risp

Ded

ilding, Da and Ate of Mscience

VEH, vehicle

B R A I N R E S E A R C H 1 1 1 3 ( 2 0 0 6 ) 2 4 3 2

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r. com/ loca te /b ra in restransport, protein metabolism, chaperone, DNA binding and cell cycle categories. Weconclude that chronic risperidone treatment accompanied by an EPS-like behavioralphenotype results in alterations in the striatal protein profile possibly subsequent toblockade of dopaminergic systems. These results suggest that possible mechanismsinvolved in APD-induced EPS include metabolic dysfunction and oxidative stress.

2006 Elsevier B.V. All rights reserved.

2DE,two-dimensional electrophoresisMALDI-TOFMS,matrix assisted laserdesorption ionization-time of flightmass spectrometryperiod compared with a vehicle (VEH) control group (n=12) (Karl et al., unpublishedobservation). Using two-dimensional gel electrophoresis (2DE), total protein extracts fromthe rat brain striatum were separated and protein expression was analyzed by Phoretix 2DExpression and Image Beta V4.02 software followed by matrix assisted laser desorptionionization-time of flight mass spectrometry (MALDI-TOF MS). 2DE gels resolved up to 450protein spots, presumably different proteins and/or their isoforms. There were 30 proteinspots showing statistically significant different densities between the RIS- and VEH-treatedgroups. All 30 proteins were successfully identified by MALDI-TOF MS, 28 of these weredivided into groups based on their known functions. These included metabolic, signaling,

Extrapyramidal symptomStriatumProteomicSpragueDawley rat

Abbreviations:APD, antipsychotic drugEPS, extrapyramidal symptomsRIS, risperidoneAntipsychotic drugRisperidone Corresponding author. Discipline of Patholo13429.

E-mail address: izuru@med.usyd.edu.au (

0006-8993/$ see front matter 2006 Elsevidoi:10.1016/j.brainres.2006.07.009of RIS (2.1 mg/kg/day, via subcutaneous osmotic minipumps) induced significant vacuouschewing movements and catalepsy in male SpragueDawley rats over a 28-day treatmentAccepted 4 July 2006Available online 30 August 2006

Keywords:eridone treatment on the striatal protein

ovaa,b, Liesl Duffyb,c, Stuart Cordwella,d, Tim Karlb,c,

06, The University of Sydney, NSW, 2006, Australiallied Disorders (NISAD), Darlinghurst, NSW, 2010, Australiaedical Research, Sydney, Australias, The University of Sydney, NSW, 2006, Australia

A B S T R A C T

Extrapyramidal symptoms (EPS) commonly occur as side effects of antipsychotic drugs(APDs) and are most likely to arise when the occupancy of dopamine D2 receptors in thestriatum by these drugs exceeds 80%. We aimed to characterize changes in the proteinexpression profile in the striatum of rats after chronic (4 week) supra-therapeutic (EPS-inducing) treatment with risperidone (RIS), an atypical antipsychotic drug. AdministrationResearch Report

Effects of chronicprofiles in rats

Elizabeth O'Briena,b, IrinaIzuru Matsumotoa,b,aDiscipline of Pathology, Blackburn BubNeuroscience Institute of SchizophrenicNeurobiology Program, Garvan InstitudSchool of Molecular and Microbial Bio

A R T I C L E I N F O

Article history:gy, Blackburn Building, D06, The University of Sydney, NSW, 2006, Australia. Fax: + 61 2 935

I. Matsumoto).

er B.V. All rights reserved.

action are dopaminergic D2 receptor and serotonergic 5-HT2

RIS-treated rodents

All 30-protein spots present in altered levels in the striatumof the RIS-treated rodents were identified using MALDI-TOFMS. Four proteins (phosphoglycerate mutase 1, triosepho-sphate isomerase, cytochrome c oxidase polypeptide Vb andannexin V) were identified in more than one spot, suggestingthat changes in post-translational modifications of theseproteins may occur. Proteins appearing with altered levels inthe striatum of RIS treated rodents were classified by functioninto metabolic, signaling, transport, protein metabolism,chaperone, DNA binding and cell cycle categories (Table 2).Of the 12 proteins in the metabolic group, four were absent inthe VEH-treated group, while the remaining eight weredecreased by the APD, including one by almost 6-fold.Seven of the eight proteins associated with signaling were

