Altered distribution and localization of organellar Na+/H+ exchangers in postmortem schizophrenia dorsolateral prefrontal cortex.

Schizophrenia is a complex and multifactorial disorder associated with altered neurotransmission as well as numerous signaling pathway and protein trafficking disruptions. The pH of intracellular organelles involved in protein trafficking is tightly regulated and impacts their functioning. The SLC9A family of Na+/H+ exchangers (NHEs) plays a fundamental role in cellular and intracellular pH homeostasis. Four organellar NHE isoforms (NHE6-NHE9) are targeted to intracellular organelles involved in protein trafficking. Increased interactions between organellar NHEs and receptor of activated protein C kinase 1 (RACK1) can lead to redistribution of NHEs to the plasma membrane and hyperacidification of target organelles. Given their role in organelle pH regulation, altered expression and/or localization of organellar NHEs could be an underlying cellular mechanism contributing to abnormal intracellular trafficking and disrupted neurotransmitter systems in schizophrenia. We thus characterized organellar NHE expression, co-immunoprecipitation with RACK1, and Triton X-114 (TX-114) phase partitioning in dorsolateral prefrontal cortex of 25 schizophrenia and 25 comparison subjects by Western blot analysis. In schizophrenia after controlling for subject age at time of death, postmortem interval, tissue pH, and sex, there was significantly decreased total expression of NHE8, decreased co-immunoprecipitation of NHE8 (64%) and NHE9 (56%) with RACK1, and increased TX-114 detergent phase partitioning of NHE6 (283%), NHE9 (75%), and RACK1 (367%). Importantly, none of these dependent measures was significantly impacted when comparing those in the schizophrenia group on antipsychotics to those off of antipsychotics for at least 6 weeks at their time of death and none of these same proteins were affected in rats chronically treated with haloperidol. In summary, we characterized organellar NHE expression and distribution in schizophrenia DLPFC and identified abnormalities that could represent a novel mechanism contributing to disruptions in protein trafficking and neurotransmission in schizophrenia.

Differential genetic associations and expression of PAPST1/SLC35B2 in bipolar disorder and schizophrenia.

Lithium's inhibitory effect on enzymes involved in sulfation process, such as inhibition of 3'(2')-phosphoadenosine 5'-phosphate (PAP) phosphatase, is a possible mechanism of its therapeutic effect for bipolar disorder (BD). 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is translocated from cytosol to Golgi lumen by PAPS transporter 1 (PAPST1/SLC35B2), where it acts as a sulfa donor. Since SLC35B2 was previously recognized as a molecule that facilitates the release of D-serine, a co-agonist of N-methyl-D-aspartate type glutamate receptor, altered function of SLC35B2 might be associated with the pathophysiology of BD and schizophrenia (SCZ). We performed genetic association analyses of the SLC35B2 gene using Japanese cohorts with 366 BD cases and 370 controls and 2012 SCZ cases and 2170 controls. We then investigated expression of SLC35B2 mRNA in postmortem brains by QPCR using a Caucasian cohort with 33 BD and 34 SCZ cases and 34 controls and by in situ hybridization using a Caucasian cohort with 37 SCZ and 29 controls. We found significant associations between three SNPs (rs575034, rs1875324, and rs3832441) and BD, and significantly reduced SLC35B2 mRNA expression in postmortem dorsolateral prefrontal cortex (DLPFC) of BD. Moreover, we observed normalized SLC35B2 mRNA expression in BD subgroups who were medicated with lithium. While there was a significant association of SLC35B2 with SCZ (SNP rs2233437), its expression was not changed in SCZ. These findings indicate that SLC35B2 might be differentially involved in the pathophysiology of BD and SCZ by influencing the sulfation process and/or glutamate system in the central nervous system.

AMPA receptor subunit localization in schizophrenia anterior cingulate cortex.

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.

mTOR kinase activity disrupts a phosphorylation signaling network in schizophrenia brain.

