Original Article | Published:

Increased Expression of c-Jun Transcription Factor in Cerebellar Vermis of Patients with Schizophrenia

Neuropsychopharmacology volume 28, pages 15061514 (2003) | Download Citation

Subjects

Abstract

In the cerebellar vermis of schizophrenic patients, our previous studies have revealed alterations in the mitogen-activated protein (MAP) kinase signaling cascade and downstream transcription factors within the c-fos promoter. Since the proteins of the Fos and Jun families of immediate-early genes dimerize to form activating protein (AP)-1, the present study was conducted to examine the expression of Jun transcription factors in schizophrenic and control subjects. Using Western blot analysis, we determined the protein levels of c-Jun, Jun B, and Jun D as well as the levels of c-jun mRNA by relative RT-PCR in post-mortem samples from cerebellar vermis. The expression of c-Jun protein and c-jun mRNA was significantly increased in the cerebellar vermis of patients with schizophrenia, whereas no significant differences were found in the expression of Jun B or Jun D proteins. Studies in rats indicated that the abnormal expression of c-Jun transcription factor observed in schizophrenic patients was not related to post-mortem intervals or chronic treatment with antipsychotic medications. This study provides new insights into cerebellar abnormalities of schizophrenia at the level of expression of c-Jun that target key genes associated with the MAP kinase cascade.

INTRODUCTION

Dysfunction of glutamatergic N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission, defect in the regulator of G-protein signaling 4 (RGS4) expression, and neurotrophic abnormality have been implicated in schizophrenia (Olney and Farber, 1995; Coyle, 1996; Mirnics et al, 2001; Takahashi et al, 2000). In neurons, extracellular stimuli, such as NMDA receptors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 can trigger activation of the mitogen-activated protein (MAP) kinase signal transduction cascades, and these signals are then relayed to the nucleus to activate transcription factors and gene expression (Fiore et al, 1993; Kurino et al, 1995; Xia et al, 1996; Vanhoutte et al, 1999; Boulton et al, 1991; Bonni et al, 1999; Kaplan and Miller, 2000; Cavanaugh et al, 2001). Three classes of MAP kinases, referred to as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38, have been identified in mammals. One of the most widely studied nuclear targets of the MAP kinase pathways is the activator protein (AP)-1 transcription factor (Whitmarsh and Davis, 1996). AP-1 is a homodimer and/or heterodimeric complex composed of members of the Jun (c-Jun, Jun B, and Jun D) and Fos (c-Fos, Fos B, Fra-1, and Fra-2) families. Dimerization can also occur with other transcription factor families, such as CREB and ATF (Herdegen and Leah, 1998).

Accumulating evidence suggests that transcriptional regulation of genes plays a key role in the molecular mechanisms of critical cellular events, including neuronal plasticity, synaptic transmission, and morphological differentiation. Recent studies have also indicated that immediate-early genes jun and fos, which are widely expressed in the central nervous system, may play an important role in long-term potentiation, learning, and memory (Dragunow, 1996). Despite the increasing number of studies linking the immediate-early genes to important brain functions, their role in various neuropsychiatric disorders including schizophrenia is still poorly understood.

Previously, we reported elevated expression of several intermediates of the ERK pathway in post-mortem brain tissue from individuals with schizophrenia. We found increased protein levels of MAP kinase ERK2 in cerebellar vermis, but not in mesopontine tegmentum or frontal pole (Brodmann area 10) (Kyosseva et al, 1999). Furthermore, we demonstrated that the levels of several transcription factors within the promoter region of c-fos gene, that is, Elk-1, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and activating transcription factor-2 (ATF-2) were elevated in the cerebellar vermis in schizophrenic patients as well (Kyosseva et al, 2000). In this study, we have determined whether members of the Jun family of transcription factors are altered in the brain of patients with schizophrenia. The studies were conducted to measure the expression of c-Jun, Jun B, and Jun D proteins by Western blot in cerebellar vermis of schizophrenic and control subjects, as well as the expression of c-jun mRNA by relative reverse transcription-polymerase chain reaction (RT-PCR). Our focus was the cerebellar vermis, since post-mortem and neuroimaging studies have described abnormalities in cerebellum in schizophrenia. Thus, patients with schizophrenia were shown to have a smaller anterior vermis (Weinberger et al, 1980) and smaller Purkinje cell size (Tran et al, 1998). Magnetic resonance imaging (MRI) studies have also described a significant reduction in the volume of the vermis as well (Nopoulos et al, 1999, 2001; Ichimiya et al, 2001). Several recent post-mortem studies have reported increased expression of nitric oxide synthase (Karson et al, 1996; Bernstein et al, 2001), decreased expression of synaptic proteins synaptophysin, complexin II (Eastwood et al, 2001b), and SNAP-25 (Mukaetova-Ladinska et al, 2001), as well as abnormalities in the expression of serotonin 5-HT2A receptors in the cerebellum in schizophrenia (Eastwood et al, 2001a). In addition, new evidence suggests that the cerebellum may play a role in cognition, behavior, and psychiatric illness (Allen et al, 1997; Rapoport et al, 2000).

