Dopamine- and cAMP-regulated phosphoprotein of 32-kDa (DARPP-32)-dependent activation of extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) signaling in experimental parkinsonism

Abstract

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.

FIGURE 1.

LID is accompanied by increased levels of phospho-Thr-34-DARPP-32 in D1R-expressing MSNs. D1-DARPP-32-FLAG/D2-DARPP-32-Myc transgenic mice were lesioned unilaterally with 6-OHDA and treated for 10 days with 20 mg/kg of l-DOPA. AIMs were determined immediately after the last injection of l-DOPA. The following day, mice were treated with vehicle or l-DOPA and killed 30 min later. Levels of phospho-Thr-34-DARPP-32 (A) and phospho-Thr-75-DARPP-32 (C) were determined in the striata of control mice (unlesioned, treated with vehicle) and mice lesioned with 6-OHDA and treated chronically with vehicle or l-DOPA. This latter group was divided into low dyskinetic (L Dys) and high dyskinetic (H Dys) (cf. “Results”). Data are expressed as percent of control (n = 4–12). **, p < 0.01 versus control; one-way ANOVA followed by Bonferroni-Dunn test. DARPP-32 was immunoprecipitated from D1R- and D2R-expressing MSNs using anti-FLAG and anti-Myc antibodies, respectively, and the levels of phospho-Thr-34-DARPP-32 (B) and phospho-Thr-75-DARPP-32 (D) were determined by Western blotting. Data are expressed as percent of control (n = 4–12). ***, p < 0.001 versus control; †††, p < 0.001 versus low dyskinetic group; one-way ANOVA followed by Bonferroni-Dunn test.

FIGURE 2.

Selective deletion of DARPP-32 in striatonigral MSNs decreases l-DOPA-induced phosphorylation of ERK, histone H3, Glu-1A, and rpS6. DARPP-32^F/FD1RCre⁺ mice, DARPP-32^F/FD2RCre⁺ mice, and DARPP-32^F/FCre⁻ littermates were lesioned unilaterally with 6-OHDA, treated for 10 days with 10 mg/kg of l-DOPA, and killed 30 min after the last injection. Top rows show representative autoradiograms obtained using antibodies against total or phosphorylated ERK (A), histone H3 (B), Glu-1A (C), and rpS6 (D and E). Bottom rows are summary of results showing means ± S.E. (n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus unlesioned (UL) DARPP-32^F/FCre⁻ and DARPP-32^F/FD2RCre⁺ mice treated with l-DOPA; †, p < 0.05; ††, p < 0.01; †††, p < 0.001 versus 6-OHDA lesioned DARPP-32^F/FCre⁻ mice treated with l-DOPA (L); two-way ANOVA, followed by Bonferroni-Dunn test. A significant interaction between treatment and genotype was found in DARPP-32^F/FD1RCre⁺ mice (F_{(1, 20)} = 8.9, p < 0.01 for phospho-ERK1; F_{(1, 20)} = 11.47, p < 0.01 for phospho-ERK2; F_{(1, 22)} = 8.32, p < 0.01 for phospho-Ser10-acetyl-Lys-14-histone H3; F_{(1, 20)} = 7.44, p < 0.05 for phospho-Ser-845-Glu-1A; F_{(1, 20)} = 7.64, p < 0.05 for phospho-Ser-235/236-rpS6; F_{(1, 20)} = 7.74, p < 0.05 for phospho-Ser-240/244-rpS6). A significant effect of the treatment was found in DARPP-32^F/FD2RCre⁺ mice (F_{(1, 20)} = 26.64, p < 0.001 for phospho-ERK1; F_{(1, 20)} = 30.95, p < 0.001 for phospho-ERK2; F_{(1, 22)} = 36.98, p < 0.001 for phospho-Ser-10-acetyl-Lys-14-histone H3; F_{(1, 20)} = 35.44, p < 0.001 for phospho-Ser-845-Glu-1A; F_{(1, 20)} = 80.3, p < 0.001 for phospho-Ser-235/236-rpS6; F_{(1, 20)} = 44.41, p < 0.001 for phospho-Ser-240/244-rpS6).

FIGURE 3.

Mutation of Thr-34 on DARPP-32 decreases LID. Wild type (WT) and T34A mutant mice were lesioned unilaterally with 6-OHDA and treated for 10 days with 10 mg/kg of l-DOPA. AIMs were determined immediately after the last injection. A, time profile of the sum of locomotive, axial, limb, and orolingual AIMs scored every 20 min over a period of 120 min after the last drug administration. B, sum of total AIMs scored during all observation periods. *, p < 0.01 versus WT, two-way ANOVA followed by Bonferroni-Dunn test. A significant effect of the genotype was found (p < 0.05, F_{(1, 20)} = 10.8).

FIGURE 4.

Mutation of Thr-34 on DARPP-32 decreases l-DOPA-induced phosphorylation of ERK, histone H3, Glu-1A, and rpS6. T34A mutant mice and wild-type littermates were lesioned with 6-OHDA, treated for 11 days with 10 mg/kg of l-DOPA, and killed 30 min after the last injection. Top rows show representative autoradiograms obtained using antibodies against total or phosphorylated ERK2 (A), Glu-1A (B), histone H3 (C), and rpS6 (D and E). Data are represented as means ± S.E. (n = 7–10). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus unlesioned mice treated with l-DOPA (open bars); †, p < 0.05; ††, p < 0.01; †††, p < 0.001 versus 6-OHDA-lesioned mice treated with l-DOPA; °, p < 0.05 versus unlesioned mice treated with l-DOPA; two-way ANOVA, followed by Bonferroni-Dunn test. A significant interaction was found between treatment and genotype (F_{(1, 26)} = 5.75, p < 0.05 for phospho-ERK2; F_{(1, 28)} = 6.82, p < 0.05 for Glu-1A; F_{(1, 28)} = 20.12, p < 0.001 for phospho-Ser-10-acetyl-Lys-14-histone H3; F_{(1, 28)} = 19.74, p < 0.001 for phospho-Ser-235/236-rpS6; F_{(1, 28)} = 4.26, p < 0.05 for phospho-Ser-240/244-rpS6).