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

Antipsychotics Alter the Protein Expression Levels of ␤-Catenin and GSK-3 in the Rat Medial Prefrontal Cortex and Striatum Heidar Alimohamad, Nagalingam Rajakumar, Yam-Hong Seah, and Walter Rushlow Background: It has been demonstrated that schizophrenics have altered levels and/or phosphorylation states of several Wnt related proteins in the brain, including ␤-catenin and GSK-3, and may represent susceptibility loci for schizophrenia. The current study was conducted to assess the effects of antipsychotics on ␤-catenin and glycogen synthase kinase-3. Methods: Western blotting and immunocytochemistry were employed to investigate the effects of antipsychotics on ␤-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 ␤-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 ␤-catenin levels were unaffected. Other drug compounds, with the exception of raclopride, had no effect on either GSK-3 or ␤-catenin protein levels or distribution. Conclusions: Targeting of ␤-catenin and GSK-3 is a common feature of antipsychotics regardless of class and appears to be mediated by D2 dopamine receptors. Therefore changes in ␤-catenin and GSK-3 may represent one of the mechanisms through which antipsychotics are able to exert behavioral changes. Key Words: Antipsychotics, Wnt, GSK-3, ␤-catenin, prefrontal cortex, striatum

A

ntipsychotics remain the only effective treatment for the symptoms of schizophrenia. Though antipsychotics are often classified as either typical or atypical and possess different receptor binding profiles, D2 dopamine (DA) receptor blockade is an essential feature of both classes of drugs (Kapur and Mamo 2003). However, atypical antipsychotics are also strong serotonin receptor (5-HT) antagonists and the combined DA/5-HT action may be responsible for improved efficacy and reduced extrapyramidal symptoms (EPS) (Ichikawa and Meltzer 1999; Meltzer 1999). Current theories suggest that the blockade of receptors in the mesolimbic DA system is responsible for the ability of antipsychotics to alleviate psychosis whereas high levels of D2 DA receptor saturation in the striatonigral system is thought to cause disabling side effects commonly referred to as EPS (Serretti et al 2004). Strong D2 DA antagonists, such as typical antipsychotics, frequently induce EPS whereas atypical antipsychotics (weaker D2 antagonists) are less likely to do so. Despite a good understanding of the receptor binding profiles of antipsychotics, the cellular consequences of repeatedly blocking D2 DA receptors remains unclear and traditional signaling pathways linked to DA receptors have not provided an easy or direct answer. Therefore other signaling pathways should be examined to ascertain their potential involvement in the antipsychotic response. Fortunately several insights have been provided recently by studies using post-mortem brain tissue obtained from schizophrenics and matched controls. One group of proteins that

From the Departments of Anatomy and Cell Biology (HA, NR, WR) and Psychiatry (NR, Y-HS, WR), University of Western Ontario; and the London Health Sciences Centre (NR, WR), London, Ontario, Canada. Address reprint requests to Dr. Walter Rushlow, Department of Psychiatry, Room 10N15, London Health Science Center-U.C., 339 Windermere Road, London, Ontario, Canada N6A 5A5; E-mail: [email protected]. Received June 2, 2004; revised November 16, 2004; accepted November 23, 2004.

0006-3223/05/$30.00 doi:10.1016/j.biopsych.2004.11.036

was identified as altered in schizophrenic brains belongs to the Wnt signal transduction pathway. Wnt is important in central nervous system (CNS) development (Cadigan and Nusse 1997; Miller and Moon 1996) and constituents of the pathway remain highly expressed in the adult brain. Wnt has also been associated with a number of CNS disorders including Alzheimer’s disease and most recently schizophrenia (De Ferrari and Inestrosa 2000; Kozlovsky et al 2002). The canonical Wnt signaling cascade is activated when a Wnt protein interacts with a Frizzled (Fz) receptor (Bhanot et al 1996). The interaction of Wnt with Fz leads to the phosphorylation of dishevelled (Dvl) (Noordermeer et al 1994). Dvl antagonizes the action of glycogen synthase kinase-3 (GSK-3) resulting in the cytoplasmic accumulation and translocation of ␤-catenin into the nucleus (van Leeuwen et al 1994). A phosphorylation regulatory site has been identified in GSK-3 (Ser 21/9 for GSK-3␣ and GSK-3␤ respectively) that inhibits the kinase activity of an otherwise constitutively active protein and may be involved in regulating cytoplasmic ␤-catenin levels (Doble and Woodgett 2003). Once in the nucleus, ␤-catenin binds to TCF/LEF (T-cell factor and lymphoid enhancer factor), forming a transcription factor complex that activates gene expression (Behrens et al 1996; Huber et al 1996). In the absence of Wnt, cytoplasmic ␤-catenin levels are maintained at low levels through regulation by GSK-3, adenomatous polyposis coli (APC) and Axin (Hamada et al 1999; Ikeda et al 1998). The Axin-APC-GSK-3 complex promotes the phosphorylation of ␤-catenin by GSK-3 targeting it for destruction by the ubiquitin-proteasome pathway (Aberle et al 1997; Polakis 1997; Siegfried et al 1992). In the absence of ␤-catenin, TCF/LEF still binds to the TCF promoter element but fails to activate transcription (Brannon et al 1997; Riese et al 1997). The purpose of the current study was to determine if antipsychotics cause alterations in the protein levels or distribution of ␤-catenin and GSK-3 in the medial prefrontal cortex (PFC) or striatum (Str). These two proteins were selected for characterization since ␤-catenin represents the key protein that transduces activation of the Wnt pathway into the nucleus while GSK-3 is BIOL PSYCHIATRY 2005;57:533–542 © 2005 Society of Biological Psychiatry

