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Increased expression of Wnt-1 in schizophrenic brains
Schizophrenia Research 38 (1999) 1–6 www.elsevier.com/locate/schres
Increased expression of Wnt-1 in schizophrenic brains Tsuyoshi Miyaoka *, Haruo Seno, Hiroshi Ishino Department of Psychiatry, Shimane Medical University, Izumo 693, Japan Accepted 9 November 1998
Abstract The regulated expression of Wnt-1, one member of the wingless/Wnt pathway, in the brain is critical for many neurodevelopmental processes. Recently, it has been reported that the wingless/Wnt pathway participates in a complex behavioral phenomenon and suggested that this pathway’s molecules are candidate genes for neuropsychiatric disorders. Thus, we investigated the expression of Wnt-1 in the hippocampal region, which is believed to be closely involved in the pathophysiology of schizophrenia, of postmortem brains from 10 schizophrenic and 10 control individuals. Immunohistochemical analysis with polyclonal antibodies recognizing Wnt-1 revealed immunoreactivity primarily in the pyramidal cell layer, particularly in CA3 and CA4 regions. We observed a significant elevation in the number of Wnt-1-immunoreactive neurons in the great majority of schizophrenic brains relative to that in controls. The expression of Wnt-1 may be related to cell adhesion, synaptic rearrangement, and plasticity. Therefore, the increase in Wnt-1 immunoreactivity in schizophrenic hippocampi suggests an altered plasticity of this structure in a large proportion of schizophrenic brains. These findings suggest an abnormality of the wingless/Wnt pathway present in the schizophrenic brain and may support the ‘neurodevelopmental hypothesis’ of schizophrenia. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Hippocampus; Immunohistochemistry; Schizophrenia; Wnt-1
1. Introduction A variety of cytoarchitectural abnormalities have been described in the limbic structures and in the frontal cortex in schizophrenic brains (Roberts, 1991; Bloom, 1993). Abnormal orientation of hippocampal neurons (Conrad et al., 1991), disturbed laminar organization of entorhinal and frontal cortices (Arnold et al., 1991), and misplacement of NADPH-diaphorase-positive neurons in both temporal and frontal cortices (Akbarian * Corresponding author. Tel.: +81-853-20-2263; Fax: +81-853-20-2260.
et al., 1993a,b) have been related to abnormal cortical development. In addition, neuronal malconnectivity resulting from these anatomical defects has been proposed as a key factor in the symptomatology of schizophrenia (Arnold and Trojanowski, 1996; Weinberger, 1987, 1995). The ‘neurodevelopmental hypothesis’ of schizophrenia (Conrad et al., 1991; Roberts, 1991; Arnold and Trojanowski, 1996) proposes that a yet-unidentified event occurring in utero or early postnatal life disturbs the normal maturation of neuronal connections in schizophrenic subjects. Therefore, it seems to be important to examine the expression of molecules of significance in neuronal development in schizophrenic brains.
0920-9964/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 9 2 0 -9 9 6 4 ( 9 8 ) 0 0 17 9 - 0
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6
Recently, it has been reported that the wingless/Wnt pathway participates in a complex behavioral phenomenon and suggested that this pathway’s molecules are candidate genes for neuropsychiatric disorders (Lijam et al., 1997). The wingless/Wnt pathway, first described in Drosophila, is a highly conserved developmental pathway involved in cell fate determination of the central nervous system in virtually all eukaryotic organisms (McMahon and Bradley, 1990; Dale, 1998). A gene-targeting study of dishevelled-1 (Dvl-1), one member of this pathway, revealed that it is a genetic factor which influences social behavior and sensorimotor gating in mice (Lijam et al., 1997). Sensorimotor-gating dysfunction is suggested to be characteristic of several human psychiatric disorders (Geyer and Braff, 1987). Therefore, we hypothesized that there is alteration in the expression of the wingless/Wnt pathway molecules in schizophrenic brains. In these molecules, we focused on Wnt-1, which is one other member of the wingless/Wnt pathway and present upstream of Dvl (including Dvl-1) in the wingless/Wnt pathway (Dale, 1998). The Wnt-1 proto-oncogene, which encodes a putative signaling molecule, is expressed in the developing central nervous system, especially, and has been proposed to be related to cell adhesion, synaptic rearrangement and plasticity (McMahon and Bradley, 1990; Parr et al., 1993; Bradley et al., 1993). We examined here the expression of Wnt-1, which is believed to be closely involved in the pathophysiology of schizophrenia, in schizophrenic hippocampi.
