Molecular signatures associated with cognitive deficits in schizophrenia: a study of biopsied olfactory neural epithelium.

Cognitive impairment is a key feature of schizophrenia (SZ) and determines functional outcome. Nonetheless, molecular signatures in neuronal tissues that associate with deficits are not well understood. We conducted nasal biopsy to obtain olfactory epithelium from patients with SZ and control subjects. The neural layers from the biopsied epithelium were enriched by laser-captured microdissection. We then performed an unbiased microarray expression study and implemented a systematic neuropsychological assessment on the same participants. The differentially regulated genes in SZ were further filtered based on correlation with neuropsychological traits. This strategy identified the SMAD 5 gene, and real-time quantitative PCR analysis also supports downregulation of the SMAD pathway in SZ. The SMAD pathway has been important in multiple tissues, including the role for neurodevelopment and bone formation. Here the involvement of the pathway in adult brain function is suggested. This exploratory study establishes a strategy to better identify neuronal molecular signatures that are potentially associated with mental illness and cognitive deficits. We propose that the SMAD pathway may be a novel target in addressing cognitive deficit of SZ in future studies.

Unique pharmacological actions of atypical neuroleptic quetiapine: possible role in cell cycle/fate control.

Quetiapine is an atypical neuroleptic with a pharmacological profile distinct from classic neuroleptics that function primarily via blockade of dopamine D2 receptors. In the United States, quetiapine is currently approved for treating patients with schizophrenia, major depression and bipolar I disorder. Despite its widespread use, its cellular effects remain elusive. To address possible mechanisms, we chronically treated mice with quetiapine, haloperidol or vehicle and examined quetiapine-specific gene expression change in the frontal cortex. Through microarray analysis, we observed that several groups of genes were differentially expressed upon exposure to quetiapine compared with haloperidol or vehicle; among them, Cdkn1a, the gene encoding p21, exhibited the greatest fold change relative to haloperidol. The quetiapine-induced downregulation of p21/Cdkn1a was confirmed by real-time polymerase chain reaction and in situ hybridization. Consistent with single gene-level analyses, functional group analyses also indicated that gene sets associated with cell cycle/fate were differentially regulated in the quetiapine-treated group. In cortical cell cultures treated with quetiapine, p21/Cdkn1a was significantly downregulated in oligodendrocyte precursor cells and neurons, but not in astrocytes. We propose that cell cycle-associated intervention by quetiapine in the frontal cortex may underlie a unique efficacy of quetiapine compared with typical neuroleptics.

Naturally occurring free D-aspartate is a nuclear component of cells in the mammalian hypothalamo-neurohypophyseal system.

It is generally believed that only L-amino acids have a physiological role in species other than bacteria. Recently, the existence of some D-amino acids, particularly D-aspartate, in various organs of several higher animals has been reported. Here we demonstrate that naturally occurring free D-aspartate is localized subcellularly to the heterochromatin in the nucleoli (but not in either the dendrites or axonal terminals) of magnocellular neurosecretory neurons in the rat hypothalamus, and also of microglia and pericytes in the posterior pituitary. Our results imply that naturally occurring free D-aspartate might have a physiological role in nuclear function in mammals. The findings provide new insight for the biological function of D-stereoisomers of amino acids as well as the organization of the nucleus of at least some eukaryotic cells.

Neurobiology of Rett syndrome: a genetic disorder of synapse development.

Rett syndrome is a developmental disorder that restricts brain growth beginning in the first year of life and evidence from neuropathology and neuroimaging indicates that axonodendritic connections are especially vulnerable. In a study of amino acid neurotransmitter receptors using receptor autoradiography in tissue slices of frontal cortex and the basal ganglia, we found a biphasic age-related pattern with relatively high receptor densities in young RS girls and lower densities at later time. Using microarray analysis of gene expression in frontal cortex, we found that some of the most prominent alterations occurred in gene products related to synapses, including the NMDA receptor NR1 subunit, the cytoskeletal protein MAP-2 and synaptic vesicle proteins. Using a new antibody that recognizes MeCP2, the transcription factor mutated in RS, we established that most neurons in the rodent brain express this transcription factor. We hypothesize that a major effect of mutations in the MeCP2 protein is to cause age-related disruption of synaptic proliferation and pruning in the first decade of life.

Postmortem brain abnormalities of the glutamate neurotransmitter system in autism.

