Striatal Subregion-selective Dysregulated Dopamine Receptor-mediated Intracellular Signaling in a Model of DOPA-responsive Dystonia

Abstract

Although the mechanisms underlying dystonia are largely unknown, dystonia is often associated with abnormal dopamine neurotransmission. DOPA-responsive dystonia (DRD) is a prototype disorder for understanding dopamine dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of dopamine and alleviated by the indirect-acting dopamine agonist l-DOPA. Although adaptations in striatal dopamine receptor-mediated intracellular signaling have been studied extensively in models of Parkinson's disease, another movement disorders associated with dopamine deficiency, little is known about dopaminergic adaptations in dystonia. To identify the dopamine receptor-mediated intracellular signaling associated with dystonia, we used immunohistochemistry to quantify striatal protein kinase A activity and extracellular signal-related kinase (ERK) phosphorylation after dopaminergic challenges in a knockin mouse model of DRD. l-DOPA treatment induced the phosphorylation of both protein kinase A substrates and ERK largely in D1 dopamine receptor-expressing striatal neurons. As expected, this response was blocked by pretreatment with the D1 dopamine receptor antagonist SCH23390. The D2 dopamine receptor antagonist raclopride also significantly reduced the phosphorylation of ERK; this contrasts with models of parkinsonism in which l-DOPA-induced ERK phosphorylation is not mediated by D2 dopamine receptors. Further, the dysregulated signaling was dependent on striatal subdomains whereby ERK phosphorylation was largely confined to dorsomedial (associative) striatum while the dorsolateral (sensorimotor) striatum was unresponsive. This complex interaction between striatal functional domains and dysregulated dopamine-receptor mediated responses has not been observed in other models of dopamine deficiency, such as parkinsonism, suggesting that regional variation in dopamine-mediated neurotransmission may be a hallmark of dystonia.

Keywords: dopamine; dystonia; extracellular signal-related kinase; protein kinase A; striatum.

Figure 1.

Baseline densities of p-PKA-sub-positive cells or p-ERK-positive cells in the dorsomedial and dorsolateral striatum of normal and DRD mice (n = 8–9 mice/group). A. The density of p-PKA-sub-positive cells was overall lower in dorsolateral striatum compared to dorsomedial striatum but there was no difference between normal and DRD mice (linear mix model with region as the repeated measure, main effect of genotype, F_1,16 = 0.071, P = 0.793; main effect of region, F_1,14 = 6.205, P = 0.0259). One sample from dorsolateral striatum of normal mice and one sample from dorsolateral striatum of DRD mice were identified as outliers and not included in the analyses. B. The density of p-ERK-positive cells in the dorsomedial striatum was reduced in DRD mice compared to normal mice (two-way repeated measures ANOVA with region as the repeated measure, genotype × region interaction effect, F_1,16 = 20.31, P = 0.0004; Holms-Sidak’s post hoc analysis P < 0.0001). Data represent means ± SEMs; ****P < 0.0001 compared to normal mice.

Figure 2.

l-DOPA-mediated PKA signaling in the dorsomedial and dorsolateral striatum of normal and DRD mice. A. Quantification of the density of p-PKA-sub-positive striatal cells after acute challenge with vehicle or l-DOPA (10 mg/kg) in normal and DRD mice (n = 7–9 mice/group). In addition, DRD mice were pretreated with either the D1R antagonist SCH 23390 (1 mg/kg) or the D2R antagonist raclopride (1 mg/kg) before l-DOPA challenge (n = 6–7 mice/group). Mice were euthanized 45 minutes after treatment and the density of p-PKA-sub-positive striatal cells was quantified in dorsomedial and dorsolateral striatum. There was no effect of l-DOPA in control mice (mixed effects model, main effect of treatment, F_1,14 = 0.316, P = 0.5829). There was a main effect of treatment for DRD mice (mixed effects model, main effect of treatment, F_5,34 = 3.702, P = 0.0088 with Holm-Sidak’s post hoc analysis) whereby l-DOPA increased the density of p-PKA-sub-positive striatal cells in dorsomedial (P = 0.0207) and dorsolateral (P = 0.0077) striatum; SCH 23390 and raclopride attenuated the effects of l-DOPA in dorsomedial (P = 0.0111 and p= 0.0556) and dorsolateral striatum (P = 0.0061 and P = 0.0099). One l-DOPA + RAC DRD mouse was not included the analysis because both dorsomedial and dorsolateral points were identified as outliers. Data represent means ± SEMs; †P < 0.05 and ††P < 0.01 compared to vehicle, *P < 0.05 and **P < 0.01 compared to l-DOPA treatment. B. Double-labeling experiments of D2R+ striatal cells (green) and p-PKA-sub-positive cells (magenta) in the dorsomedial striatum of a DRD mouse treated with l-DOPA. p-PKA-sub immunoreactivity was not observed in D2R+ cells (merged image).

