E. Santini, Department of Neuroscience, Karolinska Institutet, Retzius väg 8, 171 77 Stockholm, Sweden Fax: +46 8 320899 Tel: +46 8 524 83755 E-mail: firstname.lastname@example.org
l-3,4-Dihydroxyphenylalanine (l-dopa) remains the most effective pharmacological treatment for relief of the severe motor impairments of Parkinson’s disease. It is very effective in controlling parkinsonian symptoms in the initial phase of the disease, but its action wanes with time. Such ‘wearing-off’ imposes an escalation in the dosage of the drug, which ultimately fails to provide stable control of motor symptoms and results in the appearance of abnormal involuntary movements or dyskinesia. ‘Peak-dose’l-dopa-induced dyskinesia (LID) currently represents one of the major challenges in the treatment of Parkinson’s disease. Accumulating evidence suggests that LID derives from overstimulation of dopamine receptors located on the GABAergic medium spiny neurons (MSNs) of the dorsal striatum. These neurons form two distinct projection pathways, which exert opposite effects on motor activity: the direct, striatonigral pathway promotes locomotion, whereas the indirect, striatopallidal pathway depresses locomotion. In order to understand the mechanisms underlying LID, it is important to identify molecular adaptations produced by chronic administration of l-dopa, at the level of one or the other of these two neuronal populations. This review summarizes the results of recent studies indicating that LID is associated with abnormal dopamine D1 receptor signaling affecting the MSNs of the direct pathway. The role of this pathological adaptation and of the consequent changes in signaling in the development and expression of LID are discussed.
cAMP- and dopamine-regulated phosphoprotein of 32 kDa
extracellular signal-regulated kinase
mitogen-activated protein kinase
mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
mitogen- and stress-activated kinase-1
medium spiny neuron
cAMP-dependent protein kinase
The abnormal involuntary movements, or dyskinesia, generated by prolonged administration of l-3,4-dihydroxyphenylalanine (l-dopa) represent one of the major challenges facing current therapy for Parkinson’s disease (PD). These debilitating motor disturbances are all the more problematic because l-dopa, in spite of its introduction several decades ago, still represents the therapy of choice for the treatment of PD. Moreover, dyskinesia is clearly manifested in patients even after transplantation with fetal mesencephalic tissue [1–3]. The discovery of pharmacological interventions able to counteract l-dopa-induced dyskinesia (LID) would therefore represent an important breakthrough in the therapy for PD. The design of novel agents for the prevention and treatment of LID requires the elucidation of the adaptive changes produced in the parkinsonian brain by repeated administration of l-dopa and the assessment of their role in the development and expression of this condition.
The main targets of l-dopa are the GABAergic medium spiny neurons (MSNs) of the dorsal striatum, which have lost most of their dopaminergic innervation following degeneration of substantia nigra neurons. MSNs represent the vast majority of neurons that are present in the striatal formation and can be distinguished on the basis of the organization of their connectivity to the output stations of the basal ganglia (i.e. globus pallidus pars interna and substantia nigra pars reticulata). One set of MSNs is directly connected to these nuclei, whereas the other one projects to these structures indirectly, via the globus pallidus pars externa and subthalamic nucleus. The MSNs of the direct and indirect pathway differ also with respect to their ability to express different sets of neuropeptides and receptors. Thus, the striatonigral neurons of the direct pathway produce the neuropeptides dynorphin and substance P, whereas the neurons of the indirect pathway express enkephalin . In addition, the MSNs of the direct pathway express preferentially the D1 subtype of the dopamine (DA) receptor, whereas the MSNs of the indirect pathway are enriched in D2 DA receptors and A2A adenosine receptors [4–6].
Several lines of evidence indicate that prolonged administration of l-dopa alters the functioning of MSNs. This review summarizes the results of recent studies on the association between LID and changes in signaling affecting the MSNs. The possible role played by these changes in LID is also discussed.
Changes in cAMP signaling associated with LID
It is now well established that the depletion of DA caused by degeneration of midbrain dopaminergic neurons results in an enhancement of the responsiveness of MSNs to activation of dopaminergic receptors. For instance, the ability of DA to stimulate adenylyl cyclase via activation of DA D1 receptors (D1Rs) is enhanced in parkinsonian patients  and in experimental animals following DA depletion [8–11]. This hyper-responsiveness, which most likely represents a compensation for the lack of striatal DA, cannot be simply attributed to increased levels of receptors. In fact, studies in 6-hydroxydopamine (6-OHDA)-lesioned rodents [12–15], 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys  and parkinsonian patients [17–20] did not find significant changes in the affinity or number of D1Rs.
