Schizophrenia is the major psychiatry disorder with prevalence of approximately 1% of worldwide population 1 . It is characterized by psychosis, apathy and social withdrawal, and cognitive impairment, which results in altered functioning in many aspects of life. It is a life-long disorder and, although exact disease cause remains unknown, it is known that the disease can be triggered by a combination of genetic and environmental factors 2 .

It is well known that dopamine-mediated neurotransmission imbalance is associated with schizophrenia 3 4 5 and several studies have demonstrated altered activity of prefrontal cortex (PFC) of schizophrenics during hallucinations, delusions and cognitive tests 6 7 8 . Recent studies have demonstrated change in the expression of two proteins involved with dopaminergic signaling modulation in the schizophrenics PFC 9 10 11 . It was reported decrease of dopamine and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein of relative molecular mass 32,000 (DARPP-32) and upregulation of Neuronal Calcium Sensor-1 (NCS-1) expression 12 13 .

Dopamine receptors are G protein-coupled receptors classified into two subtypes: D₁-like receptor subtypes (D₁, D₅), positively coupled to adenylyl cyclase (G_s), and D₂-like receptor subtypes (D₂, D₃, D₄), negatively coupled to adenylyl cyclase (G_i) 14 . D₁ receptor subtype, when activated, enhances phosphorylation of DARPP-32 at threonine 34 (Thr34) by protein kinase A (PKA) 15 16 which is counteracted by the action of D₂ receptors 17 . DARPP-32, phosphorylated at Thr34, inhibits protein phosphatase-1 (PP-1), acting as a key downstream effector in transducing dopamine signaling, integrating the signaling of different neurotransmitters and neuromodulators 18 .

Desensitization and internalization of a receptor is a process that reduces cell responsiveness to neurotransmitters 19 . Dopamine D₂ receptor internalization is regulated by G-protein-coupled receptor kinase 2 (GRK2). GRK2 phosphorylates D₂ receptor, triggering the receptor sequestration by arrestin 20 . However, NCS-1, which is a member of EF-hand superfamily, forms a complex with GRK2 and D₂ receptors, inhibiting this receptor phosphorylation and consequently, inhibiting its internalization 21 . Recently, it was demonstrated the colocalization of NCS-1 and D₂ receptors in pre and post-synaptic structures of pyramidal neurons and interneurons in primate prefrontal cortex (PFC) 22 .

Antipsychotics are drugs used in pharmacological treatment to diminish symptoms of schizophrenia. Because of their differences in receptor affinities and side effects, they are classified as typical and atypical. Typical antipsychotics, such as haloperidol (HAL), are D₂ antagonists with strong affinity and slow dissociation kinetics from receptor, which is frequently associated with extrapyramidal effects 2 23 . Atypical antipsychotics, such as clozapine (CLO) and risperidone (RIS) show reduced affinity to D₂ and are antagonists of serotonin receptors. Due to these properties, lower levels of extrapyramidal effects are observed in treatments with atypical antipsychotics 23 . Although it is well known that antipsychotics modulate schizophrenia symptoms, the molecular and biochemical mechanisms implicated in this improvement is not well established.

Because of the functions of both DARPP-32 and NCS-1 in dopaminergic signaling, the main target of antipsychotics, and their alterations in PFC of schizophrenia patients, we studied the effects of typical and atypical antipsychotics in expression of DARPP-32 and NCS-1 in five regions of rat's brain: prefrontal cortex, hippocampus, striatum, cortex and cerebellum. We did not observe any alterations in both DARPP-32 and NCS-1 expression levels after chronic treatment with antipsychotics 24 . However, one of the limitations of our study was that fact of all the animals were wild type. Since PC12 cells are commonly used as a dopaminergic model, in order to investigate involvement of NCS-1 in dopaminergic intracellular signaling pathways, we established PC12 cell line overexpressing NCS-1 by stable transfection (PC12 Clone). We observed downregulation of DARPP-32 in PC12 Clone cells (Souza, submitted). Thus, our purpose was to study if upregulation of NCS-1 modulates the effects of typical and atypical antipsychotics in the expression of DARPP-32 and NCS-1. For this, we chronically treated PC12 cells wild type (PC12 WT) and PC12 cells Clone cells with antipsychotics and investigated the levels of proteins by western blot.

