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Assessing the effects of antipsychotic medications on schizophrenia functional analysis: a postmortem proteome study

Antipsychotic drugs (APDs) are effective in treating positive symptoms of schizophrenia (SCZ). However, they have a substantial impact on postmortem studies. As most cohorts lack samples from drug-naive patients, many studies, rather than understanding SCZ pathophysiology, are analyzing the drug effects. We hypothesized that comparing SCZ-altered and APD-influenced signatures derived from the same cohort can provide better insight into SCZ pathophysiology. For this, we performed LCMS-based proteomics on dorsolateral prefrontal cortex (DLPFC) samples from control and SCZ subjects and used statistical approaches to identify SCZ-altered and APD-influenced proteomes, validated experimentally using independent cohorts and published datasets. Functional analysis of both proteomes was contrasted at the biological-pathway, cell-type, subcellular-synaptic, and drug-target levels. In silico validation revealed that the SCZ-altered proteome was conserved across several studies from the DLPFC and other brain areas. At the pathway level, SCZ influenced changes in homeostasis, signal-transduction, cytoskeleton, and dendrites, whereas APD influenced changes in synaptic-signaling, neurotransmitter-regulation, and immune-system processes. At the cell-type level, the SCZ-altered and APD-influenced proteomes were associated with two distinct striatum-projecting layer-5 pyramidal neurons regulating dopaminergic-secretion. At the subcellular synaptic level, compensatory pre- and postsynaptic events were observed. At the drug-target level, dopaminergic processes influenced the SCZ-altered upregulated-proteome, whereas nondopaminergic and a diverse array of non-neuromodulatory mechanisms influenced the downregulated-proteome. Previous findings were not independent of the APD effect and thus require re-evaluation. We identified a hyperdopaminergic cortex and drugs targeting the cognitive SCZ-symptoms and discussed their influence on SCZ pathology in the context of the cortico-striatal pathway.

Hepatic kinome atlas: An in-depth identification of kinase pathways in liver fibrosis of humans and rodents

BACKGROUND AND AIMS: Resolution of pathways that converge to induce deleterious effects in hepatic diseases, such as in the later stages, have potential antifibrotic effects that may improve outcomes. We aimed to explore whether humans and rodents display similar fibrotic signaling networks. APPROACH AND RESULTS: We assiduously mapped kinase pathways using 340 substrate targets, upstream bioinformatic analysis of kinase pathways, and over 2000 random sampling iterations using the PamGene PamStation kinome microarray chip technology. Using this technology, we characterized a large number of kinases with altered activity in liver fibrosis of both species. Gene expression and immunostaining analyses validated many of these kinases as bona fide signaling events. Surprisingly, the insulin receptor emerged as a considerable protein tyrosine kinase that is hyperactive in fibrotic liver disease in humans and rodents. Discoidin domain receptor tyrosine kinase, activated by collagen that increases during fibrosis, was another hyperactive protein tyrosine kinase in humans and rodents with fibrosis. The serine/threonine kinases found to be the most active in fibrosis were dystrophy type 1 protein kinase and members of the protein kinase family of kinases. We compared the fibrotic events over four models: humans with cirrhosis and three murine models with differing levels of fibrosis, including two models of fatty liver disease with emerging fibrosis. The data demonstrate a high concordance between human and rodent hepatic kinome signaling that focalizes, as shown by our network analysis of detrimental pathways. CONCLUSIONS: Our findings establish a comprehensive kinase atlas for liver fibrosis, which identifies analogous signaling events conserved among humans and rodents.

Schizophrenia: a disorder of broken brain bioenergetics

A substantial and diverse body of literature suggests that the pathophysiology of schizophrenia is related to deficits of bioenergetic function. While antipsychotics are an effective therapy for the management of positive psychotic symptoms, they are not efficacious for the complete schizophrenia symptom profile, such as the negative and cognitive symptoms. In this review, we discuss the relationship between dysfunction of various metabolic pathways across different brain regions in relation to schizophrenia. We contend that several bioenergetic subprocesses are affected across the brain and such deficits are a core feature of the illness. We provide an overview of central perturbations of insulin signaling, glycolysis, pentose-phosphate pathway, tricarboxylic acid cycle, and oxidative phosphorylation in schizophrenia. Importantly, we discuss pharmacologic and nonpharmacologic interventions that target these pathways and how such interventions may be exploited to improve the symptoms of schizophrenia.

The active kinome: The modern view of how active protein kinase networks fit in biological research

Biological regulatory networks are dynamic, intertwined, and complex systems making them challenging to study. While quantitative measurements of transcripts and proteins are key to investigate the state of a biological system, they do not inform the “active” state of regulatory networks. In consideration of that fact, “functional” proteomics assessments are needed to decipher active regulatory processes. Phosphorylation, a key post-translation modification, is a reversible regulatory mechanism that controls the functional state of proteins. Recent advancements of high-throughput protein kinase activity profiling platforms allow for a broad assessment of protein kinase networks in complex biological systems. In conjunction with sophisticated computational modeling techniques, these profiling platforms provide datasets that inform the active state of regulatory systems in disease models and highlight potential drug targets. Taken together, system-wide profiling of protein kinase activity has become a critical component of modern molecular biology research and presents a promising avenue for drug discovery.

Editorial overview: Neuroscience: Advances in the field

KRSA: An R package and R Shiny web application for an end-to-end upstream kinase analysis of kinome array data

Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns.

Strategies to identify candidate repurposable drugs: COVID-19 treatment as a case example

Drug repurposing is an invaluable strategy to identify new uses for existing drug therapies that overcome many of the time and financial costs associated with novel drug development. The COVID-19 pandemic has driven an unprecedented surge in the development and use of bioinformatic tools to identify candidate repurposable drugs. Using COVID-19 as a case study, we discuss examples of machine-learning and signature-based approaches that have been adapted to rapidly identify candidate drugs. The Library of Integrated Network-based Signatures (LINCS) and Connectivity Map (CMap) are commonly used repositories and have the advantage of being amenable to use by scientists with limited bioinformatic training. Next, we discuss how these recent advances in bioinformatic drug repurposing approaches might be adapted to identify repurposable drugs for CNS disorders. As the development of novel therapies that successfully target the cause of neuropsychiatric and neurological disorders has stalled, there is a pressing need for innovative strategies to treat these complex brain disorders. Bioinformatic approaches to identify repurposable drugs provide an exciting avenue of research that offer promise for improved treatments for CNS disorders.

Cellular, molecular, and therapeutic characterization of pilocarpine-induced temporal lobe epilepsy

Animal models have expanded our understanding of temporal lobe epilepsy (TLE). However, translating these to cell-specific druggable hypotheses is not explored. Herein, we conducted an integrative insilico-analysis of an available transcriptomics dataset obtained from animals with pilocarpine-induced-TLE. A set of 119 genes with subtle-to-moderate impact predicted most forms of epilepsy with ~ 97% accuracy and characteristically mapped to upregulated homeostatic and downregulated synaptic pathways. The deconvolution of cellular proportions revealed opposing changes in diverse cell types. The proportion of nonneuronal cells increased whereas that of interneurons, except for those expressing vasoactive intestinal peptide (Vip), decreased, and pyramidal neurons of the cornu-ammonis (CA) subfields showed the highest variation in proportion. A probabilistic Bayesian-network demonstrated an aberrant and oscillating physiological interaction between nonneuronal cells involved in the blood-brain-barrier and Vip interneurons in driving seizures, and their role was evaluated insilico using transcriptomic changes induced by valproic-acid, which showed opposing effects in the two cell-types. Additionally, we revealed novel epileptic and antiepileptic mechanisms and predicted drugs using causal inference, outperforming the present drug repurposing approaches. These well-powered findings not only expand the understanding of TLE and seizure oscillation, but also provide predictive biomarkers of epilepsy, cellular and causal micro-circuitry changes associated with it, and a drug-discovery method focusing on these events.

Fluoxetine as an anti-inflammatory therapy in SARS-CoV-2 infection

Hyperinflammatory response caused by infections such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases organ failure, intensive care unit admission, and mortality. Cytokine storm in patients with Coronavirus Disease 2019 (COVID-19) drives this pattern of poor clinical outcomes and is dependent upon the activity of the transcription factor complex nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) and its downstream target gene interleukin 6 (IL6) which interacts with IL6 receptor (IL6R) and the IL6 signal transduction protein (IL6ST or gp130) to regulate intracellular inflammatory pathways. In this study, we compare transcriptomic signatures from a variety of drug-treated or genetically suppressed (i.e. knockdown) cell lines in order to identify a mechanism by which antidepressants such as fluoxetine demonstrate non-serotonergic, anti-inflammatory effects. Our results demonstrate a critical role for IL6ST and NF-kappaB Subunit 1 (NFKB1) in fluoxetine’s ability to act as a potential therapy for hyperinflammatory states such as asthma, sepsis, and COVID-19.

Identification of candidate repurposable drugs to combat COVID-19 using a signature-based approach

The COVID-19 pandemic caused by the novel SARS-CoV-2 is more contagious than other coronaviruses and has higher rates of mortality than influenza. Identification of effective therapeutics is a crucial tool to treat those infected with SARS-CoV-2 and limit the spread of this novel disease globally. We deployed a bioinformatics workflow to identify candidate drugs for the treatment of COVID-19. Using an “omics” repository, the Library of Integrated Network-Based Cellular Signatures (LINCS), we simultaneously probed transcriptomic signatures of putative COVID-19 drugs and publicly available SARS-CoV-2 infected cell lines to identify novel therapeutics. We identified a shortlist of 20 candidate drugs: 8 are already under trial for the treatment of COVID-19, the remaining 12 have antiviral properties and 6 have antiviral efficacy against coronaviruses specifically, in vitro. All candidate drugs are either FDA approved or are under investigation. Our candidate drug findings are discordant with (i.e., reverse) SARS-CoV-2 transcriptome signatures generated in vitro, and a subset are also identified in transcriptome signatures generated from COVID-19 patient samples, like the MEK inhibitor selumetinib. Overall, our findings provide additional support for drugs that are already being explored as therapeutic agents for the treatment of COVID-19 and identify promising novel targets that are worthy of further investigation.

