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Parkinson’s disease is caused primarily by degeneration of brain dopaminergic neurons in the substantia nigra and the consequent deficit of dopamine in the striatum. Dopamine replacement therapy with the dopamine precursor L-dopa is the mainstay of current treatment. After several years, however, the patients develop L-dopa–induced dyskinesia, or abnormal involuntary movements, thought to be due to excessive signaling via dopamine receptors. G protein–coupled receptor kinases (GRKs) control desensitization of dopamine receptors. We found that dyskinesia is attenuated by lentivirus-mediated overexpression of GRK6 in the striatum in rodent and primate models of Parkinson’s disease. Conversely, reduction of GRK6 concentration by microRNA delivered with lentiviral vector exacerbated dyskinesia in parkinsonian rats. GRK6 suppressed dyskinesia in monkeys without compromising the anti-parkinsonian effects of L-dopa and even prolonged the antiparkinsonian effect of a lower dose of L-dopa. Our finding that increased availability of GRK6 ameliorates dyskinesia and increases duration of the antiparkinsonian action of L-dopa suggests a promising approach for controlling both dyskinesia and motor fluctuations in Parkinson’s disease.
Parkinson’s disease (PD) is a neurodegenerative disorder primarily caused by the degeneration of nigral dopaminergic neurons that provide dopamine to the striatum. The best symptomatic therapeutic agent is the dopamine precursor L-dopa. Long-term treatment leads to L-dopa–induced dyskinesia (LID) or involuntary aimless movements (1). Loss of dopamine in PD causes complex alterations in cellular signaling: Numerous pathways in the dopamine-depleted striatum show exaggerated responses to stimulation by dopaminergic drugs. The signaling is further distorted by chronic L-dopa treatment (2–4). Super-sensitivity of the D1 (5) and D2 (6, 7) dopamine receptors is thought to be among the molecular mechanisms underlying LID. Because both the dyskinetic and antiparkinsonian actions of L-dopa are mediated by signaling through dopamine receptors, the molecular mechanisms of these effects are likely intertwined. Previous attempts to dissociate the detrimental and beneficial effects of the drug with pharmacological or molecular tools that inhibit the former while preserving the latter have been only moderately successful (7, 8). Underlying molecular mechanisms must be identified and selectively targeted to effectively manage LID.
A conserved desensitization mechanism terminates signaling by G protein–coupled receptors (GPCRs). The first rate-limiting step in this process is activation-dependent receptor phosphorylation by GPCR kinases (GRKs). Binding of arrestins (regulatory proteins) to phosphorylated receptors blocks further G protein activation and initiates receptor internalization (9, 10). GPCR signaling is strictly controlled by this process, and the rate and extent of desensitization depend on the availability of GRKs (11–14). In rats with dopamine depletion in one hemisphere, the concentration of GRKs in the dopamine-depleted motor striatum is reduced, and L-dopa fails to alter the GRK expression (15). In parkinsonian monkeys, loss of dopamine leads to the up-regulation of several GRKs (2), which may temper dopaminergic signaling on initial L-dopa administration and ensure a therapeutic response to the drug. However, chronic L-dopa treatment suppresses the GRK expression (2). Previously, we demonstrated elevated membrane expression and reduced internalization of D1 receptors in the striatum of dyskinetic monkeys (16), suggesting that LID is associated with deficits in D1 receptor desensitization and trafficking. Reduced GRK availability likely contributes to the exaggerated dopaminergic signaling in the dyskinetic brain. Collectively, these results suggest that increasing the capacity of the desensitization machinery in the parkinsonian striatum may ameliorate LID.
Five GRK isoforms are expressed in the brain (2, 17–19). GRK6 has been implicated in the regulation of dopaminergic signaling in the striatum (13). Here, we demonstrate that overexpression of GRK6 in the striatum facilitates receptor desensitization and ameliorates LID while preserving or even enhancing the antiparkinsonian effects of L-dopa.
We constructed lentiviruses encoding green fluorescent protein (GFP) (control) or rat GRK6 [GRK6A splice variant, which is the major messenger RNA (mRNA) variant in the brain] (20) tagged with GFP for easy detection (fig. S1A). Using an in vitro rhodopsin phosphorylation assay, we ascertained that GFP-tagged rat and human GRK6 is functional (fig. S1B). By subcellular fractionation, we found that the exogenous GRK6-GFP was enriched in synaptic membranes, similarly to the endogenous GRK6 (fig. S1C).