1 1receptors (Leysen et al., 1988). RIS has several advantages overolder neuroleptics including a broader therapeutic effective-ness, in modulating both positive and negative symptoms ofthe disease and a lower tendency for inducing extrapyramidalsymptoms (EPS) (Glick and Berg, 2002; Marder et al., 2003).

The advantageous EPS profile of RIS is based on its loweraffinity for the D2 receptor compared to the typical drughaloperidol (Leysen et al., 1992). The effect of this lower affinityis its reduced likelihood of blocking more than 80% of D2receptors, a threshold linkedwith EPS (Wadenberg et al., 2000).At doses blocking over 80% of D2 receptors, the advantageousEPS profile of RIS subsides (Kapur, 1998; Knable et al., 1997).

To date, the vast majority of molecular and anatomicalexamination of EPS have focused on the striatum due to itsinvolvement in both motor coordination and dopaminergicinnervation (Andreassen et al., 1998; Mitchell et al., 2002). Thestriatum forms dense connective circuits with other parts ofthe basal ganglia including the globus pallidus, subthalamicnucleus and substantia nigra; with impaired neurotransmis-sion in the nigrostriatal system being associated with EPS(Arnt, 1998; Schwarcz, 1982), affecting the ability to processmotor information. Stereological studies of the effects of APDson the brain have found the atypical drug clozapine to reducethe size of caudate nucleus in the striatum (Scheepers et al.,2001). It is likely that such structural change is accompaniedby changes on a molecular level and that some of thesechanges may be related to clinical efficacy of the drug or sideeffects such as acute and/or chronic EPS. Gene and/orexpression studies provide some clues to explain underlyingmechanism of EPS subsequent to dopaminergic transmissionblockade. A recent study by Feher et al. investigated the effectsof chronic RIS on gene expression in the rat cortex, finding thatmany metabolic, signaling, protein metabolism and iontransport gene were significantly altered (Feher et al., 2005).

Using proteomics, the present study aimed to investigatethe molecular effects of RIS on the total protein expressionprofile in the rat brain striatum following fourweeks of chronicsupra-therapeutic drug administration. Behavioral testing re-vealed that chronic RIS treatment induced significant changesin the behavioral profile of rats consistent with EPS (i.e. signi-ficant vacuous chewing movements and catalepsy) and af-fected all behavioral domains: motor activity and explorationdiminished, working memory performances impaired andanxiety levels increased (Karl et al., unpublished observation).

2. Results

2.1. 2DE comparison of striatal tissue from VEH andRIS-treated rats1. Introduction

Risperidone (RIS) is an antipsychotic drug (APD) commonlyused in themanagement of schizophrenia. Classed among theatypical or second generation APDs, major targets for RIS

B R A I N R E S E A R C HSignificantly altered proteins in the striatum of post-mortemtissue from VEH- and RIS-treated rats (change in mean spotabundance of 1.3-fold) were established by comparing theaverage gel from each group. Each of these average gels wasbased on the constituent 24 subgels representing eachtreatment group, of which two gels represented each brainsample (n=12 samples/group). The average gel for VEH andRIS displayed 443 and 477 spots, respectively, with each ofthese spots appearing in at least 70% of the subgels in itsgroup. More than 97% of all protein spots were matched tothe reference gel, ensuring that protein expression displayedin the average gel was representative of the subgels within itsconstituent group. Each identified protein spot was issued aunique identification number to enable cross-referencingbetween groups.

