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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., (© 2023. The Author(s).)

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 GβL, 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.

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.

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.

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.

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.

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.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) has been linked to multiple neurological and psychiatric disorders including schizophrenia, but its involvement in the pathophysiology of these disorders is unclear. Experiments in mice have revealed a set of developmentally-regulated cortical PGC-1α-dependent transcripts involved in calcium buffering (parvalbumin, PV), synchronous neurotransmitter release (synaptotagmin 2, Syt2; complexin 1, Cplx1) and axonal integrity (neurofilamaent heavy chain, Nefh). We measured the mRNA expression of PGC-1α and these transcripts in postmortem cortical tissue from control and schizophrenia patients and found a reduction in PGC-1α-dependent transcripts without a change in PGC-1α. While control subjects with high PGC-1α expression exhibited high PV and Nefh expression, schizophrenia subjects with high PGC-1α expression did not, suggesting dissociation between PGC-1α expression and these targets in schizophrenia. Unbiased analyses of the promoter regions for PGC-1α-dependent transcripts revealed enrichment of binding sites for the PGC-1α-interacting transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 mRNA expression was reduced in schizophrenia, and its transcript levels predicted that of PGC-1α-dependent targets in schizophrenia. Interestingly, the positive correlation between PGC-1α and PV, Syt2, or Cplx1 expression was lost in schizophrenia patients with low NRF-1 expression, suggesting that NRF-1 is a critical predictor of these genes in disease. These data suggest that schizophrenia involves a disruption in PGC-1α and/or NRF-1-associated transcriptional programs in the cortex and that approaches to enhance the activity of PGC-1α or transcriptional regulators like NRF-1 should be considered with the goal of restoring normal gene programs and improving cortical function., (© The Author 2015. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness.

Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs.

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 (P < 0.001; 67%) in schizophrenia subjects compared with controls. There was no significant change in other EAAT variants. EAAT2 exon9skipping mRNA was increased (P < 0.05; 38%) at region level in the anterior cingulate cortex with no significant change in other EAAT variants at region level. EAAT2 single-nucleotide polymorphisms were significantly associated with changes in EAAT2 isoform expression. Haloperidol decanoate-treated animals, acting as controls for possible antipsychotic effects, did not have significantly altered neuronal EAAT2b mRNA levels. The novel finding that EAAT2b levels are increased in populations of anterior cingulate cortex pyramidal cells further demonstrates a role for neuronal glutamate transporter splice variant expression in schizophrenia.

Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species.

The molecular mechanisms of schizophrenia have been under investigation for decades; however, the exact causes of this debilitating neuropsychiatric disorder are still unknown. Previous studies have identified multiple affected neurotransmitter systems, brain regions, and cell types, each making a unique contribution to symptom presentation and pathophysiology. Numerous studies have identified gene and protein expression changes in schizophrenia, but the role of post-translational modifications, specifically N-glycosylation, has only recently become a target of investigation. N-glycosylation of molecules associated with glutamatergic neurotransmission is disrupted in schizophrenia, but it was unknown if these alterations are exclusive to the glutamatergic system or due to a more generalized deficit.In normal human cortex, we found evidence for N-glycosylation of the α1, β1, and β2 γ-aminobutyric type A receptor (GABAAR) subunits using deglycosylation protein shift assays. This was confirmed with lectin affinity assays that revealed glycan attachment on the α1, α4, and β1-3 GABAAR subunits. Examining GABAAR subunit N-glycosylation in matched pairs of schizophrenia (N=14) and comparison (N=14) of superior temporal gyrus revealed a smaller molecular mass of immature N-glycans on the α1 subunit, more immature N-glycosylation of the 49-kDa β1 subunit isoform, and altered total N-glycosylation of the β2 GABAAR subunit in schizophrenia. Measures of altered N-glycosylation of the β1 and β2 subunits were confounded by an increased apparent molecular mass of all β1 and β2 subunit isoforms in schizophrenia. Although N-glycosylation of α1, β1, and β2 were all changed in schizophrenia, the concentrations of GABAAR subunits themselves were unchanged. These findings suggest that disruptions of N-glycosylation in schizophrenia are not exclusive to glutamate and may indicate a potential disruption of a central cell signaling process in this disorder.

