Apoptotic mechanisms in the pathophysiology of schizophrenia

Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 846 – 858 www.elsevier.com/locate/pnpbp

Review article

Apoptotic mechanisms in the pathophysiology of schizophrenia L. Fredrik Jarskog*, Leisa A. Glantz, John H. Gilmore, Jeffrey A. Lieberman Department of Psychiatry, Schizophrenia Research Center, University of North Carolina—Chapel Hill, CB# 7160, Chapel Hill, NC 27599-7160, USA Accepted 1 March 2005 Available online 23 May 2005

Abstract While schizophrenia is generally considered a neurodevelopmental disorder, evidence for progressive clinical deterioration and subtle neurostructural changes following the onset of psychosis has led to the hypothesis that apoptosis may contribute to the pathophysiology of schizophrenia. Apoptosis (a.k.a. programmed cell death) is a mechanism of cell death that operates in normal neurodevelopment and is increasingly recognized for its role in diverse neuropathological conditions. Activation of apoptosis can lead to rapid and complete elimination of neurons and glia in the central nervous system. Studies also show that in certain settings, pro-apoptotic triggers can lead to non-lethal and localized apoptotic activity that produces neuritic and synaptic loss without causing cell death. Given that the neuropathology of schizophrenia is subtle and includes reduced neuropil (especially synaptic elements), limited and often layer-specific reductions of neurons, as well as neuroimaging data suggesting progressive loss of cortical gray matter in first-episode psychosis, a role for apoptosis in schizophrenia appears plausible. Studies that have examined markers of apoptosis and levels of apoptotic regulatory proteins in postmortem schizophrenia brain tissue will be reviewed in context of this hypothesis. Overall, the data seem to indicate a dysregulation of apoptosis in several cortical regions in schizophrenia, including evidence that the apoptotic vulnerability is increased. Although the exact role of apoptosis in schizophrenia remains uncertain, the potential involvement of non-lethal localized apoptosis is intriguing, especially in earlier stages of the illness. D 2005 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Bcl-2; Caspase; Neurodegeneration; Neurodevelopment; Schizophrenia

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Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Underlying mechanism . . . . . . . . . . . . . . . . . . . . . 2.2. Synaptic apoptosis. . . . . . . . . . . . . . . . . . . . . . . . Neuropathology of schizophrenia. . . . . . . . . . . . . . . . . . . . 3.1. Postmortem studies of neuronal and glial cell numbers . . . . . 3.2. Postmortem studies of neuropil and synaptic markers. . . . . . 3.3. Neuroimaging studies: evidence of progressive volume changes 3.4. Functional neuroimaging and spectroscopy . . . . . . . . . . .

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Abbreviations: BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DSBs, double-stranded DNA breaks; ER, endoplasmic reticulum; fMRI, functional MRI; GFAP, glial fibrillary acidic protein; IAP, inhibitor-of-apoptosis protein; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate; NT-3, neurotrophin-3; NMDA, N-methyl-d-aspartate; PET, positron emission tomography; 1H, proton; SSBs, singlestranded DNA breaks; TUNEL, TdT-mediated dUTP nick end-labeling; U, units. * Corresponding author. Tel.: +1 919 966 8035. E-mail address: [email protected] (L.F. Jarskog). 0278-5846/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2005.03.010

L.F Jarskog et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 29 (2005) 846 – 858

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Apoptosis in schizophrenia . . . . . . . . . 4.1. Apoptotic regulatory proteins . . . . 4.2. DNA fragmentation . . . . . . . . . 4.3. Pro-apoptotic stress in schizophrenia 5. Conclusion . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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1. Introduction The neurodevelopmental hypothesis of schizophrenia has substantially advanced our understanding of the important roles that genes and environmental insults exert in the etiology and pathophysiology of the disorder (McDonald and Murray, 2000; Harrison and Weinberger, 2005). However, the neurodevelopmental perspective does not readily account for several important features of schizophrenia including the protracted period of symptomatic dormancy between the putative insult and the emergence of symptoms, the progressive clinical deterioration that affects at least a subgroup of patients, and emerging evidence for progressive neurostructural changes in certain ventricular and cortical brain structures (Lieberman, 1999). In an effort to identify a pathophysiological mechanism that could account for the progressive elements of schizophrenia and dovetail with neurodevelopmental processes, the potential role of apoptosis has increasingly been considered (Margolis et al., 1994; Lewis and Lieberman, 2000; Berger et al., 2003). Apoptosis is a highly regulated form of cell death that is often likened to cellular suicide. Apoptosis is pervasive during early development of the central nervous system (CNS) – over half of all developing neurons die by apoptosis (Burek and Oppenheim, 1996) – and it also serves to eliminate injured or diseased neurons throughout life. Apoptosis occurs rapidly (an apoptotic cell is typically cleared within 24 h) and proceeds without incurring a gliotic response. Furthermore, although apoptosis is often thought of as a terminal event in a given cell, the emerging concept of synaptic (a.k.a. neuritic) apoptosis suggests that activation of apoptosis in neurons can be localized to synapses or distal neurites without inducing immediate neuronal death (Mattson et al., 1998). Given that the neuropathology of schizophrenia includes evidence of shorter dendrites, reduced neuropil, limited reductions in neuronal and glial cell numbers, lack of gliosis, and in vivo neuroimaging evidence of progressive gray matter loss early in the disorder, a potential role for apoptosis appears increasingly plausible. This paper will provide an overview of apoptosis to include the major apoptotic pathways and review the evidence for localized synaptic apoptosis. A review of the neuropathology of schizophrenia will target evidence for neuronal and glial cell loss as well as reduced neuropil. Studies of apoptosis in schizophrenia will be assessed in context of the hypothesis that apoptotic mechanisms

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contribute to the neuropathology of schizophrenia. This will include a discussion on the evidence for pro-apoptotic triggers in schizophrenia in both early and late pathophysiological stages.

