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The glutamate hypothesis of schizophrenia suggests that altered glutamatergic transmission occurs in this illness, although precise mechanisms of dysregulation remain elusive. AMPA receptors (AMPARs), a subtype of ionotropic glutamate receptor, are the main facilitators of fast, excitatory neurotransmission in the brain, and changes in AMPAR number or composition at synapses can regulate synaptic strength and plasticity. Prior evidence of abnormal expression of transmembrane AMPAR regulatory proteins (TARPs) in schizophrenia suggests defective trafficking of AMPARs, which we propose could lead to altered AMPAR expression at excitatory synapses. To test this hypothesis, we isolated subcellular fractions enriched for endoplasmic reticulum (ER) and synapses from anterior cingulate cortex (ACC) from schizophrenia (N = 18) and comparison (N = 18) subjects, and measured glutamate receptor subunits (GluA1, GluA2, GluA3, GluA4, NR1, NR2A, NR2B, and NR3A) and TARP member γ2 (stargazin) in homogenates and subcellular fractions by western blot analysis. We found decreased expression of stargazin and an increased ratio of GluA2:stargazin in ACC homogenates, while in the synapse fraction we identified a decrease in GluA1 and reduced ratios of GluA1:stargazin and GluA1:GluA2 in schizophrenia. The amount of stargazin in the ER fraction was not different, but the relative amount of ER/Total stargazin was increased in schizophrenia. Together, these findings suggest that associations between stargazin and AMPA subunits are abnormal, potentially affecting forward trafficking or synaptic stability of GluA1-containing AMPARs. These data provide evidence that altered interactions with trafficking proteins may contribute to glutamate dysregulation in schizophrenia., Competing Interests: Declaration of competing interest The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no conflicts of interest to disclose., (Copyright © 2020. Published by Elsevier B.V.)