2. Materials and methods 2.1. Materials Goat polyclonal anti-human Wnt-1 antibodies (Santa Cruz) were used. 2.2. Autopsied brain samples Brain tissues were obtained at autopsy from 10 patients clinically diagnosed with schizophrenia (mean age, 62.8 years) and from 10 age- and sex-
matched controls (mean age, 60.9 years) with no clinical or morphologic evidence of brain pathology. The patients, control subjects, with the concordance of their families, consented to bequeath their bodies to medical science before death (volunteers) and were autopsied at Shimane Medical University. Postmortem delays on all brains were less than 12 h ( Table 1). Clinical data and diagnoses of the cases studied were obtained from chart reviews and are listed in Table 1. The diagnosis was confirmed by retrospective analysis of the clinical history and symptoms according to DSM-IV criteria. After being fixed in 10% formalin for 2 weeks, the brains were examined grossly, cut in coronal sections, and a block containing the hippocampus and parahippocampal gyrus was taken from a slice through the lateral geniculate body. Specimens from representative areas of the brain were embedded in paraffin. Hematoxylineosin, Woelcke myelin, Holzer, Bodian, modified Bielskowsky, and methenamine silver preparations were used for overall neuropathologic evaluation to exclude dementia and other neurological diseases. 2.3. Immunohistochemistry The brains were postfixed with 10% formalin, embedded in paraffin, and cut into 10-mm-thick sections. After deparaffinization, sections were treated with alcohol containing hydrogen peroxide, exposed to 10% rabbit serum, and then incubated with goat anti-Wnt-1 antibodies (1:1000 dilution) for 24 h at 4°C in a humid chamber. All subsequent incubations were performed at room temperature. After a rinse in PBS, the sections were incubated with rabbit biotinylated anti-goat IgG for 1 h, washed again with PBS, and incubated with peroxidase-conjugated streptavidin for 1 h. Then, the color was developed with dimethylaminoazobenzene (40 mg/ml ) and 0.002% hydrogen peroxide. Tissue sections from all 20 cases were processed for immunohistochemistry together. Nissl staining of adjacent sections was performed to observe the neural shape. Controls included both the replacement of primary antibodies with normal goat serum and liquid phase preabsorption of the primary antibodies with recombinant Wnt-1 protein.
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6 Table 1 Clinical and post-mortem data of the various brains used in this study ID
Group
Age/gender (years)
Neuroleptic exposure duration, dose (years, mg)
Cause of death
Brain weight (g)
Laterality of tissue block
PMD(h)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
C C C C C C C C C C S S S S S S S S S S
54/M 56/M 50/F 57/M 62/F 61/M 63/F 63/F 73/M 70/F 45/M 53/M 66/F 59/M 61/F 65/M 67/F 68/F 71/M 73/F
– – – – – – – – – – 16 (650) 13 (200) 21 (150) 15 (500) 13 (250) 16 (150) 14 (200) 17 (165) 16 (50) –
Heart failure Brain hemorrhage Liver failure Liver failure Respiratory failure Gastric cancer Heart failure Heart failure Pneumonia Colon cancer Cholecystitis Heart failure Heart failure Respiratory failure Lung cancer Gastric ulcer Heart failure Heart failure Brain hemorrhage Heart failure
1330 1250 1250 1260 1140 1350 1250 1260 1000 1200 1120 1360 1340 990 1050 1240 910 1120 1290 1050
R R R R L L R R L R L R R R L R L R L L
10 9 7 4 12 6 7 10 12 12 11 10 9 9 7 10 11 4 6 7
Details of case number: group (C=control; S=schizophrenic); age, gender (M=male; F=female); neuroleptic exposure (chlorpromazine equivalents); cause of death, brain weight, laterality of tissue block (L=left; R=right); PMD, post-mortem delay.
Five adjacent sections from each brain were processed for immunostaining, and all of these sections were assessed and formed the basis of the final average count. Moreover, one other section was stained with hematoxylin to monitor the neuroanatomical coordinates and for nucleolated cell counting. Counts of immunoreactive nucleolated cells were performed by two independent examiners, who were unaware of the identity of the subject. The entire cytoarchitectural field was scanned, and the total number of Wnt-1 immunoreactive neurons in fields of hippocampi were counted by each of the examiners. The boundaries between sectors CA1, 2, 3, and 4 were delineated by applying the criteria of de Lorente (1937) to sections viewed with a 10× objective lens. These cell numbers were counted using an Olympus BH microscope, and the average of these counts in five adjacent sections was recorded. Reliability of the counting procedure was confirmed by quantifying a subset of the sections at a different time. Neuronal counts in these procedures consistently differed by less than 3%.