First, we tested whether overexpression of GRK6 in the dopamine-depleted striatum would suppress contralateral rotations in response to dopamine agonists in 6-hydroxydopamine (6-OHDA)–hemilesioned rats. We measured the rotation frequency induced by apomorphine in rats injected with either GFP (control) or GRK6 virus into the striatum on the lesioned side. Both the control and GRK6 groups had similarly extensive lesions (Fig. 1A). Fewer than 3% of the tyrosine hydroxylase (TH)–positive terminals remained in the lesioned caudate putamen (CPu) (Fig. 1B). The GRK6-GFP lentivirus induced GRK6 expression throughout the CPu (Fig. 1C). GRK6 expression was detected in medium spiny neurons as determined by double immunohistochemistry for GFP and the marker of these neurons, DARPP-32 (21) (Fig. 1D). Western blots (Fig. 1D) demonstrated the presence of the transgenic GRK6-GFP in the infected striatum. We used rabbit polyclonal antibody to label both endogenous and overexpressed GRK6A (Fig. 1F). The antibody specificity was demonstrated in a separate experiment (fig. S2). Quantification of the transgenic GRK6A demonstrated that the gene transfer increased the total amount of GRK6A in the lesioned striatum by a factor of ~2 (Fig. 1G).
Drug-naïve rats overexpressing GRK6 displayed significantly less frequent apomorphine-induced rotations than control animals (Fig. 2A). Rodents with unilateral 6-OHDA lesion respond to repeated administration of L-dopa with a progressive increase in the rotation frequency (3, 15, 22). Overexpression of GRK6 significantly reduced the rotation frequency after repeated L-dopa treatment as compared to the control group [P = 0.0014 by two-way repeated-measures analysis of variance (ANOVA)] (Fig. 2B), although it did not prevent the increase in the rotation frequency from session to session.
Because antidyskinetic therapy is required for already dyskinetic PD patients, we tested whether overexpression of GRK6 would influence preexisting sensitization or dyskinesia. We treated 6-OHDA–lesioned rats with L-dopa for 5 days before injection of GRK6 or control viruses (Fig. 2C). The preinjection rotation frequencies were the same in the control and GRK6 groups, with both groups showing marked behavioral sensitization. However, after the injection, rotation frequencies in the GRK6 group were reduced (P = 0.0247) (Fig. 1C).
We also studied the effect of GRK6 overexpression on a rodent analog of dyskinesia, abnormal involuntary movements (AIMs) (23, 24). Repeated administration of L-dopa to 6-OHDA–lesioned rats leads to a progressive increase in the AIM score (Fig. 2D). The score was markedly reduced in animals expressing GRK6 (Fig. 2D). Thus, the increased availability of GRK6 alleviates already established dyskinesia.
To evaluate the role of endogenous GRK6, we tested whether knockdown of GRK6 with lentivirus-delivered microRNA (miRNA) would influence the behavioral effects of L-dopa. We have constructed a lentivirus carrying two miRNA sequences directed against different regions of GRK6 and co-cistronic GFP to label infected cells (fig. S3A). A lentivirus encoding nonsense miRNA and GFP served as control.
6-OHDA–lesioned rats were treated with L-dopa for 5 days before virus injection. The preinjection rotation frequencies were the same in the control and knockdown groups, but after the virus injection, the rotation frequencies in the GRK6 knockdown group were significantly increased (P = 0.0057 by two-way repeated-measures ANOVA) (Fig. 2E). The effect was most evident on the last testing days because there was a significant increase in the sensitization slope in the knockdown group (P = 0.001). Similarly, GRK6 knockdown increased the frequency of AIMs and exacerbated the progressive increase in AIM score (Fig. 2F).
Postmortem examination of the infected striatum revealed miRNA expression in DARPP-32–positive medium spiny striatal neurons (fig. S4A). The expression of GRK6A was significantly decreased in the lesioned as compared to the intact striatum (fig. S4, B and C), in agreement with our previous report (15). We found that the decrease was largely due to the GRK6A variant, whereas the change in GRK6B was much smaller, albeit significant (fig. S4, D and E). The GRK6 concentration was significantly decreased by the GRK6 miRNA as compared to the control lentivirus (fig. S4, B to E). Because the miRNA sequences were directed against the regions common to GRK6A and GRK8B isoforms (fig. S3B), both isoforms were down-regulated to a similar extent (38.5% GRK6A and 36% GRK6B) (fig. S4, C and E). Thus, decreased availability of GRK6 exacerbates dyskinesia.