Though the pathophysiology of schizophrenia remains poorly understood, altered brain energy metabolism is increasingly implicated. Here, we conduct meta-analyses of the available human studies measuring lactate or pH in schizophrenia brain and discuss the accumulating evidence for increased lactate and decreased pH in schizophrenia brain and evidence linking these to negative and cognitive symptom severity. Meta-analysis of six postmortem studies revealed a significant increase in lactate in schizophrenia brain while meta-analysis of 14 magnetic resonance spectroscopy studies did not reveal a significant change in brain pH in schizophrenia. However, only five of these studies were likely sufficiently powered to detect differences in brain pH, and meta-analysis of these five studies found a nonsignificant decrease in pH in schizophrenia brain. Next, we discuss evidence for altered brain energy metabolism in schizophrenia and how this may underlie a buildup of lactate and decreased pH. This alteration, similar to the Warburg effect extensively described in cancer biology, involves diminished tricarboxylic acid cycle and oxidative phosphorylation along with a shift toward increased reliance on glycolysis for energy production. We then explore the role that mitochondrial dysfunction, oxidative stress, and hypoxia-related changes in gene expression likely play in this shift in brain energy metabolism and address the functional consequences of lowered brain pH in schizophrenia including alterations in neurotransmitter regulation, mRNA stability, and overall patterns of gene expression. Finally, we discuss how altered energy metabolism in schizophrenia brain may serve as an effective target in the treatment of this illness., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Abnormalities in posttranslational protein modifications (PTMs) that regulate protein targeting, trafficking, synthesis, and function have been implicated in the pathophysiology of schizophrenia. The endoplasmic reticulum (ER) contains specialized machinery that facilitate protein synthesis, ER entry and exit, quality control, and post-translational processing, steps required for protein maturation. Dysregulation of these systems could represent potential mechanisms for abnormalities of neurotransmitter associated proteins in schizophrenia. We hypothesized that expression of ER processing pathways is dysregulated in schizophrenia. We characterized protein and complex expression of essential components from protein folding, ER quality control (ERQC), and ER associated degradation (ERAD) processes in the dorsolateral prefrontal cortex of 12 matched pairs of elderly schizophrenia and comparison subjects. We found increased expression of proteins associated with recognizing and modifying misfolded proteins, including UDP-glucose/glycoprotein glucosyltransferase 2 (UGGT2), ER degradation enhancing alpha-mannosidase like protein 2 (EDEM2), and synoviolin (SYVN1)/HRD1. As SYVN1/HRD1 is a component of the ubiquitin ligase HRD1-SEL1L complex that facilitates ERAD, we immunoprecipitated SEL1L and measured expression of other proteins in this complex. In schizophrenia, SYVN1/HRD1 and OS-9, ERAD promoters, have increased association with SEL1L, while XTP3-B, which can prevent ERAD of substrates, has decreased association. Abnormal expression of proteins associated with ERQC and ERAD suggests dysregulation in ER localized protein processing pathways in schizophrenia. Interestingly, the deficits we found are not in the protein processing machinery itself, but in proteins that recognize and target incompletely or misfolded proteins. These changes may reflect potential mechanisms of abnormal neurotransmitter associated protein expression previously observed in schizophrenia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glycosylation is a post-translational modification that is an essential element in cell signaling and neurodevelopmental pathway regulation. Glycan attachment can influence the tertiary structure and molecular interactions of glycosylated substrates, adding an additional layer of regulatory complexity to functional mechanisms underlying central cell biological processes. One type of enzyme-mediated glycan attachment, fucosylation, can mediate glycoprotein and glycolipid cell surface expression, trafficking, secretion, and quality control to modulate a variety of inter- and intracellular signaling cascades. Building on prior reports of glycosylation abnormalities and evidence of dysregulated glycosylation enzyme expression in schizophrenia, we examined the protein expression of 5 key fucose-modifying enzymes: GDP-fucose:protein