2.4. Statistical analysis Statistical analysis was performed by the Mann–Whitney U test and Pearson’s product moment correlation coefficient. 3. Results 3.1. Global group characteristics Table 1 lists the details of ages and brain weights among schizophrenic and control groups. There were no significant differences in brain weights (both male and female) between the groups by the Mann–Whitney U test. Also listed are details of laterality of available blocks for schizophrenic and control groups. 3.2. Distribution of Wnt-1 in control and schizophrenic brains Immunohistochemistry was used to examine the distribution of Wnt-1 in the control and schizo-
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6
phrenic brains. In general, immunoreactivity for Wnt-1 was present within neuronal cells in both control and schizophrenic brains. In the hippocampus, immunoreactivity of neuronal cells was observed, especially in CA3 and CA4; however, immunoreactivity in CA1, CA2, and the subiculum were very weak in both schizophrenic and control brains. Therefore, we examined these areas of CA3 and CA4 closely. In schizophrenic brains, Wnt-1 immunoreactivity within pyramidal cells was increased relative to that in controls. Fig. 1 shows
an example of immunostaining for Wnt-1 in CA4 in control and schizophrenic brains. The cellular labeling observed in CA3 and CA4 of the hippocampus allowed us to semiquantitatively estimate the density of Wnt1-immunoreactive cells in this region. A significant increase (2.5–4 times) in Wnt-1-immunoreactive cells was observed in the CA3 and CA4 regions of 90% of schizophrenic hippocampi studied (Fig. 2). The means±SD of the schizophrenic and control groups (CA3: control 60.6±8.3, schizophrenic 167.1±25.1 and CA4: control 65.9±6.2, schizophrenic 275.4±50.1) differed significantly (CA3: p=0.002, and CA4: p=0.002, Mann–Whitney U test). Although Wnt-1-immunoreactive cell density was found to be increased in the majority of schizophrenic brains, the labeling pattern in immunoreactive cells did not differ from that of the controls (Fig. 1). Moreover, the number of immunoreactive cells did not correlate with age, postmortem delay, neuroleptic exposure duration and dose, sex, or hemispheric laterality by the Mann–Whitney U test and Pearson’s product moment correlation coefficient. Cell counting in CA3 and CA4 regions performed on hematoxylinstained adjacent sections of all hippocampi stained revealed no significant difference in the total cell numbers between schizophrenic and control brains by the Mann–Whitney U test (CA3: control 213.8±15.9, schizophrenic 211.4±21.6, and CA4: control 341.2±25.8, schizophrenic 353.9±20.1).
4. Discussion
Fig. 1. Immunohistochemical localization of Wnt-1 in control and schizophrenic sections. Wnt-1 immunoreactivity in CA4 (a and b) regions of the hippocampus obtained from control (a) and schizophrenic (b) brains. In schizophrenic samples, the number of Wnt-1-immunoreactive cells is clearly larger than in controls (240×).
We observed significant increases in the number of Wnt-1-immunoreactive cells in CA3 and CA4 regions of the hippocampus in the majority of schizophrenic brains. According to their anatomical location, these cells probably belong to the pyramidal and polymorph neurons of the hippocampus (Amaral, 1989). The increase in Wnt1-immunoreactive cells in schizophrenic brains does not appear to be related to an altered cell number since the neuronal count in these regions failed to reveal a significant change in cell number in these areas, as was previously observed ( Heckers, 1991).
T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6
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Fig. 2. Wnt-1-immunoreactive cells in CA3 and CA4 regions of the hippocampus obtained from normal control (C ) and schizophrenic (S ) brains. Each dot represents the mean of Wnt-1-immunoreactive cell count performed on each hippocampus studied. The mean for each group is illustrated by the horizontal lines (CA3: control 60.6±8.3, schizophrenic 167.1±25.1; and CA4: control 65.9±6.2, schizophrenic 275.4±50.1). The asterisk represents a significant difference ( p=0.002, both CA3 and CA4) between the mean of the two groups by the Mann–Whitney U test for nonparametric values.
