Inhibition of mTOR Signaling in Parkinson’s Disease Prevents L-DOPAInduced Dyskinesia
Emanuela Santini, Myriam Heiman, Paul Greengard, Emmanuel Valjent andGilberto Fisone (21 July 2009)
Science Signaling 2 (80), ra36. [DOI: 10.1126/scisignal.2000308]

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RESEARCH ARTICLE
NEUROSCIENCE

Inhibition of mTOR Signaling in Parkinson’s
Disease Prevents L-DOPA–Induced Dyskinesia
Emanuela Santini,1 Myriam Heiman,2 Paul Greengard,2Emmanuel Valjent,1,3,4,5 Gilberto Fisone1,2*(Published 21 July 2009; Volume 2 Issue 80 ra36)

INTRODUCTION

The main pathological feature of Parkinson’s disease (PD) is the degeneration of midbrain dopaminergic neurons located in the substantia nigra,
which project to the dorsal striatum (1). The loss of nigrostriatal input
is accompanied by profound modifications in the response of GABAergic
medium spiny neurons (MSNs) to drugs that modulate dopaminergic
signaling, including L-DOPA, the most effective anti-Parkinsonian medication (2–4). Prominent among these changes is the activation of thetwo mitogen-activated protein kinases (MAPKs), extracellular signal–
regulated kinase (ERK) 1 and 2 (5–8), which are involved in various
types of synaptic plasticity (9). Persistent activation of ERK in striatal
MSNs leads to the development of dystonic and choreic motor complications, or dyskinesia, which limits the use of L-DOPA as an antiParkinsonian therapy (7, 10). The ability to interfere with the striatal
signaling machinery controlled by ERK presents a key target for thedesign of anti-Parkinsonian therapies.
Accumulating evidence indicates that ERK participates in the activation of the mammalian target of rapamycin (mTOR), a key regulator ofprotein synthesis, which promotes 5′ cap-dependent initiation of messenger RNA (mRNA) translation (11). mTOR regulates translation after
the formation of an mTOR complex 1 (mTORC1) containing mTOR
and the rapamycin-sensitive adaptor protein of mTOR, Raptor (11). In
the hippocampus, mTORC1-dependent signaling has been implicated in
the development of the late phase of long-term potentiation, throughincreased activity of the dendritic translation machinery (12, 13). Moreover, L-DOPA–induced dyskinesia (LID) has been associated with lossof depotentiation at the corticostriatal synapses of MSNs, which resultsin persistent enhancement of synaptic efficiency (14). These observations
led us to hypothesize that some of the adaptive processes responsible fordyskinesia may involve dysregulation of mTORC1 by L-DOPA.
1
Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 171 77
Stockholm, Sweden. 2Laboratory of Molecular and Cellular Neuroscience,
The Rockefeller University, New York, NY 10021, USA. 3INSERM, UMR-S839,
75005 Paris, France. 4Université Pierre et Marie Curie, 75005 Paris, France. 5Institut
du Fer à Moulin, 75005 Paris, France.
*To whom correspondence should be addressed. E-mail: gilberto.fisone@ki.se

Here, we show that lesion of the nigrostriatal pathway, a well-establishedmodel of Parkinsonism, conferred to L-DOPA the ability to activate mTOR
signaling in a subset of dopamine D1 receptor (D1R)–positive MSNs that
directly innervate the output structures of the basal ganglia. In addition,
mTORC1-mediated phosphorylation was associated with the dyskinesia
produced by chronic treatment with L-DOPA, and administration of rapamycin, an mTORC1 inhibitor, counteracted the development of this motor
complication.
RESULTSL-DOPA induces phosphorylation of S6 kinase andribosomal protein S6 in the dopamine-depleted striatum

mTOR has been implicated in the response to a variety of cell signaling
molecules, but at present, little is known about the ability of dopaminergic
drugs to affect mTOR signaling in the striatum. We examined the effect of
L-DOPA on the mTORC1 signaling cascade in mice lesioned unilaterallywith 6-hydroxydopamine (6-OHDA), a toxin used to produce a Parkinsonian model in rodents (15, 16). The choice of this model was based on
previous evidence showing that repeated administration of L-DOPA tohemi-Parkinsonian mice (which have unilateral lesions of striatal dopaminergic fibers) results in the appearance of asymmetrical abnormal involuntary
movements (AIMs), which represent a behavioral correlate for LID (17).
mTORC1 activates the p70 S6 kinase (S6K), which is responsible forthe phosphorylation of the ribosomal protein S6 (S6), a component of
the 40S ribosomal subunit (18). Administration of L-DOPA (20 mg/kg)
in combination with benserazide (12 mg/kg) to 6-OHDA–lesioned miceincreased S6K phosphorylation at Thr389, a site directly regulated by
mTOR, thereby increasing kinase activity (19) (Fig. 1). This effect wasdependent on depletion of striatal dopamine, because L-DOPA did notmodify S6K phosphorylation in sham-lesioned mice. Moreover, lesion
with 6-OHDA in the absence of L-DOPA did not affect the amount ofphospho-Thr389-S6K (Fig. 1A). Total amounts of S6K were unaffectedby treatment with 6-OHDA or L-DOPA (Fig. 1A). Similar results wereobtained when phosphorylation of S6 was assessed at Ser240 and Ser244(Ser240/244), sites specifically regulated by S6K, and at Ser235 and Ser236