2. Apoptosis 2.1. Underlying mechanism The cytomorphological features associated with apoptosis are distinctly different from necrosis, the other form of cell death. Apoptosis is characterized by cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and cellular disintegration with phagocytosis (Bredesen, 1995). Apoptosis occurs without inflammation and typically requires the formation of new gene products to proceed. The mechanism of apoptosis is focused on regulating the activation of cysteine-dependent aspartatedirected proteases known as caspase proteins (Friedlander, 2003). Caspases have a very stringent cleavage requirement for the carboxyl side of aspartate residues. Initiator caspases (e.g. caspase-8, -9, and -10) are responsible for promoting the cleavage of downstream effector caspases (e.g. caspase3, -6, and -7), and caspase-3 is the effector caspase most often associated with apoptosis in the CNS (Yuan and Yankner, 2000). Effector caspases are activated by cleavage and active caspase-3 in turn cleaves a number of specific structural and functional proteins, leading to the characteristic apoptotic morphology (Boatright and Salvesen, 2003). At least three distinct mechanisms have been identified that lead to caspase-3 activation: the mitochondrial (intrinsic) pathway, the death receptor (extrinsic) pathway and the inflammatory (caspase-1-mediated) pathway, Fig 1. The mitochondrial pathway regulates caspase activity through mitochondrial release of cytochrome c. Cytochrome c forms a complex with caspase-9 and the adaptor protein Apaf-1 to produce an apoptosome. The apoptosome cleaves procaspase-3 into active caspase-3 that then begins the structural breakdown of the cell (Adams and Cory, 2002). An important upstream checkpoint for cytochrome c release involves interactions of pro- and anti-apoptotic members of the Bcl-2 family of proteins. These proteins interact through dimerization in the mitochondrial membrane. The ratio of specific pro-apoptotic (e.g. Bax, Bak, Bid, Bcl-XS) to antiapoptotic (e.g. Bcl-2, Bcl-XL) protein levels determines whether a given pro-apoptotic stimulus will lead to

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Fig. 1. Overview of the mitochondrial, death receptor and inflammatory pathways of apoptosis. The mitochondrial pathway involves the regulation of mitochondrial cytochrome c (Cyto c) release by Bcl-2 family proteins (e.g. Bax, Bcl-2) in response to pro-apoptotic stimuli. Cyto c forms a complex with Apaf-1 and procaspase-9 to form the apoptosome. This complex cleaves procaspase-3 into activated caspase-3, leading to cleavage of key functional and structural proteins. The caspase cascade is modulated in part by inhibitor-of-apoptosis proteins (IAPs) that can inhibit caspase-9 and caspase-3. The death receptor pathway involves TNFa or Fas ligand binding to the death receptor. Adaptor molecules in turn recruit procaspase-8 which undergoes autocatalytic cleavage and then activates procaspase-3. Alternatively, activated caspase-8 can impact the mitochondrial pathway at an upstream level by promoting Baxmediated release of Cyto c via BID. Finally, the inflammatory pathway is initiated by cleavage of procaspase-1 into activated caspase-1. While caspase-1 is a well-known activator of prointerleukin-1h, active caspase-1 can promote mitochondrial Cyto c release via BID.

mitochondrial cytochrome c release and caspase activation. The Bax to Bcl-2 ratio represents a prototype for such regulatory control with high Bax/Bcl-2 ratios promoting apoptosis and low Bax/Bcl-2 ratios inhibiting apoptosis (Oltvai et al., 1993). Importantly, Bcl-2 protein also has neurotrophic properties that function independently of the cell death mechanism. Overexpression of Bcl-2 can enhance neurite outgrowth (Oh et al., 1996; Zhang et al., 1996) and promote regeneration of severed CNS axons (Chen et al., 1997), while Bcl-2 heterozygosity slows neuronal maturation (Middleton et al., 1998). Finally, the inhibitor-ofapoptosis protein (IAP) family represents a powerful downstream inhibitory checkpoint of the mitochondrial pathway that can directly inhibit the activation of caspase9 and caspase-3. The death receptor pathway involves the binding of specific receptor ligands (e.g. TNF-a, FasL) to the tumor necrosis factor (TNF) receptor, leading to the activation of caspase-8 and caspase-10 (Boatright and Salvesen, 2003). These initiator caspases can then activate caspase-3, a common downstream effector caspase. In addition, caspase-

