Directory UMM :Data Elmu:jurnal:B:Biological Psichatry:Vol48.Issue7.2000:

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Regulation in Schizophrenia

L. Fredrik Jarskog, John H. Gilmore, Elzbieta S. Selinger, and

Jeffrey A. Lieberman

Background: The etiology of schizophrenia remains un-known; however, a role for apoptosis has been hypothe-sized. Bcl-2 is a potent inhibitor of apoptosis and also exerts neurotrophic activity in the central nervous system (CNS). Bcl-2 expression is increased in the CNS of several neurodegenerative disorders. Given that schizophrenia has certain features of a limited neurodegenerative disor-der, it was hypothesized that cortical Bcl-2 expression is increased in schizophrenia.

Methods: Postmortem temporal cortex was obtained from the Stanley Foundation Neuropathology Consortium with matched control, schizophrenic, bipolar, and depressed subjects. Bcl-2 protein was measured by enzyme-linked immunoassay (ELISA) and Western blot. Primary analysis was limited to schizophrenia versus control subjects. Results: The ELISA demonstrated 25% less Bcl-2 protein in schizophrenia (p 5 .046), supported by Western blot results. A secondary analysis of schizophrenic and bipolar subjects revealed twofold higher mean Bcl-2 in antipsy-chotic-treated versus neuroleptic-naive subjects.

Conclusions: Contrary to our hypothesis, cortical Bcl-2 was reduced in schizophrenia. This supports the notion that schizophrenia is not a classic neurodegenerative disorder; however, less Bcl-2 protein may signal neuronal vulnerability to proapoptotic stimuli and to neuronal atrophy. Also, the association between neuroleptic expo-sure and higher Bcl-2 levels could underlie the favorable long-term outcomes of patients who receive maintenance

antipsychotic treatment. Biol Psychiatry 2000;48:

Key Words: Bcl-2, schizophrenia, apoptosis,

neurodegen-eration, neurodevelopment, neuroprotection

Introduction

S

chizophrenia is a complex neuropsychiatric disorder for which the etiology has remained stubbornly elu-sive. The neurodevelopmental hypothesis states that schizophrenia is acquired through early-life neurobiolog-ical insults that produce permanent brain deficits, mani-festing as psychosis in early adulthood (Weinberger 1987); however, a neurodegenerative hypothesis has also been proposed, primarily based on clinical grounds, given the protracted period of symptomatic dormancy and the progressive deterioration that frequently follows the first episode of psychosis (Lieberman 1999; McGlashan 1988). This is further supported by the progressive neurocogni-tive impairment found in schizophrenia (Bilder et al 1992), particularly in elderly patients (Davidson et al 1995; Purohit et al 1998). Given the extensive articulation of the neurodevelopmental and neurodegenerative hypotheses, it is notable that specific mechanisms by which developmen-tal and degenerative processes could cause cytoarchitec-tural and neurochemical changes have received relatively little attention. Previously, investigators have hypothe-sized a role for apoptosis in the pathophysiology of schizophrenia, particularly as related to neurodevelopmen-tal insults (Akbarian et al 1996; Catts and Catts 2000; Margolis et al 1994; Woods 1998). We suggest that limited neurodegeneration may occur in concert with a neurodevelopmental disorder and that a dysregulation of apoptosis could underlie both of these seemingly divergent processes.

Apoptosis is a form of cell death that is dependent on new gene expression. It occurs with hallmark morphologic features including cell shrinkage, nuclear condensation, and DNA strand breaks, followed by cellular fragmenta-tion and phagocytosis by adjacent cells (Steller 1995). The process is rapid and does not generate an inflammatory response. Apoptosis occurs extensively in normal early neurodevelopment (Oppenheim 1991); however, apoptotic neurons also have been documented in adulthood in certain neurodegenerative disorders such as Alzheimer’s and Huntington’s diseases (Bredesen 1995; Dragunow et al 1995). Apoptosis can be activated experimentally by a From the Departments of Psychiatry (LFJ, JHG, ESS, JAL), Pharmacology (JAL),

and Radiology (JAL) and UNC Mental Health and Neuroscience Clinical Research Center (LFJ, JHG, JAL), University of North Carolina School of Medicine, Chapel Hill.

Address reprint requests to L. Fredrik Jarskog, M.D., University of North Carolina, Department of Psychiatry, CB# 7160, Chapel Hill NC 27599.

Received March 27, 2000; revised June 29, 2000; accepted July 6, 2000.