Changes in the relative abundance of 30 protein spotswere found between the VEH and RIS groups (>1.3-folddifference, p1.7-fold

change

1.51.7-fold

change

1.31.5-fold

change

Number ofspot

changes

Increase 5 2 1 8Decrease 1 0 14 15Absent 7

Total 30

The table shows the magnitude and direction of altered striatalprotein expression by RIS. Those proteins identified as significantby one-way ANOVA were required to display a mean n-fold changebetween RIS and VEH gels of 1.3 in order to be excised foridentification by mass spectrometry.

251 3 ( 2 0 0 6 ) 2 4 3 2up-regulated by RIS, including three increased by more than4-fold and a further three absent in the VEH-group, while all

w1 1Table 2 The list of identified proteins whose spot densityfollowing chronic (4 week) treatment with RIS

Functionalclass

Spot no. Fold change Identification

26 B R A I N R E S E A R C Hthree transport related proteins were down-regulated by RIS.RIS also decreased the expression of one protein from each ofthe protein metabolism, cell cycle and DNA binding groups,while the single protein in the chaperone group wasincreased in the order of 60% by the drug. A further twoproteins were identified, however, their function is not yetknown.

Metabolic 2186 Absent Tyrosine hydroxylase262 Absent Phosphoglycerate mutase 12030 Absent Phosphoglycerate mutase 1267 1.48 Phosphoglycerate mutase 11668 Absent Triosephosphate isomerase2082 1.38 Triosephosphate isomerase1827 5.95 Cytochrome c oxidase polype

Vb mitochondrial [precursor]576 1.48 Cytochrome c oxidase polype

Vb mitochondrial [precursor]2014 1.47 Cytochrome c oxidase polype

Vb mitochondrial [precursor]181 1.48 Creatine kinase, mitochondri

ubiquitous1106 1.44 Fructose bisphosphate aldola2072 1.36 ATP synthase chain, mitoch

[precursor]Signaling 182 Absent 40S ribosomal protein SA

2162 Absent ADP ribosylation factor 52183 Absent Neuron-specific calcium bind

protein hippocalcin2116 4.68 Annexin V2117 4.77 Annexin V2184 1.48 Calcium binding protein 72298 1.53 Dihydropyrimidinase-related

protein 2 (DRP-2)Protein

metabolism1670 1.46 Protein-L-isoaspartate-O-met

transferase313 1.33 Alpha synuclein

Transport 318 1.38 Mitochondrial solute carrierprotein homolog

2179 1.43 Potassium voltage gated chansubfamily S member 1

339 1.33 Hemoglobin beta chain, majoChaperone 1024 1.64 Chaperonin containing TCP 1

subunit 3DNA binding 975 1.46 Synaptonemal complex proteCell cycle 290 1.38 Translationally controlled

tumor protein2086 4.48 G1/S-specific cyclin E1

Unknownfunction

2076 2.16 Unnamed protein product

1991 1.76 Golgi complex-associated pro(GCP) 360

The table displays the identifications of protein spots significantly altegenerated for each protein spot by entering its experimentally determineusing the proteomic servers Aldente (http://au.expasy.org/) and MASCOTof the most likely candidate from each protein spot list was validated usinweight. The MOWSE score was calculated using the formula 10*Log (P),random event. A score of at least 55 was necessary to correlate to a signias significantly altered on 2DE gels of the rat brain striatum

Accession % Sequence/no.peptidesmatched

pI/Mass Mascot score

1 3 ( 2 0 0 6 ) 2 4 3 23. Discussion

Chronic administration of RIS at a dose of 2.1 mg/kg/dayresulted in behavioral changes in rats consistent with EPS(Karl et al., unpublished observation). In humans, documentedside-effects of high doses of RIS include EPS (Yoshimura et al.,