8 can activate the mitochondrial pathway at an upstream level by promoting Bax-initiated release of cytochrome c through cleavage of Bid protein to form tBid (Antonsson and Martinou, 2000). The inflammatory pathway involves the formation of the ‘‘inflammasome,’’ an analogous complex to the apoptosome. The inflammasome consists of caspase-1, caspase-5, and an adaptor protein NALP-1 and it is responsible for converting the inflammatory cytokine pro-IL-1h into its biologically active form (Martinon et al., 2002). It has recently been demonstrated that while caspase-1 is not required for normal developmental apoptosis, it exerts an important role in hypoxic/ischemic-induced neuronal death. Upstream, caspase-1 is regulated by Rip2 and downstream it mediates Bid cleavage to tBid which then facilitates Bax mediated cytochrome c release (Zhang et al., 2003). Finally, certain data have emerged that caspase-12, an inflammatory-like caspase found in the endoplasmic reticulum (ER), may participate as an initiator caspase for ERstress mediated apoptosis (Nakagawa et al., 2000). However, these data remain inconclusive and will require further

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study to establish the exact role of caspase-12 (Lamkanfi et al., 2004). 2.2. Synaptic apoptosis Although caspase activation is generally considered a precursor to rapid cell death, the emerging concept of synaptic apoptosis suggests that apoptotic activation can be localized to synapses or distal neurites without inducing immediate neuronal death (Mattson et al., 1998). Several studies have found that the apoptotic cascade can be activated in synaptosomes (preparations that lack nuclear membranes but are enriched with synaptic elements) by diverse pro-apoptotic stimuli (Mattson and Duan, 1999; Gylys et al., 2002). Furthermore, focal application of glutamate to distal dendrites in vitro can cause a localized increase in caspase-3 activity in synaptic terminals without propagation to the neuronal soma (Mattson et al., 1998). Similarly, h-amyloid applied to distal hippocampal neurites in compartmented cultures produced localized neurite degeneration with apoptotic morphology and positive annexin V binding (a marker for apoptotic activity) without inducing cell death; pretreatment with the caspase inhibitor zVAD-fmk blocked these effects (Ivins et al., 1998). Using in vivo and in vitro models of HIV neurodegeneration, caspase-3 activity was associated with neurite damage without initiating an irreversible apoptotic cascade (Garden et al., 2002). It has been hypothesized that the spread of localized caspase activity is suppressed by neurotrophic factors that are also present in neurite terminals (Mattson and Duan, 1999). Synaptic apoptosis represents a potential mechanism underlying synaptic remodeling and elimination in both physiological and pathological conditions and could also contribute to neuronal plasticity (Gilman and Mattson, 2002; Garden et al., 2002). In Alzheimer’s disease, this form of synaptic loss is thought to have direct clinical relevance by contributing to the early cognitive decline that predates the onset of large-scale neuronal death (Mattson et al., 2001).

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regions did not differ and that the increased density was secondary to reductions in neuropil (Selemon et al., 1995, 1998). Conversely, other studies have demonstrated significant layer-specific reductions in the density of neuronal subpopulations including interneurons in layer II of prefrontal cortex and in layers II – VI of the anterior cingulate cortex (Benes et al., 1991) and pyramidal neurons in layer IV of anterior cingulate cortex (Benes et al., 2001). Furthermore, substantial reductions of neurons have been identified in several subcortical regions including nucleus accumbens (Pakkenberg, 1987; Young et al., 2000; Popken et al., 2000; Byne et al., 2002) but see Cullen et al. (2003) and DorphPetersen et al. (2004). 40% reductions in non-pyramidal neurons have also been found in CA2 of hippocampus (Benes et al., 1998). Thus, while large-scale cortical neuronal loss appears to be absent in schizophrenia, it appears that discrete reductions in cortical neuronal populations may occur with laminar and regional specificity, as well as more substantial reductions in subcortical neuron numbers. While certain older studies indicated that schizophrenia may be associated with cortical gliosis (Stevens, 1982), most recent studies show an absence of cortical gliosis (Roberts et al., 1987; Benes et al., 1991; Purohit et al., 1998; Arnold et al., 1998). Interestingly, a growing number of studies indicate that schizophrenia may in fact be associated with reduced glia. These reports include evidence of fewer oligodendrocytes in prefrontal cortex (Hof et al., 2003), fewer glial cells in prefrontal cortex (Cotter et al., 2002), reduced glial fibrillary acidic protein (GFAP)-area fraction (cell bodies + processes) in prefrontal cortex (Rajkowska et al., 2002), and fewer glial cells in prefrontal, motor, and anterior cingulate cortices (Benes et al., 1986; Stark et al., 2004). Given that glial cells represent a critical source of neurotrophic support for the surrounding axo-dendritic arbors and synapses, a reduction in the number of glial cells may significantly reduce the viability of neurons in schizophrenia. 3.2. Postmortem studies of neuropil and synaptic markers