We propose that postmortem tissue is an underutilized substrate that may be used to translate genetic and/or preclinical studies, particularly for neuropsychiatric illnesses with complex etiologies. Postmortem brain tissues from subjects with schizophrenia have been extensively studied, and thus serve as a useful vehicle for illustrating the challenges associated with this biological substrate. Schizophrenia is likely caused by a combination of genetic risk and environmental factors that combine to create a disease phenotype that is typically not apparent until late adolescence. The complexity of this illness creates challenges for hypothesis testing aimed at understanding the pathophysiology of the illness, as postmortem brain tissues collected from individuals with schizophrenia reflect neuroplastic changes from a lifetime of severe mental illness, as well as treatment with antipsychotic medications. While there are significant challenges with studying postmortem brain, such as the postmortem interval, it confers a translational element that is difficult to recapitulate in animal models. On the other hand, data derived from animal models typically provide specific mechanistic and behavioral measures that cannot be generated using human subjects. Convergence of these two approaches has led to important insights for understanding molecular deficits and their causes in this illness. In this review, we discuss the problem of schizophrenia, review the common challenges related to postmortem studies, discuss the application of biochemical approaches to this substrate, and present examples of postmortem schizophrenia studies that illustrate the role of the postmortem approach for generating important new leads for understanding the pathophysiology of severe mental illness.

Protein expression abnormalities have been implicated in the pathophysiology of schizophrenia, but the underlying cause of these changes is not known. We sought to investigate ubiquitin and ubiquitin-like (UBL) systems (SUMOylation, NEDD8ylation, and Ufmylation) as putative mechanisms underlying protein expression abnormalities seen in schizophrenia. For this, we performed western blot analysis of total ubiquitination, free ubiquitin, K48- and K63-linked ubiquitination, and E1 activases, E2 conjugases, and E3 ligases involved in ubiquitination and UBL post-translational modifications in postmortem brain tissue samples from persons with schizophrenia (n=13) and comparison subjects (n=13). We studied the superior temporal gyrus (STG) of subjects from the Mount Sinai Medical Center brain collection that were matched for age, tissue pH, and sex. We found an overall reduction of protein ubiquitination, free ubiquitin, K48-linked ubiquitination, and increased K63 polyubiquitination in schizophrenia. Ubiquitin E1 activase UBA (ubiquitin activating enzyme)-6 and E3 ligase Nedd (neural precursor cell-expressed developmentally downregulated)-4 were decreased in this illness, as were E3 ligases involved in Ufmylation (UFL1) and SUMOylation (protein inhibitor of activated STAT 3, PIAS3). NEDD8ylation was also dysregulated in schizophrenia, with decreased levels of the E1 activase UBA3 and the E3 ligase Rnf7. This study of ubiquitin and UBL systems in schizophrenia found abnormalities of ubiquitination, Ufmylation, SUMOylation, and NEDD8ylation in the STG in this disorder. These results suggest a novel approach to the understanding of schizophrenia pathophysiology, where a disruption in homeostatic adaptation of the cell underlies discreet changes seen at the protein level in this illness.

Purpose: Increased interictal concentrations of extracellular hippocampal glutamate have been implicated in the pathophysiology of temporal lobe epilepsy (TLE). Recent studies suggest that perturbations of the glutamate metabolizing enzymes glutamine synthetase (GS) and phosphate activated glutaminase (PAG) may underlie the glutamate excess in TLE. However, the molecular mechanism of the enzyme perturbations remains unclear. A better understanding of the regulatory mechanisms of GS and PAG could facilitate the discovery of novel therapeutics for TLE., Methods: We used in situ hybridization on histologic sections to assess the distribution and quantity of messenger RNA (mRNA) for GS and PAG in subfields of hippocampal formations from the following: (1) patients with TLE and concomitant hippocampal sclerosis, (2) patients with TLE and no hippocampal sclerosis, and (3) nonepilepsy autopsy subjects., Key Findings: GS mRNA was increased by ~50% in the CA3 in TLE patients without hippocampal sclerosis versus in TLE patients with sclerosis and in nonepilepsy subjects. PAG mRNA was increased by >100% in the subiculum in both TLE patient categories versus in nonepilepsy subjects. PAG mRNA was also increased in the CA1, CA2, CA3, and dentate hilus in TLE without hippocampal sclerosis versus in TLE with sclerosis. Finally, PAG mRNA was increased in the dentate gyrus in TLE with sclerosis versus in nonepilepsy subjects, and also increased in the hilus in TLE without sclerosis versus in TLE with sclerosis., Significance: These findings demonstrate complex changes in the expression of mRNAs for GS and PAG in the hippocampal formation in TLE, and raise the possibility that both transcriptional and posttranscriptional mechanisms may underlie the regulation of GS and PAG proteins in the epileptic brain., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia.