Q78E18 100/6 8.31/3181 61P25113 39/9 6.67/28,814 66P25113 65/20 6.7/28,814 69P25113 83/27 6.67/28,814 101P48500 49/12 6.51/26,773 69P48500 47/9 6.51/26,773 63

ptide P12075 34/4 6.46/12,681 61

ptide P12075 49/7 6.08/16,119 70

ptide P12075 17/26 6.1/16,031

al 1, Q5BJT9 52/220 8.4/47,004

se C P09117 59/19 6.67/39,259 101ondrial A35730 44/25 9.22/58,790 161

P38983 29/7 4.8/32,803 65P84083 56/7 6.36/20,386 64

ing P84076 20/39 4.9/22,428

O70371 57/19 4.99/33,944 136O70371 76/26 4.99/33,944 160Q66H96 15/34 4.6/24,453P47942 61/34 5.95/62,239 89

hyl P22062 40/13 7.31/24,479 55

P37377 36/5 4.74/14,506 56P16261 27/10 9.97/35,033 58

nel O88758 16/12 7.56/54,880 57

r form P02091 43/6 7.98/15,838 56, Q6P502 47/35 5.86/60,322 172

in 1 Q03410 33/38 5.63/116,439 65P63029 41/7 4.8/19,462

P39949 56/4 5.51/8388 55CAD35215 43/10 6.08/23,503 60

tein Q63714 26/86 5.01/364075 62

red by RIS treatment. A list of potential protein identifications wasd peptide masses into the SWISS-PROT, NCBI and TrEMBL databases(http://www.matrixscience.com/cgi/search_form). The identificationg the MOWSE probability score, sequence coverage, pI and molecularwhere P represented the probability that the observed match was aficant p value

1 1B R A I N R E S E A R C H2001) and weight gain (Chue and Cheung, 2004), the latter didnot occur in this study. The molecular mechanisms respon-sible for these adverse consequences of antipsychotic drugtreatment are not well understood. We used SpragueDawleyrats to model APD-induced side effects to subsequentlyidentify specific proteomic changes in the striatum by 2DE.The striatum is one of the key brain structures targeted byantipsychotic drugs (Tejedor-Real et al., 2003), hence wepostulated that the altered proteins found in this regioncould be involved in the molecular pathways responsible forthe RIS mode of action including its side-effects such as EPS.The identified 28 altered proteins belong to the followingfunctional classes: metabolic (n=12), signaling (n=7), proteinmetabolism (n=2), transport (n=3), cell cycle (n=2), DNA-binding (n=1) and chaperone (n=1). However, this subdivisionis introduced for the purposes of the discussion only, as withinthe cell, all changes in protein expression are most likely theresult of interrelated and possibly synergistic cascade me-chanisms. Some of the possible biological implications ofaltered expression of these proteins in the brain are discussedbelow.

3.1. Metabolism

RIS altered the expression of 11 protein spots identified asenzymes involved in energy transduction. These included

Fig. 1 (A) A typical 2-DE gel image of proteins spots separatedspots are circled. Intact lines circle proteins of higher expressionexpression in the VEH group compared to RIS. (B) This image is amas golgi complex assisted protein and dihydropyrimidinase-relaproteinswas increased by RIS. (C) Differences in the relative expre1991 and 2298 in terms of normalized volume. The normalized vo(p

