Reduced prefrontal cortex DARPP-32 mRNA in completed suicide victims with schizophrenia

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Abstract

Dopamine-and-cAMP-regulated neuronal phosphoprotein (32 kDa) (DARPP-32), encoded by PPP1R1B, is expressed in brain regions receiving dopaminergic projections, including the prefrontal cortex (PFC), and is implicated in the pathophysiology of schizophrenia. The broad functional capacity of DARPP-32 has potential relevance to both psychotic and negative symptoms of schizophrenia. We wished to determine if DARPP-32 gene expression and variation at selected SNPs correlated significantly with patient phenotypes. We performed RT-PCR to quantify DARPP-32 mRNA from brain samples (Brodmann Area 46) donated by the Stanley Medical Research Institute (SMRI, Array Collection): 35 from unaffected controls (UC), 35 from patients with schizophrenia (SCZ), and 35 with bipolar disorder (BP). Relative mRNA expression was calculated in relation to the housekeeping gene Cyclophilin. SNP genotyping was conducted by PCR on DNA obtained from Brodmann Area 46. We found a significant difference in gene expression levels between SCZ patients who died by suicide (SCZ-S) (n = 6) vs. other causes of death (SCZ-NS) (P < 0.004), as well as between SCZ-S and UC (P < 0.04). We genotyped the intron SNP rs907094 and found that the SCZ-S group was more similar to UC than to the SCZ-NS population. DARPP-32 expression differences between SCZ-S, SCZ-NS, and UC populations are consistent with previous literature suggesting that serotonin system components are also altered in suicide. Work in a larger sample is needed to confirm these findings.

Introduction

Schizophrenia affects 0.5–1% of the population worldwide, and the etiology remains unknown (Wong and Van Tol, 2003). Death by suicide among individuals with schizophrenia is significantly higher than that of the general population and rates have been reported to be as high as 10%; 18–55% of patients with schizophrenia attempt suicide at some time in their lives (Radomsky et al., 1999). Risk factors for suicidal ideation and completed suicide have been observed from epidemiological studies, but the serotonin system has been the main candidate molecular pathway implicated in suicidal behavior (Mann, 2003).

One major downstream component of serotonin receptor signaling is dopamine-and-cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) (Fig. 1). DARPP-32 acts as a central molecular switch in the regulation of neuronal signaling, interacting with a variety of neurotransmitters, such as dopamine (DA), serotonin (5-HT), glutamate and other neuropeptides, neuromodulators, voltage-dependent ion channels, opiate receptors, and steroid hormones in dopaminergic, serotonergic, GABAergic and glutamatergic neurons (Reis et al., 2007; Svenningsson et al., 2004). It is encoded by PPP1R1B and is localized to regions that receive dopaminergic projections (Svenningsson et al., 2004). DARPP-32 expression is enriched in the prefrontal cortex (PFC), striatum (caudate and putamen), and in GABAergic medium sized spiny neurons (Meyer-Lindenberg et al., 2007), all of which are implicated in the pathophysiology of schizophrenia (Ross et al., 2006).

In particular, reduced DARPP-32 protein has been reported in postmortem dorsolateral prefrontal cortex (DLPFC) from schizophrenia patients (Albert et al., 2002). The DLPFC is involved in aspects of cognition that are impaired in schizophrenia: reward-related learning, working memory, context processing, and executive function. (Meyer-Lindenberg et al., 2007). Although schizophrenia has not been directly associated with PPP1R1B variation in core promoter and exon regions (Li et al., 2006, Yoshimi et al., 2008), a decrease in DARPP-32 protein expression has been correlated with reduced function of the DLPFC circuit in schizophrenic (Li et al., 2006) and bipolar patients (Ishikawa et al., 2007). Also, the chromosomal region in which PPP1R1B is located (17q12), shows genetic linkage to both schizophrenia and bipolar disorder (reviewed in Yoshimi et al., 2008).

Selective serotonin reuptake inhibitor (SSRI) antidepressants such as fluoxetine, have been shown to enhance the cAMP-dependent protein kinase A (PKA) pathway at several levels in the hippocampus and frontal cortex (Svenningsson et al., 2002), through DARPP-32 modification of AMPA receptor GluA1 subunit phosphorylation, and amplified AMPA receptor signaling (Alt et al., 2006). Repeated administration of fluoxetine has been shown to increase binding at dopamine D2 receptors and D2 receptor mRNA in the nucleus accumbens of rats, as well as D3 receptor mRNA in the shell of the nucleus accumbens (reviewed in Sekine et al., 2007). In mouse knockout models of DARPP-32 the efficacy of fluoxetine is significantly reduced, indicating that DARPP-32 mediates the effect of this particular antidepressant. In particular, fluoxetine regulates multiple-site phosphorylation of DARPP-32 at Thr34 and Thr75 in mice, which show similar brain distributions of DARPP-32 to that of humans (Svenningsson et al., 2002). Fluoxetine appears to mediate DARPP-32 phosphorylation through the 5-HT2, 5-HT4, and 5-HT6 receptor subtypes (Svenningsson et al., 2002a) by increasing levels of synaptic 5-HT, and elevating intracellular cAMP and PKA (Fig. 1).

