Phosphodiesterase 4 inhibition enhances the dopamine D1 receptor/PKA/DARPP-32 signaling cascade in frontal cortex

Abstract

Rationale: Alteration of dopamine neurotransmission in the prefrontal cortex, especially hypofunction of dopamine D1 receptors, contributes to psychotic symptoms and cognitive deficit in schizophrenia. D1 receptors signal through the cAMP/PKA second messenger cascade, which is modulated by phosphodiesterase (PDE) enzymes that hydrolyze and inactivate cyclic nucleotides. Though several PDEs are expressed in cortical neurons, the PDE4 enzyme family (PDE4A-D) has been implicated in the control of cognitive function. The best studied isoform, PDE4B, interacts with a schizophrenia susceptibility factor, disrupted in schizophrenia 1 (DISC1).

Objectives: We explore the control of mouse frontal cortex dopamine D1 receptor signaling and associated behavior by PDE4.

Results: Inhibition of PDE4 by rolipram induced activation of cAMP/PKA signaling in cortical slices and in vivo, leading to the phosphorylation of DARPP-32 and other postsynaptic and presynaptic PKA-substrates. Rolipram also enhanced DARPP-32 phosphorylation invoked by D1 receptor activation. Immunohistochemical studies demonstrated PDE4A, PDE4B, and PDE4D expression in DARPP-32-positive neurons in layer VI of frontal cortex, most likely in D1 receptor-positive, glutamatergic corticothalamic pyramidal neurons. Furthermore, the ability of rolipram treatment to improve the performance of mice in a sensorimotor gating test was DARPP-32-dependent.

Conclusions: PDE4, which is co-expressed with DARPP-32 in D1 receptor-positive cortical pyramidal neurons in layer VI, modulates the level of D1 receptor signaling and DARPP-32 phosphorylation in the frontal cortex, likely influencing cognitive function. These biochemical and behavioral actions of PDE4 inhibitors may contribute to the hypothesized antipsychotic actions of this class of compounds.

Fig. 1. Effects of a PDE4 inhibitor, rolipram, on DARPP-32 phosphorylation at Thr34 and Thr75 in cortical slices

Time-course and dose-response effects for rolipram on phospho-Thr34 (A, B) and phospho-Thr75 (C, D) DARPP-32 are shown. Mouse cortical slices were incubated with rolipram (1 μM) for the indicated times (A, C) or with the indicated concentrations of rolipram for 60 min (B) or 5 min (D). Typical immunoblots for phospho-Thr34, phospho-Thr75 and total DARPP-32 are shown with quantitation. Data represent means ± SEM for 4-8 experiments. *p < 0.05, ***p < 0.001 compared with untreated slices; one-way ANOVA followed by Newman-Keuls test

Fig. 2. Effect of rolipram on the phosphorylation of GluR1, NR1, PDE4, ERK2, TH and synapsin I in cortical slices

Cortical slices were treated with rolipram (1 μM) for the indicated times. Quantitation of phosphorylation of GluR1 at Ser845 (the PKA-site) (A), NR1 at Ser897 (the PKA-site) (B), PDE4 at Ser133 (the PKA-site) (C), ERK2 at Thr202/Tyr204 (the MEK-site) (D), TH at Ser40 (the PKA-site) (E), and synapsin I at Ser9 (the PKA/CaMKI-site) (F) is shown. Data represent means ± SEM for 4-15 experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated slices; one-way ANOVA followed by Newman-Keuls test

Fig. 3. Effect of rolipram on dopamine D₁ receptor/DARPP-32 signaling in cortical slices

Cortical slices were treated with (A) rolipram (1 μM) and/or a dopamine D₁ receptor agonist, SKF81297 (1 μM), or (B) rolipram (1 μM) and/or a dopamine D₁ receptor antagonist, SCH23390 (1 μM), for 10 min. Quantitation of phospho-Thr34 DARPP-32 is shown. Data represent means ± SEM for 4 experiments. ***p < 0.001 compared with control; ^†††p < 0.001 compared with rolipram alone; one-way ANOVA followed by Newman-Keuls test

Fig. 4. Effect of rolipram on the phosphorylation of DARPP-32, GluR1, NR1 and synapsin I in the frontal cortex in vivo

Mice were injected with vehicle or rolipram (10 mg/kg). Frontal cortex was dissected 15 min later and analyzed for the phosphorylation of DARPP-32 at Thr34 (B) and Thr75 (C), GluR1 at Ser845 and Ser831 (D), NR1 at Ser897 (E), and synapsin I at Ser9, Ser549 and Ser603 (F). Typical immunoblots for detection of phospho-Thr34, phospho-Thr75 and total DARPP-32 are shown in (A). Data represent means ± SEM for 12 experiments. *p <0.05, **p < 0.01, ***p < 0.001 compared with vehicle treated mice; Student’s t-test

Fig. 5. Expression of PDE4B in the cortex

Double immunostaining of mouse brain tissues (A) and cortical tissues (cingulate cortex, area 1) (B) with DARPP-32 and PDE4B antibodies. High magnification pictures in cingulate cortex, area 1 (B) correspond to the boxed area in the merged picture of brain tissues (A). Arrows indicate neurons expressing PDE4B, but not DARPP-32. Scale bars, 250 μm for A and 10 μm for B

Fig. 6. Expression of DARPP-32 in cortical and striatal tissues from GAD67-GFP knock-in mice

Immunostaining of brain tissues (A), cortical tissues (B), and striatal tissues (C) from GAD67-GFP knock-in mice with a DARPP-32 antibody. Fluorescence of GFP and rhodamine-red for the DARPP-32 antibody was detected. In brain tissues (A), striatal, but not cortical, signals for GAD-EGFP and DARPP-32 are saturated, and therefore images of striatum (Str) and nucleus accumbens (NAc) obtained with reduced sensitivity conditions are shown (A, insets). High magnification pictures in cingulate cortex, area 1 (B) and dorsal striatum (C) correspond to the boxed areas in the merged picture of brain tissues (A and inset). Arrows indicate DARPP-32-negative/GAD67-EGFP-positive neurons, and an arrowhead indicates DARPP-32-positive/GAD67-EGFP-negative neurons. Scale bars, 250 μm for A, 100 μm for A inset and 10 μm for B and C

Fig. 7. Effect of rolipram on prepulse inhibition (PPI) of the startle response in wild-type (A) and DARPP-32 knockout (B) mice

Mice were injected intraperitoneally with vehicle or rolipram (3.0 mg/kg) and tested 15 min later. The effect of stimulus intensity on % PPI is shown. Data represent means ± SEM for 15-19 mice in each experimental group. * p < 0.05, ***p < 0.001 compared with vehicle-treated mice; two-way repeated measures ANOVA followed by Bonferroni test