Chemotherapy-induced cognitive changes is a major burden on a substantial number of cancer survivors. The mechanism of this sequel is unknown. In this study, we followed long-term effects of early in life mithramycin (MTR) treatment on behaviour and on the normal course of alterations of gene expression in brain. Between post-natal days (PND) 7 and 10, male rats were divided into 2 groups, 1 receiving MTR (0.1 mg/kg s.c. per day) and the other receiving saline. At PND11, frontal cortex tissue samples were dissected from 4 rats from each group. At PND 65 the remaining rats underwent behavioural tests after which all the rats were decapitated and their prefrontal cortex incised. Rats treated transiently with MTR early in life, showed impairments in spatial working memory and anxious-like behaviour in adulthood. The immediate molecular effect of MTR was expressed in a limited number of altered genes of different unconnected trajectories, which were simultaneously distorted by the drug. In contrast, 3 months later we observed a change in the expression of more than 1000 genes that converged into specific cellular processes. Time-dependent gene expression dynamics of several genes was significantly different between treated and untreated rats. The differences in the total number of altered genes and in gene expression trends, immediately and long after MTR treatment cessation, suggest the evolution of a new cellular homeostatic set point, which can lead to behavioural abnormalities following chemotherapy treatment.

Transcription factors play important roles in the control of neuronal function in physiological and pathological conditions. We previously reported reduced levels of transcription factor SP4 protein, but not transcript, in the cerebellum in bipolar disorder and associated with more severe negative symptoms in schizophrenia. We have recently reported phosphorylation of Sp4 at S770, which is regulated by membrane depolarization and NMDA receptor activity. The aim of this study was to investigate SP4 S770 phosphorylation in bipolar disorder and its association with negative symptoms in schizophrenia, and to explore the potential relationship between phosphorylation and protein abundance. Here we report a significant increase in SP4 phosphorylation in the cerebellum, but not the prefrontal cortex, of bipolar disorder subjects (n=10) (80% suicide) compared to matched controls (n=10). We found that SP4 phosphorylation inversely correlated with SP4 levels independently of disease status in both areas of the human brain. Moreover, SP4 phosphorylation in the cerebellum positively correlated with negative symptoms in schizophrenia subjects (n=15). Further, we observed that a phospho-mimetic mutation in truncated Sp4 was sufficient to significantly decrease Sp4 steady-state levels, while a non-phosphorylatable mutant showed increased stability in cultured rat cerebellar granule neurons. Our results indicate that SP4 S770 phosphorylation is increased in the cerebellum in bipolar disorder subjects that committed suicide and in severe schizophrenia subjects, and may be part of a degradation signal that controls Sp4 abundance in cerebellar granule neurons. This opens the possibility that modulation of SP4 phosphorylation may contribute to the molecular pathophysiology of psychotic disorders.

Altered levels of transcription factor specificity protein 4 (SP4) and 1 (SP1) in the cerebellum, prefrontal cortex and/or lymphocytes have been reported in severe psychiatric disorders, including early psychosis, bipolar disorder, and chronic schizophrenia subjects who have undergone long-term antipsychotic treatments. SP4 transgenic mice show altered hippocampal-dependent psychotic-like behaviours and altered development of hippocampal dentate gyrus. Moreover, NMDAR activity regulates SP4 function. The aim of this study was to investigate SP4 and SP1 expression levels in the hippocampus in schizophrenia, and the possible effect of antipsychotics and NMDAR blockade on SP protein levels in rodent hippocampus. We analysed SP4 and SP1 expression levels in the postmortem hippocampus of chronic schizophrenia (n = 14) and control (n = 11) subjects by immunoblot and quantitative RT-PCR. We tested the effect of NMDAR blockade on SP factors in the hippocampus of mouse treated with an acute dose of MK801. We also investigated the effect of subacute treatments with haloperidol and clozapine on SP protein levels in the rat hippocampus. We report that SP4 protein and both SP4 and SP1 mRNA expression levels are significantly increased in the hippocampus in chronic schizophrenia. Likewise, acute treatment with MK801 increased both SP4 and SP1 protein levels in mouse hippocampus. In contrast, subacute treatment with haloperidol and clozapine did not significantly alter SP protein levels in rat hippocampus. These results suggest that SP4 and SP1 upregulation may be part of the mechanisms deregulated downstream of glutamate signalling pathways in schizophrenia and might be contributing to the hippocampal-dependent cognitive deficits of the disorder.

