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Harmane Alters the Expression of a Subset of Genes with Diverse Functions in Primary Human Astrocytes

ABSTRACT: Essential tremor (ET) is the most common movement disorder in adults, but little is known about the molecular mechanisms underlying ET pathogenesis. Harmane is a member of a group of tremorogenic chemicals. In humans, increased blood harmane concentration is associated with increased risk of ET. Astrocytes are essential for brain function, and astrocyte dysfuctions are associated with many neurodegenerative diseases. Therefore, we identified the molecular targets of harmane in primary human astrocytes by using microarray gene expression profiling and computational analysis algorithms. We found that harmane altered the expression of a limited number of genes encoding diverse functions. Notably, the transcript levels of two GABA receptors and a GABA transporter were altered by harmane, consistent with previous evidence suggesting that the GABAergic neurotransmission system may be disrupted in ET. Also, we found that the transcript levels of two prominent proinflammatory enzymes, the inducible nitric oxide synthase NOS2A and the cyclooxygenase COX2, and 10 other targets of the proinflammatory IFN-gamma signaling pathway were up-regulated by harmane. These results together raise the possibility that perturbation of the expression of functions involved in neurotransmission and inflammatory activation of astrocytes might be important mechanisms underlying the neurotoxicity of harmane and ET pathogenesis. Keywords: treatment comparison

ORGANISM(S): Homo sapiens

PROVIDER: GSE5080 | GEO | 2007/06/14

SECONDARY ACCESSION(S): PRJNA95377

REPOSITORIES: GEO

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Project description:Essential tremor (ET) is the most common movement disorder in adults, but little is known about the molecular mechanisms underlying ET pathogenesis. Harmane is a member of a group of tremorogenic chemicals. In humans, increased blood harmane concentration is associated with increased risk of ET. Astrocytes are essential for brain function, and astrocyte dysfuctions are associated with many neurodegenerative diseases. Therefore, we identified the molecular targets of harmane in primary human astrocytes by using microarray gene expression profiling and computational analysis algorithms. We found that harmane altered the expression of a limited number of genes encoding diverse functions. Notably, the transcript levels of two GABA receptors and a GABA transporter were altered by harmane, consistent with previous evidence suggesting that the GABAergic neurotransmission system may be disrupted in ET. Also, we found that the transcript levels of two prominent proinflammatory enzymes, the inducible nitric oxide synthase NOS2A and the cyclooxygenase COX2, and 10 other targets of the proinflammatory IFN-gamma signaling pathway were up-regulated by harmane. These results together raise the possibility that perturbation of the expression of functions involved in neurotransmission and inflammatory activation of astrocytes might be important mechanisms underlying the neurotoxicity of harmane and ET pathogenesis. Experiment Overall Design: Primary human astrocytes (passage 4) were treated with or without 1 uM harmane for 7 days. Total RNA was extracted by using TRIzol reagent (GIBCOBRL Life Technologies). The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data (see below). We isolated RNA from three independent batches of human astrocytes, which were from different subjects. No identifiable information from these subjects was available, in keeping with the guidelines on human subjects. Three independent batches of astrocytes from three different subjects, not the same subject, were used to ensure that our data and conclusions would not totally rely on one unidentified subject, who may have experienced influential environmental or genetic conditions. The synthesis of cDNAs and biotin-labeled cRNAs were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000). The human genome U133 plus 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Initial data analysis was performed by using the Affymetrix Microarray suite.

Project description:The rostral ventrolateral medulla (RVLM), a part of the medullary reticular formation, plays a major role in several physiological responses, including cardiovascular and sympathetic nervous system functions. Although aging causes disturbances in the responses of these physiological systems, RVLM involvement in these age-related changes is not clear. Previous work using high-throughput gene expression analysis of the RVLM in aged animals suggested that chemical neurotransmission-related genes might be downregulated with advancing age. Since the RVLM function involves a balance of inhibitory and excitatory inputs, which is largely mediated by gamma-aminobutyric acid (GABA) and excitatory amino acid (EAA) neurotransmission, we hypothesized that aging is associated with altered excitatory and/or inhibitory neurotransmission-related gene expression in the RVLM. To test this hypothesis, we micropunched an RVLM-containing area from young (3–5 months), middle-aged (12–14 months), and aged (22–26 months) Fischer 344 male rats. RNA purified from these micropunches was analyzed using GABA and Glutamate RT2 Profiler PCR arrays (n= 8–10). In addition, the expression of selected genes was validated at the RNA level using TaqMan® based- qPCR and at the protein level using western blotting. All the genes that displayed significant differential expression (1.5-fold, p < .05, FDR < .05) were identified to be downregulated in the RVLM of aged and middle-aged rats compared to young rats. Among the downregulated genes, the percentage of glutamate neurotransmission-related genes was higher than GABA neurotransmission-related genes. Solute carrier family 1 member 6 (Slc1a6) gene showed the highest fold downregulation at the RNA level in the RVLM of aged compared to young rats, and its protein product, Excitatory amino acid transporter 4 (EAAT4), showed a downregulatory trend in the RVLM of aged and middle-aged rats. These results suggest that molecular constituents of both GABA and glutamate neurotransmission might be altered in the RVLM of aged and middle-aged rats, and the changes in glutamate neurotransmission might be more prominent. Investigating age-associated anatomical and functional changes in RVLM GABA and glutamate neurotransmission might provide a foundation for understanding the effects of aging on physiological function.

Project description:Exposure of adult humans to manganese (Mn) has long been known to cause neurotoxicity. Recent evidence also suggests that exposure of children to Mn is associated with developmental neurotoxicity. Astrocytes are critical for the proper functioning of the nervous system, and they play active roles in neurogenesis, synaptogenesis and synaptic neurotransmission. In this report, to help elucidate the molecular events underlying Mn neurotoxicity, we systematically identified the molecular targets of Mn in primary human astrocytes by using microarray gene expression profiling and computational data analysis algorithms. We found that Mn altered the expression of diverse genes ranging from those encoding cytokines and transporters to signal transducers and transcriptional regulators. Particularly, 28 genes encoding proinflammatory chemokines, cytokines and related functions were up-regulated whereas 15 genes encoding functions involved in DNA replication and repair and cell cycle checkpoint control were down-regulated. These results are further supported by data from real-time RT-PCR, Western blotting and flow cytometric analyses. In addition, analysis of common regulators revealed that 16 targets known to be positively affected by the IFN-gamma signaling pathway were up-regulated by Mn, suggesting that the proinflammatory IFN-gamma signaling pathway was likely activated. These results raise the possibility that inflammatory activation of astrocytes and the increased expression of proinflammatory cytokines and chemokines and/or the activation of related signaling pathways might be an important mechanism leading to Mn neurotoxicity. Experiment Overall Design: Primary human astrocytes (passage 4) were treated with or without 200 uM MnCl2 for 7 days. Total RNA was extracted by using TRIzol reagent (GIBCOBRL Life Technologies). The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data (see below). We isolated RNA from three independent batches of human astrocytes, which were from different subjects. No identifiable information from these subjects was available, in keeping with the guidelines on human subjects. Three independent batches of astrocytes from three different subjects, not the same subject, were used to ensure that our data and conclusions would not totally rely on one unidentified subject, who may have experienced influential environmental or genetic conditions. The synthesis of cDNAs and biotin-labeled cRNAs were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000). The human genome U133 plus 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Initial data analysis was performed by using the Affymetrix Microarray suite.

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Harmane Alters the Expression of a Subset of Genes with Diverse Functions in Primary Human Astrocytes

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