1 1system responsible for fast regeneration of ATP (Berg et al.,2002), has been positively correlated with synaptic densityand energy requirements (Kaldis et al., 1996; Shen et al.,2002). Hence the down-regulation of this enzyme in thepresent study may indicate possible reduction of synapticdensity in the striatum due to RIS. Furthermore, chronic RIStreatment possibly leads to impaired mechanisms of neuro-protection as uMiCK is fundamental in facilitating cellularactivities such as ion and neurotransmitter transport (Hem-mer and Wallimann, 1993) and the maintenance of mito-chondrial functional and structural integrity (Beal, 2003). Aloss of cell viability has been linked with the down-regulationof two other enzymes, MiATPase- and COX, in oxidativestress induced in Alzheimer's disease (Manczak et al., 2004).Both enzymes are involved in the mitochondrial respiratorychain: COX being complex IV, while MiATPase- is a keycomponent in the F0F1-ATPase/ATP system proton channel inmitochondria, where it plays a crucial role in the transloca-tion of protons across the membrane and catalyses the ATP-dependent expulsion of hydrogen ions the internal matrixduring oxidative phosphorylation (Howitt et al., 1988; Zhengand Ramirez, 2000). The down-regulation of this importantelement of the proton pump is likely to contribute to asignificant impairment of energy metabolism in the striatum.The three spots identified as COX in this study possiblyrepresent different isoforms of this protein. Decreasedexpression of COX in the striatum has also been noted inassociation with schizophrenia (Cavelier et al., 1995), sug-gesting the possibility that this change in human striatumresulted from the APDs taken by patients in life rather thanthe disease process. Interestingly, impaired function of themitochondrial respiratory chain has also been linked withside effects of APDs including EPS (Maurer and Moller, 1997).As neurons are reliant on the mitochondrial respiratorychain for ATP supply, disruption of this system may conferincreased risk for chronic EPS such as tardive dyskinesia dueto the effects of an energy deficit and free radical productionon neuronal viability in the striatum (Maurer et al., 2001;Prince et al., 1997).

Tyrosine hydroxylase (TH; absent in VEH) is the rate-limiting enzyme in the catecholamine synthesis pathwaythat produces dopamine. The increased presence of THfollowing RIS treatment is in line with previous reports ofAPDs increasing neuronal activity, dopamine release and THactivity in the striatum in response to antagonism of dopami-nergic receptors by these drugs (Aghajanian and Bunney, 1977;Bunney et al., 1973).

3.2. Signaling

RIS altered the expression of seven signaling proteins in thestriatum. These included 40S ribosomal protein SA (absent inVEH), ADP ribosylation factor 5 (absent in VEH), calcium-binding protein 7 (CBP7; 1.48), annexin V (4.68 and 4.77),neuron-specific calcium binding protein hippocalcin (hippo-calcin; absent inVEH) anddihydropyrimidase-relatedprotein 2(DRP-2; 1.53). Interestingly, three of these proteins, CBP7,annexin V and hippocalcin, relate to calciumbinding. Annexin

28 B R A I N R E S E A R C HV is aCa2+-dependent bindingprotein that formsvoltage-gatedCa2+ channels in the phospholipid bilayers of membranesthroughout the body. Due to its extensive expression, annexinV is used as a marker of apoptosis in the central nervoussystem (Waltonet al., 1997). Hence, the increasedexpressionofannexin V may indicate that RIS increases apoptosis in thestriatum. Hippocalcin belongs to a family of neuron-specificcalcium binding proteins (Burgoyne et al., 2004) and isexpressed primarily in hippocampal pyramidal neurons, butalso appears in cerebral, cerebellar and striatal neurons(Kobayashi et al., 1992). The structure of this protein includesa unique N-terminal myristoylated end that may enable it totarget specific organelles and modulate neurotransmitterrelease in response to raised intracellular calcium levels(O'Callaghan et al., 2005). The reduction in hippocalcinexpression in the total protein extracts from RIS-treatedstriatum is in conformation with a recent genomic study thatrevealed down-regulated hippocalcin in the fronto-temporo-parietal cortex of SpragueDawley rats following acute (96 h)treatment with RIS (Feher et al., 2005). In the cell, hippocalcinmay act in the regulatory control of the potentially excitotoxicneurotransmitter glutamate, and when absent or down-regulated, glutamate may be released in excess, causingoxidative stress and neurodegeneration. This hypothesis isimplicated in the pathogenesis of diseases such asAlzheimer'sandHuntington's (Butterfield et al., 2003; Choo et al., 2005; Sianet al., 1994). In addition, the markers of glutamatergicneurotransmission have been found to be raised in the CSF ofpatients suffering from tardive dyskinesia (Tsai et al., 1998).Thus, the decreased expression of hippocalcin in the striatalproteome of RIS-treated rats suggests that possible alterationsin glutamate release pathways trigger neurotoxicity viaoxidative stress. Another signaling protein of interest is DRP-2, a protein involved in the repair of damaged neurons and inthe development and maintenance of neuronal networks(Boyd-Kimball et al., 2005). Altered levels of DRP-2 have beenobserved in Alzheimer's Disease (Lubec et al., 1999). The higherlevels of DRP-2 induced by RISmay reflect an initial attempt byneural cells to meet increased requirements for repairmechanisms, resulting from oxidative stress created by themetabolic dysfunction discussed in the previous section.