MATERIALS AND METHODS

Subjects

Post-mortem brain tissue was obtained after consent for autopsy from the Central Veterans Healthcare System (Little Rock, AR) and the University of Arkansas for Medical Sciences (UAMS). All the procedures were approved by the Human Research Advisory Committee of UAMS.

Psychiatric diagnosis was made post mortem by using the Diagnostic Evaluation After Death (Salzman et al, 1983). This is a structured chart review for which the main source of information is the medical record. In addition, information obtained from family members and treating psychiatrists was incorporated when available. Diagnostic Evaluation After Death was used whether the patient was being evaluated pre- or post mortem. Many of the elderly psychiatric patients were interviewed pre mortem, and diagnoses were established independently by two trained psychiatrists according to DSM-III-R criteria. Clinical and pathological data for schizophrenic and control subjects are summarized in Table 1. Post-mortem brain tissue was obtained from nine patients with schizophrenia (eight men and one woman) and 12 control subjects (10 men and two women). Most patients with schizophrenia were receiving antipsychotic medications at the time of death. Two patients with schizophrenia had histories of alcohol abuse. Medical records from control subjects were reviewed to determine whether they had an active psychiatric disorder at the time of their death and/or earlier in their lives. Two control subjects had histories that were consistent with alcohol dependence, which ceased 8–9 years prior to death. A third subject had an uncertain history of alcoholism that may have been ongoing at the time of his death; two control subjects were prescribed very low doses of amitriptyline (50 mg at bedtime) and mesoridazine, respectively, for sleep or pain control. In both the groups (schizophrenic and control), the cause of death was generally cancer, cardiac failure, or a terminal respiratory condition.

Table 1: Clinical and pathological data for schizophrenic and control subjects

Neuropathology

All brain specimens were subjected to standard gross and microscopic neuropathological examination by a board-certified neuropathologist. In addition to the routine hematoxylin and eosin staining, selected sections were stained with the Sevier–Munger modification of Bielschowski's stain to evaluate neuritic plaques and neurofibrillary tangles. All cases used in this study were free of significant neuropathological change, including Alzheimer's disease as determined by using CERAD criteria (Mirra et al, 1991). Mild senile changes (normal for age) were presented in six subjects (two schizophrenic and four controls), and one schizophrenic showed a small infarction in the occipital lobe (Table 1). After collection during autopsy, the brain tissue was dissected and stored at −80°C. Cerebellar vermis was taken from the anterior vermis just of midline.

Effect of Post-Mortem Interval (PMI)

Adult male Sprague–Dawley rats (approximately 250 g) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN) and were allowed to acclimate to their new environment for several days before the experiments. Animal use procedures were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of UAMS. The rats were euthanized by carbon dioxide, and then decapitated either immediately or after 3, 6, 9, 12, and 24 h. All the killed rats were kept 1 h at room temperature and then transferred to a refrigerator maintained at a temperature of 4–8°C, in which they remained until decapitation and dissection. This procedure is analogous to our post-mortem human procedures, in which all bodies are transferred to the morgue within 1.5 h of death and kept under refrigeration until autopsied. The rat brains were rapidly removed and cerebellar vermis was dissected, and stored at −80°C until use.

Effect of Haloperidol and Risperidone

Sprague–Dawley rats (approximately 250 g) were given subcutaneous injections of haloperidol in concentrations of 0.15 or 1.5 mg/kg, rispredone in concentrations of 0.05 or 0.5 mg/kg, or saline daily for 21 days. High doses for both drugs were representative of human treatments for severe psychosis. The rats were killed by decapitation 24 h after the last injection. The brains were rapidly removed and cerebellar vermis was dissected, and stored at −80°C until use.

Isolation of Nuclear Fractions and Western Blot Analysis

Nuclear fractions from human and rat tissue were isolated as previously described (Kyosseva et al, 2000). Nuclear extracts (25 μg protein) were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the proteins were electrophoretically transferred to nitrocellulose membranes in a buffer containing 25 mM Tris-HCl, pH 7.4, 190 mM glycine, and 20% methanol for 2 h. The nonspecific binding of proteins was performed by blocking the membranes with 5% nonfat dry milk in Tris-buffered saline/0.1% Tween-20 (TBST), pH 7.5 for 1 h. The blots were incubated with a primary c-Jun polyclonal antibody corresponding to residues 55–67 of human c-Jun from Cell Signaling Technology (Beverly, MA), diluted 1 : 1000 in TBST containing 5% bovine serum albumin overnight at 4°C or polyclonal Jun B (N-17), and Jun D (329) antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) diluted 1 : 1000 in TBST containing 5% milk. After several washings with TBST, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG, diluted 1 : 2000 in TBST containing 5% milk. After further washings, the immunoblots were detected using the enhanced chemiluminescence (ECL) detection system provided with the antibody kit. The blots were stripped of detection antibodies by washing the membranes several times with water and then incubated in a buffer containing 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7 at 55°C for 30 min. After several washes in TBST, the blots were reprobed with a polyclonal actin (I-19) antibody from Santa Cruz Biotechnology, Inc. The autoradiograms were scanned with a Computerized Laser Densitometer model 300A (Molecular Dynamics, Sunnyvale, CA). Densities of the immunoreactive bands were analyzed with Image Quant software (Molecular Dynamics). The optical density of each band is expressed in arbitrary densitometric units. All the experiments were performed at least three times with similar results.

