Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Selective Increases in Phosphoinositide Signaling Activityand G Protein Levels in Postmortem Brain from Subjects

with Schizophrenia or Alcohol Dependence

Richard S. Jope, Ling Song, Carol A. Grimes, Mary A. Pacheco, *Gjnny E. Dilley,Xiaohua Li, *Herbe~Y. Meltzer, tJames C. Overholser, and *craig A. Stockmeier

Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama,and Departments of * Psychiatry and fPsychology, Case Western Reserve University, Cleveland, Ohio, U.S.A.

Abstract: Comparisons of the activity of the G protein-mediated phosphoinositide signal transduction systemand of G protein levels were made in two regionsof frontalcortex from eight schizophrenic, alcohol-dependent, andcontrol subjects. G protein-mediated phosphoinositidehydrolysis was measured by stimulating cortical mem-branes incubated with [3H]phosphatidylinositol with 0.3—10

1uM guanosine 5 ‘-0- (3-thio)triphosphate (GTPyS). Infrontal cortex areas 8/9, GTPyS-induced phosphoinosi-tide hydrolysis was 50% greater in schizophrenic thancontrol or alcohol-dependent subjects, whereas therewere no differences among these groups of subjects inthe response to GTPYS in frontal cortex area 10.Agonistsfor dopaminergic, cholinergic, purinergic, serotonergic,histaminergic, and glutamatergic receptors coupled tothe phosphoinositide signaling system increased [

3H]-phosphatidylinositol hydrolysis in a GTPyS-dependentmanner. Responses to most agonists were similar in allthree subject groups in both cortical regions, with thelargest difference being a 40% greater response to dopa-minergic receptor stimulation in frontal cortex 8/9 fromschizophrenic subjects. Measurements of the levels ofphospholipase C-~3,and of a-subunits of Gq, G

0, G11,G2, and G9, made by immunoblot analyses revealed nodifferences among the groups of subjects except for in-creased Ga0 in schizophrenic subjects and increased Ga0and Ga11 in alcohol-dependent subjects. These resultsdemonstrate that schizophrenia is associated with in-creased activity of the phosphoinositide signal transduc-tion system and increased levels of Ga0, whereas thephosphoinositide system was unaltered in alcohol depen-dence, but Ga0 and Ga,1 were increased. Key Words: Gproteins — Phosphoinositide — Schizophrenia — Alcoholdependence— Inositol—Postmortem human brain.J. Neurochem. 70, 763—771 (1998).

Effective activation and regulation of signal trans-duction systems that support interneuronal communi-cation are necessary for maintenance of normal cogni-tive and emotive functions. One of the primary meansused by cells to transduce extracellular signals to intra-

cellular responses is heterotrimeric G proteins (con-sisting of a-, /3-, and y-subunits), which coupleplasmamembrane receptors to intracellular effectors (Gilman,1987). The phosphoinositide second messenger sys-tem is one of the major G protein-linked signal trans-duction processes that is known to operate in the CNS(Fisher, 1995). This signaling system consists of threeprimary protein components, receptors, heterotrimericO proteins, and phospholipase C. Signal transductionis initiated on receptor activation by an appropriateagonist. The stimulated receptor then activates a Gprotein that is coupled to it by enhancing the exchangeof GDP previously bound to the inactive a-subunit forGTP. Through mechanisms not completely under-stood, the GTP-activated G protein then stimulatesphospholipase C, which cleaves phosphoinositides inthe plasma membrane to produce second messengerswithin the cell.

The phosphoinositide signal transduction system hasbeen studied extensively in animal tissues and culturedcells, but less so in human brain due to methodologicaldifficulties in using postmortem tissue (Pacheco andJope, 1996). However, in recent years (Wallace andClaro, 1993), it has become possible to assess theactivity of the phosphoinositide signal transductionsystem in postmortem human brain by measuring thehydrolysis of exogenous labeled phosphoinositides,such as [

3Hlphosphatidylinositol ([3H]PI), incubatedwith membranes and appropriate stimuli [i.e., receptor

Received May 27, 1997; revised manuscript received September15, 1997; accepted September 30, 1997.

Address correspondence and reprint requests to Dr. R. S. Jopeat Sparks Center 1057, Department of Psychiatry and BehavioralNeurobiology, University of Alabama at Birmingham, Birmingham,AL 35294-0017, U.S.A.

Abbreviations used: ACPD, trans-i -aminocyclopentyl- 1 ,3-dicar-boxylic acid; ANOVA, analysis of variance; FC8/9 and FC1O, fron-tal cortex areas 8/9 and 10, respectively; GAP-43, growth-associatedprotein-43; GTPyS, guanosine 5 ‘-O-(3-thiotriphosphate); P1, phos-phatidylinositol; SDS, sodium dodecyl sulfate.