3.3. Protein metabolism

Two proteins involved in protein metabolism were altered byRIS-treatment. These were protein-L-isoaspartate-O-methyl-transferase (PIMT; 1.46) and -synuclein (1.33). PIMT plays arole in the repair and/or degradation of damaged proteinsconverting L-isoaspartyl residues in damaged proteins tonormal L-aspartyl residues. PIMT-deficient mice reportedlydevelop neurodegenerative changes due to the neurotoxiceffects of accumulated aspartate (Shimizu et al., 2005). Hence,the increase in PIMT in the present study may suggest anincreased demand for protein repair mechanisms followingRIS-treatment. It is interesting that the expression of -synuclein, a protein that associated with neurodegeneration,apoptosis and oxidative stress (Xu et al., 2002) through itsinvolvement in Parkinson's and Alzheimer's diseases, wasaltered by RIS. Alpha-synuclein may have a role in protectingnerve terminals against injury and act as a molecular

1 3 ( 2 0 0 6 ) 2 4 3 2chaperone (Kim et al., 2000; Souza et al., 2000), particularly inthe folding of synaptic proteins involved in neurotransmitter

animals were allocated to two different experimental groups

1 1(n=12/group): (1) VEH: osmotic pump implants loaded withVEH solution; and (2) RIS: osmotic pump implants loaded withRIS. Animals were kept group-housed (three rats of similartreatment per cage). All research and animal care procedureswere approved by the Garvan Institute/St. Vincent's HospitalAnimal Experimentation Ethics Committee and were inagreement with the Australian Code of Practice for the Careand Use of Animals for Scientific Purpose.

4.2. Chemicals and RIS preparation

RIS was purchased from Janssen Pharmaceutica (Beerse,Belgium). Sterile water for drug preparation was obtainedfrom Baxter Healthcare (Sydney, Australia) and both glacialacetic acid and sodium hydroxide pellets were sourced fromSpectrum (Gardena, USA). Urea, Thiourea, C7Bz0, Tris,Iodoacetamide, Acrylamide monomer, Dithiothreitol (DTT)and Acetone were sourced from Sigma-Aldrich, St. Louis,USA. Molecular grade citric acid was obtained from BDH,Poole, England. All water for laboratory use in this projectwas purified by reverse osmosis and ion exchange/organicfiltration (Millipore RO and Milli Q, Billerica, USA).release and synaptic integrity known as SNARE complexes(Chandra et al., 2005). Hence a reduction in -synuclein by RISmay conform to the hypothesis that supra-therapeutic dosesof this APD have neurotoxic effects on dopaminergic neuronsin the rat brain striatum.

In conclusion, these proteins of altered expression by RISare likely to act synergistically to induce cellular cascades thatimpair the functional integrity of neurons. Most of theidentified proteins can be divided into two groups: firstlythose that may contribute to oxidative stress by way of anenergydeficit (alterations tometabolic enzymes) and/or failureof neuroprotection against excitatory amino acids such asglutamate (hippocalcin) and aspartate (PIMT); and secondlythose proteinswhose increased expressionmaybe in responseto increased requirements for cell repair and degradationmechanisms resulting from this oxidative stress (DRP-2, PIMTand-synuclein). Hence, theseproteomic findings suggest thatsupra-therapeutic doses of RIS impair cell viability within thestriatum which may contribute to subsequent motor sideeffects such as EPS due to the importance of the striatum inmotor control.