3. Neuropathology of schizophrenia 3.1. Postmortem studies of neuronal and glial cell numbers Reduced numbers of cortical neurons have not consistently been found in postmortem studies. Using an unbiased stereological approach, total cortical neuronal number was unchanged in subjects with schizophrenia compared to controls (Pakkenberg, 1993). Regionally, investigators report no evidence of neuronal loss in prefrontal cortex (Akbarian et al., 1995a; Thune et al., 2001). Another group found small but significant increases in neuronal density in prefrontal and occipital cortex in schizophrenia compared to controls; however, they concluded that the total number of neurons in these

Several studies have demonstrated increased cortical neuronal density in schizophrenia (Pakkenberg, 1993; Selemon et al., 1995; Selemon et al., 1998). The ‘‘reduced neuropil hypothesis’’ suggests that increased neuronal density may reflect a reduction in cortical neuropil (Selemon and Goldman-Rakic, 1999). Cortical neuropil is primarily composed of axons, dendrites, and the pre- and postsynaptic terminals between them. Supporting a potential reduction of cortical neuropil in schizophrenia is evidence of reduced dendritic spines and total dendritic length of pyramidal neurons (Garey et al., 1998; Glantz and Lewis, 2000; Black et al., 2004), fewer parvalbumin- and GAT-1-immunoreactive varicosities (Pierri et al., 1999; Lewis et al., 2001), reductions of presynaptic marker proteins including synap-

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tophysin (Eastwood and Harrison, 1995; Perrone-Bizzozero et al., 1996; Glantz and Lewis, 1997; Karson et al., 1999) and SNAP-25 (Thompson et al., 1998; Karson et al., 1999); but see (Gabriel et al., 1997). Altered synaptic findings also occur in hippocampus including reduced synaptophysin (Eastwood and Harrison, 1995; Eastwood et al., 1995; Vawter et al., 1999), reduced SNAP-25 (Young et al., 1998) and in temporal cortex in which SNAP-25 is altered (Thompson et al., 1998). These data are also supported by an elegant gene array study that identified reduced expression of multiple genes coding for synaptic gene products (Mirnics et al., 2000). Taken together, these findings suggest that synaptic abnormalities are implicated in the pathophysiology of schizophrenia. 3.3. Neuroimaging studies: evidence of progressive volume changes In schizophrenia, cross-sectional magnetic resonance imaging (MRI) studies have demonstrated evidence of reduced cortical gray matter volume, both globally (Zipursky et al., 1992; Gur et al., 1998; Hulshoff Pol et al., 2002) and in cortical subregions including prefrontal (Gur et al., 1998; Hulshoff Pol et al., 2002), temporal (Schlaepfer et al., 1994; Gur et al., 1998), and parietal (Schlaepfer et al., 1994) areas. This data is generally consistent with the postmortem evidence of reduced cortical neuropil. However, one of the limitations of cross-sectional studies is that these do not adequately address whether a change in brain volume is active or is antecedent to the scan. To address this issue, investigators have increasingly focused on longitudinal structural MRI studies. Interestingly, progressive brain volume changes have been identified in several different patient cohorts including in patients with prodromal signs of psychosis, childhood-onset schizophrenia, and in new-onset schizophrenia. In a single study of prodromal patients judged at high risk to transition to psychosis, a progressive loss of gray matter in certain hippocampal and frontal regions was found only in those patients who later developed psychosis (Pantelis et al., 2003). In childhood-onset schizophrenia, several progressive volume changes have been reported including ventricular enlargement (Rapoport et al., 1997), cortical gray matter loss in frontal and temporal areas (Jacobsen et al., 1998; Rapoport et al., 1999) and cerebellar volume loss (Keller et al., 2003) compared to normal controls. In newonset schizophrenia, progressive ventricular enlargement has been identified by several groups (DeLisi et al., 1997; Lieberman et al., 2001; Cahn et al., 2002), but not others (Gur et al., 1998). Progressive volume loss has been reported in first-episode schizophrenia in cerebral hemispheres bilaterally (DeLisi et al., 1997), total cerebral gray matter (Cahn et al., 2002), frontal cortex (Gur et al., 1998), and in superior temporal gyrus (Kasai et al., 2003), while Lieberman et al. (2001) did not detect cortical volume differences.

3.4. Functional neuroimaging and spectroscopy Reduced metabolic activity in the prefrontal cortex (hypofrontality) has been demonstrated using positron emission tomography (PET) and functional MRI (fMRI) studies in schizophrenia (Weinberger et al., 1986; Andreasen et al., 1997). Studies of phosphorus (31P) magnetic resonance spectroscopy (MRS) in the prefrontal cortex found decreased phosphomonoesters (Pettegrew et al., 1991; Stanley et al., 1995; Hinsberger et al., 1997) and increased phosphodiesters (Pettegrew et al., 1991; Stanley et al., 1995) in first-episode schizophrenia. A profile of reduced phosphomonoesters and increased phosphodiesters is thought to reflect an accelerated breakdown of membrane phospholipids (Pettegrew et al., 1993). In addition, several studies using proton (1H) MRS have reported reduced Nacetylaspartate (NAA) levels in temporal and frontal cortex in schizophrenia (Bertolino et al., 1998; Cecil et al., 1999). NAA is generally considered a measure of neuronal metabolism and reduced NAA levels may therefore reflect reduced neuronal viability.

