Altered levels of glutamatergic receptors and Na+/K+ ATPase-α1 in the prefrontal cortex of subjects with schizophrenia

Evidence has accumulated over the past years that dysregulation of glutamatergic neurotransmission maybe implicated in the pathophysiology of schizophrenia. Glutamate acts on two major classes of receptors: ionotropic receptors, which are ligand-gated ion channels, and metabotropic receptors (mGluRs), coupled to heterotrimeric G-proteins. Although several pharmacological evidences point to abnormal glutamatergic transmission in schizophrenia, changes in the expression of glutamatergic receptors in the prefrontal cortex of patients with schizophrenia remains equivocal. In the present work, we have investigated glutamatergic neurotransmission in schizophrenia by assessing the expression in Brodmann Area 10 of mGluR5, the AMPA receptor subunits GluR1 and GluR2, and Na(+)/K(+) ATPase-α1, a potential modulator of glutamate uptake in the brain. Semiquantitative analysis of the expression of these proteins from postmortem brains revealed a particularly prominent reduction of GluR1 and GluR2 expression in patients with schizophrenia vs the control group. Conversely, we observed an up-regulation in the levels of Na(+)/K(+) ATPase-α1 expression. Finally, no change in the protein levels of mGluR5 was observed in schizophrenia. Our findings support and expand the hypothesis of glutamatergic dysfunction in prefrontal cortex in the pathophysiology of schizophrenia.     

A polymorphism in the PDLIM5 gene associated with gene expression and schizophrenia.

BACKGROUND: Results of recent DNA microarray analyses of postmortem brains of patients with schizophrenia revealed that expression of the PDZ and LIM domain 5 gene (PDLIM5) is increased. In the present study, we examined whether polymorphisms in PDLIM5 are associated with schizophrenia. METHODS: We screened for mutations in PDLIM5 in 24 Japanese patients with schizophrenia and evaluated the associations of the identified polymorphisms with schizophrenia in a Japanese case-control population (total samples, 278 schizophrenia patients and 462 control subjects). Expression of PDLIM5 was quantified by real-time quantitative polymerase chain reaction (PCR) in postmortem prefrontal cortex of 34 schizophrenia patients. Electrophoretic mobility shift assay (EMSA) was performed to examine whether a polymorphism influences nuclear protein binding. RESULTS: We identified 27 polymorphisms in PDLIM5 and found associations between polymorphisms (rs2433320 and rs2433322) in the 5' region of the gene and schizophrenia (p = .004). Real-time quantitative PCR revealed that these polymorphisms influenced gene expression (p = .007). An EMSA showed that the different alleles of the rs2433320 polymorphism bound differently to nuclear proteins. CONCLUSIONS: These results suggest that PDLIM5 might play a role in genetic susceptibility to schizophrenia.     

Prenatal exposure to PCP produces behavioral deficits accompanied by the overexpression of GLAST in the prefrontal cortex of postpubertal mice

Altered glutamatergic neurotransmission in the prefrontal cortex (PFC) has been implicated in a myriad of neuropsychiatric disorders. We previously reported that prenatal exposure to PCP produced long-lasting behavioral deficits, accompanied by the abnormal expression and dysfunction of NMDA receptors. In addition, these behavioral changes were attenuated by clozapine treatment. However, whether the prenatal exposure adversely affects pre-synaptic glutamatergic neurotransmission in postpubertal mice remains unknown. In the present study, we investigated the involvement of prefrontal glutamatergic neurotransmission in the impairment of cognitive and emotional behavior after prenatal PCP treatment (5mg/kg/day) from E6 to E18 in mice. The PCP-treated mice showed an impairment of recognition memory in a novel object recognition test and enhancement of immobility in a forced swimming test at 8 weeks of age. Moreover, the prenatal treatment reduced the extracellular glutamate level, but increased the expression of a glial glutamate transporter (GLAST) in the PFC. The microinjection of DL-threo-β-benzyloxyaspartate (DL-TBOA, 10 nmol/site/bilaterally), a potent blocker of glutamate transporters, reversed these behavioral deficits by enhancing the prefrontal glutamatergic neurotransmission. Taken together, prenatal exposure to PCP produced impairments of long-term memory and emotional function which are associated with abnormalities of pre-synaptic glutamate transmission in the PFC of postpubertal mice. These findings suggest the prenatal inhibition of NMDA receptor function to contribute partly to the pathophysiology of neurodevelopment-related disorders, such as schizophrenia.     