534 BIOL PSYCHIATRY 2005;57:533–542 the central regulator of cytoplasmic ␤-catenin levels (Novak and Dedhar 1999).

Methods and Materials Animals For all aspects of the study, adult female Sprague-Dawley rats 14⫹ weeks of age (Charles River, Quebec, Canada) were used. Female rats were selected over male rats because they maintained a more stable body weight and body mass index over the course of the study. Rats were housed in pairs with free access to food and water in a room scheduled on a 12-hour light/dark cycle. All efforts were made to minimize the number of animals used in the current study and to eliminate pain and suffering. Use of animals for the current study was reviewed and approved by the University of Western Ontario Animal Research Ethics Committee in accordance with the guidelines developed by the Canadian Council on Animal Care. Drugs and Drug Paradigms For the western blotting studies all rats received intramuscular or subcutaneous, injections of .25 mg/kg or 1.0 mg/kg haloperidol, .9 mg/kg or 2 mg/kg risperidone, 25 mg/kg clozapine or appropriate vehicles (n ⫽ 5 rats/treatment group) for 7 days (sub-chronic treatment) and sacrificed 2 hours post-injection. Two doses of haloperidol were selected for the study since a controversy has recently arisen concerning appropriate dosing in rats. One mg/kg is the dose that has often been used for antipsychotic studies but has been shown to induce catalepsy, a rat measure of EPS liability. A lower dose results in saturation of central DA receptors in the rat brain that more closely approximates what is observed in treated patients without significant signs of EPS and may not induce catalepsy in rats (Kapur et al 2000a; Wadenberg et al 2001). However, the receptor occupancy and behavioral studies were only conducted following acute drug administration and it is not clear if the results can be extrapolated to a subchronic or chronic injection paradigm. Therefore both doses of haloperidol were used in the study. Two doses of risperidone were also chosen, for similar reasons. The higher dose of risperidone (2 mg/kg) would be expected to break the threshold of atypicality and induce catalepsy whereas the lower dose (.9 mg/kg) is less likely to do so (Wadenberg et al 2001). Consistent with the behavioral observations, higher doses of risperidone induce immediate early genes in the lateral caudate-putamen (CPu) whereas the lower doses of risperidone do not (Robertson et al 1994). Finally, the dose of clozapine was selected based on a receptor occupancy study showing comparable D2 DA receptor saturation between 25 mg/kg of clozapine and the lower doses of risperidone and haloperidol (Schotte et al 1993). Only a single dose of clozapine was selected since it is generally accepted that clozapine does not induce EPS in rats or humans and does not induce immediate early genes in the lateral CPu of rats (Robertson et al 1994). Antipsychotics only alleviate psychosis clinically in humans following repeated treatment and stabilization can take weeks or months. To determine if changes in ␤-catenin and GSK-3 followed a similar pattern, rats (n ⫽ 5/treatment group) were also injected with haloperidol (1 mg/kg) or risperidone (.9 mg/kg) acutely (single injection) or chronically for 28 days (28 daily injections) and sacrificed 2 hours post-injection. Only the PFC was examined following acute and chronic antipsychotic administration (and for most of the remaining portions of the study) since the results of the subchronic experiments suggested that www.elsevier.com/locate/biopsych

H. Alimohamad et al the medial prefrontal cortex is commonly affected by all three drugs and both doses of haloperidol and risperidone whereas the changes in the Str are dependent on dose and/or drug class (i.e. typical versus atypical). In addition to the single daily bolus injection experiments, rats were also injected with a single dose of haloperidol decanoate (1 mg/kg/day), a slow continuous release formulation of haloperidol lasting 14-21 days, or sesame seed oil vehicle and sacrificed 14 days following injection. The haloperidol decanoate was used to ensure the results were not the consequence of delivery method or other factors such as sedation. To examine receptor and drug specificity properties, rats (n ⫽ 5 rats/treatment group) were injected with raclopride (DA D2/D3 receptor antagonist, 3 mg/kg, 7 days), fluoxetine (selective serotonin reuptake inhibitor, 10 mg/kg, 9 days), ritanserin (serotonin 5-HT2a, 5-HT1c receptor antagonist, 1 mg/kg, 7 days), amphetamine (psychotomimetic, 2.5 and 5 mg/kg, 7 days), apomorphine (DA D1/D2 receptor agonist, .5 and 2.5 mg/kg, 7 days), quinpirole (DA D2 receptor agonist, 2.5 mg/kg, 7 days), SKF-82958 (DA D1/D5 receptor agonist, 1 mg/kg, 7 days), valproic acid (mood stabilizer, 300 mg/kg, 10 days) or appropriate vehicles and sacrificed 2 or 4 hours following the final injection. The dose selected for each of the drugs was based on published work including behavioral studies involving pre-pulse inhibition (Geyer et al 2001). The length of treatment was chosen to match the subchronic antipsychotic injection paradigm (7 days) with the exception of valproic acid and fluoxetine (9 days). The treatment interval was extended for these two agents since several published studies used a slightly longer injection period. For example, a recent study examining the effects of lithium on GSK-3 used a nine day treatment interval for valproic acid (Gould et al 2004). For the immunocytochemistry portion of the study, rats (n ⫽ 3/treatment) received intramuscular or subcutaneous injections of haloperidol (1 mg/kg), risperidone (.9 mg/kg or 2 mg/kg), amphetamine (5 mg/kg), apomorphine (2.5 mg/kg), quinpirole (2.5 mg/kg), valproic acid (300 mg/kg), ritanserin (1 mg/kg), fluoxetine (10 mg/kg), SKF-82958 (1 mg/kg) or vehicle daily for 7-10 days (as indicated above) and were killed 2 hours postinjection. All drugs used throughout the study were obtained from Sigma-Aldrich (Mississauga, Ontario, Canada). Western Blot Following treatment the rats were decapitated, the brains rapidly removed and dissected to obtain the Str (CPu and caudal nucleus accumbens) and the PFC (medial prefrontal cortex plus anterior cingulate cortex). Tissue from individual rats was immediately homogenized on ice in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris (pH 8.0), 1% NP-40, 10% glycerol and .1% sodium dodecyl sulfate) to which a protease inhibitor tablet (Roche, Laval, Quebec, Canada) was added using a dounce homogenizer. For the phosphorylation state portion of the study, Ser/Thr phosphatase inhibitors (Sigma) were included in the lysis buffer. The homogenized tissue was sonicated for 15 seconds, mixed with 5x loading buffer (125 mM Tris pH 6.8, 10% glycerol, 2% SDS, 2% 2-mercaptoethanol, .01% xylene cyanol and .01% bromophenol blue) and boiled for 5-7 minutes. The extracts were stored at -80°C until needed. Protein concentrations were determined using a bicinchoninic acid Protein Assay Kit (Pierce Chemical Co., Rockford, Illinois) and a ␮-Quant plate reader. For western blot analysis, 15-25 ␮g of protein from drug