In the striatum, D1Rs are coupled to a Gαolf protein that activates adenylyl cyclase, thereby stimulating cAMP-dependent signaling [21,22]. Studies performed in 6-OHDA-lesioned rats and post-mortem samples from parkinsonian patients demonstrated that striatal DA depletion results in increased levels of Gαolf [8,23]. It is therefore likely that the sensitized response to DA produced by degeneration of dopaminergic innervation to the striatum is caused by enhanced activation of Gαolf, following binding of DA to D1Rs. Interestingly, studies performed in MPTP-lesioned monkeys have found an association between LID and increased coupling between D1Rs and Gαolf . Taken together, these various observations suggest that dyskinesia may be produced by persistent hyperactivation of cAMP signaling in the MSNs of the direct striatonigral pathway (Fig. 1).
Abnormal regulation of cAMP- and DA-regulated phosphoprotein of 32 kDa (DARPP-32) is implicated in LID
Increased D1R transmission results in enhanced activation of cAMP-dependent protein kinase (PKA), which phosphorylates various downstream proteins involved in short- and long-term changes in the activity of MSNs. One important mediator of cAMP signaling is the DA- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). PKA-catalyzed phosphorylation at Thr34 converts DARPP-32 into an inhibitor of protein phosphatase-1 (PP-1) , which is abundantly expressed in MSNs . This, in turn, prevents dephosphorylation of downstream target proteins regulated by PKA, thereby intensifying cAMP-mediated responses. DARPP-32 is known to play a critical role in D1R-mediated transmission .
In 6-OHDA-lesioned rodents, DARPP-32 phosphorylation at Thr34 is dramatically enhanced [27,28]. This effect is partly normalized by repeated administration of l-dopa. However, the ability of this drug to increase the levels of phosphoThr34-DARPP-32 persists in animals affected by dyskinesia . This observation raises the possibility that DARPP-32 plays a permissive role in LID by exacerbating the sensitized response of D1Rs to l-dopa. A mouse model of LID , combined with the use of transgenic mice, has recently been employed to test this possibility. 6-OHDA-lesioned wild-type and DARPP-32 knockout mice were treated chronically with l-dopa, and dyskinesia was evaluated by determining the severity of specific abnormal involuntary movements. Using this approach, it was found that genetic inactivation of DARPP-32 results in a significant attenuation of LID .
What are the consequences of the sensitized cAMP/PKA/DARPP-32 signaling that occurs during dyskinesia? One immediate effect is upregulation of the phosphorylation of downstream effector proteins potentially involved in the development of LID. For example, a positive correlation has been established between LID and increased phosphorylation of the GluR1 subunit of the glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor at the PKA site, Ser845 . Phosphorylation of GluR1 at Ser845 promotes glutamatergic transmission by increasing the probability of AMPA channels being open  and surface expression . Augmented AMPA receptor transmission appears to be implicated in dyskinesia. For instance, in MPTP-lesioned monkeys, LID is increased by administration of an AMPA receptor agonist and decreased by an AMPA receptor antagonist . DARPP-32 participates in the increase in GluR1 phosphorylation at Ser845 produced by l-dopa, most likely via inhibition of PP-1-mediated dephosphorylation . It is therefore possible that DARPP-32 promotes dyskinesia, by facilitating the phosphorylation/activation of AMPA receptors (Fig. 1).
Abnormal extracellular signal-regulated kinase (ERK) signaling is implicated in LID
The hyperphosphorylation of DARPP-32 produced by l-dopa may also have important consequences for the changes in gene expression associated with dyskinesia (see below). Phosphorylation of DARPP-32 at Thr34 is implicated in the increase in expression of immediate early genes, such as c-fos, zif268 and arc, produced by activation of D1R [33,34]. PhosphoThr34-DARPP-32 is also involved in the accumulation of ΔFosB produced by chronic treatment with cocaine, a psychostimulant able to increase the release of DA . The positive effect of cocaine on DA transmission, even if exerted through a different mechanism (i.e. interference with the monoamine reuptake system), is similar to that produced by l-dopa, which also acts by elevating the concentration of neurotransmitter.
One possible mechanism by which DARPP-32 may participate in the changes in gene expression associated with dyskinesia is via regulation of the state of phosphorylation of the two mitogen-activated protein kinases (MAPKs), ERK1 and ERK2. Activation of ERK occurs via phosphorylation catalyzed by the MAPK/ERK kinase (MEK) and leads, via different mechanisms, to the activation of the transcription factors cAMP-responsive element-binding protein (CREB) and Elk-1 [35,36].