O-fucosyltransferase 1 (POFUT1), GDP-fucose:protein O-fucosyltransferase 2 (POFUT2), fucosyltransferase 8 (FUT8), fucosyltransferase 11 (FUT11), and plasma α-l-fucosidase (FUCA2) in postmortem superior temporal gyrus of schizophrenia (N=16) and comparison (N=14) subjects. We also used the fucose binding protein, Aleuria aurantia lectin (AAL), to assess α-1,6-fucosylated N-glycoprotein abundance in the same subjects. In schizophrenia, we found increased expression of POFUT2, a fucosyltransferase uniquely responsible for O-fucosylation of thrombospondin-like repeat domains that is involved in a non-canonical endoplasmic reticulum quality control pathway. We also found decreased expression of FUT8 in schizophrenia. Given that FUT8 is the only α-1,6-fucosyltransferase expressed in mammals, the concurrent decrease in AAL binding in schizophrenia, particularly evident for N-glycoproteins in the ~52-58kDa and ~60-70kDa molecular mass ranges, likely reflects a consequence of abnormal FUT8 expression in the disorder. Dysregulated FUT8 and POFUT2 expression could potentially explain a variety of molecular abnormalities in schizophrenia., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent reports suggest abnormalities of neurotransmitter receptor trafficking, targeting, dendritic localization, recycling, and degradation in the brain in schizophrenia. We hypothesized that a potential explanation for these findings may be abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of protein palmitoylation is a strong candidate for such a process. S-palmitoylation is a reversible thioesterification of palmitoyl-groups to cysteine residues that can regulate trafficking and targeting of intracellular proteins. Using a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain, we identified a pattern of palmitoylated proteins that cluster into 17 bands of discrete molecular masses, including numerous proteins associated with receptor signal transduction. Using mass spectrometry, we identified 219 palmitoylated proteins in human frontal cortex, and individually validated palmitoylation status of a subset of these proteins. Next, we assayed protein palmitoylation in dorsolateral prefrontal cortex from 16 schizophrenia patients and paired comparison subjects. S-palmitoylation was significantly reduced for proteins in most of the 17 schizophrenia bands. In rats chronically treated with haloperidol, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that the diminution in protein palmitoylation in schizophrenia is not due to chronic antipsychotic treatment. These results indicate there are changes in the extent of S-palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification, these data suggest a potential mechanism reconciling previous observations of abnormal intracellular targeting and trafficking of neurotransmitter receptors in this illness., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Changes in the extent of the posttranslational modification glycosylation have been previously reported in several brain regions in schizophrenia. Quality control within the endoplasmic reticulum and Golgi, branching of glycans, intracellular trafficking and targeting, protein-protein interactions, and endocytosis are processes regulated by both N-linked and O-linked glycosylation. Previous studies in schizophrenia have found altered glycan biosynthesis and abnormal glycan levels in cerebrospinal fluid (CSF) and plasma, as well as altered expression in frontal cortex of glycosyltransferase transcripts encoding proteins associated with both N- and O-linked glycosylation. The N-acetylglucosaminyltransferases (GlcNAcTs) are glycosylating enzymes that play a key role in adding N-acetylglucosamine (GlcNAc) to substrates to facilitate their proper trafficking, intracellular targeting, and cellular function. Given previous results indicating abnormal glycosylation in schizophrenia, we hypothesized that these GlcNAcTs may be abnormally expressed in this illness. We measured protein expression of nine distinct GlcNAcTs by Western blot analysis in postmortem samples of dorsolateral prefrontal cortex (DLPFC) from twelve pairs of elderly patients with schizophrenia and comparison subjects. We found decreased protein expression of UDP-GlcNAc:BetaGal Beta-1,3 GlcNAcT 8 (B3GNT8) and mannosyl (alpha-1,3-)-glycoprotein beta-1,4 GlcNAcT (MGAT4A) expression in schizophrenia. These data provide further evidence that glycosylation is dysregulated in schizophrenia, and suggest a potential mechanism associated with alterations in protein function, trafficking, and intracellular targeting in this illness., (Published by Elsevier B.V.)

