Glutamate regulation of DARPP-32 phosphorylation in neostriatal neurons involves activation of multiple signaling cascades

Abstract

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca2+/protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca2+/protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.

Fig. 1.

Regulation of DARPP-32 phosphorylation at Thr-34 and Thr-75 by glutamate and the role of ionotropic glutamate receptors in neostriatal slices. Neostriatal slices were incubated in the absence (A) or presence (B–D) of an AMPA receptor antagonist, CNQX (20 μM) (B), an NMDA receptor antagonist, MK801 (100 μM) (C), or CNQX plus MK801 (D) for a total of 20 min, with addition of l-glutamic acid HCl (5 mM) for the indicated times at the end of incubation. (Upper) Phosphorylation at Thr-34. (Lower) Phosphorylation at Thr-75. A.U., arbitrary units. Data represent means ± SEM for 4–10 experiments. *, P < 0.05; **, P < 0.01, compared with 0 min. (E) Typical immunoblots for detection of phospho-Thr-34, phospho-Thr-75, and total DARPP-32 in the same membrane.

Fig. 2.

Role of mGlu receptors in the regulation of DARPP-32 phosphorylation by glutamate. Neostriatal slices were preincubated in the presence of an mGlu5 receptor antagonist, MPEP (10 μM) (A), an mGlu1 receptor antagonist, LY367385 (100 μM) (B), or MPEP plus LY367385 (C) for a total of 20 min, with addition of glutamate (5 mM) for the indicated times. Data represent means ± SEM for 6–12 experiments. *, P < 0.05; **, P < 0.01, compared with 0 min.

Fig. 3.

Role of mGlu receptors in the glutamate-induced increase in DARPP-32 phosphorylation under conditions of NMDA and AMPA receptor blockade. Neostriatal slices were preincubated in the presence of MK801 (100 μM)/CNQX (20 μM) (A), MK801/CNQX plus MPEP (10 μM) (B), or MK801/CNQX plus LY367385 (100 μM) (C) for a total of 20 min, with addition of glutamate (5 mM) for the indicated times. Data represent means ± SEM for 4–7 experiments. *, P < 0.05; **, P < 0.01, compared with 0 min. A is reproduced from Fig. 1D for comparison.

Fig. 4.

Role of ERK and PLCβ in the glutamate-induced increase in DARPP-32 phosphorylation under conditions of NMDA and AMPA receptor blockade. Neostriatal slices were preincubated in the presence of MK801 (100 μM)/CNQX (20 μM), MK801/CNQX plus U0126 (40 μM), or MK801/CNQX plus U73122 (25 μM) for a total of 70 min, with addition of glutamate (5 mM) for 10 min. The quantified data were normalized to values obtained from slices treated with glutamate in the presence of MK801/CNQX. Data represent means ± SEM for 5–8 experiments. *, P < 0.05; **, P < 0.01, compared with MK801/CNQX alone; †, P <0.05; ‡, P < 0.01, compared with glutamate in the presence of MK801/CNQX; §§, P < 0.01, compared with MK801/CNQX plus U0126.

Fig. 5.

Role of nNOS/NO/cGMP/PKG signaling in the regulation of DARPP-32 phosphorylation at Thr-34 by glutamate. Neostriatal slices were preincubated in the presence of a dopamine D1 receptor antagonist, SCH23390 (1 μM) (A), an adenosine A_2A receptor antagonist, ZM241385 (1 μM) (B), an nNOS inhibitor, 7-NINA (10 μM) (C), or a soluble guanylyl cyclase inhibitor, ODQ (30 μM) (D), for a total of 15 min, with addition of glutamate (5 mM) for the indicated times. Data represent means ± SEM for 3–5 experiments. *, P < 0.05; **, P < 0.01, compared with 0 min.

Fig. 6.

Role of protein phosphatases in the regulation of DARPP-32 phosphorylation by glutamate. Neostriatal slices were preincubated in the presence of cyclosporin A (1 μM) (A) or okadaic acid (1 μM) (B) for a total of 65 min, with addition of glutamate (5 mM) for 5 min. Data represent means ± SEM for 4–7 experiments. *, P < 0.05; **, P < 0.01, compared with 0 min.

Fig. 7.

Regulation of nNOS Ser-847 dephosphorylation by PP inhibitors in neostriatal slices. (Right) Neostriatal slices were incubated with calyculin A (CalyA, 200 nM), okadaic acid (OKA, 200 nM and 1 μM), or cyclosporin A (CyA, 10 μM) for 60 min. Immunoblots for detection of phospho-Ser-847 nNOS and total nNOS in the same membrane are shown (Left). Data represent means ± SEM for 4–7 experiments. **, P < 0.01, compared with control; ††, P < 0.01, compared with CalyA (200 nM); §§, P < 0.01, compared with OKA (200 nM).

Fig. 8.

Signaling cascades activated by glutamate NMDA/AMPA and mGlu receptors. Glutamate activates multiple signaling cascades by activation of NMDA/AMPA and mGlu1/5 receptors (A), leading to the modulation of DARPP-32 phosphorylation at Thr-34 (B) and Thr-75 (C) and the regulation of PP-1 and PKA activities. Pathways leading to increased phospho-Thr-34 DARPP-32 and increased PP-1 inhibition are shown in blue, and those leading to decreased phospho-Thr-34 DARPP-32 and decreased PP-1 inhibition are shown in red. Inhibition of PP-1 by phospho-Thr-34 DARPP-32 and inhibition of PKA by phospho-Thr-75 DARPP-32 are shown in orange. (B) In the initial 15–30 s of incubation, activation of NMDA/AMPA and mGlu5 receptors in nNOS-positive interneurons stimulates the synthesis and release of NO, leading to activation in medium spiny neurons of PKG and DARPP-32 Thr-34 phosphorylation. At 1–5 min of incubation, activation of NMDA/AMPA receptors induces Ca²⁺-dependent activation of PP-2B, resulting in the dephosphorylation of Thr-34. At 5–10 min of incubation, there is a gradual increase in DARPP-32 Thr-34 phosphorylation. This increase is due, in part, to activation of a pathway involving mGlu5 receptor/PLC/CK1/phospho-Ser-130 DARPP-32, resulting in inhibition of dephosphorylation of phospho-Thr-34 by PP-2B, and, in part, to activation of signaling through the adenosine A_2A receptor pathway. A synergistic activation of adenosine A_2A receptor signaling involves (i) activation of an mGlu5 receptor/ERK pathway and, through some yet unknown mechanism, increased efficiency of A_2A receptor-induced cAMP formation, and (ii) increased extracellular adenosine levels arising from both medium spiny neurons and presynaptic terminals (15). (C) In the initial 5 min of incubation, activation of NMDA/AMPA receptors induces Ca²⁺-dependent activation of PP-2A, resulting in the dephosphorylation of Thr-75. Subsequently, activation of a pathway involving mGlu1 receptor/PLC/CK1/Cdk5 increases the phosphorylation of Thr-75.