Schizophrenia is a devastating psychiatric illness that afflicts 1% of the population worldwide, resulting in substantial impact to patients, their families, and health care delivery systems. For many years, schizophrenia has been felt to be associated with dysregulated dopaminergic neurotransmission as a key feature of the pathophysiology of the illness. Although numerous studies point to dopaminergic abnormalities in schizophrenia, dopamine dysfunction cannot completely account for all of the symptoms seen in schizophrenia, and dopamine-based treatments are often inadequate and can be associated with serious side effects. More recently, converging lines of evidence have suggested that there are abnormalities of glutamate transmission in schizophrenia. Glutamatergic neurotransmission involves numerous molecules that facilitate glutamate release, receptor activation, glutamate reuptake, and other synaptic activities. Evidence for glutamatergic abnormalities in schizophrenia primarily has implicated the NMDA and AMPA subtypes of the glutamate receptor. The expression of these receptors and other molecules associated with glutamate neurotransmission has been systematically studied in the brain in schizophrenia. These studies have generally revealed region- and molecule-specific changes in glutamate receptor transcript and protein expression in this illness. Given that glutamatergic neurotransmission has been implicated in the pathophysiology of schizophrenia, recent drug development efforts have targeted the glutamate system. Much effort to date has focused on modulation of the NMDA receptor, although more recently other glutamate receptors and transporters have been the targets of drug development. These efforts have been promising thus far, and ongoing efforts to develop additional drugs that modulate glutamatergic neurotransmission are underway that may hold the potential for novel classes of more effective treatments for this serious psychiatric illness.

Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites. We hypothesized that alterations in AMPA receptor trafficking through the endoplasmic reticulum (ER) may also be altered in schizophrenia. Accordingly, we developed a technique to isolate and measure content of the ER from postmortem brain tissue. We used Western blot and electron microscopy to show that we isolated an ER enriched fraction. We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia. These data suggest that AMPA receptor trafficking through the ER is largely intact in schizophrenia.

Several lines of evidence point to alterations of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking in schizophrenia. Multiple proteins, including synapse-associated protein 97 (SAP97), glutamate receptor-interacting protein 1 (GRIP1), and N-ethylmaleimide sensitive factor (NSF), facilitate the forward trafficking of AMPA receptors toward the synapse. Once localized to the synapse, AMPA receptors are trafficked in a complex endosomal system. We hypothesized that alterations in the expression of these proteins and alterations in the subcellular localization of AMPA receptors in endosomes may contribute to the pathophysiology of schizophrenia. Accordingly, we measured protein expression of SAP97, GRIP1, and NSF in the dorsolateral prefrontal cortex and found an increase in the expression of SAP97 and GRIP1 in schizophrenia. To determine the subcellular localization of AMPA receptor subunits, we developed a technique to isolate early endosomes from post-mortem tissue. We found increased GluR1 receptor subunit protein in early endosomes in subjects with schizophrenia. Together, these data suggest that there is an alteration of forward trafficking of AMPA receptors as well as changes in the subcellular localization of an AMPA receptor subunit in schizophrenia.

The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) is a master regulator of metabolism in peripheral tissues, and it has been proposed that PGC-1alpha plays a similar role in the brain. Recent evidence suggests that PGC-1alpha is concentrated in GABAergic interneurons, so we investigated whether male and female PGC-1alpha -/- mice exhibit abnormalities in interneuron gene expression and/or function. We found a striking reduction in the expression of the Ca(2+)-binding protein parvalbumin (PV), but not other GABAergic markers, throughout the cerebrum in PGC-1alpha +/- and -/- mice. Furthermore, PGC-1alpha overexpression in cell culture was sufficient to robustly induce PV expression. Consistent with a reduction in PV rather than a loss of PV-expressing interneurons, spontaneous synaptic inhibition was not altered in PGC-1alpha -/- mice. However, evoked synaptic responses displayed less paired-pulse depression and dramatic facilitation in response to repetitive stimulation at the gamma frequency. PV transcript expression was also significantly reduced in retina and heart of PGC-1alpha -/- animals, suggesting that PGC-1alpha is required for proper expression of PV in multiple tissues. Together these findings indicate that PGC-1alpha is a novel regulator of interneuron gene expression and function and a potential therapeutic target for neurological disorders associated with interneuron dysfunction.

