Regulation of Signal Transduction in the Striatum by Typical and Atypical Antipsychotic Drugs

The only effective therapy for schizophrenia is based on the use of antipsychotic drugs. These substances act as dopamine D2 receptor antagonists and can be classified as typical or atypical. Typical antipsychotics, e.g. haloperidol, reduce the positive symptoms of schizophrenia, but have no effect in treating the negative symptoms of the disease. In addition, prolonged use of these drugs often causes extrapyramidal side effects (EPS). Treatment with the atypical antipsychotic, clozapine, ameliorates both the negative and the positive symptoms of schizophrenia with a much lower incidence of EPS. However, the use of clozapine is limited by the occurrence of agranulocytosis in about one percent of the patients. The aim of this thesis is to examine the effects produced by a haloperidol and clozapine on the state of phosphorylation of proteins involved in postsynaptic and presynaptic transmission within the basal ganglia, a group of brain structures critically involved in motor control. The results of these studies show the existence of differences in the effects produced by haloperidol and clozapine, which may help to identify specific molecular determinants involved in the generation of EPS. Acute administration of haloperidol or clozapine increases the phosphorylation of the dopamineand cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at the cAMP-dependent protein kinase (PKA) site (Thr34). This phosphorylation converts DARPP-32 into an inhibitor of protein phosphatase-1 (PP-1), thereby amplifying cAMP/PKAmediated responses. Haloperidol also stimulates the phosphorylation of the two mitogenactivated protein kinases (MAPK), extracellular signal-regulated kinases 1 and 2 (Erk1/2). In addition, haloperidol phosphorylates and activates the transcription factors cAMP response element binding protein (CREB) and Elk-1. In contrast, clozapine significantly reduces the phosphorylation and activation of Erk1/2, CREB and Elk-1. The stimulation of Erk1/2 phosphorylation produced by haloperidol is not prevented by genetic inactivation of DARPP-32, indicating that the regulation exerted by haloperidol on the MAPK cascade is independent of activation of the cAMP/PKA/DARPP-32 cascade. Repeated administration of haloperidol for 14 days followed by a challenge dose of the same drug on day 15, led to a reduction in the stimulation of Erk1/2 phosphorylation, suggesting the development of tolerance after chronic treatment. In contrast, the ability of clozapine to decrease Erk 1/2 phosphorylation was not affected by prolonged administration. AMPA receptors are widely distributed in the brain, and are regulated by phosphorylation on different subunits. The GluR1 subunit is phosphorylated by PKA at Ser845, and by calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) at Ser 831. Haloperidol produced a transient and dose-dependent increase GluR1 phosphorylation at Ser845, without affecting Ser831 phosphorylation. In the striatum, PKA is activated via dopamine D1 receptors in striatonigral neurons and via adenosine A2A receptors in striatopallidal neurons. Blockade of A2A, but not of D1, receptors prevented haloperidol from increasing Ser845 phosphorylation. The ability of haloperidol to phosphorylate Ser845 was also abolished by genetic inactivation of DARPP-32. This indicates that haloperidol regulates the state of phosphorylation of GluR1 at Ser845 in striatopallidal neurons and that this effect depends both on PKA activation and PP-1 inhibition (achieved via DARPP-32 phosphorylation at Thr34). Clozapine had no effect on Ser845, but it produced a long-lasting (≥120 min) decrease in Ser831 phosphorylation. In the basal ganglia, antipsychotics block dopamine D2 autoreceptors, thereby stimulating dopamine synthesis and release. The turnover of dopamine depends on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, which is regulated by phosphorylation at multiple seryl residues. Acute administration of haloperidol, but not clozapine, stimulated the phosphorylation of TH at Ser19, Ser 31 and Ser40. This effect was abolished in dopamine D2-receptor knockout mice. Moreover, blockade of Erk1/2 activation by SL327 prevented haloperidol induced phosphorylation of TH at Ser 31 and Ser40, showing that MAPK are involved in TH regulation in vivo.