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Parkinson’s disease (PD), a disorder caused by degeneration of the dopaminergic input to the basal
ganglia, is commonly treated with L-DOPA. Use of this drug, however, is severely limited by motor side
effects, or dyskinesia. We show that administration of L-DOPA in a mouse model of Parkinsonism led
to dopamine D1 receptor–mediated activation of the mammalian target of rapamycin (mTOR) complex
1 (mTORC1), which is implicated in several forms of synaptic plasticity. This response occurred selectively in the GABAergic medium spiny neurons that project directly from the striatum to the output
structures of the basal ganglia. The L-DOPA–mediated activation of mTORC1 persisted in mice thatdeveloped dyskinesia. Moreover, the mTORC1 inhibitor rapamycin prevented the development of dyskinesia without affecting the therapeutic efficacy of L-DOPA. Thus, the mTORC1 signaling cascade represents a promising target for the design of anti-Parkinsonian therapies.

RESEARCH ARTICLE(Ser235/236), sites regulated by S6K and ERK (20) (Fig. 1A). Phosphorylation at both sets of sites is thought to activate S6 (20).
L-DOPA phosphorylates S6 in D1R-containing
MSNs of the direct pathway

The effects of L-DOPA are secondary to its conversion to dopamine. In the
striatum, increased dopamine concentrations activate D1Rs and dopamine
D2 receptors (D2Rs), which are abundant in MSNs (21). We examined thepossible involvement of these receptors in the phosphorylation of S6K and
S6 induced by L-DOPA. Administration of the D1R antagonist, SCH23390(0.125 mg/kg ip), abolished the L-DOPA–induced increases in phosphorylation of both proteins. In contrast, blockade of D2Rs with raclopride

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Fig. 1. L-DOPA induces D1Rdependent phosphorylation of
S6K and S6 in the striata of 6OHDA–lesioned mice. (A andB) Quantification of phospho(P)-Thr389-S6K, P-Ser 240/244 S6, and P-Ser 235/236-S6 in
the striata of sham-lesioned
mice (Sham) or 6-OHDA–
lesioned mice treated withsaline, L-DOPA, L-DOPA plus
SCH23390 (SCH), or L-DOPAplus raclopride (Rac). Representative Western blots ofphosphorylated and total
proteins are shown (left). Error bars represent SEM [(A)n = 12 to 24 mice per treatment, (B) n = 7 to 14 mice
per treatment] of data calculated as percent of Shamtreated with saline. Statistical significance was determined by two-way (A) andone-way ANOVA (B) followedby the Bonferroni–Dunn test.
***P < 0.001 versus Shamtreated with saline. (C and D)
Immunofluorescent detection(C) and quantification (D) ofcells positive for P-Ser240/244S6 and P-Ser235/236-S6 in the
striata of Sham and 6-OHDA–
lesioned mice treated with
L-DOPA alone or in combination with SCH23390 (SCH)or raclopride (Rac). Scalebar, 40 mm. Error bars represent SEM (n = 3 mice pertreatment). Statistical significance was determined by
one-way ANOVA followedby the Bonferroni–Dunn test.
***P < 0.001 versus Shamtreated with L-DOPA.

(0.2 mg/kg ip) (22) did not change the effects produced by L-DOPA (Fig.
1B). These results were confirmed by immunofluorescence analysis of
S6 phosphorylated at Ser240/244 and Ser235/236 (Fig. 1, C and D) and indicated a prominent role of D1Rs in the ability of L-DOPA to activate mTOR
signaling in PD.
D1Rs and D2Rs are present in distinct populations of striatal MSNs,
which project, directly or indirectly, respectively, to the output structuresof the basal ganglia (21). We used transgenic mice expressing enhanced
green fluorescent protein (EGFP) under the control of the promoter for
the D1R (Drd1a-EGFP mice) or the D2R (Drd2-EGFP mice) (23) to
identify the specific subset of MSNs in which L-DOPA regulates S6
phosphorylation.

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RESEARCH ARTICLE

In Drd1a-EGFP mice, administration of L-DOPA increased S6 phosphorylation at Ser240/244 and Ser235/236 mainly in EGFP-positive MSNs
(which corresponded to D1R-containing projection neurons) (Fig. 2). Immunoreactivity for phospho-S6 was also detected in a few EGFP-negative
cells (Fig. 2, A and B, upper panels). Some of these cells were positive for
DARPP-32 [dopamine- and adenosine 3′,5′-monophosphate (cAMP)–
regulated phosphoprotein, 32 kD] and corresponded to striatopallidal
MSNs, whereas others were devoid of DARPP-32 and most likely represented aspiny cholinergic interneurons (which do not contain D1Rs). In
both types of cells, S6 phosphorylation was not affected by administration of L-DOPA (Fig. 2, C and D). In Drd2-EGFP mice, L-DOPA produced
an increase in immunoreactivity corresponding to S6 phosphorylated at
Ser240/244 and Ser235/236, which was confined to EGFP-negative cells

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Fig. 2. L-DOPA activates mTOR signaling in striatonigral MSNs. (A and B)
Immunofluorescence for P-Ser240/244S6 (A) or P-Ser235/236-S6 (B) (red),
EGFP (green), and DARPP-32 (blue)in the striata of 6-OHDA–lesioned
Drd1a-EGFP (top) or Drd2-EGFP mice(bottom) treated with L-DOPA. Scalebar, 40 mm. (C and D) Quantificationof P-Ser240/244-S6 (C) or P-Ser235/236-S6(D) positive cells among EGFP-positive(D1+ or D2+) and EGFP-negative (D1−
or D2−) neurons in the dorsal striata of
Sham and 6-OHDA–lesioned Drd1a(top) and Drd2-EGFP mice (bottom) after administration of L-DOPA. Error bars
represent SEM (n = 4 mice per treatment). Statistical significance was determined by two-way ANOVA followedby the Bonferroni–Dunn test. ***P <0.001 versus respective Sham treatedwith saline.