4. Apoptosis in schizophrenia The data reviewed above indicate that the postmortem neuropathology of schizophrenia is characterized by synaptic and dendritic deficits in cortex and hippocampus, findings that may be related to neuroimaging evidence of reduced gray matter volume. Many (but not all) studies have found reduced neuronal numbers in mediodorsal thalamus while most cortical regions appear to have no overall neuronal reductions. Some evidence suggests that layerspecific reductions of neuronal subpopulations occur in cortex and hippocampus. Increasingly, studies indicate that cortical glial cell numbers are reduced in schizophrenia. Given accumulating MRI evidence for progressive volume changes occurring early in psychosis, the fact that postmortem studies reveal an absence of cortical gliosis, and that apoptotic mechanisms can produce either cell loss or synaptic/dendritic loss, the involvement of an apoptotic mechanism in the pathophysiology of schizophrenia appears increasingly plausible. The following sections will review postmortem studies of apoptosis in schizophrenia, divided into studies of apoptotic regulatory factors and studies of DNA fragmentation. Findings will be considered in relation to the hypothesis that apoptotic mechanisms contribute to the pathophysiology of schizophrenia. 4.1. Apoptotic regulatory proteins The first study to examine the potential role of apoptosis in schizophrenia measured levels of the anti-apoptotic regulatory protein Bcl-2 in postmortem brain tissue (Jarskog et al., 2000). This study found that Bcl-2 was reduced by

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¨25% in middle temporal gyrus in schizophrenia compared to control, Fig 2. Bcl-2 is potently anti-apoptotic, as demonstrated using in vivo and in vitro systems where Bcl-2 overexpression was found to protect against proapoptotic stimuli such as ischemia, growth factor withdrawal and glutamate toxicity (Zhong et al., 1993a, b; Lawrence et al., 1996). Thus, reduced Bcl-2 level may limit the protection that neurons have against pro-apoptotic insults, a potentially important deficit in the pathophysiology of schizophrenia. On the other hand, reduced Bcl-2 in schizophrenia is in contrast to findings in classic neurodegeneration such as Alzheimer’s disease where cortical Bcl-2 expression is upregulated and neuronal apoptosis is accelerated. Higher Bcl-2 levels in classic neurodegeneration are thought to represent a compensatory upregulation in response to the neurodegenerative process (Satou et al., 1995), suggesting that the pathophysiology of schizophrenia is distinct from that of classic neurodegenerative disorders. However, while low Bcl-2 may signal that apoptosis is not active in temporal cortex in chronic schizophrenia, it may alternatively represent a deficit in the ability to mount a compensatory pro-survival response, as suggested by Benes et al. (2003). This may significantly increase the vulnerability of neurons and glia to pro-apoptotic stimuli. Furthermore, as described earlier, Bcl-2 can exert independent neurotrophic properties at the dendrite level as evidenced by enhanced neurite outgrowth and enhanced neuronal regeneration in Bcl-2 overexpressing neurons.

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Fig. 2. (A) Bcl-2 protein levels in middle temporal gyrus (Brodmann’s area 21) in healthy control and schizophrenia subjects, measured by enzymelinked immunoassay (ELISA). Bcl-2 was reduced by 25% in schizophrenia (21.9 T 2.8 Units (U)/mg, mean T S.E., n = 15) as compared to control subjects (29.3 T 2.1 U/mg, n = 15) by Student’s t-test (p < 0.05). (B) Representative Western blot of Bcl-2 protein expression in temporal cortex (30 Ag per lane) in control (lanes 2,3), schizophrenia (lanes 4,5), bipolar disorder (lanes 6,7) and major depression (lanes 8,9) subjects with Jurkat lysate in lane 1 as a positive control for Bcl-2. (Adapted from Jarskog et al., 2000; Reprinted with permission from the Society of Biological Psychiatry).