Comparative analysis of group II metabotropic glutamate receptor immunoreactivity in Brodmann's area 46 of the dorsolateral prefrontal cortex from patients with schizophrenia and normal subjects

Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system, and a key neurotransmitter in prefrontal cortical function. Converging lines of evidence implicate prefrontal cortical dysfunction in the neurobiology of schizophrenia. Thus, aberrant glutamate neurotransmission may underlie schizophrenia and other complex disorders of behavior. Group II metabotropic receptors (mGluRs) are important modulators of glutamatergic and non-glutamatergic neurotransmission. Moreover, in an animal model, an agonist for group II mGluRs has been shown to reverse the behavioral, locomotor, and cognitive effects of the psychotomimetic drug phencyclidine. Accordingly, group II mGluRs constitute attractive targets for the pharmacotherapeutics and study of schizophrenia. Using immunocytochemistry and Western immunoblotting, we compared the localization and levels of group II mGluRs in Brodmann's area 46 of the dorsolateral prefrontal cortex from patients with schizophrenia and normal subjects. Consistent with previous reports, we found that immunolabeling of group II mGluRs is prominent in Brodmann's area 46. The majority of labeling was present on axon terminals distributed in a lamina-specific fashion. No apparent difference in the cellular localization or laminar distribution of immunoreactive group II mGluRs was noted between the two diagnostic groups. Similarly, the levels of receptor immunoreactivity determined by quantitative Western immunoblotting were comparable between schizophrenic patients and normal subjects. We conclude that while the function of group II mGluRs in Brodmann's area 46 of dorsolateral prefrontal cortex may be altered in patients with schizophrenia, this is not evident at the level of protein expression using an antibody against mGluR2 and mGluR3.     

Increased prefrontal and hippocampal glutamate concentration in schizophrenia: evidence from a magnetic resonance spectroscopy study.

BACKGROUND: Glutamatergic dysfunction has been implicated in the pathophysiology of schizophrenia. However, so far there is limited direct evidence of altered in vivo glutamate concentrations in the brains of patients with schizophrenia. To test the hypothesis that altered glutamatergic neurotransmission might play a role in the pathogenesis of schizophrenia, we measured glutamate and glutamine concentrations in the prefrontal cortex and the hippocampus of patients with chronic schizophrenia using high-field magnetic resonance spectroscopy. METHODS: Twenty-one patients with schizophrenia and 32 healthy volunteers were examined clinically and by means of short echo time single voxel magnetic resonance spectroscopy of the dorsolateral prefrontal cortex and the hippocampus. Absolute concentrations of neurometabolites were calculated. RESULTS: Absolute concentrations of glutamate were significantly higher in the prefrontal cortex and the hippocampus in the patient group. Factorial analysis of variance (ANOVA) revealed no significant interactions between duration of schizophrenia, number of hospitalizations, or type of antipsychotic medication and glutamate concentrations. Increased prefrontal glutamate concentrations were associated with poorer global mental functioning. CONCLUSIONS: This is the first study that reports increased levels of glutamate in prefrontal and limbic areas in patients with schizophrenia. Our data support the hypothesis of glutamatergic dysfunction in schizophrenia.     