Using protein turnover to expand the applications of transcriptomics

RNA expression and protein abundance are often at odds when measured in parallel, raising questions about the functional implications of transcriptomics data. Here, we present the concept of persistence, which attempts to address this challenge by combining protein half-life data with RNA expression into a single metric that approximates protein abundance. The longer a protein’s half-life, the more influence it can have on its surroundings. This data offers a valuable opportunity to gain deeper insight into the functional meaning of transcriptome changes. We demonstrate the application of persistence using schizophrenia (SCZ) datasets, where it greatly improved our ability to predict protein abundance from RNA expression. Furthermore, this approach successfully identified persistent genes and pathways known to have impactful changes in SCZ. These results suggest that persistence is a valuable metric for improving the functional insight offered by transcriptomics data, and extended application of this concept could advance numerous research fields.

Similarities and dissimilarities between psychiatric cluster disorders

The common molecular mechanisms underlying psychiatric disorders are not well understood. Prior attempts to assess the pathological mechanisms responsible for psychiatric disorders have been limited by biased selection of comparable disorders, datasets/cohort availability, and challenges with data normalization. Here, using DisGeNET, a gene-disease associations database, we sought to expand such investigations in terms of number and types of diseases. In a top-down manner, we analyzed an unbiased cluster of 36 psychiatric disorders and comorbid conditions at biological pathway, cell-type, drug-target, and chromosome levels and deployed density index, a novel metric to quantify similarities (close to 1) and dissimilarities (close to 0) between these disorders at each level. At pathway level, we show that cognition and neurotransmission drive the similarity and are involved across all disorders, whereas immune-system and signal-response coupling (cell surface receptors, signal transduction, gene expression, and metabolic process) drives the dissimilarity and are involved with specific disorders. The analysis at the drug-target level supports the involvement of neurotransmission-related changes across these disorders. At cell-type level, dendrite-targeting interneurons, across all layers, are most involved. Finally, by matching the clustering pattern at each level of analysis, we showed that the similarity between the disorders is influenced most at the chromosomal level and to some extent at the cellular level. Together, these findings provide first insights into distinct cellular and molecular pathologies, druggable mechanisms associated with several psychiatric disorders and comorbid conditions and demonstrate that similarities between these disorders originate at the chromosome level and disperse in a bottom-up manner at cellular and pathway levels.

Role of Astrocytes in Major Neuropsychiatric Disorders

Astrocytes are the primary homeostatic cells of the central nervous system, essential for normal neuronal development and function, metabolism and response to injury and inflammation. Here, we review postmortem studies examining changes in astrocytes in subjects diagnosed with the neuropsychiatric disorders schizophrenia (SCZ), major depressive disorder (MDD), and bipolar disorder (BPD). We discuss the astrocyte-related changes described in the brain in these disorders and the potential effects of psychotropic medication on these findings. Finally, we describe emerging tools that can be used to study the role of astrocytes in neuropsychiatric illness.

Emerging Kinase Therapeutic Targets in Pancreatic Ductal Adenocarcinoma and Pancreatic Cancer Desmoplasia

Kinase drug discovery represents an active area of therapeutic research, with previous pharmaceutical success improving patient outcomes across a wide variety of human diseases. In pancreatic ductal adenocarcinoma (PDAC), innovative pharmaceutical strategies such as kinase targeting have been unable to appreciably increase patient survival. This may be due, in part, to unchecked desmoplastic reactions to pancreatic tumors. Desmoplastic stroma enhances tumor development and progression while simultaneously restricting drug delivery to the tumor cells it protects. Emerging evidence indicates that many of the pathologic fibrotic processes directly or indirectly supporting desmoplasia may be driven by targetable protein tyrosine kinases such as Fyn-related kinase (FRK); B lymphoid kinase (BLK); hemopoietic cell kinase (HCK); ABL proto-oncogene 2 kinase (ABL2); discoidin domain receptor 1 kinase (DDR1); Lck/Yes-related novel kinase (LYN); ephrin receptor A8 kinase (EPHA8); FYN proto-oncogene kinase (FYN); lymphocyte cell-specific kinase (LCK); tec protein kinase (TEC). Herein, we review literature related to these kinases and posit signaling networks, mechanisms, and biochemical relationships by which this group may contribute to PDAC tumor growth and desmoplasia.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

A role for endothelial NMDA receptors in the pathophysiology of schizophrenia

Numerous genetic and postmortem studies link N-methyl-d-aspartate receptor (NMDAR) dysfunction with schizophrenia, forming the basis of the popular glutamate hypothesis. Neuronal NMDAR abnormalities are consistently reported from both basic and clinical experiments, however, non-neuronal cells also contain NMDARs, and are rarely, if ever, considered in the discussion of glutamate action in schizophrenia. We offer an examination of recent discoveries elucidating the actions and consequences of NMDAR activation in the neuroendothelium. While there has been mixed literature regarding blood flow alterations in the schizophrenia brain, in this review, we posit that some common findings may be explained by neuroendothelial NMDAR dysfunction. In particular, we emphasize that endothelial NMDARs are key mediators of neurovascular coupling, where increased neuronal activity leads to increased blood flow. Based on the broad conclusions that hypoperfusion is a neuroanatomical finding in schizophrenia, we discuss potential mechanisms by which endothelial NMDARs contribute to this disorder. We propose that endothelial NMDAR dysfunction can be a primary cause of neurovascular abnormalities in schizophrenia. Importantly, functional MRI studies using BOLD signal as a proxy for neuron activity should be considered in a new light if neurovascular coupling is impaired in schizophrenia. This review is the first to propose that NMDARs in non-excitable cells play a role in schizophrenia.

KEOPS complex expression in the frontal cortex in major depression and schizophrenia

OBJECTIVES: Recently, the presence of a complete five subunit Kinase, Endopeptidase and Other Proteins of small Size (KEOPS) complex was confirmed in humans. The highly conserved KEOPS protein complex has established roles in tRNA modification, protein translation and telomere homeostasis in yeast, but little is known about KEOPS mRNA expression and function in human brain and disease. Here, we characterise KEOPS expression in post-mortem tissue from subjects diagnosed with major depression (MDD) and schizophrenia and assess whether KEOPS is associated with telomere length dysregulation in neuropsychiatric disorders. METHODS: We assessed mRNA expression of KEOPS complex subunits TP53RK, TPRKB, GON7, LAGE3, OSGEP, and OSGEP mitochondrial ortholog OSGEPL1 in the dorsolateral prefrontal cortex (DLPFC) of subjects with MDD, schizophrenia and matched non-psychiatrically ill controls (n = 20 per group) using qPCR. We conducted bioinformatic analysis using Kaleidoscope, data mining post-mortem transcriptomic datasets to characterise KEOPS expression in human brain. Finally, we assayed relative telomere length in the DLPFC using a qPCR-based assay and carried out correlation analysis with KEOPS subunit mRNA expression to determine if the KEOPS complex is associated with telomere length dysregulation in neuropsychiatric disorders. RESULTS: There were no significant changes in KEOPS mRNA expression in the DLPFC in MDD or schizophrenia compared to non-psychiatrically ill controls. Relative telomere length was not significantly altered in MDD or schizophrenia, nor was there an association between relative telomere length and KEOPS subunit gene expression in these subjects. CONCLUSIONS: This study is the first to describe KEOPS complex expression in post-mortem brain and neuropsychiatric disorders. KEOPS subunit mRNA expression is not significantly altered in the DLPFC in MDD or schizophrenia. Unlike in yeast, the KEOPS complex does not appear to play a role in telomere length regulation in humans or in neuropsychiatric disorders.

KEOPS complex expression in the frontal cortex in major depression and schizophrenia

OBJECTIVES: Recently, the presence of a complete five subunit Kinase, Endopeptidase and Other Proteins of small Size (KEOPS) complex was confirmed in humans. The highly conserved KEOPS protein complex has established roles in tRNA modification, protein translation and telomere homeostasis in yeast, but little is known about KEOPS mRNA expression and function in human brain and disease. Here, we characterise KEOPS expression in post-mortem tissue from subjects diagnosed with major depression (MDD) and schizophrenia and assess whether KEOPS is associated with telomere length dysregulation in neuropsychiatric disorders. METHODS: We assessed mRNA expression of KEOPS complex subunits TP53RK, TPRKB, GON7, LAGE3, OSGEP, and OSGEP mitochondrial ortholog OSGEPL1 in the dorsolateral prefrontal cortex (DLPFC) of subjects with MDD, schizophrenia and matched non-psychiatrically ill controls (n = 20 per group) using qPCR. We conducted bioinformatic analysis using Kaleidoscope, data mining post-mortem transcriptomic datasets to characterise KEOPS expression in human brain. Finally, we assayed relative telomere length in the DLPFC using a qPCR-based assay and carried out correlation analysis with KEOPS subunit mRNA expression to determine if the KEOPS complex is associated with telomere length dysregulation in neuropsychiatric disorders. RESULTS: There were no significant changes in KEOPS mRNA expression in the DLPFC in MDD or schizophrenia compared to non-psychiatrically ill controls. Relative telomere length was not significantly altered in MDD or schizophrenia, nor was there an association between relative telomere length and KEOPS subunit gene expression in these subjects. CONCLUSIONS: This study is the first to describe KEOPS complex expression in post-mortem brain and neuropsychiatric disorders. KEOPS subunit mRNA expression is not significantly altered in the DLPFC in MDD or schizophrenia. Unlike in yeast, the KEOPS complex does not appear to play a role in telomere length regulation in humans or in neuropsychiatric disorders.

Consequences of NMDA receptor deficiency can be rescued in the adult brain

N-methyl-D-aspartate receptors (NMDARs) are required to shape activity-dependent connections in the developing and adult brain. Impaired NMDAR signalling through genetic or environmental insults causes a constellation of neurodevelopmental disorders that manifest as intellectual disability, epilepsy, autism, or schizophrenia. It is not clear whether the developmental impacts of NMDAR dysfunction can be overcome by interventions in adulthood. This question is paramount for neurodevelopmental disorders arising from mutations that occur in the GRIN genes, which encode NMDAR subunits, and the broader set of mutations that disrupt NMDAR function. We developed a mouse model where a congenital loss-of-function allele of Grin1 can be restored to wild type by gene editing with Cre recombinase. Rescue of NMDARs in adult mice yields surprisingly robust improvements in cognitive functions, including those that are refractory to treatment with current medications. These results suggest that neurodevelopmental disorders arising from NMDAR deficiency can be effectively treated in adults.