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H. Alimohamad et al treated and control animals was subjected to electrophoresis on SDS polyacrylamide minigels (Bio-Rad Laboratories, Mississauga, Ontario, Canada) along with sizing standards (Bio-Rad Laboratories). Once electrophoresis was complete, the separated proteins were transferred to nitrocellulose membrane (Bio-Rad Laboratories). To detect the protein of interest, membranes were blocked in Carnation 5% nonfat dry milk in Tris buffered saline (TBS) with .05% Tween-20 (TBST) and then probed with a series of antibodies raised against ␤-catenin, GSK-3 or ␣-tubulin. Antibodies were added to fresh nonfat dry milk solution (5%) in TBST except for the phosphorylation state specific antibodies which were added to TBST containing 5% bovine serum albumin (VWRCanlab, Mississauga, Ontario, Canada, fraction V) in place of milk. The sources, species and dilutions for the antibodies used for western blotting were as follows; ␤-catenin (SigmaAldrich, rabbit, c2206, 1:20,000; Santa Cruz Biotechnology (Santa Cruz, California), mouse, sc7963, 1:400), GSK-3 (Cell Signaling Technology, Beverly, Massachusetts, rabbit, #9332, 1:1,000; Santa Cruz Biotechnology, mouse, sc7291, 1:300), ␣-tubulin (SigmaAldrich, mouse, T9026, 1:60,000), phosphorylated GSK-3␤ (Ser9) (Cell Signaling Technology, rabbit, #9336, 1:1000) and phosphorylated ␤-catenin (Ser33/37/Thr41) (Cell Signaling Technology, rabbit, #9561, 1:1,000). The blots were incubated in primary antibody solution for 1 hour at room temperature, except for the phosphorylation specific antibodies which were incubated overnight at 4oC. Membranes were then incubated in the appropriate horseradish peroxidase (HRP) conjugated secondary antibodies (Pierce Chemical Co., Rockford, Illinois); goat anti-mouse IgG HRP, 1:50,000; goat antirabbit IgG HRP antibody 1:50,000) diluted in 5% nonfat dry milk or 5% bovine serum albumin. Proteins were visualized using Supersignal (WestPico, Pierce Chemical Co.) and X-ray film (Kodak X-Omat LS). Multiple exposures were obtained to ensure that the bands on the X-ray films used for quantification were not saturated. Results were quantified by densitometry and band intensity was corrected for background by subtraction (Kodak software). Endogenous ␣-tubulin was used as a control to ensure that all lanes within a blot contained approximately the same quantity of protein. The data were statistically analyzed for differences between the groups using a two-way analysis of variance (ANOVA), followed by Tukey’s Multiple Comparison Test or a Student’s t-test. Differences of p ⬍ .05 (two-tailed) were considered to be significant. Immunocytochemistry Rats were injected intraperitoneally with sodium pentobarbital (65 mg/kg; MTC Pharmaceutical, Toronto, Ontario, Canada) and perfused transcardially using 200 ml of saline solution followed by 500 ml of 2% paraformaldehyde (SigmaAldrich) in .1M phosphate buffered saline (PBS, pH 7.4). The brains were immediately removed and cryoprotected in 15% sucrose solution for 24 hours. Cryoprotected brains were cut at 40 ␮m on a freezing microtome (Leitz, Wetzlar, Germany). Sections through the PFC and Str were collected in .1M PBS and then placed in blocking serum, composed of 10% normal goat serum (NGS) (Sigma-Aldrich) and .1% Triton X-100 in PBS for one hour. To qualitatively compare the protein levels and distribution of ␤-catenin and GSK-3 following drug administration, immunofluorescence was employed. Immunofluorescence staining was conducted as outlined previously (Rushlow et al 1996) using appropriate Alexa-568 conjugated secondary antibodies (1:200; Molecular Probes, Eugene, Oregon). Following fluorescence labeling the sections were also counterstained with Hoechst. In