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broad array of stimuli including ischemia, hypoxia, and proinflammatory cytokines (Charriaut-Marlangue et al 1996; Thompson 1995). Interestingly, these stimuli have also been implicated as neurodevelopmental insults asso-ciated with schizophrenia (Geddes and Lawrie 1995; Gilmore and Jarskog 1997; Mednick et al 1988).

The molecular mechanisms underlying apoptosis have recently become the focus of intense research. Members of the Bcl-2 family of proteins are emerging as primary regu-lators of apoptosis (Adams and Cory 1998). The best char-acterized is Bcl-2, a membrane-bound 26 kD protein that strongly inhibits apoptosis. It appears to exert its antiapop-totic function through homo- and heterodimerization with other Bcl-2 family members (e.g., Bax and Bcl-xL) to

regulate the passage of oxidative mediators (e.g., cytochrome c) through the mitochondrial membrane (Green and Reed 1998). As an antiapoptotic regulatory protein, Bcl-2 exerts a powerful neuroprotective effect. Cells that overexpress Bcl-2 demonstrate considerable resistance to a variety of proapop-totic insults (Zhong et al 1993). Importantly, Bcl-2 also has a neurotrophic property that appears to be independent of its antiapoptotic function—it can promote dendritic branching and produce regeneration of damaged central nervous system (CNS) neurons (Chen et al 1997). Therefore, it is interesting that those neurodegenerative disorders with evidence of apoptotic neurons generally have altered Bcl-2 expression as well. For example, Bcl-2 protein is upregulated in striatum of Parkinson’s disease (Marshall et al 1997; Mogi et al 1996) and also in frontal and temporal cortices of Alzheimer’s disease (Kitamura et al 1998; Satou et al 1995). In addition, Bcl-2 demonstrates developmental upregulation in human frontal cortex across the life span, increasing from early childhood into adulthood (Jarskog and Gilmore 2000). Both in normal aging and in neurodegenerative disease, Bcl-2 upregulation is thought to represent a compensatory neuro-protective response (Satou et al 1995; Vyas et al 1997).

Although considerable neuropathologic evidence exists to support a neurodevelopmental etiology in schizophre-nia, some data also indicate progressive and deteriorative changes that suggest a limited neurodegenerative process. A selected review of data will follow, relating the potential involvement of apoptosis to developmental and degener-ative hypotheses.

Evidence for a neurodevelopmental process includes neuroimaging studies that demonstrate increased ventric-ular size (reviewed by Lawrie and Abukmeil 1998), smaller temporal lobes (Dauphinais et al 1990; Gur et al 1998; Suddath et al 1989) and generalized cortical reduc-tions primarily affecting gray matter in schizophrenia (Zipursky et al 1992, 1998). Because these volume changes have been found even at the onset of psychosis, it frequently has been hypothesized that they derived from an early neurodevelopmental event. Considered

indepen-dently, the neuroimaging data would be consistent with a role for apoptosis in schizophrenia, particularly because evidence of inflammation and scarring is absent; however, structural neuroimaging offers limited direct insight into the mechanism of tissue loss. Postmortem studies are somewhat more revealing, but they are also more conflict-ing (reviewed in Harrison 1999). Investigators have used rigorous stereologic cell counting techniques and did not find evidence of cortical cell loss (Pakkenberg 1993; Selemon et al 1995); however, Selemon et al (1995) acknowledged that neuronal loss in schizophrenic frontal cortex could not definitively be ruled out. Nonetheless, although cell numbers appeared unchanged, clear evidence of neuronal atrophy was documented (Rajkowska et al 1998; Selemon et al 1995). On the other hand, significant neuronal reductions have been found in the thalamus and nucleus accumbens (Pakkenberg 1990; Young et al 2000). This provides stronger evidence that apoptotic cell loss may have occurred in these subcortical brain structures. Another line of evidence implicating apoptosis in schizo-phrenia is the report of neuronal maldistribution in tem-poral cortex (Akbarian et al 1993) and prefrontal cortex (Akbarian et al 1996). These authors argued that certain neurons did not die at developmentally appropriate stages, resulting in maldistribution. Finally, supporting the neu-roimaging studies, postmortem investigations have not demonstrated excess gliosis or other common markers of neurodegeneration (Arnold et al 1998; Benes et al 1991; Purohit et al 1998). Thus, neuropathologic evidence con-sistent with a neurodevelopmental etiology of schizophre-nia also appears moderately supportive of a limited role for apoptosis.

