadrants (Fig 3) but not in the ventrolateral
quadrant of the putamen, where MPTP alone had a
significant effect on the D1 mRNA levels (see Fig 3).
In this region, MPTP decreased the D1 mRNA expression (see Fig 3). Even if, in other subregions of the
striatum, the MPTP had not a significant effect, a decrease in the D1 mRNA levels was found in MPTPlesioned group compared with control group (⫺34%
in mean). L-Dopa treatment, in nondyskinetic NHP,
did not induce modifications of D1 mRNA expression.
The level was similar to D1 mRNA level observed in
the MPTP-lesioned group. In dyskinetic NHP, the D1
mRNA level displayed a tendency to increase, and the
difference with control animals became less pronounced (see Figs 3A, B).
Similar to the D1 mRNA level, D1 binding was not
affected by MPTP treatment alone. However, L-dopa
administration increased D1 binding in the striata of all
MPTP-lesioned animals (eg, nondyskinetic and dyskinetic monkeys) (see Figs 3C, D).
Overall, these results showed increased levels of D1
protein in the striatum after L-dopa treatment. In addition, in MPTP-lesioned animals, the expression of
D1 mRNA is decreased, though not significantly
(⫺34%), in comparison with control animals and was
normalized by further L-dopa treatment. This decrease
is more marked in the ventrolateral putamen. How-
Aubert et al: Increased D1 Signaling in Dyskinesia
21
Fig 3. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and L-dopa treatment on (A, B) striatal D1
messenger RNA (mRNA) expression (group effect: F(3,119) ⫽ 39.46; p ⬍ 0.0001; interaction between group and striatal subregion: F(21,119) ⫽ 0.51), and (C, D) D1 binding levels (group effect: F(3,135) ⫽ 34.79; p ⬍ 0.0001; interaction between group
and striatal subregion: F(21,135) ⫽ 1.12). The results are expressed as the percentage of control animals. For example, in the
putamen dorsolateral of control animals, messenger RNA (mRNA) level was of 78.28 ⫾ 10.59 fmol/mg of tissue equivalent,
and binding level was 5.74 ⫾ 0.62 cpm/␮m2 (* ⫽ statistically significant difference compared with control animals; p ⬍
0.05). (B) Examples of D1 mRNA expression autoradiographs and (D) of D1 binding ␤-imager images at the rostral level of
striatum.
ever, the D1 binding is not affected by the DA denervation.6,29 Whereas the expression of D1 mRNA is not
affected by the L-dopa therapy, this treatment provokes
a strong increase in the D1 binding in both nondyskinetic and dyskinetic NHP. These results show dissociation between D1 mRNA and protein, indicating that
modification of the levels in D1 mRNA is not necessarily correlated with a comparable change in D1 protein levels, and they suggest a modification of D1
mRNA transcription regulation after chronic L-dopa
treatment.
Changes in Overall D1 Agonist–Stimulated
GTP␥S Binding
Although no significant differences in receptor binding
were found between nondyskinetic and dyskinetic
NHPs, it could still be possible that dyskinesia was due
to more subtle changes at the level of dopamine D1
receptor transmission. We have previously hypothesized that D1 receptors are subjected to a differential
trafficking13 by showing that L-dopa induces a cytoplasmic localization of D1 receptors in striatal neurons
of 6-hydroxydopamine–treated rats and parkinsonian
patients, although none of the rats or PD cases were
22
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dyskinetic at the time of their death.13 We also wondered whether those D1 receptors have the same sensitivity to pharmacological stimulation. To assess the difference of D1 sensitivity between dyskinetic and
nondyskinetic groups, we studied D1 agonist–stimulated GTP␥S binding. The [35S]GTP␥S autoradiography detects functionally active receptors by indicating
their ability to interact with G proteins.