Oxytocin's anti-inflammatory and proimmune functions in COVID-19: a transcriptomic signature-based approach

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic, infecting over 16 million people worldwide with a significant mortality rate. However, there is no current Food and Drug Administration-approved drug that treats coronavirus disease 2019 (COVID-19). Damage to T lymphocytes along with the cytokine storm are important factors that lead to exacerbation of clinical cases. Here, we are proposing intravenous oxytocin (OXT) as a candidate for adjunctive therapy for COVID-19. OXT has anti-inflammatory and proimmune adaptive functions. Using the Library of Integrated Network-Based Cellular Signatures (LINCS), we used the transcriptomic signature for carbetocin, an OXT agonist, and compared it to gene knockdown signatures of inflammatory (such as interleukin IL-1beta and IL-6) and proimmune markers (including T cell and macrophage cell markers like CD40 and ARG1). We found that carbetocin’s transcriptomic signature has a pattern of concordance with inflammation and immune marker knockdown signatures that are consistent with reduction of inflammation and promotion and sustaining of immune response. This suggests that carbetocin may have potent effects in modulating inflammation, attenuating T cell inhibition, and enhancing T cell activation. Our results also suggest that carbetocin is more effective at inducing immune cell responses than either lopinavir or hydroxychloroquine, both of which have been explored for the treatment of COVID-19.

Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.

Identification of new drug treatments to combat COVID19: A signature-based approach using iLINCS

The COVID-19 pandemic caused by the novel SARS-CoV-2 is more contagious than other coronaviruses and has higher rates of mortality than influenza. As no vaccine or drugs are currently approved to specifically treat COVID-19, identification of effective therapeutics is crucial to treat the afflicted and limit disease spread. We deployed a bioinformatics workflow to identify candidate drugs for the treatment of COVID-19. Using an “omics” repository, the Library of Integrated Network-Based Cellular Signatures (LINCS), we simultaneously probed transcriptomic signatures of putative COVID-19 drugs and signatures of coronavirus-infected cell lines to identify therapeutics with concordant signatures and discordant signatures, respectively. Our findings include three FDA approved drugs that have established antiviral activity, including protein kinase inhibitors, providing a promising new category of candidates for COVID-19 interventions.

Signature-based approaches for informed drug repurposing: targeting CNS disorders

CNS disorders, and in particular psychiatric illnesses, lack definitive disease-altering therapeutics. The limited understanding of the mechanisms driving these illnesses with the slow pace and high cost of drug development exacerbates this issue. For these reasons, drug repurposing - both a less expensive and time-efficient practice compared to de novo drug development - has been a promising strategy to overcome the paucity of treatments available for these debilitating disorders. While empirical drug-repurposing has been a routine practice in clinical psychiatry, innovative, informed, and cost-effective repurposing efforts using big data (“omics”) have been designed to characterize drugs by structural and transcriptomic signatures. These strategies, in conjunction with ontological integration, provide an important opportunity to address knowledge-based challenges associated with drug development for CNS disorders. In this review, we discuss various signature-based in silico approaches to drug repurposing, its integration with multiple omics platforms, and how this data can be used for clinically relevant, evidence-based drug repurposing. These tools provide an exciting translational avenue to merge omics-based drug discovery platforms with patient-specific disease signatures, ultimately facilitating the identification of new therapies for numerous psychiatric disorders.

Bilirubin remodels murine white adipose tissue by reshaping mitochondrial activity and the coregulator profile of peroxisome proliferator-activated receptor alpha

Activation of lipid-burning pathways in the fat-storing white adipose tissue (WAT) is a promising strategy to improve metabolic health and reduce obesity, insulin resistance, and type II diabetes. For unknown reasons, bilirubin levels are negatively associated with obesity and diabetes. Here, using mice and an array of approaches, including MRI to assess body composition, biochemical assays to measure bilirubin and fatty acids, MitoTracker-based mitochondrial analysis, immunofluorescence, and high-throughput coregulator analysis, we show that bilirubin functions as a molecular switch for the nuclear receptor transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Bilirubin exerted its effects by recruiting and dissociating specific coregulators in WAT, driving the expression of PPARalpha target genes such as uncoupling protein 1 (Ucp1) and adrenoreceptor beta 3 (Adrb3). We also found that bilirubin is a selective ligand for PPARalpha and does not affect the activities of the related proteins PPARgamma and PPARdelta. We further found that diet-induced obese mice with mild hyperbilirubinemia have reduced WAT size and an increased number of mitochondria, associated with a restructuring of PPARalpha-binding coregulators. We conclude that bilirubin strongly affects organismal body weight by reshaping the PPARalpha coregulator profile, remodeling WAT to improve metabolic function, and reducing fat accumulation.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter

The astrocytic cystine/glutamate antiporter system xc(-) represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc(-), xCT (xCT(-/-) mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT(-/-) mice. A proteomic and kinomic screen of the striatum of xCT(-/-) mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT(-/-) mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc(-) plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Adenosine Kinase Expression in the Frontal Cortex in Schizophrenia

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

A bioinformatic inquiry of the EAAT2 interactome in postmortem and neuropsychiatric datasets

Altered expression and localization of the glutamate transporter EAAT2 is found in schizophrenia and other neuropsychiatric (major depression, MDD) and neurological disorders (amyotrophic lateral sclerosis, ALS). However, the EAAT2 interactome, the network of proteins that physically or functionally interact with EAAT2 to support its activity, has yet to be characterized in severe mental illness. We compiled a list of “core” EAAT2 interacting proteins. Using Kaleidoscope, an R-shiny application, we data mined publically available postmortem transcriptome datasets to determine whether components of the EAAT2 interactome are differentially expressed in schizophrenia and, using Reactome, identify which interactome-associated biological pathways are altered. Overall, these “look up” studies highlight region-specific, primarily frontal cortex (dorsolateral prefrontal cortex and anterior cingulate cortex), changes in the EAAT2 interactome and implicate altered metabolism pathways in schizophrenia. Pathway analyses also suggest that perturbation of components of the EAAT2 interactome in animal models of antipsychotic administration impact metabolism. Similar changes in metabolism pathways are seen in ALS, in addition to altered expression of many components of the EAAT2 interactome. However, although EAAT2 expression is altered in a postmortem MDD dataset, few other components of the EAAT2 interactome are changed. Thus, “look up” studies suggest region- and disease-relevant biological pathways related to the EAAT2 interactome that implicate glutamate reuptake perturbations in schizophrenia, while providing a useful tool to exploit “omics” datasets.

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.

Neuronal impact of patient-specific aberrant NRXN1alpha splicing

NRXN1 undergoes extensive alternative splicing, and non-recurrent heterozygous deletions in NRXN1 are strongly associated with neuropsychiatric disorders. We establish that human induced pluripotent stem cell (hiPSC)-derived neurons well represent the diversity of NRXN1alpha alternative splicing observed in the human brain, cataloguing 123 high-confidence in-frame human NRXN1alpha isoforms. Patient-derived NRXN1(+/-) hiPSC-neurons show a greater than twofold reduction in half of the wild-type NRXN1alpha isoforms and express dozens of novel isoforms from the mutant allele. Reduced neuronal activity in patient-derived NRXN1(+/-) hiPSC-neurons is ameliorated by overexpression of individual control isoforms in a genotype-dependent manner, whereas individual mutant isoforms decrease neuronal activity levels in control hiPSC-neurons. In a genotype-dependent manner, the phenotypic impact of patient-specific NRXN1(+/-) mutations can occur through a reduction in wild-type NRXN1alpha isoform levels as well as the presence of mutant NRXN1alpha isoforms.

Synergistic effects of common schizophrenia risk variants

The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.

Pioglitazone improves working memory performance when administered in chronic TBI

Traumatic brain injury (TBI) is a leading cause of long-term disability in the United States. Even in comparatively mild injuries, cognitive and behavioral symptoms can persist for years, and there are currently no established strategies for mitigating symptoms in chronic injury. A key feature of TBI-induced damage in acute and chronic injury is disruption of metabolic pathways. As neurotransmission, and therefore cognition, are highly dependent on the supply of energy, we hypothesized that modulating metabolic activity could help restore behavioral performance even when treatment was initiated weeks after TBI. We treated rats with pioglitazone, a FDA-approved drug for diabetes, beginning 46days after lateral fluid percussion injury and tested working memory performance in the radial arm maze (RAM) after 14days of treatment. Pioglitazone treated TBI rats performed significantly better in the RAM test than untreated TBI rats, and similarly to control animals. While hexokinase activity in hippocampus was increased by pioglitazone treatment, there was no upregulation of either the neuronal glucose transporter or hexokinase enzyme expression. Expression of glial markers GFAP and Iba-1 were also not influenced by pioglitazone treatment. These studies suggest that targeting brain metabolism, in particular hippocampal metabolism, may be effective in alleviating cognitive symptoms in chronic TBI.

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.

RNA sequencing in human HepG2 hepatocytes reveals PPAR-alpha mediates transcriptome responsiveness of bilirubin

Bilirubin is a potent antioxidant that reduces inflammation and the accumulation of fat. There have been reports of gene responses to bilirubin, which was mostly attributed to its antioxidant function. Using RNA sequencing, we found that biliverdin, which is rapidly reduced to bilirubin, induced transcriptome responses in human HepG2 hepatocytes in a peroxisome proliferator-activated receptor (PPAR)-alpha-dependent fashion (398 genes with >2-fold change; false discovery rate P < 0.05). For comparison, a much narrower set of genes demonstrated differential expression when PPAR-alpha was suppressed via lentiviral shRNA knockdown (23 genes). Gene set enrichment analysis revealed the bilirubin-PPAR-alpha transcriptome mediates pathways for oxidation-reduction processes, mitochondrial function, response to nutrients, fatty acid oxidation, and lipid homeostasis. Together, these findings suggest that transcriptome responses from the generation of bilirubin are mostly PPAR-alpha dependent, and its antioxidant function regulates a smaller set of genes.