BACKGROUND: Studies examining the brains of individuals with autism have identified anatomic and pathologic changes in regions such as the cerebellum and hippocampus. Little, if anything, is known, however, about the molecules that are involved in the pathogenesis of this disorder. OBJECTIVE: To identify genes with abnormal expression levels in the cerebella of subjects with autism. METHOD: Brain samples from a total of 10 individuals with autism and 23 matched controls were collected, mainly from the cerebellum. Two cDNA microarray technologies were used to identify genes that were significantly up- or downregulated in autism. The abnormal mRNA or protein levels of several genes identified by microarray analysis were investigated using PCR with reverse transcription and Western blotting. alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)- and NMDA-type glutamate receptor densities were examined with receptor autoradiography in the cerebellum, caudate-putamen, and prefrontal cortex. RESULTS: The mRNA levels of several genes were significantly increased in autism, including excitatory amino acid transporter 1 and glutamate receptor AMPA 1, two members of the glutamate system. Abnormalities in the protein or mRNA levels of several additional molecules in the glutamate system were identified on further analysis, including glutamate receptor binding proteins. AMPA-type glutamate receptor density was decreased in the cerebellum of individuals with autism (p < 0.05). CONCLUSIONS: Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.

Microarray analysis of differential gene expression in lead-exposed astrocytes.

The toxic metal lead is a widespread environmental health hazard that can adversely affect human health. In an effort to better understand the cellular and molecular consequences of lead exposure, we have employed cDNA microarrays to analyze the effects of acute lead exposure on large-scale gene expression patterns in immortalized rat astrocytes. Our studies identified many genes previously reported to be differentially regulated by lead exposure. Additionally, we have identified novel putative targets of lead-mediated toxicity, including members of the family of calcium/phospholipid binding annexins, the angiogenesis-inducing thrombospondins, collagens, and tRNA synthetases. We demonstrate the ability to distinguish lead-exposed samples from control or sodium samples solely on the basis of large-scale gene expression patterns using two complementary clustering methods. We have confirmed the altered expression of candidate genes and their encoded proteins by RT-PCR and Western blotting, respectively. Finally, we show that the calcium-dependent phospholipid binding protein annexin A5, initially identified as a differentially regulated gene by our microarray analysis, is directly bound and activated by nanomolar concentrations of lead. We conclude that microarray technology is an effective tool for the identification of lead-induced patterns of gene expression and molecular targets of lead.

Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification.

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.

Assessment of neural cell adhesion molecule (NCAM) in autistic serum and postmortem brain.

Studies have identified structural abnormalities in areas of the autistic brain, with a pattern suggesting that a neurodevelopmental anomaly took place. Neural cell adhesion molecule (NCAM), which is involved in development of the central nervous system, was previously shown to be decreased in the serum of autistic individuals. In the present study, we measured NCAM protein in the sera from controls, patients with autism, siblings of autistic patients, and individuals with other neurologic disorders, but found no significant differences. We also measured NCAM protein in autistic postmortem brain samples and found the longest isoform, NCAM-180, to be significantly decreased. In addition, we investigated the mRNA expression of NCAM in these brain samples using cDNA microarrays and RT-PCR. Results show that NCAM mRNA levels are not altered in autism.

Emerging technologies for large-scale screening of human tissues and fluids in the study of severe psychiatric disease.

Neuropsychiatric diseases such as schizophrenia and bipolar disorder are major causes of morbidity throughout the world. Despite extensive searches, no single gene, RNA transcript, or protein has been found which can, on its own, account for these disorders. Recently, the availability of genomic tools such as cDNA microarrays, serial analysis of gene expression (SAGE) and large-scale sequencing of cDNA libraries has allowed researchers to assay biological samples for a large number of RNA transcripts. Similarly, proteomic tools allow for the quantitation of a large number of peptides and proteins. These methods include two-dimensional electrophoresis and surface-enhanced laser desorption/ionization (SELDI). We have initiated experiments which apply these techniques to the comparison of RNAs and proteins expressed in clinical samples obtained from individuals with psychiatric diseases and controls. These methods have the potential to identify pathways that are involved in the pathogenesis of complex psychiatric disorders. The characterization of these pathways may allow for the development of new methods for the diagnosis and treatment of schizophrenia, bipolar disorder, and other human psychiatric diseases.

Synaptotagmin I is a molecular target for lead.

Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with specific molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.

Control of fusion pore dynamics during exocytosis by Munc18.

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. All vesicle transport steps also have an essential requirement for a member of the Sec1 protein family, including the neuronal Munc18-1 (also known as nSec1) in regulated exocytosis. Here, in adrenal chromaffin cells, we expressed a Munc18 mutant with reduced affinity for syntaxin, which specifically modified the kinetics of single-granule exocytotic release events, consistent with an acceleration of fusion pore expansion. Thus, Munc18 functions in a late stage in the fusion process, where its dissociation from syntaxin determines the kinetics of postfusion events.

The abnormal regulation of gene expression in autistic brain tissue.

Autism is a pervasive developmental disorder of unknown etiology. It is likely caused by mutations in one or more genes. One approach to understanding the molecular changes that occur in autism is to measure gene expression in post-mortem brain samples from individuals diagnosed with autism. This may be accomplished with techniques such as cDNA microarrays or subtractive hybridization. In general, gene expression is regulated as a function of body region, developmental time, and physiological state. A premise of the approaches we describe is that gene expression is regulated in cells from autistic individuals as a consequence of the disease process. It may be useful to detect such changes in order to identify selective biological markers for autism. Additionally, the abnormal regulation of gene expression may reveal cellular pathways that have been disrupted, suggesting strategies for therapeutic intervention.

Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals.

Little evidence is available for the physiological function of D-amino acids in species other than bacteria. Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus. The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation. Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo. Similar changes were observed in the vasopressin system. These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology. The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages.

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

Induction of vascular endothelial growth factor in human astrocytes by lead. Involvement of a protein kinase C/activator protein-1 complex-dependent and hypoxia-inducible factor 1-independent signaling pathway.

The mechanism(s) underlying lead neurotoxicity are not fully elucidated. cDNA expression microarray analysis identified lead-sensitive genes in immortalized human fetal astrocytes (SV-FHA). Of the represented genes expressed, vascular endothelial growth factor (VEGF) was one of the most sensitive. Lead induced VEGF mRNA 3-fold and VEGF protein approximately 2-fold with maximum mRNA induction following incubation with 10 micrometer lead acetate for 24 h. Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C (PKC) activator, increased VEGF mRNA 2-fold and PKC inhibition by GF-109203 completely blocked VEGF induction by lead. Expression of dominant-negative PKC-epsilon, but not PKC-alpha, completely inhibited VEGF mRNA induction by lead. Lead activated the transcription factor AP-1 and increased AP-1-dependent luciferase expression >2-fold. Transfection of cells with a c-jun dominant-negative effectively inhibited both AP-1 activation and VEGF mRNA induction by lead. Hypoxia-inducible factor 1 (HIF-1) activity in SV-FHAs was moderately increased by lead (86%) and PMA (96%). Pretreatment with GF-109203 completely inhibited these effects of lead and PMA. However, lead did not alter HIF-1-dependent luciferase expression and a HIF-1alpha dominant-negative had no effects on the induction of VEGF mRNA by lead. These findings indicate that lead induces VEGF expression in SV-FHAs via a PKC/AP-1-dependent and HIF-1-independent signaling pathway.

High throughput analysis of gene expression in the human brain.

The human brain is thought to have the greatest complexity of gene expression of any region of the body, reflecting the diverse functions of neurons and glia. Studies of gene expression in the human brain may yield fundamental information about the phenotype of brain cells in different stages of development, in different brain regions, and in different physiological and pathological states. As the human genome project nears completion, several technological advances allow the analysis of thousands of expressed genes in a small brain sample. This review describes available sources of human brain material, and several high throughput techniques used to measure the expression of thousands of genes. These techniques include expressed sequence tag (EST) sequencing of cDNA libraries; differential display; subtractive hybridization; serial analysis of gene expression (SAGE); and the emerging technology of high density DNA microarrays. Measurement of gene expression with microarrays and other technologies has potential applications in the study of human brain diseases, including cognitive disorders for which animal models are typically not available. Gene expression measurements may be used to identify genes that are abnormally regulated as a secondary consequence of a disease state, or to identify the response of brain cells to pharmacological treatments.

DRAGON: Database Referencing of Array Genes Online.

UNLABELLED: "Database Referencing of Array Genes ONline" or "DRAGON" is a web-accessible database that aids in the analysis of differential gene expression data as a biological annotation tool. Users of DRAGON can submit data sets containing large lists of genes and then choose particular characteristics that DRAGON supplies for all genes on the list rapidly and simultaneously. AVAILABILITY: The DRAGON database is available for queries on the DRAGON web site www.kennedykrieger.org/pevsnerlab/dragon.htm. CONTACT: pevsner@kennedykrieger.org or cbouton@jhmi.edu

Effects of lead on gene expression.

Lead poisoning is a worldwide, environmental health-hazard that affects children and adults. In this review we discuss the effects of lead on gene expression due to both general and specific mechanisms. In particular we focus on the ability of lead to substitute for biologically essential metals such as calcium and zinc in metal-binding domains of cytoplasmic enzymes, nuclear transcription factors and other proteins. The binding of lead to these proteins causes an alteration of their activity resulting in aberrant expression of their own genes and in some cases their target genes. Finally, we discuss the impact of microarray technology on the study of the genome-wide effects of lead and other toxicants on gene expression.

The biology of the mammalian Krüppel-like family of transcription factors.

Recent advances in molecular cloning have led to the identification of a large number of mammalian zinc finger-containing transcription factors that exhibit homology to the Drosophila melanogaster protein, Krüppel. Although the amino acid sequences in the zinc finger domains of these Krüppel-like factors (KLFs) are closely related to one another, the regions outside the zinc fingers of the proteins are usually unique. KLFs display seemingly different and broad biological properties with each functioning as an activator of transcription, a repressor or both. This review article provides a current phylogenetic classification of the identified KLFs to date. More importantly, the currently known biological activities of the KLFs in regulating transcription, cell proliferation, differentiation and development are summarized and compared. Further characterization of this interesting protein family should provide additional insights into the their respective regulatory role in various important biological processes.