Background: Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand-bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that the expression of GRK proteins is altered in schizophrenia, consistent with previous findings of alterations upstream and downstream from this family of molecules that facilitate intracellular signaling processes., Methods: In this study, we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (n=36) and a comparison group (n=33). To control for antipsychotic treatment, we measured these same targets in haloperidol-treated vs. untreated rats (n=10 for both)., Results: We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months., Conclusion: These data suggest that increased GRK5 expression may contribute to the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Excitatory amino acid transporter 2 (EAAT2) belongs to a family of Na(+) dependent glutamate transporters that maintain a low synaptic concentration of glutamate by removing glutamate from the synaptic cleft into astroglia and neurons. EAAT2 activity depends on Na(+) and K(+) gradients generated by Na(+)/K(+) ATPase and ATP. Hexokinase 1 (HK1), an initial enzyme of glycolysis, binds to mitochondrial outer membrane where it couples cytosolic glycolysis to mitochondrial oxidative phosphorylation, producing ATP utilized by the EAAT2/Na(+)/K(+) ATPase protein complex to facilitate glutamate reuptake. In this study, we hypothesized that the protein complex formed by EAAT2, Na(+)/K(+) ATPase and mitochondrial proteins in human postmortem prefrontal cortex may be disrupted, leading to abnormal glutamate transmission in schizophrenia. We first determined that EAAT2, Na(+)/K(+) ATPase, HK1 and aconitase were found in both EAAT2 and Na(+)/K(+) ATPase interactomes by immunoisolation and mass spectrometry in human postmortem prefrontal cortex. Next, we measured levels of glutamate transport complex proteins in subcellular fractions in the dorsolateral prefrontal cortex and found increases in the EAAT2B isoform of EAAT2 in a fraction containing extrasynaptic membranes and increased aconitase 1 in a mitochondrial fraction. Finally, an increased ratio of HK1 protein in the extrasynaptic membrane/mitochondrial fraction was found in subjects with schizophrenia, suggesting that HK1 protein is abnormally partitioned in this illness. Our findings indicate that the integrity of the glutamate transport protein complex may be disrupted, leading to decreased perisynaptic buffering and reuptake of glutamate, as well as impaired energy metabolism in schizophrenia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Abnormal synaptic plasticity has been implicated in the cognitive deficits seen in schizophrenia, where alterations have been found in neurotransmission, signaling and dendritic dynamics. Rapid rearrangement of the actin cytoskeleton is critical for plasticity and abnormalities of molecular regulators of this process are candidates for understanding mechanisms underlying these changes in schizophrenia. The myristoylated, alanine-rich C-kinase substrate (MARCKS) is crucial for many roles associated with synaptic plasticity, including facilitation of neurotransmission, dendritic branching and in turn cognitive function. Accordingly, we hypothesized that this protein is abnormally expressed or regulated in schizophrenia. We measured protein expression of MARCKS by Western blot analysis in postmortem samples of dorsolateral prefrontal cortex (DLPFC) from elderly schizophrenia patients (N=16) and a comparison group (N=20). We also assayed phosphorylated-MARCKS (pMARCKS), given the role of phosphorylation in reversing membrane association by MARCKS. We found decreased expression of both MARCKS and pMARCKS in schizophrenia. Altered myristoylation may be a mechanism that explains this down-regulation of MARCKS, so we also assayed expression of the two isoforms of the key myristoylation enzyme, NMT, and an enzymatic inhibitor of this enzyme, NMT-inhibitor protein (NIP71) by Western blotting in these same subjects. Expression did not change between groups for these proteins, suggesting a mechanism other than myristoylation is responsible for decreased MARCKS expression in schizophrenia. These data suggest a potential mechanism underlying aspects of altered synaptic plasticity observed in schizophrenia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The glutamate hypothesis of schizophrenia proposes that abnormal glutamatergic neurotransmission occurs in this illness, and a major contribution may involve dysregulation of the AMPA subtype of ionotropic glutamate receptor (AMPAR). Transmembrane AMPAR regulatory proteins (TARPs) form direct associations with AMPARs to modulate the trafficking and biophysical functions of these receptors, and their dysregulation may alter the localization and activity of AMPARs, thus having a potential role in the pathophysiology of schizophrenia. We performed comparative quantitative real-time PCR and Western blot analysis to measure transcript (schizophrenia, N=25; comparison subjects, N=25) and protein (schizophrenia, N=36; comparison subjects, N=33) expression of TARPs (γ subunits 1-8) in the anterior cingulate cortex (ACC) in schizophrenia and a comparison group. TARP expression was also measured in frontal cortex of rats chronically treated with haloperidol decanoate (28.5mg/kg every three weeks for nine months) to determine the effect of antipsychotic treatment on the expression of these molecules. We found decreased transcript expression of TARP γ-8 in schizophrenia. At the protein level, γ-3 and γ-5 were increased, while γ-4, γ-7 and γ-8 were decreased in schizophrenia. No changes in any of the molecules were noted in the frontal cortex of haloperidol-treated rats. TARPs are abnormally expressed at transcript and protein levels in ACC in schizophrenia, and these changes are likely due to the illness and not to the antipsychotic treatment. Alterations in the expression of TARPs may contribute to the pathophysiology of schizophrenia, and represent a potential mechanism of glutamatergic dysregulation in this illness., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Numerous studies have demonstrated brain region- and subunit-specific abnormalities in the expression of subunits of the AMPA subtype of glutamate receptors in schizophrenia. In addition, abnormalities in the expression of proteins that regulate the forward trafficking of AMPA receptors through the cell have been reported. These findings suggest abnormal trafficking of AMPA receptors as a mechanism underlying dysregulated glutamate neurotransmission in schizophrenia. AMPA receptor subunits (GluR1-4) assemble to form AMPA receptor complexes in the lumen of the endoplasmic reticulum (ER). These subunits undergo the posttranslational modification of N-linked glycosylation in the ER and the Golgi apparatus before the assembled receptors are transported to the plasma membrane. In this study, we measured expression of AMPA receptors and the extent of their N-glycosylation using Western blot analysis in the dorsolateral prefrontal cortex in subjects with schizophrenia (N = 35) and a comparison group (N = 31). N-glycosylation was assessed using molecular mass shift assays following digestion with endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and with peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. Of the four AMPA receptor subunits, only GluR4 was significantly increased in schizophrenia. GluR2 and GluR4 were both sensitive to Endo H and PNGase F treatment. Endo H-mediated deglycosylation of GluR2 resulted in a significantly smaller pool of GluR2 protein to shift in schizophrenia, reflecting less N-linked high mannose and/or hybrid sugars on the GluR2 protein in this illness. This was confirmed by immunoisolation of GluR2 and probing with Concanavalin A, a mannose specific lectin; in subjects with schizophrenia GluR2 was significantly less reactive to Concanavalin A. Altered N-linked glycosylation of the GluR2 subunit in schizophrenia suggests abnormal trafficking of AMPA receptors from the ER to the synaptic membrane in schizophrenia., (Published by Elsevier B.V.)