Although the majority of schizophrenic brains demonstrated a significant increase in the hippocampal Wnt-1-immunoreactive cell number compared with controls, one schizophrenic brain did not show an increase (Fig. 2). This may be due to the heterogeneity of the disease process itself or to the inherent problems in the diagnosis of this complex disorder. The contribution of long-term neuroleptic treatment in the observed increase in Wnt-1 expression in schizophrenic brains cannot be ruled out. It is, however, unlikely to have had a major influence. An increase in expression of Wnt-1 confined to the CA3 and CA4 regions is difficult to explain solely by prior neuroleptic exposure. Hippocampal formation is believed to be closely involved in the pathophysiology of schizophrenia (Akbarian et al., 1993b; Conrad et al., 1991; Cotter et al., 1997). Recently, reduced expression of synapsin and a neural cell adhesion molecule was described in the hippocampus of postmortem schizophrenic brains (Barbeau et al., 1995; Glantz and Lewis, 1997). Moreover, the expression of Wnt-1 may be related to cell adhesion, synaptic rearrangement and plasticity (McMahon and Bradley, 1990; Bradley et al., 1993; Parr et al., 1993). Experimental evidence from magnetic resonance imaging and cerebral blood flow studies
suggests a dysfunction of a prefrontal–hippocampal network in schizophrenia ( Weinberger, 1995), and recent data obtained in rats suggest that the integrity of the ventral hippocampus is essential for adequate development of the prefrontal cortex (Glantz and Lewis, 1997). Therefore, an increase of Wnt-1-immunoreactive cells in this area in the schizophrenic brain, as observed in the present study, may induce and/or reflect aberrant connections between hippocampal neurons with potential impact on hippocampal efferents to cortical and subcortical structures. These findings may partly explain some of the deficits in hippocampal functions, including memory, observed in schizophrenic subjects ( Weinberger, 1987, 1995). However, the question as to whether increased expression of Wnt-1-immunoreactive neurons in the schizophrenic hippocampus is a cause or merely a consequence of the disease cannot be readily answered. Recently, Dvl-1, a downstream member of the wingless/Wnt pathway in mutant mice, provided a model for aspects of several human psychiatric disorders (Lijam et al., 1997). Moreover, interestingly, Cotter et al. (1998) recently reported abnormalities of Wnt signaling in the hippocampus in schizophrenia. Considering the key function of Wnt-1 in brain development in general, and in the hippocampus in particular,
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its increase in schizophrenic brains may be a central element in the pathophysiology of this disorder.
Acknowledgment Part of this work was supported by a Grant-inAid for Scientific Research on Priority Areas, 09770741, from the Ministry of Education, Science, Sports and Culture of Japan.
References Akbarian, S., Bunney, W.E., Potkin, S.G., Wigal, S.B., Hagman, J.O., Sandman, C.A., Jones, E.G., 1993a. Altered distribution of nicotinamide-adenine dinucleotide phosphatediaphorase cells in frontal lobe of schizophrenics implies disturbances of cortical development. Arch. Gen. Psychiatry 50, 169–177. Akbarian, S., Vinuela, A., Kim, J.J., Potkin Jr., S.G., Bunney, W.E., Jones, E.G., 1993b. Distorted distribution of nicotinamide-adenine dinucleotide phosphate-diaphorase neurons in temporal lobe of schizophrenics implies anomalous cortical development. Arch. Gen. Psychiatry 50, 178–187. Amaral, D., 1989. The three-dimensional organization of the hippocampal formation: a review of anatomical data. Neuroscience 31, 571–591. Arnold, S.E., Trojanowski, J.Q., 1996. Recent advances in defining the neuropathology of schizophrenia. Acta Neuropathol. 92, 217–231. Arnold, S., Hyman, B., Hoesen, G., Damasio, A., 1991. Some cytoarchitectural abnormalities of the entorhinal cortex in schizophrenia. Arch. Gen. Psychiatry 48, 625–632. Barbeau, D., Liang, J.J., Robitaille, Y., Quirion, R., Srivastava, L.K., 1995. Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brain. Proc. Natl. Acad. Sci. USA. 92, 2785–2789. Bloom, F.E., 1993. Advancing a neurodevelopmental origin for schizophrenia. Arch. Gen. Psychiatry 50, 224–227. Bradley, R.S., Cowin, P., Brown, A.M.C., 1993. Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and