The hypothesis of N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia was initially based on observations that blockade of the NMDA subtype of glutamate receptor by noncompetitive antagonists, such as phencyclidine and ketamine, can lead to clinical symptoms similar to those present in schizophrenia. Recently, glutamate has also been implicated in the pathophysiology of the mood disorders. As impaired NMDA receptor activity may be the result of a primary defect in the NMDA receptors themselves, or secondary to dysfunction in the protein complexes that mediate their signaling, we measured expression of both NMDA subunits and associated postsynaptic density (PSD) proteins (PSD95, neurofilament-light (NF-L), and SAP102) transcripts in the dorsolateral prefrontal cortex in subjects with schizophrenia, bipolar disorder, major depression, and a comparison group using tissue from the Stanley Foundation Neuropathology Consortium. We found decreased NR1 expression in all three illnesses, decreased NR2A in schizophrenia and major depression, and decreased NR2C in schizophrenia. We found no changes of NR2B or NR2D. Receptor autoradiography revealed no alterations in receptor binding in any of the illnesses, indicating no change in total receptor number, but taken with the subunit data suggests abnormal receptor stoichiometry. In the same subjects, PSD95 was unchanged in all three illnesses, while reduced NF-L expression was found in schizophrenia, especially in large cells of layer V. SAP102 expression was reduced in bipolar disorder restricted to small cells of layer II and large cells of layer III in bipolar disorder. These alterations likely reflect altered signaling cascades associated with glutamate-mediated neurotransmission within specific cortical circuits in these psychiatric illnesses.

Altered glutamate signaling contributes to a myriad of neural disorders, including schizophrenia. While synaptic levels are intensely studied, nonvesicular release mechanisms, including cystine-glutamate exchange, maintain high steady-state glutamate levels in the extrasynaptic space. The existence of extrasynaptic receptors, including metabotropic group II glutamate receptors (mGluR), pose nonvesicular release mechanisms as unrecognized targets capable of contributing to pathological glutamate signaling. We tested the hypothesis that activation of cystine-glutamate antiporters using the cysteine prodrug N-acetylcysteine would blunt psychotomimetic effects in the rodent phencyclidine (PCP) model of schizophrenia. First, we demonstrate that PCP elevates extracellular glutamate in the prefrontal cortex, an effect that is blocked by N-acetylcysteine pretreatment. To determine the relevance of the above finding, we assessed social interaction and found that N-acetylcysteine reverses social withdrawal produced by repeated PCP. In a separate paradigm, acute PCP resulted in working memory deficits assessed using a discrete trial t-maze task, and this effect was also reversed by N-acetylcysteine pretreatment. The capacity of N-acetylcysteine to restore working memory was blocked by infusion of the cystine-glutamate antiporter inhibitor (S)-4-carboxyphenylglycine into the prefrontal cortex or systemic administration of the group II mGluR antagonist LY341495 indicating that the effects of N-acetylcysteine requires cystine-glutamate exchange and group II mGluR activation. Finally, protein levels from postmortem tissue obtained from schizophrenic patients revealed significant changes in the level of xCT, the active subunit for cystine-glutamate exchange, in the dorsolateral prefrontal cortex. These data advance cystine-glutamate antiporters as novel targets capable of reversing the psychotomimetic effects of PCP.

