GSK-3 and the neurodevelopmental hypothesis of schizophrenia

European Neuropsychopharmacology 12 (2002) 13–25 www.elsevier.com / locate / euroneuro

Review

GSK-3 and the neurodevelopmental hypothesis of schizophrenia a a,b a, Nitsan Kozlovsky , R.H. Belmaker , Galila Agam * a

Stanley Foundation Research Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beersheva, Israel b Ministry of Health, Mental Health Center, Beersheva, Israel Received 17 April 2001; received in revised form 27 September 2001; accepted 2 October 2001

Abstract The Neurodevelopmental Hypothesis of schizophrenia suggests that interaction between genetic and environmental events occurring during critical early periods in neuronal growth may negatively influence the way by which nerve cells are laid down, differentiated and selectively culled by apoptosis. Recent advances offer insights into the regulation of brain development. The Wnt family of genes plays a central role in normal brain development. Activation of the Wnt cascade leads to inactivation of glycogen synthase kinase-3b (GSK-3b), accumulation and activation of b-catenin and expression of genes involved in neuronal development. Alteration in the Wnt transduction cascade, which may represent an aberrant neurodevelopment in schizophrenia, is discussed. Programmed cell death is also an essential component of normal brain development. Abnormal neuronal distribution found in schizophrenic patients’ brains may imply aberrant programmed cell death. GSK-3 participates in the signal transduction cascade of apoptosis. The possible role of aberrant GSK-3 in the etiology of schizophrenia is discussed.  2002 Elsevier Science B.V. / ECNP. All rights reserved. Keywords: Schizophrenia; Neurodevelopmental hypothesis; Glycogen synthase kinase-3 (GSK-3); Wnt signaling; Apoptosis

1. Introduction The dopamine hypothesis of schizophrenia dominated research in this area for over two decades. This hypothesis was based on the discovery that all anti-psychotic drugs in clinical use are dopamine receptor blockers (Arnt and Skarsfeldt, 1998; Creese et al., 1976; Meltzer, 1994; Snyder, 1976). The dopaminergic hypothesis was heuristic in the development of new anti-psychotic compounds, however, it failed to generate findings relating to the etiology and natural course of schizophrenia. An alternative and increasingly attractive hypothesis addresses the etiology and natural course of schizophrenia as its central concern. The Neurodevelopmental Hypothesis suggests that interaction between genetic and environmental events occurring during critical early periods in neuronal growth may negatively influence the way by which nerve cells are laid down, differentiated, selectively culled by apoptosis *Corresponding author. Psychiatry Research Unit, Mental Health Center, PO Box 4600, Beersheva 84170, Israel. Fax: 1972-8-640-0737. E-mail address: [email protected] (G. Agam).

and remodeled by expansion and retraction of dendrites and synaptic connections (Bloom, 1993; Murray, 1994; Roberts, 1990; Weinberger, 1987). These changes begin in utero, are affected by perinatal events around birth, and become fully expressed in early adulthood (Arnold and Trojanowski, 1996; Bunney et al., 1995; Harrison, 1999). Representative evidence for the neurodevelopmental hypothesis of schizophrenia are: (1) Morphological findings such as enlarged ventricles and cortical volume loss (Lieberman et al., 1992; Weinberger et al., 1982; Zigun et al., 1992), reduced neuronal count in selected brain regions (Benes et al., 1986; Benes et al., 1991) and abnormal laminar organization that seems to predate overt illness (Arnold et al., 1995). (2) Behavioral, neuromotor and other functional abnormalities, which occur in childhood and predict schizophrenia, such as low IQ (David et al., 1997), poor motor skills (Jones et al., 1994), poor development of language and word skills (Crow, 1995), and poor development of social skills (Benes et al., 1991; Fish, 1987). (3) Obstetric complications during pregnancy and the perinatal period, such as low birth weight and hypoxia

0924-977X / 02 / $ – see front matter  2002 Elsevier Science B.V. / ECNP. All rights reserved. PII: S0924-977X( 01 )00131-6

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(Brixey et al., 1993; Heun and Maier, 1993; Lewis and Murray, 1987), viral infection and / or starvation, particularly during the second trimester when substantial neuronal growth takes place and a considerable number of neurons migrate from the ventricular walls of the cortical plate to form cortical connections, are associated with increased risk of schizophrenia (Mednick et al., 1988; Torrey et al., 1993). (4) Minor non-specific physical developmental anomalies which occur at a higher rate among schizophrenic patients than in the normal population include high palate, low-set ears and webbed digits (Green et al., 1989; Lohr and Flynn, 1993). Taken together, these phenomena support the concept of genetic predisposition for schizophrenia, the expression of which is influenced by events in utero and during early life, interacting with later childhood malmaturation events such as impaired synaptic pruning (Stevens, 1992). It has been suggested that genetic factors may affect brain development in this disorder (Gottesman and Bertelsen, 1989; Guidry and Kent, 1999). Since approximately 30% of the genome is expressed in brain, and many genes are turned on and off during discrete phases of brain development, there are many potential candidates which could be abnormally expressed during this period and affect the pathogenesis of schizophrenia. There is no clear data linking schizophrenia with a defect in any known gene related to brain development (Tsuang, 2000). However, the existence of a genetic basis for schizophrenia suggests a biochemical substrate through which these events are expressed.