763

764 R. S. JOPE ET AL.

agonists and guanosine 5’-O- (3-thiotriphosphate)(GTPyS ) I (for review, see Pacheco and Jope, 1996).Such studieshave revealedthat the activity of the phos-phoinositide system is selectively impaired in discretebrain regions in samples from subjects with certainneurological(e.g., Alzheimer’s disease; for review, seeJope, 1996) and psychiatric (e.g., major depressionand bipolar mood disorder; Jope et al., 1996; Pachecoet al., 1996) illnesses. These initial findings in studiesusing human brain suggest that subtle alterations inthe activity of the phosphoinositide signal transductionsystem may influence cognition, mood, and behaviorand, therefore, may be associated with other such dis-orders. Therefore, the first goal of this investigationwas to assess the functional state of the phosphoinosi-tide signal transduction system in cerebral corticalsamples from control, schizophrenic, and alcohol-de-pendent subjects.

As described above for the phosphoinositide system,O proteins play a central role in coupling receptorsexposed to the extracellular milieu to intracellular ef-fector enzymes. 0 proteins have been categorized intofour families based on the functional properties andsequence homologies of the a-subunits (Simon et al.,1991) and these include the Gq, O~,G~,0,and G12families. Cellular functions linked to these 0 proteinsinclude activation of phospholipase C (Gq), stimula-tion of adenylyl cyclase (Gb), inhibition of adenylylcyclase (01), and modulation of ion channels (G0),among others. Assessments of tissue 0 protein levelsare amenable to quantitative analysis by using immu-noblot procedures, and several G proteins, including0q’ O~,0u. G

12, and G~,have been found to be rela-tively stable in postmortem human brain (Li et al.,1996). Because of their central role in signal transduc-tion processes, potential alterations in 0 proteins asso-ciated with a number of psychiatric disorders havebeen investigated in postmortem human brain (Manji,1992; Hudson et al., 1993), but little information isavailable concerning G protein levels in brain regionsfrom schizophrenic or alcohol-dependent subjects(Nishino et al., 1993; Okada et al., 1994). Therefore,the second goal of this investigation was to determineif the levels of several 0 protein a-subunits in tworegions of the cerebral cortex were altered in subjectswith schizophrenia or alcohol dependence.

MATERIALS AND METHODS

SubjectsBrain tissue was obtained at autopsy from the Cuyahoga

County Coroner’s Office, Cleveland, OH, U.S.A. The studywas performed in compliance with policies of an institutionalreview board and informed written consent was obtainedfrom the next of kin for all subjects. The cause of death forthe subjects was ruled by the County Coroner (Table 1).Tissue samples were collected from eight subjects withschizophrenia (six males and two females; 41 ± 11 yearsof age; 16 ±6 h postmortem interval; mean ±SD), eightsubjects with alcohol dependence (six males and two fe-

males; 41 ±12 years of age; 14 ±9 h postmortem interval),and eight psychiatrically normal comparison subjects (eightmales; 41 ±12 years of age; 17 ±8 h postmortem interval).Subjects with schizophrenia died from natural causes (n= 3), suicide (n = 4), or homicide (n = 1). Subjects withalcohol dependence died from natural causes (n = 2), sui-cide (n = 4), ethanol abuse plus cirrhosis (n = 1), orhomicide (n = 1). Comparison subjects died from naturalcauses (n = 5) or homicide (n = 3). Analysis of variance(ANOVA) revealed no significant differences in age andpostmortem intervals between groups. Matched cohorts ofsubjects were grouped according to age (<6 years).

Samples of blood and urine from all of the subjects wereexamined by the toxicology laboratory of the County Coro-ner’ s Office. Qualitative and quantitative assays were usedto detect several classes of psychoactive compounds, as pre-viously described (Pacheco et al., 1996). The results of thetoxicology testing are presented in Table 1. Information isalso presented in Table 1 on current and previous prescrip-tions for medications. All subjects with schizophrenia had aprescription for an antipsychotic drug at some time in theirlives, and three had such a prescription within the last monthof life. Although three subjects with alcohol dependence hadprescriptions for psychotropic medications, none of theseprescriptions was written within the last month of life.

Retrospective psychiatric assessmentsRetrospective psychiatric assessments were used to evalu-

ate psychiatric diagnoses among the subjects. Based on diag-nostic interviews, the followingthree groups were identified:(1) subjects with a current diagnosis of schizophrenia, (2)subjects with a diagnosis of alcohol dependence at the timeof death, and(3) normal controls with no psychiatric history.A trained interviewer met with a knowledgeable informant3—4 months after the death. A structured diagnostic inter-view was used to collect information needed for evaluatingthe presence or absence of Axis I psychiatric syndromes.For 22 of the 24 subjects, psychiatric diagnosis was basedon a structured interview with a knowledgeable informantwho either lived with or had frequent weekly contact withthe subjects before death. Dataon lifetime and current mentalillness were gathered with a modified Schedule for AffectiveDisorders and Schizophrenia; lifetime version (SADS-L;Spitzer and Endicott, 1978), as previously described (Pa-.checo et al., 1996). For two subjects with schizophrenia,only hospitalization records were available for making theAxis I diagnosis.