Pharmacological and anatomical evidence suggests that abnormal glutamate neurotransmission may be associated with the pathophysiology of schizophrenia and mood disorders. Medial temporal lobe structural alterations have been implicated in schizophrenia and to a lesser extent in mood disorders. To comprehensively examine the ionotropic glutamate receptors in these illnesses, we used in situ hybridization to determine transcript expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor subunits in the medial temporal lobe of subjects with schizophrenia, bipolar disorder (BD), or major depression (MDD). We used receptor autoradiography to assess changes in glutamate receptor binding in the same subjects. Our results indicate that there are region- and disorder-specific abnormalities in the expression of ionotropic glutamate receptor subunits in schizophrenia and mood disorders. We did not find any changes in transcript expression in the hippocampus. In the entorhinal cortex, most changes in glutamate receptor expression were associated with BD, with decreased GluR2, GluR3, and GluR6 mRNA expression. In the perirhinal cortex we detected decreased expression of GluR5 in all three diagnoses, of GluR1, GluR3, NR2B in both BD and MDD, and decreased NR1 and NR2A in BD and MDD, respectively. Receptor binding showed NMDA receptor subsites particularly affected in the hippocampus, where MK801 binding was reduced in schizophrenia and BD, and MDL105,519 and CGP39653 binding were increased in BD and MDD, respectively. In the hippocampus AMPA and kainate binding were not changed. We found no changes in the entorhinal and perirhinal cortices. These data suggest that glutamate receptor expression is altered in the medial temporal lobe in schizophrenia and the mood disorders. We propose that disturbances in glutamate-mediated synaptic transmission in the medial temporal lobe are important factors in the pathophysiology of these severe psychiatric illnesses.

Abnormal expression of the N-methyl-D-Aspartate (NMDA) receptor and its interacting molecules of the postsynaptic density (PSD) are thought to be involved in the pathophysiology of schizophrenia. Frontal regions of neocortex including dorsolateral prefrontal (DLPFC) and anterior cingulate cortex (ACC) are essential for cognitive and behavioral functions that are affected in schizophrenia. In this study, we have measured protein expression of two alternatively spliced isoforms of the NR1 subunit (NR1C2 and NR1C2') as well as expression of the NR2A-D subunits of the NMDA receptor in DLPFC and ACC in post-mortem samples from elderly schizophrenic patients and a comparison group. We found significantly increased expression of NR1C2' but not of NR1C2 in ACC, suggesting altered NMDA receptor cell membrane expression in this cortical area. We did not find significant changes in the expression of either of the NR1 isoforms in DLPFC. We did not detect changes of any of the NR2 subunits studied in either cortical area. In addition, we studied expression of the NMDA-interacting PSD molecules NF-L, SAP102, PSD-95 and PSD-93 in ACC and DLPFC at both transcriptional and translational levels. We found significant changes in the expression of NF-L in DLPFC, and PSD-95 and PSD-93 in ACC; increased transcript expression was associated with decreased protein expression, suggesting abnormal translation and/or accelerated protein degradation of these molecules in schizophrenia. Our findings suggest abnormal regional processing of the NMDA receptor and its associated PSD molecules, possibly involving transcription, translation, trafficking and protein stability in cortical areas in schizophrenia.

Post-mortem studies have yet to produce consistent findings on cortical glutamatergic markers in schizophrenia; therefore, it is not possible to fully understand the role of abnormal glutamatergic function in the pathology of the disorder. To better understand the changes in cortical glutamatergic markers in schizophrenia, we measured the binding of radioligands to the ionotropic glutamate receptors (N-methyl D-aspartate, [3H]CGP39653, [3H]MK-801), amino-3-hydroxy-5-methyl-4-isoxazole ([3H]AMPA), kainate ([3H]kainate), and the high-affinity glutamate uptake site ([3H]aspartate) using in situ radioligand binding with autoradiography and levels of mRNA for kainate receptors using in situ hybridization in the dorsolateral prefrontal cortex from 20 subjects with schizophrenia and 20 controls matched for age and sex. Levels of [3H]kainate binding were significantly decreased in cortical laminae I-II (p = 0.01), III-IV (p < 0.05), and V-VI (p < 0.01) from subjects with schizophrenia. By contrast, levels of [3H]MK-801, [3H]AMPA, [3H]aspartate, or [3H]CGP39653 binding did not differ between the diagnostic cohorts. Levels of mRNA for the GluR5 subunit were decreased overall (p < 0.05), with no changes in levels of mRNA for GluR6, GluR7, KA1, or KA2 in tissue from subjects with schizophrenia. These data indicate that the decreased number of kainate receptors in the dorsolateral prefrontal cortex in schizophrenia may result, in part, from reduced expression of the GluR5 receptor subunits.