2. Neurodevelopment

2.1. Differentially expressed CNS proteins supporting the neurodevelopmental hypothesis of schizophrenia In the search for molecular correlates of the alteration of

brain structure and function in schizophrenia numerous studies focus on neuronal and synaptic proteins in specific brain regions (Table 1)

2.1.1. GAP-43 Growth-associated protein-43 (GAP-43) is a neuronal membrane phosphoprotein enriched in growth cones and axons localized in association cortices and in the hippocampus. It has been suggested that this protein marks circuits involved in the acquisition, processing and storage of new information (Neve et al., 1987; Perrone-Bizzozero et al., 1996), processes altered in schizophrenia, GAP-43 mRNA levels were found decreased in medial temporal lobe, primary visual cortex and anterior cingulate gyrus (Eastwood and Harrison, 1998) and its protein levels were found to be increased in the visual association and prefrontal cortex of schizophrenic patients (Sower et al., 1995). These findings suggest that the pathophysiology of schizophrenia affects hippocampal and cortical circuitry and that one manifestation of these abnormalities is reflected in altered expression of GAP-43. 2.1.2. Synaptic terminal proteins Aberrant synaptic elimination or synaptic reinnervation have been proposed to be a key feature of schizophrenia in the light of profound changes in synaptic organization and density which take place during cortical development (Bourgeois and Rakic, 1993; Eckenhoff and Rakic, 1991). The detection and measurement of synaptic terminal proteins have become established as means to investigate synapses in human brain, and the abundance of these gene products provide a tool to estimate relative synaptic density. Thus, proteins such as synaptophysin, synapsin and synaptic-associated protein-25 (SNAP-25) are used in a quantitative or semiquantitative fashion to demonstrate significant synaptic alteration in post mortem human brain of schizophrenic patients. Synaptophysin mRNA and protein levels were found to be reduced in CA3, CA4, subiculum and parahippocampal gyrus of schizophrenic

Table 1 Possible molecular markers of schizophrenia Marker

Characteristic

Changes in brain

References

Growth-associated protein-43 (GAP-43)

Structural synaptic vesicle protein

mRNA levels ↓ Protein levels ↑

Eastwood and Harrison, 1998 Sower et al., 1995

Synaptophysin

Structural synaptic vesicle protein

mRNA and protein levels ↓

Glantz and Lewis, 1997 Eastwood et al., 1995

Synaptosomal associated protein-25 (SNAP-25)

Presynaptic protein

mRNA and protein levels a

Thompson et al., 1998 Young et al., 1998 Karson et al., 1999

Nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d)

Marker of prefrontal cortex neurons

No. of neurons containing NADPH-d b

Akbarian et al., 1993a Akbarian et al., 1993b

a b

Inconsistent. Abnormal distribution.

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patients (Eastwood et al., 1995) and its protein levels were also reported low in two regions of the prefrontal cortex of subjects with schizophrenia (Glantz and Lewis, 1997). SNAP-25, a presynaptic-terminal protein restrictedly expressed in a subset of terminals was shown to serve as a marker of disrupted patterns of connectivity in the hippocampus (Duc and Catsicas, 1995). Immunocytochemical studies in post mortem brain samples of schizophrenic patients indicate reduced SNAP-25 protein levels in the terminal fields of entorhinal cortex projections (Young et al., 1998), an increase by 30% in Brodmann’s area 9 prefrontal cortex, and a decrease by 55% and 33% in areas 10 and 20, respectively (Thompson et al., 1998). Although the finding of low SNAP-25 protein levels in Brodmann’s area 10 was replicated, mRNA levels were not found to be decreased (Karson et al., 1999).

2.1.3. NADPH-d The enzyme nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d) is present in a small population of cortical subplate neuronal cells thus serving as their marker. Since these neurons are resistant to neurodegeneration and neurotoxicity, deviances from their normal distribution is consistent with neurodevelopmental disturbances (Bunney and Bunney, 1999). The dorsolateral prefrontal cortex of schizophrenic patients showed a significant decline in NADPH-d neurons in the superficial white matter and in the overlying cortex but a significant increase in these neurons in white matter below the cortex (Akbarian et al., 1993a). Distorted distribution of NADPHd neurons was also found in the hippocampal formation and the neocortex of the lateral temporal lobe (Akbarian et al., 1993a). These results may imply that a fundamental mechanism of cortical development, such as neuronal migration and / or programmed cell death of transitory white matter neurons are altered in the prefrontal cortex of schizophrenic patients (Akbarian et al., 1993a). Alteration in ontogenesis, as reflected in the abnormal distribution of NADPH-d neurons, appears to be particularly important because dysfunction of the association fields of the prefrontal cortex is thought to underlie the negative symptoms (Tamminga et al., 1992) and the associative disturbances commonly observed in this disorder (Goldberg and Weinberger, 1986). As summarized in Table 1, the findings concerning different proteins as well as findings related to a given protein are inconsistent. It is therefore difficult to draw an integrative hypothesis based on this information. These markers and many others cannot be interpreted as related to a molecular mechanism leading to aberrant neurodevelopment. Perhaps they reflect the outcome. Although the exact developmental mechanisms involved in schizophrenia remain unresolved, recent advances in molecular cloning and related techniques, such as in situ hybridization and immunohistochemistry based on recombinant antigens, have resulted in massive new information related to these mechanisms in the vertebrate central nervous