Diagnoses for Axis I disorders were independently evalu-ated based on information from the SADS-L. A consensusdiagnosis was obtained when both a clinical psychologistand a psychiatrist evaluated available information from theknowledgeable informants, coroner’s office, previous hospi-talizations, and doctors’ records (Table 1).The final diagno-sis was compatible with the Diagnostic and Statistical Man-ual of Mental Disorders, third edition, revised (DSM-III-R; American Psychiatric Association, 1987) classification.Among the subjects with schizophrenia, one had a historyof bulimia nervosa and anotherhad a history of psychoactivesubstanceabuse. Among subjects meeting criteria for alcoholdependence, three had comorbid major depression and onehad comorbid adjustment disorder with depressed mood. Forthe four alcohol-dependent subjects with a comorbid mooddisorder, the symptoms of alcohol dependence emerged be-fore the symptoms of the mood disorder. The control subjectsnever met diagnostic criteria for schizophrenia, psychoactive

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G PROTEIN LEVELS IN SCHIZOPHRENIA AND ALCOHOLISM 765

TABLE 1. Characteristics of subjects

Age (yr)/Sample Axis 1 diagnosis gender PM! pH Cause of death Toxicology Medication history

Control No diagnosis 23/M 19 6.75 Gsw-thorax Cannabinoids NoneScz SCZ (chronic, paranoid) 23/M 19 6.71 Suicide, hanging Nothing detected Loxapine, cogentin, enalapnl, duramedAlcohol Alcohol dependence, major 26/M 6 6.63 Suicide, overdose Propoxyphene, None

depression acetaminophen,ethanol (0.1%)

Control No diagnosis 26/M 13 6.66 Gsw-neck Nothing detected NoneScz SCZ (chronic, paranoid); Hx: 32/M 10 6.78 Suicide, Sigsw- Ethanol (0.04%) Haloperidol, cogentin (noncompliant)

cannabis, cocaine use headAlcohol Alcohol and cocaine 291M 9 6.73 Suicide, hanging Cocaine, ethanol None

dependence, major (0.09%)depression

Control No diagnosis 37/M 26 6.00 Heart disease Lidocaine NoneScz SCZ (chronic, paranoid); Hx: 35/F 15 6.71 Suicide, Sigsw- Nothing detected Thioridazine (noncompliant),

bulimia nervosa head furosemide. dyazideAlcohol Alcohol dependence, 32/M 14 6.54 Suicide, Sigsw- Ethanol (0.3%) None

cannabis abuse, adjustment thoraxdisorder with depressedmood

Control No diagnosis; Hx: alcohol 40/M 22 ND Heart disease Lidocaine Noneabuse

Scz SCZ~ 43/M 16 6.77 Sigsw-abdomen Nothing detected Fluphenazine, Hx thiothixeneAlcohol Alcohol and anxiolytic 40/M 15 6.70 Sigsw-chest Nothing detected Fluoxetine, bupropion, buspirone,

dependence, major lithium, flurazepam, clonazepamdepression (singleepisode); Hx: dysthymia

Control No diagnosis 44/M 6 6.67 Aneurism Ephedrine,chlorpheniramine,phenylpropanolamine

None

Scz SCZ (chronic, undiff.) 45/F 6 6.58 Heart disease Loxapine, amoxapine,carbamazepine

LOXAPINE, AMOXAPINE,RISPERIDONE, CARBAMAZEPINE,BENZTROPINE

Alcohol Alcohol dependence, alcohol 40/M 7 ND Gsw-neck Ethanol (0.31%), Nonehallucinosis cocaine metabolite

Control No diagnosis 46/M 10 ND Heart disease Nothing detected Furosemide, lisinopril, captopril,glipizide

Scz SCZ (chronic, disorgan.)~ 47/M 16 6.62 Pulmonary disease Nothing detected ChlorpromazineAlcohol Alcohol dependence 45/F 28 5.82 Bronchopneumonia,

pleuritisNothing detected Chlordiazepoxide, insulin, glyburide.

ranitidine, phenobarbitalControl No diagnosis 51/M 16 6.71 Heart disease Nothing detected NoneScz SCZ (chronic, undiff.) 51/M 18 6.76 Suicide, Sigsw-

headNothing detected TRIFLUOPERAZINE, LOXAPINE,

CLONAZEPAM, BENZTROPINE,Hx: electroconvulsive therapy

Alcohol Alcohol dependence 55/F 7 ND Alcohol abuse,cirrhosis

Ethanol (0.05%) Lithium, chlorpromazine, valproate,thiamine, triamterene

Control No diagnosis 60/M 27 6.75 Gsw-thorax Nothing detected NoneScz SCZ (chronic, paranoid) 55/M 24 6.76 Heart disease Nothing detected ChiorpromazineAlcohol Alcohol dependence 6l/M 27 6.68 Heart disease Nothing detected Nitroglycerin

Medications that are in all capital letters were prescribed within the last month of life. disorgan., disorganized; F, female; Gsw, gunshot wound; Hx, historyof; M, male; ND, not determined; PM!, postmortem interval (rounded to the nearest hour); S Z, schizophrenia; Sigsw, self-inflected gunshot wound; undiff.,undifferentiated.