Relative Reverse Transcription Polymerase Chain Reaction

Total RNA was isolated from 0.1 g samples of frozen (−80°C) human brain tissue taken from the cerebellar vermis using RNease Mini Kit (Qiangen, Valencia, CA). RNA concentrations were calculated from the optical density at 260 nm, and the purity was determined by A260/A280. The integrity of the isolated RNA was evaluated by gel electrophoresis on 1.25% precast agarose gel (Sigma) in MOPS buffer stained with ethidium bromide. Total RNA (300 ng) from each sample was reverse transcribed using ‘Ready-to-Go You-Prime-First Strand’ Beads (Amersham Pharmacia Biotech, Piscataway, NJ) and oligo d(T)15 (Promega, Madison, WI). A volume of 2 μl of each RT products were PCR amplified in a final volume of 20 μl using Taq PCR master mix (Qiagen, Valencia, CA) and 0.5 μl of each c-jun gene-specific primers (final concentration 1 μM). The levels of 28S rRNA served as an endogenous control. The sequences of c-jun and 28S rRNA primers were as follows: c-jun 5′-primer: bases 2514–2540; c-jun 3′ primer: bases 2675–2651; expected PCR fragment: 162 bp; 28S rRNA 5′-primer: bases 4535–4564; 28S rRNA 3′-primer: bases 4667–4638, expected PCR fragment: 133 bp (Puntschart et al, 1998).

The reactions were run on a Gene Amp PCR System 2400. After initial denaturing at 94°C for 2 min, the subsequent cycles for c-jun were as follows: denaturing for 10 s at 94°C; annealing for 90 s at 37°C (five cycles), at 45°C (five cycles), and at 65°C (23 cycles); extension for 30 s at 72°C; and final extension for 5 min at 72°C. In all 16 cycles of PCR, each consisting of denaturing at 94°C for 10 s, annealing at 60°C for 90 s, and extension at 72°C for 10 s were performed for 28S rRNA. Total PCR products were electrophoresed on 1.25% agarose gel in TBE and stained with ethiduim bromide. The area and density of PCR product bands were measured using Scion Image Program for IBM (Scion Corporation) and the resulting measures were expressed as arbitrary densitometry units and were statistically analyzed.

Statistical Analysis

Results are presented as mean±standard deviation (SD) values. For human post-mortem studies, data from experimental groups were compared using analysis of variances ANOVA post hoc test. Correlations were carried out using the Spearman rank correlation. For experiments examining the effects of antipsychotic medications in rats, data were analyzed using a Friedman two-way analysis of variance test and the results are expressed as χ2 statistic. The statistical analysis was conducted using software StatView for Windows, version 4.5 (Abacus Concepts, Inc., Berkley, CA). p-Values less than 0.05 were considered to be statistically significant.

RESULTS

Effect of Age, PMI, and Duration of Illness on C-Jun Expression

Demographic data for schizophrenic and control subjects are shown in Table 1. Schizophrenic patients did not significantly differ from controls in age (mean±SD, 63±14 vs 67±9 years; p=0.39), PMI (mean±SD, 7.2±4.7 vs 6.7±4.1 h; p=0.77), or brain weight (mean±SD, 1309.2±120.9 vs 1294±132 g; p=0.81). There was no significant correlation between c-Jun protein levels and age (r=−0.25, p=0.27) or PMI (r=−0.08, p=0.74). Similarly, there was no significant correlation between c-jun mRNA and age (r=0.16, p=0.51) or PMI (r=−0.19, p=0.45). We also did not observe a correlation between duration of illness and c-Jun protein (r=0.24, p=0.43) or mRNA expression (r=−0.22, p=0.53).