4. Experimental procedures

4.1. Animals

Twenty-four age-matched adult, male SpragueDawley ratsobtained from the animal facility of the University of Adelaide,Laboratory Animal Services, Adelaide, Australia (910weeks atarrival, 35775 g body weight) were kept under standardlaboratory conditions with a 12:12 h light:dark schedule (lightphase: white light with illumination of 70 lxdark phase: redlight with illumination of

1 1resulting mixture was centrifuged (15,000g, 4 C, 20 min;Biofuge primo R, Heraeus, Kendro, Asheville, USA) and thesupernatant collected. Iodoacetamide and acrylamide mono-mer (final concentrations of 5 mM and 10 mM, respectively)were added to reduce and alkylate the sample. After 2 h atroom temperature (RT), the reactionwas stopped using DTT toa final concentration of 10 mM. Five volumes of acetone in15mM citric acidwere used to induce precipitation for 5min atRT before the solutions were centrifuged at 2500g and 15 Cfor 15 min (Megafuge 1.0, Heraeus, Kendro, Asheville, USA).The pellet was left to air dry for 5 min. These acetone,centrifugation and drying steps were repeated once againbefore the resultant pellet was resuspended in 0.5 ml Buffer 2(7Murea, 2M thiourea, 1% C7Bz0). This solutionwas sonicated(50% intensity, 100% cycle, 8 s single burst). The mixture wasseparated by centrifugation (15,000g, 4 C, 20 min) to removeundissolved particles, and the supernatant decanted andstored at 80 C until use.

4.7. Two-dimensional electrophoresis (2DE)

4.7.1. Determination of protein concentrationThe protein concentration of rat brain striatal samples wasdetermined following the procedure described by Bradford(1976) using a kit from Sigma-Aldrich, St. Louis, USA.

4.7.2. Isoelectric focusingIPG strips, run in duplicate for each sample, were re-hydratedin 200 l of protein extract (containing 200 g protein) mixedwith 2 l tracking dye for 6 h at room temperature. Oncehydrated, the strips were run using the ElectrophoretIQ3

system (Proteome Systems, Billerica, USA). In this protocol,an 8-h first phase of increasing voltage (100 to 10,000 V)preceded a 9-h constant voltage (10,000 V) second phase withcurrent constant at 50 A/strip and running temperature of14 C. Each IPG strip was equilibrated with 2 ml EquilibrationBuffer for 10 min on a shaker, and this step was repeated onceagain after washing strips in Milli Q water.

ProteomIQ Tracking dye, Wicks, IPG Cover Fluid, IPGstrips (linear gradient, pH 310, 11 cm) and Equilibrium Buffer(6 M urea, 2% SDS, 50 mm Trisacetate pH 7, bromophenolblue) were obtained from Proteome Systems (Billerica, USA).

4.7.3. 2D SDS-PAGE gel electrophoresisIPG strips were loaded onto precast 10 cm15 cm SDS-PAGEgels andplaced four to a tank filledwith running buffer (dilutedto 10% stock concentration). Gels were run in the seconddimension using the ElectrophoretIQ3 system for between 90and 110 min until the front line reached the bottom of the gel.The running temperature was set at 25 C, and power, currentand voltage were set at 18.7 W/gel, 30 mA/gel and 250 W,respectively. Gelswere removed fromthe tank, rinsed inMilli Qwater, cut for identification and placed into fixative solution(25%methanol, 10%acetic acid) for 30minona shaker. Fixativewas decanted and gels were rinsed with 100 ml water beforebeing stained with Coomassie blue stain for approximately16 h. Gels were stored in storage solution.

ProteomIQ Running Buffer Stock (Tris/Glycine/SDS), SDS-

30 B R A I N R E S E A R C HPAGE gels (Gel ChIP 2D: TrisHCl 816% linear gradient,10 cm15 cm), Coomaisse Blue Gel Stain Kit, and StorageSolution were obtained from Proteome Systems (Billerica,USA). Methanol was sourced from Fronine (Sydney, Australia)and acetic acid from Spectrum (Gardena, USA).