(Fig. 2, A and B, lower panels). This effect was seen in MSNs, as shownby the presence of DARPP-32 in neurons positive for phospho-Ser240/244-S6
and phospho-Ser235/236-S6 (Fig. 2, A and B). Together with the pharmacological studies, these results indicate that in 6-OHDA–lesioned mice,
L-DOPA activates mTOR signaling in a discrete group of striatal neurons
corresponding to the D1R-containing MSNs of the direct pathway.
L-DOPA induces D1R-mediated phosphorylationof 4E-binding protein

We next examined the effect of L-DOPA on the phosphorylation of the
eukaryotic translation initiation factor (eIF) 4E-binding protein (4E-BP),
which is another major target of mTORC1 (24). Lesion with 6-OHDA
or administration of L-DOPA did not change the amounts of total or phos-

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and is thought to promote phosphorylation of eIF4E on Ser209 by the MAPKinteracting kinases (Mnks) (25), which are phosphorylated and activatedby ERK (26). In 6-OHDA–lesioned mice, treatment with L-DOPA increased phosphorylation of Mnks at Thr197 and Thr202 (phospho-Thr197/202Mnks) by 62 ± 8% compared to that of vehicle-treated mice (table S1). Thiseffect was accompanied by a concomitant increase in the phosphorylation ofeIF4E, which was prevented by administration of the D1R antagonist
SCH23390 (Fig. 3B).

ERK is involved in L-DOPA–mediated activationof mTORC1

Activation of mTORC1 signaling is associated with LID

Fig. 3. L-DOPA regulates 4E-BP and eIF4E in 6-OHDA–lesioned mice.
(A and B) Quantification of P-Ser65-4E-BP (A) and P-Ser209-eIF4E (B)in sham-lesioned mice (Sham) or 6-OHDA–lesioned mice treated with
L-DOPA alone or in combination with SCH23390 (SCH) or raclopride(Rac). Representative Western blots of phosphorylated and total proteins
are shown (top). Error bars represent SEM (left, n = 12 to 24 mice pertreatment; right, n = 7 to 14 mice per treatment); data were calculated
as percent of Sham treated with saline. Statistical significance wasdetermined by two-way (left) and one-way ANOVA (right) followed by
the Bonferroni–Dunn test. ***P < 0.001 versus Sham treated with saline.

phorylated 4E-BP (Fig. 3A). However, when combined, these treatments
produced a large increase in phospho-Ser65-4E-BP without affecting total
protein abundance. Moreover, the increase in 4E-BP phosphorylation wasprevented by systemic administration of SCH23390, but not of raclopride(Fig. 3A).
L-DOPA increases MAPK interacting kinase–dependent
phosphorylation of eukaryotic initiation factor 4E

mTOR-catalyzed phosphorylation of 4E-BP abrogates binding to eukaryotic initiation factor 4E (eIF4E) and leads to the formation of the eIF4F′
multiprotein complex composed of eIF4E, eIF4G, and eIF4A. The assembly of eIF4F′ is the major rate-limiting step in initiation of translation

The results of the biochemical experiments suggested that, during PD, shortterm administration of L-DOPA results in the activation of multiple targetsof mTOR and in the formation of the eIF4F′ complex, the latter of which isthe critical step involved in the regulation of cap-dependent translation. Increased translational efficiency may be involved in long-term adaptive responses to L-DOPA, such as the development of dyskinesia.
To test the possibility that enhanced mTORC1 signaling was associatedwith dyskinesia, 6-OHDA–lesioned mice were injected for 9 days with
L-DOPA (20 mg/kg, one injection per day) in combination with benserazide(12 mg/kg), a procedure that induces dyskinesia (17). LID was evaluatedby scoring four types of AIMs immediately after the last injection of L-DOPA(17). At each time point, the scores for all four types of AIMs were totaled,
and the average score was 25.0 ± 0.5 (over a possible range of 0 to 96, withlower scores indicating lower degrees of dyskinesia). The following day,the mice were killed 30 min after L-DOPA administration and striatal tissue
was analyzed by Western blotting and immunohistochemistry. Simple regression analysis showed a significant correlation between the severity ofAIMs and L-DOPA–induced phosphorylation of S6K and S6 (Fig. 5, A
and B). These data were confirmed by immunofluorescence analyses performed in Drd2-EGFP mice, in which severely dyskinetic mice showed
higher phospho-Ser240/244-S6 immunoreactivity in EGFP-negative cells(corresponding to D1R-positive MSNs) compared to sham-lesioned miceor to mice showing low dyskinesia (Fig. 5E). There was also a significant
correlation between AIMs and L-DOPA–induced phosphorylation of 4E-BP
and eIF4E (Fig. 5, C and D). Thus, activation of mTORC1 in striatal MSNs
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ERK-mediated phosphorylation of the guanosine triphosphatase–activating
protein, tuberous sclerosis complex 2 (TSC2) (27, 28), is thought to stimulate the small heterotrimeric GTP (guanosine 5′-triphosphate)–binding protein Rheb, which activates mTOR (29). It has been reported that ERK
stimulates mTORC1 through activation of the p90 ribosomal S6K and phosphorylation of Raptor (30). Thus, we tested the involvement of ERK in
L-DOPA–mediated activation of mTORC1. Striatal slices from 6-OHDA–
lesioned mice were incubated with the D1R agonist SKF81297 (1 mM)
in the presence or absence of the MAPK kinase (MEK) inhibitor, U0126(25 mM). SKF81297 increased the phosphorylation of both S6 (Fig. 4A)
and 4E-BP (Fig. 4B), confirming the critical role played by D1Rs in LDOPA–induced activation of mTORC1 signaling. This effect was paralleledby increased phosphorylation of ERK2, which was blocked by U0126 (Fig.
4C). The MEK inhibitor also prevented SKF81297-induced phosphorylation of S6 and 4E-BP (Fig. 4, A and B) without affecting cAMP-mediatedphosphorylation of the glutamate receptor 1 (GluR1) subunit of the AMPA
(a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate)-type glutamate receptor at Ser845 (Fig. 4D). The notion that ERK plays a critical role in promoting
dopamine-dependent mTORC1 signaling was supported by immunostaining showing colocalization of phosphorylated ERK and S6 in striatal MSNs
after short-term administration of L-DOPA (Fig. 4E).