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system. Although most of these discoveries have been made in non-primate models, existing evidence suggests that the cellular and molecular mechanisms of neurodevelopment are highly conserved among vertebrate species.

2.2. The Wnt family of genes Recent studies offer insights into possible regulatory mechanisms of developmental brain changes. The Wnt family of genes is a major component in these studies. These genes encode a family of cysteine-rich, secreted glycoproteins involved in embryonic developmental processes such as cell adhesion and rearrangement of synapses (Hall et al., 2000; Patapoutian and Reichardt, 2000). The term Wnt is an amalgam of ‘Wingless’ (Wg) and ‘Int’. The Wg gene was identified as a locus in Drosophila required in each segment for the establishment of normal pattern (Pfeiffer and Vincent, 1999). The Int loci were originally identified in mice as sites of frequent integration of the Moloney murine leukemia virus. Subsequently, it was shown that the murine Int-1 locus was homologous to the Drosophila Wg and the family name was changed to Wnt (Thomas and Capecchi, 1990). The Wnt proteins are capable of inducing cells to proliferate, differentiate and survive by signaling through autocrine and paracrine pathways. They exert these effects on cells during early development by interacting with specific cell-surface receptors, which in turn initiate a signaling cascade culminating with changes in gene expression (reviewed by Nusse and Varmus, 1992; Parr and McMahon, 1994). During embryogenesis extracellularly secreted Wnt molecules interact with cell-membrane receptors of the frizzled family. The interaction is regulated by glycosaminoglycans, molecules for which Wnt proteins have a high affinity and thus serve as co-receptors. This agonist– receptor interaction induces the hyperphosphorylation and activation of the protein Disheveled (Dvl) and thereby its assembly with three other proteins — Axin, Frat-1 / GBP (frequently rearranged in advance T-cell lymphomas 1 / glycogen synthase kinase-3 binding protein) and glycogen synthase kinase-3b (GSK-3b). The four proteins merge into a heterotetramer complex. This assembly leads to the suppression of GSK-3b’s activity, which is constitutively active under resting conditions. While active, GSK-3b facilitated by Axin and adenomatous polyposis coli (APC, a scaffold protein coordinating the interaction of b-catenin and GSK-3b) phosphorylates b-catenin, thereby promoting its assembly with phosphorylated APC leading to degradation of b-catenin through the ubiquitin proteolytic pathway. Inactivation of GSK-3 upon activation of the Wnt cascade protects b-catenin against degradation and allows its translocation into the nucleus. In the nucleus, in the absence of Wnt signaling, TCF / LEF-1 (T-cell factor / lymphocyte enhancer factor-1) which belongs to the highmobility group (HMG) box architectural transcription factors represses the expression of Wnt target genes. In the presence of Wnt signaling, the rise of b-catenin steady-

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state levels enables its accumulation in the nucleus of embryonic cells where it interacts with TCF and forms a transcriptional complex that activates gene transcription of crucial development regulatory genes (Moon et al., 1997) (Fig. 1). An additional cascade which possibly interacts with GSK-3b via dishevelled is the Notch pathway that is involved in cell-fate determination (Anderton et al., 2000).

2.3. The Wnt signaling cascade and the development of the CNS Wnt signal transduction cascades are important mediators of axis establishment when the primary patterns of the embryo are determined (Sokol, 1999). In situ hybridization studies have suggested that the Wnt cascade plays an important role during early patterning of the neuronal tube (Lee and Jessell, 1999). In mouse embryos, the timing of Wnt gene expression in the central nervous system indicates that these genes might assist in regional specification of the neuronal tube, particularly in the forebrain region and in the spinal cord. Wnt gene expression within the developing spinal cord exhibits particular patterns of restriction along the dorsal–ventral axis prior to differentiation of neurons (Wodarz and Nusse, 1998). Wnt-1 and

Fig. 1. A scheme of the Wnt signaling pathway. Wnt glycoproteins bind to the frizzeled (FZl) receptor to transduce a signal through disheveled (Dsh). Upon activation, disheveled assembles with three other proteins, Axin, GBP/ Frat and GSK-3b leading to inhibition of GSK-3b activity. While active, GSK-3b phosphorylates b-catenin, promoting its assembly with phosphorylated APC, leading to degradation of b-catenin. Inactivation of GSK-3b upon activation of the Wnt cascade allows b-catenin to accumulate and translocate into the nucleus were it interacts with the transcription factor TCF / LEF-1 to remove the suppression of expression of Wnt target genes.