Glutamatergic signaling through N-methyl-d-aspartate receptors (NMDARs) is important for neuronal development and plasticity and is often dysregulated in psychiatric disorders. Mice mutant for the transcription factor Sp4 have reduced levels of NMDAR subunit 1 (NR1) protein, but not mRNA, and exhibit behavioral and memory deficits (Zhou et al., [2010] Human Molecular Genetics 19: 3797-3805). In developing cerebellar granule neurons (CGNs), Sp4 controls dendrite patterning (Ramos et al., [2007] Proc Natl Acad Sci USA 104: 9882-9887). Sp4 target genes that regulate dendrite pruning or NR1 levels are not known. Here we report that Sp4 activates transcription of Nervous Wreck 2 (Nwk2; also known as Fchsd1) and, further, that Nwk2, an F-BAR domain-containing protein, mediates Sp4-dependent regulation of dendrite patterning and cell surface expression of NR1. Knockdown of Nwk2 in CGNs increased primary dendrite number, phenocopying Sp4 knockdown, and exogenous expression of Nwk2 in Sp4-depleted neurons rescued dendrite number. We observed that acute Sp4 depletion reduced levels of surface, but not total, NR1, and this was rescued by Nwk2 expression. Furthermore, expression of Nr1 suppressed the increase in dendrite number in Sp4- or Nwk2- depleted neurons. We previously reported that Sp4 protein levels were reduced in cerebellum of subjects with bipolar disorder (BD) (Pinacho et al., [2011] Bipolar Disorders 13: 474-485). Here we report that Nwk2 mRNA and NR1 protein levels were also reduced in postmortem cerebellum of BD subjects. Our data suggest a role for Sp4-regulated Nwk2 in NMDAR trafficking and identify a Sp4-Nwk2-NMDAR1 pathway that regulates neuronal morphogenesis during development and may be disrupted in bipolar disorder.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Mutation in TAR DNA binding protein 43 (TDP-43) is a causative factor of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurodegeneration may not require the presence of pathogenic TDP-43 in all types of relevant cells. Rather, expression of pathogenic TDP-43 in neurons or astrocytes alone is sufficient to cause cell-autonomous or non-cell-autonomous neuron death in transgenic rats. How pathogenic TDP-43 in astrocytes causes non-cell-autonomous neuron death, however, is not clear. Here, we examined the effect of pathogenic TDP-43 on gene expression in astrocytes. Microarray assay revealed that pathogenic TDP-43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down-regulated, neurotoxic genes were up-regulated. Representative genes Lcn2 and chitinase-3-like protein 1 were markedly up-regulated in astrocytes from primary culture and intact transgenic rats. Furthermore, synthetic chitinase-3-like protein 1 induced neuron death in a dose-dependent manner. Our results suggest that TDP-43 pathogenesis is associated with the simultaneous induction of multiple neurotoxic genes in astrocytes, which may synergistically produce adverse effects on neuronal survival and contribute to non-cell-autonomous neuron death. Restricted expression of pathogenic TDP-43 in astrocytes causes non-cell-autonomous motor neuron death in transgenic rats. As revealed by microarray assay, pathogenic TDP-43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down-regulated, neurotoxic genes were up-regulated. Therefore, TDP-43 pathogenesis is associated with simultaneous induction of neurotoxic genes and repression of neurotrophic genes in astrocytes.

It is currently accepted that complex behavior and mental disorder results from a combination of biological susceptibility and exposure to environmental stimuli. Most of the gene-environment interaction models focus on the interaction between the stimuli and a single candidate gene. We suggest that an alternative approach is interference with the expression of multiple genes followed by exposure to environmental insults.

Early interference with gene transcription was performed by treatment of 7 days old Wistar male rats for 4 days with the Sp1/DNA binding inhibitor, mithramycin. Environmental insult was mimicked by exposing these rats during adulthood (34 days) to sub-chronic (12 days, n=30) or chronic stress (28 days, n=48). The effects of mithramycin and stress treatment on the behavioral response and serum corticosterone concentration were assessed.

Exposure of mithramycin treated rats to sub-chronic stress led to anxious behavior in the open field test, high startle response, low sucrose preference, indifference to novel objects and high serum corticosterone concentration. However, exposure to chronic stress resulted in normal sucrose preference, startle response and serum corticosterone, novelty seeking behavior and reduced anxiety. In saline treated rats the extension of stress duration led to behavioral and hormonal adaptation to stress.

