The leukocytes expressing DARPP-32 are reduced in patients with schizophrenia and bipolar disorder

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Abstract

Bipolar disorder (BPD) and schizophrenia (SCZ) are severe disorders representing an enormous social, familiar and individual burden, being SCZ the most disabling psychiatric disorder characterized by psychosis and cognitive impairment. It is well known that SCZ and BPD are associated with abnormalities in dopamine signaling pathway. Recent data in the literature have demonstrated altered expression levels of some proteins involved in the modulation of this pathway in both brain and peripheral tissues. It was shown that protein and mRNA levels of dopamine and cAMP regulated phosphoprotein (DARPP-32) were downregulated in dorsolateral prefrontal cortex (DLPFC) of patients with SCZ or BPD when compared to controls. Due to the difficulty to access brain tissue and the absence of objective laboratory tests for bio-markers, we measured DARPP-32 expression in blood cell sub-populations (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) taking advantage of the close relation of nervous and immune systems. Using flow cytometry as the analytical method, our results have shown that the DARPP-32 expression was diminished in CD4+ T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and was also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that DARPP-32 expression in immune cells agrees with reports of reduced DARPP-32 protein in the DLPFC of BPD or SCZ patients. Our data suggest that DARPP-32 expression in PBMC could be used as a source of bio-markers to help in the treatment response of neuropsychiatry disorders as a window to the changes in the brain of those patients.

Introduction

Schizophrenia (SCZ) is the most disabling psychiatry disorder characterized by psychotic positive symptoms, negative symptoms and cognitive impairment. It is a life-long disorder with a world-wide prevalence of 1% (McGurk et al., 2003). Bipolar disorder (BPD) is in turn a frequent and severe disease which affects mood, behavior, and thinking, switching between mania and depression (Goodwin and Jamison, 1990).

Evidence indicates abnormalities in the dopamine system in both SCZ and BPD (Manji and Lenox, 2000, Gao et al., 2005, Seeman et al., 2006, Horacek et al., 2006). Recently, alterations in intracellular signal integrating proteins associated with both disorders were shown (Emamian et al., 2004, Souza et al., 2006a, Souza et al., 2006b, Reis et al., 2007, Volkow et al., 2007). It was demonstrated, in post-mortem brain tissue, decreased levels of dopamine and cyclic adenosine 3:5-monophosphate-regulated phosphoprotein of relative molecular mass 32,000 (DARPP-32) expression in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia (Albert et al., 2002, Ishikawa et al., 2007).

DARPP-32 is a major player in the transduction of dopaminergic signaling, integrating signals from different converging pathways in neurons (Svenningsson et al., 2004). Activation of protein kinase A (PKA) results in phosphorylation of DARPP-32 at Thr-34 (Nishi et al., 1997) and (Svenningsson et al., 2000). In this phosphorylated state, DARPP-32 shows an inhibitory effect on protein phosphatase 1 (PP1) which regulates the activity of receptors, channels and transcriptional factors (Huang et al., 1999) and (Svenningsson et al., 2004). DARPP-32 effect is terminated by dephosphorylation at Thr-34 residue by protein phosphatase 2B (PP-2B, calcineurin) (Hernandez-Lopez et al., 2000). Several functions of this phosphoprotein were recently reviewed such as involvement in motor control, drug abuse, cell differentiation, tissue development, morphology and function of several organs, and anti-apoptotic effects (Souza et al., 2006a, Souza et al., 2006b, Nairn et al., 2004), (Reis et al., 2007) and (Svenningson et al., 2004).

There is accumulating evidence showing the bi-directional correlation between the central nervous system (CNS) and the immune system (IS), and lymphocytes have a central role in this inter-communication (Gladkevich et al., 2004). Currently, due to the lack of specific biomarkers, the understanding of the development of mental disorders, including SCZ and BPD is still unknown. Thus, the identification of markers in cells from peripheral blood of the patients would be a convenient and accessible way to study the signaling pathways involved in these disorders as a window to the changes in the brain of those patients.

Various studies showed similarities, between the nervous and immune systems, in receptor expression and intracellular biochemical pathways of neurons, glia and leukocytes. Given that, lymphocytes are being used to identify peripheral bio-markers in neuropsychiatric disorders (Gladkevich et al., 2004, Meredith et al., 2005, Du et al., 2006, Pellicano et al., 2007).

Then, in this work, DARPP-32 was chosen as a candidate in the search of peripheral bio-markers of SCZ and BPD. Using flow cytometry, we measured the intracellular levels of DARPP-32 in monocytes, NK cells and T and B lymphocytes. We have shown that DARPP-32 expression is decreased in immune cells of patients with SCZ and BPD. Our data suggest that DARPP-32 expression in lymphocytes and monocytes could be used as a source of bio-markers to help in the treatment response of neuropsychiatry disorders as a window to the changes in the brain of those patients.

Section snippets

Materials

The following materials were purchased from indicated commercial sources: Ficoll/Hypaque (Sigma, St. Louis, MO, USA); anti-CD3 monoclonal antibodies (PharMingen-Becton Dickinson, San Diego CA, USA); anti-CD28 monoclonal antibodies (PharMingen-Becton Dickinson, San Diego CA, USA); RPMI 1640, Sigma, St. Louis, MO, USA) bovine fetal serum (GIBCO, long Island, NY, USA); l-glutamine penicillin/streptomycin (GIBCO, long Island, NY, USA); PBS (Sigma St. Louis, MO, USA). Anti-CD19, anti-CD14 and

Comparison of leukocyte sub-populations among patients with schizophrenia or bipolar disorder

To investigate the expression of DARPP-32, in leukocyte sub-populations, the frequency of CD4+ T and CD19+ B cells, CD14+ monocytes and of CD56+ NK cells in patients with SCZ and BPD was analyzed for standardization.

The expression of CD4+ T lymphocytes of patients with SCZ (39.55 ± 10.50%) did not differ from controls (40.98 ± 6.09%), however, it was increased in samples from patients with BPD (54.32 ± 4.17%) compared to SCZ and controls (Fig. 2a). The expression of CD19+ B lymphocytes did not vary

Discussion

In this work we tried to characterize DARPP-32 as a putative peripheral bio-marker for SCZ or BPD, measuring its expression in PBMC (CD4+ T cells, CD19+ B cells, CD56+ NK cells and CD14+ monocytes). The identification of markers in cells from peripheral blood of the patients would be helpful as a model to study intracellular cascades related to neuropsychiatric disorders. Despite the apparent lower number of studied subjects and controls the flow cytometry has consistent results shown in the

Conclusion

These results reinforce the idea that lymphocytes may therefore function as an easily accessible model to study the dopamine intracellular signaling in the cells of each patient and the expression of proteins on blood cells as possible peripheral biomarkers of prognostic and treatment response of psychiatric disorders.

Acknowledgments

The authors thank Vitor Bortolo Rezende for his help in improving the artwork quality of Fig. 1. This work was funded by FAPEMIG and CNPq. We are very grateful to all those individuals who have participated in this study and for the Guggenheim Foundation.

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    Present address: Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Antonio Carlos av, 6627, Belo Horizonte, Minas Gerais, Brazil.

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