c-Jun, Jun B, and Jun D Levels in Post-Mortem Cerebelar Vermis

Nuclear fractions of cerebellar vermis from patients with schizophrenia and age-matched control subjects were electrophoresed on SDS polyacrylamide gel electrophoresis and blots were probed with antibodies to c-Jun, Jun B, and Jun D. The c-Jun antibody recognized a protein of approximately 46 kDa, whereas for Jun B and Jun D, proteins of about 39 kDa were detected on the autoradiograms. Representative immunoblots showing data for c-Jun, Jun B, and Jun D levels from one schizophrenic and one control subject are presented in Figure 1a. The immunoblots were reprobed with the housekeeping protein actin to verify equal loading and to help control for nonspecific differences. Differences in the levels of Jun proteins were quantified by densitometry of immunoblots from nine patients with schizophrenia and 12 control subjects. As seen in Figure 1b, the schizophrenics had c-Jun levels significantly higher than those of control subjects (mean±SD, 1044±469 vs 350±346 arbitrary densitometric units, p=0.0009, respectively). Four patients with schizophrenia showed relatively low expression of c-Jun protein, but there was no direct association with any demographic characteristics or antipsychotic treatment (eg one patient was on haloperidol, second received haloperidol in combination with carbamazepine, the third was on loxapine, and the fourth received trifluoperazine and trihexylphenidyl). In contrast to the elevated c-Jun levels, the protein expression of the other two members of Jun family, Jun B (mean±SD, 724±124 vs 660±116 arbitrary densitometric units, p=0.24), and Jun D (mean±SD, 2410±334 vs 2389±254 arbitrary densitometric units, p=0.87) appeared to be unaltered in schizophrenic patients as compared to the control subjects.

Figure 1
Figure 1

Representative immunoblots of c-Jun, Jun B, and Jun D (a) in cerebellar vermis from one patient with schizophrenia (s) and one control subject (c). Samples (25 μg protein) from nuclear fractions were subjected to SDS polyacrylamide gel electrophoresis, and the resulting gels were processed for Western blot analysis as described in ‘Materials and methods’ section. The blots were stripped and reprobed with actin antibody. Actin was used as a loading control. Autoradiograms from nine schizophrenic and 12 control subjects were scanned by densitometer and data are expressed in densitometry units (b). Values are means±SD.

c-jun mRNA Expression in Post-Mortem Cerebelar Vermis

To further determine whether the increased protein levels of c-Jun were due to increased gene expression, we examined the mRNA levels of c-jun in nine patients with schizophrenia and nine control subjects by relative RT-PCR. Representative gel electrophoresis of PCR products from one schizophrenic and one control subject using gene-specific c-jun (162 bp) and 28S rRNA (133 bp) primers is presented in Figure 2a. The levels of 28S rRNA served as an internal standard to ensure equal loading of RNA. The results showed statistically significant elevation of c-jun mRNA in the vermis of schizophrenic brains (2853±646 vs 1573±488, p<0.0002, Figure 2b) and thus confirmed the established elevation of c-Jun protein levels.

Figure 2
Figure 2

Relative RT-PCR of c-jun mRNA in cerebellar vermis from patients with schizophrenia and control subjects. Total RNA (300 ng) were reverse transcribed and amplified with polymerase chain reaction using gene-specific c-jun (162 bp) and 28S rRNA (133 bp) primers. 28S rRNA served as an internal standard. The upper panel (a) shows representative electrophoresis on a 1.25% agarose gel stained with ethidium bromide of PCR products from 1 schizophrenic (s) and one control (c) subject. Data for c-jun mRNA from nine schizophrenic and nine control subjects were analyzed by measuring the area and density of electrophoretic bands, and are expressed in densitometry units (b). Values are means±SD.

Effect of PMI and Antipsychotic Medications on c-Jun Expression

We have previously shown that the protein levels of transcription factors Elk-1, CREB and ATF-2 did not change significantly in rat brain during storage of the tissue for 24 h at 4°C (Kyosseva et al, 2000). In this study, we also determined the post-mortem stability of c-Jun protein and mRNA levels at time intervals of 0, 3, 6, 9, 12, and 24 h. As shown in Figure 3, we did not detect significant changes of c-jun expression after the rat brains were left for 24 h at 4°C.

Figure 3
Figure 3

Effect of PMI on c-Jun protein and mRNA levels in rat cerebellum. Rats (three per experimental group) were killed and their brains were removed for dissection at 0 time, 3, 6, 9, 12, 24 h. Nuclear fractions (25 μg protein) were subjected to SDS polyacrylamide gel electrophoresis and Western blot analysis. The c-jun mRNA levels were determined by relative RT-PCR using gene-specific primers for c-jun (162 bp) and 28 S rRNA (133 bp).

Next, we determined whether the increased protein levels of transcription factor c-Jun could result from treatment with antipsychotic drugs, since most of the patients included in this study had an extensive lifetime exposure to medications. The typical antipsychotic haloperidol (0.15 and 1.5 mg/kg) or the atypical antipsychotic risperidone (0.05 and 0.5 mg/kg) was administered to rats (six per experimental group) by injection for 21 days. Densitometry of the immunoblots presented in Figure 4 shows that chronic treatment with two different doses of either haloperidol or risperidone does not have any effect on the protein levels of c-Jun (χ42=2.67, p=0.61).