Glutamate transporters facilitate the buffering, clearance and cycling of glutamate and play an important role in maintaining synaptic and extrasynaptic glutamate levels. Alterations in glutamate transporter expression may lead to abnormal glutamate neurotransmission contributing to the pathophysiology of schizophrenia. In addition, alterations in the architecture of the superior temporal gyrus and hippocampus have been implicated in this illness, suggesting that synapses in these regions may be remodeled from a lifetime of severe mental illness and antipsychotic treatment. Thus, we hypothesize that glutamate neurotransmission may be abnormal in the superior temporal gyrus and hippocampus in schizophrenia. To test this hypothesis, we examined protein expression of excitatory amino acid transporter 1-3 and vesicular glutamate transporter 1 and 2 in subjects with schizophrenia (n=23) and a comparison group (n=27). We found decreased expression of EAAT1 and EAAT2 protein in the superior temporal gyrus, and decreased EAAT2 protein in the hippocampus in schizophrenia. We didn't find any changes in expression of the neuronal transporter EAAT3 or the presynaptic vesicular glutamate transporters VGLUT1-2. In addition, we did not detect an effect of antipsychotic medication on expression of EAAT1 and EAAT2 proteins in the temporal association cortex or hippocampus in rats treated with haloperidol for 9 months. Our findings suggest that buffering and reuptake, but not presynaptic release, of glutamate is altered in glutamate synapses in the temporal lobe in schizophrenia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A growing body of evidence suggests that abnormal elements of the cytoskeleton may be associated with the pathophysiology of schizophrenia. Isoforms of a major cytoskeleton protein, β-tubulin, were recently demonstrated to have distinct roles in neuronal differentiation and cell viability. For these reasons, we tested the hypothesis that there are differences in the expression of β-tubulin isoforms (βI-βIV) in the brain in schizophrenia, using western blot analysis in an elderly group of subjects with this illness and a control group. We found that βI-tubulin protein expression was decreased in the anterior cingulate cortex and increased in the dorsolateral prefrontal cortex, but not changed in superior temporal gyrus or hippocampus in schizophrenia. Our data supports the growing body of evidence suggesting abnormalities of the cytoskeleton in schizophrenia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have implicated alterations in the endosomal trafficking of AMPA receptors in this illness. We hypothesized that late endosome content of AMPA receptor subunits is altered in schizophrenia. Accordingly, we developed a technique to isolate and measure contents of late endosomes from postmortem human tissue. We found no changes in the expression of the AMPA subunits, GluR1-4, in late endosomes from the dorsolateral prefrontal cortex in schizophrenia. We also hypothesized that proteins involved in the sorting and trafficking of AMPA receptors between endosomal compartments would be altered in schizophrenia. We found no changes in expression of multiple proteins associated with these processes (dynamin3, Arc/ARG3.1, NEEP21, GRASP1, liprin α, and syntaxin13). Together, these data suggest that endosomal trafficking of AMPA receptors in the prefrontal cortex may be largely intact in schizophrenia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dysregulated glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. In particular, hypofunction of the NMDA glutamate receptor has been proposed to play an important role in mediating cognitive deficits in patients. The two NMDA receptor subunits, NR2A and NR2B, are distinctly regulated during development and are associated with different intracellular pathways and functions, which suggest that these receptors play separate roles in the control of higher cognitive functions such as learning and memory. Trafficking of the NR2B subunit-containing receptor is regulated by a microtubule-associated trafficking complex consisting of the KIF17, APBA1, CASK, and mLin7 proteins. Several studies have demonstrated an integrated functional regulation of this trafficking complex with NR2B receptor subunit expression, which in turn has been linked to higher cognitive functions. In the present work, we investigated whether expression of this NR2B-associated trafficking complex might be abnormal in schizophrenia. We analyzed the expression of KIF17, APBA1, CASK, mLin7A and mLin7C in postmortem brain from patients with schizophrenia a comparison group. Analysis of transcripts for all of these proteins revealed particularly prominent expression in cortical layer III and layer IV, which overlapped with NR2B but not NR2A transcripts. We found altered expression of transcripts for the CASK, ABPA1, and mLin7 molecules and the CASK, mLin7 proteins, suggesting that NR2B-containing NMDA receptor transport could be selectively compromised in schizophrenia, and that these changes likely involve altered NR2B function in a subset of cortical neurons., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The excitatory amino acid transporters (EAATs) are a family of molecules that are essential for regulation of synaptic glutamate levels. The EAATs may also be regulated by N-glycosylation, a posttranslational modification that is critical for many cellular functions including localization in the plasma membrane. We hypothesized that glycosylation of the EAATs is abnormal in schizophrenia. To test this hypothesis, we treated postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortices of patients with schizophrenia and comparison subjects with deglycosylating enzymes. We then measured the resulting shifts in molecular weight of the EAATs using Western blot analysis to determine the mass of glycans cleaved from the transporter. We found evidence for less glycosylation of both EAAT1 and EAAT2 in schizophrenia. We did not detect N-linked glycosylation of EAAT3 in either schizophrenia or the comparison subjects in these regions. Our data suggest an abnormality of posttranslational modification of glutamate transporters in schizophrenia that suggests a decreased capacity for glutamate reuptake., ((c) 2009 Elsevier B.V. All rights reserved.)