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In light of the relatively consistent findings of neuropa-thology in the temporal lobe including volume reduction by MRI as well as the neurocognitive importance of this brain region in schizophrenia, the current study was designed to examine apoptotic regulation in schizophrenia by assessing Bcl-2 expression in temporal cortex. Altered regulation of Bcl-2 may provide insight into the nature of the pathophysiology in schizophrenia. Because Bcl-2 pro-tein has frequently been upregulated in neurodegenerative disorders and schizophrenia has certain features consistent with neurodegeneration, we tested the hypothesis that Bcl-2 protein levels would be elevated in postmortem temporal cortex in schizophrenia.

Methods and Materials

Postmortem Samples

This study was approved by the Institutional Review Board of the University of North Carolina School of Medicine. Postmor-tem Postmor-temporal cortex (Brodmann’s area 21) was obtained from 60 subjects from the Stanley Foundation Neuropathology Consor-tium (Bethesda, MD) as a set of control, schizophrenic, bipolar, and depressed subjects, n515 per group. Brains were collected from four state medical examiners under supervision of the Stanley Foundation using standardized protocols for tissue pro-curement and processing across all sites. Subjects over age 68 were excluded to avoid comorbid neurologic disorders. Two senior psychiatrists established DSM-IV diagnoses using infor-mation from all available medical records and from family interviews. Details regarding the subject selection, diagnostic process, and tissue processing is described by Torrey et al (2000). Samples were matched for age, gender, ethnicity, side of brain, brain pH, and postmortem interval (PMI; Table 1). Table 2 presents demographic and clinical characteristics for individual subjects, including cumulative antipsychotic medication expo-sure and medications at time of death. All samples were stored at

280°C until use. As a condition for supplying its brain tissue, the Stanley Foundation requires that all diagnostic groups are stud-ied; however, schizophrenia was the primary focus of this study.

All experiments and data collection were performed in a blinded manner.

Tissue Homogenization

Tissue (100 –300 mg) was placed in 10 volumes of 10 mmol/L HEPES buffer (pH 7.0) with 0.32 mol/L sucrose, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10mg/mL aprotinin, 5

mg/mL pepstatin A, 1 mmol/L benzamidine, 0.1 mmol/L benze-thonium chloride. Samples were homogenized (PowerGen 125, Fisher Scientific, Pittsburgh) on ice for 30 sec and sonicated (Sonic Dismembrator 60, Fisher Scientific) for 10 sec at 10 mV. Samples were centrifuged for 15 min at 3000 rpm and 4°C. Supernatants were assayed for total protein by the BCA method (Pierce, Rockford, IL). All chemicals were obtained from Sigma (St. Louis).

Enzyme-Linked Immunoassay (ELISA)

Samples were assayed for Bcl-2 using a commercially available ELISA kit (Endogen, Woburn, MA), using previously described methods (Jarskog and Gilmore 2000). Briefly, 96-well micro-plates were precoated with a mouse monoclonal antihuman Bcl-2 antibody (Ab; Endogen), and samples (50mL) were diluted 1:1 (vol/vol) with fluorescein isothiocyanate (FITC)-labeled second-ary Ab and applied in triplicate. The plates were incubated for 2 hours at room temperature, washed, and then all wells received horseradish peroxidase-labeled anti-FITC Ab. Following 30 min incubation, plates were washed, and TMB/peroxide was added for color development. The reaction was stopped with sulfuric acid, and the optical density was measured at 450 nm using a microplate reader (Vmax, Molecular Devices, Sunnyvale, CA). A Bcl-2 standard curve was generated to quantitate the amount of Bcl-2 in Units/mg total protein. One unit is defined as the amount of Bcl-2 protein in 1000 lysed cells of an internal control cell line (Endogen). Intra-assay coefficients of variance were,9.3% and the interassay coefficient of variance was 6.9%.

Semiquantitative Western Blot

Samples were separated on 12% Tris-glycine polyacrylamide gels using a minicell electrophoresis unit (Xcell II, NOVEX, San Diego), as previously described (Jarskog and Gilmore 2000). Equal amounts of protein (30mg) were boiled for 5 min in Tris-glycine SDS sample buffer (NOVEX) and applied to the gels. Gels were run with a low-range molecular weight ladder (Rainbow MW Marker, Amer-sham Pharmacia, Piscataway, NJ) and a Jurkat cell lysate (Trans-duction Laboratories, Lexington, KY) for Bcl-2 control. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, MA) at 25 V for 2 hours, and complete transfer was ascertained by staining duplicate gels with Coomassie Blue and membranes with Ponceau S (data not shown). Nonspecific protein binding was blocked for 1 hour with 5% blocking reagent (ECL, Amersham Pharmacia) in 0.1% Tween TBS (TBST). Membranes were incubated for 1 hour at 25°C with a mouse monoclonal antihuman Bcl-2 primary Ab (1:500, Transduc-tion Laboratories) followed by 1 hour incubaTransduc-tion with a secondary sheep antimouse HRP-labeled Ab (1:1000, ECL, Amersham Phar-Table 1. Summary of Demographic Characteristics of Human