An association study between PPP1R1B gene and schizophrenia in the Chinese population

Schizophrenia has been linked with dysfunctions of glutamatergic, dopaminergic, and serotonergic neurotransmission. Dopamine- and cAMP-regulated phosphoprotein of relative molecular mass 32 kDa (DARPP-32), encoded by PPP1R1B (protein phosphatase 1, regulatory/inhibitor subunit 1B) gene, is enriched in neostriatal medium spiny neurons. It plays a key regulator role in dopaminergic and glutamatergic signaling pathways. The combined evidence from reduced DARPP-32 expression in the dorsolateral prefrontal cortex (DLPFC) in schizophrenic patients and from abnormalities in mice with a genetic deletion of DARPP-32 or with point mutations in phosphorylation sites of DARPP-32 suggested that it would be worthwhile to investigate the association between DARPP-32 and schizophrenia. In the present study, we genotyped five single nucleotide polymorphisms (SNPs) in the PPP1R1B gene and conducted a case-control study involving 520 schizophrenic patients and 386 healthy subjects drawn from the Chinese population. No allelic, genotypic or haplotypic association was found. However, our results do not preclude the possibility that the PPP1R1B is a susceptibility gene for schizophrenia in the Chinese population, since, as a central molecular switch, PPP1R1B may contribute to schizophrenia by interacting with other genes. Further functional analysis and genetic association studies are needed to determine the potential roles of PPP1R1B and other related genes in the pathophysiology of schizophrenia.     

Evidence for glutamatergic neuronal dysfunction in the prefrontal cortex in chronic but not in first-episode patients with schizophrenia: a proton magnetic resonance spectroscopy study

Based upon pharmacological challenge and postmortem studies, schizophrenia has been hypothesized to be caused by decreased glutamatergic neurotransmission. We investigated the glutamatergic neuronal metabolism of the dorsolateral prefrontal cortex with localized 1H magnetic resonance spectroscopy in 18 first-episode patients, 21 chronic patients with schizophrenia, and 21 age-matched controls. Chronic patients had significantly lower levels of glutamate/glutamine (Glx) and N-acetylaspartate (NAA) compared to healthy controls and first-episode patients. Reduced metabolite levels were not correlated with duration of illness or medication. Our results indicate glutamatergic dysfunction in chronic schizophrenia that could be evidence of a progressive brain disorder.     

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.     

Chronic phencyclidine administration reduces the expression and editing of specific glutamate receptors in rat prefrontal cortex

Phencyclidine (PCP) induces a form of psychosis that mimics naturally occurring schizophrenia in the most relevant domains of the psychopathology. In this report, we investigated the effect of chronic treatment with PCP on expression and RNA editing of alpha-amino-propionic acid (AMPA) and kainate (KA) glutamate receptor (GluR), in the rat prefrontal cortex and the hippocampus. We found that chronic, but not acute, PCP treatment decreased GluRs expression in the rat prefrontal cortex but not in the hippocampus. In particular, the mRNA coding for GluR2 and GluR3 subunits were reduced by 50%, whereas those coding for KA GluR5 and GluR6 were decreased by 30%. In addition, we observed a decrease of the editing levels of the R/G site in the flop form of both GluR2 and GluR3 and a significant increase in the editing level of GluR6 Q/R site. The variation in the editing level of the R/G sites suggests that chronic PCP treatment induced the formation of glutamate receptor subunits with slower resensitization kinetics and, with respect to kainate receptors, an increase in the Q/R editing level might generate receptor channels with a lower permeability to cations. Combining all the data, it can be inferred that the PCP treatment induced a specific and site-selective reduction of glutamatergic neurotransmission in the prefrontal cortex but not in the hippocampus.     

Effect of Aging on Gene Expression of Metabotropic Glutamate Receptor 5 and Glutamate Transporter-1 in the Prefrontal Cortex of Schizophrenics