We examined the subcellular localization of three GPCRs, D1 and D2 dopamine receptors and mGluR5 glutamate receptor, after acute L-dopa challenge in rats expressing GRK6 or GFP (Fig. 3). Although D1 immunostaining was restricted to the plasma membrane in the GFP group (Fig. 3, top right panel) (16, 25), it was clearly intracellular in GRK6 animals (Fig. 3, top left panel), indicating greater D1 receptor internalization. Conversely, as a substantial proportion of the D2 receptor was already internalized in GFP-expressing animals (Fig. 3, right middle panel) (16), GRK6 did not further promote the D2 receptor internalization (Fig. 3, left middle panel). For comparison, we examined internalization of the mGluR5 receptor in both D1-and D2-expressing medium spiny neurons and found no differences in mGluR5 internalization between the GFP- and GRK6-expressing animals (Fig. 3, lower panels).
Next, we tested whether overexpression of GRK6 in the lesioned striatum blunted dopaminergic signaling after L-dopa administration. The amount of dynorphin, which is largely coexpressed with D1 receptors (26, 27), is reduced by the loss of dopamine but significantly up-regulated by subsequent chronic L-dopa treatment (3, 22). Similarly, D3 receptors are reduced by dopamine depletion but up-regulated by chronic L-dopa in the CPu (3, 22). GRK6 expression suppressed L-dopa–induced up-regulation of prodynorphin mRNA (Fig. 4, A and B) and D3 receptor binding (Fig. 4, C and D) in the CPu. Collectively, these data suggest that expressed GRK6 normalizes the D1 signaling by promoting receptor desensitization.
Enkephalin, which is usually coexpressed with D2 receptors (26, 27), is up-regulated in the dopamine-depleted striatum (3, 28). GRK6 significantly reduced the expression of preproenkephalin mRNA in the striatum in both saline- and L-dopa–treated lesioned rats (Fig. 4, E and F). Thus, GRK6 also normalizes the signaling in D2 receptor–bearing indirect pathway neurons.
Next, we investigated the effectiveness of GRK6 in the gold standard model of LID, L-dopa–treated, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)–lesioned macaque monkeys (29). L-Dopa–treated dyskinetic macaques received GFP (n = 6) or GRK6-GFP (n = 6) virus in the motor striatum. Upon completion of the behavioral experiments, all monkeys were tested to evaluate the lesion and GRK6 expression. Both groups had similar extensive dopamine depletion, as evidenced by the marked decrease in the TH immunoreactivity and dopamine transporter (DAT) binding (Fig. 5, A and B). GRK6-GFP was readily detectable in the motor putamen by immunohistochemistry (Fig. 5C). Using Western blotting, we further confirmed the presence of GRK6-GFP at the level targeted during surgery [anterior commissural, 0 mm (AC0)], whereas there was no expression in the adjacent nontargeted area (AC + 3 mm) (Fig. 5D).
Before GRK6 administration, parkinsonian disability scores in both OFF (before L-dopa administration) and ON (after L-dopa administration) states (Fig. 6A), LID scores in the ON state (Fig. 6B), and the time course of L-dopa–induced locomotor activity (Fig. 6C) were indistinguishable between the two groups. Starting at 6 weeks after surgery, when behavioral experiments resumed, the antiparkinsonian efficacy of L-dopa was comparable in GRK6 and GFP animals (Fig. 6, A and D). There was an overall positive effect of GRK6 on the parkinsonian disability score, suggesting that GRK6 animals benefited longer from L-dopa than controls (Fig. 6, A and D). In agreement with our rodent data, monkeys expressing GRK6 had significantly less intense LID (Fig. 6, B and E). There was also a significant decrease in locomotor activity during the ON state in GRK6 animals in comparison to the GFP group (Fig. 6, C and F). Thus, GRK6 expression in the macaque motor striatum diminished LID intensity without interfering with the antiparkinsonian action of L-dopa.