It has been shown that depletion of striatal DA produced by injection of 6-OHDA in rats , or by genetic inactivation of tyrosine hydroxylase (the rate-limiting enzyme in the synthesis of catecholamines) in mice , is accompanied by a remarkable potentiation of the ability of D1R to activate ERK. Moreover, it has been found that this potentiation subsides following repeated administration of l-dopa . Experiments in 6-OHDA-lesioned mice have confirmed and extended these observations. It was shown that administration of l-dopa, which normally does not affect ERK phosphorylation, strongly activates ERK following DA depletion . Whereas this effect tends to decline after repeated administration of l-dopa, it is largely preserved in animals with severe dyskinesia . It therefore appears that dyskinesia correlates with the inability of MSNs to normalize their response to l-dopa.
The persistent increase in ERK phosphorylation produced by l-dopa in 6-OHDA-lesioned mice, and correlated with the appearance of dyskinesia, depends on DARPP-32. In fact, the ability of l-dopa to activate ERK is markedly attenuated in DARPP-32-null mice . Thus, the enhancement of cAMP/PKA/DARPP-32 signaling associated with dyskinesia appears to be involved in the concomitant increase in ERK signaling. The mechanism by which DARPP-32 regulates the state of phosphorylation of ERK is still unknown. However, it has been proposed that the activation of ERK produced by cocaine via stimulation of D1R involves DARPP-32-mediated regulation of MEK and of the striatal-enriched tyrosine phosphatase . A similar mechanism may also be involved in the l-dopa -induced increase in ERK phosphorylation observed in dyskinetic mice.
The occurrence of sensitized ERK signaling in association with dyskinesia is confirmed by the observation that the striata of dyskinetic mice also contain higher levels of phosphorylated mitogen- and stress-activated kinase-1 (MSK-1), which is directly activated by ERK. One important target of MSK-1 is CREB, a transcription factor involved in the regulation of numerous genes, including those coding for Fos and dynorphin . Previous work has shown that 6-OHDA lesion of the substantia nigra promotes the ability of DA to phosphorylate and activate CREB . It is likely that this effect of DA depends, at least in part, on concomitant activation of MSK-1, which occurs under identical experimental conditions  (Fig. 1).
LID is also accompanied by increased phosphorylation of histone H3 , which is another important target of MSK-1. Phosphorylation of acetylated histone H3 is a critical step for chromatin rearrangements and transcriptional regulation. It is therefore evident that the abnormal activation of ERK signaling observed in dyskinesia is implicated in changes in gene expression associated with this condition (see above).
ERK hyperphosphorylation not only correlates with dyskinesia, but is also implicated in the development of this condition. In 6-OHDA-lesioned mice, administration of SL327, an inhibitor of MEK that prevents the phosphorylation/activation of ERK, strongly reduces the ability of chronic l-dopa to generate LID. Treatment with SL327 also prevents the activation of MSK-1 and the consequent phosphorylation of histone H3 occurring downstream of ERK activation. Taken together, these various findings suggest that blockade of ERK signaling is likely to prevent many of the changes in gene expression associated with and potentially involved in the manifestation of dyskinesia (Fig. 1). Chronic inhibition of ERK, however, is likely to affect basic physiological processes and is not very suitable as a clinical strategy. It will therefore be important to identify targets located downstream of ERK activation, in order to increase the specificity of therapeutic interventions and avoid major side-effects.
LID is associated with changes in gene expression in striatonigral neurons
What are the long-term effects produced by persistent activation of cAMP and ERK signaling, which ultimately results in the development of dyskinesia? During recent years, studies performed in different animal models have shown that LID is associated with changes in the expression of numerous genes that are potentially involved in the development or expression of dyskinesia. It also appears that, in most cases, these changes affect specifically the striatonigral neurons of the direct pathway.
Experiments in hemiparkinsonian rats have demonstrated the existence of a correlation between dyskinesia and increased expression of transcription factors belonging to the fos family of immediate early genes. The levels of FosB, along with those of its alternatively spliced isoforms, collectively named ΔFosB, are enhanced in the dorsolateral striata of rats that develop dyskinesia . The enhancement of fos gene expression occurs in dynorphin-expressing MSNs , which belong to the direct striatonigral pathway , and appears to be causally related to the development of dyskinesia. Thus, striatal infusion of a fosB antisense oligonucleotide reduces the severity of dyskinetic movements in a rat model of LID .
