Critical involvement of cAMP/DARPP-32 and extracellular signal-regulated protein kinase signaling in L-DOPA-induced dyskinesia - PubMed (original) (raw)

Critical involvement of cAMP/DARPP-32 and extracellular signal-regulated protein kinase signaling in L-DOPA-induced dyskinesia

Emanuela Santini et al. J Neurosci. 2007.

Abstract

The molecular basis of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), one of the major hindrances in the current therapy for Parkinson's disease, is still unclear. We show that attenuation of cAMP signaling in the medium spiny neurons of the striatum, achieved by genetic inactivation of the dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces LID. We also show that, in dyskinetic mice, sensitized cAMP/cAMP-dependent protein kinase/DARPP-32 signaling leads to phosphorylation/activation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). The increase in ERK1/2 phosphorylation associated with dyskinesia results in activation of mitogen- and stress-activated kinase-1 (MSK-1) and phosphorylation of histone H3, two downstream targets of ERK involved in transcriptional regulation. In line with these observations, we found that c-Fos expression is abnormally elevated in the striata of mice affected by LID. Persistent enhancement of the ERK signaling cascade is implicated in the generation of LID. Thus, pharmacological inactivation of ERK1/2 achieved using SL327 (alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile), an inhibitor of the mitogen-activated kinase/ERK kinase, MEK, during chronic L-DOPA treatment counteracts the induction dyskinesia. Together, these results indicate that a significant proportion of the abnormal involuntary movements developed in response to chronic L-DOPA are attributable to hyperactivation in striatal medium spiny neurons of a signaling pathway including sequential phosphorylation of DARPP-32, ERK1/2, MSK-1, and histone H3.

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Figures

Figure 1.

Effects of 6-OHDA lesion and

-DOPA administration. a, Left forelimb use was determined using the cylinder test (see Materials and Methods) in sham-lesioned mice (Sham) and in 6-OHDA-lesioned mice before (6-OHDA) or after (6-OHDA/L-DOPA) administration of 20 mg/kg

-DOPA plus 12 mg/kg benserazide. Data are means ± SEM (n = 9–36). ***p < 0.001 versus Sham and 6-OHDA/L-DOPA, one-way ANOVA (F(1,83) = 37.48), followed by Bonferroni–Dunn test. b, 6-OHDA-lesioned mice were treated for 10 d with 20 mg/kg

-DOPA plus 12 mg/kg benserazide and total axial, limb, orolingual, and locomotive AIMs were scored every 20 min over a period of 140 min after the last drug administration. c, Simple linear regression analysis showing absence of correlation between depletion of TH and AIMs score in 6-OHDA-lesioned mice with a reduction in TH immunoreactivity ≥80% (r = 0.219; p = 0.304).

Figure 2.

PKA-mediated phosphorylation of DARPP-32 and GluR1 is associated with LID. Sham- or 6-OHDA-lesioned mice were treated with saline, acute

-DOPA, or chronic

-DOPA. Phospho-Thr34–DARPP-32 (a, b) and phospho-Ser845–GluR1 (c, d) were determined by Western blotting (see Materials and Methods). a, c, 6-OHDA-lesioned mice were divided into low dyskinetic (L Dys) and highly dyskinetic (H Dys) according to their total AIM score (see Materials and Methods) using the median value of 28 as cutoff. Top row shows representative autoradiograms. Bottom row is a summary of data represented as means ± SEM (n = 9–13). ***p < 0.001 versus Sham treated with saline; †††p < 0.001 versus H Dys; one-way ANOVA (a, F(4,52) = 38.44; b, F(4,52) = 69.85), followed by Bonferroni–Dunn test. b, d, Simple regression analysis indicating a significant correlation between AIMs and levels of phospho-Thr34–DARPP-32 (b) (r = 0.921; p < 0.001) and phospho-Ser845–GluR1 (d) (r = 0.953; p < 0.001).

Figure 3.