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haem-binding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haem-regulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

Several lines of investigation support a hypothesis of glutamatergic dysfunction in schizophrenia, including our recent reports of altered NMDA receptor subunit and associated intracellular protein transcripts in the thalamus of elderly patients with schizophrenia. In the present study, we used in situ hybridization to measure the expression of NMDA subunits (NR1, NR2A-D), and associated intracellular proteins (NF-L, PSD95, and SAP102) in a second, younger cohort from the Stanley Foundation Neuropathology Consortium, which included patients with both schizophrenia and affective disorders. We wanted to determine whether glutamatergic abnormalities in the thalamus in schizophrenia are present at younger ages, and whether these abnormalities occur in other psychiatric illnesses. In the present work, we observed increased expression of NMDA NR2B subunit transcripts, and decreased expression of all three associated postsynaptic density protein transcripts in schizophrenia. We also found evidence of glutamatergic dysfunction in the thalamus in affective disorders, particularly in bipolar disorder. In particular, we found decreased NF-L, PSD95, and SAP102 transcripts in bipolar disorder, and decreased SAP102 levels in major depression. Interestingly, one of the most consistent findings across diagnostic groups was an abnormality of intracellular signaling molecules that are linked to the NMDA receptor, rather than changes in the receptor subunits themselves. PSD95 and similar scaffolding molecules link the NMDA receptor with intracellular enzymes that mediate signaling, and also provide a physical link between different neurotransmitter systems to coordinate and integrate information from multiple effector systems. Abnormalities of PSD95-like molecules and other intracellular signaling machinery may contribute to dysregulated communication between multiple neurotransmitter systems (such as glutamatergic and dopaminergic systems) that are potentially involved in the neurobiology of schizophrenia and affective disorders., (Copyright 2004 Nature Publishing Group)

Because abnormalities of glutamatergic neurotransmission in psychiatric illness are likely not limited to glutamate receptor expression, we investigated expression of excitatory amino acid transporters (EAATs) in the striatum. The EAATs, normally expressed in both glia (EAAT1 and EAAT2) and neurons (EAAT3 and EAAT4), have previously been implicated in Huntington's disease, amyotrophic lateral sclerosis, and schizophrenia. In this study, we investigated striatal expression of transcripts encoding EAATs in tissue from mood disordered and schizophrenic subjects. With probes designed for the human EAAT1, EAAT2, EAAT3, and EAAT4 transcripts, we performed in situ hybridization and detected decreased expression of EAAT3 and EAAT4 transcripts in the striatum in bipolar disorder. We also detected decreased EAAT3 transcript expression in schizophrenia and decreased EAAT4 transcript expression in major depressive disorder. These results suggest that changes in striatal transporter mRNA expression are restricted to neuronal EAATs and extend the body of evidence implicating abnormal glutamatergic neurotransmission in schizophrenia and mood disorders.

Abnormalities of molecules associated with the glutamate synapse have been implicated in the pathophysiology of schizophrenia. Of the many glutamate receptors, those most commonly suggested to be involved in schizophrenia are the ionotropic subtypes, the NMDA, AMPA, and kainate receptors. Both the NMDA and AMPA subtypes have been extensively studied in postmortem brains of individuals with schizophrenia, but relatively little is known about the expression of the kainate subtype of glutamate receptor. In this study, we have determined cortical and striatal kainate receptor expression in brains from persons with schizophrenia and a comparison group, using both in situ hybridization and receptor autoradiography. At the level of subunit mRNA expression, a shift in subunit stoichiometry was evident in multiple regions of the prefrontal cortex, with increased expression of gluR7 mRNA and decreased expression of KA2 mRNA. Decreased kainate receptor binding was also observed in the subjects with schizophrenia, but was restricted to infragranular laminae of the prefrontal cortex. No differences in kainate receptor binding or subunit mRNA levels were found in striatum or occipital cortex, suggesting that these findings may be restricted to association cortex. These data add to the growing literature implicating ionotropic glutamate receptor disturbances in schizophrenia, and indicate that in addition to AMPA and NMDA receptors, the kainate receptors are also abnormally expressed in this illness.