The diagnosis of schizophrenia for these subjects was made by review of medical records from psychiatric hospitalizations.

substance dependence, or a depressive disorder during theirlifetimes. Cannabinoids were present in blood from one con-trol subject, but thesubject did not meet criteria for apsycho-active substance use disorder. Another control subject metcriteria for alcohol abuse for several months at 3 years beforehis death. This subject did not meet criteria for alcohol abuseat the time of death or in the 3 years before death. Aftertissue collection and consensus diagnosis in Cleveland, tis-sue samples were coded to conceal psychiatric diagnosis andshipped to Birmingham for biochemical analysis.

NeuropathologySections of prefrontal cortex (area 10) were stained with

hematoxylin and eosin and examined by a board-certifiedneuropathologist to rule out majorpathology. Further studieswere done in some subjects with a mouse monoclonal anti-

body to glial fibrillary acidic protein (GA5, Sigma Chemi-cal) to rule out gliosis, or with 4G8, a monoclonal antibodyto A~3amyloid plaques, to rule out Alzheimer’s disease.

pH measurementsSamples of cerebellum from most subjects were used for

measurement of pH as described by Harrison et al. (1995).The pH values are listed in Table 1. The average pH values(±SD) for the groups were as follows: control subjects,6.59 ±0.29; subjects with schizophrenia, 6.71 ±0.07; andsubjects with alcohol dependence, 6.52 ±0.35. ANOVAindicated there were no significant differences in pH amongthe three groups of subjects.

[3H]PI hydrolysis assayMembranes were prepared as described previously (Jope

et al., 1996) from right human frontal cortex Brodmann areas

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766 R. S. JOPE ET AL

8 and 9 combined (FC8/9) and Brodmann area 10 (FC1O)and were stored at —80°C.Membrane samples were resus-pended by homogenization in 10 mM Tris-maleate (pH 6.4).After a 30-mm preincubation at room temperature, mem-branes (0.1 mg of protein) were incubated at 37°Cfor 60mm in 10 mM Tris-maleate, pH 6.4, 6 mM MgCl2, 8 mMLiC1, 3 mM EGTA, 3.25 mM CaC12, 1 mM deoxycholate,0.1 mM [

3H]PI (5—10)< io~cpmllO nmol; Du Pont), andGTPyS and other stimulants as indicated. The reaction wasstopped by the addition of 1.2 ml CHC1

3/CH3OH (1:2; vol/vol), followed by 0.5 ml CHC13 and 0.5 ml 0.25 M HC1.Samples were incubated on ice for 20 mm, phases wereseparated by centrifugation, and radioactivity in the aqueousphase was quantitated by liquid scintillation spectrometry.

Assays were performed blind to subject diagnoses, andmembranes from a matched cohort of one control, oneschizophrenic, and one alcohol-dependent subject were as-sayed (each in triplicate) in each experiment. The data werecalculated as nanomoles of [

3H 1 P1 hydrolyzed per milligramofprotein per hour after subtraction of basal values that wereobtained for each tissue by incubation in the absence ofadded stimulant (i.e., no GTPyS or receptor agonist). Foragonists that stimulated appreciable [3H1 P1 hydrolysis in theabsence of GTPyS (see Pacheco and Jope, 1996), whichincluded serotonin, ATP, and SKF 38393, values obtainedwith each agonist in the absence of GTP

7S were subtractedfrom those obtained in the presence of GTPyS to calculatethe G protein-dependent responses. TheGTPyS-independentvalues did not vary among subject groups (data not shown).Statistical significance was determined by using ANOVAwith a post hoc Bonferroni test.

Western blottingSubtypes of G proteins and phospholipase C-13 were mea-

sured by using quantitative western blot methods describedpreviously (Greenwood et al., 1995; Li et al., 1996). Tissuesamples were solubilized in Laemmli sample buffer con-taining 1% sodium dodecyl sulfate (SDS) (Laemmli, 1970),proteins were resolved by gel electrophoresis on 10% SDS-polyacrylamide gels, and proteins were transferred to nitro-cellulose membranes. Before analyses, different amounts ofprotein wereprobed with each antibody to ascertain therangethat produced a linear response. Assays were performedblind to subject diagnosis, and extracts from each cohortof matched schizophrenic, alcohol-dependent, and controlsubjects were run together in triplicate on the same immu-noblot. In cases where significant differences among subjectgroups were identified, this procedure of triplicate measure-ments of the entire cohort was repeated to reconfirm theanalysis. In addition, some blots of the G0 a-subunit werereprobed with an antibody to tubulin to confirm that equiva-lent amounts of protein were present in each lane. In addi-tion, urea gradient SDS-polyacrylamide gels were used toseparate subtypes of Ga0 as described previously (Li et al.,1995). Tomeasure 0 protein levels, blots were probed withpolyclonal antisera for Gaqiti (10 ~ig of protein/lane;1:5,000 antibody dilution: New England Nuclear) or Ga~(20 ~.eg;1:800; Upstate Biotechnology), or with monoclonalantisera (Li et al., 1995) for Ga~1(10 ~sg; 1:1,000), Ga12(10 gig; 1:500), or Ga0(2 ,ug; 1:15,000). To measurephos-pholipase C-/3 levels, blots were probed with a monoclonalantibody specific for the phospholipase C-/3 subtype (30 ~sg;1:500; Upstate Biotechnology). The secondary antibody washorseradish peroxidase-conjugated anti-IgG (1:3,000; Gib-c0BRL) and the protein bands were visualized by a color

reaction with 0.05% 3,3 ‘-diaminobenzidine and 0.01% H202in 50 mM Tris/ 10 mM imidazole, pH 7.2. Western blotswere quantitated by densitometry and data were analyzedfor statistical significance by using ANOVA with post hocDunnett’ s multiple comparison test.