The AKT-mTOR signaling transduction pathway plays an important role in neurodevelopment and synaptic plasticity. mTOR is a serine/threonine kinase that modulates signals from multiple neurotransmitters and phosphorylates specific proteins to regulate protein synthesis and cytoskeletal organization. There is substantial evidence demonstrating abnormalities in AKT expression and activity in different schizophrenia (SZ) models. However, direct evidence for dysregulated mTOR kinase activity and its consequences on downstream effector proteins in SZ pathophysiology is lacking. Recently, we reported reduced phosphorylation of mTOR at an activating site and abnormal mTOR complex formation in the SZ dorsolateral prefrontal cortex (DLPFC). Here, we expand on our hypothesis of disrupted mTOR signaling in the SZ brain and studied the expression and activity of downstream effector proteins of mTOR complexes and the kinase activity profiles of SZ subjects. We found that S6RP phosphorylation, downstream of mTOR complex I, is reduced, whereas PKCα phosphorylation, downstream of mTOR complex II, is increased in SZ DLPFC. In rats chronically treated with haloperidol, we showed that S6RP phosphorylation is increased in the rat frontal cortex, suggesting a potential novel mechanism of action for antipsychotics. We also demonstrated key differences in kinase signaling networks between SZ and comparison subjects for both males and females using kinome peptide arrays. We further investigated the role of mTOR kinase activity by inhibiting it with rapamycin in postmortem tissue and compared the impact of mTOR inhibition in SZ and comparison subjects using kinome arrays. We found that SZ subjects are globally more sensitive to rapamycin treatment and AMP-activated protein kinase (AMPK) contributes to this differential kinase activity. Together, our findings provide new insights into the role of mTOR as a master regulator of kinase activity in SZ and suggest potential targets for therapeutic intervention.

Dysregulation of the unfolded protein response (UPR) in the dorsolateral prefrontal cortex in elderly patients with schizophrenia.

Abnormalities in protein localization, function, and posttranslational modifications are targets of schizophrenia (SCZ) research. As a major contributor to the synthesis, folding, trafficking, and modification of proteins, the endoplasmic reticulum (ER) is well-positioned to sense cellular stress. The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction to environmental and pathological perturbation in ER function. The UPR is a highly orchestrated and complex cellular response, which is mediated through the ER chaperone protein, BiP, three known ER transmembrane stress sensors, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), inositol requiring enzyme 1α (IRE1α), and their downstream effectors. In this study, we measured protein expression and phosphorylation states of UPR sensor pathway proteins in the dorsolateral prefrontal cortex (DLPFC) of 22 matched pairs of elderly SCZ and comparison subjects. We observed increased protein expression of BiP, decreased PERK, and decreased phosphorylation of IRE1α. We also observed decreased p-JNK2 and increased sXBP1, downstream targets of the IRE1α arm of the UPR. The disconnect between decreased p-IRE1α and increased sXBP1 protein expression led us to measure sXbp1 mRNA. We observed increased expression of the ratio of sXbp1/uXbp1 transcripts, suggesting that splicing of Xbp1 mRNA by IRE1α is increased and drives upregulation of sXBP1 protein expression. These findings suggest an abnormal pattern of UPR activity in SCZ, with specific dysregulation of the IRE1α arm. Dysfunction of this system may lead to abnormal responses to cellular stressors and contribute to protein processing abnormalities previously observed in SCZ.

Evidence for altered energy metabolism, increased lactate, and decreased pH in schizophrenia brain: A focused review and meta-analysis of human postmortem and magnetic resonance spectroscopy studies.

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.

Post-translational protein modifications in schizophrenia.