Wnt-3 are the earliest markers expressed along the dorsal midline of the developing spinal cord (Roelink and Nusse, 1991). Wnt-5a and Wnt-7a are expressed in the overlapping domains in the ventral and lateral diencephalon (Dealy et al., 1993). Target disruption of the mouse Wnt-3a gene results in a severe truncation of the body axis posterior to the forelimbs (Shum et al., 1999). Wnt gene products and proteins that transduce the Wnt signal are centrally involved in brain development, and may have a role in developmental brain disorders in man. Since schizophrenia involves developmental brain changes and abnormal neuronal and synaptic organization, it is possible that a shared mechanism underlies these changes. Alteration in the transduction of the Wnt signaling cascade may represent such a mechanism. It has been shown that mice completely deficient of Dvl1 display abnormalities in social behavior and sensorimotor gating characteristic to several psychiatric disorders including schizophrenia (Lijam et al., 1997).

2.4. GSK-3b mediates Wnt and other signaling cascades GSK-3, a downstream component of the Wnt signaling cascade, is a ubiquitous serine / threonine kinase initially identified as an enzyme negatively regulating the activity of glycogen synthase (Cohen, 1985). It is highly abundant in brain tissues (Pei et al., 1999; Woodgett, 1990) and highly conserved during evolution (Yu and Yang, 1993). It was first purified from skeletal muscle (Woodgett, 1991). Two isoforms with 85% sequence homology and 98% identity in the catalytic domains (Woodgett, 1990) referred to as GSK-3 a and b encoded by separate genes mapped to chromosomes 19 and 3, respectively (Shaw et al., 1998) have been identified in mammals. Recent studies demonstrated that GSK-3b is also involved in the regulation of growth and development. The GSK-3b homolog in Xenopus plays a critical role in axis pattern formation by regulating the establishment of dorsoventral polarity (Dominguez et al., 1995). The Drosophila GSK-3b homolog, designated shaggy or zeste-white 3 (sgg /zw3), has a critical role in cell fate determination in the Wnt signalling cascade (Wodarz and Nusse, 1998). But the role of GSK-3 is not restricted to the Wnt system. GSK-3 is a juncture of three signal transduction cascades, the Wnt, the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI-3K) (Srivastava and Pandey, 1998). Its multiple targets are spread extracellularly and at various subcellular organelles — membranal, cytoskeletal, nuclear and cytosolic (Table 2) and include transcription factors (Plyte et al., 1992), regulatory enzymes (Rubinfeld et al., 1996) and structural proteins (Hanger et al., 1992; Mandelkow et al., 1992). Thus, GSK-3 mediates metabolic, developmental, differential and proliferative processes (He et al., 1995; Siegfried et al., 1992).

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Table 2 GSK-3 substrates Cell compartment

Substrates

References

Nuclear

Developmental regulatory genes b Proto-oncogenes: c-jun / c-fos a,b Jun D, Jun B, c-myb, c-myc a,b Embryonic initiation factor-2B a,b CREB-P a,b

Plyte et al., 1992 deGroot et al., 1993 Boyle et al., 1991 Welsh and Proud, 1993 Fiol et al., 1994

Cytosolic

Glycogen synthase a,b RII (regulatory subunit of PKA)a ATP cytrate lyase a Mg–ATP-dependent protein phosphatase-1 a

Vandenheede et al., 1981 Hemmings et al., 1982 Hughes et al., 1992 Yang and Fong, 1985

Extracellular

Myelin basic protein a Neuronal cell adhesion molecule (NCAM)a

Yang, 1986 Mackie et al., 1989

a b

Phosphorylated by GSK-3a. Phosphorylated by GSK-3b.

2.5. Neuronal proteins regulated by GSK-3 The role of GSK-3 in developmental brain changes, in general, and in schizophrenia, in particular, may not be restricted to the Wnt signaling cascade. Some neuronal proteins implicated to play a role in neurodevelopment are regulated by GSK-3. GSK-3a, the ATP–Mg-dependent type-1 protein phosphatase activating factor (FA), was shown to be the kinase that phosphorylates synapsin I. The latter is a neuronal protein coating synaptic vesicles which binds to the cytoskeleton and is involved in the modulation of neurotransmission (Thiel, 1993). GSK-3a phosphorylates and thereby inhibits cross-linking of synapsin I with microtubules in a unique tail region (Yang et al., 1992). This suggests a possible involvement of GSK-3a in the regulation of axonal transport processes of synaptic vesicles via the promotion of vesicle mobility. The neuronal cell adhesion molecule (N-CAM) mediates cell–cell interaction throughout development and in adulthood. Alterations in levels of N-CAM expression play a crucial role at sites of embryonic induction (Edelman, 1986; Thiery et al., 1982). GSK-3a and casein kinase I (CK-I) isolated from mammalian and avian brain, were found to rapidly phosphorylate purified chicken N-CAM. Prior phosphorylation of N-CAM at other common phosphorylation sites by cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase or protein kinase C, is a prerequisite for the phosphorylation by GSK-3a and CK-I (Mackie et al., 1989). Cytoskeleton re-organization may be accomplished by modulating microtubule dynamics (Mitchison and Kirschner, 1988). Phosphorylation of cytoskeletal components and associated proteins plays a major role in the regulation of the cytoskeleton. For example, microtubule assembly and stability are regulated by the phosphorylation state of microtubule-associated proteins (MAPs) (Mandell and Banker, 1995). MAP-1B, a major phosphoprotein