Measurement of lactate levels in postmortem brain, iPSCs, and animal models of schizophrenia

Converging evidence suggests bioenergetic defects contribute to the pathophysiology of schizophrenia and may underlie cognitive dysfunction. The transport and metabolism of lactate energetically couples astrocytes and neurons and supports brain bioenergetics. We examined the concentration of lactate in postmortem brain (dorsolateral prefrontal cortex) in subjects with schizophrenia, in two animal models of schizophrenia, the GluN1 knockdown mouse model and mutant disrupted in schizophrenia 1 (DISC1) mouse model, as well as inducible pluripotent stem cells (iPSCs) from a schizophrenia subject with the DISC1 mutation. We found increased lactate in the dorsolateral prefrontal cortex (p = 0.043, n = 16/group) in schizophrenia, as well as in frontal cortical neurons differentiated from a subject with schizophrenia with the DISC1 mutation (p = 0.032). We also found a decrease in lactate in mice with induced expression of mutant human DISC1 specifically in astrocytes (p = 0.049). These results build upon the body of evidence supporting bioenergetic dysfunction in schizophrenia, and suggests changes in lactate are a key feature of this often devastating severe mental illness.

Connectivity Analyses of Bioenergetic Changes in Schizophrenia: Identification of Novel Treatments

We utilized a cell-level approach to examine glycolytic pathways in the DLPFC of subjects with schizophrenia (n = 16) and control (n = 16) and found decreased mRNA expression of glycolytic enzymes in pyramidal neurons, but not astrocytes. To replicate these novel bioenergetic findings, we probed independent datasets for bioenergetic targets and found similar abnormalities. Next, we used a novel strategy to build a schizophrenia bioenergetic profile by a tailored application of the Library of Integrated Network-Based Cellular Signatures data portal (iLINCS) and investigated connected cellular pathways, kinases, and transcription factors using Enrichr. Finally, with the goal of identifying drugs capable of “reversing” the bioenergetic schizophrenia signature, we performed a connectivity analysis with iLINCS and identified peroxisome proliferator-activated receptor (PPAR) agonists as promising therapeutic targets. We administered a PPAR agonist to the GluN1 knockdown model of schizophrenia and found it improved long-term memory. Taken together, our findings suggest that tailored bioinformatics approaches, coupled with the LINCS library of transcriptional signatures of chemical and genetic perturbagens, may be employed to identify novel treatment strategies for schizophrenia and related diseases.

Role of glutamatergic system and mesocorticolimbic circuits in alcohol dependence

Emerging evidence demonstrates that alcohol dependence is associated with dysregulation of several neurotransmitters. Alterations in dopamine, glutamate and gamma-aminobutyric acid release are linked to chronic alcohol exposure. The effects of alcohol on the glutamatergic system in the mesocorticolimbic areas have been investigated extensively. Several studies have demonstrated dysregulation in the glutamatergic systems in animal models exposed to alcohol. Alcohol exposure can lead to an increase in extracellular glutamate concentrations in mesocorticolimbic brain regions. In addition, alcohol exposure affects the expression and functions of several glutamate receptors and glutamate transporters in these brain regions. In this review, we discussed the effects of alcohol exposure on glutamate receptors, glutamate transporters and glutamate homeostasis in each area of the mesocorticolimbic system. In addition, we discussed the genetic aspect of alcohol associated with glutamate and reward circuitry. We also discussed the potential therapeutic role of glutamate receptors and glutamate transporters in each brain region for the treatment of alcohol dependence. Finally, we provided some limitations on targeting the glutamatergic system for potential therapeutic options for the treatment alcohol use disorders.

Neurotransmitter changes after traumatic brain injury: an update for new treatment strategies

Traumatic brain injury (TBI) is a pervasive problem in the United States and worldwide, as the number of diagnosed individuals is increasing yearly and there are no efficacious therapeutic interventions. A large number of patients suffer with cognitive disabilities and psychiatric conditions after TBI, especially anxiety and depression. The constellation of post-injury cognitive and behavioral symptoms suggest permanent effects of injury on neurotransmission. Guided in part by preclinical studies, clinical trials have focused on high-yield pathophysiologic mechanisms, including protein aggregation, inflammation, metabolic disruption, cell generation, physiology, and alterations in neurotransmitter signaling. Despite successful treatment of experimental TBI in animal models, clinical studies based on these findings have failed to translate to humans. The current international effort to reshape TBI research is focusing on redefining the taxonomy and characterization of TBI. In addition, as the next round of clinical trials is pending, there is a pressing need to consider what the field has learned over the past two decades of research, and how we can best capitalize on this knowledge to inform the hypotheses for future innovations. Thus, it is critically important to extend our understanding of the pathophysiology of TBI, particularly to mechanisms that are associated with recovery versus development of chronic symptoms. In this review, we focus on the pathology of neurotransmission after TBI, reflecting on what has been learned from both the preclinical and clinical studies, and we discuss new directions and opportunities for future work.

Chronic Dysregulation of Cortical and Subcortical Metabolism After Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. Although chronic disability is common after TBI, effective treatments remain elusive and chronic TBI pathophysiology is not well understood. Early after TBI, brain metabolism is disrupted due to unregulated ion release, mitochondrial damage, and interruption of molecular trafficking. This metabolic disruption causes at least part of the TBI pathology. However, it is not clear how persistent or pervasive metabolic injury is at later stages of injury. Using untargeted (1)H-NMR metabolomics, we examined ex vivo hippocampus, striatum, thalamus, frontal cortex, and brainstem tissue in a rat lateral fluid percussion model of chronic brain injury. We found altered tissue concentrations of metabolites in the hippocampus and thalamus consistent with dysregulation of energy metabolism and excitatory neurotransmission. Furthermore, differential correlation analysis provided additional evidence of metabolic dysregulation, most notably in brainstem and frontal cortex, suggesting that metabolic consequences of injury are persistent and widespread. Interestingly, the patterns of network changes were region-specific. The individual metabolic signatures after injury in different structures of the brain at rest may reflect different compensatory mechanisms engaged to meet variable metabolic demands across brain regions.

Sex differences in DEK expression in the anterior cingulate cortex and its association with dementia severity in schizophrenia

DEK is a chromatin-remodeling phosphoprotein found in most human tissues, but its expression and function in the human brain is largely unknown. DEK depletion in vitro induces cellular and molecular anomalies associated with cognitive impairment, including down-regulation of the canonical Wnt/beta-catenin signaling pathway. ToppGene analyses link DEK loss to genes associated with various dementias and age-related cognitive decline. To examine the role of DEK in cognitive impairment in severe mental illness, DEK protein expression was assayed by immunoblot in the anterior cingulate cortex (ACC) of subjects with schizophrenia. Cognitive impairment is a core feature of schizophrenia and cognitive function in subjects was assessed antemortem using the clinical dementia rating (CDR) scale. DEK protein expression was not significantly altered in schizophrenia (n=20) compared to control subjects (n=20). Further analysis revealed significant reduction in DEK protein expression in women with schizophrenia, and a significant increase in expression in men with schizophrenia, relative to their same-sex controls. DEK protein expression levels were inversely correlated with dementia severity in women. Conversely, in men, DEK protein expression and dementia severity were positively correlated. Notably, there was no sex difference in DEK protein expression in the control group, suggesting that this sex difference is specific to schizophrenia and not due to inherent differences in DEK expression between males and females. These results suggest a novel, sex-specific role for DEK in cognitive performance and highlight a putative sex-specific link between central nervous system DEK protein expression and a neuropsychiatric disease that is commonly associated with cognitive impairment.

Neuron-specific deficits of bioenergetic processes in the dorsolateral prefrontal cortex in schizophrenia

Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.

Cell-subtype-specific changes in adenosine pathways in schizophrenia

Prior work in animal models implicates abnormalities of adenosine metabolism in astrocytes as a possible pathophysiological mechanism underlying the symptoms of schizophrenia. In the present study, we sought to reverse-translate these findings back to the human brain in schizophrenia, focusing on the following questions: (1) Which components of the adenosine system are dysregulated in schizophrenia, and (2) are these changes limited to astrocytes? To address these questions, we captured enriched populations of DLPFC pyramidal neurons and astrocytes from schizophrenia and control subjects using laser capture microdissection and assessed expression of adenosine system components using qPCR. Interestingly, we found changes in enriched populations of astrocytes and neurons spanning metabolic and catabolic pathways. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) and ENTPD2 mRNA levels were significantly decreased (p < 0.05, n = 16 per group) in enriched populations of astrocytes; in pyramidal neurons equilibrative nucleoside transporter 1 (ENT1) and adenosine A1 receptor mRNA levels were significantly decreased, with an increase in adenosine deaminase (ADA) (p < 0.05, n = 16 per group). Rodent studies suggest that some of our findings (A1R and ENTPD2) may be due to treatment with antipsychotics. Our findings suggest changes in expression of genes involved in regulating metabolism of ATP in enriched populations of astrocytes, leading to lower availability of substrates needed to generate adenosine. In pyramidal neurons, changes in ENT1 and ADA mRNA may suggest increased catabolism of adenosine. These results offer new insights into the cell-subtype-specific pathophysiology of the adenosine system in this illness.

Defects in Bioenergetic Coupling in Schizophrenia

Synaptic neurotransmission relies on maintenance of the synapse and meeting the energy demands of neurons. Defects in excitatory and inhibitory synapses have been implicated in schizophrenia, likely contributing to positive and negative symptoms as well as impaired cognition. Recently, accumulating evidence has suggested that bioenergetic systems, important in both synaptic function and cognition, are abnormal in psychiatric illnesses such as schizophrenia. Animal models of synaptic dysfunction demonstrated endophenotypes of schizophrenia as well as bioenergetic abnormalities. We report findings on the bioenergetic interplay of astrocytes and neurons and discuss how dysregulation of these pathways may contribute to the pathogenesis of schizophrenia, highlighting metabolic systems as important therapeutic targets.

The role of glutamate transporters in the pathophysiology of neuropsychiatric disorders

Altered glutamate transporter expression is a common feature of many neuropsychiatric conditions, including schizophrenia. Excitatory amino acid transporters (EAATs) are responsible for the reuptake of glutamate, preventing non-physiological spillover from the synapse. Postmortem studies have revealed significant dysregulation of EAAT expression in various brain regions at the cellular and subcellular level. Recent animal studies have also demonstrated a role for glutamate spillover as a mechanism of disease. In this review, we describe current evidence for the role of glutamate transporters in regulating synaptic plasticity and transmission. In neuropsychiatric conditions, EAAT splice variant expression is altered. There are changes in the localization of the transporters and disruption of the metabolic and structural protein network that supports EAAT activity. This results in aberrant neuroplasticity and excitatory signaling, contributing to the symptoms associated with neuropsychiatric disease. Understanding the complex functions of glutamate transporters will clarify the relevance of their role in the pathophysiology of neuropsychiatric disorders.