RESEARCH ARTICLE
severity of AIMs, raising the possibility that the mTORC1 cascade isinvolved in the development of dyskinesia. Therefore, we examined the effect of rapamycin, the prototypical mTORC1 inhibitor, on AIMs.

Administration of rapamycin reduces LID

DISCUSSION

Fig. 4. D1R-mediated activation of mTOR signaling depends on ERK phosphorylation. (A to D) Striatal slices from sham- and 6-OHDA–lesioned mice
were incubated with the D1R agonist SKF81297 (1 mM) for 5 min in theabsence or presence of the MEK inhibitor U0126 (25 mM, applied 15 min
before SKF81297). Error bars represent SEM, n = 4 to 6 pairs of slices pertreatment; data were calculated as percent of control (for example, slices
from sham-lesioned mice incubated in the absence of drugs). Phosphorylation of S6 (A), 4E-BP (B), and ERK (C) decreased in parallel. In contrast,
U0126 did not affect SKF81297-induced phosphorylation of GluR1 at
Ser845, a site regulated by cAMP-dependent protein kinase (D). Statistical significance was determined by one-way ANOVA followed by the Bonferroni–
Dunn test. ***P < 0.001 versus control. (E) Immunostaining for P-ERK and
P-Ser240/244-S6 (top) or P-Ser235/236-S6 (bottom) in the dorsal striatum of a 6OHDA–lesioned mouse treated with L-DOPA. Scale bar, 40 mm.

Here, we show that, in a mouse model of PD, administration of L-DOPA
activated mTORC1 signaling in a well-defined population of striatal projection MSNs belonging to the direct pathway. This effect was exerted
through activation of D1Rs and represents, to the best of our knowledge,
the first connection between dopaminergic transmission and mTOR signaling in the striatum. In addition, persistent activation of the mTORC1 cascade during chronic administration of L-DOPA was associated with thedevelopment of dyskinesia and administration of the mTORC1 inhibitor
rapamycin reduced dyskinesia.
In the striatum, loss of dopaminergic innervation results in the development of a sensitization to D1R agonists, which has been implicated in thedevelopment of LID (2–4). We showed that the ability of L-DOPA to activate mTORC1 signaling was due to this hypersensitivity of D1Rs. Thus,
the increases in phosphorylation of S6K, S6, and 4E-BP that were causedby administration of L-DOPA to 6-OHDA–lesioned mice were blocked by
a D1R antagonist. Moreover, the L-DOPA–induced increase in S6 phosphorylation was localized to the MSNs of the direct, striatonigral pathway,
which contain D1Rs but not D2Rs (21, 23).
It has been reported that the substantia nigra of PD patients and ofmice treated with the dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, contains higher amounts of the stress-regulated protein RTP801 (31, 32), which is an mTOR inhibitor (33, 34). We did not
examine the effect of 6-OHDA on the abundance of RTP801; however,
6-OHDA did not modify basal mTORC1 activity in striatal MSNs, as indicated by unchanged amounts of phosphorylated downstream targets.
Moreover, in striatal MSNs, 6-OHDA promoted L-DOPA–induced activation