Background : The involvement of the glutamatergic system in the patho-physiology of schizophrenia is attracting interest because an antagonist of N-methyl-D-aspartate (NMDA) glutamate receptor has been demonstrated to induce schizophrenic-like symptoms in normal subjects. The results of previous studies have also suggested that glutamatergic function is reduced in the prefrontal cortex (PFC) of diagnosed schizophrenics. In order to provide additional evidence for this, we investigated whether expression of metabotropic glutamate receptor 5 (mGluR5) and glutamate transporter-1 (GLT-1) in the PFC is altered in schizophrenia.
Methods : We compared the expression of mGluRS and GLT-1 by in situ hybridization in Brodmann area 9 (B9) and 10 (B10) of the prefrontal cortex in 5 normal individuals and 5 schizophrenics that were younger than in previous studies, and examined the relationship between age and mRNA expression.
Results : There were no significant differences in either mGluR5 or GLT-1 mRNA expression between the schizophrenics and controls, however, there was a significant correlation to increase mGluR5 mRNA levels in the schizophrenics in both layer III and layer V of B9 with age, a finding not observed in the controls.
Conclusion : The results suggest that the brains of schizophrenics may be vulnerable to aging and that the glutamatergic dysfunctions previously reported in schizophrenics may be partly explained by the aging process.     

GABA transporter-1 mRNA in the prefrontal cortex in schizophrenia: decreased expression in a subset of neurons

OBJECTIVE: Within the prefrontal cortex of schizophrenic subjects, alterations in markers of gamma-aminobutyric acid (GABA) neurotransmission, including decreased immunoreactivity for the GABA membrane transporter GAT-1, may be most prominent in a subset of inhibitory neurons. In the present study, the authors sought to determine whether the alterations in GAT-1 protein could be attributed to a reduction in GAT-1 mRNA expression. METHOD: Tissue sections containing prefrontal cortex area 9 from 10 matched pairs of schizophrenic and comparison subjects were processed for in situ hybridization histochemistry with (35)S-oligonucleotide probes for GAT-1 mRNA. RESULTS: In the schizophrenic subjects, the relative density of labeled neurons was 21%-33% lower in layers 1-5 of the prefrontal cortex but was unchanged in layer 6. In contrast, cellular levels of GAT-1 mRNA expression, as reflected in grain density per labeled neuron, did not differ by more than 11% between subject groups in any layer. These findings indicate that GAT-1 mRNA expression is relatively unaltered in the majority of prefrontal cortex GABA neurons in schizophrenic subjects but is reduced below a detectable level in a subset of GABA neurons. Furthermore, the magnitude and laminar pattern of these results were strikingly similar to those found in a previous study of mRNA expression for the synthesizing enzyme of GABA, glutamic acid decarboxylase(67), in the same subjects. CONCLUSIONS: Both GABA synthesis and reuptake appear to be altered at the level of gene expression in a subset of GABA neurons, and the resulting changes in GABA neurotransmission may contribute to prefrontal cortex dysfunction in schizophrenia.     

Is the G72/G30 locus associated with schizophrenia? single nucleotide polymorphisms, haplotypes, and gene expression analysis.

BACKGROUND: The genes G72/G30 were recently implicated in schizophrenia in both Canadian and Russian populations. We hypothesized that 1) polymorphic changes in this gene region might be associated with schizophrenia in the Ashkenazi Jewish population and that 2) changes in G72/G30 gene expression might be expected in schizophrenic patients compared with control subjects. METHODS: Eleven single nucleotide polymorphisms (SNPs) encompassing the G72/G30 genes were typed in the genomic deoxyribonucleic acid (DNA) from 60 schizophrenic patients and 130 matched control subjects of Ashkenazi ethnic origin. Case-control comparisons were based on linkage disequilibrium (LD) and haplotype frequency estimations. Gene expression analysis of G72 and G30 was performed on 88 postmortem dorsolateral prefrontal cortex samples. RESULTS: Linkage disequilibrium analysis revealed two main SNP blocks. Haplotype analysis on block II, containing three SNPs external to the genes, demonstrated an association with schizophrenia. Gene expression analysis exhibited correlations between expression levels of the G72 and G30 genes, as well as a tendency toward overexpression of the G72 gene in schizophrenic brain samples of 44 schizophrenic patients compared with 44 control subjects. CONCLUSIONS: It is likely that the G72/G30 region is involved in susceptibility to schizophrenia in the Ashkenazi population. The elevation in expression of the G72 gene coincides with the glutamatergic theory of schizophrenia.     