Glutamate cycling is critically important for neurotransmission, and may be altered in schizophrenia. The excitatory amino acid transporters (EAATs) facilitate the reuptake of glutamate from the synaptic cleft and have a key role in glutamate cycling. We hypothesized that expression of the EAATs and the EAAT regulating proteins ARHGEF11, JWA, G-protein suppressor pathway 1 (GPS1), and KIAA0302 are altered in the brain in schizophrenia. To test this, we measured expression of EAAT1, EAAT2, EAAT3, and EAAT interacting proteins in postmortem tissue from the dorsolateral prefrontal and anterior cingulate cortex of patients with schizophrenia and a comparison group using in situ hybridization and Western blot analysis. We found increased EAAT1 transcripts and decreased protein expression, increased EAAT3 transcripts and protein, and elevated protein expression of both GPS1 and KIAA0302 protein. We did not find any changes in expression of EAAT2. These data indicate that proteins involved in glutamate reuptake and cycling are altered in the cortex in schizophrenia, and may provide potential targets for future treatment strategies.

Altered expression of structural and functional molecules expressed by astrocytes may play a role in the pathophysiology of schizophrenia. We investigated the hypothesis that the astrocytic enzyme glutamine synthetase, involved in maintaining the glutamate-glutamine cycle, and the cytoskeletal molecule glial fibrillary acidic protein (GFAP) are abnormally expressed in schizophrenia. We used Western blot analysis to measure levels of glutamine synthetase and GFAP in several brain regions of subjects with schizophrenia and a comparison group. We found that glutamine synthetase protein expression was significantly decreased in the superior temporal gyrus, and both glutamine synthetase and GFAP were significantly reduced in the anterior cingulate cortex in schizophrenia. Neither molecule demonstrated altered expression in the dorsolateral prefrontal cortex, primary visual cortex, or hippocampus. Chronic treatment with haloperidol did not alter the expression of these molecules in the rat brain, suggesting that our findings are not due to a medication effect. These data support an astrocytic component to the pathophysiology of schizophrenia and suggest that astrocytic molecules involved in enzymatic activity and cytoskeletal integrity may have a role in disease-related abnormalities in this illness.

Several recent studies have found changes in the expression of genes functionally related to myelination and oligodendrocyte homeostasis in schizophrenia. These studies utilized microarrays and quantitative PCR (QPCR), methodologies which do not permit direct, unamplified examination of mRNA expression. In addition, these studies generally only examined transcript expression in homogenates of gray matter. In the present study, we examined the expression of myelination-related genes previously implicated in schizophrenia by microarray or QPCR. Using in situ hybridization, we measured transcript expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), transferrin (TF), quaking (QKI), gelsolin, myelin oligodendrocyte glycoprotein, v-erb-b2 erythroblastic leukemia viral oncogene homolog 3, erbb2 interacting protein, motility-related protein-1, SRY-box containing gene 10, oligodendrocyte transcription factor 2, peripheral myelin protein 22, and claudin-11 in both gray and white matter of the anterior cingulate cortex (ACC) in subjects with schizophrenia (n=41) and a comparison group (n=34). We found decreased expression of MAG, QKI, TF, and CNP transcripts in white matter. We did not find any differences in expression of these transcripts between medicated (n=31) and unmedicated (n=10) schizophrenics, suggesting that these changes are not secondary to treatment with antipsychotics. Finally, we found significant positive correlations between QKI and MAG or CNP mRNA expression, suggesting that the transcription factor QKI regulates MAG and CNP expression. Our results support the hypothesis that myelination and oligodendrocyte function are impaired in schizophrenia.

As ceruloplasmin and copper abnormalities have been implicated in schizophrenia, we investigated the role of a second copper-containing non-ceruloplasmin protein, the iron oxidase ferroxidase II, in a prospective study of ten inpatients with schizophrenia and a comparison group. Ferroxidase II is a protein known to reciprocally regulate with ceruloplasmin in Wilson's disease, an illness characterized by psychotic symptoms, decreased ceruloplasmin, and increased copper deposition in tissues. Ferroxidase II plays a key role in the maintenance of near-normal iron metabolism in Wilson's disease, but its role in schizophrenia has never been studied. In this study, we assayed ceruloplasmin by two enzymatic assays, a standard clinical laboratory p-phenylenediamine oxidation assay and a second assay based on the rate of the oxidation and incorporation of iron (Fe3+) into transferrin; we assayed ferroxidase II activity using this second iron oxidation assay. We found that ceruloplasmin levels as measured by both enzymatic methods, but not ferroxidase II, were elevated in schizophrenia. The increased ceruloplasmin also correlated with elevated serum copper as assayed by atomic absorption spectrophotometry, which was unsurprising as the majority of copper in blood is bound to ceruloplasmin. It has been proposed that copper, as a component of several enzymes linked to dopamine synthesis, may play a role in schizophrenia by exacerbating or perpetuating dopaminergic dysregulation. Our study suggests that the ceruloplasmin elevation in schizophrenia is specific, and not simply an elevation of plasma copper-containing oxidative enzymes. Increases in ceruloplasmin may result in increased levels of copper, which ultimately proves deleterious in schizophrenia.