E-cadherin and increased cellular adhesion. J. Cell Biol. 123, 1857–1865. Conrad, A., Abebe, T., Austin, R., Forsythe, S., Scheibel, A., 1991. Hippocampal pyramidal cell disarray in schizophrenia as a bilateral phenomenon. Arch. Gen. Psychiatry 48, 413–417. Cotter, D., Kerwin, R., Doshi, B., Martin, C.S., Everall, I.P., 1997. Alteration in hippocampal non-phosphorylated MAP2 protein expression in schizophrenia. Brain Res. 756, 238–246. Cotter, D., Kerwin, R., Al-Sarraji, S., Brion, J.P., Chadwich, A., Lovestone, S., Anderton, B., Everall, I., 1998. Abnormalities of Wnt signalling in schizophrenia—evidence for neurodevelopmental abnormality. Neuroreport 9, 1379–1383. Dale, T.C., 1998. Signal transduction by the Wnt family of ligands. Biochem. J. 329, 209–223. de Lorente, R., 1937. Studies on the structure of cerebral cortex. II. Continuation of the study of the amnonic system. J. Psychol. Neurol. 46, 113–177. Geyer, M.A., Braff, D.L., 1987. Startle habituation and sensorimotor gating in schizophrenia and related animal models. Schizophr. Bull. 13, 643–668. Glantz, L.A., Lewis, D.A., 1997. Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia. Arch. Gen. Psychiatry 54, 660–669. Heckers, S., 1991. Hippocampal neuron number in schizophrenia. A stereological study. Arch. Gen. Psychiatry 48, 1002–1008. Lijam, N., Paylor, R., McDonald, M.P., Crawley, J.N., Deng, C.X., Herrup, K., Stevens, K.E., Maccaferri, G., McBain, C.J., Sussman, D.J., Wynshaw-Boris, A., 1997. Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 90, 895–905. McMahon, A.P., Bradley, A., 1990. The Wnt-1 (int-1) protooncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085. Parr, B.A., Shea, M.J., Vassileva, G., McMahon, A.P., 1993. Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds. Development 1993, 247–261. Roberts, G., 1991. Schizophrenia: a neuropathological perspective. Br. J. Psychiatry 158, 8–17. Weinberger, D., 1987. Implications of normal brain development for the pathogenesis of schizophrenia. Arch. Gen. Psychiatry 44, 660–669. Weinberger, D., 1995. Schizophrenia, from neuropathology to neurodevelopment. The Lancet 346, 552–557.
Increased expression of Wnt-1 in schizophrenic brains Tsuyoshi Miyaoka *, Haruo Seno, Hiroshi Ishino Department of Psychiatry, Shimane Medical University, Izumo 693, Japan Accepted 9 November 1998
Abstract The regulated expression of Wnt-1, one member of the wingless/Wnt pathway, in the brain is critical for many neurodevelopmental processes. Recently, it has been reported that the wingless/Wnt pathway participates in a complex behavioral phenomenon and suggested that this pathway’s molecules are candidate genes for neuropsychiatric disorders. Thus, we investigated the expression of Wnt-1 in the hippocampal region, which is believed to be closely involved in the pathophysiology of schizophrenia, of postmortem brains from 10 schizophrenic and 10 control individuals. Immunohistochemical analysis with polyclonal antibodies recognizing Wnt-1 revealed immunoreactivity primarily in the pyramidal cell layer, particularly in CA3 and CA4 regions. We observed a significant elevation in the number of Wnt-1-immunoreactive neurons in the great majority of schizophrenic brains relative to that in controls. The expression of Wnt-1 may be related to cell adhesion, synaptic rearrangement, and plasticity. Therefore, the increase in Wnt-1 immunoreactivity in schizophrenic hippocampi suggests an altered plasticity of this structure in a large proportion of schizophrenic brains. These findings suggest an abnormality of the wingless/Wnt pathway present in the schizophrenic brain and may support the ‘neurodevelopmental hypothesis’ of schizophrenia. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Hippocampus; Immunohistochemistry; Schizophrenia; Wnt-1
1. Introduction A variety of cytoarchitectural abnormalities have been described in the limbic structures and in the frontal cortex in schizophrenic brains (Roberts, 1991; Bloom, 1993). Abnormal orientation of hippocampal neurons (Conrad et al., 1991), disturbed laminar organization of entorhinal and frontal cortices (Arnold et al., 1991), and misplacement of NADPH-diaphorase-positive neurons in both temporal and frontal cortices (Akbarian * Corresponding author. Tel.: +81-853-20-2263; Fax: +81-853-20-2260.
et al., 1993a,b) have been related to abnormal cortical development. In addition, neuronal malconnectivity resulting from these anatomical defects has been proposed as a key factor in the symptomatology of schizophrenia (Arnold and Trojanowski, 1996; Weinberger, 1987, 1995). The ‘neurodevelopmental hypothesis’ of schizophrenia (Conrad et al., 1991; Roberts, 1991; Arnold and Trojanowski, 1996) proposes that a yet-unidentified event occurring in utero or early postnatal life disturbs the normal maturation of neuronal connections in schizophrenic subjects. Therefore, it seems to be important to examine the expression of molecules of significance in neuronal development in schizophrenic brains.