Research investigating the pathophysiology of schizophrenia has not yet precisely defined the molecular phenotype of this disorder. Many studies have investigated cellular dysfunction by examining expression levels of molecular targets in postmortem patient brain; however, inconsistencies between transcript and protein measures in schizophrenia are common in the field and represent a challenge to the identification of a unified model of schizophrenia pathogenesis. In humans, >4800 unique proteins are expressed, and the majority of these are modified by glycans and/or lipids. Estimates indicate ~70% of all eukaryotic proteins are modified by at least one type of glycosylation, while nearly 20% of all proteins are known to be lipid-modified. Protein post-translational modification (PTM) by glycosylation and lipidation rely on the spatiotemporal colocalization of enzyme, substrate, and glycan or lipid donor molecule and do not require an upstream "blueprint" or specialized processing machinery for synthesis. Glycan and lipid PTMs can thus facilitate cellular adaptation to environmental signals more rapidly than changes of gene or protein expression, and can significantly impact the localization, function, and interactions of modified substrates, though relatively few studies in schizophrenia have evaluated the PTM status of target proteins. A growing body of literature reports glycosylation and lipidation abnormalities in schizophrenia brain as well as in patient peripheral fluids. In this review, we explain the functional significance of key glycan and lipid PTMs and summarize current findings associated with abnormal glycosylation and lipidation in this illness.

Protein expression of prenyltransferase subunits in postmortem schizophrenia dorsolateral prefrontal cortex.

The pathophysiology of schizophrenia includes altered neurotransmission, dysregulated intracellular signaling pathway activity, and abnormal dendritic morphology that contribute to deficits of synaptic plasticity in the disorder. These processes all require dynamic protein-protein interactions at cell membranes. Lipid modifications target proteins to membranes by increasing substrate hydrophobicity by the addition of a fatty acid or isoprenyl moiety, and recent evidence suggests that dysregulated posttranslational lipid modifications may play a role in multiple neuropsychiatric disorders, including schizophrenia. Consistent with these emerging findings, we have recently reported decreased protein S-palmitoylation in schizophrenia. Protein prenylation is a lipid modification that occurs upstream of S-palmitoylation on many protein substrates, facilitating membrane localization and activity of key intracellular signaling proteins. Accordingly, we hypothesized that, in addition to palmitoylation, protein prenylation may be abnormal in schizophrenia. To test this, we assayed protein expression of the five prenyltransferase subunits (FNTA, FNTB, PGGT1B, RABGGTA, and RABGGTB) in postmortem dorsolateral prefrontal cortex from patients with schizophrenia and paired comparison subjects (n = 13 pairs). We found decreased levels of FNTA (14%), PGGT1B (13%), and RABGGTB (8%) in schizophrenia. To determine whether upstream or downstream factors may be driving these changes, we also assayed protein expression of the isoprenoid synthases FDPS and GGPS1 and prenylation-dependent processing enzymes RCE and ICMT. We found these upstream and downstream enzymes to have normal protein expression. To rule out effects from chronic antipsychotic treatment, we assayed FNTA, PGGT1B, and RABGGTB in the cortex from rats treated long-term with haloperidol decanoate and found no change in the expression of these proteins. Given the role prenylation plays in localization of key signaling proteins found at the synapse, these data offer a potential mechanism underlying abnormal protein-protein interactions and protein localization in schizophrenia.

Downregulated AKT-mTOR signaling pathway proteins in dorsolateral prefrontal cortex in Schizophrenia.

Abnormal neurotransmission is central to schizophrenia (SZ). Alterations across multiple neurotransmitter systems in SZ suggest that this illness may be associated with dysregulation of core intracellular processes such as signaling pathways that underlie the regulation and integration of these systems. The AKT-mTOR signaling cascade has been implicated in SZ by gene association, postmortem brain and animal studies. AKT and mTOR are serine/threonine kinases which play important roles in cell growth, proliferation, survival, and differentiation. Both AKT and mTOR require phosphorylation at specific sites for their complete activation. mTOR forms two functionally distinct multiprotein complexes, mTOR Complex 1 (mTORC1) and Complex 2 (mTORC2). mTORC1 mediates ribosome biogenesis, protein translation, and autophagy, whereas mTORC2 contributes to actin dynamics. Altered protein synthesis and actin dynamics can lead to an abnormal neuronal morphology resulting in deficits in learning and memory. Currently, there is a lack of direct evidence to support the hypothesis of disrupted mTOR signaling in SZ, and we have addressed this by characterizing this signaling pathway in SZ brain. We found a reduction in AKT and mTOR protein expression and/or phosphorylation state in dorsolateral prefrontal cortex (DLPFC) from 22 pairs of SZ and matched comparison subjects. We also found reduced protein expression of GßL, a subunit protein common to both mTOR complexes. We further investigated mTOR complex-specific subunit composition and phosphorylation state, and found abnormal mTOR expression in both complexes in SZ DLPFC. These findings provide evidence that proteins associated with the AKT-mTOR signaling cascade are downregulated in SZ DLPFC.