As in the rat, prodynorphin mRNA expression detected by in situ hybridization was reduced in the GRK6-GFP group in comparison to the GFP group (Fig. 5E), with the latter showing the enhanced prodynorphin expression typical for dyskinetic animals in comparison with non–L-dopa–treated MPTP monkeys (Fig. 5E) (30). Collectively, these data indicate that expressed GRK6 improves LID by normalizing D1 receptor signaling.
One strategy for controlling LID severity is to decrease the L-dopa dosage. In the L-dopa–treated, MPTP-lesioned macaque monkey, a dose corresponding to 50% of the optimal yields shorter antiparkinsonian effect. Control monkeys displayed this phenomenon (Fig. 7, A and C), with the corresponding decrease in LID severity and duration (Fig. 7, B and D). In contrast, in GRK6-expressing monkeys treated with the 50% L-dopa dose, the duration of the antiparkinsonian effect was comparable to that of the 100% dose (Fig. 7, A and C) but without LID (Fig. 7, B and D). The overall locomotor activity of control animals treated with the 50% dose of L-dopa was reduced by 50%, reflecting shorter duration of L-dopa effect. The GRK6-expressing monkeys showed a less pronounced decrease in locomotor activity due to longer L-dopa effect (Fig. 7E).
Because L-dopa is a D1 or D2 indirect agonist, we investigated how GRK6 expression modifies the effects of D1 and D2 agonists. GRK6 monkeys displayed a shortening of the D1 agonist–mediated anti-parkinsonian action compared to controls (P < 0.05) (Fig. 7, F and H), which was accompanied by a significant reduction in LID severity and duration (P < 0.05) (Fig. 7, G and I). We also detected a significant difference in the antiparkinsonian action of a D2 agonist between the two groups (P < 0.05) (Fig. 7, F and H), with GRK6 monkeys displaying lower LID intensity than controls (P < 0.05) (Fig. 7, G and I). Thus, the anti-LID effect of GRK6 is mediated by reduced super-sensitivity of both D1 and D2 receptors.
Loss of dopamine in PD causes multiple changes in the dopamine-mediated signaling (2, 3, 26). The initial dysregulation of signaling pathways is further aggravated by chronic L-dopa treatment, eventually leading to dyskinesia and other motor complications. Although the exact molecular mechanisms of LID remain to be elucidated, exaggerated signaling of the striatal D1 (4, 5, 23), D2 (7), and D3 (3, 8, 22) receptors has been implicated in LID in rodents and primates, suggesting that normalization of this excessive signaling may be beneficial. The challenge is to reduce the signaling in a way that alleviates LID while preserving the antiparkinsonian activity of the drug, which is also mediated by dopamine receptors.
Our data demonstrate that promoting GPCR desensitization in the dopamine-depleted striatum via virus-mediated overexpression of GRK6 attenuates LID in both primate and rodent models. GRK6 suppresses LID in dyskinetic monkeys without compromising the antiparkinsonian effects of L-dopa. GRK6 prolongs the antiparkinsonian effect, especially at the lower L-dopa dose. The duration of the anti-parkinsonian effect of the half-dose in GRK6-expressing animals was even slightly longer than that of the full L-dopa dose in controls. The additional time afforded by GRK6 was LID-free. In the rodent model, GRK6 consistently reduced the rotation frequency and the appearance of AIMs. The inhibition of the rotations and AIMs in rats by GRK6 parallels its potent antidyskinetic activity in the primate model of PD, suggesting an overlap between the molecular mechanisms underlying LID in primates and dyskinetic behaviors in rodents. Collectively, these data demonstrate that increased availability of GRK6 helps to control LID without sacrificing the antiparkinsonian benefits of L-dopa.
The rat knockdown studies demonstrated that reduced availability of GRK6 promoted rotational behavior and increased AIM scores, in agreement with our finding (15) that dopamine depletion reduces the expression of GRK6 and L-dopa treatment does not reverse this reduction. Here, we showed that GRK6A splice variant is most affected by the lesion. The loss of GRK6 in the lesioned hemisphere suggests a link between lower GRK6 availability and dyskinesia. MiRNA-mediated GRK6 knockdown exacerbated the decrease in the GRK6 expression in the lesioned hemisphere and aggravated the behavioral consequences of dopamine depletion and L-dopa treatment, supporting the role of low GRK6 in dyskinesia. Conversely, via overexpression of GRK6A, the splice variant most affected by the lesion, we significantly ameliorated dyskinetic behavior. The lesion reduced the GRK6 concentration by ~40%, and lentiviral knockdown further reduced it by 36 to 40%, whereas overexpression doubled GRK6 concentration. These numbers are in good agreement with the work by Gainetdinov et al. (13), who found in GRK6 hemizygous mice (with ~50% reduction in the GRK6 concentration) a behavioral phenotype close to that of knockout animals. Thus, even a modest modulation of GRK6 concentration seems to have critical impact on dopaminergic signaling and dopamine-dependent behavior. These data underscore an important functional role of GRK6 in signaling mechanisms underlying dyskinesia.
