Walter J. Rushlow

Antipsychotics alter the protein expression levels of β-catenin and GSK-3 in the rat medial prefrontal cortex and striatum

BACKGROUND It has been demonstrated that schizophrenics have altered levels and/or phosphorylation states of several Wnt related proteins in the brain, including beta-catenin and GSK-3, and may represent susceptibility loci for schizophrenia. The current study was conducted to assess the effects of antipsychotics on beta-catenin and glycogen synthase kinase-3. METHODS Western blotting and immunocytochemistry were employed to investigate the effects of antipsychotics on beta-catenin and glycogen synthase kinase-3 following acute, subchronic and chronic drug administration. Specificity of the response was tested using additional drugs such as fluoxetine, amphetamine and valproic acid. RESULTS Significant increases in the levels of beta-catenin and glycogen synthase kinase-3 total protein were identified following administration of clozapine, haloperidol or risperidone. The phosphorylation state of GSK-3 was also increased but phosphorylated beta-catenin levels were unaffected. Other drug compounds, with the exception of raclopride, had no effect on either GSK-3 or beta-catenin protein levels or distribution. CONCLUSIONS Targeting of beta-catenin and GSK-3 is a common feature of antipsychotics regardless of class and appears to be mediated by D(2) dopamine receptors. Therefore changes in beta-catenin and GSK-3 may represent one of the mechanisms through which antipsychotics are able to exert behavioral changes.

Activation of the canonical Wnt pathway by the antipsychotics haloperidol and clozapine involves dishevelled-3

Protein kinase B (Akt), glycogen synthase kinase‐3 (GSK‐3) and members of the Wnt signal transduction pathway were recently found to be altered in schizophrenia and targeted by antipsychotic drugs. In the current study, selected Wnt signalling proteins were investigated to determine if they are altered by the antipsychotics clozapine or haloperidol in the rat prefrontal cortex. Pheochromocytoma (PC12) and neuroblastoma (SH‐SY5Y) cells were also used to elucidate how antipsychotics generated the pattern of changes observed in vivo. Western blotting (WB) revealed that treatment with haloperidol or clozapine caused an up‐regulation of Wnt‐5a, dishevelled‐3, Axin, total and phosphorylated GSK‐3 and β‐catenin protein levels. Treatment of PC12 and SH‐SY5Y cells with a variety of pharmacological agents as well as the over‐expression of several Wnt related proteins failed to mimic the pattern observed in vivo following antipsychotic treatment. However, the over‐expression of dishevelled‐3 nearly perfectly duplicated the changes observed in vivo. Immunoprecipitations (IP) conducted using protein isolated from the rat prefrontal cortex indicated that dishevelled‐3 is associated with the D2 dopamine receptor thereby suggesting that antipsychotics may act on dishevelled‐3 via D2 dopamine receptors to initiate a cascade of downstream changes involving Axin, GSK‐3 and β‐catenin that may help to alleviate psychosis in schizophrenic patients.

The effects of antipsychotics on β‐catenin, glycogen synthase kinase‐3 and dishevelled in the ventral midbrain of rats

Protein kinase B and glycogen synthase kinase‐3 have been identified as susceptibility genes for schizophrenia and altered protein and mRNA levels have been detected in the brains of schizophrenics post‐mortem. Recently, we reported that haloperidol, clozapine and risperidone alter glycogen synthase kinase‐3 and β‐catenin protein expression and glycogen synthase kinase‐3 phosphorylation levels in the rat prefrontal cortex and striatum. In the current study, β‐catenin, adenomatous polyposis coli, Wnt1, dishevelled and glycogen synthase kinase‐3 were examined in the ventral midbrain and hippocampus using western blotting. In addition, β‐catenin and GSK‐3 were examined in the substantia nigra and ventral tegmental area using confocal and fluorescence microscopy. The results indicate that repeated antipsychotic administration results in significant elevations in glycogen synthase kinase‐3, β‐catenin and dishevelled‐3 protein levels in the ventral midbrain and hippocampus. Raclopride causes similar changes in β‐catenin and GSK‐3 in the ventral midbrain, suggesting that D2 dopamine receptor antagonism mediated the changes observed following antipsychotic administration. In contrast, amphetamine, a drug capable of inducing psychotic episodes, had the opposite effect on β‐catenin and GSK‐3 in the ventral midbrain. Collectively, the results suggest that antipsychotics may exert their beneficial effects through modifications to proteins that are associated with the canonical Wnt pathway.