Drugs that target dopamine D₂ receptors are commonly used in the treatment of schizophrenics 2 , which is an evidence of the involvement of dopaminergic signaling pathway in this disorder. However, because of several studies suggest that there are no changes in D₂ receptor expression in brains of schizophrenic patients 25 , it was postulated that changes in receptor-associated signaling complex and second messengers might be involved in the dopamine disturbance in these patients 9 25 .

NCS-1 inhibits phosphorylation of dopamine D₂ receptor by GRK2 and consequently D₂-Arrestin-GRK2 complex formation, which is responsible for internalization and desensitization of D₂ 26 27 28 . Also, it was reported a colocalization of NCS-1 and D₂ in PFC of primates 29 . Since it was previously shown that NCS-1 is upregulated in PFC of schizophrenic subjects 12 13 , and that there was no alteration in NCS-1 levels in brain of rats chronically treated with typical or atypical antipsychotics 24 , we hypothesized if upregulation of NCS-1 could be modulated by antipsychotic treatment. Thus, we investigated the levels of NCS-1 in PC12 Clone cells, which overexpressed NCS-1, after 14 days of typical or atypical antipsychotics treatments. We observed that there was no alteration in the levels of NCS-1 in PC12 wt and PC12 Clone cells after chronic antipsychotic treatment (Figure 1).

Recently, it was showed that levels of DARPP-32, a key downstream effector in transducing dopamine signaling, is decreased in PFC of schizophrenia subjects 10 11 and that there are DARPP-32 genetic variations associated with PFC cognitive functions 30 . However, we did not find alterations in the expression of DARPP-32 in brain of rats after chronic treatment with typical or atypical antipsychotics 24 . In a previous study we demonstrated that DARPP-32 levels are donwregulated in PC12 cells overexpressing NCS-1 (Souza, submitted). Thus, because the rats used were wild type, we addressed if the downregulation of DARPP-32 could be modulated by antipsychotic treatments. Therefore, we treated PC12 Clone cells with typical or atypical antipsychotics for 14 days. We observed that there was no change in the levels of DARPP-32 in both PC12 wt and PC12 Clone cells after chronic antipsychotic treatment (Figure 2).

We previously demonstrated that levels of NCS-1 and DARPP-32 are not altered in brains of rats chronically treated with typical and atypical antipsychotics 24 . However, one of the limitations of the study was the fact of all rats were wild type. Thus, we used PC12 cells overexpressing NCS-1, which we observed a decreased levels of DARPP-32 (Souza, submitted), to investigate if there was alteration in levels of both NCS-1 and DARPP-32 protein expression after 14 days treatment with typical or atypical antipsychotics. We demonstrated that the levels of both proteins are not modulated by antipsychotics in PC12 Clone cells. Therefore, our findings reinforce the suggestion that both downregulation of DARPP-32 and upregulation of NCS-1 reported to occur in the PFC of schizophrenia patients, might be associated with the psychopathology of the disorder but not with antipsychotic treatment (Figure 3). Taking into consideration that there is no good model able to mimic the major characteristics of schizophrenia, this is an idea difficult to be tested. Thus, only an extensive investigation of intracellular integrators and modulators will shed more light on the signaling mechanisms involved in this serious psychiatric disorder and its treatment.

The authors declare that they have no competing interests.

BRS, BSM, ESM, MMG and DSC performed the experiment. BRS, DVFR and RPS analyzed the data. BRS, KCL, DMM and HJR wrote the manuscript. AJ developed a new research tool. MARS conceived the study. All authors read and approved the final manuscript.