GRK6 likely alters dopamine-dependent behavior by facilitating desensitization of dopamine receptors. Previous work with mice has demonstrated that behavioral supersensitivity to psychostimulants caused by GRK6 knockout is due to modified signaling through the D2 but not the D1 receptor (13). However, dopamine depletion and subsequent development of LID in the course of L-dopa treatment precipitates multiple marked changes in the striatal signaling pathways (29, 31). Although both receptor subtypes are involved in LID, the D1 receptor seems to play a particularly important role (5, 16, 25). Thus, we expected that in the dyskinetic brain, GRK6 might act on both major dopamine receptor subtypes, which proved to be the case. GRK6 reduced LID caused by selective D1 and D2 agonists, indicating that desensitization of both receptor subtypes was facilitated. In this respect, the effect of GRK6 is qualitatively different from our previous results with RGS9-2 (7), which affected D2 receptors coupled to its target Gαi/o but not D1 receptors coupled to Gαs (32). In hemiparkinsonian rats, GRK6 promoted D1 receptor internalization and suppressed the L-dopa–induced up-regulation of prodynorphin and D3 receptor attributed to the enhanced D1 receptor signaling (22). Similarly, GRK6 reduced the prodynorphin expression in dyskinetic monkeys. Although we did not detect any increase in D2 receptor internalization, GRK6 reduced the up-regulation of preproenkephalin mRNA expressed in D2 receptor–bearing neurons (26–28). Thus, the pattern of behavioral and molecular effects is consistent with the conclusion that GRK6 alleviated LID by facilitating the desensitization of both major dopamine receptor subtypes.
In animals treated with selective D1 or D2/D3 agonists, GRK6 not only suppressed LID but also shortened the overall duration of their effects, including the antiparkinsonian activity, which is consistent with faster receptor desensitization due to increased GRK6 availability. Conversely, in GRK6-expressing animals, L-dopa–induced antiparkinsonian effect lasted longer than in control monkeys. Because of the high selectivity of GRKs for active receptors (33), we expected the anti-LID effect of GRK6 to be coupled with the preservation of the anti-parkinsonian activity. The receptor must be activated, allowing the signal to go through, before it is desensitized by GRK-mediated phosphorylation. Apparently, this initial signaling is sufficient for the antiparkinsonian effect but not for LID. Unique effects of L-dopa might arise from its simultaneous action at both D1 and D2 receptors. The presence of GRK6 is likely to shift the balance in favor of D2-like receptors because they do not desensitize as readily as D1 receptors (34, 35). This conclusion is consistent with our finding that in monkeys GRK6 had only a modest effect on the duration of D2-mediated effects, whereas it substantially shortened that of the D1 agonist (Fig. 7). Such rebalancing of the activity of D1 and D2 receptors and, consequently, of the direct and indirect pathways might contribute to extended antiparkinsonian benefits.
Our results demonstrate that a targeted enhancement of GPCR desensitization machinery substantially relieves dyskinesia in two animal models. This amelioration of LID is combined with a longer duration of the antiparkinsonian benefits of L-dopa, offering the hope of achieving the elusive goal of controlling both LID and motor fluctuations. These results pave the way for the development of treatments for dyskinesia in Parkinson’s patients based on judicious manipulation of receptor signaling. Our data identify the receptor desensitization machinery as a therapeutic target in numerous disorders associated with aberrant signaling via GPCRs, including schizophrenia and drug abuse.
The full-length coding sequence of the rat or human GRK6A C-terminally tagged with GFP or GFP alone (control) was cloned into the lentiviral vector pLenti6/V5-DEST, and the virus was produced using ViraPower system (Invitrogen) (fig. S1A). MiRNA sequences targeting rat GRK6 were selected with Invitrogen Block-iT RNAi Designer software (fig. S3), and miRNA-encoding viruses were produced with Block-iT HiPerform Lentiviral Pol II RNAi expression system (Invitrogen).