all instances, master mixes of antibody solutions were prepared and the same number of sections from comparable levels of the brain were simultaneously labeled to facilitate direct comparison between treated and untreated rats. In addition, regions not thought to be targeted by antipsychotics were examined along with the PFC and Str to ensure that sections were comparably stained. The fluorescence-labeled sections were examined and images captured using an LSM410 scanning laser confocal microscope (Zeiss, North York, Ontario, Canada). Antibody Specificity All of the antibodies used in the study generated the expected banding pattern on western blots. The ␤-catenin and ␣-tubulin antibodies generated a single distinct band while the GSK-3 antibody generated 2 distinct bands representing the ␣ and ␤ isoforms of the protein. The relative size of ␤-catenin, ␣-tubulin and GSK-3, determined by molecular weight (MW) standards was as expected for each of the proteins. In addition, 2 different antibodies directed against GSK-3 and ␤-catenin were used to probe western blots and produced identical results. Finally, blots generated and probed without the primary antibody failed to show any positive labeling.

Results GSK- and ␤-catenin Protein Levels Following Antipsychotic Drug Administration as Revealed by Western Blotting Sub-chronic (7 days) administration of haloperidol (1 mg/kg) and risperidone (2 mg/kg) caused a significant increase in ␤-catenin and GSK-3 protein levels in the Str two hours following the last injection of antipsychotic. A lower dose of haloperidol (.25 mg/kg), and risperidone (.9 mg/kg) as well as clozapine (25 mg/kg) had no effect on ␤-catenin or GSK-3 protein levels (Figure 1A). All three drugs, however, caused significant up-regulation of ␤-catenin and GSK-3 in the PFC at all doses examined (Figure 1B). Despite significant changes in overall ␤-catenin proteins levels, no change in the level of phosphorylated ␤-catenin was observed in the PFC. However, significant increases in the phosphorylation state of GSK-3␤ were detected in the PFC paralleling increases in total protein levels (Figure 1C). The results suggest that antipsychotics, independent of class, have a profound effect on both ␤-catenin and GSK-3 in a region of the brain (PFC) that is innervated by the mesolimbic DA system and believed to play a vital role in both schizophrenia and the action of antipsychotics. The Str (CPu in particular), on the other hand, is innervated by the striatonigral DA system and only shows a clear response at doses of haloperidol and risperidone expected to exceed the threshold for EPS (Kapur et al 2000a; Wadenberg et al 2000). The protein levels of GSK-3 and ␤-catenin were also examined following acute (1 injection) and chronic (28 daily injections) administration of risperidone (.9 mg/kg), haloperidol (1 mg/kg) or vehicle in the PFC. Acute treatment had no effect on ␤-catenin or GSK-3 protein levels while chronic administration resulted in an identical pattern to that observed following subchronic administration (Figure 2). Therefore the results show that changes in ␤-catenin and GSK-3 require repeated drug administration to be manifested and persist with long term drug treatment. GSK-3 and ␤-catenin Protein Levels Following Administration of Other Drug Compounds as Revealed by Western Blotting To determine if the results obtained following antipsychotic administration are specific to antipsychotics, a variety of different www.elsevier.com/locate/biopsych

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Figure 1. Changes in ␤-catenin and GSK-3 protein levels following sub-chronic administration of antipsychotics. (A)-(B) Left Panels: Representative western blots showing ␤-catenin, GSK-3␤ and ␤ and ␣-tubulin generated using protein isolated from the Str (A) or PFC (B) of rats sub-chronically injected with haloperidol (.25 mg/kg and 1.0 mg/kg), risperidone (.9 mg/kg and 2.0 mg/kg), clozapine (25 mg/kg) or appropriate vehicles. In each case, the left band represents the vehicle while the right band shows the expression level of a given protein following antipsychotic treatment. Band(s) of equal intensity means there is no change in the expression level of the protein. If the right band(s) is more intense than the left, antipsychotics caused an up-regulation in the expression level of that particular protein. All of the antibodies used generated a single band except the GSK-3 antibody which yields two bands; GSK-3␣ (upper band) and GSK-3␤ (lower band). (A)-(B) Right Panels: Graphs comparing ␤-catenin and GSK-3␤ and ␤ protein levels in the Str (A) or PFC (B) following sub-chronic antipsychotic treatment (n ⫽ 5). (C) Left Panel: Representative western blots showing phosphorylated ␤-catenin, phosphorylated GSK-3␤ or ␣-tubulin (loading control) in the PFC following subchronic administration of haloperidol (1 mg/kg) or risperidone (.9 mg/kg). (C) Right Panel: Graph showing phosphorylated ␤-catenin (Ser33/37/Thr41) and GSK-3␤ (Ser 9) protein levels in the PFC compared with total protein levels following sub-chronic administration of haloperidol (1 mg/kg) or risperidone (.9 mg/kg and 2.0 mg/kg) relative to controls (n ⫽ 5). For all of the graphs, the band intensity data for each western blot was quantified by densitometry and statistically analyzed for differences within the groups using a two-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. Differences of p ⬍ .05 were considered statistically significant and are denoted by an asterisk. The standard errors of the means are indicated for each treatment. p, phosphorylated; Veh, vehicle; Hal, haloperidol; Risp, risperidone; Cloz, Clozapine.