Phosphorylation of ERK1/2 is associated with LID. Sham- or 6-OHDA-lesioned mice were treated with saline, acute

-DOPA, or chronic

-DOPA. Phospho-Thr202/Tyr204–ERK1 (a, b, e) and ERK2 (c–e) were determined by Western blotting (see Materials and Methods). a, c, 6-OHDA-lesioned mice were divided into low dyskinetic (L Dys) and high dyskinetic (H Dys) according to their total AIM score (see Materials and Methods) using the median value of 28 as cutoff. Data are means ± SEM (n = 9–13). ***p < 0.001 versus Sham treated with saline; †††p < 0.001 and †p < 0.05 versus H Dys; one-way ANOVA (a, F(4,52) = 31.64; c, F(4,52) = 26.96), followed by Bonferroni–Dunn test. b, d, Simple regression analysis indicating a significant correlation between AIMs and levels of phospho-Thr202/Tyr204–ERK1 (b) (r = 0.917; p < 0.001) and phospho-Thr202/Tyr204–ERK2 (d) (r = 0.931; p < 0.001). e, Representative autoradiogram showing phospho-ERK1 (P-ERK1) and phospho-ERK2 (P-ERK2) immunoreactivity. f–h, Immunocytochemical detection of phospho-Thr202/Tyr204–ERK (P-ERK) in the dorsal striata of sham-lesioned mice treated with vehicle (Sham) (f) and of 6-OHDA-lesioned mice treated with

-DOPA showing low (L Dys) (g) and high (H Dys) (h) dyskinesia. Scale bar, 40 μm.

Figure 4.

LID is attenuated in DARPP-32 knock-out mice. Wild-type (WT) and DARPP-32 knock-out (DARPP-32 KO) mice received unilateral injections of 6-OHDA (see Materials and Methods) and were treated for 10 d with 20 mg/kg

-DOPA combined with 12 mg/kg benserazide. a, Time profile of total axial, limb, orolingual, and locomotive AIMs scored every 20 min over a period of 140 min after the last drug administration. b, Sum of total axial, limb, and orolingual (ALO) AIMs scored during all observation periods. c, Sum of locomotive AIMs scored during all observation periods. Data are expressed as means ± SEM (n = 13–19). a, Repeated-measure ANOVA, significant effect of genotype (F(1,180) = 4.64; p < 0.05), time (F(6,180) = 50.16; p < 0.001), and genotype × time interaction (F(6,180) = 2.47; p < 0.05). b, *p < 0.05 versus WT; Student's t test.

Figure 5.

-DOPA-induced phosphorylation of GluR1 and ERK1/2 is reduced in DARPP-32 knock-out mice. Sham- or 6-OHDA-lesioned wild-type (WT) and DARPP-32 knock-out (DARPP-32 KO) mice were treated for 10 d with saline (SHAM/Saline) or

-DOPA (20 mg/kg) plus benserazide (12 mg/kg) (6-OHDA/

-DOPA) and were killed 30 min after the last injection (see Materials and Methods). Phospho-Ser845–GluR1 (a), phospho-Thr202/Tyr204–ERK1 (b), and phospho-Thr202/Tyr204–ERK2 (c) were determined by Western blotting (see Materials and Methods). Top row shows representative autoradiograms. Bottom row shows summary of data represented as means ± SEM (n = 6–19). **p < 0.01 and ***p < 0.001 versus respective SHAM/Saline group; †††p < 0.001 versus 6-OHDA/L-DOPA WT; two-way ANOVA, followed by Bonferroni–Dunn test. A significant interaction was found between genotype and treatment (a, F(1,41) = 5.24, p < 0.05; b, F(1,41) = 4.54, p < 0.05; and c, F(1,41) = 4.96, p < 0.05).

Figure 6.