Temporal Cortex Specimens

N Gender Ethnicity

Age (years)

Postmortem

interval (hours) Brain pH

Control 15 9 M 14 W 48.1610.7 23.769.9 6.360.2 6 F 1 AA

Schizophrenia 15 9 M 13 W 44.5613.1 33.7614.6 6.260.3 6 F 2 As

Bipolar 15 9 M 14 W 42.3611.7 32.4615.9 6.260.2 disorder 6 F 1 AA

Major 15 9 M 15 W 46.569.3 27.5610.7 6.260.2 depression 6 F

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Table 2. Demographic and Clinical Characteristics of Individual Subjects in the Stanley Foundation Neuropathology Consortium

Subject no. Diagnosis Age/gen/ethnicity Cause of death PMI (hours) Medications at time of death Lifetime fluph. eq Substance abuse history

1 N 52/M/W CPD 28 None 0 Alc(p)

2 N 44/F/W CPD 25 None 0 None

3 N 59/M/W CPD 26 None 0 None

6 N 53/M/W CPD 28 None 0 Alc(p)

9 N 41/M/AA CPD 11 None 0 None

14 N 29/F/W Accident 42 None 0 None

15 N 57/F/W Accident 26 None 0 None

16 SCZ, D 30/F/W Suicide 60 Thx, Des 6000 Cnb 17 SCZ, U 52/M/W CPD 61 None 9000 None 18 SCZ, U 30/M/W CPD 32 Ris, Tdz 50,000 None 19 SCZ, P 62/F/As Accident 26 None 50,000 None

20 SCZ, U 60/F/W CPD 40 None 0 None

21 SCZ, U 60/M/W Accident 31 Tdz, Ami 80,000 None 22 SCZ, U 32/M/W Other 19 Clz 15,000 Alc, Amp 23 SCZ, U 31/M/W Suicide 14 Clz 4000 None 24 SCZ, P 58/F/W CPD 26 Hal, Dh 35,000 Alc(p) 25 SCZ, U 25/M/W Suicide 32 Ris, Par 4000 Alc(p) 26 SCZ, U 44/M/W CPD 50 Hal, Cbz, Fx, Cz, Bz 100,000 None 27 SCZ, P 44/M/W CPD 29 Clz, Cpz, Li 130,000 Alc, Poly(p) 28 SCZ, U 56/F/As Suicide 12 Hal, Li, Dh, CH 150,000 None 29 SCZ, P 35/M/W CPD 35 Clz, Cpz, Ma, Bz, Dh 50,000 Poly 30 SCZ, U 49/F/W CPD 38 Hal, Clz, Cz .200,000 None 31 BD, P 25/F/W Suicide 24 Thx, Cbz, Li, Trz 7500 Alc 32 BD, P 48/F/W CPD 22 Val, Ser, Cpx, Cbz 32,000 Alc, Met 33 BD, P 37/F/W Suicide 29 Li, Bup, Cz, Lz 1200 None 34 BD, woP 54/M/W Other 39 Li, Cbz 2500 Alc(p) 35 BD, P 30/M/W CPD 31 Li, Clz 60,000 None 36 BD, woP 30/M/W Suicide 56 None 0 None 37 BD, P 57/M/W CPD 19 Hal, Dh 60,000 Alc(p) 38 BD, P 34/M/W Suicide 23 Ris, Val, Vfx 7000 Alc(p) 39 BD, P 48/M/W Suicide 13 None 200 None 40 BD, P 31/M/W Suicide 28 Hal, Trz, Trx 30,000 Poly 41 BD, P 30/M/W Suicide 45 Val, Bup 0 None 42 BD, woP 50/F/AA Other 18 None 12,000 Cnb, Coc 43 BD, P 61/F/W Suicide 60 Fx, Val 40,000 None 44 BD, P 50/M/W Suicide 19 Val, Clz, Fz, Bz 60,000 None 45 BD, woP 50/F/W CPD 62 Val, Cmi 0 None 46 MD 32/F/W Suicide 47 Imi, Ami, Ntp, Cz 0 None 47 MD 53/F/W Other 40 Li, Trz 0 Alc 48 MD 44/F/W Suicide 32 Fx, Imi, Lz 0 None

