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Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32(1,2,3).

Polito M, Guiot E, Gangarossa G, Longueville S, Doulazmi M, Valjent E, Hervé D, Girault JA, Paupardin-Tritsch D, Castro LR, Vincent P - eNeuro (2015)

Bottom Line: PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8256 "Biological Adaptation and Ageing", Institut de Biologie Paris-Seine (IBPS) , F-75005 Paris, France ; Université Pierre et Marie Curie (UPMC, Paris 6), Sorbonne Universités , Paris, F-75005, France.

ABSTRACT
Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

No MeSH data available.


The DARPP-32 T34 residue is required for a TP-10-induced increase of histone H3 phosphorylation in the striatum in adult mice in vivo. Wild type (WT) and DARPP-32 T34A mutant mice were treated with TP-10 (3 mg/kg) or vehicle. A, Examples of PH3 immunofluorescence, showing the dramatic reduction of TP-10 effects in the DARPP-32 T34A mutant mice. Scale bar, 20 µm. B, Quantification of the number of PH3-positive neurons in striatal coronal sections. Error bars indicate the SEM. Data were analyzed by a two-way ANOVA: genotype effect, F(1,12) = 13.7, p < 0.01; TP-10 effect, F(1,12) = 16.1, p < 0.01; genotype × TP-10 interaction, F(1,12) = 14.8, p < 0.01. Bonferroni’s post hoc test, ***p < 10−3.
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Figure 6: The DARPP-32 T34 residue is required for a TP-10-induced increase of histone H3 phosphorylation in the striatum in adult mice in vivo. Wild type (WT) and DARPP-32 T34A mutant mice were treated with TP-10 (3 mg/kg) or vehicle. A, Examples of PH3 immunofluorescence, showing the dramatic reduction of TP-10 effects in the DARPP-32 T34A mutant mice. Scale bar, 20 µm. B, Quantification of the number of PH3-positive neurons in striatal coronal sections. Error bars indicate the SEM. Data were analyzed by a two-way ANOVA: genotype effect, F(1,12) = 13.7, p < 0.01; TP-10 effect, F(1,12) = 16.1, p < 0.01; genotype × TP-10 interaction, F(1,12) = 14.8, p < 0.01. Bonferroni’s post hoc test, ***p < 10−3.

Mentions: Mice, 8-10 weeks old, were treated with the drug for 60 min and then rapidly anesthetized with pentobarbital (500 mg/kg, i.p.; Sanofi-Aventis) and were transcardially perfused with 4% (w/v) paraformaldehyde in 0.1 m PBS, pH 7.5. Brains were post-fixed overnight in the same solution and stored at 4°C. The 30-μm-thick sections were cut with a vibratome and stored at −20°C in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol and 0.1 m sodium phosphate buffer, until they were processed for immunofluorescence. Sections were processed as described in Bertran-Gonzalez et al. (2009). Sodium fluoride 0.1 mm was included in all buffers and incubation solutions. Histone H3 phosphorylation was revealed with a rabbit polyclonal antibody against phospho-Ser10-H3 (1:1000; catalog #06570; Millipore) the specificity of which was confirmed in a previous study (Jordi et al., 2013). GFP was detected using chicken antibody against GFP (1:500; catalog #A10262; Life Technologies). Following incubation with primary antibodies, sections were rinsed three times for 10 min in TBS and incubated for 45–60 min with goat Cy3-coupled (1:500; Jackson ImmunoResearch; Fig. 5) and goat A488 (1:500; Life Technologies; Fig. 6). Sections were rinsed for 10 min twice in Tris-buffered saline and twice in Tris buffer (0.25 m Tris) before mounting in 1,4-diazabicyclo-[2. 2. 2]-octane (Sigma-Aldrich).


Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32(1,2,3).

Polito M, Guiot E, Gangarossa G, Longueville S, Doulazmi M, Valjent E, Hervé D, Girault JA, Paupardin-Tritsch D, Castro LR, Vincent P - eNeuro (2015)

The DARPP-32 T34 residue is required for a TP-10-induced increase of histone H3 phosphorylation in the striatum in adult mice in vivo. Wild type (WT) and DARPP-32 T34A mutant mice were treated with TP-10 (3 mg/kg) or vehicle. A, Examples of PH3 immunofluorescence, showing the dramatic reduction of TP-10 effects in the DARPP-32 T34A mutant mice. Scale bar, 20 µm. B, Quantification of the number of PH3-positive neurons in striatal coronal sections. Error bars indicate the SEM. Data were analyzed by a two-way ANOVA: genotype effect, F(1,12) = 13.7, p < 0.01; TP-10 effect, F(1,12) = 16.1, p < 0.01; genotype × TP-10 interaction, F(1,12) = 14.8, p < 0.01. Bonferroni’s post hoc test, ***p < 10−3.
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Figure 6: The DARPP-32 T34 residue is required for a TP-10-induced increase of histone H3 phosphorylation in the striatum in adult mice in vivo. Wild type (WT) and DARPP-32 T34A mutant mice were treated with TP-10 (3 mg/kg) or vehicle. A, Examples of PH3 immunofluorescence, showing the dramatic reduction of TP-10 effects in the DARPP-32 T34A mutant mice. Scale bar, 20 µm. B, Quantification of the number of PH3-positive neurons in striatal coronal sections. Error bars indicate the SEM. Data were analyzed by a two-way ANOVA: genotype effect, F(1,12) = 13.7, p < 0.01; TP-10 effect, F(1,12) = 16.1, p < 0.01; genotype × TP-10 interaction, F(1,12) = 14.8, p < 0.01. Bonferroni’s post hoc test, ***p < 10−3.
Mentions: Mice, 8-10 weeks old, were treated with the drug for 60 min and then rapidly anesthetized with pentobarbital (500 mg/kg, i.p.; Sanofi-Aventis) and were transcardially perfused with 4% (w/v) paraformaldehyde in 0.1 m PBS, pH 7.5. Brains were post-fixed overnight in the same solution and stored at 4°C. The 30-μm-thick sections were cut with a vibratome and stored at −20°C in a solution containing 30% (v/v) ethylene glycol, 30% (v/v) glycerol and 0.1 m sodium phosphate buffer, until they were processed for immunofluorescence. Sections were processed as described in Bertran-Gonzalez et al. (2009). Sodium fluoride 0.1 mm was included in all buffers and incubation solutions. Histone H3 phosphorylation was revealed with a rabbit polyclonal antibody against phospho-Ser10-H3 (1:1000; catalog #06570; Millipore) the specificity of which was confirmed in a previous study (Jordi et al., 2013). GFP was detected using chicken antibody against GFP (1:500; catalog #A10262; Life Technologies). Following incubation with primary antibodies, sections were rinsed three times for 10 min in TBS and incubated for 45–60 min with goat Cy3-coupled (1:500; Jackson ImmunoResearch; Fig. 5) and goat A488 (1:500; Life Technologies; Fig. 6). Sections were rinsed for 10 min twice in Tris-buffered saline and twice in Tris buffer (0.25 m Tris) before mounting in 1,4-diazabicyclo-[2. 2. 2]-octane (Sigma-Aldrich).

Bottom Line: PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear.The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition.These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8256 "Biological Adaptation and Ageing", Institut de Biologie Paris-Seine (IBPS) , F-75005 Paris, France ; Université Pierre et Marie Curie (UPMC, Paris 6), Sorbonne Universités , Paris, F-75005, France.

ABSTRACT
Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

No MeSH data available.