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Mice lesioned with 6-OHDA were treated for 9 days with L-DOPA (10 mg/kg)
in combination with benserazide (7.5 mg/kg) in the absence or presence of
rapamycin (2 and 5 mg/kg). Treatment with L-DOPA (10 mg/kg) increased
phosphorylation of S6K, S6, 4E-BP, and eIF4E (Fig. 6A). Rapamycin prevented these increases (Fig. 6A) without affecting cAMP or ERK signaling (fig. S1). On day 10, both groups of mice were treated with L-DOPA
alone and were scored for AIMs. Mice that had received only L-DOPAplus vehicle during the chronic treatment displayed a robust dyskinetic
response. The average score of total AIMs in this experimental groupwas 19.8 ± 3.5. In contrast, in mice treated with L-DOPA plus rapamycin(2 or 5 mg/kg), the average score of total AIMs was reduced to 7.0 ± 2.2
and 6.0 ± 0.7, respectively (Fig. 6B). In this group of animals, administration of rapamycin in combination with L-DOPA on the day of the test
did not produce any additional decrease of dyskinesia; the average scoreof total AIMs was 7.0 ± 2.6 and 6.3 ± 1.1 in mice treated with L-DOPAplus rapamycin (2 and 5 mg/kg, respectively). These data indicated thatblockade of mTORC1 signaling reduced the development of dyskinesia
produced by chronic administration of L-DOPA.
The ability of rapamycin to counteract the action of L-DOPA might
have affected the anti-Parkinsonian properties of this drug. We examined this possibility by comparing the effect of combined administration of L-DOPA plus rapamycin with that of L-DOPA plus vehicle inthe cylinder test. Rapamycin did not diminish the efficacy of L-DOPA tocounteract forelimb akinesia produced by the 6-OHDA lesion (Fig. 6C).
Therefore, blockade of mTORC1 signaling does not appear to affect thetherapeutic (anti-akinetic) action of L-DOPA.

RESEARCH ARTICLE

of mTORC1. Therefore, increased RTP801 or its negative effect on
mTOR signaling appears to be limited to midbrain dopaminergic neurons.
Our data show that repeated administration of L-DOPA led to a normalization of sensitized mTORC1 signaling. However, such normalization
occurred only in mice with undetectable or low dyskinesia, whereas inhighly dyskinetic mice, the ability of L-DOPA to activate mTORC1 was fully
preserved (Fig. 5 and table S1). Thus, LID was associated with persistentenhancement of mTORC1 signaling, which appears to result from theinability of the MSNs of the direct pathway to normalize their response
to L-DOPA during the course of chronic administration. An identical pattern of regulation with respect to L-DOPA–induced activation of ERK
has been described in 6-OHDA–lesioned mice and rats (4, 8, 35). In
the mouse, ERK phosphorylation in response to L-DOPA occurs in
the MSNs of the direct pathway (35), that is, in the same group of neurons in which mTORC1 signaling is increased. In agreement with thisfinding, we found almost complete colocalization in striatal neurons
between L-DOPA–induced phospho-ERK and phospho-S6. Furthermore,
blockade of ERK, achieved with the MEK inhibitor U0126, prevented theability of a D1R agonist to phosphorylate S6 and 4E-BP. Together, theseobservations indicate that activation of ERK is implicated in the increase
in mTORC1 signaling produced by L-DOPA in dyskinetic mice. This regulation may be exerted by ERK through inhibition of TSC2 and sequential
activation of Rheb and mTOR (27, 28) or through phosphorylation and activation of the mTORC1 component, Raptor (30).
The ability of ERK to promote mTORC1 signaling in striatal MSNs isfurther supported by the observation that administration of L-DOPA, as
well as dyskinesia, was accompanied by increased phosphorylation ofMnks (table S1), which are direct targets of ERK (26). Mnks bind to
eIF4G and catalyze the phosphorylation of eIF4E (25). In line with thisnotion, we found increased amounts of phosphorylated eIF4E in the striata
of dyskinetic mice. Although the role of eIF4E phosphorylation remains
to be fully understood (36), this event has been proposed to occur during
the cap-dependent initiation process (37). Thus, the increase in eIF4E

phosphorylation associated with LID may reflect increased formation of
the initiation complex eIF4F′. In support of this idea, we found that inhibition of mTORC1 by rapamycin, which prevents phosphorylation of
4E-BP, thereby promoting the sequestration of eIF4E and reducing theformation of eIF4F′, leads to concomitant abolishment of L-DOPA–
induced eIF4E phosphorylation.
The observation that abnormal phosphorylation of several components of the mTORC1 signaling cascade correlates with the severity of
LID raises the question of the possible antidyskinetic effects producedby suppression of mTORC1 signaling. Previous studies have shown thatblockade of ERK, which is required for mTORC1 signaling, attenuates
dyskinetic behavior in mice and rats (7, 10). However, because of the involvement of ERK in basic physiological processes, including transcriptional regulation, ERK inhibitors in the treatment of dyskinesia are not
likely to be used clinically. In this regard, one key finding of this study
is that LID was reduced by inhibiting mTORC1, which is located downstream of ERK and is not involved in ERK-mediated transcriptional control.
Indeed, administration of rapamycin counteracts LID without interferingwith the regulation of nuclear targets of ERK, such as histone H3, which
is highly phosphorylated in LID (7, 35) (fig. S2). Rapamycin and its derivatives are currently tested in humans as anticancer therapeutics (38).
The present data suggest that these drugs and other classes of mTORC1
inhibitors are potential antidyskinetic agents for the treatment of PD.
In conclusion, this study implicates mTORC1 signaling in dopaminergictransmission in the MSNs of the direct, striatonigral pathway. Moreover, suppression of mTORC1 signaling in this neuronal population is paralleled by areduction of dyskinesia caused by L-DOPA. It is possible that abnormal activation of the mTORC1 signaling machinery during chronic administration of
L-DOPA results in changes in mRNA translation and protein production.
These changes may be involved, for instance, in dysfunctions of long-term
potentiation or depotentiation at corticostriatal synapses, which have been
associated with PD and LID (14). Further studies will be necessary to assess
the role played by mTORC1 signaling in striatal transmission and plasticity.