Figure 4
Figure 4

Effect of haloperidol and risperidone on c-Jun protein levels in rat cerebellum. Rats (six per treatment group) received haloperidol by injections at low (HL, 0.15 mg/kg/day) or high (HH, 1.5 mg/kg/day), risperidone at low (RL, 0.05 mg/kg/day) or high (RH, 0.5 mg/kg/day) doses, or saline for 21 days. Nuclear fractions were isolated and subjected to SDS polyacrylamide gel electrophoresis and Western blot analysis. The resulting autoradiograms were scanned by densitometer and data are expressed as densitometry units. Values are means±SD.

DISCUSSION

We have recently provided evidence that schizophrenia is associated with alterations of the ERK signal transduction pathway in the cerebellar vermis in both patients and a rodent phencyclidine (PCP) model of schizophrenia (Kyosseva et al, 1999, 2001). We also showed that downstream transcription factor targets within the promoter of c-fos were upregulated in post-mortem cerebellar vermis (Kyosseva et al, 2000). These earlier findings prompted us to elucidate the possible involvement of Jun transcription factors in the cerebellar abnormalities in schizophrenia, since proteins of jun and fos leucine zipper families dimerize to form AP-1 transcription factor that has been shown to be involved in CNS disorders and development (Pennypacker, 1995). In this study, we examined by Western blot analysis the protein levels of c-Jun, Jun B, and Jun D in cerebellar vermis of patients with schizophrenia and control subjects. We found significant elevation in the expression of c-Jun protein in schizophrenic patients. We also showed, by relative RT-PCR, that the expression of c-jun mRNA was significantly increased in the vermis of schizophrenic subjects compared with control subjects. Neither Jun B nor Jun D protein was increased in the cerebellar vermis from patients with schizophrenia. Such selective alterations of Jun family of transcription factors in post-mortem brain of schizophrenics may be of major interest, since the transcriptional regulation of genes plays an important role in the disease process.

Our findings of increased protein levels of c-Jun in patients with schizophrenia could suggest increased phosphorylation of this transcription factor. However, using commercially available antibodies, we were unable to detect the phosphorylated active form of c-Jun. The phosphorylation of proteins cannot be reliably determined in post-mortem tissue due to rapid dephosphorylation, which can affect the expression of phophorylated active forms. At this point, however, we cannot comment if the observed overexpression of c-Jun protein levels is caused by increased phosphorylation.

Treatment with the typical antipsychotic drug haloperidol is shown to increase the expression of the immediate-early genes in the striatum and nucleus accumbens and the therapeutic affect of this agent is modulated by NMDA-dopamine D2 receptor interactions (Leveque et al, 2000; Hussain et al, 2001). The atypical antipsychotic drugs including risperidone have different affinities for several neurotransmitter receptors, such as dopamine, serotonin, muscarinic, and adrenergic receptors (Lieberman et al, 1998; Meltzer, 1999; Svensson, 2000). Therefore, in the present study, we tested whether the induced c-Jun expression in the cerebellar vermis could be increased by effects of antipsychotic treatment, because most of the schizophrenic patients included in our study (Table 1) had received antipsychotic medications. However, in contrast to the observations in humans, there were no significant differences in the levels of c-Jun protein in haloperidol- or risperidone-treated rats and controls. A very recent study has shown that chronic 17-day treatment with haloperidol and clozapine increased the expression levels of jun and fos family immediate-early genes in the rat prefrontal cortex using in situ hybridization (Kontkanen et al, 2002). Taken together, these results indicate a differential and brain region-specific regulation of jun and fos genes by antipsychotic drug treatment. Although further work is required to determine the precise mechanisms by which different antipsychotics exert their effects on gene expression, the results of elevated c-jun expression in cerebellar vermis in schizophrenic patients do not seem to be directly related to the chronic antipsychotic drug treatment and thus may represent a neuropathological feature of the disease itself.

The finding of abnormal protein and gene expression of c-Jun in cerebellar vermis of patients with schizophrenia may be relevant to the notion that the cerebellum may be an important neroanatomic region in the pathophysiology in psychiatric disorders, including schizophrenia (Katsetos et al, 1997). Until recently, little attention has been paid to the role of the cerebellum, presumably due to its primary role in motor control (Martin and Albers, 1995). However, increasing numbers of studies have found evidence that links the cerebellum with nonmotor functions (Middleton and Strick, 1994, 1998; Paradiso et al, 1997; Schmahmann, 1998). These findings support a role for the cerebellum in diverse cognitive functions, including attention, working memory, verbal learning, and memory. Recent evidence derived from anatomical, physiological, and functional neuroimaging studies has suggested cognitive function impairments in schizophrenic patients (Jacobsen et al, 1997; Wassink et al, 1999). In addition, several PET studies have found abnormalities in cerebellar blood flow in a variety of cognitive tasks in patients with schizophrenia (Andreasen et al, 1996, 1997). Based on the above new evidence, it has been hypothesized that in schizophrenia, there is a disturbance in the cortical–cerebellar–thalamic–cortical circuit, producing a fundamental cognitive deficit in the disease (Wassink et al, 1999). In addition, cerebellar abnormalities, particularly a decrease in tissue volume, abnormalities in cerebellar blood volume and expression of tyrosine receptor kinase B, reelin, and glutamic acid decarboxylase (GAD) 67 mRNA levels have been found in patients with mood disorder (Soares and Mann, 1997; Loeber et al, 2002; Bayer et al, 2000; Guidotti et al, 2001).