0920-9964/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 9 2 0 -9 9 6 4 ( 9 8 ) 0 0 17 9 - 0
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6
Recently, it has been reported that the wingless/Wnt pathway participates in a complex behavioral phenomenon and suggested that this pathway’s molecules are candidate genes for neuropsychiatric disorders (Lijam et al., 1997). The wingless/Wnt pathway, first described in Drosophila, is a highly conserved developmental pathway involved in cell fate determination of the central nervous system in virtually all eukaryotic organisms (McMahon and Bradley, 1990; Dale, 1998). A gene-targeting study of dishevelled-1 (Dvl-1), one member of this pathway, revealed that it is a genetic factor which influences social behavior and sensorimotor gating in mice (Lijam et al., 1997). Sensorimotor-gating dysfunction is suggested to be characteristic of several human psychiatric disorders (Geyer and Braff, 1987). Therefore, we hypothesized that there is alteration in the expression of the wingless/Wnt pathway molecules in schizophrenic brains. In these molecules, we focused on Wnt-1, which is one other member of the wingless/Wnt pathway and present upstream of Dvl (including Dvl-1) in the wingless/Wnt pathway (Dale, 1998). The Wnt-1 proto-oncogene, which encodes a putative signaling molecule, is expressed in the developing central nervous system, especially, and has been proposed to be related to cell adhesion, synaptic rearrangement and plasticity (McMahon and Bradley, 1990; Parr et al., 1993; Bradley et al., 1993). We examined here the expression of Wnt-1, which is believed to be closely involved in the pathophysiology of schizophrenia, in schizophrenic hippocampi.
2. Materials and methods 2.1. Materials Goat polyclonal anti-human Wnt-1 antibodies (Santa Cruz) were used. 2.2. Autopsied brain samples Brain tissues were obtained at autopsy from 10 patients clinically diagnosed with schizophrenia (mean age, 62.8 years) and from 10 age- and sex-
matched controls (mean age, 60.9 years) with no clinical or morphologic evidence of brain pathology. The patients, control subjects, with the concordance of their families, consented to bequeath their bodies to medical science before death (volunteers) and were autopsied at Shimane Medical University. Postmortem delays on all brains were less than 12 h ( Table 1). Clinical data and diagnoses of the cases studied were obtained from chart reviews and are listed in Table 1. The diagnosis was confirmed by retrospective analysis of the clinical history and symptoms according to DSM-IV criteria. After being fixed in 10% formalin for 2 weeks, the brains were examined grossly, cut in coronal sections, and a block containing the hippocampus and parahippocampal gyrus was taken from a slice through the lateral geniculate body. Specimens from representative areas of the brain were embedded in paraffin. Hematoxylineosin, Woelcke myelin, Holzer, Bodian, modified Bielskowsky, and methenamine silver preparations were used for overall neuropathologic evaluation to exclude dementia and other neurological diseases. 2.3. Immunohistochemistry The brains were postfixed with 10% formalin, embedded in paraffin, and cut into 10-mm-thick sections. After deparaffinization, sections were treated with alcohol containing hydrogen peroxide, exposed to 10% rabbit serum, and then incubated with goat anti-Wnt-1 antibodies (1:1000 dilution) for 24 h at 4°C in a humid chamber. All subsequent incubations were performed at room temperature. After a rinse in PBS, the sections were incubated with rabbit biotinylated anti-goat IgG for 1 h, washed again with PBS, and incubated with peroxidase-conjugated streptavidin for 1 h. Then, the color was developed with dimethylaminoazobenzene (40 mg/ml ) and 0.002% hydrogen peroxide. Tissue sections from all 20 cases were processed for immunohistochemistry together. Nissl staining of adjacent sections was performed to observe the neural shape. Controls included both the replacement of primary antibodies with normal goat serum and liquid phase preabsorption of the primary antibodies with recombinant Wnt-1 protein.