component of the neuronal cytoskeleton, is involved in axonal extension. The ability of MAP-1B to bind and stabilize microtubules depends on its phosphorylation state (Brugg et al., 1993). MAP-1B phosphorylated state levels increase during axonal extension and then decline to a low level at the end of axogenesis (Fischer and RomanoClarke, 1990). GSK-3b phosphorylates MAP-1B in postmitotic neurons that are extending axons (Goold et al., 1999). Wnt signaling molecules induce cytoskeletal changes in developing axons (Nusse, 1997). Thus, expression of Wnt-3 and Wnt-7a coincides with the period of dendritic maturation, axonal extension and formation of synapses (Lucas and Salinas, 1997). Wnt-7a induces changes in cytoskeletal dynamics by inhibiting GSK-3b leading to changes in the phosphorylation state of MAP-1B (Nusse, 1997). The neurofilament (NF) is an intermediate filament class associated with the neuronal cytoskeletal system in neuronal cells (Hoffman and Lasek, 1975). The mature neuronal NF is comprised of a three-subunit protein — NF-L (low), NF-M (medium) and NF-H (high) which differ in their molecular masses (Kaufmann et al., 1984). Phosphorylated NFs appear to be mainly in axons where the dephosphorylated forms are found in the cell body and dendrites (Sternberger and Sternberger, 1983), suggesting a role of NF phosphorylation in NF transport and interaction. GSK3a is the protein kinase responsible for the in vivo phosphorylation of NF-M in a site flanked by a carboxyterminal proline residue (Guidato et al., 1996). GSK-3a phosphorylates NF-H side-arms in vivo and in vitro (Guan et al., 1991; Yang et al., 1995a). These data support the role of GSK-3 in the mechanism regulating NF’s phosphorylation in axons.

2.6. Expression of GSK-3 during brain development The central role of GSK-3 in the regulation of neurode-

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velopmental processes is further supported by the finding of Leroy and Brion (Leroy and Brion, 1999) who studied the expression and the neuroanatomical distribution of GSK-3b immunoreactivity in rat brain from embryonic up to adult stage. GSK-3b is expressed in most brain regions, although significant local variations were observed. e.g., GSK-3b immunoreactivity was present in cortical and subcortical structures, in the cerebellum and in the brainstem. This widespread expression may suggest that in the adult brain GSK-3b is involved in regulatory signaling pathways common to many neurons. GSK-3b is expressed in the developing brain from embryonic day 10 (E10), with the highest expression observed from E18 up to 10 days of postnatal life. Its expression decreases thereafter and is lowest in the adult. GSK-3b is strongly expressed in developing neurons, weakly in layers containing neuroblasts, present in perikarya, proximal dendrites and axonal tracts. In adult human brain it is abundant in all brain regions but not in astrocytes. Since the increased expression of GSK-3b could be associated with increased enzymatic activity, the results suggest that increased phosphorylation of selected brain substrates by GSK-3b is critical during the intense period of brain development. Aberrant expression of GSK-3b during this period might significantly affect the position and function of neurons. An intriguing recent study found that disruption of the GSK-3b gene in mice results in embryonic lethality caused by severe liver degeneration during mid-gestation (Hoeflich et al., 2000). GSK-3a is also highly abundant in rabbit brain tissue (Yu and Yang, 1993) although no microanatomical studies of its expression levels during brain development have been reported. Yet, since GSK-3a is involved in the regulation of diverse brain functions such as microtubule assembly and disassembly, neurotransmission and myelin function, it is reasonable to assume that its expression levels, similarly to those of GSK-3b, undergo changes during brain development, and that GSK-3a is differentially expressed in different parts of adult brain regions.