compounds were administered and the protein levels of ␤-catenin and GSK-3 examined in the PFC. None of the compounds tested, including fluoxetine, ritanserin, valproic acid, apomorphine, amphetamine, SKF-82958 or quinpirole, had an appreciable effect on ␤-catenin or GSK-3 protein levels in the PFC two or four hours following the final injection (data not shown). However, both the D2/D3 DA receptor antagonist raclopride and haloperidol decanoate caused significant elevations in ␤-catenin and GSK-3 protein levels in the PFC and Str respectively (Figure 3). Therefore the results suggest that up-regulation of ␤-catenin and GSK-3 protein levels are specific to antipsychotics, likely mediated through D2 DA receptor antagonism and not the consequence of extraneous factors such as method of drug delivery or sedation. www.elsevier.com/locate/biopsych

Distribution of ␤-catenin and GSK-3 Following Drug Administration The cellular localization of ␤-catenin and GSK-3 is at least as important as the quantity or phosphorylation state of the protein. Both ␤-catenin and GSK-3 have dramatically different functions depending on their localization within the cell. Therefore the distribution of ␤-catenin and GSK-3 was also examined in sections obtained from rats treated with antipsychotics and compared to controls. An overview of the qualitative data is presented in Table 1. Following administration of high dose haloperidol (1.0 mg/ kg) and risperidone (2.0 mg/kg), increased ␤-catenin and GSK-3 labeling was observed across the CPu (Figure 4). Specifically, GSK-3 showed increased labeling intensity in the cell body and

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H. Alimohamad et al proximal dendrites following haloperidol (1.0 mg/kg) or risperidone (2.0 mg/kg) administration whereas more intense nuclear and neuropil labelling could be seen for ␤-catenin following treatment. No apparent change in either ␤-catenin or GSK-3 could be readily detected in the nucleus accumbens core (NAc) or nucleus accumbens shell (NAs) following haloperidol administration though risperidone did appear to increase the levels of both proteins in the NAs (Figure 5). The lower dose of risperidone (.9 mg/kg) and clozapine showed no changes in the CPu or NAc but small changes in the NAs, similar to what is depicted in Figure 5 (E-H). More intense ␤-catenin and GSK-3 labelling could also be seen in the deeper layers of the PFC following haloperidol (1.0 mg/kg), risperidone (.9 and 2.0 mg/kg) and to a lesser extent clozapine relative to controls (Figure 6). Apomorphine,

Figure 3. Representative western blots showing ␤-catenin, GSK-3␣ and ␤, and ␣-tubulin generated using protein isolated from the Str or PFC of rats injected with a slow release depot preparation of haloperidol (⬃1.0 mg/kg/ day), the D2/D3 specific antagonist raclopride (3 mg/kg) or appropriate vehicles. In each case, the left band represents the vehicle while the right band shows the expression level of a given protein following drug treatment. Right Panel: Graph comparing ␤-catenin and GSK-3␣ and ␤ protein levels in the Str following haloperidol decanoate or raclopride treatment (n ⫽ 5). Band intensity for each western blot was quantified by densitometry. The data was statistically analyzed for differences within the groups using a Student’s t-test. Differences of p ⬍ .05 were considered statistically significant and are denoted by an asterisk. The standard errors of the means are indicated for each treatment/vehicle. Racl, raclopride; Hal.dec, haloperidol decanoate; Str, striatum; PFC, prefrontal cortex.

quinpirole, SKF-82958, valproic acid, ritanserin and fluoxetine, in contrast to antipsychotics, had no apparent effect in the PFC or Str (data not shown). The qualitative results confirm the findings obtained for western blotting and support the premise that the antipsychoticinduced changes are specific. In addition the confocal data also revealed that changes in the PFC are confined mainly to the deeper layers, a region known to contain DA D2 receptors and project to structures such as the limbic portion of the Str (NAs) (Berendse et al 1992; Larson and Ariano 1995). The limbic portion of the Str, which is also a target of the mesolimbic DA projection system (Gerfen et al 1987), also showed some changes following atypical but not typical antipsychotic administration. At the level of individual cells, neurons responsive to antipsychotics could be described as showing more intense cell labeling overall when compared with sections from vehicle treated animals. No major redistribution or translocation of GSK-3 could be seen, however more intense nuclear ␤-catenin labeling could be observed following antipsychotic treatment (Figure 7). Increased ␤-catenin staining in the nucleus is intriguing since the presence of nuclear ␤-catenin has been shown to regulate TCF/LEF transcription (Behrens et al 1996; Huber et al 1996).

Discussion Figure 2. Changes in ␤-catenin and GSK-3 protein levels following acute and chronic administration of antipsychotics in the PFC. Upper Panel: Representative western blots showing ␤-catenin, GSK-3␣ and ␤, and ␣-tubulin generated using protein isolated from the PFC of rats injected acutely (single injection) or chronically (28 days) with haloperidol (1.0 mg/kg), risperidone (.9 mg/kg), or appropriate vehicles. In each case, the left band represents the vehicle while the right band shows the expression level of a given protein following antipsychotic treatment. Lower Panel: Graph comparing ␤-catenin and GSK-3␣ and ␤ protein levels in the PFC following acute or chronic drug treatment (n ⫽ 5). Band intensity for each western blot was quantified by densitometry. The data was statistically analyzed for differences within the groups using a two-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. Differences of p ⬍ .05 were considered statistically significant and are denoted by an asterisk. The standard errors of the means are indicated for each treatment/vehicle. Veh, vehicle; Hal, haloperidol; Risp, Risperidone.