Our study suggests that postnatal temporal interference with multiple gene expression can lead to hyper-responsiveness to environmental stimuli, the features of which affects the phenotypic outcomes. Such a paradigm may be used to model gene-environmental interaction in the etiology of behavioral disorders.

N-Methyl-d-aspartate (NMDA) receptors are major glutamatergic receptors involved in most excitatory neurotransmission in the brain. The transcriptional regulation of NMDA receptors is not fully understood. Previously, we found that the GluN1 and GluN2B subunits of the NMDA receptor are regulated by nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2). NRF-1 and NRF-2 also regulate all 13 subunits of cytochrome c oxidase (COX), a critical energy-generating enzyme, thereby coupling neuronal activity and energy metabolism at the transcriptional level. Specificity protein (Sp) is a family of transcription factors that bind to GC-rich regions, with Sp1, Sp3, and Sp4 all binding to the same cis- motifs. Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 expression is restricted to neurons and testicular cells. Recently, we found that the Sp1 factor regulates all subunits of COX. The goal of the present study was to test our hypothesis that the Sp factors also regulate specific subunits of NMDA receptors, and that they function with NRF-1 and NRF-2 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches we found that Sp4 functionally regulated GluN1, GluN2A, and GluN2B, but not GluN2C. On the other hand, Sp1 and Sp3 did not regulate these subunits as previously thought. Our data suggest that Sp4 operates in a complementary and concurrent/parallel manner with NRF-1 and NRF-2 to mediate the tight coupling between energy metabolism and neuronal activity at the molecular level.

Negative symptoms are the most resilient manifestations in schizophrenia. An imbalance in dopamine and glutamate pathways has been proposed for the emergence of these symptoms. SP1, SP3 and SP4 transcription factors regulate genes in these pathways, suggesting a possible involvement in negative symptoms. In this study, we characterized Sp factors in the brains of subjects with schizophrenia and explored a possible association with negative symptoms. We also included analysis of NR1, NR2A and DRD2 as Sp target genes. Postmortem cerebellum and prefrontal cortex from an antemortem clinically well-characterized and controlled collection of elderly subjects with chronic schizophrenia (n = 16) and control individuals (n = 14) were examined. We used the Positive and Negative Syndrome and the Clinical Global Impression Schizophrenia scales, quantitative PCR and immunoblot. SP1 protein and mRNA were reduced in the prefrontal cortex in schizophrenia whereas none of Sp factors were altered in the cerebellum. However, we found that SP1, SP3 and SP4 protein levels inversely correlated with negative symptoms in the cerebellum. Furthermore, NR2A and DRD2 mRNA levels correlated with negative symptoms in the cerebellum. In the prefrontal cortex, SP1 mRNA and NR1 and DRD2 inversely correlated with these symptoms while Sp protein levels did not. This pilot study not only reinforces the involvement of SP1 in schizophrenia, but also suggests that reduced levels or function of SP1, SP4 and SP3 may participate in negative symptoms, in part through the regulation of NMDA receptor subunits and/or Dopamine D2 receptor, providing novel information about the complex negative symptoms in this disorder.

Glial reaction is a common feature of neurodegenerative diseases. Recent studies have suggested that reactive astrocytes gain neurotoxic properties, but exactly how reactive astrocytes contribute to neurotoxicity remains to be determined. Here, we identify lipocalin 2 (lcn2) as an inducible factor that is secreted by reactive astrocytes and that is selectively toxic to neurons. We show that lcn2 is induced in reactive astrocytes in transgenic rats with neuronal expression of mutant human TAR DNA-binding protein 43 (TDP-43) or RNA-binding protein fused in sarcoma (FUS). Therefore, lcn2 is induced in activated astrocytes in response to neurodegeneration, but its induction is independent of TDP-43 or FUS expression in astrocytes. We found that synthetic lcn2 is cytotoxic to primary neurons in a dose-dependent manner, but is innocuous to astrocytes, microglia, and oligodendrocytes. Lcn2 toxicity is increased in neurons that express a disease gene, such as mutant FUS or TDP-43. Conditioned medium from rat brain slice cultures with neuronal expression of mutant TDP-43 contains abundant lcn2 and is toxic to primary neurons as well as neurons in cultured brain slice from WT rats. Partial depletion of lcn2 by immunoprecipitation reduced conditioned medium-mediated neurotoxicity. Our data indicate that reactive astrocytes secrete lcn2, which is a potent neurotoxic mediator.