49 MD 65/M/W CPD 19 Pht 0 None

50 MD 52/M/W CPD 12 None 0 None

51 MD 46/M/W Suicide 26 Cz, Dh 0 None

52 MD 42/F/W CPD 25 Li, Fx 0 None

53 MD 51/M/W Suicide 26 Nef, Hxz 0 Alc (p) 54 MD 39/M/W Suicide 23 None 0 Alc, Amp

55 MD 42/M/W Suicide 7 None 0 None

56 MD 56/M/W CPD 23 Ser 0 None

57 MD 56/F/W CPD 28 Vfx, Bus, Az 0 None 58 MD 30/F/W Suicide 33 Ntp, Az, Cmi 0 None

59 MD 43/M/W CPD 43 Tri 0 Alc

60 MD 47/M/W CPD 28 Fx, Nef 0 None

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macia). Membranes were developed using chemiluminescence (ECL, Amersham Pharmacia), and the protein bands were detected on radiographic film (Hyperfilm ECL, Amersham Pharmacia) after 30 to 120 sec exposure. Optical densitometry of Bcl-2 bands was performed using NIH Image 1.62, with all measures falling in the linear portion of the curve. Band densities were normalized to a control applied to all gels. In addition, 10 to 80mg of the control sample were immunoblotted to ascertain that all samples fell within the linear portion of the densitometric curve (data not shown).

Statistical Analysis

A priori, the primary analysis was limited to control versus

schizophrenic specimens, given our underlying hypothesis. Bcl-2 levels between these groups were compared using a Student t

test, with two-tailed p values considered significant at .05. The following secondary analyses were also performed, given the exploratory nature of this study:

1. Bcl-2 levels for all diagnostic groups were analyzed by one-way analysis of variance (ANOVA), with significance at p,.05.

2. A linear correlation analysis was performed between age and Bcl-2 in the control group.

3. Bcl-2 levels were analyzed by gender across all samples using Student t test and by gender and diagnosis using two-way ANOVA.

4. Bcl-2 levels for schizophrenic and bipolar subjects were compared on neuroleptic-naive versus neuroleptic-ex-posed status using a Student t test, with two-tailed p values significant at p,.05.

5. A linear correlation analysis was performed in schizophre-nia and bipolar disorder between lifetime fluphenazine equivalents and Bcl-2 concentrations.

6. In bipolar disorder, Bcl-2 levels were compared on lithium and valproic acid treatment status at time of death.

Results

Contrary to our hypothesis, ELISA demonstrated a 25% reduction in mean Bcl-2 levels in the temporal cortex of subjects with schizophrenia (21.962.8 Units/mg protein, mean6SEM) compared with control subjects (29.362.1 Units/mg protein) by Student t test (p5.046; Figure 1 and Table 3). These results provide quantitative evidence of reduced Bcl-2 protein in subjects with schizophrenia. Inspection of Figure 2 indicates that subjects with bipolar disorder and major depression had lower mean Bcl-2 levels compared with control subjects (schizophrenia , bipolar,depressed); however, the overall ANOVA was not significant. In the control group, Bcl-2 levels had a nonsignificant positive correlation with age (R25 .0392,

Figure 1. Quantitative Bcl-2 protein expression (U/mg total protein) in temporal cortex (Brodmann’s area 21) in control and schizophrenic (SCZ) subjects, measured by enzyme-linked im-munoassay. Bcl-2 levels were significantly reduced by 25% in schizophrenic subjects (21.962.8 U/mg, mean6 SEM, n5

15), as compared with control subjects (29.362.1 U/mg, n5

15) by Student t test (*p5.046).