Intracellular compartment-specific proteasome dysfunction in postmortem cortex in schizophrenia subjects.

Protein homeostasis is an emerging component of schizophrenia (SZ) pathophysiology. Proteomic alterations in SZ are well-documented and changes in transcript expression are frequently not associated with changes in protein expression in SZ brain. The underlying mechanism driving these changes remains unknown, though altered expression of ubiquitin proteasome system (UPS) components have implicated protein degradation. Previous studies have been limited to protein and transcript expression, however, and do not directly test the function of the proteasome. To address this gap in knowledge, we measured enzymatic activity associated with the proteasome (chymotrypsin-, trypsin-, and caspase-like) in the superior temporal gyrus (STG) of 25 SZ and 25 comparison subjects using flourogenic substrates. As localization regulates which cellular processes the proteasome contributes to, we measured proteasome activity and subunit expression in fractions enriched for nucleus, cytosolic, and membrane compartments. SZ subjects had decreased trypsin-like activity in total homogenate. This finding was specific to the nucleus-enriched fraction and was not associated with changes in proteasome subunit expression. Interestingly, both chymotrypsin-like activity and protein expression of 19S RP subunits, which facilitate ubiquitin-dependent degradation, were decreased in the cytosol-enriched fraction of SZ subjects. Intracellular compartment-specific proteasome dysfunction implicates dysregulation of protein expression both through altered ubiquitin-dependent degradation of cytosolic proteins and regulation of protein synthesis due to degradation of transcription factors and transcription machinery in the nucleus. Together, these findings implicate proteasome dysfunction in SZ, which likely has a broad impact on the proteomic landscape and cellular function in the pathophysiology of this illness.

Kinase network dysregulation in a human induced pluripotent stem cell model of DISC1 schizophrenia.

Protein kinases orchestrate signal transduction pathways involved in central nervous system functions ranging from neurodevelopment to synaptic transmission and plasticity. Abnormalities in kinase-mediated signaling are involved in the pathophysiology of neurological disorders, including neuropsychiatric disorders. Here, we expand on the hypothesis that kinase networks are dysregulated in schizophrenia. We investigated changes in serine/threonine kinase activity in cortical excitatory neurons differentiated from induced pluripotent stem cells (iPSCs) from a schizophrenia patient presenting with a 4 bp mutation in the disrupted in schizophrenia 1 (DISC1) gene and a corresponding control. Using kinome peptide arrays, we demonstrate large scale abnormalities in DISC1 cells, including a global depression of serine/threonine kinase activity, and changes in activity of kinases, including AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), and thousand-and-one amino acid (TAO) kinases. Using isogenic cell lines in which the DISC1 mutation is either introduced in the control cell line, or rescued in the schizophrenia cell line, we ascribe most of these changes to a direct effect of the presence of the DISC1 mutation. Investigating the gene expression signatures downstream of the DISC1 kinase network, and mapping them on perturbagen signatures obtained from the Library of Integrated Network-based Cellular Signatures (LINCS) database, allowed us to propose novel drug targets able to reverse the DISC1 kinase dysregulation gene expression signature. Altogether, our findings provide new insight into abnormalities of kinase networks in schizophrenia and suggest possible targets for disease intervention.

Fractionation of Subcellular Compartments from Human Brain Tissue.

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.