The wild-type (WT) Cpr(lox/lox) (cytochrome P(450) oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P(450) enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)-dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cpr(lox/lox) mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P(450)1B1-associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14-E17) exposure to B(a)P (100 μg/m(3)), Cpr(lox/lox) offspring exhibited: (1) elevated B(a)P metabolite and F(2)-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype.

CYP46A1 is a neuron-specific cytochrome P450 that plays a pivotal role in maintaining cholesterol homeostasis in the CNS. However, the molecular mechanisms underlying human CYP46A1 expression are still poorly understood, partly because of the lack of a cellular model that expresses high levels of CYP46A1. Our previous studies demonstrated that specificity protein (Sp) transcription factors control CYP46A1 expression, and are probably responsible for cell-type specificity. Herein, we have differentiated Ntera2/cloneD1 cells into post-mitotic neurons and identified for the first time a human cell model that expresses high levels of CYP46A1 mRNA. Our results show a decrease in Sp1 protein levels, concomitant with the increase in CYP46A1 mRNA levels. This decrease was correlated with changes in the ratio of Sp proteins associated to the CYP46A1 proximal promoter. To examine if the increase in (Sp3+Sp4)/Sp1 ratio was observed in other Sp-regulated promoters, we have selected four genes--reelin, glutamate receptor subunit zeta-1, glutamate receptor subunit epsilon-1 and μ-opioid receptor--known to be expressed in the human brain and analyzed the Sp proteins binding pattern to the promoter of these genes, in undifferentiated and differentiated Ntera2/cloneD1. Our data indicate that the dissociation of Sp1 from promoter regions is a common feature amongst Sp-regulated genes that are up-regulated after neuronal differentiation.

Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.

Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca(2+) concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.

Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca(2+) entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of alpha7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca(2+) entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca(2+) entry induced by donepezil.

Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.

Multiple factors are involved in the glutamate-induced excitotoxicity phenomenon, such as overload of ionotropic and metabotropic receptors, excess Ca(2+) influx, nitric oxide synthase activation, oxidative damage due to increase in free radicals, and release of endogenous polyamine, among others. In order to attempt a more integrated approach to address this issue, we established, by microarray analysis, the hippocampus gene expression profiles under glutamate-induced excitotoxicity conditions. Increased gene expression is mainly related to excitotoxicity (CaMKII, glypican 2, GFAP, NCX3, IL-2, and Gmeb2) or with cell damage response (dynactin and Ecel1). Several genes that augmented their expression are related to glutamatergic system modulation, in particular with NMDA receptor modulation and calcium homeostasis (IL-2, CaMKII, acrosin, Gmeb2, hAChE, Slc83a, and SP1 factor). Conversely, among genes that diminished their expression, we found the Syngap 1, which is downregulated by CaMKII, and the MHC II, which is downregulated by glutamate. Changes observed in gene expression induced by monosodium glutamate (MSG) neonatal treatment in the hippocampus are consistent with the activation of the mechanisms that modulate NMDA receptor function as well as with the implementation of plastic response to cell damage and intracellular calcium homeostasis. Regarding this aspect, we report here that NCX3/Slc8a3, a Na(+)/Ca(2+) membrane exchanger, is highly expressed in astrocytes, both in vitro and in vivo, in response to glutamate-induced excitotoxicity. Hence, the results of this analysis present a broad view of the expression profile elicited by MSG neonatal treatment, and lead us to suggest the possible molecular pathways of action and reaction involved under this experimental model of excitotoxicity.

Gene by environment interactions (G × E) are thought to underlie neurodevelopmental disorder, etiology, neurodegenerative disorders, including the multiple forms of autism spectrum disorder. However, there is limited biological information, indicating an interaction between specific genes and environmental components. The present study focuses on a major component of airborne pollutants, polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene [B(a)P], which negatively impacts cognitive development in children who have been exposed in utero. In our study, prenatal exposure of Cpr(lox/lox) timed-pregnant dams to B(a)P (0, 150, 300, and 600 μg/kg body weight via oral gavage) on embryonic day (E14-E17) consistent with our susceptibility-exposure paradigm was combined with the analysis of a replicated autism risk gene, the receptor tyrosine kinase, Met. The results demonstrate a dose-dependent increase in B(a)P metabolite generation in B(a)P-exposed Cpr(lox/lox) offspring. Additionally, a sustained persistence of hydroxy metabolites during the onset of synapse formation was noted, corresponding to the peak of Met expression. Prenatal B(a)P exposure also downregulated Met RNA and protein levels and dysregulated normal temporal patterns of expression during synaptogenesis. Consistent with these data, transcriptional cell-based assays demonstrated that B(a)P exposure directly reduces human MET promoter activity. Furthermore, a functional readout of in utero B(a)P exposure showed a robust reduction in novel object discrimination in B(a)P-exposed Cpr(lox/lox) offspring. These results confirm the notion that common pollutants, such as the PAH B(a)P, can have a direct negative impact on the regulated developmental expression of an autism risk gene with associated negative behavioral learning and memory outcomes.