Enhanced Neurite Outgrowth in PC12 Cells Mediated by Connexin Hemichannels and ATP

Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.

Identification of genes differentially expressed in C6 glioma cells transfected with connexin43

Astrocytes are characterized by extensive gap junctional intercellular communication (GJIC) mediated primarily by channels composed of connexin43. In contrast, C6 glioma cells are deficient in connexin expression and gap junctional communication. Transfection of these glioma cells with connexin cDNAs results in changes in cellular phenotype following increased GJIC. Specifically, connexin expression correlates with reduced cellular proliferation and tumorigenicity. To characterize the role of gap junctions in this growth control, we have screened for changes in gene expression by differential display. We have observed that these changes in GJIC are associated with changes in expression of several genes, including those coding for a number of secreted factors which may play a role in modulating the tumor phenotype of these cells. These include the immediate early gene cyr61, ostoepontin and the KC gene (murine homologue of the human gro gene).

Dose-Dependent Differential Upregulation of CCN1 /Cyr61 and CCN3/NOV by the Gap Junction Protein Connexin43 in Glioma Cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction‐independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6‐glioma cells (C6‐Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma‐overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1‐overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6‐Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43. J. Cell. Biochem. 103: 1772–1782, 2007.

Prefrontal cortical D1 dopamine receptors modulate subcortical D2 dopamine receptor-mediated stress responsiveness

Increased responsiveness to stress plays an important role in the manifestation of schizophrenia symptoms. Evidence indicates that the prefrontal cortex (PFC), and dopamine neurotransmission in the PFC in particular, is involved in the modulation of stress responsiveness. Decreased dopaminergic activity and loss of dopamine fibres have been reported in PFC in schizophrenia patients. Consequently, it was hypothesized that depletion of dopamine in PFC may facilitate increased stress responsiveness. Adult Sprague-Dawley rats received injections of 6-hydroxydopamine or saline bilaterally into the medial PFC (mPFC) following desipramine pretreatment to selectively deplete dopaminergic fibres. Following a 3-wk recovery period, the lesioned and control rats received injections of a D1 or D2 dopamine receptor agonist or vehicle into the mPFC and were immediately subjected to forced swimming as a stressor. Results showed that frequency of locomotion and rearing, behavioural measures indicative of increased dopaminergic activity in the nucleus accumbens (NAc), were significantly increased following stress in prefrontal cortical dopamine-depleted rats. This effect was significantly ameliorated by infusions of a D1 dopamine receptor agonist directly into the mPFC in a dose-dependent manner but not by infusion of a D2 dopamine receptor agonist. In addition, stress-induced behavioural changes in prefrontal cortical dopamine-depleted rats were significantly reduced following selective discrete infusions of a D2 dopamine receptor antagonist into the NAc shell. The results suggest that dopaminergic transmission via D1 receptors in the mPFC modulates D2 dopamine receptor-mediated stress responsiveness in the NAc, a feature that may be disrupted in schizophrenia patients.

Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics

Calcineurin (CN) was recently identified as a susceptibility gene for schizophrenia as well as showing altered RNA expression levels in the post‐mortem brains of individuals with schizophrenia. CN knockout mice show a number of behaviours associated with schizophrenia, including deficits in sensorimotor gating, suggesting a link between CN and psychosis. Concurrently, we found, using genome screening techniques, that antipsychotics alter CN expression levels. Therefore, western blotting, in situ hybridization, immunocytochemistry and phosphatase assays were employed to determine what effect antipsychotics have on CN. The results indicate that clozapine, risperidone and haloperidol cause substantial reductions in the A subunit of CN but not CN B at both the RNA and protein levels in the striatum and prefrontal cortex. The changes could only be observed after repeated treatment with antipsychotics but not after acute administration. The alterations in CN protein levels were specific to antipsychotics and mediated by D2 dopamine receptor antagonism. However, despite reductions in CN protein levels, the phosphatase activity of CN was significantly elevated after treatment with antipsychotics. Collectively the results suggest that CN may be a common target for antipsychotics and that antipsychotic‐induced alterations in CN may represent one of the mechanisms by which antipsychotics alleviate psychosis.