RESULTS

G protein function associated with phosphoinositidesignaling was examined by incubating cortical mem-branes with [

3H]PT and the stable analogue of GTP,GTP

7S, to stimulate directly G protein activation ofphosphoinositide hydrolysis. ti

3HIPT hydrolysis wasstimulated by 0.3—10 pM GTP

7S in a concentration-dependent manner in membranes prepared from eachregion of frontal cortex (FC8/9 and FC1O) from con-trol, schizophrenic, and alcohol-dependent subjects(Fig. 1). In FC8/9, GTP’yS-stimulated Ii

3H]PI hydro-lysis was significantly greater in schizophrenic subjectsthan in control subjects, with elevations of 65, 50, 39,and 44% above controls obtained with 0.3, 1.0, 3.0,and 10 ~tM GTPyS, respectively. This differenceamounted to an average response to G protein stimula-tion in FC8/9 from schizophrenic subjects that was150% of that in control subjects. There were no sig-nificant differences in GTP’yS-stimulated [3HI P1 hy-drolysis between alcohol-dependent and control sub-jects in FC8/9 or among any of the three groups ofsubjects in FC1O.

The next goal was to compare receptor-coupledphosphoinositide hydrolysis in samples from control,schizophrenic, and alcohol-dependent subjects. Thestimulation of [3H1 P1 hydrolysis was measured in re-sponse to incubation of membranes with 3

1uM GTP7S(to support 0 protein-mediated responses) and six dif-ferent receptor agonists that have been reported tostimulate phosphoinositide hydrolysis in human brainmembranes, including SKF 38393 (dopamine Dl re-ceptor), ATP (P2 purinergic receptor), carbachol(cholinergic muscarinic receptor), serotonin, hista-~nine, and trans-i -aminocyclopentyl- 1 ,3-dicarboxylicacid (ACPD; glutamatergic metabotropic receptor). Inthe presence of 3 1uM GTPyS, each of these receptoragonists induced greater [

3H]PI hydrolysis than thatobtained with GTP

7S alone in membranes preparedfrom each of the brain regions (Fig. 2). In FC8/9, 3ptM GTPyS-stimulated [

3H)P1 hydrolysis was greaterin schizophrenic than control subjects, and this wasreflected in a slightly greater hydrolysis stimulated bymost of the agonists, with borderline significant (p<0.06) increases observed with SKF 38393 (140%of control) and serotonin (134% of control). Agonist-induced [3H]PI hydrolysis in FC8/9 from alcohol-dependent subjects did not differ from control subjectsexcept with ACPD, which induced a 20% smaller re-sponse in alcohol-dependent subjects. In FC1O, equiva-lent stimulation of [3H1 P1 hydrolysis was induced byeach of the agonists in each of the subject groups ex-cept for histamine and ACPD, which produced 33%

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G PROTEIN LEVELS IN SCHIZOPHRENIA AND ALCOHOLISM 767

FIG. 2. Agonist-stimulated [3H]Plhydrolysis in membranes pre-pared from FC8/9 (A) and FC1O (B). Membranes were incu-bated with 3 ,aM GTPYS alone or with 1 mM SKF 38393, ATP,carbachol (Carb), serotonin (5HT), histamine (Hist), or ACPDas described in Materials and Methods. [3H]P1 hydrolysis is givenas nanomoles per milligram of protein per hour; mean ±SEMvalues (n = 8). °p 0.06, compared with values from controlsubjects.

FIG. 1. GTPYS concentration-dependent stimulation of [3H]P1 hy-drolysis in membranes prepared from FC8/9 (A and B) and FC1 0(C) from control, schizophrenic, and alcohol-dependent subjects.Membranes were incubated with [3H]Pland 0.3—10 pM GTPYS, asdescribed in Materials and Methods. [3H]P1 hydrolysis is given asnanomoles per milligram of protein per hour; mean ±SEM values(n = 8). °p 0.05, compared with values from control subjects.

lower responses (p 0.06) in schizophrenic comparedwith control subjects.

The difference in the GTP7S-stimulated activity of

the phosphoinositide signal transduction system be-tween schizophrenic and control subjects could be re-lated to altered levels of the G protein (Gq) or thephospholipase (phospholipase C-~3)mediating this ac-tivity. To test this hypothesis, quantitative immunoblotanalysis was used to measure the levels of Gaq andphospholipase C-/3. Representative immunoblots foreach protein are shown in Fig. 3. These experimentsrevealed that there were no differences between schizo-phrenic and control subjects in the levels of the Gq a-subunit or phospholipase C-/.3 in either areaof thefron-tal cortex (Table 2).