Phosphorylation of MSK-1 is associated with LID. a–d, Immunocytochemical detection of phospho-Thr581–MSK-1 (P-MSK1) in sham-lesioned mouse treated with vehicle (a; SHAM), 6-OHDA-lesioned mouse treated with

-DOPA (20 mg/kg) in combination with SL327 (b; SL327) (75 mg/kg), 6-OHDA-lesioned mouse with low dyskinesia (c; L Dys), and 6-OHDA-lesioned mouse with high dyskinesia (d; H Dys). Note the abolishment of phospho-MSK-1 immunoreactivity in b compared with c and d. e, Phospho-Thr202/Tyr204–ERK (P-ERK) immunoreactivity in the dorsal striatum of a mouse with high dyskinesia. f, Double immunolabeling showing colocalization between phospho-ERK and phospho-MSK-1 in a mouse with high dyskinesia. g, 6-OHDA-lesioned mice were divided into low dyskinetic (L Dys) and high dyskinetic (H Dys) according to their total AIM score (see Materials and Methods) using the median value of 29 as cutoff. Histograms show the number of phospho-MSK-1-positive cells in the dorsal striata of sham-lesioned mice (SHAM), mice with low (L Dys) and high (H Dys) dyskinesia, and 6-OHDA-lesioned mice treated with

-DOPA in combination with SL327 (75 mg/kg) (SL327). Data are means ± SEM (n = 3–7). ***p < 0.001 versus L Dys; one-way ANOVA, followed by Bonferroni–Dunn test. h, Simple regression analysis showing significant correlation (r = 0.858; p < 0.05) between the number of phospho-MSK-1-positive cells and AIM score. Scale bar: a–f, 40 μm.

Figure 7.

Phosphorylation of histone H3 is associated with LID. a–d, Immunocytochemical detection of phospho-Ser10–histone H3 (P-H3) in sham-lesioned mouse treated with vehicle (a; Sham), 6-OHDA-lesioned mouse treated with

-DOPA (20 mg/kg) in combination with SL327 (75 mg/kg) (b; SL327), 6-OHDA-lesioned mouse with low dyskinesia (c; L Dys), and 6-OHDA-lesioned mouse with high dyskinesia (d; H Dys). Note the abolishment of phospho-Ser10–histone H3 immunoreactivity in b compared with c and d. e, Phospho-Thr202/Tyr204–ERK (P-ERK) immunoreactivity in the dorsal striatum of a mouse with high dyskinesia. f, Double immunolabeling showing colocalization between Phospho-Thr202/Tyr204–ERK and phospho-Ser10–histone H3 in a mouse with high dyskinesia. g, 6-OHDA-lesioned mice were divided into low dyskinetic (L Dys) and high dyskinetic (H Dys) according to their total AIM score (see Materials and Methods) using the median value of 29 as cutoff. Histograms show the number of phospho-Ser10–histone H3-positive cells in the dorsal striata of sham-lesioned mice (SHAM), mice with low (L Dys) and high (H Dys) dyskinesia, and 6-OHDA-lesioned mice treated with

-DOPA in combination with SL327 (75 mg/kg) (SL327). Data are means ± SEM (n = 3–7). ***p < 0.001 versus L Dys; one-way ANOVA, followed by Bonferroni–Dunn test. h, Simple regression analysis showing significant correlation (r = 0.803; p < 0.05) between the number of phospho-Ser10–histone H3-positive cells and AIM score. Scale bar: a–f, 40 μm.

Figure 8.