Differential gene expression in premalignant human trophoblast: Role of IGFBP-5

Tumorigenesis results from genetic alterations that occur in a stepwise manner giving rise to cells with increasingly cancer‐like characteristics. We used in vitro propagated first trimester human extravillous trophoblast (EVT) cells to identify genetic changes responsible for the transition of the EVT from a normal to premalignant stage. The model used consisted of a normal invasive EVT (HTR8) cell line and its premalignant derivative (RSVT2/C) generated by transfection with the SV40 Tag and selected using a forced crisis regimen. RSVT2/C display increased proliferative, migratory and invasive behavior, unresponsiveness to anti‐proliferative and anti‐invasive signals of TGFβ and a deficiency in gap junctional intercellular communication. These cells, however, were unable to form colonies on soft agar or tumors in nude mice and are thus defined as premalignant. Differential display revealed 18 gene sequences, 7 with unknown and 11 with known identity, showing altered expression between the normal HTR8 and premalignant RSVT2/C cell lines. The known sequences include the potential tumor suppressors insulin‐like growth factor binding protein (IGFBP)‐5 and fibronectin (FN) and potential protooncogenes such as chromokinesin (KIF4), alternative splicing factor (SF2), dynein, DNA polymerase ϵ (DNApol ϵ) and NF‐κB activating kinase (NAK). The role of the remaining 4 genes upregulated in the premalignant EVT is presently unknown and these are FK506 binding protein (FKBP) 25, histone protein (HP1Hs)‐γ, nucleoporin (Nup) 155 and an 82 kDa acidic human protein. The functional role of IGFBP‐5 was examined in the control of proliferation, migration and invasiveness of RSVT2/C cells measured in vitro. IGFBP‐5 alone had no effect on these properties of RSVT2/C cells. Furthermore, unlike normal EVT cells, RSVT2/C cells exhibited refractoriness to the migration stimulating signals of IGF‐II, which was explained by the loss or downregulation of the IGF type 2 receptor (IGF‐R2). RSVT2/C cells, however, expressed the IGF type 1 receptor (IGF‐R1) and responded to IGF‐I by increased proliferation. This response was blocked with increasing concentrations of IGFBP‐5. These results suggest that the loss of IGFBP‐5 and possibly IGF‐R2, both of which can sequester IGF‐I from IGF‐R1, permits unhindered proliferation of the premalignant EVT in an IGF‐I rich environment of the fetal‐maternal interface. The functions of the other differentially expressed genes, some of which are essential for cell cycle progression or cell survival require further investigation.

The dopamine D2 receptor regulates Akt and GSK-3 via Dvl-3.

The dopamine D2 receptor (D2DR) regulates Akt and may also target the Wnt pathway, two signalling cascades that inhibit glycogen synthase kinase-3 (GSK-3). This study examined whether the Wnt pathway is regulated by D2DR and the role of Akt and dishevelled-3 (Dvl-3) in regulating GSK-3 and the transcription factor β-catenin in the rat brain. Western blotting showed that subchronic treatment of raclopride (D2DR antagonist) increase phosphorylated Akt, Dvl-3, GSK-3, phosphorylated GSK-3 and β-catenin, whereas subchronic treatment of quinpirole (D2DR agonist) induced the opposite response. Co-immunopreciptations revealed an association between GSK-3 and the D2DR complex that was altered following raclopride and quinpirole, albeit in opposite directions. SCH23390 (D1DR antagonist) and nafadotride (D3DR antagonist) were also used to determine if the response was specific to the D2DR. Neither subchronic treatment affected Dvl-3, GSK-3, Akt nor β-catenin protein levels, although nafadotride altered the phosphorylation state of Akt and GSK-3. In addition, in-vitro experiments were conducted to manipulate Akt and Dvl-3 activity in SH-SY5Y cells to elucidate how the pattern of change observed following manipulation of D2DR developed. Results indicate that Akt affects the phosphorylation state of GSK-3 but has no effect on β-catenin levels. However, altering Dvl-3 levels resulted in changes in Akt and the Wnt pathway similar to what was observed following raclopride or quinpirole treatment. Collectively, the data suggests that the D2DR very specifically regulates Wnt and Akt signalling via Dvl-3.