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While the pathophysiology of schizophrenia has been extensively investigated using homogenized postmortem brain samples, few studies have examined changes in brain samples with techniques that may attribute perturbations to specific cell types. To fill this gap, we performed microarray assays on mRNA isolated from anterior cingulate cortex (ACC) superficial and deep pyramidal neurons from 12 schizophrenia and 12 control subjects using laser-capture microdissection. Among all the annotated genes, we identified 134 significantly increased and 130 decreased genes in superficial pyramidal neurons, while 93 significantly increased and 101 decreased genes were found in deep pyramidal neurons, in schizophrenia compared to control subjects. In these differentially expressed genes, we detected lamina-specific changes of 55 and 31 genes in superficial and deep neurons in schizophrenia, respectively. Gene set enrichment analysis (GSEA) was applied to the entire pre-ranked differential expression gene lists to gain a complete pathway analysis throughout all annotated genes. Our analysis revealed overrepresented groups of gene sets in schizophrenia, particularly in immunity and synapse-related pathways, suggesting the disruption of these pathways plays an important role in schizophrenia. We also detected other pathways previously demonstrated in schizophrenia pathophysiology, including cytokine and chemotaxis, postsynaptic signaling, and glutamatergic synapses. In addition, we observed several novel pathways, including ubiquitin-independent protein catabolic process. Considering the effects of antipsychotic treatment on gene expression, we applied a novel bioinformatics approach to compare our differential expression gene profiles with 51 antipsychotic treatment datasets, demonstrating that our results were not influenced by antipsychotic treatment. Taken together, we found pyramidal neuron-specific changes in neuronal immunity, synaptic dysfunction, and olfactory dysregulation in schizophrenia, providing new insights for the cell-subtype specific pathophysiology of chronic schizophrenia.

While the pathophysiology of schizophrenia has been extensively investigated using homogenized postmortem brain samples, few studies have examined changes in brain samples with techniques that may attribute perturbations to specific cell types. To fill this gap, we performed microarray assays on mRNA isolated from anterior cingulate cortex (ACC) superficial and deep pyramidal neurons from 12 schizophrenia and 12 control subjects using laser capture microdissection. Among all the annotated genes, we identified 134 significantly increased and 130 decreased genes in superficial pyramidal neurons, while 93 significantly increased and 101 decreased genes were found in deep pyramidal neurons, in schizophrenia compared to control subjects. In these differentially expressed genes, we detected lamina-specific changes of 55 and 31 genes in superficial and deep neurons in schizophrenia, respectively. Gene set enrichment analysis (GSEA) was applied to the entire pre-ranked differential expression gene lists to gain a complete pathway analysis throughout all annotated genes. Our analysis revealed over-represented groups of gene sets in schizophrenia, particularly in immunity and synapse related pathways in pyramidal neurons, suggesting the disruption of these pathways plays an important role in schizophrenia. We also detected pathways previously demonstrated in schizophrenia pathophysiology, including cytokine and chemotaxis, post-synaptic signaling, and glutamatergic synapses. In addition, we observed several novel pathways, including ubiquitin-independent protein catabolic process. By comparing our differential expression gene profiles with 51 antipsychotic treatment datasets, we demonstrated that our results were not influenced by antipsychotic treatment of our subjects. Taken together, we found pyramidal neuron-specific changes in neuronal immunity, synaptic dysfunction, and olfactory dysregulation in schizophrenia, providing new insights for the cell-subtype specific pathophysiology of chronic 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.

Excitatory amino-acid transporters (EAATs) bind and transport glutamate, limiting spillover from synapses due to their dense perisynaptic expression primarily on astroglia. Converging evidence suggests that abnormalities in the astroglial glutamate transporter localization and function may underlie a disease mechanism with pathological glutamate spillover as well as alterations in the kinetics of perisynaptic glutamate buffering and uptake contributing to dysfunction of thalamo-cortical circuits in schizophrenia. We explored this hypothesis by performing cell- and region-level studies of EAAT1 and EAAT2 expression in the mediodorsal nucleus of the thalamus in an elderly cohort of subjects with schizophrenia. We found decreased protein expression for the typically astroglial-localized glutamate transporters in the mediodorsal and ventral tier nuclei. We next used laser-capture microdissection and quantitative polymerase chain reaction to assess cell-level expression of the transporters and their splice variants. In the mediodorsal nucleus, we found lower expression of transporter transcripts in a population of cells enriched for astrocytes, and higher expression of transporter transcripts in a population of cells enriched for relay neurons. We confirmed expression of transporter protein in neurons in schizophrenia using dual-label immunofluorescence. Finally, the pattern of transporter mRNA and protein expression in rodents treated for 9 months with antipsychotic medication suggests that our findings are not due to the effects of antipsychotic treatment. We found a compensatory increase in transporter expression in neurons that might be secondary to a loss of transporter expression in astrocytes. These changes suggest a profound abnormality in astrocyte functions that support, nourish and maintain neuronal fidelity and synaptic activity.