Table 3. Enzyme-Linked Immunoassay Bcl-2 Levels by Diagnosis and Subject

Normal control subjects Schizophrenic subjects Subjects with bipolar disorder Subjects with major depression

Subject No. Bcl-2 U/mg Subject No. Bcl-2 U/mg Subject No. Bcl-2 U/mg Subject No. Bcl-2 U/mg

1 18.2 16 18.5 31 35.2 46 16.7

2 28.4 17 36.3 32 7.7 47 27.0

3 27.9 18 17.0 33 24.2 48 9.0

4 42.1 19 28.9 34 33.2 49 37.2

5 42.0 20 12.7 35 18.1 50 25.5

6 44.3 21 46.7 36 8.3 51 9.7

7 25.0 22 18.0 37 23.6 52 25.7

8 28.7 23 17.2 38 31.3 53 34.8

9 29.2 24 10.8 39 14.0 54 19.7

10 24.2 25 21.7 40 34.7 55 33.4

11 15.2 26 33.6 41 4.5 56 29.1

12 28.9 27 8.2 42 32.2 57 13.0

13 27.2 28 24.9 43 31.3 58 27.1

14 32.3 29 9.1 44 24.1 59 29.1

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p. .05). There was no evidence of gender differences when analyzing by gender and diagnosis using two-way ANOVA or across all samples using a Student t test.

Semiquantitative Western blotting in temporal cortex confirmed the ELISA results. Bcl-2 protein was 34%

lower in subjects with schizophrenia (Figures 3 and 4), although this did not quite reach statistical significance (p 5 .09). Taken together, the ELISA and Western blot methodologies provide consistent evidence that Bcl-2 protein is reduced in schizophrenia.

When the schizophrenic and bipolar subjects were combined and analyzed by neuroleptic-naive (n 5 4) versus neuroleptic-treated (n 5 26) status, mean Bcl-2 levels in the treated group were 96% higher (p 5 .033) than in the untreated group (Figure 5); however, among neuroleptic-exposed subjects, Bcl-2 levels did not corre-late with cumulative neuroleptic exposure (p . .05). Among bipolar subjects, Bcl-2 levels were 29% higher in lithium-treated patients, but this difference was not signif-icant. Likewise, no significant difference emerged for valproic acid in this group.

Figure 2. Bcl-2 protein concentrations (U/mg protein) in tempo-ral cortex across all diagnostic groups of Stanley Foundation brain collection as measured by enzyme-linked immunoassay. Mean6 SEM Bcl-2 levels were 29.36 2.1 U/mg in control subjects, 21.9 6 2.8 U/mg in schizophrenic (SCZ) subjects, 23.16 2.7 U/mg in subjects with bipolar disorder (BD), and 25.062.4 U/mg in subjects with major depression (MD); n5

15 per group. Secondary analysis of variance across all groups was not significant (p..05).

Figure 3. Bcl-2 protein levels in the temporal cortex across all diagnostic groups of Stanley Foundation brain collection as measured by semiquantitative Western blot. Bcl-2 bands were measured using optical densitometry and data were normalized to a control sample (defined as 1.00). Normalized mean optical densities (OD) are presented as mean6SEM, with 1.8060.29 in control samples, 1.1860.19 in schizophrenic (SCZ) samples, 1.5160.72 in bipolar disorder (BD) samples, and 1.5660.28 in major depression (MD) samples; n515 per group. Although not quite significant (p5.09), the mean for SCZ was 34% lower than the mean for control subjects.

Figure 4. Representative Western blot of Bcl-2 protein expres-sion in the temporal cortex from the Stanley Foundation brain collection. An equal amount of total protein (30mg) was added to each lane. The transfer membrane was incubated with a monoclonal antihuman Bcl-2 Ab. The bands shown migrated to 26 kd using molecular weight markers. Lane 1 contained Jurkat cell lysate for Bcl-2 control. Other lanes comprised control subjects (2 and 3), schizophrenic subjects (4 and 5), subjects with bipolar disorder (6 and 7), and subjects with major depression (8 and 9).

Figure 5. Bcl-2 protein concentrations by enzyme-linked immu-noassay in bipolar and schizophrenic patients as analyzed on exposure to antipsychotic medication. Patients treated with antipsychotic medications had 96% higher Bcl-2 levels (24.26

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Discussion

This is the first study to demonstrate that the apoptotic-regulatory protein Bcl-2 is reduced in the CNS in schizo-phrenia. Our results did not support the initial hypothesis that Bcl-2 protein would be elevated as a reflection of a neurodegenerative property of the illness, similar to ele-vations of Bcl-2 previously observed in the CNS of individuals with Alzheimer’s or Parkinson’s disease. This implies that if schizophrenia does encompass a neurode-generative component, then the deneurode-generative mechanism differs substantially from classic neurodegeneration. The reduction of Bcl-2 protein in schizophrenia has several potential pathophysiologic implications. First, Bcl-2 is a potent inhibitor of apoptosis, and a reduction of this protein would suggest that the temporal cortex in schizo-phrenia is more vulnerable to proapoptotic stimuli, whether those stimuli are products of normal physiology and aging (Mrak et al 1997) or from a pathologic process. Second, because Bcl-2 protein has neurotrophic properties that are independent of apoptosis (Chen et al 1997), a limited reduction of Bcl-2 could promote neuronal atrophy and reduced axodendritic branching, without effects on cell death. Thus, we propose that both apoptotic and nonapoptotic mechanisms could subserve some of the subtle neuropathologic findings in schizophrenia, medi-ated through reduced Bcl-2.