It is increasingly recognized that transcription factors play a critical role in the development and functioning of the normal nervous system, as well as adaptation of the nervous system to various stimuli such as injury, growth factors, and drug treatment, as well as physiological and pathological events (Morgan and Curran, 1991; Herdegen and Leah, 1998; Duman et al, 1999). Transcription factors are nuclear proteins recognizing specific DNA sequences in gene promoters that modulate transcriptional activity in response to extracellular stimuli. The MAP kinase pathways provide a common route by which signals from different receptors, including heterotrimeric G-protein coupled receptors, converge at major regulatory elements of promoters of the immediate-early genes including jun and fos. Several studies have shown changes in G-protein levels in post-mortem brain in schizophrenics, although the results are somewhat contradictory (Okada et al, 1990, 1991, 1994; Nishino et al, 1993; Yang et al, 1998). A very recent study provides evidence of elevated dopamine receptor-coupled G proteins measured in mononuclear leukocytes of patients with schizophrenia (Avissar et al, 2001). Moreover, abnormalities were found in the expression of the regulator of RGS4 in schizophrenia using cDNA microarrays (Mirnics et al, 2001).

Despite a growing body of data demonstrating that immediate-early genes play an important physiological role in CNS, there is no evidence to our knowledge about the role of Jun family of transcription factors in psychiatric disorders. Whether the observed c-jun alterations are specific to schizophrenia or also can be found in major psychosis is still unclear. We are currently designing experiments to evaluate the expression of immediate-early genes jun and fos in patients with mood disorders, since schizophrenia and bipolar disorder are similar in several epidemiological aspects, such as age of onset, lifetime risk, course of illness, and genetic susceptibility (Berrettini, 2000). It has been shown that Jun proteins are differentially expressed in human neurological diseases. For example, several studies reported increased expression of c-Jun protein in Alzheimer's disease (Anderson et al, 1994, 1996; MacGibbon et al, 1997; Marcus et al, 1998) and multiple sclerosis (Martin et al, 1996), while decreased Jun D protein was found in brains of patients with Down's syndrome (Labudova et al, 1998). The lack of c-Jun elevation in the cerebellum of Alzheimer's and multiple sclerosis subjects, in contrast to that observed in patients with schizophrenia, suggests a regional and disease-specific alteration in c-Jun expression.

In conclusion, we found evidence of specific overexpression of c-Jun protein and mRNA levels in cerebellar vermis in schizophrenia. These results show that c-Jun may be involved in the neuropathological changes in the brains of patients with schizophrenia because the immediate-early genes encode transcription factors that serve to regulate the expression of other so-called late respond genes and therefore can trigger a cascade of events that may lead to functional and morphological alterations of neurons (Morgan and Curran, 1991). The above data, as well as data from our previous studies suggest the involvement of novel target genes and signal transduction pathways in cerebellar abnormalities in schizophrenia. In an attempt to elucidate the molecular mechanisms underlying these alterations in the brains of schizophrenic patients, future studies will concentrate on determining the mRNA expression and DNA binding activity of the corresponding transcription factors forming the AP-1 complex in both patients and PCP model of schizophrenia.

References

  1. , , , (1997). Attentional activation of the cerebellum independent of motor involvement. Science 275: 1940–1943.

  2. , , (1994). Increased immunoreactivity for Jun- and Fos-related proteins in Alzheimer's disease: association with pathology. Exp Neurol 125: 286–295.

  3. , , (1996). DNA damage and apoptosis in Alzheimer's disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay. J Neurosci 16: 1710–1719.

  4. , , , , , et al (1996). Schizophrenia and cognitive dysmetria: a PET study of dysfunctional prefrontal–thalamic–cerebellar circuitry. Proc Natl Acad Sci 93: 9985–9990.

  5. , , , , , et al (1997). Hypofrontality in schizophrenia: disturbed dysfunctional circuits in neuroleptic naïve patients. Lancent 349: 1730–1734.

  6. , , , , (2001). Elevated dopamine receptor-coupled G(s) protein measures in mononuclear leukocytes of patients with schizophrenia. Schizophr Res 47: 37–47.