Antipsychotics alleviate psychosis by inducing ill-defined changes in target neurons. The changes are likely mediated via the repeated blockade of DA D2 receptors or possibly D2 and 5-HT receptors in the case of atypicals. Different antipsychotic drugs show dramatic differences in their binding profiles and affinities for the D2 DA family as well as other potentially important receptors such as serotonin, histaminergic, adrenergic and muscarinic receptors (Richelson 1999). However, despite the many differences, all antipsychotics by their very definition are able to alleviate psychosis suggesting that a common mechanism(s) of action may unite them. In addition to alleviating psychosis, antipsychotics have proven to be effective in the treatment of mood disorders and may also share some common features with mood stabilizers such as lithium and valproic acid www.elsevier.com/locate/biopsych

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Table 1. Summary of Qualitative Changes Induced by Antipsychotics in Defined Brain Regions as Observed Using Confocal Microscopy

Drug Hal. (1 mg/kg) Risp. (.9 mg/kg) Risp. (2.0 mg/kg) Cloz. (25 mg/kg) Other Drugs

Protein

␤-catenin GSK-3 ␤-catenin GSK-3 ␤-catenin GSK-3 ␤-catenin GSK-3 ␤-catenin GSK-3

Medial Prefrontal Cortex

Medial CaudatePutamen

Lateral CaudatePutamen

Nucleus Accum. Core

Nucleus Accum. Shell

⫹⫹⫹

⫹⫹⫹

⫹⫹⫹

-

-

⫹⫹⫹

-

-

-

⫹

⫹⫹⫹

⫹⫹⫹

⫹⫹⫹

-

⫹⫹

⫹⫹

-

-

-

⫹

-

-

-

-

-

Summary of the qualitative observations obtained from sections stained using immunofluorescence for ␤-catenin and GSK-3 following treatment with various drugs relative to vehicle treated sections (n ⫽ 3). Areas of the brain outside the regions of interest were also examined to ensure the changes were caused by the drugs and not experimental artefact. ⫹ ⫽ small increase in protein levels, ⫹⫹ ⫽ moderate increase in protein levels, ⫹⫹⫹ ⫽ large increase in protein levels and - ⫽ no change in protein levels. Hal, haloperietol; Risp, rispericlone; Cloz, clozapine; Accum, accumbens.

(Macqueen and Young 2003; McIntyre and Katzman 2003; Sharma 2003; Yatham 2003). Several recent publications have identified common signaling targets for drugs that induce similar behavioral changes but are themselves very different chemically. For example, the mood stabilizers lithium, carbamazepine and valproic acid were all shown to cause inositol depletion in primary cultures of rat dorsal root ganglia while the psychomimetic drugs amphetamine, lysergic acid diethylamide (LSD) and phencyclidine (PCP) were all shown to alter the phosphorylation state of DARPP32 with concomitant downstream regulation of GSK-3, CREB and c-fos (Svenningsson et al 2003; Williams et al 2002). Such studies are valuable since they provide a common thread to begin to elucidate how different compounds that impact the brain might work. In a similar light, the results of the present study suggest that alterations in ␤-catenin and GSK-3 might be a common feature of antipsychotic drugs downstream of the D2 receptor. A number of recent findings in humans make this hypothesis particularly attractive. For example, decreased levels of ␤-catenin and GSK-3␤ protein and decreased levels of GSK-3 kinase activity have been reported in the brains of schizophrenic patients post-mortem (Beasley et al 2001; Cotter et al 1998; Kozlovsky et al 2000; Kozlovsky et al 2001). Decreased levels of AKT-1, a kinase that regulates the phosphorylation state and hence activity of GSK-3␤, and altered phosphorylation levels of GSK-3␤ were described by western blotting using protein isolated from the postmortem brain of schizophrenics compared with matched control brains (Emamian et al 2004). AKT was also identified by the authors as a potential schizophrenia susceptibility locus. Calcineurin, too, was recently recognized as a possible schizophrenia susceptibility locus and although calcineurin has several functions within neurons, it also serves as a key regulator of DARPP-32 and possibly GSK-3 (Gerber et al 2003; Nishi et al 1999; Svenningsson et al 2003). Finally, the Wnt receptor frizzled-3 has been linked to schizophrenia as a potential susceptibility locus (Katsu et al 2003). Corroborating evidence for the involvement of ␤-catenin and GSK-3 has also been provided by several animal studies. Decreased GSK-3␤ protein levels have been described in the frontal cortex of the neurodevelopmental hippocampal lesion model of schizophrenia and altered phosphorylation levels of GSK-3 were www.elsevier.com/locate/biopsych