Another immediate early gene whose expression is affected during dyskinesia is zif268 (or NGFI-A/krox24/egr1). The ability of l-dopa to increase the amount of zif268 mRNA in MSNs is dramatically enhanced in both striatopallidal and striatonigral neurons, following DA depletion. Interestingly, repeated administration of l-dopa to 6-OHDA-lesioned rats normalizes the levels of zif268 mRNA in the neurons of the striatopallidal pathway, but not in those of the striatonigral pathway . Prolonged overexpression of zif268 during l-dopa administration may result in permanent changes in the functional features of striatonigral MSNs, which may be implicated in LID. More studies are needed to understand the nature of these changes.
The increases in fosB and zif268 expression observed in nigrostriatal neurons and associated with LID are paralleled by enhanced levels of activity-regulated cytoskeletal-associated protein, an important regulator of long-term potentiation and neuronal excitability .
It has been reported that LID is accompanied by increased levels of nur77 (or NGFI-B) mRNA in dynorphin-expressing MSNs . This finding is in contrast with experiments showing that, in 6-OHDA-lesioned rats, behavioral sensitization to l-dopa (a response generally regarded as predictive of dyskinesia) is accompanied by reduced expression of nur77 mRNA in nigostriatal neurons . Recent studies performed in Nur77 knockout mice, however, have shown that the behavioral sensitization to l-dopa developed by these animals is indistinguishable from that of wild-type mice . Therefore, it appears that modifications in the expression of Nur77 are not implicated in LID.
The changes in immediate early gene expression found in association with LID raise the question of their role in the regulation of downstream target proteins that are ultimately involved in dyskinesia. In this regard, it has been found that the increase in FosB-like immunoreactivity associated with dyskinesia is involved in the upregulation of mRNA coding for prodynorphin . A precise assessment of the role played by increased opioid transmission in dyskinesia, however, is complicated by contrasting data, some studies reporting a beneficial effect exerted on dyskinesia by opioid receptor antagonism, and others indicating that blockade of opioid receptors exacerbates LID . Further work will be necessary to identify other proteins regulated by FosB, which may play a role in the development of LID.
Studies in rodents  and nonhuman primates  have demonstrated that dyskinesia is accompanied by increased expression of the DA D3 receptor (D3R) subtype. Experiments performed in 6-OHDA-lesioned rats have shown that this increase occurs mainly in dynorphin-expressing MSNs and depends on activation of D1Rs . Moreover, treatment with a partial agonist selective for D3R alleviates LID in MPTP-intoxicated monkeys . The increase in D3R expression is triggered by enhanced levels of brain-derived neurotrophic factor . It remains to be assessed whether the changes in D3R levels observed in dyskinesia are due to the ability of brain-derived neurotrophic factor to affect the expression of specific immediate early genes.
The studies presented in this review exemplify the importance of elucidating the molecular mechanisms underlying LID, in order to provide better treatment for this disorder. The identification of specific components of the signal transduction machinery involved in the development and expression of dyskinesia would offer the possibility of designing novel therapeutic strategies aimed at prolonging the use of l-dopa in the treatment of PD.
The availability of nonhuman primate and rat models of LID has been paramount in the identification of alterations in gene expression associated with dyskinesia. However, the importance of these changes for the development and/or expression of LID as well as for the identification of the transduction pathways involved in these adaptive responses has been more difficult to investigate. In this regard, the recent development of a mouse model of LID, combined with the use of transgenic mice, represents an important tool for the investigation of signaling abnormalities implicated in dyskinesia.
The picture emerging from these studies supports the idea that LID is generated as a consequence of persistent hyper-responsiveness of striatal MSNs to l-dopa. Such sensitization is a consequence of DA depletion and is attributable to overexpression of specific components (e.g. Gαolf) of the D1R signal transduction machinery. If protracted, this condition may ultimately lead to long-term changes in gene expression, which will permanently affect the function of striatonigral MSNs.
This review focuses on signal transduction in the striatum. Several studies, however, have shown that LID is associated with molecular changes (particularly changes in gene expression) affecting other components of the basal ganglia, such as the globus pallidus, substantia nigra pars reticulata, and subthalamic nucleus. These changes are likely to reflect abnormal transmission at the striatal level, as these structures are heavily innervated by the MSNs, of the direct and indirect pathway. Future studies will be necessary to further characterize these molecular abnormalities and to understand their relationship to dysfunctions of signal transduction occurring in striatal MSNs.
GF is supported by Swedish Research Council grants 13482 and 14862, the Björn Oscarssons donation, Parkinson Fonden and Hjärnfonden. EV is supported by the Institut national de la santé et de la recherche médicale (INSERM).