There is increasing evidence that the activity of midbrain DA neurons in the ventral tegmental area (VTA) is mediated by their interaction with afferent serotonergic projections from axon terminals originating in the midbrain raphe nuclei (Di Mascio and Esposito, 1997, Dhir and Kulkarni, 2007). The dorsal and ventral striatum, including the nucleus accumbens, are densely innervated by nigral DA fibers, 5-HT fibers, as well as cortical glutamatergic afferent fibers (reviewed in Muñoz et al., 2003). Fluoxetine has been shown to reduce the basal firing rate of DA cells in the VTA by increasing serotonergic transmission (Di Mascio and Esposito, 1997). Fluoxetine effects are enhanced when administered in combination with a selective D3-preferring D2/D3 receptor agonist, suggesting a functional interaction between the dopamine and serotonin systems (Dhir and Kulkarni, 2007). DARPP-32 is highly concentrated in the neostriatum and the nucleus accumbens (Greengard, 2001) and several studies have suggested that acute fluoxetine treatment may decrease extracellular DA levels in the striatum and nucleus accumbens (reviewed in Penttila et al., 2004).

Depression and depressive symptoms are often present in patients with schizophrenia, and these are typically treated with antidepressant drugs (Siris et al., 2001). A variety of changes in the 5-HT system are associated with suicidal behavior in both mood and psychotic disorders (De Luca et al., 2005, De Luca et al., 2007a, Mann, 2003). Because serotonin receptors are part of the complex interactome of DARPP-32, it is plausible that altered expression of DARPP-32 may also be important in suicidal behavior.

There is also evidence to support that DARPP-32-dependent signaling mediates the action of multiple drugs of abuse that affect dopamine, serotonergic neurotransmission in the forebrain, working memory, and motivation (Svenningsson et al., 2005). DARPP-32 may also play a role in the pathophysiology of symptoms in schizophrenia by modulating D2 (Meyer-Lindenberg et al., 2007) and D1 receptor signaling (Fernandez et al., 2006). DARPP-32 integrates glutamatergic and dopaminergic signaling by transmission of the signal from any one of its input receptors through protein phosphatase-1 (PP-1) which has multiple downstream phosphorylation targets (Fig. 1). (Greengard, 2001). Recently, glutamatergic receptor variants (GRIK2 and GRIA3) have been associated with increased suicidal ideation in patients with depression being treated with the SSRI citalopram (Laje et al., 2007). Serotonergic regulation of DARPP-32 phosphorylation appears to be counteracted by the application of the atypical antipsychotic clozapine, which has high affinity for 5-HT2 and 5-HT6 receptors (Svenningsson et al., 2002a).

Based on the association of serotonin system changes with suicide, previous reports of DARPP-32 alterations in schizophrenia, and the interactions between serotonin signaling and DARPP-32, we hypothesized that DARPP-32 might also be altered in patients with schizophrenia who died by suicide. Here we describe the analysis of DARPP-32 mRNA levels and polymorphic variation in the PPP1R1B gene in postmortem brain samples from the Stanley Medical Research Institute (SMRI) Array Collection. These samples were donated by patients with schizophrenia or bipolar disorder and by unaffected controls. Some of these subjects died by suicide, allowing us to test our hypothesis by comparing these cases to subjects who died from other causes.

Section snippets

Postmortem brain RNA and DNA samples

The SMRI Array Collection consists of mRNA and genomic DNA samples that have been extracted from dorsolateral prefrontal cortex (Brodmann Area 46) from individuals diagnosed with schizophrenia, bipolar disorder, and unaffected controls (35 in each group; total 105 samples) (Higgs et al., 2006). The samples from patients with schizophrenia and unaffected controls are uniformly Caucasian. There is one bipolar sample of Native American origin; the remaining bipolar samples are Caucasian. The

Results

SNP genotypes rs907094 and rs9532 were selected to cover both major haplotype blocks (Fig. 2) in the Caucasian HapMap population. We found that SNP rs907094 genotype frequencies in SCZ-S group more closely resembled that of the UC group than the SCZ-NS group (Fig. 3, Table 3, Table 4) although the variability between SCZ-S vs. SCZ-NS did not quite reach statistical significance (P = 0.078). SCZ-NS patients have a greater than fourfold difference in the C/T genotype frequency of rs907094 (3.6%) in

Discussion

The main limitation of our study is the sample size, in which we report comparisons with a small group of schizophrenia patients who died by suicide (n = 6). Given the inherent variability of human post-mortem samples arising from differences in disease course, treatment and tissue acquisition, replication in other independent samples is needed before making more general conclusions about molecular findings. However, our data show significant differences in DARPP-32 mRNA levels between SCZ-S and

Role of funding source

Funding for this study was provided by the Canadian Institutes for Health Research (CIHR) grant numbers GMH-79044 and MOP 79525, the National Alliance for Research in Schizophrenia and Depression (NARSAD), and CNPq-Brazil. Each of these sources had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report and the decision to submit the paper for publication.

Contributors

A.H.C.W. designed the study. L.A.F. conducted gene expression assays and statistical analysis, managed literature searches and analyses, and wrote the first draft of the manuscript as well as the protocol pertaining to her laboratory work. R.P.S. conducted genotyping and statistical analysis, and wrote the protocol pertaining to his laboratory work. All authors have contributed to and have approved the final manuscript.

Conflict of interest

J.L.K. is a consultant for GlaxoSmithKline. All other authors deny any conflicts of interest.

Acknowledgements

Postmortem brain tissue mRNA was donated by The Stanley Medical Research Institute's Array Collection courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, Serge Weis, and Robert H. Yolken.

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