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.

Contrasting the Role of xCT and GLT-1 Upregulation in the Ability of Ceftriaxone to Attenuate the Cue-Induced Reinstatement of Cocaine Seeking and Normalize AMPA Receptor Subunit Expression

Long-term treatment with ceftriaxone attenuates the reinstatement of cocaine seeking while increasing the function of the glutamate transporter 1 (GLT-1) and system xC- (Sxc) in the nucleus accumbens core (NAc). Sxc contributes the majority of nonsynaptic extracellular glutamate in the NAc, while GLT-1 is responsible for the majority of glutamate uptake. Here we used antisense to decrease the expression of GLT-1 and xCT (a catalytic subunit of Sxc) to determine the relative importance of both proteins in mediating the ability of ceftriaxone to prevent cue-induced reinstatement of cocaine seeking and normalize glutamatergic proteins in the NAc of rats. Intra-NAc xCT knockdown prevented ceftriaxone from attenuating reinstatement and from upregulating GLT-1 and resulted in increased surface expression of AMPA receptor subunits GluA1 and GluA2. Intra-NAc GLT-1 knockdown also prevented ceftriaxone from attenuating reinstatement and from upregulating xCT expression, without affecting GluA1 and GluA2 expression. In the absence of cocaine or ceftriaxone treatment, xCT knockdown in the NAc increased the expression of both GluA1 and GluA2 without affecting GLT-1 expression while GLT-1 knockdown had no effect. PCR and immunoprecipitation of GLT-1 revealed that ceftriaxone does not upregulate GLT-1 and xCT through a transcriptional mechanism, and their coregulation by ceftriaxone is not mediated by physical interaction. These data support important and distinct roles for xCT and GLT-1 in the actions of ceftriaxone and add to a body of literature finding evidence for coregulation of these transporters. Our results also point to xCT expression and subsequent basal glutamate levels as being a key mediator of AMPA receptor expression in the NAc.SIGNIFICANCE STATEMENT Ceftriaxone attenuates the reinstatement of cocaine, alcohol, and heroin seeking. The mechanism of action of this behavioral effect has been attributed to glutamate transporter 1 (GLT-1) and xCT (a catalytic subunit of Sxc)/Sxc upregulation in the nucleus accumbens core. Here we used an antisense strategy to knock down GLT-1 or xCT in the nucleus accumbens core and examined the behavioral and molecular consequences. While upregulation of both xCT and GLT-1 are essential to the ability of ceftriaxone to attenuate cue-induced reinstatement of cocaine seeking, each protein uniquely affects the expression of other glutamate receptor and transporter proteins. We also report that reducing basal glutamate levels through the manipulation of xCT expression increases the surface expression of AMPA receptor subunits, providing insight to the mechanism by which cocaine alters AMPA surface expression.

Postsynaptic Density-95 Isoform Abnormalities in Schizophrenia

Background: Postsynaptic density-95 (PSD-95) protein expression is dysregulated in schizophrenia in a variety of brain regions. We have designed experiments to examine PSD-95 mRNA splice variant expression in the dorsolateral prefrontal cortex from subjects with schizophrenia. Methods: We performed quantitative PCR and western blot analysis to measure PSD-95 expression in schizophrenia vs control subjects, rodent haloperidol treatment studies, rodent postmortem interval studies, and GluN1 knockdown (KD) mice vs controls. Results: We found decreased mRNA expression of beta (t = 4.506, df = 383, P < .0001) and truncated (t = 3.378, df = 383, P = .0008) isoforms of PSD-95, whereas alpha was unchanged. Additionally, we found decreased PSD-95 protein expression in schizophrenia (t = 2.746, df = 71, P = .0076). We found no correlation between PSD-95 protein and alpha, beta, or truncated mRNA isoforms in schizophrenia. PSD-95 beta transcript was increased (t = 3.346, df = 14, P < .05) in the GluN1 KD mouse model of schizophrenia. There was an increase in PSD-95 alpha mRNA expression (t = 2.905, df = 16, P < .05) in rats following long-term haloperidol administration. Conclusions: Our findings describe a unique pathophysiology of specific PSD-95 isoform dysregulation in schizophrenia, chronic neuroleptic treatment, and a genetic lesion mouse model of drastically reduced N-methyl-d-aspartate receptor (NMDAR) complex expression. These data indicate that regulation of PSD-95 is multifaceted, may be isoform specific, and biologically relevant for synaptic signaling function. Specifically, NMDAR-mediated synaptic remodeling, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking and interaction may be impaired in schizophrenia by decreased PSD-95 beta and truncated expression (respectively). Further, increased PSD-95 beta transcript in the GluN1 KD mouse model poses a potential compensatory rescue of NMDAR-mediated function via increased postsynaptic throughput of the severely reduced GluN1 signal. Together, these data propose that disruption of excitatory signaling complexes through genetic (GluN1 KD), pharmacologic (antipsychotics), or disease (schizophrenia) mechanisms specifically dysregulates PSD-95 expression.

Traumatic Brain Injury Induces Alterations in Cortical Glutamate Uptake without a Reduction in Glutamate Transporter-1 Protein Expression

We hypothesize that the primary mechanism for removal of glutamate from the extracellular space is altered after traumatic brain injury (TBI). To evaluate this hypothesis, we initiated TBI in adult male rats using a 2.0 atm lateral fluid percussion injury (LFPI) model. In the ipsilateral cortex and hippocampus, we found no differences in expression of the primary glutamate transporter in the brain (GLT-1) 24 h after TBI. In contrast, we found a decrease in glutamate uptake in the cortex, but not the hippocampus, 24 h after injury. Because glutamate uptake is potently regulated by protein kinases, we assessed global serine-threonine protein kinase activity using a kinome array platform. Twenty-five kinome array peptide substrates were differentially phoshorylated between LFPI and controls in the cortex, whereas 19 peptide substrates were differentially phosphorylated in the hippocampus (fold change >/= +/- 1.15). We identified several kinases as likely to be involved in acute TBI, including protein kinase B (Akt) and protein kinase C (PKC), which are well-characterized modulators of GLT-1. Exploratory studies using an inhibitor of Akt suggest selective activation of kinases in LFPI versus controls. Ingenuity pathway analyses of implicated kinases from our network model found apoptosis and cell death pathways as top functions in acute LFPI. Taken together, our data suggest diminished activity of glutamate transporters in the prefrontal cortex, with no changes in protein expression of the primary glutamate transporter GLT-1, and global alterations in signaling networks that include serine-threonine kinases that are known modulators of glutamate transport activity.

Glutamate Neurotransmission in Rodent Models of Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and disability in people younger than 45 and is a significant public health concern. In addition to primary mechanical damage to cells and tissue, TBI involves additional molecular mechanisms of injury, termed secondary injury, that continue to evolve over hours, days, weeks, and beyond. The trajectory of recovery after TBI is highly unpredictable and in many cases results in chronic cognitive and behavioral changes. Acutely after TBI, there is an unregulated release of glutamate that cannot be buffered or cleared effectively, resulting in damaging levels of glutamate in the extracellular space. This initial loss of glutamate homeostasis may initiate additional changes in glutamate regulation. The excitatory amino acid transporters (EAATs) are expressed on both neurons and glia and are the principal mechanism for maintaining extracellular glutamate levels. Diffusion of glutamate outside the synapse due to impaired uptake may lead to increased extrasynaptic glutamate signaling, secondary injury through activation of cell death pathways, and loss of fidelity and specificity of synaptic transmission. Coordination of glutamate release and uptake is critical to regulating synaptic strength, long-term potentiation and depression, and cognitive processes. In this review, we will discuss dysregulation of extracellular glutamate and glutamate uptake in the acute stage of TBI and how failure to resolve acute disruptions in glutamate homeostatic mechanisms may play a causal role in chronic cognitive symptoms after TBI.

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

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.

Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright (c) 2015 John Wiley & Sons, Ltd.

Tyrosine hydroxylase localization in the nucleus accumbens in schizophrenia

The nucleus accumbens (NAcc) has been implicated in schizophrenia (SZ) pathology, based on antipsychotic action therein. However, recent imaging studies suggest that the NAcc may not be a locus of dopamine dysregulation in SZ. This study examined postmortem human tissue to determine if abnormalities are present in dopamine synthesis in the NAcc in SZ. We compared the immunohistochemical localization of tyrosine hydroxylase (TH), the rate-limiting synthesizing enzyme of dopamine, in postmortem tissue from SZ subjects and demographically matched controls. To study the effects of chronic antipsychotic drug (APD) treatment on TH immunolabeling in the NAcc, rats were treated for 6 months with haloperidol or olanzapine. In the NAcc, TH immunolabeling was similar in control and SZ subjects, in both the core and shell. Rats had similar TH optical density levels across treatment groups in both the core and shell. Similar levels of TH suggest DA synthesis may be normal. These findings provide further insight into the role of the NAcc in SZ.

Cortical PGC-1alpha-Dependent Transcripts Are Reduced in Postmortem Tissue From Patients With Schizophrenia

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) 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-1alpha-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-1alpha and these transcripts in postmortem cortical tissue from control and schizophrenia patients and found a reduction in PGC-1alpha-dependent transcripts without a change in PGC-1alpha. While control subjects with high PGC-1alpha expression exhibited high PV and Nefh expression, schizophrenia subjects with high PGC-1alpha expression did not, suggesting dissociation between PGC-1alpha expression and these targets in schizophrenia. Unbiased analyses of the promoter regions for PGC-1alpha-dependent transcripts revealed enrichment of binding sites for the PGC-1alpha-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-1alpha-dependent targets in schizophrenia. Interestingly, the positive correlation between PGC-1alpha 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-1alpha and/or NRF-1-associated transcriptional programs in the cortex and that approaches to enhance the activity of PGC-1alpha or transcriptional regulators like NRF-1 should be considered with the goal of restoring normal gene programs and improving cortical function.

Cell-specific abnormalities of glutamate transporters in schizophrenia: sick astrocytes and compensating relay neurons?

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.

Glutamate transporter splice variant expression in an enriched pyramidal cell population in schizophrenia

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.

