Schizophrenia is the major psychiatry disorder with prevalence of approximately 1% of worldwide population . 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 .
It is well known that dopamine-mediated neurotransmission imbalance is associated with schizophrenia [3–5] and several studies have demonstrated altered activity of prefrontal cortex (PFC) of schizophrenics during hallucinations, delusions and cognitive tests [6–8]. Recent studies have demonstrated change in the expression of two proteins involved with dopaminergic signaling modulation in the schizophrenics PFC [9–11]. 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 (Gs), and D2-like receptor subtypes (D2, D3, D4), negatively coupled to adenylyl cyclase (Gi) . 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 . 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 .
Desensitization and internalization of a receptor is a process that reduces cell responsiveness to neurotransmitters . Dopamine D2 receptor internalization is regulated by G-protein-coupled receptor kinase 2 (GRK2). GRK2 phosphorylates D2 receptor, triggering the receptor sequestration by arrestin . 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 . 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) .
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 . 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 . 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.