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.

Shaping plasticity: Alterations in glutamate transporter localization as a pathophysiological mechanism in severe mental illness

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.

Background Impairment of glutamate neurons that relay sensory and cognitive information from the medial dorsal thalamus to the dorsolateral prefrontal cortex and other cortical regions may contribute to the pathophysiology of schizophrenia. In this study, we have assessed the cell-specific expression of glutamatergic transcripts in the medial dorsal thalamus. Methods We used laser capture microdissection to harvest two populations of medial dorsal thalamic cells, one enriched with glutamatergic relay neurons and the other with gamma-aminobutyric acidergic neurons and astroglia, from postmortem brains of subjects with schizophrenia ( n = 14) and a comparison group ( n = 20). Quantitative polymerase chain reaction of extracted RNA was used to assay gene expression in the different cell populations. Results The transcripts encoding the ionotropic glutamate receptor subunits NR2D, GluR3, GluR6, GluR7, and the intracellular proteins GRIP1 and SynGAP1 were significantly decreased in relay neurons but not in the mixed glial and interneuron population in schizophrenia. Conclusions Our data suggest that reduced ionotropic glutamatergic expression occurs selectively in neurons, which give rise to the cortical projections of the medial dorsal thalamus in schizophrenia, rather than in thalamic cells that function locally. Our findings indicate that glutamatergic innervation is dysfunctional in the circuitry between the medial dorsal thalamus and cortex.

RNA editing is a posttranscriptional process which critically modulates the function of several neurotransmitter receptors regulating mood, anxiety, learning, and memory. Data from several postmortem studies have shown increased 5-hydroxytryptamine-2C receptor RNA editing in mood disorders and suicide, and therefore the 5-hydroxytryptamine-2C receptor might be expected to have reduced signal transduction in these patients. In this study, we have tested the hypothesis that the expression levels of the enzymes which catalyze RNA editing, adenosine deaminase acting on RNA 1 (ADAR1) and ADAR2, are also abnormal in suicide. Gene expression was measured in the dorsolateral prefrontal cortex of individuals from the Stanley Consortium Brain series, which includes patients with schizophrenia (n=15), major depression (n=15), bipolar disorder (n=15), and a comparison group (n=14). Of the psychiatric patients, 20 were suicide victims. ADAR1 expression was found to be significantly increased in major depressive suicide victims compared with patients who did not commit suicide. Neither ADAR1 nor ADAR2 expression was altered in any of the other diagnostic groups. These data indicate that ADAR1 could play a role in the pathophysiology of suicide in patients with major depression.

Schizophrenia has been proposed to be associated with abnormal glutamatergic neurotransmission. The AMPA subtype of glutamate receptors (AMPARs) mediates fast excitatory synaptic transmission in the brain, and their trafficking and function is regulated in part by AMPAR auxiliary proteins including the cornichons (CNIH) and transmembrane AMPAR-regulatory proteins. Abnormal regulation of AMPARs through altered expression of these auxiliary proteins could induce changes in glutamatergic neurotransmission and thus the pathophysiology of schizophrenia. In this study, transcript expression of cornichon homologs 1-4 was measured in the dorsolateral prefrontal cortex from schizophrenia (N=25) and comparison (N=25) patient groups by comparative quantitative real-time PCR. Significant upregulation of CNIH-1, CNIH-2, and CNIH-3 mRNA expression was found in schizophrenia, with no change in CNIH-4 expression. To determine the effect of antipsychotic treatment on the expression of these genes, cornichon mRNA expression was assayed in the frontal cortex of rats treated chronically with haloperidol decanoate and no changes in any of the cornichon transcripts were found. Abnormal expression of the CNIH family of genes is consistent with cornichon-mediated AMPAR trafficking abnormalities in schizophrenia, and suggests a new mechanism contributing toward the pathophysiology of this illness.

Dysregulation of the glutamate transporters EAAT1 and EAAT2 and their isoforms have been implicated in schizophrenia. EAAT1 and EAAT2 expression has been studied in different brain regions but the prevalence of astrocytic glutamate transporter expression masks the more subtle changes in excitatory amino acid transporters (EAATs) isoforms in neurons in the cortex. Using laser capture microdissection, pyramidal neurons were cut from the anterior cingulate cortex of postmortem schizophrenia (n=20) and control (n=20) subjects. The messenger RNA (mRNA) levels of EAAT1, EAAT2 and the splice variants EAAT1 exon9skipping, EAAT2 exon9skipping and EAAT2b were analyzed by real time PCR (RT-PCR) in an enriched population of neurons. Region-level expression of these transcripts was measured in postmortem schizophrenia (n=25) and controls (n=25). The relationship between selected EAAT polymorphisms and EAAT splice variant expression was also explored. Anterior cingulate cortex pyramidal cell expression of EAAT2b mRNA was increased (PP