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

Schizophrenia is currently believed to result from variations in multiple genes, each contributing a subtle effect, which combines with each other and with environmental stimuli to impact both early and late brain development. At present, schizophrenia clinical heterogeneity as well as the difficulties in relating cognitive, emotional and behavioral functions to brain substrates hinders the identification of a disease-specific anatomical, physiological, molecular or genetic abnormality. Mitochondria play a pivotal role in many essential processes, such as energy production, intracellular calcium buffering, transmission of neurotransmitters, apoptosis and ROS production, all either leading to cell death or playing a role in synaptic plasticity. These processes have been well established as underlying altered neuronal activity and thereby abnormal neuronal circuitry and plasticity, ultimately affecting behavioral outcomes. The present article reviews evidence supporting a dysfunction of mitochondria in schizophrenia, including mitochondrial hypoplasia, impairments in the oxidative phosphorylation system (OXPHOS) as well as altered mitochondrial-related gene expression. Abnormalities in mitochondrial complex I, which plays a major role in controlling OXPHOS activity, are discussed. Among them are schizophrenia specific as well as disease-state-specific alterations in complex I activity in the peripheral tissue, which can be modulated by DA. In addition, CNS and peripheral abnormalities in the expression of three of complex I subunits, associated with parallel alterations in their transcription factor, specificity protein 1 (Sp1) are reviewed. Finally, this review discusses the question of disease specificity of mitochondrial pathologies and suggests that mitochondria dysfunction could cause or arise from anomalities in processes involved in brain connectivity.

Neuronal activity and energy metabolism are tightly coupled processes. Regions high in neuronal activity, especially of the glutamatergic type, have high levels of cytochrome c oxidase (COX). Perturbations in neuronal activity affect the expressions of COX and glutamatergic NMDA receptor subunit 1 (NR1). The present study sought to test our hypothesis that the coupling extends to the transcriptional level, whereby NR1 and possibly other NR subunits and COX are coregulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), which regulates all COX subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutations, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Grin 1 (NR1), Grin 2b (NR2b) and COX subunit genes, but not of Grin2a and Grin3a genes. These transcripts were upregulated by KCl and downregulated by tetrodotoxin (TTX) in cultured primary neurons. However, silencing of NRF-1 with small interference RNA blocked the upregulation of Grin1, Grin2b, and COX induced by KCl, and overexpression of NRF-1 rescued these transcripts that were suppressed by TTX. NRF-1 binding sites on Grin1 and Grin2b genes are also highly conserved among mice, rats, and humans. Thus, NRF-1 is an essential transcription factor critical in the coregulation of NR1, NR2b, and COX, and coupling exists at the transcriptional level to ensure coordinated expressions of proteins important for synaptic transmission and energy metabolism.

The prevailing hypothesis regards schizophrenia as a polygenic disease, in which multiple genes combine with each other and with environmental stimuli to produce the variance of its clinical symptoms. We investigated whether the ubiquitous transcription factor Sp1 is abnormally expressed in schizophrenia, and consequently can affect the expression of genes implicated in this disorder.

mRNA of Sp1 and of mitochondrial complex I subunits (NDUFV1, NDUFV2) was analyzed in three postmortem brain regions obtained from the Stanley Foundation Brain Collection, and in lymphocytes of schizophrenic patients and controls. Sp1 role in the transcription of these genes was studied as well. Sp1 was abnormally expressed in schizophrenia in both brain and periphery. Its mRNA alteration pattern paralleled that of NDUFV1 and NDUFV2, decreasing in the prefrontal cortex and the striatum, while increasing in the parieto-occipital cortex and in lymphocytes of schizophrenic patients as compared with controls. Moreover, a high and significant correlation between these genes existed in normal subjects, but was distorted in patients. Sp1 role in the regulation of complex I subunits, was demonstrated by the ability of the Sp1/DNA binding inhibitor, mithramycin, to inhibit the transcription of NDUFV1 and NDUFV2, in neuroblastoma cells. In addition, Sp1 activated NDUFV2 promoter by binding to its three GC-boxes. Both activation and binding were inhibited by mithramycin.