The novel serotonin receptor subtypes, 5-HT6 and 5-HT7, are located in limbic regions and have nanomolar affinities for atypical antipsychotics. These factors have led some to speculate about the involvement of 5-HT6 and 5-HT7 receptors in schizophrenia. However, relatively little is known about these receptor subtypes, including the regulation of their expression in limbic regions. In particular, the regulation of extracellular serotonin levels in the striatum and hippocampal formation by glutamate receptors led us to examine the effects of systemic ionotropic glutamate receptor modulator treatment on 5-HT6 and 5-HT7 receptor expression in these regions. MK-801 treatment induced a dose-dependent decrease in striatal 5-HT6 receptor mRNA levels; similarly, both aniracetam and NBQX treatments also led to decreases in striatal 5-HT6 receptor mRNA levels. Hippocampal 5-HT6 and 5-HT7 receptor expression were not dramatically affected by any of the treatments. To our knowledge, this is the first demonstration of the regulation of striatal 5-HT6 receptor mRNA expression, and provides neurochemical anatomical evidence for the interaction of serotonergic and glutamatergic systems. Furthermore, although these two neurotransmitter systems are separately implicated in schizophrenia, the glutamatergic regulation of the expression of a receptor subtype associated with schizophrenia suggests that alterations in serotonin receptor expression in schizophrenia may result, in part, from altered glutamatergic activity.

The prevalence of smoking is markedly elevated in schizophrenia. Low smoking cessation rates and reports that some smokers with schizophrenia experience an acute increase in symptoms during attempts to quit smoking, suggest a self-medication model. Alternatively, smoking may modulate medication side effects. The effects of treated and untreated smoking abstinence on psychotic symptoms and medication side effects were examined in this study. Nineteen outpatients with schizophrenia or schizoaffective disorder participated in a randomized, double-blind, balanced crossover study: 1 day of ad libitum smoking followed by 3 days of acute smoking abstinence while wearing 22 mg/day active or placebo transdermal nicotine patches, with a return to 3 days of smoking between patch conditions. Daily symptom and side-effect ratings, nicotine and cotinine blood levels were collected. Twelve subjects completed the study. Neither positive symptoms nor mood symptoms changed. An increase in negative symptoms during the first abstinent day occurred in both placebo and active patch conditions, but was not sustained over subsequent abstinent days. Despite physiological signs of withdrawal, completers did not endorse increased nicotine withdrawal symptoms. Dropouts reported higher withdrawal symptoms, but also had no increase in psychiatric symptoms in either phase of the study. Of note, dyskinesias decreased during abstinence and placebo patch treatment, but increased during abstinence and the active patch conditions. Acute exacerbation of psychiatric symptoms is an unlikely explanation for any difficulty smokers with schizophrenia have in early abstinence.

The dopamine hypothesis of schizophrenia has recently evolved into a model of dysfunctional integration between cortical and subcortical dopaminergic activity. Anatomical data suggest that regional alterations in dopaminergic activity may be linked by means of the rich glutamatergic innervation of the striatum by corticostriatal projections, suggesting a potential role for glutamatergic dysfunction in schizophrenia. Although pharmacological data have implicated the NMDA subtype of glutamate receptor in this illness, disturbance in AMPA receptor expression could potentially lead to the NMDA receptor hypoactivity hypothesized in schizophrenia. To address this possibility, we examined AMPA receptor binding and subunit mRNA levels in prefrontal cortex and striatum of schizophrenics and matched controls. There were no significant differences in AMPA receptor binding or subunit mRNA levels in either prefrontal cortical or striatal regions of schizophrenics. Furthermore, AMPA receptor expression did not seem to be regulated by chronic antipsychotic drug exposure, when neuroleptic treated and drug-free schizophrenics were analyzed separately. These data do not support a role for altered AMPA receptor expression in cortex and striatum in schizophrenia.

The dopamine (DA) hypothesis of schizophrenia, which was based largely on evidence that pharmacological manipulations of DA systems influence the symptoms of schizophrenia, is undergoing a transformation due to our knowledge of the anatomy and pharmacology of additional subtypes of dopamine receptors. New research links the multiplicity of D2-like receptors to divergent neuroanatomic sites of suspected pathology in schizophrenia. We hypothesize that this research suggests that D2 receptors in the basal ganglia are the likely site of extrapyramidal symptoms and not antipsychotic effects. Rather, D3 receptors of the mesolimbic system are a likely site of antipsychotic effects, and D2 and D4 receptors in the medial temporal lobe and limbic cortical areas are the sites of additional antipsychotic effects. This work also suggests that divergent DA receptor circuits are likely associated with the pathophysiology of this disorder.