Previous studies have described abnormal expression of molecules involved in glutamatergic signaling in psychiatric illnesses, including proteins associated with receptor signaling complexes in the postsynaptic density (PSD). In particular the N-methyl-d-aspartate (NMDA) receptor complex has been associated with these illnesses. Several subcortical structures including the striatum are innervated by direct glutamatergic projections from the prefrontal cortex, and these connections may be affected in severe psychiatric illnesses. Abnormal expression of molecules critical for glutamatergic signaling in subcortical structures may thus be associated with the pathophysiology of severe psychiatric illnesses. In the present study postmortem tissue from patients with schizophrenia, bipolar disorder and major depression was used to examine striatal expression of transcripts encoding NMDA receptor interacting proteins of the PSD required for trafficking, membrane targeting and synaptic function of this receptor. We found decreased striatal expression of transcripts encoding PSD-95 and SAP-102 in bipolar disorder and of SAP-102 in major depression and schizophrenia, while no significant changes in NF--L and PSD-93 mRNAs were observed. Abnormal expression of SAP-102 in schizophrenia and SAP-102 and PSD-95 in mood disorders in subcortical structures receiving afferent glutamatergic innervation from frontal cortex suggests dysregulation of cortical-subcortical circuitry in these illnesses.

Numerous molecules enable the handling of glutamate that is destined for neurotransmitter release, including transporters, receptors and glutamatergic enzymes. Previous work in our lab has shown altered levels of transcript expression of excitatory amino acid transporters and a vesicular glutamate transporter in the thalamus in schizophrenia. These changes suggest that molecules that facilitate the release and reuptake of glutamate may be abnormal in schizophrenia. In this study we determined the levels of expression of phosphate activated glutaminase (PAG), which converts glutamine to glutamate, and glutamine synthetase (GS), which converts glutamate to glutamine, with the hypothesis that thalamic PAG and GS transcript expression is altered in schizophrenia. We investigated expression of PAG and GS mRNA using in situ hybridization in six different thalamic nuclei (anterior, dorsomedial, centromedial, ventral anterior, ventral and reticular) from 13 persons with schizophrenia and 8 comparison subjects and found that transcripts for PAG and GS were significantly increased in schizophrenia. Increased PAG and GS transcripts suggest enhanced glutamatergic neurotransmission in the thalamus and its efferent targets in schizophrenia.

Growing evidence suggests that NMDA receptor (NMDAR) dysfunction may be involved in schizophrenia. The NMDAR is a multimeric assembly derived from seven different genes (NR1, NR2A-2D and NR3A-3B). While region-specific changes in the expression of most NMDAR subunits have been reported in schizophrenia, possible abnormalities of NR3A expression have not been investigated. Both electrophysiological and anatomical data in rodents, however, suggest that NR3A subunits could play a role in this disorder. In this study, we measured NR3A transcript levels in the dorsolateral prefrontal cortex (DLPFC) and inferior temporal neocortex in the brains of people with schizophrenia, bipolar disorder, depression, and a comparison group. This transcript was elevated by 32% in schizophrenia relative to controls, but only in the DLPFC and not inferior temporal cortical regions. Interestingly, this effect was restricted to gyral aspects of the DLPFC and did not involve sulcal areas. NR3A mRNA was significantly decreased by 12% in bipolar disorder relative to the comparison group in DLPFC, although there were no gyral versus sulcal differences. As was the case in schizophrenia, no changes in NR3A expression were observed in the inferior temporal cortex in bipolar disorder. These data indicate that the NR3A subunit is abnormally expressed in both schizophrenia and bipolar disorder.

