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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.


Related in: MedlinePlus

DARPP-32-mediated phosphatase inhibition favors PKA signaling in D2 MSNs. A, PP-1 and PP-2A were inhibited with cantharidin. Cantharidin (30 µm) alone did not change the basal ratio but strongly increased the AKAR3 response to PQ-10 (100 nm) in all MSNs. These responses were not reversible, making the final identification of D1 and D2 MSNs impossible (gray bars in F, which represent the responses of all MSNs). B, D2 MSNs responded selectively to PQ-10 (100 nm) even when the PP-2A inhibitor fostriecin (200 nm) was applied (n = 4, paired Student’s t test; **p < 0.01). C–E, Mutation of the Thr34 to Ala in DARPP-32 (DARPP-32 T34A) strongly reduced the effect of PQ-10 (100 nm) in D2 MSNs, whereas the selective effect of PQ-10 on D2 MSNs remained in brain slices from animals bearing the Thr75 to Ala mutation in DARPP-32 (DARPP-32 T75A). C, D, Representative experiments performed with DARPP-32 T34A (C) and DARPP-32 T75A (D) knock-in mice. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and is identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. E, The data expressed as the mean ± SEM were analyzed by two-way ANOVA: genotype effect, F(2,72) = 71.12, p < 10−4; D1/D2 effect, F(1,72) = 333.07, p < 10−4; genotype × D1/D2 interaction, F(2, 72) = 49.53, p < 10−4. Bonferroni’s post hoc test: ***p < 0.001. F, In wild-type (WT) mice and DARPP-32 T34A mutants, and in the presence of cantharidin (30 µm), all MSNs responded to PQ-10 (100 nm) with an increase in AKAR3 ratio such that D1 and D2 MSNs could not be distinguished (n = 5 for both). No significant difference was obtained between wild-type and DARPP-32 T34A mutant (unpaired Student's t test, p > 0.05). G, D2 MSNs responded selectively to PQ-10 (100 nm) even when the Cdk5 inhibitor roscovitine (10 µm) was applied (n = 4, paired Student’s t test; *p < 0.05).
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Figure 4: DARPP-32-mediated phosphatase inhibition favors PKA signaling in D2 MSNs. A, PP-1 and PP-2A were inhibited with cantharidin. Cantharidin (30 µm) alone did not change the basal ratio but strongly increased the AKAR3 response to PQ-10 (100 nm) in all MSNs. These responses were not reversible, making the final identification of D1 and D2 MSNs impossible (gray bars in F, which represent the responses of all MSNs). B, D2 MSNs responded selectively to PQ-10 (100 nm) even when the PP-2A inhibitor fostriecin (200 nm) was applied (n = 4, paired Student’s t test; **p < 0.01). C–E, Mutation of the Thr34 to Ala in DARPP-32 (DARPP-32 T34A) strongly reduced the effect of PQ-10 (100 nm) in D2 MSNs, whereas the selective effect of PQ-10 on D2 MSNs remained in brain slices from animals bearing the Thr75 to Ala mutation in DARPP-32 (DARPP-32 T75A). C, D, Representative experiments performed with DARPP-32 T34A (C) and DARPP-32 T75A (D) knock-in mice. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and is identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. E, The data expressed as the mean ± SEM were analyzed by two-way ANOVA: genotype effect, F(2,72) = 71.12, p < 10−4; D1/D2 effect, F(1,72) = 333.07, p < 10−4; genotype × D1/D2 interaction, F(2, 72) = 49.53, p < 10−4. Bonferroni’s post hoc test: ***p < 0.001. F, In wild-type (WT) mice and DARPP-32 T34A mutants, and in the presence of cantharidin (30 µm), all MSNs responded to PQ-10 (100 nm) with an increase in AKAR3 ratio such that D1 and D2 MSNs could not be distinguished (n = 5 for both). No significant difference was obtained between wild-type and DARPP-32 T34A mutant (unpaired Student's t test, p > 0.05). G, D2 MSNs responded selectively to PQ-10 (100 nm) even when the Cdk5 inhibitor roscovitine (10 µm) was applied (n = 4, paired Student’s t test; *p < 0.05).

Mentions: Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.