Adult Sprague-Dawley rats (Charles River) were used. The animals were housed at the Vanderbilt University animal facility in a 12:12 light-dark cycle with free access to food and water. All procedures followed the National Institutes of Health guidelines and were approved by the Vanderbilt University Institutional Animal Care and Use Committee. The 6-OHDA lesion was performed as described (3, 15). The viruses were injected either at the time of the 6-OHDA lesion or after the behavioral pretesting. The animals were tested for rotations in an automated rotometer (AccuScan Instruments) as described (3, 15). In the experiments with AIMs, rats were assessed for AIMs on a 0 to 4 AIM rating scale (24).
All experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/ EEC) for the care of laboratory animals in an American Association for Accreditation of Laboratory Animal Care–accredited facility. Veterinarians skilled in the healthcare and maintenance of nonhuman primates supervised animal care. Experiments followed published procedures (5, 7, 8, 31). After parkinsonian syndrome stabilized, all 12 MPTP-treated monkeys were treated with Modopar for 6 months to develop dyskinesia. The improved Horsley-Clarke stereotactic technique was used as described (2, 5, 7, 8, 36, 37). Either GRK6-GFP (n = 6) or GFP (n = 6) lentivirus was injected into the dorsolateral putamen as described (7). The response of monkeys to L-dopa was assessed as described (7, 8, 31, 36).
The rotation data were analyzed by two-way repeated-measures ANOVA, with Group (GFP versus GRK6) as the between-group factor and Session as the repeated-measures factor. If the significant effect of Group was observed, the data for individual sessions were compared by unpaired Student’s t test. The AIM scores were compared for each session with the Mann-Whitney nonparametric test. Neurochemical data were analyzed by repeated-measures ANOVA, with Hemisphere (intact versus lesioned) as the within-group factor and Group (GRK6 versus GFP) as the between-group factor, or by Mann-Whitney test where appropriate. The value of P < 0.05 was considered significant. Detailed description of rat experiments is given in Supplementary Material. Monkey behavioral data were analyzed by two-way repeated-measures ANOVA, with Group (GRK6 versus GFP) as the between-group factor and Session (before and after surgery) as the within-group factor. Additional analysis was performed with Wilcoxon matched-pairs signed-ranks test or Mann-Whitney test where appropriate.
We thank R. Baishen, J. Li, and H. Li for excellent technical assistance; J.-M. Elalouf and J. L. Benovic for rat and human GRK6 cDNAs; D. Levesque and S. Sabol (NIH, Bethesda, MD) for prodynorphin and preproenkephalin RNA probes; and J. L. Benovic for purified human GRK6A protein.
Funding: Agence Nationale de la Recherche, France (E. Bezard); Biothèque Primate–Centre National de la Recherche Scientifique Life Sciences Department (E. Bezard); NIH grants EY011500 (V.V.G.), NS45117 and NS065868 (E.V.G.), and GM077561 and GM081756 (V.V.G.); and Michael J. Fox Foundation for Parkinson Research (E. Bezard and E.V.G.).
Author contributions: E. Bezard and E.V.G. designed and organized the experiments; E.V.G., V.V.G., M.R.A., Y.T.C., and S.K. designed, cloned, and produced viral vectors and viruses; E.V.G., M.R.A., Y.T.C., E. Bychkov, S.K., A.B., and G.P. performed rat behavioral, neurochemical, and histological experiments; E. Bezard, A.B., G.P., Q.L., B.H.B., B.B., I.A., S.D., and E.D. performed monkey behavioral, neurochemical, and histological experiments; E. Bezard and E.V.G. analyzed the data; E. Bezard, V.V.G., and E.V.G. wrote the paper.
Competing interests: The authors have declared no competing interests.
Materials and Methods
Fig. S1. The GFP-tagged GRK6 is functional and has the subcellular localization of the endogenous GRK6.
Fig. S2. Antibodies to GRK6 selectively recognize GRK6A or GRK6B splicing variants.
Fig. S3. The lentivirus carrying two chained miRNAs targets both GRK6A and GRK6B splice variants.
Fig. S4. Infection of the rat striatum with the miRNA lentivirus induces the GRK6 knockdown.