Abnormal ER quality control of neural GPI-anchored proteins via dysfunction in ER export processing in the frontal cortex of elderly subjects with schizophrenia.

Abnormalities of posttranslational protein modifications (PTMs) have recently been implicated in the pathophysiology of schizophrenia. Glycosylphosphatidylinositols (GPIs) are a class of complex glycolipids, which anchor surface proteins and glycoproteins to the cell membrane. GPI attachment to proteins represents one of the most common PTMs and GPI-associated proteins (GPI-APs) facilitate many cell surface processes, including synapse development and maintenance. Mutations in the GPI processing pathway are associated with intellectual disability, emphasizing the potential role of GPI-APs in cognition and schizophrenia-associated cognitive dysfunction. As initial endoplasmic reticulum (ER)-associated protein processing is essential for GPI-AP function, we measured protein expression of molecules involved in attachment (GPAA1), modification (PGAP1), and ER export (Tmp21) of GPI-APs, in homogenates and in an ER enriched fraction derived from dorsolateral prefrontal cortex (DLPFC) of 15 matched pairs of schizophrenia and comparison subjects. In total homogenate we found a significant decrease in transmembrane protein 21 (Tmp21) and in the ER-enriched fraction we found reduced expression of post-GPI attachment protein (PGAP1). PGAP1 modifies GPI-anchors through inositol deacylation, allowing it to be recognized by Tmp21. Tmp21 is a component of the p24 complex that recognizes GPI-anchored proteins, senses the status of the GPI-anchor, and regulates incorporation into COPII vesicles for export to the Golgi apparatus. Together, these proteins are the molecular mechanisms underlying GPI-AP quality control and ER export. To investigate the potential consequences of a deficit in export and/or quality control, we measured cell membrane-associated expression of known GPI-APs that have been previously implicated in schizophrenia, including GPC1, NCAM, MDGA2, and EPHA1, using Triton X-114 phase separation. Additionally, we tested the sensitivity of those candidate proteins to phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves GPI from GPI-APs. While we did not observe a difference in the amount of these GPI-APs in Triton X-114 phase separated membrane fractions, we found decreased NCAM and GPC1 within the PI-PLC sensitive fraction. These findings suggest dysregulation of ER-associated GPI-AP protein processing, with impacts on post-translational modifications of proteins previously implicated in schizophrenia such as NCAM and GPC1. These findings provide evidence for a deficit in ER protein processing pathways in this illness.

Abnormal expression of ER quality control and ER associated degradation proteins in the dorsolateral prefrontal cortex in schizophrenia.

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.

Actin polymerization is reduced in the anterior cingulate cortex of elderly patients with schizophrenia.

Recent reports suggest abnormalities in the regulation of actin cytoskeletal dynamics in schizophrenia, despite consistent evidence for normal actin expression. We hypothesized that this may be explained by changes in the polymerization state of actin, rather than in total actin expression. To test this, we prepared filamentous actin (F-actin, polymeric) and globular actin (G-actin, monomeric) fractions from postmortem anterior cingulate cortex from 16 patients with schizophrenia and 14 comparison subjects. Additionally, binding of fluorescently-labeled phalloidin, a selectively F-actin-binding peptide, was measured in unfractionated samples from the same subjects. Western blot analysis of fractions revealed decreased F-actin, increased G-actin, and decreased ratios of F-actin/total actin and F-actin/G-actin in schizophrenia. Decreased phalloidin binding to F-actin in parallel experiments in the same subjects independently supports these findings. These results suggest a novel aspect of schizophrenia pathophysiology and are consistent with previous evidence of reduced dendritic spine density and altered synaptic plasticity in schizophrenia, both of which have been linked to cytoskeletal abnormalities.

Abnormalities of signal transduction networks in chronic schizophrenia.