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This suggests that low Bcl-2 levels in temporal cortex in schizophrenia could contribute to reduced axo-dendritic branching, independent of the apoptotic mechanism. To provide another context within which to understand low Bcl-2 levels in schizophrenia, a follow-up study measured the ratio of Bax to Bcl-2 in the same temporal region. The Bax/Bcl-2 ratio is a key determinant regulating the release of cytochrome c from the mitochondrial membrane and high Bax/Bcl-2 ratios promote cytochrome c release (Oltvai et al., 1993). Interestingly, the Bax/Bcl-2 ratio in schizophrenia was increased by 50% compared to control (Jarskog et al., 2004), Fig 3. A higher mean Bax/Bcl-2 ratio in schizophrenia is similar to several conditions in which neuronal apoptosis is known to be accelerated including in temporal cortex in Down Syndrome (Sawa et al., 1997), in in vitro cultures modeling h-amyloid toxicity (Paradis et al., 1996) and in Bax-overexpressing mouse cortex (Oltvai et al., 1993). Furthermore, given the putative involvement of glutamatergic mechanisms in the pathophysiology of schizophrenia, it is notable that the ratio of Bax to Bcl-XL (another anti-apoptotic member of the Bcl-2 gene family) is substantially increased in neonatal rat cortex following brief Nmethyl-d-aspartate (NMDA) receptor antagonism (Wang et al., 2001). A high Bax/Bcl-2 ratio provides further evidence that neurons and glia in temporal cortex in schizophrenia are more susceptible to pro-apoptotic stimuli. However, in contrast to classic neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases where high caspase-3 levels are found in postmortem brain tissue (Masliah et al., 1998; Hartmann et al., 2000), caspase-3 levels were essentially unchanged and actually trended toward a slight decrease in schizophrenia temporal cortex (Jarskog et al., 2004). Since caspase-3 activation is thought to occur as a final common pathway of apoptosis in the CNS and serves as a useful marker of such activation (Krajewska et al., 1997), this provides relatively unambiguous evidence that apoptosis is, in fact, not active in temporal cortex in chronic schizophrenia. Thus, while it appears that the temporal cortex in schizophrenia may have increased vulnerability to proapoptotic stimuli by virtue of higher Bax/Bcl-2 ratio and an absolute reduction in Bcl-2 levels, it does not appear that apoptotic activity – whether at cellular or synaptic levels –is increased in this brain region in chronic stages of the disorder. This, however, does not preclude a role for apoptosis in earlier stages of the illness. We hypothesize that apoptotic activity could contribute to the evidence for progressive neurostructural changes in prodromal and firstepisode psychosis and that the principal substrate affected by this process is cortical neuropil and that glia and certain neuronal subpopulations may also be affected, as suggested by postmortem data reviewed earlier. It is proposed that caspase-3 and/or other effector caspases could be more susceptible to activation at the onset of psychosis in response to a time-limited increase in pro-apoptotic stimuli (e.g. oxidative stress, glutamate excitotoxicity), especially if

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Fig. 3. Bax/Bcl-2 ratio in middle temporal gyrus (Brodmann’s area 21) in healthy control and schizophrenia subjects. Bax and Bcl-2 levels were determined by semiquantitative Western blot. The mean Bax/Bcl-2 ratio was 50% higher in schizophrenia (1.65 T 0.23, mean T S.E., n = 15) compared to control (1.10 T 0.10, n = 15) subjects ( p < 0.05). (Adapted from Jarskog et al., 2004; reprinted with permission from the American Journal of Psychiatry, Copyright 2004. American Psychiatric Association).

the Bax/Bcl-2 ratio is already increased at this stage. Then, after the period of clinical and neurostructural deterioration, the pro-apoptotic stress attenuates and caspase activity returns to baseline. Given the paucity of available postmortem brain tissue from patients with first-episode psychosis, alternative approaches are needed to test this hypothesis. Possibilities include a longitudinal study measuring apoptotic markers in cerebrospinal fluid in firstepisode psychosis patients as well as using PET imaging for annexin V (a protein that binds membrane-bound phosphatidylserine which is externalized during apoptosis) in this same patient group. Increased apoptotic activity in several neurodegenerative disorders has been suggested by cerebrospinal fluid levels of apoptotic markers (Vermes et al., 1999; Cid et al., 2003) and by annexin V imaging (D’Arceuil et al., 2000; Spence et al., 2003). While the specific mechanisms by which apoptotic activity could be activated in schizophrenia are unknown, potential sources of pro-apoptotic stress in schizophrenia are discussed in the last section. Several possibilities exist for integrating apoptotic mechanisms into the neurodevelopmental hypothesis of schizophrenia. First, the synaptic pruning hypothesis suggests that abnormalities in normal synaptic pruning and related neuromaturational processes such as cortical myelination contribute to schizophrenia (Feinberg, 1982; Weinberger, 1987; Keshavan et al., 1994). Reductions in synaptic density have been demonstrated in human prefrontal cortex during normal adolescence and early adulthood (Huttenlocher, 1979; Huttenlocher and Dabholkar, 1997), overlapping the age of highest incidence of first-episode psychosis. While the physiological basis for normal synaptic pruning is unknown, a potential role for apoptosis is suggested by evidence that apoptosis can be activated locally in synapses and terminal dendrites. The possible roles of apoptosis in the pruning hypothesis of schizophrenia include a contribution to a genetically mediated