The levels of four other 0 protein a-subunit sub-types were measured in both areas of frontal cortex todetermine if any of these 0 proteins that mediate other

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768 R. S. JOPE ET AL.

significantly higher in FC8/9 from alcohol-dependentsubjects than in control subjects. Otherwise, the phos-phoinositide C-/3 and the G protein a-subunit levelsin alcohol-dependent subjects did not differ from con-trols in either brain region.

DISCUSSION

FIG. 3. Representative immunoblots of proteins in FC8/9 fromcontrol (C), schizophrenic (Scz), and alcohol-dependent (Al)subjects. Western blots were prepared as described in Materialsand Methods. Therewere two immunoreactive bands for Ga0 (52and 45 kDa). Quantitative comparisons of protein levels amongsubject groups are presented in Table 2.

signal transduction systems were altered in schizophre-nia. Immunoblot analyses (Fig. 3; Table 2) revealedthat in schizophrenic subjects there was a large, selec-tive increase in the level of Ga0 in FC8/9, whereaslevels of the other G protein a-subunits were not differ-ent between schizophrenic and control subjects. Sepa-ration of Ga,~subtypes, using a urea gradient SDS-polyacrylamide gel, revealed that two major bands rep-resenting the two subtypes of Ga0 were detected (Fig.3). Both Ga0 subtypes were elevated equivalently inFC8/9 from schizophrenic compared withcontrol sub-jects. In FC1O, no significant differences betweenschizophrenic and control subjects were detected.

The levels of phospholipase C-~3and of five G pro-tein a-subunits were also compared in the two frontalcortical areas from alcohol-dependent and control sub-jects (Fig. 3; Table 2). The levels of two G proteina-subunits, 0. (including both subtypes) and G~1,were

This investigation examined whether the activity ofthe 0 protein-mediated phosphoinositide signaling sys-tem or the levels of several 0 protein a-subunits weredifferent in two regions of frontal cortex obtained fromschizophrenic or alcohol-dependent subjects comparedwith psychiatrically normal control subjects. With re-gard to [

3H I PT hydrolysis, two intriguing findings wereobserved, (1) a 50% increase in the GTPyS-inducedstimulation of 0 protein-mediated activation of phos-pholipase C in FC8/9 in schizophrenia, and (2) anearly absolute lack of effect of alcohol dependenceon the phosphoinositide signaling system. The moststriking observation from the measurements of 0 pro-teins was the dramatic elevation in the level of Ga

0 inFC8/9 associated with both schizophrenia and alcoholdependence. These findings indicate that there are se-lective alterations in G protein-mediated signaling sys-tems in the prefrontal cortex that distinguish subjectswith schizophrenia or alcohol dependence from con-trols.

Phosphoinositide signaling in schizophreniaAlthough potential alterations in signal transduction

systems in schizophrenia havebegun tobe investigatedin recent years, reports on this topic remain sparse(Hudson et al., 1993). Minimal attention has beendirected toward examining the phosphoinositide sys-tem in brain samples from subjects with schizophrenia,in part because of previous methodological limitationsin studying the phosphoinositide systemin postmortemhuman brain. Techniques have recently been devel-oped (for review, see Pacheco and Jope, 1996) that

TABLE 2. Quantitative analysis of G protein a-subin FC8/9 and FCJOfrom control, schizophrenic,

units and phospholipase C-8 levelsand alcohol-dependent subjects

Protein

FC8/9 FC1O

Ct! Scz Al Ct! Scz Al

Gctq 100±2 125±12 115±13 100±4 114±9 102±11PLC-/I 100±7 100±11 99±7 100±6 97±7 94± 8

Ga0 100±3 214±22° 240±31° 100±5 117±18 96±11Ga~1 100 ±6 129 ±10 134 ±13” 100 ±7 118 ±9 109 ±11Get,2 100 ±2 116 ±16 105 ±13 100 ±9 119 ±12 97 ±13Ga,52kDa 100~2 115±9 111±8 100±3 94±9 110±164SkDa 100±4 92±13 87±10 100±5 131±18 112±27

Data are percentages of levels in matched control subjects and are measured at least in triplicate.Mean ±SEM; n = 8. Ctl, control; Scz, schizophrenic; Al, alcohol dependent; PLC-/I, phospholipaseC-/I.

“p < 0.05.