Changes in cortical N-methyl-D-aspartate receptors and post-synaptic density protein 95 in schizophrenia, mood disorders and suicide

OBJECTIVES: In humans, depending on dose, blocking the N-methyl-D-aspartate receptor (NMDAR) with ketamine can cause psychomimetic or antidepressant effects. The overall outcome for drugs such as ketamine depends on dose and the number of its available binding sites in the central nervous system, and to understand something of the latter variable we measure NMDAR in the frontal pole, dorsolateral prefrontal, anterior cingulate and parietal cortices from people with schizophrenia, bipolar disorder, major depressive disorders and age/sex matched controls. METHOD: We measured levels of NMDARs (using [(3)H]MK-801 binding) and NMDAR sub-unit mRNAs (GRINs: using in situ hybridisation) as well as post-synaptic density protein 95 (anterior cingulate cortex only; not major depressive disorders: an NMDAR post-synaptic associated protein) in bipolar disorder, schizophrenia and controls. RESULTS: Compared to controls, levels of NMDAR were lower in the outer laminae of the dorsolateral prefrontal cortex (-17%, p = 0.01) in people with schizophrenia. In bipolar disorder, levels of NMDAR binding (laminae IV-VI; -19%, p < 0.01) and GRIN2C mRNA (laminae I-VI; -27%, p < 0.05) were lower in the anterior cingulate cortex and NMDAR binding was lower in the outer lamina IV of the dorsolateral prefrontal cortex (-19%, p < 0.01). In major depressive disorders, levels of GRIN2D mRNA were higher in frontal pole (+22%, p < 0.05). In suicide completers, levels of GRIN2B mRNA were higher in parietal cortex (+20%, p < 0.01) but lower (-35%, p = 0.02) in dorsolateral prefrontal cortex while post-synaptic density protein 95 was higher (+26%, p < 0.05) in anterior cingulate cortex. CONCLUSION: These data suggest that differences in cortical NMDAR expression and post-synaptic density protein 95 are present in psychiatric disorders and suicide completion and may contribute to different responses to ketamine.

Purinergic signaling and energy homeostasis in psychiatric disorders

Purinergic signaling regulates numerous vital biological processes in the central nervous system (CNS). The two principle purines, ATP and adenosine act as excitatory and inhibitory neurotransmitters, respectively. Compared to other classical neurotransmitters, the role of purinergic signaling in psychiatric disorders is not well understood or appreciated. Because ATP exerts its main effect on energy homeostasis, neuronal function of ATP has been underestimated. Similarly, adenosine is primarily appreciated as a precursor of nucleotide synthesis during active cell growth and division. However, recent findings suggest that purinergic signaling may explain how neuronal activity is associated neuronal energy charge and energy homeostasis, especially in mental disorders. In this review, we provide an overview of the synaptic function of mitochondria and purines in neuromodulation, synaptic plasticity, and neuron-glia interactions. We summarize how mitochondrial and purinergic dysfunction contribute to mental illnesses such as schizophrenia, bipolar disorder, autism spectrum disorder (ASD), depression, and addiction. Finally, we discuss future implications regarding the pharmacological targeting of mitochondrial and purinergic function for the treatment of psychiatric disorders.

Decreased chloride channel expression in the dorsolateral prefrontal cortex in schizophrenia

Alterations in GABAergic neurotransmission are implicated in several psychiatric illnesses, including schizophrenia. The Na-K-Cl and K-Cl cotransporters regulate intracellular chloride levels. Abnormalities in cotransporter expression levels could shift the chloride electrochemical gradient and impair GABAergic transmission. In this study, we performed Western blot analysis to investigate whether the Na-K-Cl and K-Cl cotransporter protein is abnormally expressed in the dorsal lateral prefrontal cortex and the anterior cingulate cortex in patients with schizophrenia versus a control group. We found decreased K-Cl cotransporter protein expression in the dorsal lateral prefrontal cortex, but not the anterior cingulate cortex, in subjects with schizophrenia, supporting the hypothesis of region level abnormal GABAergic function in the pathophysiology of schizophrenia. Subjects with schizophrenia off antipsychotic medication at the time of death had decreased K-Cl cotransporter protein expression compared to both normal controls and subjects with schizophrenia on antipsychotics. Our results provide evidence for KCC2 protein abnormalities in schizophrenia and suggest that antipsychotic medications might reverse deficits of this protein in the illness.

Postmortem brain: an underutilized substrate for studying severe mental illness

Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia

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

Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: a light and electron microscopic study

The process of glutamate release, activity, and reuptake involves the astrocyte, the presynaptic and postsynaptic neurons. Glutamate is released into the synapse and may occupy and activate receptors on both neurons and astrocytes. Glutamate is rapidly removed from the synapse by a family of plasma membrane excitatory amino acid transporters (EAATs), also localized to neurons and astrocytes. The purpose of the present study was to examine EAAT labeling in the postmortem human cortex at the light and electron microscopic (EM) levels. The postmortem prefrontal cortex was processed for EAAT1 and EAAT2 immunohistochemistry. At the light microscopic level, EAAT1 and EAAT2 labeling was found in both gray and white matter. Most cellular labeling was in small cells which were morphologically similar to glia. In addition, EAAT1-labeled neurons were scattered throughout, some of which were pyramidal in shape. At the EM level, EAAT1 and EAAT2 labeling was found in astrocytic soma and processes surrounding capillaries. EAAT labeling was also found in small astrocytic processes adjacent to axon terminals forming asymmetric (glutamatergic) synapses. While EAAT2 labeling was most prevalent in astrocytic processes, EAAT1 labeling was also present in neuronal processes including the soma, axons, and dendritic spines. Expression of EAAT1 protein on neurons may be due to the hypoxia associated with the postmortem interval, and requires further confirmation. The localization of EAATs on the astrocytic plasma membrane and adjacent to excitatory synapses is consistent with the function of facilitating glutamate reuptake and limiting glutamate spillover. Establishment that EAAT1 and EAAT2 can be measured at the EM level in human postmortem tissues will permit testing of hypotheses related to these molecules in diseases lacking analogous animal models.

Altered serine/threonine kinase activity in schizophrenia

Converging evidence implicates alterations in multiple signaling pathways in the etiology of schizophrenia. Previously, these studies were limited to the analysis of one or a few phosphoproteins at a time. Here, we use a novel kinase array platform to simultaneously investigate the convergence of multiple signaling cascades implicated in schizophrenia. This technology uses consensus peptide substrates to assess activity levels of a large number (>100) of serine/threonine protein kinases. 19 peptide substrates were differentially phosphorylated (>15% change) in the frontal cortex in schizophrenia. These peptide substrates were examined using Ingenuity Pathway Analysis to group them according to the functions and to identify processes most likely affected in schizophrenia. Pathway analysis placed 14 of the 19 peptides into cellular homeostatic pathways, 10 into pathways governing cytoskeletal organization, and 8 into pathways governing ion homeostasis. These data are the first to simultaneously investigate comprehensive changes in signaling cascades in a severe psychiatric disorder. The examination of kinase activity in signaling pathways may facilitate the identification of novel substrates for drug discovery and the development of safer and more effective pharmacological treatment for schizophrenia.

Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia

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

Global signaling effects of a schizophrenia-associated missense mutation in neuregulin 1: an exploratory study using whole genome and novel kinome approaches

Aberrant neuregulin 1-ErbB4 signaling has been implicated in schizophrenia. We previously identified a novel schizophrenia-associated missense mutation (valine to leucine) in the NRG1 transmembrane domain. This variant inhibits formation of the NRG1 intracellular domain (ICD) and causes decreases in dendrite formation. To assess the global effects of this mutation, we used lymphoblastoid cell lines from unaffected heterozygous carriers (Val/Leu) and non-carriers (Val/Val). Transcriptome data showed 367 genes differentially expressed between the two groups (Val/Val N = 6, Val/Leu N = 5, T test, FDR (1 %), alpha = 0.05, -log10 p value >1.5). Ingenuity pathway (IPA) analyses showed inflammation and NRG1 signaling as the top pathways altered. Within NRG1 signaling, protein kinase C (PKC)-eta (PRKCH) and non-receptor tyrosine kinase (SRC) were down-regulated in heterozygous carriers. Novel kinome profiling (serine/threonine) was performed after stimulating cells (V/V N = 6, V/L N = 6) with ErbB4, to induce release of the NRG1 ICD, and revealed significant effects of treatment on the phosphorylation of 35 peptides. IPA showed neurite outgrowth (six peptides) as the top annotated function. Phosphorylation of these peptides was significantly decreased in ErbB4-treated Val/Val but not in Val/Leu cells. These results show that perturbing NRG1 ICD formation has major effects on cell signaling, including inflammatory and neurite formation pathways, and may contribute significantly to schizophrenia pathophysiology.

Postmortem brain: an underutilized substrate for studying severe mental illness

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.

Update on the neurobiology of schizophrenia: a role for extracellular microdomains

The glutamate system includes presynaptic glutamatergic terminals, complex post-synaptic densities found on diverse types of neurons expressing glutamate receptors, as well as glutamate transporters and enzymes that facilitate the glutamate/glutamine cycle. Abnormalities of this system have been implicated in schizophrenia based on an accumulating body of evidence from postmortem, imaging, and preclinical studies. However, recent work has suggested that astrocytes may have more than a bystander role in the synchronization of neuronal responses in the brain. Converging evidence suggests that extrasynaptic glutamate microdomains are formed by astrocytes and may facilitate neuroplasticity via the modulation of extra-synaptic glutamate receptors on neuronal membranes within these domains. In this article the authors propose that the composition and localization of protein complexes in glutamate microdomains is abnormal in schizophrenia, leading to pathological neuroplastic changes in the structure and function of glutamate circuits in this illness.

Abnormal N-linked glycosylation of cortical AMPA receptor subunits in schizophrenia

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.

Gene expression of glutamate metabolizing enzymes in the hippocampal formation in human temporal lobe epilepsy

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.

Abnormal expression of glutamate transporters in temporal lobe areas in elderly patients with schizophrenia

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

AMPA receptor subunit expression in the endoplasmic reticulum in frontal cortex of elderly patients with schizophrenia

Several lines of evidence indicate altered trafficking of alpha-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.