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6 Table 1 Clinical and post-mortem data of the various brains used in this study ID
Group
Age/gender (years)
Neuroleptic exposure duration, dose (years, mg)
Cause of death
Brain weight (g)
Laterality of tissue block
PMD(h)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
C C C C C C C C C C S S S S S S S S S S
54/M 56/M 50/F 57/M 62/F 61/M 63/F 63/F 73/M 70/F 45/M 53/M 66/F 59/M 61/F 65/M 67/F 68/F 71/M 73/F
– – – – – – – – – – 16 (650) 13 (200) 21 (150) 15 (500) 13 (250) 16 (150) 14 (200) 17 (165) 16 (50) –
Heart failure Brain hemorrhage Liver failure Liver failure Respiratory failure Gastric cancer Heart failure Heart failure Pneumonia Colon cancer Cholecystitis Heart failure Heart failure Respiratory failure Lung cancer Gastric ulcer Heart failure Heart failure Brain hemorrhage Heart failure
1330 1250 1250 1260 1140 1350 1250 1260 1000 1200 1120 1360 1340 990 1050 1240 910 1120 1290 1050
R R R R L L R R L R L R R R L R L R L L
10 9 7 4 12 6 7 10 12 12 11 10 9 9 7 10 11 4 6 7
Details of case number: group (C=control; S=schizophrenic); age, gender (M=male; F=female); neuroleptic exposure (chlorpromazine equivalents); cause of death, brain weight, laterality of tissue block (L=left; R=right); PMD, post-mortem delay.
Five adjacent sections from each brain were processed for immunostaining, and all of these sections were assessed and formed the basis of the final average count. Moreover, one other section was stained with hematoxylin to monitor the neuroanatomical coordinates and for nucleolated cell counting. Counts of immunoreactive nucleolated cells were performed by two independent examiners, who were unaware of the identity of the subject. The entire cytoarchitectural field was scanned, and the total number of Wnt-1 immunoreactive neurons in fields of hippocampi were counted by each of the examiners. The boundaries between sectors CA1, 2, 3, and 4 were delineated by applying the criteria of de Lorente (1937) to sections viewed with a 10× objective lens. These cell numbers were counted using an Olympus BH microscope, and the average of these counts in five adjacent sections was recorded. Reliability of the counting procedure was confirmed by quantifying a subset of the sections at a different time. Neuronal counts in these procedures consistently differed by less than 3%.
2.4. Statistical analysis Statistical analysis was performed by the Mann–Whitney U test and Pearson’s product moment correlation coefficient. 3. Results 3.1. Global group characteristics Table 1 lists the details of ages and brain weights among schizophrenic and control groups. There were no significant differences in brain weights (both male and female) between the groups by the Mann–Whitney U test. Also listed are details of laterality of available blocks for schizophrenic and control groups. 3.2. Distribution of Wnt-1 in control and schizophrenic brains Immunohistochemistry was used to examine the distribution of Wnt-1 in the control and schizo-
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T. Miyaoka et al. / Schizophrenia Research 38 (1999) 1–6
phrenic brains. In general, immunoreactivity for Wnt-1 was present within neuronal cells in both control and schizophrenic brains. In the hippocampus, immunoreactivity of neuronal cells was observed, especially in CA3 and CA4; however, immunoreactivity in CA1, CA2, and the subiculum were very weak in both schizophrenic and control brains. Therefore, we examined these areas of CA3 and CA4 closely. In schizophrenic brains, Wnt-1 immunoreactivity within pyramidal cells was increased relative to that in controls. Fig. 1 shows
an example of immunostaining for Wnt-1 in CA4 in control and schizophrenic brains. The cellular labeling observed in CA3 and CA4 of the hippocampus allowed us to semiquantitatively estimate the density of Wnt1-immunoreactive cells in this region. A significant increase (2.5–4 times) in Wnt-1-immunoreactive cells was observed in the CA3 and CA4 regions of 90% of schizophrenic hippocampi studied (Fig. 2). The means±SD of the schizophrenic and control groups (CA3: control 60.6±8.3, schizophrenic 167.1±25.1 and CA4: control 65.9±6.2, schizophrenic 275.4±50.1) differed significantly (CA3: p=0.002, and CA4: p=0.002, Mann–Whitney U test). Although Wnt-1-immunoreactive cell density was found to be increased in the majority of schizophrenic brains, the labeling pattern in immunoreactive cells did not differ from that of the controls (Fig. 1). Moreover, the number of immunoreactive cells did not correlate with age, postmortem delay, neuroleptic exposure duration and dose, sex, or hemispheric laterality by the Mann–Whitney U test and Pearson’s product moment correlation coefficient. Cell counting in CA3 and CA4 regions performed on hematoxylinstained adjacent sections of all hippocampi stained revealed no significant difference in the total cell numbers between schizophrenic and control brains by the Mann–Whitney U test (CA3: control 213.8±15.9, schizophrenic 211.4±21.6, and CA4: control 341.2±25.8, schizophrenic 353.9±20.1).
