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.

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.

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.

Stable association of presenilin derivatives and absence of presenilin interactions with APP.

Mutations in two related genes, presenilin 1 and 2 presenilin 2 (PS1 and PS2), cosegregate with Alzheimer's disease. PS1 and PS2 are highly homologous polytopic membrane proteins that are subject to endoproteolytic cleavage in vivo. The resulting N- and C-terminal derivatives are the preponderant PS-related species that accumulate in cultured cells and tissue. In earlier studies, we demonstrated that PS1 N- and C-terminal derivatives accumulate to 1:1 stoichiometry and that the absolute levels of fragments are established by a tightly regulated and saturable mechanism. These findings led to the suggestion that the levels of PS1 derivatives might be determined by their association with limiting cellular components. In this study, we use in situ chemical cross-linking and coimmunoprecipitation analyses to document that the N- and C-terminal derivatives of either PS1 or PS2 can be coisolated. Moreover, and in contrast to published reports which documented that PS1 and PS2 form stable heteromeric assemblies with the beta-amyloid precursor protein (APP), we have failed to provide evidence for physiological complexes between PS1 and PS2 holoproteins or their derivatives with APP.