While abnormalities of the prefrontal cortex and temporal lobe structures have typically been associated with the pathophysiology of schizophrenia, recent findings implicate thalamic dysfunction in this illness as well. The thalamus plays a critical role in processing and integrating sensory information relevant to emotional and cognitive functions. Neuropathological and in vivo imaging studies in schizophrenia have identified several structural and metabolic abnormalities in the thalamus, which may contribute to a deficit in sensory processing and be related to psychotic symptomatology. In addition to these postmortem and in vivo imaging studies indicating structural and metabolic changes in the thalamus in schizophrenia, more recent studies have examined the neurochemical substrates that accompany these changes. Much of this work to date has focused on glutamatergic abnormalities in the thalamus, in part because it is a predominant neurotransmitter used in the thalamus, and because glutamatergic dysfunction has been hypothesized to be involved in schizophrenia. Several studies, however, have also examined markers of gamma-aminobutyric acid (GABA) and dopaminergic neurotransmission in the thalamus in schizophrenia. We review these neurochemical findings, as well as the growing body of postmortem and in vivo imaging evidence that supports the hypothesis of thalamic dysfunction in schizophrenia.

Frontal lobe dysfunction in individuals with schizophrenia has frequently been detected using both neuroimaging and neuropsychological testing. Results from previous studies vary in the findings of regional specificity vs. generalized frontal cortical dysfunction. We sought to examine potential regional differences in frontal cortical functioning among patients with schizophrenia vs. a comparison group using two different neurocognitive tasks: the Gambling Task (GT) and the Wisconsin Card Sorting Test (WCST). In general, the GT is thought to reflect function of the ventromedial prefrontal cortex (VMPFC), while the WCST reflects function of the dorsolateral prefrontal cortex (DLPFC). Twenty individuals with schizophrenia or schizoaffective disorder and 15 nonpsychiatrically ill comparison subjects underwent an assessment battery consisting of the GT, WCST, and positive and negative symptom ratings. Patients with schizophrenia performed worse on the GT with respect to total monetary gain (p=0.05) and total monetary loss (p<0.05). They also preferred disadvantaged vs. advantaged cards (p<0.04). Surprisingly, WCST performance was poor in both groups and was not significantly different between groups. These findings are at some variance with those in the previously reported literature, but nonetheless support the idea that prefrontal cortical areas mediating different cognitive tasks may be distinguished by specific neurocognitive assessments.

The high affinity of 5-HT6 receptors for atypical antipsychotic drugs, and their localization in limbic and cortical regions of the brain, suggest that they might play a role in the pathophysiology of schizophrenia. To determine if this receptor is regulated by antipsychotics, rats were injected with clozapine (20 mg/kg/day), haloperidol (2 mg/kg/day), or vehicle daily for 2 weeks, and 5-HT6 receptor mRNA levels were measured by in situ hybridization. Clozapine but not haloperidol significantly decreased 5-HT6 expression in all subfields of the hippocampus. No drug effects were observed in cortical or forebrain structures. These results suggest that downregulation of this receptor in the hippocampus might be a characteristic of atypical antipsychotic drugs, although this hypothesis will require testing with other atypical antipsychotics.

Rates of substance-use disorders among psychiatric patients are consistently higher than in the general population, yet there is no clear specificity to the relationship between types of substance use and psychiatric diagnoses. Cigarette smoking may represent a substance-use behavior which has greater specificity for major psychiatric diagnoses. We examined the self-reported history of cigarette smoking vs marijuana, alcohol and cocaine use among 83 male veteran psychiatric patients with primary mood (major depression or bipolar disorder; n = 20) or schizophrenic (schizophrenia or schizoaffective; n = 63) disorders. Those in the SCZ group compared to those in the AFF group were more likely to be ever-smokers (OR 8.5, 95% CI [2.2, 32.3]), and current smokers (OR 12.0%, 95% CI [3.6, 40]), independent of age differences between the groups. There were no significant differences in marijuana, alcohol or cocaine use between the two groups when age differences were controlled. Generalizability of the findings is limited by small number of subjects, male gender and veteran status; however, the significantly higher prevalence of smoking among individuals with schizophrenic disorders may support the growing evidence of linkages between the effects of nicotine and the neurobiology of schizophrenia.

Our efforts to study and understand the complex clinical, behavioral, neurochemical and neuroanatomical manifestations of the schizophrenias are plagued by the heterogeneity of findings, and lack of ability to define subtypes within the syndrome. Theoretically, a behavior associated with schizophrenia among a majority of those affected might provide more broadly applicable information about the illness. Cigarette smoking represents such a behavior. Herein we present an overview of evidence linking cigarette smoking and schizophrenia, and describe three cases suggesting that nicotine withdrawal leads to an exacerbation of schizophrenic symptoms.