Expression of equilibrative nucleoside transporter type 1 protein in elderly patients with schizophrenia

Alterations in glutamatergic neurotransmission are thought to be involved in several psychiatric disorders, including schizophrenia. Equilibrative nucleoside transporter type 1 (ENT1) regulates glutamate levels by regulating excitatory amino acid transporter expression and activity in the brain. In this study, we investigated whether ENT1 is abnormally expressed in the brain of elderly patients with schizophrenia. We measured protein expression of ENT1 in the superior temporal gyrus (STG) and anterior cingulate cortex (ACC) in patients with schizophrenia (STG, n=22; ACC, n=34) and a comparison group (STG, n=24; ACC, n=29). We found decreased ENT1 expression in the STG in patients with schizophrenia, supporting the hypothesis of altered glutamate transport in this illness.

Recent advances in targeting the ionotropic glutamate receptors in treating schizophrenia

The treatment of schizophrenia has been focused on modulation of dopamine receptors for over 50 years. Recent developments have implicated other neurotransmitter systems in the pathophysiology of this illness. The discovery and characterization of glutamate receptors and their roles in the brain has lead to novel approaches for the treatment of schizophrenia. In this article, we review drugs that modulate ionotropic gluamate receptors and discuss their efficacy for the treatment of this often debilitating severe mental illness.

Regional differences in expression of beta-tubulin isoforms in schizophrenia

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

Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia

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.

Endosomal trafficking of AMPA receptors in frontal cortex of elderly patients with schizophrenia

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

Glutamatergic gene expression is specifically reduced in thalamocortical projecting relay neurons 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.

Novel approaches to the study of postmortem brain in psychiatric illness: old limitations and new challenges

Biological psychiatry has made significant advances through the development of postmortem studies, animal models, and studies with living humans. Although these approaches each have advantages and disadvantages, the postmortem field is undergoing a significant shift toward more complex and informative methodologies. In the first part of this review, we summarize the long-standing methodologic challenges facing this field. In the second part of the article, we discuss the innovative approaches being used for postmortem studies, including laser capture microdissection and subcellular fractionization. These techniques will permit scientists working in the postmortem field to ask and answer the largest possible questions, providing new targets for drug discovery and improved treatments for severe mental illness.

Evidence for abnormal forward trafficking of AMPA receptors in frontal cortex of elderly patients with 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.

Vesicular glutamate transporter mRNA expression in the medial temporal lobe in major depressive disorder, bipolar disorder, and schizophrenia

BACKGROUND: Altered glutamate transmission has been found in the medial temporal lobe in severe psychiatric illnesses, including major depressive disorder (MDD) and bipolar disorder (BD). The vesicular glutamate transporters (VGLUTs) have a pivotal role in presynaptic release of glutamate into the synaptic cleft. We investigated this presynaptic marker in major psychiatric illness by measuring transcript expression of the VGLUTs in the medial temporal lobe. METHODS: The study sample comprised four groups of 13 subjects with MDD, BD, or schizophrenia (SCZ), and a comparison group from the Stanley Foundation Neuropathology Consortium. In situ hybridization was performed to quantify messenger RNA (mRNA) expression of VGLUT 1, 2, and 3 in medial temporal lobe structures. We also examined the same areas of rats treated with antidepressants, a mood stabilizer, and antipsychotics to assess the effects of these medications on VGLUT mRNA expression. RESULTS: We found decreased VGLUT1 mRNA expression in both MDD and BD in the entorhinal cortex (ERC), decreased VGLUT2 mRNA expression in MDD in the middle temporal gyrus, and increased VGLUT2 mRNA expression in SCZ in the inferior temporal gyrus (ITG). We also found a negative correlation between age and VGLUT1 mRNA expression in BD in the ERC and ITG. We did not find any changes in VGLUT mRNA expression in the hippocampus in any diagnostic group. We found decreased VGLUT1 mRNA expression in rats treated with haloperidol in the temporal cortex. CONCLUSIONS: These data indicate region-specific alterations of presynaptic glutamate innervation in the medial temporal lobe in the mood disorders.

Abnormal glycosylation of EAAT1 and EAAT2 in prefrontal cortex of elderly patients with schizophrenia

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

Decreased expression of NMDA receptor-associated proteins in frontal cortex of elderly patients with schizophrenia

Converging evidence suggests too few activation-ready N-methyl-D-aspartic acid (NMDA) receptor complexes in the postsynaptic density in schizophrenia. Postsynaptic density protein 95 (PSD95), Synaptic GTPase-activating protein (SynGAP), and Multiple PDZ domain protein (MUPP1) are integral components of the NMDA receptor signaling complex, and help facilitate signaling, trafficking, and stabilization. We hypothesized that deficits involving these molecules may contribute to the pathophysiology of schizophrenia. To test our hypothesis, we measured protein expression of PSD95, SynGAP, and MUPP1 in the anterior cingulate cortex and dorsolateral prefrontal cortex. We found decreased PSD95 expression in the anterior cingulate cortex. Antipsychotic medication analyses showed decreased SynGAP expression in the anterior cingulate cortex in patients off medication when analyzed against our comparison group. These data suggest that NMDA receptor complex formation, localization, and downstream signaling may be abnormal in schizophrenia.

Expression of four housekeeping proteins in elderly patients with schizophrenia

We compared protein expression by Western blot analysis in four areas of postmortem brain from patients with schizophrenia and control subjects for several proteins that are often used as controls for Western blot studies: beta-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and valosin-containing protein. We did not detect any differences in expression between subjects with schizophrenia and a comparison group. These results suggest that all four proteins are suitable loading controls for postmortem studies of schizophrenia.

High-affinity Na+/K+-dependent glutamate transporter EAAT4 is expressed throughout the rat fore- and midbrain

Excitatory amino acid transporter 4 (EAAT4), a member of the high-affinity Na+/K+-dependent glutamate transporter family, is highly enriched in Purkinje cells of the cerebellum, although it is not restricted to these cells. The detailed expression of EAAT4 protein in different adult rat fore- and midbrain regions was examined. Despite moderate expression levels compared with the cerebellum, EAAT4 protein was omnipresent throughout the fore- and midbrain. With antibodies raised against the N-terminal mouse EAAT4 sequence, the highest protein expression levels were observed in the substantia nigra pars compacta, ventral tegmental area, paranigral nucleus, habenulo-interpeduncular system, supraoptic nucleus, lateral posterior thalamic nucleus, subiculum, and superficial layers of the superior colliculus. Relatively high levels of EAAT4 protein were also detected in the hippocampal principal cells, in the glutamatergic, gamma-aminobutyric acid (GABA)ergic, dopaminergic and most likely cholinergic cells of all nuclei of the basal ganglia, and in neurons of layers II/III and V of the cerebral cortex. The expression of EAAT4 was confirmed at the mRNA level in some important fore- and midbrain structures by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) and estimated to range from 6.7 to 1.6% of the amount in the cerebellum as measured by real-time PCR.

Abnormal expression of glutamate transporter and transporter interacting molecules in prefrontal cortex in elderly patients with schizophrenia

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

Cortical expression of glial fibrillary acidic protein and glutamine synthetase is decreased in schizophrenia

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

Altered vesicular glutamate transporter expression in the anterior cingulate cortex in schizophrenia

BACKGROUND: Schizophrenia is a chronic, severe mental illness with profound emotional and economic burdens for those afflicted and their families. An increasing number of studies have found that schizophrenia is marked by dysregulation of glutamatergic neurotransmission. While numerous studies have found alterations of postsynaptic molecules in schizophrenia, a growing body of evidence implicates presynaptic factors. Vesicular glutamate transporters (VGLUTs) have been identified and are known to package glutamate into vesicles in the presynaptic terminal for subsequent release into the synaptic cleft. Recent studies have shown that VGLUTs regulate synaptic activity via the amount of glutamate released. Accordingly, we hypothesized that VGLUTs are altered in schizophrenia, contributing to dysfunction of presynaptic activity. METHODS: Using in situ hybridization and Western blot analysis, we investigated alterations in VGLUT1 and VGLUT2 transcript and protein expression in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and a comparison group. RESULTS: We found increased VGLUT1 transcript and reduced VGLUT1 protein expression in the ACC, but not DLPFC, in schizophrenia. Vesicular glutamate transporter 2 was unchanged at both levels of gene expression. We did not find changes in VGLUT1 messenger RNA (mRNA) or protein levels following 28-day treatment of rats with haloperidol (2 mg/kg/day), suggesting that our findings in schizophrenia are not due to an effect of antipsychotic treatment. CONCLUSIONS: Overall, our data suggest decreased glutamate release in the ACC, as well as discordant regulation of VGLUT1 expression at different levels of gene expression.

Abnormal glutamate receptor expression in the medial temporal lobe in schizophrenia and mood disorders

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.

Expression of transcripts for myelination-related genes in the anterior cingulate cortex in schizophrenia

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

Decreased NR1, NR2A, and SAP102 transcript expression in the hippocampus in bipolar disorder

OBJECTIVES: Schizophrenia is associated with dysfunction of glutamatergic neurotransmission, and several studies have suggested glutamatergic abnormalities in bipolar disorder. Recent data suggest involvement of the NMDA receptor signaling complex, which includes NMDA receptor subunits as well as associated intracellular interacting proteins critical for NMDA receptor assembly, trafficking, and activation; the most well-characterized being PSD93, PSD95, SAP102, and NF-L. Previously, studies from our laboratories have described changes in glutamate receptor subunit transcript and binding site expression in schizophrenia and changes in NMDA receptor binding site expression in bipolar disorder in postmortem brain tissue. In the present work, we focus on the expression of these molecules in hippocampus in schizophrenia and bipolar affective disorder I. METHODS: We performed in situ hybridization to assess hippocampal expression of the transcripts encoding NMDA receptor subunits NR1, 2A, 2B, 2C and 2D, and the transcripts for the NMDA receptor associated PSD proteins PSD95, PSD93, NF-L, and SAP102 in subjects with schizophrenia, bipolar affective disorder I, and a comparison group. We also measured [(3)H]CGP39653 and [(3)H]MK-801 binding site expression in the hippocampus in schizophrenia. RESULTS: There was a significant decrease in the expression of transcripts for NR1 and NR2A subunits and SAP102 in bipolar disorder. We did not detect any changes in these transcripts or in binding site expression in the hippocampus in schizophrenia. CONCLUSIONS: We propose that the NMDA receptor signaling complex, including the intracellular machinery that is coupled to the NMDA receptor subunits, is abnormal in the hippocampus in bipolar disorder. These data suggest that bipolar disorder might be associated with abnormalities of glutamate-linked intracellular signaling and trafficking processes.