acceleration of normal synaptic pruning or to a pathological potentiation of normal pruning by one or more pro-apoptotic stimuli. A second potential overlap between the neurodevelopmental hypothesis of schizophrenia and apoptosis could occur in very early development. A fetal or perinatal insult could transiently alter normal developmental apoptosis to yield enduring cytoarchitectural deficits and altered synaptic connectivity leading to deficits in brain circuitry. Interestingly, a number of pro-apoptotic stimuli such as ischemia, hypoxia, and pro-inflammatory cytokines (Thompson, 1995) have been implicated as early life insults that increase the risk of developing schizophrenia (Geddes and Lawrie, 1995; Gilmore and Jarskog, 1997; Urakubo et al., 2001). Finally, it should be noted that the evidence for progressive neurostructural changes in the early stages of schizophrenia may be due to variables that are not pathophysiologically related to schizophrenia. It has been argued that the progressive neurostructural changes in schizophrenia are probably reversible and represent normal plastic changes in response to confounding factors such as the impact of antipsychotic medication, concomitant substance use, poor health and nutrition and lack of environmental stimulation (Weinberger and McClure, 2002). Further studies are needed to clarify the impact of such factors on brain structure. The potential confounding effects of psychotropic medications and postmortem stability of apoptotic protein levels have been considered. One week of haloperidol did not influence Bax, Bcl-2 or caspase-3 levels in rat frontal cortex (Jarskog et al., 2004); however, another study found that 1 month of olanzapine and clozapine both increase Bcl2 mRNA and protein by 30 –50% in rat frontal cortex (Bai et al., 2004). Overall, these data suggest that antipsychotic treatment does not contribute to low Bcl-2 levels in schizophrenia, although it could be masking even lower Bcl-2 levels. In another study, 1 month of haloperidol, quetiapine and clozapine led to 40– 50% higher activated caspase-3 levels in rat frontal cortex (German et al., 2004). Since caspase-3 levels were not elevated in schizophrenia cortex, this again does not appear to confound the postmortem findings other than possibly mask even lower caspase-3 levels. Finally, high 24-h postmortem stability of Bax, Bcl-2 and caspase-3 proteins has been determined (Jarskog and Gilmore, 2000; Jarskog et al., 2004). Taken together, these data indicate that several apoptotic regulatory proteins are altered in schizophrenia and that the vulnerability to apoptotic activation may be increased as evidenced by high Bax/Bcl-2 ratio and low Bcl-2 levels. Caspase-3 levels are not increased, indicating a distinct contrast with the pathophysiology of classic neurodegenerative disorders. The absence of elevated caspase-3 in postmortem cortex suggests that apoptosis is not active in chronic schizophrenia. However, we hypothesize that caspase-3 and other downstream markers of apoptosis may be upregulated during the period of clinical and neuro-

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structural progression that has been observed around the new onset of psychosis. 4.2. DNA fragmentation A common method for visualizing cells undergoing apoptosis is the TdT-mediated dUTP nick end-labeling (TUNEL) method. Double-stranded DNA breaks (DSBs) are associated with irreversible activation of apoptosis and TUNEL uses in situ hybridization to detect DSBs. TUNELpositive cells in the CNS have been detected in human brain in settings where large-scale neuronal cell death is known to be occurring including in early normal development (Spreafico et al., 1999), in stroke and in Alzheimer’s disease (Adamec et al., 1999). However, given that apoptotic cells are generally cleared rapidly (within 24 h) and do not leave a residue (Hetts, 1998), TUNEL-positive cells may be difficult to detect in conditions where cell death occurs infrequently. Another approach used to study disorders with lower rates of neuronal death is to measure single-stranded DNA breaks (SSBs) using the Klenow technique. SSBs are typically more numerous than DSBs, are potentially reversible and represent a sensitive indicator of cells at risk for undergoing apoptosis (Jin et al., 1999). As reviewed earlier, the anterior cingulate cortex in schizophrenia represents an area where layer-specific reductions in neuronal subtypes have been identified (Benes et al., 1991; Benes et al., 2001). A study using the Klenow method in anterior cingulate cortex found that, compared to matched controls, subjects with schizophrenia but not bipolar disorder had a reduction in a distinct subset of Klenow-positive neurons that exhibited chromatin clumping (Benes et al., 2003). This finding suggests that the regulation of apoptosis is altered in anterior cingulate cortex in schizophrenia, but that the rate of apoptosis may actually be reduced rather than increased. In contrast, preliminary experiments from our laboratory using both the Klenow and TUNEL methods in temporal cortex do not show altered rates in the total number of SSBs or DSBs in schizophrenia or bipolar disorder, suggesting that the rates of DNA fragmentation may vary by brain region (Glantz et al., 2003). Given the absence of increased DNA fragmentation, these data are consistent with the caspase-3 findings suggesting that cortical apoptosis is not increased in chronic schizophrenia. Benes et al. (2003) proposed that their Klenow findings might either represent a compensatory response to promote cell survival or, alternatively, a failure of cingulate neurons to mount an appropriate apoptotic response to an oxidative challenge. If one posits a baseline increase in pro-apoptotic stimuli (e.g. oxidative stress) in cortical brain regions in schizophrenia, then the evidence for low Bcl-2 and low/ normal caspase-3 levels could be consistent with a failure of apoptotic signaling in response to pro-apoptotic stress. Alternatively, if pro-apoptotic stress is transiently increased around the onset of psychosis and largely absent in