found in the DA transporter knockout mouse (a putative model of schizophrenia) (Beaulieu et al 2004; Nadri et al 2003). Finally, Dvl-1 knockout mice displayed deficits in sensorimotor gating and reduced social interactions. Social withdrawal and deficits in sensorimotor gating are two key behavioral characteristics of schizophrenia (Lijam et al 1997). The results of the current study are particularly relevant in light of the recent findings since they suggest that antipsychotics may compensate for deficits caused by schizophrenia. Schizophrenia results in less GSK-3 and/or reduced GSK-3␤ (Ser 9) phosphorylation leading to reduced GSK-3 activity while antipsychotics increase the amount of phosphorylated GSK-3␤ (Ser 9) and GSK-3. It should be noted, however, that there are some differences between the data presented in the current study and two other recent publications with respect to GSK-3. One study reported increases in phosphorylated GSK-3␤ (Ser 9) but no changes in total GSK-3␤ levels following chronic haloperidol administration (Emamian et al 2004). Another study found no change in either GSK-3␤ protein levels or GSK-3 activity following chronic administration of haloperidol of clozapine (Kozlovsky et al 2003). Though the explanation for the discrepancies is not clear, both of the other studies examined protein isolated from the frontal cortex whereas in the current study protein was isolated specifically from the PFC. With respect to ␤-catenin, the consequences of schizophrenia on the protein have not been well investigated though there is one publication reporting decreased levels in the brain of schizophrenics (Cotter et al 1998). Again, based on the data obtained, it would appear that antipsychotics may also reverse this change. How antipsychotics alter ␤-catenin, GSK-3 and phosphorylated GSK-3␤ (Ser9) protein levels remains to be elucidated though it is likely that D2 DA receptors mediate the process since the D2/D3 specific antagonist raclopride induced similar changes in the PFC compared to antipsychotics. Possible mechanisms that may be responsible for the observed protein changes include AKT and Dvl. Increased phosphorylation of AKT has been reported following haloperidol treatment in the frontal cortex of mice and increased phosphorylation of AKT and Dvl following treatment of SH-SY5Y cells in culture with clozapine (Emamian et al 2004; Kang et al 2004). Furthermore a recent study showed that

H. Alimohamad et al

BIOL PSYCHIATRY 2005;57:533–542 539 antagonize DA D2 receptors are capable of satisfactorily alleviating psychosis (Kapur and Remington 2001). The results of the current study are very much in agreement with both hypotheses. All 3 antipsychotics tested caused significant up-regulation of ␤-catenin and GSK-3 in the PFC and perhaps the nucleus accumbens (atypicals only); both regions innervated by mesolimbic DA fibers. The observation that only atypical antipsychotics altered ␤-catenin and GSK-3 in the NA was unexpected since other studies, such as those that examined immediate early gene induction following acute antipsychotic administration, found that both classes of drugs induced changes in the NA (Robertson and Fibiger 1992; Robertson et al 1994). The difference in response between haloperidol and risperidone or clozapine in the NA might represent an important distinction between typical and atypical antipsychotics that may be related

Figure 4. Representative confocal images taken from sections stained for ␤-catenin (A)-(D) or GSK-3 (E)-(H) using immunofluorescence. All of the captured images were obtained from the lateral aspect of the CPu following administration of 1.0 mg/kg haloperidol (B) and (F), 2.0 mg/kg risperidone (D) and (H) or appropriate vehicles (A, C, E and G) for 7 days. ␤-catenin staining is dramatically enhanced in both the neuroplexis (arrows) and what appear to be nuclei of cells (filled arrowheads) following administration of haloperidol and risperidone. GSK-3 labeling is also enhanced following antipsychotic administration but is mainly confined to the cytoplasm (open arrowheads). Magnification bar ⫽ 50 ␮m. CPu, caudate-putamen; Hal, haloperidol; Risp, risperidone.

AKT phosphorylation (Thr 308) could be regulated by D1 (SKF-38393) and D2 (quinpirole) DA receptor agonists in striatal primary cultures (Brami-Cherrier et al 2002) suggesting that the link between the observations reported in the current study and DA receptors may be AKT. From a signaling perspective the data generated conforms nicely with recent publications by other investigators related to schizophrenia or models of schizophrenia. However, it is also important to determine if the data makes sense based on what is known about antipsychotics. Despite a number of controversies surrounding antipsychotics, it is generally accepted that they alleviate psychosis by modifying the mesolimbic DA system while EPS is caused by alterations in the striatonigral DA system (Broich et al 1998; Kapur et al 2000b; Kapur and Mamo 2003). It is also known that only drugs that possess some ability to

Figure 5. Representative confocal images taken from sections stained for ␤-catenin using immunofluorescence. The captured images were obtained from either the NAc (A, B, G and H) or the NAs (C, D, G and H) following administration of 1.0 mg/kg haloperidol (B and D), 2.0 mg/kg risperidone (F and H) or appropriate vehicles (A, C, E and G) for 7 days. No obvious changes are apparent in either the NAc or NAs following haloperidol treatment. Following risperidone administration, however, more intense ␤-catenin staining can be observed in the NAs though the NAc appears to be unaffected. Magnification bar ⫽ 50 ␮m. Nac, nucleus accumbens core; Nas, nucleus accumbens shell; Hal, haloperidol; Risp, risperidone.