Phosphorylation of acetyl-histone H3 is associated with LID. a–d, Immunocytochemical detection of phospho-Ser10–acetylLys14 histone H3 (P-AcH3) in sham-lesioned mouse treated with vehicle (a; SHAM), 6-OHDA-lesioned mouse treated with

-DOPA (20 mg/kg) in combination with SL327 (75 mg/kg) (b; SL327), 6-OHDA-lesioned mouse with low dyskinesia (c; L Dys), and 6-OHDA-lesioned mouse with high dyskinesia (d; H Dys). Note the abolishment of phospho-Ser10–acetylLys14 histone H3 immunoreactivity in b compared with c and d. e, Phospho-Thr202/Tyr204–ERK (P-ERK) immunoreactivity in the dorsal striatum of a mouse with high dyskinesia. f, Double immunolabeling showing colocalization between Phospho-Thr202/Tyr204–ERK and phospho-Ser10–acetylLys14 histone H3 in a mouse with H Dys. g, 6-OHDA-lesioned mice were divided into low dyskinetic (L Dys) and high dyskinetic (H Dys) according to their total AIMs score (see Materials and Methods) using the median value of 29 as cutoff. Histograms show the number of phospho-Ser10–acetylLys14 histone H3-positive cells in the dorsal striata of sham-lesioned mice (SHAM), mice with low (L Dys) and high (H Dys) dyskinesia, and 6-OHDA-lesioned mice treated with

-DOPA in combination with SL327 (75 mg/kg) (SL327). Data are means ± SEM (n = 3–7). ***p < 0.001 versus L Dys; one-way ANOVA, followed by Bonferroni–Dunn test. h, Simple regression analysis showing significant correlation (r = 0.835; p < 0.05) between the number of phospho-Ser10–acetylLys14 histone H3-positive cells and AIM score. Scale bar: a–f, 40 μm.

Figure 9.

Pretreatment with SL327 reduces LID. 6-OHDA-lesioned mice were treated for 9 d with 20 mg/kg

-DOPA alone (white symbols) or in combination with SL327 (75 mg/kg) (SL; black and gray symbols). At the end of this period, AIMs were determined after administration of

-DOPA alone (open and black symbols) or

-DOPA plus SL327 (gray symbols). a, Time profile of total axial, limb, orolingual, and locomotive AIMs scored every 20 min over a period of 140 min after the last drug administration. b, Sum of total axial, limb, and orolingual (ALO) AIMs scored during all observation periods. c, Sum of locomotive AIMs scored during all observation periods. Data are expressed as means ± SEM (n = 8). a, Repeated-measure ANOVA, significant effect of SL327 treatment (F(2,126) = 4.59; p < 0.05), time (F(6,126) = 14.95; p < 0.001), and SL327 treatment × time interaction (F(12,126) = 2.23; p < 0.05). b, *p < 0.05 and **p < 0.01 versus L-DOPA/L-DOPA group; one-way ANOVA, followed by Bonferroni–Dunn test.

Figure 10.

Schematic diagram illustrating the involvement of the cAMP/DARPP-32 and ERK pathways in LID. In Parkinson's disease, depletion of dopamine results in increased responsiveness of dopamine D1 receptors, which are expressed by a large proportion of striatal medium spiny neurons. Administration of

-DOPA leads to D1 receptor-mediated activation of PKA, which phosphorylates the GluR1 subunit of AMPA receptors at Ser845 and DARPP-32 at Thr34. PKA-mediated phosphorylation of GluR1 promotes glutamatergic transmission and is intensified by phospho-Thr34–DARPP-32 via inhibition of PP-1. In addition, increased PKA/DARPP-32 signaling leads to activation of MEK, which is facilitated by inhibition of PP-1 and is likely to depend on other signals induced by cAMP and Ca2+ (not shown). Phospho-ERK translocation to the nucleus results in sequential phosphorylation/activation of MSK-1 and histone H3. This, in turn, leads to chromatin rearrangements and transcription of immediate early genes, such as c-fos (supplemental Fig. 2, available at

www.jneurosci.org

as supplemental material). Persistent abnormal activation of these pathways by

-DOPA appears to be involved in long-term changes responsible for dyskinesia. Thus, LID is attenuated by genetic inactivation of DARPP-32 or by blockade of ERK signaling, achieved with SL327.

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Critical involvement of cAMP/DARPP-32 and extracellular signal-regulated protein kinase signaling in L-DOPA-induced dyskinesia - PubMed (original) (raw)