These findings suggest that abnormality in Sp1, which can be the main activator/repressor or act in combination with additional transcription factors and is subjected to environmental stimuli, can contribute to the polygenic and clinically heterogeneous nature of schizophrenia.

Metabotropic glutamate receptor 1 (mGlu1) has a discrete distribution in the central nervous system restricted to neurons. Its expression undergoes important changes during development and in response to physiological and pathological modifications. Here, we have determined the structure of the mGlu1 gene and demonstrated that mGlu1 transcription takes places at alternative first exons. Moreover, we have identified active promoter regions upstream from the two most expressed first exons by means of luciferase reporter gene assays performed in primary cerebellar granule neurons. Targeted mutations of active elements constituting the core promoter and electrophoretic mobility shift assays demonstrated that the factors thyroid transcription factor-1 and CCAAT/enhancer-binding proteins beta act synergistically to promote mGlu1 transcription. We have also elucidated the molecular bases for the neuron-specific expression of mGlu1 identifying a neural restrictive silencing element and a regulatory factor for X box element, which suppressed mGlu1 expression in nonneuronal cells. These results reveal the molecular bases for cell- and context-specific expression of an important glutamate receptor critically involved in synaptogenesis, neuronal differentiation, synaptic transmission, and plasticity.

The aims of this project were to investigate whether inflammation in the orofacial muscle alters mu opioid receptor (MOR) mRNA and protein expressions in trigeminal ganglia (TG), and to assess the contribution of peripheral MORs under acute and inflammatory muscle pain conditions. mRNA and protein levels for MOR were quantified by reverse-transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, from the TG of naïve rats, and compared with those from the rats treated with complete Freund's adjuvant (CFA) in the masseter. TG was found to express mRNA and protein for MOR, and CFA significantly up-regulated both MOR mRNA and protein by 3 days following the inflammation. The MOR protein up-regulation persisted to day 7 and returned to the baseline level by day 14. We then investigated whether peripheral application of a MOR agonist, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin acetate salt (DAMGO), attenuates masseter nociception induced by masseteric infusion of hypertonic saline (HS) in lightly anesthetized rats. DAMGO (1, 5, 10 microg) or vehicle was administered directly into the masseter 5-10 min prior to the HS infusion. The DAMGO effects were assessed on mean peak counts (MPC) and overall magnitude as calculated by the area under the curve (AUC) of the HS-evoked behavioral responses. Under this condition, only the highest dose of DAMGO (10 microg) significantly reduced MPC, which was prevented when H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective MOR antagonist, was co-administered. DAMGO pre-treatment in the contralateral masseter did not attenuate MPC. The same doses of DAMGO administered into CFA-inflamed rats, however, produced a greater attenuation of both MPC and AUC of HS-evoked nocifensive responses. These results demonstrated that activation of peripheral MOR provides greater anti-nociception in inflamed muscle, and that the enhanced MOR effect can be partly explained by significant up-regulation of MOR expression in TG.

The molecular mechanism regulating serotonin 5-HT(7) receptor expression is still unclear. In this study we provide evidence that transcription of the 5-HT(7) gene is at least partly regulated by Sp1 and Sp3. We isolated and sequenced >3000 bp of the upstream sequences and identified by RACE a number of transcriptional initiation sites over a region of 300 bp upstream of the coding region. This region has a high GC content, but contains no obvious TATA or a CAAT box. Besides a Sp1/Sp3 consensus motif, regulatory elements for AP2, Egr-1 and MAZ are also present. Transient transfection assays using deletion variants indicated that the GC-rich region is essential for full promoter activity. The role of Sp1 in this was confirmed by transient overexpression of both wild type Sp1 or dominant-negative forms. By gel shift and supershift analyses, targeting the Sp1 consensus sequence and the GC-rich region just upstream of the transcription initiation sites, binding of Sp1 and Sp3 was demonstrated. Both in vitro as well as in vivo experiments, using a cell line which endogenously expresses the 5-HT(7) receptor, indicated that mithramycin A, an inhibitor of Sp1/3 transcription factor binding, was able to inhibit 5-HT(7) promoter activity. Taken together these results support the essential role of Sp factors in regulating 5-HT(7) promoter activity.