  7. , , , , (2000). Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder. Prog Neuropsychopharmacol Biol Psychiatry 24: 881–887.

  8. , , , , , et al (2001). Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia. J Neurocytol 60: 661–670.

  9. (2000). Are schizophrenic and bipolar disorders related? A review of family and molecular studies. Biol Psychiatry 48: 531–538.

  10. , , , , , (1999). Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286: 1358–1362.

  11. , , , , , et al (1991). ERKs: a family of protein-serine/treonine kinases that are activated and tyrosine phosphorylated in responses to insulin and NGF. Cell 65: 663–675.

  12. , , , , , (2001). Differential regulation of mitogen-activated protein kinases ERK1/ERK2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons. J Neurosci 21: 434–443.

  13. (1996). The glutamatergic hypothesis for schizophrenia. Harvard Rev Psychiatry 3: 241–253.

  14. (1996). A role for immediate-early transcription factors in learning and memory. Behav Genet 26: 293–299.

  15. , , (1999). Neural plasticity and antidepressant treatment. Biol Psychiatry 46: 1181–1191.

  16. , , , , (2001a). Expression of serotonin 5-HT2A receptors in the human cerebellum and alterations in schizophrenia. Synapse 42: 104–114.

  17. , , (2001b). Cerebellar synaptic protein expression in schizophrenia. Neuroscience 105: 219–229.

  18. , , , , (1993). Activation of p42 mitogen-activated protein kinase by glutamate receptor stimulation in rat primary cortical cultures. J Neurochem 61: 1626–1633.

  19. , , , , , et al (2001). Decrease in reelin and glutamic acid decarboxylase 67 (GAD) 67 expression in schizophrenia and bipolar disorder. Arch Gen Psychiatry 57: 1061–1069.

  20. , (1998). Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res Rev 27: 370–490.

  21. , , (2001). Glutamatergic regulation of haloperidol-induced c-fos expression in rat striatum and nucleus accumbens. Neuroscience 102: 391–399.

  22. , , , (2001). Reduced volume of the cerebellar vermis in neuroleptic-naïve schizophrenia. Biol Psychiatry 49: 20–27.

  23. , , , , , et al (1997). Quantitative morphology of the cerebellum and fourth ventricle in childhood-onset schizophrenia. Am J Psychiatry 154: 1663–1669.

  24. , (2000). Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10: 381–391.

  25. , , , , , et al (1996). Nitric oxide synthase (NOS) in schizophrenia. Increase in cerebellar vermis. Mol Chem Neuropathol 27: 275–284.

  26. , , (1997). Neuropathology of the cerebellum in schizophrenia—and update: 1996 and future directions. Biol Psychiatry 42: 213–224.

  27. , , , (2002). Chronic antipsychotic drug treatment induces long-lasting expression of fos and jun family genes and activator protein 1 complex in the rat prefrontal cortex. Neuropsychopharmacology 27: 152–162.

  28. , , , (1995). Activation of mitogen-activated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors. J Neurochem 65: 1282–1289.

  29. , , , , , (1999). Mitogen-activated protein kinases in schizophrenia. Biol Psychiatry 46: 689–696.

  30. , , , , , et al (2000). Increased levels of transcription factors Elk-1, cyclic adenosine monophosphate response element-binding protein, and activating transcription factor 2 in the cerebellar vermis of schizophrenic patients. Arch Gen Psychiatry 57: 685–691.

  31. , , , (2001). Differential and region-specific activation of mitogen-activated protein kinases following chronic administration of phencyclidine in rat brain. Neuropsychopharmacology 24: 267–277.

  32. , , , , , et al (1998). Decreased transcription factor jun D in brains of patients with Down syndrome. Neurosci Lett 252: 159–162.

  33. , , , , , et al (2000). Intracellular modulation of NMDA receptor function by antipsychotic drugs. J Neurosci 20: 4011–4020.

  34. , , , , , et al (1998). Serotonergic basis of antipsychotic drug effects in schizophrenia. Biol Psychiatry 44: 1099–1117.

  35. , , , , , (2002). Cerebellar blood volume in bipolar patients correlates with medication. Biol Psychiatry 51: 370–376.

  36. , , , , , et al (1997). Expression of Fos, Jun, and Krox family proteins in Alzheimer's disease. Exp Neurol 147: 316–332.

  37. , , , , , et al (1998). Quantitative neuronal c-fos and c-jun expression in Alzheimer's disease. Neurobiol Aging 19: 393–400.

  38. , (1995). Cerebellum and schizophrenia: a selective review. Schizophr Bull 21: 241–250.

  39. , , , , , (1996). Jun expression is found in neurons located in the vicinity of subacute plaques in patients with multiple sclerosis. Neurosci Lett 212: 95–98.

  40. (1999). The role of serotonin in antipsychotic drug action. Neuropsychopharmacology 21: 106S–115S.