2.7. The role of GSK-3 in mediating apoptosis Regulation of cell survival is crucial for normal brain development. Perturbation of cell survival mechanisms may lead to either excessive or insufficient cell death, which may result in pathological conditions. Apoptosis, or programmed cell death, is an evolutionary conserved form of cell death critical for tissue homeostasis (ChaleckaFranaszek and Chuang, 1999). GSK-3b has recently been associated with apoptosis through the phosphatidyl inositol 3-kinase (PI-3K) /Akt cascade (Pap and Cooper, 1998). The PI-3K /Akt pathway is activated by insulin and growth factors and inactivated by environmental stressors such as heat shock or oxidative stress and inhibitory neurotransmission such as glutamate toxicity (Burgering and Coffer, 1995). Akt, also known as

Fig. 2. Involvement of GSK-3b in apoptosis. The PI-3-kinase / akt pathway is inactivated by environmental stressors and inhibitory neurotransmission such as glutamate toxicity leading to GSK-3b activation which promotes apoptosis.

protein kinase B (PKB) is a multi serine / threonine kinase, a downstream target of PI-3K. Activation of Akt requires phosphorylation by an upstream PI-3-dependent kinase. Subsequently, Akt phosphorylates Ser 9 of GSK-3b, inhibiting its activity (Cross et al., 1995) (Fig. 2). Activation of GSK-3b contributes to pro-apoptotic signaling, evidenced by the recent finding that over-expression of GSK-3b in Rat-1 and PC12 cells stimulates apoptosis (Pap and Cooper, 1998). Another line of evidence regarding the potentially important role of GSK-3b in regulating apoptosis is the fact that lithium, a selective inhibitor of GSK-3 (Klein and Melton, 1996; Stambolic et al., 1996), protects cultured neurons against glutamate-induced apoptosis mediated by N-methyl-D-aspartate (NMDA) receptors in a PI-3K-dependent manner (Chalecka-Franaszek and Chuang, 1999).

3. Neurodevelopment and schizophrenia

3.1. Differences in Wnt cascade markers in schizophrenia The Wnt family of genes plays a fundamental role in neuronal development through the control of migration and differentiation. There are indications that a mutation in one or more of these genes may lead to abnormal cerebral development in mice (McMahon and Bradley, 1990). The role of this pathway in the regulation of neuronal migration

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during development suggests that alteration of this pathway may be involved in producing the cytoarchitectural defects observed in schizophrenia (Cotter et al., 1998). Both GSK-3a and GSK-3b play a role in neurodevelopment, hypothesized to be altered in schizophrenic patients. Since GSK-3b is a component of the Wnt pathway, its reduced levels (details in Section 3.2) corroborate recent reports of abnormal Wnt cascade markers in schizophrenia. Miyaoka et al. (1999) reported an increase in Wnt-1 containing neurons in the hippocampal pyramidal cell layers CA3 and CA4 of schizophrenic patients. Reduction in b- and g-catenin in the CA3 and CA4 regions of the hippocampus have also been noted in this disorder (Cotter et al., 1998). In the study by Beasley et al. (2001) showing reduced GSK-3b levels in the frontal cortex of schizophrenic patients there was no significant alteration in b-catenin or dishevelled protein levels. Upon Wnt cascade activation b-catenin translocates from the cytosol to the nucleus (Moon et al., 1997), but the assay of Moon et al. (1997) uses whole-cell homogenates and thus determines only total cell levels. Abnormalities of social interaction and sensorimotor gating have been reported in mice lacking the dishevelled-1 gene (Lijam et al., 1997). In humans, a deletion of this gene characterizes the DiGeorge syndrome. Indeed, approximately 25% of the adults suffering from this syndrome, which presents with learning disabilities, craniofacial abnormalities and congenital heart defects, develop schizophrenia (Pizzuti et al., 1996). The possible involvement of elements of the Wnt signaling pathway genes in bipolar disorder and schizophrenia was also studied by high-resolution radiation hybrid mapping (Rhoads et al., 1999). Frizzled 3, expressed in highest levels in the adult brain (Wang et al., 1996) is located on chromosome 7q11.23 shown to be associated with the developmental disorder Williams syndrome (Robinson et al., 1996). A potential susceptibility locus at 7q11 has recently been reported for schizophrenia (Blouin et al., 1998) and linkage disequilibrium mapping has recently shown that NOTCH4 (see Section 2.2 above) is highly associated with schizophrenia (Wei and Hemmings, 2000).

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ability, a calibration standard curve of known amounts of recombinant GSK-3b units was run in each gel and used to derive the absolute value of each sample band run on the same gel. Each sample was analyzed at least three times and at two different protein concentrations, both within the linear range of detection. Fig. 3A shows that frontal cortex samples of schizophrenic patients have 41% reduced GSK3b protein levels compared with normal controls. There was no correlation between GSK-3b levels of all groups and post mortem interval, or age. In the schizophrenic patients’ group there was no correlation between GSK-3b levels and their estimated lifetime anti-psychotic drugs consumption (Kozlovsky et al., 2000). We next measured the levels of GSK-3b in the post mortem occipital cortex specimens from the same subjects of the four diagnostic groups. There was no difference between their GSK-3b protein levels, suggesting that the reduction in frontal cortex does not represent a general alteration of GSK-3b protein levels in these patients’