The K(+) channel TASK-3 is highly expressed in cerebellar granule neurons where it encodes the K(+) current IKso. Besides the role of TASK-3 in controlling cellular excitability and shaping neuronal responses, it has recently been proposed to contribute to the development and maturation of neurons in the cerebellum. K(+) dependent apoptosis and tumorigenesis have also been attributed to TASK-3 over-expression. Transcription of TASK-3 is strongly dependent on depolarization-induced Ca(2+)-entry. To understand the mechanisms involved in TASK-3 regulation, we have characterized a minimal promoter which specifically expresses in cellular backgrounds expressing endogenous TASK-3. Moreover, we have cloned and characterized the 5' and 3' untranslated regions of TASK-3. Both regions contribute to inhibit expression of a reporter gene. Given the direct consequence of membrane potential on TASK-3 expression, this is an important first step towards the understanding of the complex regulation of this gene.

Calcineurin (protein phosphatase 2B) is a calcium-dependent serine-threonine phosphatase. It has diverse roles and is centrally involved in synaptic plasticity. The catalytic A subunit of calcineurin has three isoforms, alpha, beta and gamma. Their expression and ontogeny in the brain has not been systematically investigated; such data become important with a report that PPP3CC, the gene encoding calcineurin Agamma, is a susceptibility gene for schizophrenia, and the finding that its expression is decreased in the disorder. We used in situ hybridization histochemistry to measure the relative transcript abundance of calcineurin Agamma and the other catalytic isoforms, Aalpha and Abeta, during development of the Sprague-Dawley rat hippocampus and cerebellum. All three isoforms are present in both regions at all time points [embryonic day 19 (E19) to postnatal day 42 (P42)] and undergo developmental regulation, but differ in their ontogenic profile. Calcineurin Aalpha and Abeta mRNAs increased from E19 through to adulthood, whereas Agamma mRNA was most highly expressed during early developmental stages. Calcineurin Aalpha and Abeta mRNAs positively correlated with synaptophysin mRNA (a synaptic marker), whilst Agamma mRNA was either unrelated to, or negatively correlated, with this transcript. These data confirm that all three calcineurin A subunits are expressed in the rodent brain, and indicate that calcineurin Agamma may have different roles than Aalpha and Abeta. The data also suggest a potential importance of calcineurin Agamma in neurodevelopment, and in the genetically influenced neurodevelopmental disturbance that is thought to underlie schizophrenia.

Certain kinds of developmental neurotoxicants are considered to act by affecting the levels of thyroid hormones, which are essential for the brain development of both humans and experimental animals. Hypothyroidism experimentally induced in rats with propylthiouracil (PTU) offers a useful animal model for developmental neurotoxicity. The purpose of the present study was to clarify developmental alterations in gene expression caused by PTU in this model, with the focus on eight genes implicated in neural network formation or synaptic functions, such as the brain-derived neurotrophic factor (BDNF) and NMDA receptors 2A/2B. First, we measured the developmental profile of gene expression in vehicle-dosed rat cerebellum by quantitative RT-PCR and then examined the effects of PTU on mRNA levels on postnatal day (PND) 22, when most of the cerebellar structures in mature animals are already formed. PTU induced up-regulation of NR2B mRNA and down-regulation of NR2A and BDNF mRNAs in the cerebellum on PND 22, but there were no changes in the other genes (growth associated protein-43, L1, neuronal cell adhesion molecule, synaptophysin, post synaptic density-95). Examination of the effects of PTU on maturation of NMDAR subunits (NR2A/NR2B) demonstrated changes in relative expression on PND 14, but not on PND 4, with recovery after maturation. The profile of NMDAR subunits in vehicle-dosed rats showed a shift from NR2B to NR2A during development. These results suggest PTU can delay this switching from NR2B to NR2A subunits in the maturation of NMDA receptors.

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.

NMDA receptor is an important player in neuronal plasticity, including cortical reorganization. In the adult cerebral cortex, the receptor properties are regulated by relative expression of NR2A and NR2B subunits. We have previously found that 3 days of sensory conditioning, in which stimulation of whiskers was paired with a tail shock, induce NMDA-receptor-dependent expansion of metabolically labeled cortical representations of the stimulated vibrissae. Here, we examined the effect of learning-induced cortical reorganization upon expression of NR2A and NR2B NMDA receptor subunits. An increase in NR2A mRNA expression in the barrel of the "trained" row of vibrissae was observed with in situ hybridization 24 h after sensory conditioning. NR2B mRNA expression level did not change. Protein level of both regulatory subunits and obligatory NR1 subunit were examined in P2 fraction. NR2A protein level was found elevated 1 h and 24 h after the sensory conditioning, but not in controls which received only whisker stimulation, signifying that the change was associated with cortical map reorganization. NR2B protein level was transiently elevated in both trained and stimulated control groups. NR1 protein level did not change. The results show that simple sensory learning induces a change in expression of regulatory NMDA receptor subunits, indicating a potential for receptor channel properties modification.