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Fig. 5. Dyskinesia is associated with increased striatal mTORC1 signaling in
striatonigral MSNs. (A to D) Simple regression analysis indicating a significant
correlation between AIMs and L-DOPA–induced phosphorylation of (A) S6K (r =0.9; P < 0.001), (B) S6 (r = 0.8; P < 0.001), (C) 4E-BP (r = 0.83; P < 0.001), and (D)eIF4E (r = 0.87; P < 0.001). Representative autoradiograms (top) obtained fromthe striata of a sham-lesioned mouse (Sham), a mouse displaying high dyskinesia(H Dys), and a mouse displaying low dyskinesia (L Dys). (E) Immunostaining for
P-Ser240/244-S6 (red) and EGFP (green) in the dorsal striatum of a Sham, a H
Dys, and a L Dys Drd2-EGFP mouse. Scale bar, 40 mm. Quantification (right) of
P-Ser240/244-S6 positive cells among EGFP-positive (D2+) and EGFP-negative(D2−) neurons in the dorsal striata of Sham (open bars) and H Dys Drd2-EGFP
mice (filled bars).

RESEARCH ARTICLE

MATERIALS AND METHODS

Animals
Male C57BL/6J mice (30 g) were purchased from Taconic (Tornbjerg,
Denmark). Bacterial artificial chromosome transgenic mice expressing
EGFP under the control of the promoter for D2R (Drd2-EGFP) or D1R(Drd1a-EGFP) were generated by the GENSAT (Gene Expression Nervous System Atlas) program at the Rockefeller University (23) and werecrossed on a C57BL/6 background for three generations. The animals
were housed in groups of five under standardized conditions with a
12-hour light-dark cycle and stable temperature (20°C) and humidity(40 to 50%).

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Fig. 6. Inhibition of mTOR signaling reduces
dyskinesia without affecting the anti-akinetic
properties of L-DOPA. (A) Rapamycin prevents
L-DOPA–induced phosphorylation of S6K, S6,
4E-BP, and eIF4E. Representative Western
blotting of phosphorylated and total proteinsis shown (top). Error bars in graphs represent
SEM (n = 8 to 16 mice per treatment) of data
calculated as percent of Sham treated with
L-DOPA. Statistical significance was determined by one-way ANOVA followed by the
Bonferroni–Dunn test. ***P < 0.001 versus Shamtreated with L-DOPA. °°P < 0.01 and °°°P <0.001 versus 6-OHDA–lesioned mice treatedwith L-DOPA. (B) Time profile of total axial, limb,
orolingual, and locomotive AIMs scored every
20 min over a period of 120 min after the last
drug administration (left). Sum of total AIMsscored during all observation periods (right).
Statistical significance was determined by oneway ANOVA followed by the Bonferroni–Dunn
test. ***P < 0.001 versus mice treated chronicallywith L-DOPA plus vehicle (Veh). (C) Left forelimb use determined by the cylinder test in
6-OHDA–lesioned mice before (left) and after(right) administration of L-DOPA plus vehicle
or rapamycin (2 and 5 mg/kg). Broken line
shows the percent of left forelimb use determinedin sham-lesioned mice (47 ± 3). Statistical significance was determined by repeated-measures
ANOVA. ***P < 0.001 versus untreated 6-OHDA–
lesioned mice.

Drugs
All drugs, with the exception of rapamycin (see below), were purchasedfrom Sigma-Aldrich Sweden AB (Stockholm), dissolved in saline (0.9%NaCl), and injected intraperitoneally in a volume of 10 ml per kilogram ofbody weight. L-DOPA was injected at a dose of 10 or 20 mg/kg in combination with the peripheral DOPA decarboxylase inhibitor, benserazidehydrochloride (7.5 or 12 mg/kg). SCH23390 (0.125 mg/kg) and raclopride(0.2 mg/kg) were administered 10 min before administration of L-DOPA.
The doses of SCH23390 and raclopride were chosen on the basis of previous studies in C57BL/6 mice showing their ability to effectively antagonize
D1Rs and D2Rs, respectively (22, 35, 39). Rapamycin (2 and 5 mg/kg) (LCLaboratories, Woburn, MA) was dissolved in a solution of 5% dimethyl

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RESEARCH ARTICLE
sulfoxide (DMSO), 15% PEG-400 (polyethylene glycol, molecular weight400), and 5% Tween-20 and was administered once per day in a volume of
2 ml per kilogram body weight, starting 3 days before the beginning of thetreatment with L-DOPA. For the combined treatment, rapamycin was
injected 1 hour before administration of L-DOPA. Mice that did not receive
rapamycin were injected with an equivalent amount of vehicle (5% DMSO,
15% PEG-400, and 5% Tween-20). This treatment did not produce any adverse effect or any change in the intensity of LID, even when repeated over
10 days; the average scores for total AIMs were 19.5 ± 0.5 for mice treatedwith L-DOPA plus vehicle and 18.0 ± 5.8 for mice treated with L-DOPA
without vehicle. For the experiments on slices, SKF81297 (1 mM) andU0126 (25 mM) (Sigma-Aldrich Sweden AB, Stockholm) were dissolved in saline and DMSO, respectively, and diluted 1:100 in incubationbuffer.