3.2. GSK-3a and b are altered in schizophrenic patients We have previously reported that GSK-3 is altered in schizophrenic patients’ brains. The study was carried out in frozen post mortem brain specimens obtained from the Stanley Foundation Brain Bank (Torrey et al., 2000). The 60 samples of frontal and occipital cortex consisted of 15 schizophrenic patients, 15 bipolar patients, 15 unipolar depressed patients and 15 normal controls. The four groups were matched for age, sex, race, post mortem interval and side of the brain. Quality of preservation was monitored by two factors — mRNA degree of degradation and pH. GSK-3b protein levels were quantified by Western immunoblotting. To minimize the effect of interblot vari-

Fig. 3. GSK-3b protein levels (A) and total GSK-3 activity (B) in post mortem frontal cortex of schizophrenic patients vs. bipolar and unipolar patients and normal controls. (A) Translation of the arbitrary densitometric values of the immunoreactive levels of GSK-3b into absolute units was carried out by calculation from the standard curve run in each gel. The results are means6S.E.M. Each specimen was assayed at least three independent times. *ANOVA: F53.0903; d.f.53,54; P50.0346; post-hoc LSD, P50.008 for schizophrenic patients compared with controls. (B) Horizontal lines depict mean values. Kruskal–Wallis analysis: chisquare58.27; d.f.53; P50.04. Mann–Whitney U-test between the control group and the schizophrenic patients P50.039.

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brains (Kozlovsky et al., 2001). This is consistent with other neuronal-specific molecules, such as synaptophysin (Glantz and Lewis, 1997) and GAP-43 (Sower et al., 1995), also found altered in prefrontal and frontal cortical areas but unchanged in occipital cortex of schizophrenic patients. Since GSK-3b is unusual among protein kinases in being constitutively active under non-stimulated conditions in the cell and exerting repression on its substrates (Stambolic and Woodgett, 1994), the next question raised was whether low frontal cortex GSK-3b levels are accompanied by low enzymatic activity. To answer this question GSK-3 activity was measured in the same frontal cortex specimens that were used for quantifying protein levels. GSK-3 activity was assayed in homogenates by quantifying the phosphorylation of a phospho-CREB peptide, a specific GSK-3 substrate (Wang et al., 1994). We utilized the fact that GSK-3 activity is specifically inhibited by lithium to discriminate between other kinases activities and that of GSK-3. Fig. 3B shows that mean GSK-3 activity in the frontal cortex of schizophrenic patients was 45% lower than that of normal controls. The other two diagnostic groups showed no difference from the control group (Kozlovsky et al., 2001). There was no correlation between GSK-3 activity and any demographic parameter. Frontal cortex GSK-3 activity and GSK-3b protein levels did not correlate in the specimens of all four diagnostic groups, nor in the specimens of the schizophrenic patients only (Kozlovsky et al., 2001). Since the phospho-CREB substrate does not distinguish the forms of GSK-3, and lithium ions used to discriminate GSK-3 activity from other kinases inhibit both GSK-3a and b, our assay does not distinguish between the activities of the two GSK-3 isoforms. Therefore, it is not unreasonable that no correlation was found between the GSK-3b protein levels and the GSK-3 activity values. Beasley et al. (2001) supported our finding in frontal cortex samples from the MRC Brain Bank. They found a significant 19% reduction in GSK-3b protein levels in schizophrenic patients compared to control subjects. They however were not able to replicate their own and our finding in the Stanley Foundation Collection, apparently due to subtle differences in the methodology (Beasley et al., submitted for publication). Yang et al. (1995b) measured the levels of GSK-3a in peripheral tissue of 48 schizophrenic patients. They found that both enzymatic activity and protein levels of GSK-3a were considerably reduced in lymphocytes of schizophrenic patients as compared to controls. The activity of GSK-3a was less than 20% of that of normal controls suggesting that patients with schizophrenia may have a dysfunction common to GSK-3a and GSK-3b. It remains to be investigated whether this dramatically lower GSK-3a activity in peripheral cells reflects the central nervous system and, if so, whether it is brain region specific. If the reduction in

GSK-3b in frontal cortex of schizophrenic patients will be found to be reflected in peripheral tissue as well, it could serve as a diagnostic marker.