4. Discussion
Fig. 1. Immunohistochemical localization of Wnt-1 in control and schizophrenic sections. Wnt-1 immunoreactivity in CA4 (a and b) regions of the hippocampus obtained from control (a) and schizophrenic (b) brains. In schizophrenic samples, the number of Wnt-1-immunoreactive cells is clearly larger than in controls (240×).
We observed significant increases in the number of Wnt-1-immunoreactive cells in CA3 and CA4 regions of the hippocampus in the majority of schizophrenic brains. According to their anatomical location, these cells probably belong to the pyramidal and polymorph neurons of the hippocampus (Amaral, 1989). The increase in Wnt1-immunoreactive cells in schizophrenic brains does not appear to be related to an altered cell number since the neuronal count in these regions failed to reveal a significant change in cell number in these areas, as was previously observed ( Heckers, 1991).
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Fig. 2. Wnt-1-immunoreactive cells in CA3 and CA4 regions of the hippocampus obtained from normal control (C ) and schizophrenic (S ) brains. Each dot represents the mean of Wnt-1-immunoreactive cell count performed on each hippocampus studied. The mean for each group is illustrated by the horizontal lines (CA3: control 60.6±8.3, schizophrenic 167.1±25.1; and CA4: control 65.9±6.2, schizophrenic 275.4±50.1). The asterisk represents a significant difference ( p=0.002, both CA3 and CA4) between the mean of the two groups by the Mann–Whitney U test for nonparametric values.
Although the majority of schizophrenic brains demonstrated a significant increase in the hippocampal Wnt-1-immunoreactive cell number compared with controls, one schizophrenic brain did not show an increase (Fig. 2). This may be due to the heterogeneity of the disease process itself or to the inherent problems in the diagnosis of this complex disorder. The contribution of long-term neuroleptic treatment in the observed increase in Wnt-1 expression in schizophrenic brains cannot be ruled out. It is, however, unlikely to have had a major influence. An increase in expression of Wnt-1 confined to the CA3 and CA4 regions is difficult to explain solely by prior neuroleptic exposure. Hippocampal formation is believed to be closely involved in the pathophysiology of schizophrenia (Akbarian et al., 1993b; Conrad et al., 1991; Cotter et al., 1997). Recently, reduced expression of synapsin and a neural cell adhesion molecule was described in the hippocampus of postmortem schizophrenic brains (Barbeau et al., 1995; Glantz and Lewis, 1997). Moreover, the expression of Wnt-1 may be related to cell adhesion, synaptic rearrangement and plasticity (McMahon and Bradley, 1990; Bradley et al., 1993; Parr et al., 1993). Experimental evidence from magnetic resonance imaging and cerebral blood flow studies
suggests a dysfunction of a prefrontal–hippocampal network in schizophrenia ( Weinberger, 1995), and recent data obtained in rats suggest that the integrity of the ventral hippocampus is essential for adequate development of the prefrontal cortex (Glantz and Lewis, 1997). Therefore, an increase of Wnt-1-immunoreactive cells in this area in the schizophrenic brain, as observed in the present study, may induce and/or reflect aberrant connections between hippocampal neurons with potential impact on hippocampal efferents to cortical and subcortical structures. These findings may partly explain some of the deficits in hippocampal functions, including memory, observed in schizophrenic subjects ( Weinberger, 1987, 1995). However, the question as to whether increased expression of Wnt-1-immunoreactive neurons in the schizophrenic hippocampus is a cause or merely a consequence of the disease cannot be readily answered. Recently, Dvl-1, a downstream member of the wingless/Wnt pathway in mutant mice, provided a model for aspects of several human psychiatric disorders (Lijam et al., 1997). Moreover, interestingly, Cotter et al. (1998) recently reported abnormalities of Wnt signaling in the hippocampus in schizophrenia. Considering the key function of Wnt-1 in brain development in general, and in the hippocampus in particular,
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its increase in schizophrenic brains may be a central element in the pathophysiology of this disorder.
Acknowledgment Part of this work was supported by a Grant-inAid for Scientific Research on Priority Areas, 09770741, from the Ministry of Education, Science, Sports and Culture of Japan.
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