The distributions of the transcripts encoding the five dopamine receptors have been determined in the human striatum and selected regions of the neocortex. In the striatum significant levels of dopamine receptor expression are restricted to the D1, D2, and D3 receptors. D1 and D2 receptor messenger ribonucleic acids (mRNAs) are homogeneously distributed throughout the caudate, putamen, and nucleus accumbens. D3 receptor mRNA is particularly enriched in the nucleus accumbens, with moderate levels in the ventral putamen. In the prefrontal cortex D1 and D4 receptor mRNAs are the most abundant, although the other three transcripts are seen at lower levels. A similar pattern is seen in the temporal neocortex. In the occipital cortex, D1 receptor mRNA is the most abundant, D3 the rarest, while the other three transcripts are present at modest levels of expression. These data add to a growing understanding of the neuroanatomical distribution of these transcripts in the human brain. They are essential to understand in the context of the limbic circuitry of the brain, as new hypotheses of dysfunction of dopaminergic neurotransmission are advanced in psychiatry and as these receptor subtypes are targeted for development of novel pharmacological treatments.

The tone and regulation of the brain dopaminergic projections are, in part, determined by the presence or absence of dopamine (DA) autoreceptors: rate of DA synthesis and turnover, as well as both pattern and rate of neuronal firing, are modulated by the expression and activity of these autoreceptors. The expression of dopaminergic receptors in the midbrain DA cell groups, presumably reflecting DA autoreceptors, was determined in the brains of the rat, Old World monkey, and human. In the rat, both the substantia nigra (A9) and the ventral tegmental area (A10) appear to express DA autoreceptors. In the monkey and human, however, only the projections arising from the substantia nigra express these receptors; the limbic projections originating in the ventral tegmental area lack this substrate for DA autoregulation. These results indicate that in the human, the nigrostriatal and mesocorticolimbic dopamine systems may be differentially autoregulated.

The distributions of the messenger RNA molecules encoding the five known dopamine receptors have been determined in the medial temporal lobe of postmortem human brain. All five receptor mRNAs are present in temporal lobe structures, although their distributions are heterogeneous. The D1-like receptors, D1 and D5, have strikingly dissimilar distributions. D1 receptor mRNA is abundant in temporal neocortex but is rare elsewhere. D5 receptor message, however, is seen in the hippocampus, subicular complex, and in temporal cortex. The D2-like receptors have similar distributions: D2, D3, and D4 receptor mRNAs are all identifiable in the hippocampal formation and in the cortical regions of the medial temporal lobe. Distinct patterns of relative regional concentrations for each message are observed, however, suggesting a neuroanatomical substrate for potential differences in dopaminergic regulation within discrete regions of the medial temporal lobe. These results provide a description of the distribution of these receptor mRNAs in normal humans and suggest multiple levels of complexity as well as regulation of the medial temporal lobe dopamine projection.

Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired.

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.

The filling defects of a particular lymphosarcoma of the colon or rectum may present an appearance identical to that of a carcinoma in the same location. In general, lymphosarcomas tend to be longer, more distensible, less sharply demarcated, more frequently polypoid, and to feel larger as palpable masses. The filling defects are infrequently of the annular "napkin ring" appearance, and occasionally the involved segment is dilated.

In a survey of its use in 198 cases of penetrating and nonpenetrating abdominal trauma causing various kinds of lesions, roentgenographic examination was found to be a considerable diagnostic aid. Consultation with a radiologist before, during and after the examination would seem indicated. The general plan of examination included anteroposterior projections with the patient erect, recumbent and in the left lateral decubitus positions, plus a posteroanterior film of the chest with the patient erect. Techniques varied with the condition of the patient and the nature of the lesion clinically suspected. Special procedures were done as dictated by the clinical and radiologic findings. Studies designed to demonstrate displacement of gastrointestinal and urinary organs should be made in anteroposterior and lateral projections. Factors that were important in determining the site of internal lesions from radiographic evidence were the site of associated fractures, reactive ileus, the position of foreign bodies, and by far the most important the roentgen abnormalities caused by escaped gas, blood, other fluids or contrast media, which caused abnormal densities, displacements of organs and blurring or obliteration of outlines normally visible.

Ascaris infestations may be found in California, particularly in patients who have migrated from endemic regions. Clinical manifestations include vague abdominal pains, unexplained fever, anemia, malaise and upper respiratory tract infections. Intestinal obstruction and infections are among the severe complications that can occur. Diagnosis is made by the observation of worms or ova in the feces, and occasionally by roentgenographic manifestations.