6-OHDA lesioning

Cylinder test
The cylinder test (41) was used to monitor the anti-akinetic effect of L-DOPA.
Mice were placed in individual glass cylinders (diameter, 12 cm) and movements were recorded for 5 min. Each 6-OHDA–lesioned mouse was tested
before L-DOPA treatment and 1 hour after the first injection of L-DOPA or
L-DOPA plus rapamycin (2 and 5 mg/kg). The number of contacts of either
forelimb with the wall was counted by an observer blind to the mouse treatment. To discriminate between accidental touches and meaningful physiological movements, only wall contacts where the animal supported its bodyweight on the paw with extended digits were counted. The use of the impaired (left) forelimb was calculated as a percentage of the total number of
supporting wall contacts. Sham-lesioned mice, which showed 47 ± 3%
(broken line in Fig. 6C) left forelimb use, were used as control.

Abnormal involuntary movements
6-OHDA–lesioned mice were treated for 9 days with one injection per dayof L-DOPA (20 or 10 mg/kg) plus benserazide (12 or 7.5 mg/kg). The effectof rapamycin was examined by treating 6-OHDA–lesioned mice for 9 days
with L-DOPA–benserazide plus rapamycin (2 and 5 mg/kg) and, on day 10,
by administering L-DOPA in combination with vehicle or rapamycin. AIMs
were assessed with a pharmacologically validated mouse model of LID after
the last injection of L-DOPA (day 10) by an observer blind to the mouse
treatment (17). Briefly, 20 min after L-DOPA administration, mice wereplaced in separate cages and individual dyskinetic behaviors were assessed
for a 1-min monitoring period every 20 min, over a period of 120 min.
Purposeless movements, distinguished from natural stereotyped behaviors(such as grooming, sniffing, rearing, and gnawing), were classified into four

In vitro experiments
Sham- or 6-OHDA–lesioned mice were killed by decapitation and thebrains were rapidly removed. Coronal slices (250 mm) were prepared withthe use of a vibratome (Leica, Nussloch, Germany). Dorsal striata weredissected out from each slice under a microscope. Two slices were placed
in individual 5-ml polypropylene tubes containing 2 ml of Krebs-Ringer
bicarbonate buffer [KRB; 118 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2,
1.5 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.7 mM glucose equilibrated with 95% O2–5% CO2 (v/v) (pH 7]. The samples were
equilibrated at 30°C for two 30-min intervals, each followed by replacement of the medium with 2 ml of fresh KRB. Slices were first incubatedfor 15 min in the presence of U0126 or vehicle and then for 5 min in thepresence of SKF81297 (1 mM). After incubation, the solutions were rapidly removed, the slices were sonicated in 1% SDS, and the samples wereanalyzed by Western blotting as described below.

In vivo experiments
Mice were treated with L-DOPA and benserazide alone or in combination with SCH23390, raclopride, or rapamycin and killed by decapitation 30 min later. The heads of the animals were cooled in liquid
nitrogen for 6 s and the brains were removed. The striata were dissected
out on an ice-cold surface, sonicated in 750 ml of 1% SDS, and boiled
for 10 min. The effectiveness of this extraction procedure in preventing
protein phosphorylation and dephosphorylation, hence ensuring thatthe state of phosphorylation of phosphoproteins measured ex vivo reflects the in vivo situation, has previously been shown (39).

Western blotting

Aliquots (5 ml) of the homogenate were used for protein determination with a BCA (bicinchoninic acid) assay kit (Pierce Europe, Oud
Beijerland, the Netherlands). Equal amounts of protein (30 mg) for
each sample were loaded onto 10% polyacrylamide gels. Proteins wereseparated by SDS–polyacrylamide gel electrophoresis and transferredovernight to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) (42). The membranes were immunoblotted with antibodies against phospho-Thr389-S6K (1:1000), S6K
dually phosphorylated at Thr421/424 (1:1000), S6 dually phosphorylated
at Ser240/244-S6 (1:1000), or Ser235/236-S6 (1:1000), phospho-Ser65-4E-BP(1:1000), Mnks dually phosphorylated at phospho-Thr197/202, phosphoSer209-eIF4E (1:1000), ERK1-2 dually phosphorylated at Thr202 andTyr204 (1:2000) (Cell Signaling Technology, Beverly, MA), and phosphoSer845-GluR1 (PhosphoSolutions, Aurora, CO) (1:750). Antibodies against
S6, S6K, 4E-BP, Mnks, eIF4E, ERK, and GluR1 (1:1000, Cell Signaling
Technology) that are not phosphorylation state–specific were used toestimate the total amount of proteins. An antibody against tyrosine hydroxylase (1:1000, Chemicon International, Temecula, CA) was used to assess
the severity of 6-OHDA lesions. Detection was based on fluorescentsecondary antibody binding and quantified with a Li-Cor Odyssey infrared
fluorescent detection system (Li-Cor, Lincoln, NE). The amount of each
phosphoprotein was normalized for the amount of the corresponding total
protein detected in the sample.