The Astrocyte-Derived α7 Nicotinic Receptor Antagonist Kynurenic Acid Controls Extracellular Glutamate Levels in the Prefrontal Cortex

The cognitive deficits seen in schizophrenia patients are likely related to abnormal glutamatergic and cholinergic neurotransmission in the prefrontal cortex. We hypothesized that these impairments may be secondary to increased levels of the astrocyte-derived metabolite kynurenic acid (KYNA), which inhibits α7 nicotinic acetylcholine receptors (α7AChR) and may thereby reduce glutamate release. Using in vivo microdialysis in unanesthetized rats, we show here that nanomolar concentrations of KYNA, infused directly or produced in situ from its bioprecursor kynurenine, significantly decrease extracellular glutamate levels in the prefrontal cortex. This effect was prevented by the systemic administration of galantamine (3 mg/kg) but not by donepezil (2 mg/kg), indicating that KYNA blocks the allosteric potentiating site of the α7AChR, which recognizes galantamine but not donepezil as an agonist. In separate rats, reduction of prefrontal KYNA formation by (S)-4-ethylsulfonyl benzoylalanine, a specific inhibitor of KYNA synthesis, caused a significant elevation in extracellular glutamate levels. Jointly, our results demonstrate that fluctuations in endogenous KYNA formation bidirectionally influence cortical glutamate concentrations. These findings suggest that selective attenuation of cerebral KYNA production, by increasing glutamatergic tone, might improve cognitive function in individuals with schizophrenia.     

The emerging role of glutamate in the pathophysiology and treatment of schizophrenia

OBJECTIVE: Research has implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. This review evaluates evidence from preclinical and clinical studies that brain glutamatergic neurotransmission is altered in schizophrenia, may affect symptom expression, and is modulated by antipsychotic drugs. METHOD: A comprehensive review of scientific articles published over the last decade that address the role of glutamate in the pathophysiology of schizophrenia was carried out. RESULTS: Glutamatergic neurons are the major excitatory pathways linking the cortex, limbic system, and thalamus, regions that have been implicated in schizophrenia. Postmortem studies have revealed alterations in pre- and postsynaptic markers for glutamatergic neurons in several brain regions in schizophrenia. The N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptor may be particularly important as blockade of this receptor by the dissociative anesthetics reproduces in normal subjects the symptomatic manifestations of schizophrenia, including negative symptoms and cognitive impairments, and increases dopamine release in the mesolimbic system. Agents that indirectly enhance NMDA receptor function via the glycine modulatory site reduce negative symptoms and variably improve cognitive functioning in schizophrenic subjects receiving typical antipsychotics. CONCLUSIONS: Dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia, especially of the negative symptoms and cognitive impairments associated with the disorder, and is a promising target for drug development.     

Microarray analysis of gene expression in the prefrontal cortex in schizophrenia: a preliminary study

Microarray studies can be used to examine expression levels for large numbers of genes simultaneously and may be applied to identify genes involved in schizophrenia. A microarray with 1127 brain-relevant genes was used to screen relative gene expression in the dorsolateral prefrontal cortex (DLPFC) from three pools of patients with schizophrenia (n = 15) and three matched control pools (n = 15). Pooling of tissue samples was employed as a strategy to detect changes in gene expression that are consistently found across individual cases of schizophrenia. Differences in gene expression were examined by z-ratios in addition to traditional normalized ratios. Three genes that showed consistently decreased expression in schizophrenia by both z-ratio differences and decreased normalized numerical ratios were identified. These were histidine triad nucleotide-binding protein (HINT), ubiquitin conjugating enzyme E2N (UBE2N) and glutamate receptor, ionotropic, AMPA 2 (GRIA2). Moreover, HINT gene expression was decreased to a similar degree in a prior study. In addition, a decrease in AMPA receptor expression is consistent with a decrease in glutamate synaptic function. These results are subject to limitations based on variations inherent to human subjects and tissue samples, possible effects of neuroleptic treatment, and the requirement for verification using independent techniques.