MPTP and L-dopa treatment had a significant effect
on the [35S]GTP␥S binding in the striatum, independently of the D1 agonist concentration (Figs 4A, B). In
basal condition, that is, without D1 agonist stimulation, [35S]GTP␥S binding was similar in all four
groups studied. MPTP treatment alone increased
[35S]GTP␥S binding in the striatum, regardless of the
D1 agonist concentration (see Fig 4A). After L-dopa
treatment, the [35S]GTP␥S binding was different in
nondyskinetic and dyskinetic animals. In the nondyskinetic group, L-dopa reduced the MPTP-induced increase in [35S]GTP␥S binding, which returned to control levels (see Figs 4A, B). In the dyskinetic group,
35
L-dopa induced a strong increase of the [ S]GTP␥S
35
binding. This [ S]GTP␥S binding was higher than
Fig 4. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and L-dopa treatment on the striatal D1 agonist–stimulated GTP␥S binding. (A) Raw values (0.1␮M, F(3,14) ⫽ 26.45, p ⬍ 0.0001; 0.3␮M, F(3,14) ⫽ 64.08, p ⬍ 0.0001;
1␮M, F(3,14) ⫽ 22.49, p ⬍ 0.0001; 3␮M, F(3,14) ⫽ 58.09, p ⬍ 0.0001; 10␮M, F(3,14) ⫽ 27.64, p ⬍ 0.0001), and (C)
normalized values (raw values/D1 binding) (0.1␮M, F(3,14) ⫽ 30.26, p ⬍ 0.0001; 0.3␮M, F(3,14) ⫽ 61.92, p ⬍ 0.0001;
1␮M, F(3,14) ⫽ 21.70, p ⬍ 0.0001; 3␮M, F(3,14) ⫽ 51.06, p ⬍ 0.0001; 10␮M, F(3,14) ⫽ 15.80, p ⫽ 0.0003) of D1stimulated GTP␥S binding expressed in cpm/␮m2 (* ⫽ statistically significant difference compared with control animals, p ⬍
0.05, # ⫽ significant difference compared with MPTP-alone animals, p ⬍ 0.05; § ⫽ statistically significant difference compared
with nondyskinetic animals, p ⬍ 0.05). (B) Examples of D1-stimulated (1.0␮M) GTP␥S binding images obtained with the
␤-imager at the rostral level of striatum.
that in the control group, in the MPTP group, and in
the nondyskinetic group (see Figs 4A, B).
Normalized D1 Agonist–Stimulated GTP␥S Binding
Is Linearly Related to L-Dopa–Induced Dyskinesia
Severity
However, D1 binding levels are increased in dyskinetic NHP (see Fig 3). The increased sensitivity thus
might well be the direct consequence of the increased
number of proteins or of an increased availability of
receptor at the membrane surface,13 and not necessarily of sensitivity. Consequently, we normalized the
GTP␥S binding according to the D1 binding. Once
normalized, the results were different, even if MPTP
and L-dopa treatment still had significant effects (see
Fig 4C).
MPTP lesions induced an increase of the [35S]GTP␥S binding only for the low D1 agonist concentrations (see Fig 4C), and for the other concentration
the [35S]GTP␥S binding was similar to the control
binding. In the nondyskinetic NHP, the [35S]GTP␥S
binding was decreased compared with the control
group (see Fig 4C) and to the MPTP-lesioned group
(see Fig 4C). In the dyskinetic group, L-dopa treatment
increased the [35S]GTP␥S binding above the levels reported in the control group, showing that the sensitivity per receptor is increased in the dyskinetic situation
(see Fig 4C). Interestingly, these [35S]GTP␥S binding
values were similar to the values obtained in the
MPTP-alone group.
These results suggest that the L-dopa treatment does
not act in the same way on the D1 receptor sensitivity.
In nondyskinetic animals, L-dopa would induce a decrease of D1 sensitivity, whereas in dyskinetic animals,
D1 sensitivity is strongly increased. Moreover, the D1
agonist–stimulated GTP␥S binding levels in putamen
correlated with occurrence and severity of LID (r2 ⫽
0.96, p ⬍ 0.05, n ⫽ 8). These results show that LIDs
were accompanied by an increased responsiveness of
the D1-mediated signaling, a result to compare with
the previously demonstrated correlation between D3 receptor binding and LID in the same experimental conditions.16 Thus, these results suggest a correlation between D1 supersensitivity and LID.