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540 BIOL PSYCHIATRY 2005;57:533–542

H. Alimohamad et al other well known inhibitor of GSK-3 activity, to induce changes in ␤-catenin in vivo, however, is much less certain. No changes were found in the present study using valproic acid nor were ␤-catenin protein levels altered in bipolar patients treated with lithium for decades during life post-mortem (Lesort et al 1999). However, significant changes in ␤-catenin protein levels were recently described in the frontal cortex of adolescent rats continuously treated with rat chow containing lithium or valproic acid (Gould et al 2004) though a separate study found significant changes in the hippocampus only (O’Brien et al 2004). Therefore

Figure 6. Representative confocal images taken from sections stained for ␤-catenin (A and B) or GSK-3 (C and D) using immunofluorescence. The captured images were obtained from the deeper layers of the medial prefrontal cortex following the administration of 1.0 mg/kg haloperidol (B and D) or vehicle (A and C) for 7 days. Following antipsychotic administration a substantial increase in ␤-catenin labelling can be seen in the deeper layers (5/6) of the medial prefrontal cortex. Both the cell bodies (filled arrowheads) and the neuropil (closed arrowheads) show enhanced staining. Enhanced GSK-3 cytoplasmic staining (arrows) and fiber labeling is also evident in the lower layers of the cortex. In the higher layers little difference is evident (open arrows). The border between the gray matter of the cortex and the underlying white matter of the corpus callosum (cc) is marked with a dotted line. Magnification bar ⫽ 50 ␮m. GSK-3, glycogen synthase kinase-3.

to the improved clinical efficacy observed with the use of atypicals. Alternatively, the differences may be due to experimental factors such as dose, timing of protein isolation following the final injection or sensitivity of immunofluoresence to detect small changes. In the CPu, only a high dose of haloperidol or risperidone elicited increases in ␤-catenin and GSK-3 protein levels. High doses of both drugs would be expected to saturate D2 DA receptors in the Str and cause EPS in humans or induce catalepsy in rats (Kapur et al 2000a; Wadenberg et al 2001). Furthermore, clozapine, a drug that rarely causes EPS, elicited no response in the Str at 25 mg/kg. This is not to suggest that the changes observed in ␤-catenin and GSK-3 are responsible for EPS, but rather that the results are consistent with previously reported studies showing a correlation between high D2 DA receptor saturation, changes in protein and mRNA in the CPu and EPS. Specificity is another important issue for antipsychotics. Different drug compounds impact the brain differently though there may be some overlap in their biological effects. For example, drugs used to treat other disorders (i.e. depression) do not alleviate psychosis in human patients. However, both mood stabilizers and antipsychotics are effective for the treatment of bipolar disorder. In the current study, neither valproic acid (mood stabilizer) nor fluoxetine (anti-depressant) had an effect on GSK-3 or ␤-catenin protein levels in the PFC or Str, 2 or 4 hours post final injection. Results for valproic acid are somewhat surprising given literature reports indicating that valproic acid can upregulate ␤-catenin in vitro (Chen et al 1999). The ability of physiologically relevant doses of valproic acid or lithium, anwww.elsevier.com/locate/biopsych

Figure 7. Representative high magnification confocal images taken from sections stained for ␤-catenin (A and B) or GSK-3 (E and F) using immunofluorescence and counterstained with Hoechst to label nuclei (C, D, G and H). All of the captured images were obtained from the lateral aspect of the CPu following 7 days of treatment with vehicle (A, C, E and G) or 1 mg/kg of haloperidol (B, D, F and H). ␤-catenin staining coincides with Hoechst nuclear labelling (filled arrowheads) whereas GSK-3 immunoreactivity is confined to the cytoplasm and not present in the nucleus (arrowheads). ␤-catenin staining is more intense in the nucleus (filled arrowheads) and neuropil (open arrowheads) following haloperidol treatment relative to controls. Similarly, GSK-3 staining intensity is increased in the cytoplasm following haloperidol treatment (open arrowheads). There is also likely a relatively small increase in the intensity of fibers stained for GSK-3 following haloperidol treatment. Magnification bar ⫽ 25 ␮m. CPu, caudate-putamen; GSK-3, glycogen synthase kinase-3.

H. Alimohamad et al there may be some overlap between mood stabilizers and antipsychotics though perhaps they don’t target GSK-3/␤-catenin by the same mechanism or affect the same populations of neurons. A variety of DA agonists including quinpirole, apomorphine and SKF-82958 also failed to induce changes in ␤-catenin or GSK-3 protein levels in either the PFC or Str. The inability of the other drugs to alter ␤-catenin and GSK-3 demonstrates that the antipsychotic response is not a generic effect such as has been described for the induction of immediate early genes (Robertson et al 1994). Amphetamine also had no effect in the PFC on either GSK-3 or ␤-catenin. Again the results were unexpected since amphetamine is a known psychomimetic and can induce schizophrenic symptoms in individuals who abuse the drug and thereby might be expected to show the opposite response to antipsychotics. However, a recent study reported that amphetamine can alter the phosphorylation state of DARPP-32 and GSK-3␤ suggesting similar targets for both psychomimetics and antipsychotics though additional studies will be necessary to clarify differences in mechanism (Svenningsson et al 2003). The changes identified following antipsychotic administration are also consistent with clinical observations concerning the time course of treatment for schizophrenic patients treated with antipsychotics. It has been well established that antipsychotics do not alleviate psychosis after a single dose. Likewise antipsychotics have no effect on ␤-catenin or GSK-3 following a single injection in the rat. After repeated administration, improvement in psychotic symptoms can be observed in humans, and in the rat, changes in GSK-3 and ␤-catenin protein levels can be detected. Finally, continuous treatment for weeks to months leads to stabilization of positive symptoms in human patients and significant elevations in both ␤-catenin and GSK-3 are readily detectable after chronic treatment.

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