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)

DARPP-32-mediated phosphatase inhibition favors PKA signaling in D2 MSNs. A, PP-1 and PP-2A were inhibited with cantharidin. Cantharidin (30 µm) alone did not change the basal ratio but strongly increased the AKAR3 response to PQ-10 (100 nm) in all MSNs. These responses were not reversible, making the final identification of D1 and D2 MSNs impossible (gray bars in F, which represent the responses of all MSNs). B, D2 MSNs responded selectively to PQ-10 (100 nm) even when the PP-2A inhibitor fostriecin (200 nm) was applied (n = 4, paired Student’s t test; **p < 0.01). C–E, Mutation of the Thr34 to Ala in DARPP-32 (DARPP-32 T34A) strongly reduced the effect of PQ-10 (100 nm) in D2 MSNs, whereas the selective effect of PQ-10 on D2 MSNs remained in brain slices from animals bearing the Thr75 to Ala mutation in DARPP-32 (DARPP-32 T75A). C, D, Representative experiments performed with DARPP-32 T34A (C) and DARPP-32 T75A (D) knock-in mice. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and is identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. E, The data expressed as the mean ± SEM were analyzed by two-way ANOVA: genotype effect, F(2,72) = 71.12, p < 10−4; D1/D2 effect, F(1,72) = 333.07, p < 10−4; genotype × D1/D2 interaction, F(2, 72) = 49.53, p < 10−4. Bonferroni’s post hoc test: ***p < 0.001. F, In wild-type (WT) mice and DARPP-32 T34A mutants, and in the presence of cantharidin (30 µm), all MSNs responded to PQ-10 (100 nm) with an increase in AKAR3 ratio such that D1 and D2 MSNs could not be distinguished (n = 5 for both). No significant difference was obtained between wild-type and DARPP-32 T34A mutant (unpaired Student's t test, p > 0.05). G, D2 MSNs responded selectively to PQ-10 (100 nm) even when the Cdk5 inhibitor roscovitine (10 µm) was applied (n = 4, paired Student’s t test; *p < 0.05).
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Related In: Results  -  Collection

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Figure 4: DARPP-32-mediated phosphatase inhibition favors PKA signaling in D2 MSNs. A, PP-1 and PP-2A were inhibited with cantharidin. Cantharidin (30 µm) alone did not change the basal ratio but strongly increased the AKAR3 response to PQ-10 (100 nm) in all MSNs. These responses were not reversible, making the final identification of D1 and D2 MSNs impossible (gray bars in F, which represent the responses of all MSNs). B, D2 MSNs responded selectively to PQ-10 (100 nm) even when the PP-2A inhibitor fostriecin (200 nm) was applied (n = 4, paired Student’s t test; **p < 0.01). C–E, Mutation of the Thr34 to Ala in DARPP-32 (DARPP-32 T34A) strongly reduced the effect of PQ-10 (100 nm) in D2 MSNs, whereas the selective effect of PQ-10 on D2 MSNs remained in brain slices from animals bearing the Thr75 to Ala mutation in DARPP-32 (DARPP-32 T75A). C, D, Representative experiments performed with DARPP-32 T34A (C) and DARPP-32 T75A (D) knock-in mice. Each trace on the graph indicates the ratio measurement on MSNs expressing AKAR3 and is identified as D1 or D2 according to their response to either SKF-38393 (SKF, 1 µm) or CGS 21680 (CGS, 1 µm), respectively. The thick black line represents the average of all the traces in each group. E, The data expressed as the mean ± SEM were analyzed by two-way ANOVA: genotype effect, F(2,72) = 71.12, p < 10−4; D1/D2 effect, F(1,72) = 333.07, p < 10−4; genotype × D1/D2 interaction, F(2, 72) = 49.53, p < 10−4. Bonferroni’s post hoc test: ***p < 0.001. F, In wild-type (WT) mice and DARPP-32 T34A mutants, and in the presence of cantharidin (30 µm), all MSNs responded to PQ-10 (100 nm) with an increase in AKAR3 ratio such that D1 and D2 MSNs could not be distinguished (n = 5 for both). No significant difference was obtained between wild-type and DARPP-32 T34A mutant (unpaired Student's t test, p > 0.05). G, D2 MSNs responded selectively to PQ-10 (100 nm) even when the Cdk5 inhibitor roscovitine (10 µm) was applied (n = 4, paired Student’s t test; *p < 0.05).
Mentions: Two-photon imaging was used to separate individual neurons for a precise quantification of the amplitude of the response (Figs. 1, 2). Ratio measurements were performed on a series of 5–10 consecutive image from the image stack, centered on the cell body. With cytosolic biosensors, when visible, the nucleus was excluded from the measurement. Wide-field imaging (Figs. 3A–E) also allowed the unambiguous identification of D1 and D2 MSNs, provided that the infection level was kept low and no fluorescence overlap between neighboring neurons was observed. The optical cross-contamination resulting from out-of-focus light was evaluated by the final response to CGS 21680 and SKF-38393, applied sequentially: cells were rejected from analysis if the cross-contamination was >30%. For cGMP imaging (Fig. 3F,G), the data were quantified as relative ratio change.

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.


Related in: MedlinePlus