Increased Cyclin-Dependent Protein Kinase 5 and
DARPP-32 Levels Are Associated with D1 Receptor
Supersensitivity and L-Dopa–Induced Dyskinesia
Recent evidence indicates that Cdk5 is involved in
long-term synaptic changes. Moreover, this kinase participates in the regulation of DARPP-32, a DA- and
cAMP-regulated phosphoprotein of 32kDa that plays a
critical role in DA D1 receptor–mediated transmission
by modulating the state of phosphorylation and activity of a variety of downstream physiological effectors.30
Unfortunately, quantitative assessments of phosphorylated DARPP-32 and effector proteins are not possible
in our animals. Indeed, accurate measurement of phosphorylation state is prevented by the time required to
dissect the brains, which is known to be crucial. Microwave killing is not available for the primate, and
this technique is mandatory for studying phosphorylated proteins. We therefore determined the total levels
of Cdk5, p35, a Cdk5 activator, and DARPP-32 in the
striata of NHP by western immunoblotting. We found
that the striata of animals affected by LID contained
significantly higher levels of Cdk5 and DARPP-32
compared with the striata of control, MPTP-lesioned,
and MPTP-lesioned nondyskinetic NHP (Fig 5). No
Aubert et al: Increased D1 Signaling in Dyskinesia
23
Fig 5. Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and L-dopa treatment on the levels of Cdk5 and
DARPP-32. Lower panels show autoradiograms of Western blots for all animals tested (four in the control group, four in the
MPTP-lesioned group, four in the nondyskinetic group, and three in the dyskinetic group) obtained using polyclonal antibodies
against Cdk5 (A) or DARPP-32 (B). Upper panels show the amount of Cdk5 and DARPP-32 as median ⫾ standard deviation
and are expressed as percentage of control group. * ⫽ p ⬍ 0.01 (One-way analysis of variance followed by Newman–Keuls test;
Cdk5: F(3,14) ⫽ 8.09; DARPP-32: F(3,14) ⫽ 7.73).
difference was observed between the levels of p35 in
the different experimental groups (data not shown).
Discussion
In this study, we have utilized the MPTP-lesioned
monkey model of PD to demonstrate that the severity
of LID is linearly correlated with normalized D1 agonist–stimulated GTP␥S binding levels. Furthermore,
we provide evidence indicating that the levels of Cdk5
and DARPP-32, two pivotal players in DA signaling,
are increased in the striata of dyskinetic animals. Together, these data suggest that LID is caused by increased G protein coupling efficiency at the level of
DA D1 receptors, most likely occurring in the striatonigral neurons of the direct pathway.
Interestingly, from the point of view of D1 receptor
levels, we cannot distinguish the dyskinetic from the
nondyskinetic monkeys. MPTP intoxication induces a
significant increase of D1 agonist–stimulated [35S]GTP␥S binding without affecting the number of D1
receptors. This finding suggests that striatal DA depletion induces a sensitization of D1 receptors. In nondyskinetic animals, the D1 agonist–induced [35S]GTP␥S binding is decreased, becoming lower than
that in control animals. In the nondyskinetic situation,
the enhancement of the D1 protein expression, induced
by L-dopa, seems to offset a decrease in the GTP binding, suggesting that the D1 receptors are either desensitized or subjected to a differential trafficking.13 Although the phenomenon of receptor desensitization is
common, it cannot simply be explained by the mechanism of homologous desensitization through arrestin–
receptor interaction31 since there are many potential
regulators of receptor–G protein coupling, that is, by
definition, all scaffolding proteins present in a synapse
such as the membrane-associated guanylate kinase su-
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Annals of Neurology
Vol 57
No 1
January 2005
perfamily of synapse-associated proteins.32 It nevertheless remains that internalization and desensitization
would represent two aspects of a common mechanism
that either fails or is overactive in dyskinetic monkeys,
representing an attempt of the system to compensate
for DA receptor overstimulation. Theoretically, if desensitization is impaired or delayed, DA receptors
would appear functionally supersensitive. This hypothesis is in agreement with our results since despite the
absence of difference of D1 protein expression between
dyskinetic and nondyskinetic NHP, the D1 agonist–
induced [35S]GTP␥S binding is strongly increased,
showing a D1 supersensitivity. In fact, it is linearly correlated with LID severity. Thus, in dyskinetic animals,
internalization of the D1 receptor is probably associated with the reported increase in the activity of signal
transduction pathway of the still functional receptors
present at the membrane surface.10,11,33 Such a mechanism, though compensatory in nature, fails since
NHPs are dyskinetic.