Schizophrenia is a serious neuropsychiatric disorder characterized by disruptions of brain cell metabolism, microstructure, and neurotransmission. All of these processes require coordination of multiple kinase-mediated signaling events. We hypothesize that imbalances in kinase activity propagate through an interconnected network of intracellular signaling with potential to simultaneously contribute to many or all of the observed deficits in schizophrenia. We established a workflow distinguishing schizophrenia-altered kinases in anterior cingulate cortex using a previously published kinome array data set. We compared schizophrenia-altered kinases to haloperidol-altered kinases, and identified systems, functions, and regulators predicted using pathway analyses. We used kinase inhibitors with the kinome array to test hypotheses about imbalance in signaling and conducted preliminary studies of kinase proteins, phosphoproteins, and activity for kinases of interest. We investigated schizophrenia-associated single nucleotide polymorphisms in one of these kinases, AKT, for genotype-dependent changes in AKT protein or activity. Kinome analyses identified new kinases as well as some previously implicated in schizophrenia. These results were not explained by chronic antipsychotic treatment. Kinases identified in our analyses aligned with cytoskeletal arrangement and molecular trafficking. Of the kinases we investigated further, AKT and (unexpectedly) JNK, showed the most dysregulation in the anterior cingulate cortex of schizophrenia subjects. Changes in kinase activity did not correspond to protein or phosphoprotein levels. We also show that AKT single nucleotide polymorphism rs1130214, previously associated with schizophrenia, influenced enzyme activity but not protein or phosphoprotein levels. Our data indicate subtle changes in kinase activity and regulation across an interlinked kinase network, suggesting signaling imbalances underlie the core symptoms of schizophrenia. DISEASE MECHANISMS: A SIGNALING IMBALANCE: A study by US scientists indicates that changes in the activity of key signaling proteins may underlie core symptoms of schizophrenia. Protein kinases mediate the activation of intracellular signaling events and analyses of the kinome, the complete set of protein kinases encoded in the genome, previously revealed significant changes in phosphorylation patterns in postmortem brain tissue from patients with schizophrenia. Based on these findings, Jennifer McGuire at the University of Cincinnati and colleagues investigated the upstream regulation of these proteins. They identified both established and novel proteins associated with schizophrenia in the anterior cingulate cortex, with JNK and AKT activity being the most disrupted in schizophrenia patients. Their findings highlight how subtle changes in the activity of a small number of signaling proteins can propagate and have major consequences for mental health.

Altered fucosyltransferase expression in the superior temporal gyrus of elderly patients with schizophrenia.

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.

Decreased protein S-palmitoylation in dorsolateral prefrontal cortex in schizophrenia.

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.

Decreased expression of cortactin in the schizophrenia brain.

Schizophrenia is a severe psychiatric disorder that is characterized by a wide array of symptoms and a complex neuropathology. A well-characterized neurobiological feature of schizophrenia is abnormal synaptic plasticity, although the mechanisms underlying this are not fully understood. Numerous studies have demonstrated a link between proper functioning of the cytoskeleton and synaptic plasticity. The actin-related protein-2/3 (Arp2/3) complex is responsible for the nucleation of new actin filaments and elongation of existing actin filaments and is thus crucial to cytoskeletal dynamics, especially actin polymerization and organization. To determine whether the Arp2/3 complex is abnormally expressed in schizophrenia, we measured the protein expression of Arp2 and Arp3, as well as Arp2/3 complex binding partners and associated proteins including cortactin, neuronal-Wiskott-Aldrich syndrome protein (WASP), WASP-family verprolin homologous protein 1 (WAVE1), and Abelson interactor 1 (Abi1) in the superior temporal gyrus of paired schizophrenia and comparison participants. No changes were found in Arp2, Arp3, neuronal-WASP, WAVE1, or Abi1. However, all three isoforms of cortactin were decreased in schizophrenia. Specifically, the 62 kDa isoform was decreased by 43%; the 71 kDa isoform was decreased by 32%; and the 58 kDa isoform was decreased by 35%. Cortactin regulates branching of filamentous actin through its binding and activation of the Arp2/3 complex, and it is thus critical to the formation of stable actin networks. These findings contribute to a growing body of evidence implicating altered cytoskeletal dynamics in schizophrenia.