Sp1-like family is an expanding transcription factor family. Members of this family bind to the GC-box or GT-box elements in the promoter/enhancers and regulate the expression of the target genes. Currently, this family consists of at least nine members, which may act as a transactivator or a repressor on target promoters. Sp1-like transcription factors are expressed during development of vertebrate embryos in ubiquitous or tissue-specific manners and play various roles in embryonic development. This review mainly summarises their expression patterns and functions during vertebrate embryogenesis.

Secretin is a neuropeptide that is expressed in distinct central neurones. As there is no information on how the secretin gene is regulated in neuronal cells, a well established neuronal differentiation cell model, SH-SY5Y, was used to study transcriptional regulation of the human secretin gene. High secretin transcript and peptide levels were found in this cell, and secretin gene expression and promoter activity were up-regulated upon all-trans retinoic acid (RA) treatment. Within the promoter, a functional GC-box 1 (-131 from ATG, relative to the ATG initiation codon) was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The human secretin gene in SH-SY5Y cells is controlled by the (Sp1 + Sp4)/Sp3 ratio and the RA-induced activation is a partial result of a decrease in Sp3 levels. In addition to the GC-box 1, an N1 motif in close proximity was also responsible for RA-induced secretin gene activation. Competitive gel mobility shift and southwestern blot studies revealed binding of Nuclear Factor I (NFI) with the N1 motif. Overexpression of NFI-C increased promoter activity upon RA treatment. Consistent with this observation, NFI-C transcript levels were augmented after RA treatment. We conclude that RA induction of the secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expression.

The dopamine transporter is a plasma membrane protein that controls extracellular concentrations of the neurotransmitter dopamine. The physiological importance of the DAT provides the impetus for studies aimed at understanding the molecular mechanisms underlying regulation of the DAT gene. In this study, we identified a DAT-expressing neuroblastoma cell line (SK-N-AS) and employed it to investigate the transcriptional regulation of the human DAT gene. Two GC boxes (located at -130 and -60, respectively, relative to the transcriptional start site) were identified as important cis-acting elements mediating DAT promoter activity in dopaminergic SK-N-AS cells. Utilizing Sp-deficient Drosophila Schneider line (SL-2) cells, we showed that both Sp1 and Sp3 are strong activators of DAT transcriptional activity. Differential binding of Sp1 and Sp3 to the two GC boxes was demonstrated by electrophoretic mobility shift assays and super-shift assays. Our results indicate that the Sp1 family of proteins plays an important role in controlling the expression of the dopamine transporter gene within dopaminergic neurons.

The ionotropic receptor of glutamate activated by N-methyl-D-aspartate (iGluR-NMDA) is a multiheteromeric complex constituted by at least three different types of subunits, encoded by seven different genes. The subunits of iGluR-NMDA have a complex system of regulation of their gene expression. Their expression is specific for each type of neural cell, as well as for the age of the organism. Moreover, there are reports that iGluR-NMDA expression is species-specific. Even though this macromolecular complex is very important in physiology and pathology of the central nervous system, knowledge to date about the regulatory elements controlling expression is scarce. We present the results of an in silico prediction of potential regulatory elements, some of which coincide with the few known experimentally. We also present the important differences regarding the presence and the localization of the regulatory elements among human, rat, and mouse species.

The gamma-aminobutyric acid type A receptors (GABAA-Rs) mediate fast inhibitory synaptic transmission in the brain. The alpha4 subunit of the GABAA-R confers distinct pharmacological properties on the receptor and its expression pattern exhibits plasticity in response to physiological and pharmacological stimuli, including withdrawal from progesterone and alcohol. We have analyzed the promoter region of the mouse GABRA4 gene that encodes the alpha4 subunit and found that the promoter has multiple transcriptional initiation sites and lacks a TATA box. The minimal promoter for GABRA4 spans the region between -444 to -19 bp relative to the coding ATG and shows high activity in cultured mouse cortical neurons. Both Sp3 and Sp4 transcription factors can interact with the two Sp1 binding sites within the minimal promoter and are critical for maximal activity of the promoter in neurons.

The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.