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Mice were anesthetized with a mixture of fentanyl citrate (0.315 mg/ml),
fluanisone (10 mg/ml) (VetaPharma, Leeds, UK), midazolam (5 mg/ml)(Hameln Pharmaceuticals, Gloucester, UK), and water (1:1:2 in a volumeof 10 ml/kg) and mounted in a stereotaxic frame (David Kopf Instruments,
Tujunga, CA) equipped with a mouse adaptor. 6-OHDA-HCl (Sigma-Aldrich
Sweden AB) was dissolved in 0.02% ascorbic acid in saline at a concentration of 3 mg of free base 6-OHDA per microliter. Each mouse received
two unilateral injections of 6-OHDA (2 ml per injection) into the right striatum as previously described (7), according to the following coordinates(mm) (40): anteroposterior (AP), +1; mediolateral (ML), −2.1; dorsoventral (DV), −3.2; and AP +0.3; ML, −2.3; DV, −3.2. Animals were allowed
to recover for 3 weeks before behavioral evaluation and drug treatment
were carried out. This procedure leads to ≥80% decrease in striatal tyrosine hydroxylase immunoreactivity. The efficacy of the lesion is illustrated
by the loss of tyrosine hydroxylase immunoreactivity in both striatum andsubstantia nigra pars compacta (fig. S3).

subtypes: locomotive AIMs (tight contralateral turns), axial AIMs (contralateral dystonic posture of the neck and upper body toward the side contralateral to the lesion), limb AIMs (jerky and fluttering movements of the limb
contralateral to the side of the lesion), and orolingual AIMs (vacuous jaw
movements and tongue protrusions). Each subtype was scored on a severity
scale from 0 to 4: 0, absent; 1, occasional; 2, frequent; 3, continuous; and 4,
continuous and not interruptible by outer stimuli.

RESEARCH ARTICLEREFERENCES AND NOTES

Mice were rapidly anaesthetized with pentobarbital (100 mg/kg ip, SanofiAventis, France) and perfused transcardially with 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5). Brains were postfixed
overnight in the same solution and stored at 4°C. Thirty-micrometer-thicksections were cut with a vibratome (Leica, Nussloch, Germany) and storedat −20°C in 0.1 M sodium phosphate buffer containing 30% (v/v) ethylene
glycol and 30% (v/v) glycerol until they were processed for immunofluorescence. Free-floating sections were rinsed in tris-buffered saline[TBS; 0.25 M tris and 0.5 M NaCl (pH 7.5)], incubated for 5 min in
TBS containing 3% H2O2 and 10% methanol (v/v), and then rinsed
three times for 10 min each in TBS. After 20 min of incubation in
0.2% Triton X-100 in TBS, sections were again rinsed three times inTBS. Finally, they were incubated overnight at 4°C with the different
primary antibodies. For detection of phosphorylated proteins, 0.1 mM
NaF was included in all buffers and incubation solutions. Phosphorylation was analyzed with rabbit polyclonal antibodies that recognize histone H3 only when acetylated on Lys14 and phosphorylated at Ser10,
and S6 only when dually phosphorylated at Ser240/244 or Ser235/236(1:500, Cell Signaling Technology). In the double-labeling experiments, amonoclonal antibody against EGFP (1:500, Invitrogen) was used to detect
EGFP fluorescence in Drd2- and Drd1a-EGFP mice. A mouse monoclonal
antibody against diphospho-Thr202/Tyr204-ERK1/2 (1:400, Sigma-Aldrich
Sweden AB) was used for some double-labeling experiments. In the
triple-labeling experiments, a monoclonal antibody against DARPP-32(1:1000) was used (43). After incubation with primary antibodies, sections
were rinsed three times for 10 min in TBS and incubated for 45 min withgoat secondary antibody against rabbit or Cy3-coupled secondary antibody
against mouse (1:400, Jackson Laboratory, Bar Harbor, ME), or goat Alexa
Fluor 488– or Alexa Fluor 633–conjugated secondary antibodies againstmouse (1:400, Invitrogen AB, Stockholm, Sweden). In the triple-labelingexperiment, after incubation with the secondary antibodies, the slices werethen rinsed three times for 10 min in TBS and incubated overnight with apolyclonal antibody against EGFP directly coupled to Alexa Fluor 488(1:500, Invitrogen AB). A monoclonal antibody against tyrosine hydroxylase (1:1000, Chemicon International) was also used to assess the severityof 6-OHDA lesions. Sections were rinsed for 10 min twice in TBS andtwice in TB (0.25 M tris) before mounting in 1,4-diazabicyclo-[2. 2. 2]octane (DABCO, Sigma-Aldrich Sweden AB). Single-, double-, andtriple-labeled images from the dorsolateral striatum were obtained by sequential laser scanning confocal microscopy (Zeiss LSM). Neuronal quantification was performed in 562 × 562-mm or 375 × 375-mm images. In all
the experiments, phospho-S6 positive cells were only counted when colocalized with DARPP-32- or EGFP-positive neurons for assessment of
D2R- or D1R-positive cells.

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Statistics
Data were analyzed with one-way or two-way analysis of variance(ANOVA), where treatment and time were the independent variables,
followed by Bonferroni–Dunn post hoc test for specific comparisons.
Data expressed as percentages were subjected to arcsine square roottransformation (pi = arcsine √p) before ANOVA.
SUPPLEMENTARY MATERIALS
www.sciencesignaling.org/cgi/content/full/2/80/ra36/DC1
Fig. S1. Rapamycin does not affect L-DOPA–induced phosphorylation of GluR1 and
ERK2.
Fig. S2. Rapamycin does not affect L-DOPA–induced phosphorylation of Lys14-acetylated
histone H3.
Fig. S3. Evaluation of the degree of DA denervation after striatal 6-OHDA injection.
Table S1. Effect of 6-OHDA and L-DOPA on selected proteins involved in mTOR signaling.

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Tissue preparation and immunofluorescence

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Submitted 6 March 2009
Accepted 1 July 2009
Final Publication 21 July 200910.1126/scisignal.2000308
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