  41. , (1994). Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function. Science 266: 458–461.

  42. , (1998). Cerebellar output: motor and cognitive channels. Trends Cognit Sci 2: 338–347.

  43. , , , , (2001). Disease-specific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol Psychiatry 6: 293–301.

  44. , , , , , et al (1991). The Consortium to Establish a Registry for Alzheimer's Disease (CERAD), part II: standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 41: 479–486.

  45. , (1991). Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci 14: 421–451.

  46. , , , , (2001). Loss of synaptic but not cytoskeletal proteins in the cerebellum of chronic schizophrenics. Neurosci Lett 317: 161–165.

  47. , , , , , et al (1993). Increase in [3H]cAMP binding sites and decrease in Giα and Goα immunoreactivities in left temporal cortices from patients with schizophrenia. Brain Res 615: 41–49.

  48. , , , (1999). An MRI study of cerebellar vermis morphology in patients with schizophrenia: evidence in support of the ‘cognitive dysmetria’. Biol Psychiatry 46: 703–711.

  49. , , , (2001). An MRI study of midbrain morphology in patients with schizophrenia: relationship to psychosis, neuroleptics, and cerebellar neural circuitry. Biol Psychiatry 49: 13–19.

  50. , , (1990). G-protein (Gi, Go) in the basal ganglia of control and schizophrenic brain. J Neural Transm (Gen Sect) 79: 227–234.

  51. , , (1991). G-protein (Gi, Go) in the medial temporal lobe in schizophrenia: preliminary report of a neurochemical correlate of structural change. J Neural Transm (Gen Sect) 84: 147–153.

  52. , , , , (1994). Reduced concentrations of the α subunit of GTP-binding protein Go in schizophrenic brain. J Neural Transm (Gen Sect) 95: 95–104.

  53. , (1995). Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry 52: 998–1007.

  54. , , , , (1997). Cerebellar size and cognition: correlations with IQ, verbal memory, and motor dexterity. Neuropsychiatry Neuropsychol Behav Neurol 10: 1–8.

  55. (1995). AP-1 transcription factor complexes in CNS disorders and development. J Fla Med Assoc 82: 551–554.

  56. , , , , , et al (1998). Expression of fos and jun genes in human skeletal muscle after exercise. Am J Physiol 274: C129–C137.

  57. , , (2000). The role of the cerebellum in cognition and behavior: a selective review. J Neuropsychiatry Clin Neurosci 12: 193–198.

  58. , , , (eds) (1983). Diagnostic Evaluation After Death (DEAD). National Institute of Mental Health Neuroscience Research Branch: Rockville, MD.

  59. (1998). Dysmetria of thought: clinical consequences of cerebellar dysfunction on cognition and affect. Trends Cognit Sci 2: 362–371.

  60. , (1997). The anatomy of mood disorders—review of structural neuroimaging studies. Biol Psychiatry 41: 86–106.

  61. (2000). Dysfunctional brain dopamine systems induced by psychotomimetic NMDA-receptor antagonists and the effects of antipsychotic drugs. Brain Res Rev 31: 320–329.

  62. , , , , , et al (2000). Abnormal expression of brain derived neurotrophic factor and its receptor in the corticolimbic system of schizophrenic patients. Mol Psychiatry 5: 293–300.

  63. , , , (1998). Reduced Purkinje cell size in the cerebellar vermis of elderly patients with schizophrenia. Am J Psychiatry 155: 1288–1290.

  64. , , , , , et al (1999). Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via and extracellular signal-regulated kinase-dependent pathway in brain slices. Mol Cell Biol 19: 136–146.

  65. , , , (1999). Cerebellar morphology as a predictor of symptom and psychosocial outcome in schizophrenia. Biol Psychiatry 45: 41–48.

  66. , , , , (1980). Cerebellar pathology in schizophrenia: a controlled postmortem study. Am J Psychiatry 137: 359–561.

  67. , (1996). Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74: 589–607.

  68. , , , (1996). Calcium influx via NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J Neurosci 16: 5425–5436.

  69. , , , , (1998). Isotype-specific G protein abnormalities in the left superior temporal cortex and limbic structures of patients with chronic schizophrenia. Biol Psychiatry 43: 12–19.

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Acknowledgements

This study was supported by the National Institute of Mental Health Grant MH 60739 (SVK) and the Marie Wilson Howells Endowment of the Department of Psychiatry at the University of Arkansas for Medical Sciences. We gratefully acknowledge WS Griffin, PhD and RE Mrak, MD, PhD from University of Arkansas for Medical Sciences for providing us with autopsied human brains and for their helpful comments and suggestions.

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  1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    • Valentina K Todorova
    • , Alan D Elbein
    •  & Svetlana V Kyosseva

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Correspondence to Svetlana V Kyosseva.

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https://doi.org/10.1038/sj.npp.1300211

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