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postmortem tissue (representing patients with chronic schizophrenia), then low/normal caspase-3 levels would represent an appropriate compensatory response and that low Bcl-2 and high Bax/Bcl-2 ratio may reflect a footprint of earlier pro-apoptotic stress. 4.3. Pro-apoptotic stress in schizophrenia While many factors have been found to promote apoptosis, several pro-apoptotic stimuli have also been suggested to play a role in the pathophysiology of schizophrenia including glutamatergic excitotoxicity, excess synaptic calcium flux, oxidative stress, and reduced neurotrophin levels. Data to support the involvement of these stimuli and pathological processes in schizophrenia and their potential relationship to apoptosis will be reviewed. NMDA hypofunction, glutamate excitotoxicity and altered calcium signaling have all been implicated in the pathophysiology of schizophrenia (Javitt and Zukin, 1991; Olney and Farber, 1995; Goff and Coyle, 2001). High calcium levels, oxidative stress, and mitochondrial dysfunction can all lead to glutamate excitoxicity and each can also promote apoptotic activity (Mattson and Duan, 1999). Even very brief NMDA receptor blockade during vulnerable intervals during rat development can greatly augment normal developmental neuronal apoptosis (Ikonomidou et al., 1999). As mentioned earlier, NMDA antagonism can also produce long-term changes in Bcl-2 gene family expression including higher pro-apoptotic Bax and lower anti-apoptotic Bcl-XL as well as increase expression of the NMDA NR1 receptor subunit (Wang et al., 2001). Furthermore, altered expression of the NMDA NR1 and NR2A receptor subunits can increase the apoptotic vulnerability of neurons (Anegawa et al., 2000). Emerging data implicate changes in these NMDA subunits and others in the postmortem neuropathology of schizophrenia (MeadorWoodruff and Healy, 2000). Of particular interest, altered editing of GluR2 mRNA of the AMPA receptor– as has been identified in schizophrenia cortex (Akbarian et al., 1995b)– has been associated with significantly higher calcium flux. Such flux can lead to neuronal atrophy and apoptosis (Mattson et al., 1998; Segal et al., 2000). Thus, while the implications of glutamatergic dysfunction in schizophrenia remain uncertain, it presents as a candidate mechanism that can adversely impact neuroapototic processes. In particular, the susceptibility of cortical neurons to NMDA antagonists during neurodevelopment suggests a plausible scenario for a transient activation of apoptosis that could exert limited neurostructural effects early in the course of schizophrenia. Oxidative stress is known to cause neuronal apoptosis and has been hypothesized to contribute to the pathophysiology of schizophrenia (Mahadik et al., 2001). Serum and red blood cell markers of antioxidant defense enzymes, including superoxide dismutase, are lower in patients with first-episode and chronic schizophrenia, suggesting an inherent vulnerability to oxidative stress

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(Mukerjee et al., 1996). Other markers of increased oxidative stress in schizophrenia include increased membrane phospholipid turnover as evidenced by increased phospholipase A2 activity (Horrobin, 1998) and increased cortical phosphodiester levels by 31P MRS (Pettegrew et al., 1993). While the data on oxidative stress may be more suggestive of a chronic increase in schizophrenia, many of these are either peripheral markers or relatively non-specific in vivo neuroimaging markers. It remains possible that in the CNS, exposure to oxidative stress is either time-limited or that effective compensatory mechanisms eventually limit the effect of chronic stress, thereby creating a transient pro-apoptotic environment in the early stages of schizophrenia. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are critical to neuronal survival and maturation as well as to promoting synapse formation (McAllister, 2001), and their withdrawals are among the best studied triggers of neuronal apoptosis (Thompson, 1995). Recent studies have documented reduced levels of BDNF mRNA and protein in prefrontal cortex in schizophrenia (Hashimoto et al., 2002; Weickert et al., 2003) as well as concomitant reductions in the BDNF TrkB mRNA receptor (Hashimoto et al., 2002). While the basis for lower BDNF levels in schizophrenia is uncertain, the evidence for fewer cortical glial cells in schizophrenia may be a contributing factor, given that glia represent an important source of neurotrophins for CNS neurons. Reduced neurotrophic support represents another potential source of pro-apoptotic stress in schizophrenia.

5. Conclusion Taken together, the neuropathology of schizophrenia demonstrates reduced neuropil (including dendrites and synaptic markers) in specific cortical regions and in hippocampus. There is also evidence of layer-specific reductions of neuronal subtypes in specific cortical regions, fewer neurons in the mediodorsal thalamus and evidence for reduced cortical glial cell numbers. Postmortem data are beginning to implicate apoptotic dysregulation in the pathophysiology of schizophrenia. Specifically, levels of upstream apoptotic regulatory proteins appear to increase the vulnerability to apoptotic stimuli. On the other hand, downstream caspase-3 protein is not increased, suggesting that excess apoptotic cell death is not occurring in chronic schizophrenia. Given the neuroimaging evidence for gray matter loss in the early stages of psychosis, it is hypothesized that apoptotic processes such as synaptic apoptosis and regional and layer-specific neuronal and/or glial apoptosis occur during this phase of the illness, but that a subsequent compensatory downregulation of apoptosis occurs as illness moves into a stable chronic stage. Further studies are

needed to test this hypothesis and document that proapoptotic proteins such as caspase-3 are increased in the CNS during the early phase of the illness. While the source of pro-apoptotic stress remains uncertain, a growing body of literature suggests that glutamate excitotoxicity, oxidative stress, reduced BDNF levels and increased synaptic calcium flux contribute to the pathophysiology of schizophrenia, and that these factors, individually or in concert, can create a pro-apoptotic environment. The current review has demonstrated that apoptotic markers are altered in schizophrenia and that apoptotic mechanisms provide a plausible explanation for several neuropathological deficits associated with the disorder.

Acknowledgements This work was funded by NIMH grants MH-01752 (LFJ), MH-064065 (JAL), NARSAD Young Investigator Award (LAG).

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