The etiology and timing of lower Bcl-2 protein in adult schizophrenic brain is unclear. Although speculative, sev-eral possibilities emerge. First, Bcl-2 protein may be constitutively underexpressed in schizophrenia. A geneti-cally mediated underexpression of Bcl-2 may be less likely, given the multisystem abnormalities and acceler-ated mortality demonstracceler-ated in Bcl-2 deficient mice (Veis et al 1993). Overall, the minor physical anomalies and subtle neuropathology in schizophrenia seem inconsistent with such a mechanism. Alternatively, an environmental stimulus during development could produce an enduring yet limited downregulation of Bcl-2 expression. Such a mechanism could potentially contribute to both early and later brain development, thereby unifying neurodevelop-mental and neurodegenerative hypotheses of schizophre-nia; at this time, however, little is known of the effects of early developmental insults on the long term effects on Bcl-2 expression. Third, schizophrenia could be mediated by an as yet unidentified neurodegenerative process that induces the downregulation of Bcl-2 protein in adulthood. In Alzheimer’s disease, Bcl-2 protein expression is up-regulated overall, but there is evidence of selective Bcl-2 downregulation in neurofibrillary tangle-positive neurons (Satou et al 1995). It seems that the pathophysiologic process in schizophrenia must differ substantially from the Alzheimer’s paradigm because schizophrenia does not

have consistent evidence of large-scale neuronal loss or robust gliosis, both hallmark features of Alzheimer’s disease. A final consideration is that antipsychotic medi-cations contribute to the downregulation of Bcl-2. This possibility will be discussed in more depth below; how-ever, our data suggests a correlation between higher Bcl-2 levels and antipsychotic exposure (Figure 5).

Bcl-2 protein may exert its effects in schizophrenia by several specific mechanisms. First, Bcl-2 is strongly anti-apoptotic, and overexpression of this protein is known to confer resistance to neuronal cell death both in vitro and in vivo by a broad spectrum of proapoptotic stimuli (Yang et al 1998; Zhong et al 1993). Also, human cortical Bcl-2 expression is developmentally upregulated across the life span (Jarskog and Gilmore 2000). In adulthood, upregu-lation may continue in response to age-related accumula-tion of higher oxidized protein levels, irreversible protein glycation, lipofuscin, and DNA damage (Mrak et al 1997; Vyas et al 1997). Therefore, a reduction of Bcl-2 protein could increase the vulnerability of schizophrenic brain to proapoptotic stimuli. Second, Bcl-2 has been found to promote regeneration of damaged CNS neurons and en-hance neurite outgrowth, revealing a growth-promoting neurotrophic capacity that occurs independently of apo-ptosis (Chen et al 1997). Thus, as described earlier, lower Bcl-2 levels in schizophrenia could lead to neuronal atrophy and reduced dendritic branching. Studies that have demonstrated higher neuronal density, reduced neuropil, and reduced neuronal size in the absence of cell loss in schizophrenic cortex are consistent with this mechanism (Rajkowska et al 1998; Selemon et al 1995). In addition, magnetic resonance spectroscopy studies have found lower N-acetylaspartate levels in temporal and frontal cortex in schizophrenia, thought to reflect reduced neuro-nal viability (Bertolino et al 1998; Cecil et al 1999). Although such data does not prove Bcl-2 involvement, it is also consistent and merits further study.

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bipolar disorder who were taking lithium at the time of death also had numerically higher Bcl-2 levels (by 29%), this difference was not statistically significant. The failure to detect a difference may have been due to insufficient power or to other, as yet uncharacterized confounding variables. Future studies will be required to fully evaluate the effects of psychiatric medications on Bcl-2 protein.