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G PROTEIN LEVELS IN SCHIZOPHRENIA AND ALCOHOLISM 769

now allow the use of postmortem brain samples forexamining the functional state of the phosphoinositidesignaling system in schizophrenic subjects. The use ofGTPyS to assess the activation of the G protein (Gaq)coupled with the stimulation of phospholipase C re-vealed a regionally selective, large increase in this re-sponse in FC8/9 from schizophrenic subjects. Thisincreased activity was not due to increased levels ofGaq or phospholipase C-~3,indicating that the activity,but not the protein levels, of Gaq was increased inschizophrenia. This finding is in partial agreement withthe only previous study of phosphoinositide signalingin schizophrenic brain in which a large (67%) butnotstatistically significant increase in phosphoinositidehydrolysis stimulated by 3.3 ~aMGTPyS was observedin prefrontal cortex (area 8/9) of subjects with schizo-phrenia compared with control subjects (Wallace andClaro, 1993). It is noteworthy that two groups of inves-tigators reported increased phosphoinositide signalingin platelets obtained from subjects with schizophreniacompared with controls (Das et al., 1992; Yao et al.,1992). Thus, in conjunction with the present study,there is now substantial evidence of hyperactivity ofthe phosphoinositide signaling system in schizophrenicsubjects. Furthermore, in brain this hyperactivity wasdetected primarily at the level of the G protein, withadditional regionally selective increases in dopaminer-gic and serotonergic receptor-activated responses(FC8/9) and decreases in histaminergic and meta-botropic glutamatergic receptor-activated responses(FC1O). The large increase in G protein-activatedphosphoinositide hydrolysis in schizophreniamarkedlydistinguishes this disease from major depression andbipolar mood disorder where regionally selective de-creases in 0 protein function linked to phosphoinosi-tide hydrolysis were detected (Jope et al., 1996; Pa-checo et al., 1996). The potential for a central role ofaltered 0 protein function in a variety of diseases(Manji, 1992; Hudson et al., 1993; Pacheco and Jope,1996) emphasizes the need for further clarification ofthe mechanisms involved in regulating G protein ac-tivity.

Consideration must be given to the potential contri-butions of therapeutic drugs or other characteristicsof the subjects on the increased 0 protein-activatedphosphoinositide signaling observed in schizophrenicfrontal cortex. Although tissues were matched as wellas possible for subject age and sex, and for postmorteminterval, and tissues were subjected to extensive toxi-cological analyses, it is not known which variablesmay influence measurements of signaling activity, asrecently reviewed (Pacheco and Jope, 1996). Priordrug use is clearly a major concern, and a numberof psychoactive compounds, including antipsychoticdrugs, were detected in some of these schizophrenicbrain samples. In several previous studies in animals,treatments with antipsychotic drugs have been reported

- to decrease phosphoinositide signaling (Friedel andKnauer, 1981; Edwards et al., 1991; Li et al., 1992,

1994). If this action of antipsychotic drugs also occursin humans, these drugs would be predicted to dampenthe increased phosphoinositide signaling detected inschizophrenic brain in this study. Thus, it appears un-likely that treatment of subjects with schizophreniawith antipsychotic drugs accounts for the observed in-crease in phosphoinositide signaling activity in post-mortem tissue of subjects with schizophrenia, althoughsuch a possibility cannot entirely be discounted. Incontrast, these findings suggest that attenuation of hy-peractive phosphoinositide signaling activity in schizo-phrenic subjects represents an action that contributesto the therapeutic efficacy of the antipsychotic drugs.

Phosphoinositide signaling in alcohol dependenceIn contrast to samples from schizophrenic subjects,

the activity of the phosphoinositide signaling systemin prefrontal cortical regions 8/9 and 10 from alcohol-dependent subjects did not differ from activities inmatched control samples except for a slight decreasein the response to ACPD in FC8/9. This general lackof effect of alcohol dependence on the activity of thephosphoinositide signal transduction system wassomewhat surprising considering that much evidenceobtained by using nonhuman tissue has indicated thatthis signaling system is sensitive to ethanol (see, e.g.,Pandey et al., 1993; Kovacs et al., 1995; Larsson etal., 1995; for review, see Gandhi and Ross, 1989).The relative lack of effects of alcohol dependence mayindicate that ethanol itself influences phosphoinositidesignaling directly but the effects are reversible. Thus,in this postmortem study, factors such as withdrawaland the varied levels of ethanol when it was presentmay have contributed to the unaltered signaling activ-ity in this group of subjects.

G proteins in schizophreniaG proteins play a central role in many signaling

systems and altered brain levels have been detectedin subjects with a number of psychiatric disorders,but studies of schizophrenic and alcohol-dependentsubjects remain limited (Manji, 1992; Hudson et al.,1993). The finding that 0 protein levels in FC8/9and FC1O are generally unaffected in schizophreniais in agreement with previous studies of 0 proteinsin other brain regions, but the finding of increasedGa0 in FC8/9 is a novel observation. Reported find-ings in schizophrenia include unaltered Ga, in tem-poral cortex (Nishino et al., 1993; Young et al.,1993), decreased Ga1 in left, but not right, temporalcortex (Nishino et al., 1993), and no changes in Ga12in six brain regions (Okada et al., 1994). The generallack of differences found in this and previous studiesin the levels of other 0 protein a-subunits, betweensamples from schizophrenic and control subjects, ac-centuates the doubling that was identified in Ga0 inFC8/9 from schizophrenic subjects. In contrast, otherstudies have reported either no change or a decreasein the level of Ga0 in cortical regions from schizo-phrenic subjects. Decreases in the level of Ga0 in

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770 R. S. JOPE ET AL.

schizophrenia were reported in the left, but not right,temporal cortex (Nishino et al., 1993), and Okadaet al. (1994) reported no difference in Ga0 levels inorbital frontal cortex in schizophrenia. Thus, aswould be expected for biochemical changes inschizophrenia, it is evident that alterations in Ga,,levels are regionally selective. Further studies of Gprotein levels in schizophrenia are clearly warranted,especially with information included about currentor prior psychoactive substance use, data which areoften lacking. This information is especially im-portant when studying tissue from schizophrenic sub-jects, because they are prone to the abuse of alcoholand other psychoactive substances (Mueser et al.,1990).