We show that the signal transduction pathway is increased in dyskinetic animals. Previous work performed
in the 6-OHDA–lesioned rat model of PD showed
that, in dyskinetic animals, DARPP-32 is hyperphosphorylated at Thr34,12 but its expression levels are not
modified at all.12 These results show, however, an increased expression of DARPP-32 in dyskinetic animals,
a finding that is at odds with rodent data.12 Such a
discrepancy could be attributed to a species difference
to a certain extent, but more likely to a difference in
duration of dopamimetic treatment. Indeed, rats were
treated with L-dopa for 16 to 22 days,12 whereas our
monkeys were treated for several months, a time frame
that allows further dysregulation of signaling cascade.
We have not been able to address the phosphorylation
issue for technical reasons (see above), but we could at
least assume that the D1 receptor pulsatile stimulation
by L-dopa induces an increased phosphorylation of
DARPP-32 at Thr34 through protein kinase A (PKA)
activation.30 Cdk5 phosphorylates DARPP-32 at
Thr75,34 thereby converting this phosphoprotein into
an inhibitor of PKA.34 We hypothesize that the increase in Cdk5 expression found in dyskinetic NHP
may represent a homeostatic response to hyperactivation of the D1–PKA pathway. Interestingly, the role of
Cdk5 has been studied in another model of hyperdopaminergia, that is, in animals sensitized after chronic
exposure to cocaine.35 They found a reduction in
PKA-dependent phosphorylation of DARPP-32 (at
Thr34) in striatal tissue dissected from rats chronically
treated with cocaine. This effect was proposed to depend on increased Cdk5 expression, phosphorylation
of DARPP-32 at Thr75, and inhibition of PKA.35 We
hypothesize that a similar mechanism is present in dyskinetic animals, where Cdk5 is also overexpressed. If
Cdk5 regulates activation of the D1–PKA pathway in
the striatal neuron, it would negatively regulate DA release from DA terminals.36 Although few terminals remain in the MPTP-denervated striatum, Cdk5 might
attempt controlling newly formed DA from exogenous
L-dopa. However, these two negative feedbacks at both
presynaptic and postsynaptic levels are not efficient
enough to correct for D1 hyperactivation. The hypothesis that Cdk5 overexpression represents a compensatory mechanism remains to be experimentally addressed.
This study clearly shows that, whereas D2 receptor
levels are not significantly impaired by L-dopa treatment, the D1 receptor expression, sensitivity, and integrity of signaling cascade is modified by the chronic
pharmacological stimulation. Interestingly, we have recently shown in the very same animals that the DA D3
receptor binding level is also linearly correlated with
the severity of LID.16 D3 receptor mRNA is expressed
in the striatal medium spiny neurons of the direct
pathway, that is, those that express the D1 receptor.37
Considering that (1) the D1 receptor expression is increased after L-dopa treatment, (2) that the sensitivity
of the D1 signaling cascade is enhanced in LID, (3)
that D1 and D3 receptors are likely coexpressed in the
direct pathway neurons, and (4) that D2 receptor levels
expressed by medium spiny neurons of the indirect
pathway are neither normalized nor increased after
L-dopa treatment, these results support the hypothesis
of a predominant role for the direct pathway in LID
manifestation. Moreover, our results lead us to hypothesize that Cdk5-increased expression in LID is compensatory upon DARPP-32 activity. Unraveling of this
machinery may have tremendous consequences on the
development on therapeutic tools for the management
of LID.
This work was supported by grants from the Michael J. Fox Foundation for Parkinson Research (C.G., B.B., E.B.), the Fédération
pour la Recherche sur le Cerveau (C.E.G., E.B.) and the Fondation
pour la Recherche Médicale (E.B., C.E.G.).
The University Victor Segalen, the Centre National de la Recherche
Scientifique, the IFR of Neuroscience (Institut National de la Sante
et de la Recherche Médicale No. 8; Centre National de la Recherche
Scientifique No. 13) provided the infrastructural support. We thank
L. Cardoit and C. Imbert for technical assistance.
The authors declare that they have no competing financial interests.
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