Several potential confounding variables could have af-fected the results of this study. First, it is possible that reduced Bcl-2 protein was secondary to medication effects; however, because a significant correlation emerged between a history of antipsychotic treatment and higher Bcl-2 expression, it suggests that reduced Bcl-2 in schizophrenia was not a result of antipsychotic treatment. In fact, if antipsychotic medica-tions do upregulate Bcl-2 protein, then such treatment may have masked even lower baseline levels in the schizophrenic subjects. A second variable relates to the known develop-mental upregulation of Bcl-2 expression, as documented previously in human frontal cortex from infancy to adoles-cence and adulthood (Jarskog and Gilmore 2000). In our study, we found a nonsignificant positive correlation between age and Bcl-2 in the control group, which may have had insufficient power to detect a correlation, or expression of Bcl-2 in temporal cortex may differ from that of frontal cortex. Nonetheless, because diagnostic groups were matched for age, and age did not differ significantly between groups (Table 1), aging-related changes would be unlikely to account for lower Bcl-2 in schizophrenia. A third variable relates to postmortem stability of Bcl-2 protein. We previ-ously have determined that 24-hour postmortem stability of Bcl-2 is high (,6% loss) using a rodent model designed to approximate the human postmortem condition (Jarskog and Gilmore 2000). Because mean PMIs in this study ranged from 23.7 to 33.7 hours (Table 1) and did not differ significantly among the four diagnostic groups, postmortem degradation of Bcl-2 likely did not account for our findings. Fourth, the Bcl-2 data in this study was quantified using conventional methods based on mg total protein; however, this method does not account for potential variations of total protein per weight of brain tissue among subject groups. Theoretically, if total protein per brain weight is higher in schizophrenic compared with control cortex, then our find-ings of lower Bcl-2 in units per mg total protein could be negated on a unit per brain weight basis. In fact, in a separate experiment, schizophrenic subjects had 25% less total protein per weight of brain compared with control subjects (data not shown). This suggests that Bcl-2 protein per brain weight may be even lower in schizophrenia than the 25% reduction we measured in units per total protein. Finally, a number of other confounding variables are possible, including diagnos-tic heterogeneity, history of substance abuse, and concurrent medical illness. There are inherent limitations to ascertaining clinical information in postmortem assessed subjects;

how-ever, given these limitations, the Stanley Foundation Neuro-pathology Consortium provides a unique opportunity to study brain tissue in a relatively young group of subjects with severe psychiatric disorders (Torrey et al 2000).

Interestingly, the data also revealed lower Bcl-2 levels by ELISA in other psychiatric disorders, with reductions of 21% in individuals with bipolar disorder and of 14% in individuals with major depression compared with control subjects, al-though these changes did not reach statistical significance (Figure 2). These trends were also observed in the Western blot data (Figure 3). Neuroimaging studies in affective disorders have revealed evidence of cortical volume reduc-tions (Drevets et al 1997; Soares and Mann 1997). This suggests a potential Bcl-2 mediated mechanism of neuronal atrophy, loss, or both. Although speculative, the convergence of downregulation in Bcl-2 protein across several disorders could indicate a common downstream pathway in the patho-physiology of affective and psychotic disorders.

In summary, this study provides evidence that the patho-physiology of schizophrenia involves a dysregulation of the apoptotic-regulatory protein Bcl-2. A reduction in Bcl-2 protein suggests that neuronal apoptosis, glial apoptosis, or both may be altered through increased vulnerability to pro-apoptotic stimuli. In addition, Bcl-2 downregulation may promote neuronal atrophy and reduced dendritic branching through mechanisms unrelated to apoptosis. Unlike classic neurodegenerative disorders such as Alzheimer’s disease, specific markers of apoptotic cells have yet to be demon-strated in schizophrenia. Nonetheless, most evidence sug-gests that large-scale cell loss does not occur in schizophre-nia, and small increases in neuronal apoptosis may remain undetectable given the limitations of available techniques for visualizing this process. Bcl-2 protein can begin to provide an alternate source of information regarding the apoptotic bal-ance of CNS neurons in schizophrenia. Given the complexity of apoptotic protein regulation, a role for Bcl-2 in schizo-phrenia would likely occur in concert with other pro- and antiapoptotic proteins and related factors that affect neuronal viability. Further studies characterizing other Bcl-2 family proteins and apoptosis-effector proteins (e.g., caspases) in brain regions implicated in schizophrenia may help to clarify the pathophysiology of this disorder. Ultimately, if a role for apoptotic-regulatory proteins is established, then new ave-nues for intervention and treatment may emerge.

Supported by a National Alliance for Research on Schizophrenia and Depression Young Investigator Award (LFJ) and National Institutes of Health Center Grant No. MH-33127 (JAL).

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