The large increase in the level of Ga,, detected in thisstudy in schizophreniacould be a response to treatmentwith antipsychotic medication, rather than part of theetiology of schizophrenia per Se. Three of the subjectswith schizophrenia had prescriptions for antipsychoticdrugs within the last month of their lives, and all ofthese subjects had some exposure to antipsychoticdrugs. However, studies in rats have demonstrated thatchronic treatment with antipsychotic drugs like halo-peridol, sulpiride, or clozapine did not alter levels ofGa,, in several brain regions (Gupta and Mishra, 1992;See et al., 1993; Shin et al., 1995). Furthermore, theincrease in Ga0 levels in this study was detected onlyin FC8/9 and not in FCIO. Thus, it does not appearthat the significant increase in the levels of Ga,, inFC8/9 in schizophrenic subjects is merely a responseto prior exposure to antipsychotic medication.

G proteins in alcohol dependence0 protein levels were also found to be altered in the

frontal cortex of alcohol-dependent subjects comparedwith that in controls. The level of Ga,, was remarkablyincreased (240% of control) in FC8 /9 from alcohol-dependent subjects. In addition, the level of Ga,1 waselevated significantly in FC8/9 from alcohol-depen-dent subjects, suggesting lower production of cyclicAMP in these subjects. Ozawa et al. (1993, 1994)previously examined Ga0 levels in cerebral cortexfrom subjects classified as alcoholic (either alcoholdependence or alcohol abuse) and reported no differ-ences from control subjects in frontal, parietal, or tem-poral cortex. Alcohol dependence is distinguished fromalcohol abuse by the potential for withdrawal and toler-ance, and behavioral changes with distinct physiologi-cal and cognitive consequences (DSM-IV). Thus, itis difficult to compare directly the results of Ozawa etal. (1993, 1994) with the results of this study, becausethe increase in levels of Ga,, detected in this studyinvolving alcohol-dependent subjects may be specifi-cally related to distinct physiological correlates of tol-erance or withdrawal. However, studies in rodents tol-erant to ethanol but not withdrawn from it reveal nosignificant change in levels of Ga0 in cerebral cortex(Pellegrino et al., 1993; Tabakoff et al., 1995).

SummaryPhosphoinositide signaling activity mediated by 0

protein activation was found to be significantly ele-vated in FC8/9, but not FC1O, from schizophrenicsubjects, and no differences from controls were foundin either cortical region from alcohol-dependent sub-jects. These findings suggest that signaling abnormali-ties may contribute to the disturbances associated withschizophrenia, and it is potentially relevant that thera-peutic antipsychotic drugs dampen phosphoinositidesignaling. In contrast, the phosphoinositide signalingsystem appears to be unaffected by alcohol depen-dence. Clearly, studies in additional brain regions andin larger groups of subjects are needed to solidify theseinitial conclusions.

Although the levels of most 0 protein a-subunitswere unaltered in schizophrenic and alcohol-dependentsubjects, both groups had increased levels of Ga0 inFC8/9. The significance of large increases in Ga0 inFC8/9 in schizophrenia and alcohol dependence is notknown. Ga,, is the most abundant of the heterotrimericG proteins in mammalian brain and one of its primaryfunctions is the regulation of several ion channels (Oil-man, 1987). Thus, the large increase in the level ofGa,, identified in these two disorders indicates that Gprotein-mediated regulation of ion channels may beabnormal in schizophrenia and/or alcohol dependence.Of particular interest is the recent report that the levelsof growth-associated protein-43 (GAP-43, also knownas B50 or Fl) is twice as high in frontal cortex fromschizophrenic subjects compared with control subjects(Perrone-Bizzozero et al., 1996). GAP-43 has beenreported to colocalize with Ga0 in growth cones andto regulate the activity of 0,, directly (Strittmatter etal., 1990, 1991). Because the increased level of GAP-43 was suggested to be related to a developmentalbased disruption in synaptic connections in schizophre-nia, this may also be the basis of the large increase inGa0 in schizophrenia. Thus, it will be of interest todetermine if the elevated levels of GAP-43 and Ga,,ifi schizophrenia are related and if these findings pro-vide an important clue as to the developmental irregu-larities that may contribute to the disorder.

Acknowledgment: This study was supported by PHSgrants MH45488 (C.A.S.), MH41684 (H.Y.M.), andMH38752 (R.S.J.), a grant from the Theodore and VadaStanley Foundation, and a grant from the American Founda-tion for Suicide Prevention. We give special thanks to thecoroner, deputy coroners, toxicology laboratory, and staffat the Cuyahoga County Coroner’s Office, Cleveland, OH,U.S.A. The assistance of Pierluigi Gambetti, M.D., in theneuropathological examination of tissue samples is greatlyappreciated.

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