Expression of excitatory amino acid transporter interacting protein transcripts in the thalamus in schizophrenia

The excitatory amino acid transporters (EAATs) are a family of plasma membrane proteins that maintain synaptic glutamate concentration by removing glutamate from the synaptic cleft. EAATs are expressed by glia (EAAT1 and EAAT2) and neurons (EAAT3 and EAAT4) throughout the brain. Glutamate reuptake is regulated, in part, by EAAT-interacting proteins that modulate subcellular localization and glutamate transport activity of the EAATs. Several lines of investigation support the hypothesis of glutamatergic abnormalities in schizophrenia. Previous work in our laboratory demonstrated increased expression of EAAT1 and EAAT2 transcripts in the thalamus, suggesting that alterations in synaptic glutamate levels may contribute to the pathophysiology of schizophrenia. Since EAAT-interacting proteins regulate EAAT function, directly impacting glutamatergic neurotransmission, we hypothesized that expression of EAAT-interacting proteins may also be altered in schizophrenia. Using in situ hybridization in subjects with schizophrenia and a comparison group, we detected increased expression of JWA and KIAA0302, molecules that regulate EAAT3 and EAAT4, respectively, in the thalamus in schizophrenia. In contrast, we did not find changes in the expression of transcripts for the EAAT2 and EAAT4 regulatory proteins GPS-1 and ARHGEF11. To address prior antipsychotic treatment in our schizophrenic subjects, we treated rats with haloperidol and clozapine for 4 weeks, and found changes in transcript expression of the EAAT-interacting proteins in clozapine-, but not haloperidol-, treated rats. These findings suggest that proteins associated with the regulation of glutamate reuptake may be abnormal in this illness, supporting the hypothesis of altered thalamic glutamatergic neurotransmission in schizophrenia.

Metabotropic glutamate receptor protein expression in the prefrontal cortex and striatum in schizophrenia

We investigated the expression of metabotropic glutamate receptors (mGluR) in the prefrontal cortex (PFC) and striatum in schizophrenia. mGluRs modulate the release and reuptake of synaptic glutamate and mediate some molecular correlates of neuroplasticity, including long-term potentiation. The mGluRs are expressed widely in the PFC and striatum, regions often implicated in the pathophysiology of schizophrenia. Thus, we hypothesized that abnormal expression of mGluRs might contribute to glutamatergic dysfunction observed in the PFC and striatum in schizophrenia. Accordingly, we measured the expression of metabotropic glutamate receptors (mGluRs) in Brodmann areas 9, 11, 32, and 46 in the prefrontal cortex (PFC) and the caudate, putamen, and nucleus accumbens in schizophrenia (16 cases, 9 controls) by Western blot analysis. We found an increase in the expression of mGluR1a and mGluR2/3 immunoreactivity in the PFC in schizophrenia, while no changes in the expression of mGluR4a or mGluR5 were detected in this region. In the striatum we found no changes in the expression of any of the mGluRs studied. These results suggest that alterations of mGluR1a and mGluR2/3 expression in the PFC may contribute to the pathophysiology of schizophrenia, and support targeting these receptors for the generation of novel treatment modalities for this disabling illness.

Increased expression of glutaminase and glutamine synthetase mRNA in the thalamus in schizophrenia

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

Schizophrenia as a disorder of neuroplasticity

Schizophrenia is a devastating mental illness affecting millions worldwide with significant financial and emotional burdens for afflicted persons, their families, and society. Considering schizophrenia as a disorder of neuroplasticity permits integration of competing neurochemical and neurodevelopmental hypotheses. Recent advances have linked the pathophysiology of schizophrenia with abnormalities of the glutamate neurotransmitter system. Elements of glutamergic neurotransmission implicated in schizophrenia, including glutamate receptors and receptor-associated molecules, have critical roles in long-term potentiation, a molecular correlate of neuroplasticity. We suggest that schizophrenia can be considered a disorder of plasticity, associated with molecular abnormalities of the glutamate synapse.

Effects of 6-cyano-7-nitroquinoxaline-2,3-dione on nicotinic receptor subunit transcript expression in the rat brain

The nicotinic cholinergic system exerts potent modulatory effects on glutamatergic neurotransmission, an effect mediated in part by increased glutamate release following activation of presynaptic nicotinic cholinergic receptors. Ionotropic glutamate receptor agonists also stimulate release of acetylcholine, suggesting that these neurotransmitter systems reciprocally regulate one another. We investigated an interface between the nicotinic cholinergic and glutamatergic systems by measuring nicotinic receptor subunit transcript expression following administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the AMPA and kainate subtypes of glutamate receptors. Using [(35)S] in situ hybridization, we measured expression of alpha 2, alpha 3, alpha 4, alpha 5, alpha 7, beta 2, beta 3, and beta 4 nicotinic receptor subunit transcripts in the rat forebrain. Following 7 days of treatment with vehicle or CNQX (1 mg/kg/day or 10 mg/kg/day), changes in nicotinic receptor subunit transcript expression were restricted to subunits that form heteromeric receptors. We found increased levels of transcripts for alpha 2 and beta 2 nicotinic receptor subunits in the hippocampus, decreased alpha 4 subunit transcripts in the medial habenula and amygdala, and increased beta 2 subunit transcripts in the septum and piriform cortex. We did not detect changes in expression of transcripts for the alpha 7 subunit, which forms homomeric nicotinic receptors. Our findings indicate that expression of nicotinic cholinergic receptor subunit transcripts are regulated in a subunit- and region-specific fashion by CNQX, an antagonist of non-NMDA ionotropic glutamate receptors.

Expression of ARHGEF11 mRNA in schizophrenic thalamus

Molecular abnormalities of the glutamate synapse in the thalamus in schizophrenia

Schizophrenia has been associated with dysfunction of glutamatergic neurotransmission. Synaptic glutamate activates pre- and postsynaptic ionotropic NMDA, AMPA, and kainate and metabotropic receptors, is removed from the synapse via five cell surface-expressed transporters, and is packaged for release by three vesicular transporters. In addition, there is a family of intracellular molecules enriched in the postsynaptic density (PSD) that target glutamate receptors to the synaptic membrane, modulate receptor activity, and coordinate glutamate receptor-related signal transduction. Each family of PSD proteins is selective for a given glutamate receptor subtype, the most well characterized being the NMDA receptor binding proteins PSD93, PSD95, NF-L, and SAP102. Besides binding glutamate receptors, many of these proteins also interact with cell surface proteins like cell adhesion molecules, ion channels, cytoskeletal elements, and signal transduction molecules. Given the complexity of the glutamate neurotransmitter system, there are many locations where disruption of normal signaling could occur and give rise to abnormal glutamatergic neurotransmission in schizophrenia. Using multiple cohorts of postmortem tissue, we have examined these synaptic molecules in schizophrenic thalamus. The expression of NR1 and NR2C subunit transcripts is decreased in the thalamus in schizophrenia. Interestingly, three intracellular PSD molecules that link the NMDA receptor to signal transduction pathways are also abnormally expressed. Additionally, several of the cell surface and vesicular transporters are abnormal in the schizophrenic thalamus. While occasional findings of abnormal receptor expression are made, the most dramatic and consistent alterations that we have found in the thalamus in schizophrenia involve the family of intracellular signaling/scaffolding molecules. We propose that schizophrenia has a glutamatergic component that involves alterations in the intracellular machinery that is coupled to glutamate receptors, in addition to abnormalities of the receptors themselves. Our data suggest that schizophrenia is associated with abnormal glutamate receptor-related intracellular signaling in the thalamus, and point to novel targets for innovative drug discovery.

CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis

MCP-1, which signals via the CC chemokine receptor 2 (CCR2), is induced in lung fibrosis that is accompanied by mononuclear cell recruitment and activation of lung fibroblasts. To evaluate the role of CCR2 in lung fibrosis, CCR2 knockout (ko) mice were used in a model of bleomycin-induced lung fibrosis. Wild type (wt) and ko mice were injected endotracheally with bleomycin to induce lung injury and fibrosis, and then analyzed for degree of lung fibrosis and cytokine expression. The results showed significantly reduced fibrosis in ko mice as evidenced by decreased lung type I collagen gene expression and hydroxyproline content relative to those in wt mice. Lung TNF-alpha and TGF-beta1 expression was significantly lower in ko vs. wt mice, while MCP-1 expression was unaffected. Interestingly, lung alpha-smooth muscle actin (alpha-SMA) expression, a marker for myofibroblast differentiation, was also decreased in ko mice, which was confirmed by analysis of isolated lung fibroblasts. Fibroblasts from ko mice exhibited decreased responsiveness to TGF-beta1 induced alpha-SMA expression, which was associated with reduced expression of TGF-beta receptor II (TbetaRII) and Smad3. These findings suggest that CCR2 signaling plays a key role in bleomycin-induced pulmonary fibrosis by regulating fibrogenic cytokine expression and fibroblast responsiveness to TGF-beta.

Differential effects of antipsychotics on haloperidol-induced vacuous chewing movements and subcortical gene expression in the rat

The behavioral and neurochemical effects of switching from typical to atypical medications have not been evaluated in the rodent models of tardive dyskinesia. Thus, we treated rats with haloperidol-decanoate for 12 weeks, and assessed the effects of additional treatment with olanzapine, haloperidol, clozapine, or vehicle on vacuous chewing movements and expression of transcripts for dopamine receptors, tyrosine hydroxylase, delta-opioid receptor, prodynorphin, preproenkephalin, glutamic acid decarboxylase-65 (glutamic acid decarboxylase (GAD)-65) and GAD-67 and N-methyl-D-aspartate (NMDA) receptor subunits in the striatum and its efferent pathways. Haloperidol-decanoate induced vacuous chewing movements extinguished following an additional 4 weeks of treatment with vehicle, olanzapine or haloperidol, but not clozapine. Post-treatment, vacuous chewing movements in the clozapine group were significantly higher than the vehicle, olanzapine and haloperidol groups. GAD-67 mRNA expression in the globus pallidus was decreased following additional treatment with olanzapine or haloperidol, but not clozapine. Changes in expression of other transcripts were not detected. These findings demonstrate important differences in the effects of typical and atypical antipsychotics on chronic vacuous chewing movements.

Striatal excitatory amino acid transporter transcript expression in schizophrenia, bipolar disorder, and major depressive disorder

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.