3.3. Difference in GSK-3a -related markers in schizophrenia GSK-3a and some of its substrates were found to be altered in schizophrenia. As described above, GSK-3a protein levels and activity were reported to be reduced in lymphocytes of schizophrenic patients (Yang et al., 1995b). Synapsin I, a neuronal protein involved in modulation of neurotransmission, is phosphorylated and thereby inactivated by GSK-3a (Yang et al., 1992). Levels of synapsin I were found to be extremely low in post mortem hippocampal specimens of schizophrenic patients compared with age-matched normal controls, whereas levels of synaptophysin, another synaptic vesicle protein, were nearly normal (Browning et al., 1993). Given that synapsin I is thought to regulate neurotransmitter release, it is possible that its deficit could result in abnormal processing of neuronal information. It remains to be investigated whether GSK-3a mediates this reduction. It has been suggested that cell recognition molecules (CRMs) such as neuronal cell adhesion molecule (NCAM), mediate abnormalities and disturbances in neurodevelopment related to schizophrenia (Landmesser et al., 1990). These substances, which belong to the immunoglobulin superfamily, play an important role in neurodevelopmental processes including axonal guidance, synapse stabilization and cell migration (Hemperly et al., 1986; McClain and Edelman, 1982). N-CAM alteration in schizophrenia was proposed as an explanation for disorientation of cells in the hippocampus (Conrad and Scheibel, 1987). Indeed, decreased numbers of hippocampal neurons expressing polysialylated N-CAM were found (Barbeau et al., 1995). Cerebrospinal fluid (CSF) from schizophrenic patients contains two- to three-fold higher N-CAM levels (van Kammen et al., 1998). Higher levels of 105 to 115 kDa N-CAM were also found in the hippocampus and frontal cortex of schizophrenic patients (Vawter et al., 1998). N-CAM-modified function in the brain of schizophrenic patients may either stem from its altered protein levels or be a consequence of reduced GSK-3a levels and activity. The microtubule-associated proteins MAP2 and MAP5 (alternatively known as MAP-1B) are major phosphoprotein components of the neuronal cytoskeleton involved in axonal extension and neuronal polarity (Matus and Riederer, 1986). Altered distribution of these two proteins in subiculum and entorhinal cortex, and an increased fraction of the non-phosphorylated form of MAP2 in the subiculum were reported in schizophrenia (Arnold et al., 1991; Cotter et al., 1997). The ability of MAP1B to bind to microtubules for cytoskeletal re-organization depends upon its phosphorylation state which was found to be regulated by

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GSK-3a and b in extending axons and growth cones (Garcia-Perez et al., 1998; Goold et al., 1999).

3.4. Programmed cell death — implication for schizophrenia Abnormal neuronal loss is increasingly recognized as an important pathological feature in a variety of neuropsychiatric disorders including autism, fragile X syndrome, various chromosomal abnormalities and schizophrenia. Neuronal deficits have been observed in multiple areas of the cerebral cortex (Benes et al., 1991; Falkai et al., 1988), several regions of the hippocampus (Jeste and Lohr, 1989) and in the mediodorsal thalamic nuclei of schizophrenic patients (Pakkenberg, 1990). Abnormal neuronal distribution, recently found in the dorsolateral prefrontal cortex and in the lateral and temporal lobes, may also stem from abnormal neuronal death (Akbarian et al., 1993a,b). The evidence that abnormalities of neuronal death are associated with schizophrenia is not surprising, since neuronal death plays a fundamental role in the normal development process of the nervous system. The two most fundamental events in the genesis of the cerebral cortex are the migration of young neurons from the ventricular walls to the cortical plate occurring mainly during the second trimester of pregnancy, and the subsequent elimination of over-abundant neurons and connections by programmed cell death, mainly in the late prenatal and early postnatal period (Jones, 1995). As many as 50% of all neurons that form the developing nervous system die before reaching full maturity (Oppenheim, 1991). Programmed cell death, the cellular death consequent to the activation of signal transduction pathways in the cells, accounts for much of this neuronal pruning. With the rapidly accumulating evidence that cell death is a normal and essential process in the life of an organism it is also increasingly apparent that abnormal programmed cell death at an inappropriate place or time, or in excess or insufficient quantity, is a potential etiologic factor or mediating event in central nervous system pathology.

4. Summary What could be the consequences of decreased GSK-3 levels and activity in schizophrenic patients’ frontal cortex? Given that GSK-3 is a multi-substrate enzyme that unlike most protein kinases is constitutively active under resting conditions and exerts repressing effects on its substrates, decreased GSK-3 activity in schizophrenic patients may result in an accumulation of b-catenin which, in turn, associates with the HMG-box transcription factors Tcf / LEF family, promoting the transcription of developmentally regulated genes in an inappropriate timing during neurodevelopment (Fig. 1). Although there is a clear abnormal signal transduction

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contribution to schizophrenia, apparently of a neurodevelopmental origin, why is obvious early psychopathology absent in children who will be diagnosed with schizophrenia later in life? An etiology involving a ‘two-hit’ process has been suggested to explain this riddle (Impagnatiello et al., 1998; Manschreck et al., 2000; McCarley et al., 1999). The ‘two-hit’ process means that genetic load, adverse embryonic events and perinatal events consist of a neurodevelopmental first hit that leads to vulnerability to schizophrenia. During puberty, hormonal events such as altered neurosteroid biosynthesis, along with the demand to turn on adolescent levels of affect and thought, act as a second hit.

Acknowledgements Post mortem brains were donated by the Stanley Foundation Brain Consortium courtesy of Drs Llewellyn B. Bigelow, Juraj Cervenak, Mary M. Herman, Thomas M. Hyde, Joel E. Kleinman, Jose D. Paltan, Robert M. Post, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken. This work was supported by a Stanley Foundation Research grant to N.K. and G.A.

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