Figure 3.

l-DOPA-mediated phosphorylation of ERK in the dorsomedial and dorsolateral striatum of normal and DRD mice. A. Quantification of the density of p-ERK positive striatal cells after acute administration of vehicle or l-DOPA (10 mg/kg) in normal and DRD mice (n = 6–9 mice/group). In addition, DRD mice were pretreated with either the D1R antagonist SCH 23390 (1 mg/kg) or the D2R antagonist raclopride (1 mg/kg) before l-DOPA challenge (n=7 mice/group). Mice were euthanized 45 minutes after treatment and the density of striatal cells immunolabeled with p-ERK antibody was quantified in dorsomedial and dorsolateral striatum. There was no effect of l-DOPA on control mice (mixed effects model, main effect of treatment, F_1,14 = 2.76, P = 0.1189) but there was a main effect of region (F_1,13 = 30.75, P < 0.0001) with higher expression in the dorsomedial than dorsolateral striatum. For DRD mice there was a significant treatment × region interaction effect (mixed effects model with repeated measures for region, F_5,34 = 17.60, P < 0.0001 with Holm-Sidak’s post hoc analysis) whereby l-DOPA treatment increased the density of p-ERK positive cells in dorsomedial (P < 0.0001) but not dorsolateral (P = 0.7662) striatum. The dorsomedial-specific increase was attenuated by SCH 23390 (P < 0.0001) and raclopride (P < 0.0001) pretreatment. One sample from dorsolateral striatum of normal mice treated with l-DOPA and one sample from dorsolateral striatum of DRD mice treated with l-DOPA were identified as outliers and not included in the analyses. Data represent means ± SEMs; ††††P < 0.0001 compared to vehicle, ****P < 0.0001 compared to l-DOPA treatment. B. Double-labeling experiments of D2R+ striatal cells (green) and p-ERK-positive cells (magenta) in the dorsomedial striatum of a DRD mouse treated with l-DOPA. p-ERK immunoreactivity was not observed in D2R+ cells (merged image). C. Brightfield photomicrographs p-ERK immunoreactivity in the striatum of saline- and l-DOPA-treated DRD mice.

Figure 4.

Quantification of D1R and D2R agonist-mediated p-PKA-sub-positive or p-ERK-positive striatal cell density in normal and DRD mice. A & B. Mice were treated with vehicle or 0.2 mg/kg SKF 81297 (n = 6–9 mice/group) and euthanized 45 minutes after vehicle or drug challenge. A. SKF 81297 treatment had no effect on normal mice but significantly increased the density of p-PKA-sub-positive cells in DRD mice (three-way mixed effects model, treatment × genotype interaction effect, F_1,29 = 6.930, P = 0.0134 with Holm-Sidak’s post hoc analysis) in both dorsomedial (P = 0.0087) and dorsolateral (P = 0.0070) striatum. One sample from the dorsolateral striatum of normal mice treated with SK81297 was identified as an outlier and not included in the analysis. B. The density of p-ERK-positive cells was not affected by SKF 81297 treatment in either normal or DRD mice (three-way repeated measures ANOVA, main effect of treatment, F_1,28 = 0.1233, P = 0.7281). There was a genotype × region interaction effect (three-way RM-ANOVA, F_1,28 = 7.609, P = 0.0101), reflecting the low basal p-ERK-positive cell density in the dorsomedial striatum of DRD mice. One DRD mouse treated with SKF 81297 was excluded from the analysis because both dorsomedial and dorsolateral datapoints were identified as outliers. C & D. Normal or DRD mice were treated with vehicle or 0.1 mg/kg quinpirole (n = 5–9 mice/group) and euthanized 45 minutes after vehicle or drug challenge. C. There was no effect of quinpirole treatment on the density of p-PKA-sub-positive cells in either normal or DRD mice (three-way mixed effects model, main effect of treatment, F_1,22 = 1.310, P = 0.2647). D. There was no effect of quinpirole treatment on the density of p-ERK-positive cells in either normal or DRD mice (three-way repeated measures ANOVA, main effect of treatment, F_1,26 = 0.0046, P = 0.9463). Data represent means ± SEMs. **P < 0.01 compared to vehicle.

Figure 5.

Quantification of D1R and D2R antagonist-mediated p-PKA-sub-positive or p-ERK-positive striatal cell density in normal and DRD mice. A & B. Normal or DRD mice were treated with vehicle or 1 mg/kg SCH 23390 (n = 4–9 mice/group) and euthanized 45 minutes after vehicle or drug challenge. A. SCH 23390 treatment did not alter the density of p-PKA-sub-positive striatal cells in either normal or DRD mice (3-way mixed effects analysis, main effect of treatment, F_1,22 = 1.310, P = 0.2647). B. SCH 23390 treatment significantly reduced the density of p-ERK-positive cells in dorsomedial striatum of normal mice (P < 0.0001) but the effect was not significant (P < 0.9767) in DRD mice (three-way repeated measures ANOVA, genotype × region × treatment interaction effect F_1,22 = 6.549, P = 0.0179 with Holm-Sidak’s post hoc analysis). C & D. Normal or DRD mice were treated with vehicle or 1 mg/kg raclopride (n = 7–9 mice/group) and euthanized 45 minutes after vehicle or drug challenge. C. Raclopride treatment significantly increased the density of p-PKA-sub-positive cells in the dorsolateral striatum of normal mice (three-way mixed effects analysis, treatment × genotype interaction effect F_1,29 = 4.615, P = 0.0402; Holm-Sidak’s post hoc analysis, P = 0.0464). D. There was not a significant effect of raclopride on the density of p-ERK-positive striatal cells (three-way repeated measures ANOVA, main effect of treatment, F_1,29 = 2.109, P = 0.1571), but there was a significant region × genotype interaction effect (three-way repeated measures ANOVA, F_1,29 = 32.48, P < 0.0001; Holm-Sidak’s post hoc analysis, P < 0.0001 vehicle-treated dorsomedial normal mice versus vehicle-treated dorsomedial DRD mice). Data represent means ± SEMs. *P < 0.05 and ****P < 0.0001 compared to vehicle.