4.8. Image analysis

Gels were analyzed using Phoretix 2D Expression software (Non-linear Dynamics, Newcastle Upon Tyne, UK) after beingscanned using a transmissive, flatbed scanner (UMAX) cali-brated using an AGFA 25125 mm scale. Average gels for RISand VEH groups were created after initial editing of spots insuch a way that a spot only appeared on the average gel if itwas present in at least 70% of the 24 gels in its group. One-wayAnalysis of Variance (ANOVA) was used to identify differen-tially expressed spots of statistical significance between thetwo groups. Proteins with a mean n-fold change of betweenRIS and VEH gels of 1.3 were excised for identification bymass spectrometry.

4.9. Sample preparation for mass spectrometry

Protein spots of interest were cut from the 2DE gels and de-stained for 1 h at room temperature using a freshly preparedwash solution consisting of 100% acetonitrile/50 mM ammo-nium bicarbonate (60:40 v/v). Wash solution was removed andspots were left to dry for 30 min at 37 C. Proteins weredigested using a trypsin solution containing 12 g/ml trypsinin 50 mM ammonium bicarbonate solution. This reaction wasleft to proceed for 45 min at 4 C. Excess trypsin solution wasremoved and 15 l of 50 mM ammonium bicarbonate wasadded before gel pieces were placed in a 37 C incubatorovernight.

The transfer of samples onto a MALDI plate was a five-stepprocess: (1) the priming of C-18 purification tips (Eppendorf,Hamburg, Germany) using twowashes of buffer A (70% ACN v/v/0.1% TFA v/v) followed by two washes of buffer B (0.1% TFAv/v), (2) binding of protein sample to the C-18 tip by thetemporary uptake of sample, (3) washing the sample withbuffer B three times, (4) uptake and elution of 3 l matrixsolution (-Cyano-4-hydroxycinnamic acid 99%, 8 mg/ml in70% v/v acetonitrile/1% v/v formic acid) onto MALDI plate, (5)allowing plate to air dry and crystallize.

Sodium bicarbonate, TFA and -Cyano-4-hydroxycinnamicacid were sourced from Sigma-Aldrich (St. Louis, USA),acetonitrile from Mallinckrodt (South Oakleigh, Australia)and porcine sequencing grade trypsin from Promega (Madi-son, USA).

Samples were analyzed using an Applied BiosystemsMALDI-TOF Voyager DE-PRO (BMSF, UNSW), using positivereflector mode and delayed extraction.

4.10. Protein identification

A list of potential protein identifications was generated foreach protein spot by entering its experimentally determinedpeptide masses into the SWISS-PROT, NCBI and TrEMBLdatabases using the proteomic servers Aldente (http://au.expasy.org/) and MASCOT (http://www.matrixscience.com/

1 3 ( 2 0 0 6 ) 2 4 3 2cgi/search_form). The identification of the most likelycandidate from each protein spot list was validated using

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Burgoyne, R.D., O'Callaghan, D.W., Hasdemir, B., Haynes, L.P.,the MOWSE probability score, sequence coverage, pI andmolecular weight. The MOWSE score was calculated usingthe formula 10*Log (P), where P represented the probabi-lity that the observed match was a random event. A scoreof at least 55 was necessary to correlate to a significantp value

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Effects of chronic risperidone treatment on the striatal protein profiles in ratsIntroductionResults2DE comparison of striatal tissue from VEH and RIS-treated ratsIdentification of altered proteins in the striatum of RIS-treated rodents

DiscussionMetabolismSignalingProtein metabolism

Experimental procedureAnimalsChemicals and RIS preparationSurgeryBehavioral testingTissue dissectionProtein extractionTwo-dimensional electrophoresis (2DE)Determination of protein concentrationIsoelectric focusing2D SDS-PAGE gel electrophoresis

Image analysisSample preparation for mass spectrometryProtein identification

AcknowledgmentsReferences