DNA arrays and functional genomics in neurobiology.
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Animals↗
Biomedical subjects
M F Miles
Publications and source records attributed to M F Miles.
Microarrays: lost in a storm of data?
Microarray expression profiling is instrumental to our understanding of the function of the genome. Resolution of functionally relevant expression patterns will require the analysis of large data sets compiled from multiple investigators. For this and other reasons, I argue that it is crucial for array data to be publicly shared in a format as close to the 'raw data' as possible. Issues such as protection of intellectual property, ensuring quality of the data, and the format and timing for sharing array data are also discussed.
Animals↗
Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression.
Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.
Animals↗
Characterization of promoter elements mediating ethanol regulation of hsc70 gene transcription.
Chronic exposure to ethanol increases transcription of the molecular chaperone Hsc70 in NG108-15 neuroblastoma X glioma cells. This and other ethanol-induced changes in gene expression may contribute to central nervous system tolerance and dependence in alcoholics. Here, we characterized sequences in the hsc70 promoter that are required for ethanol-induced transcriptional regulation. Deletion analysis of the hsc70 promoter showed that the 74-base pair region proximal to the transcription start site was sufficient for ethanol responsiveness. Point mutation or deletion of a consensus Spl-binding site at -67/-61 base pairs greatly reduced the induction by ethanol. Hsc70 promoter constructs with diminished ethanol responsiveness in NG108-15 cells similarly had decreased transcriptional activation by exogenous Sp1 in Drosophila SL2 cells. Some artificial promoter constructs containing multiple Sp1 sites were highly responsive to ethanol, but others were not, suggesting that the organization of the proximal promoter region was an additional factor that affected the ethanol response. Gel mobility shift analysis confirmed that an Sp1-like protein bound to the -67/-61 consensus Sp1 site. However ethanol exposure did not alter Sp1 DNA-binding activity. Together, our findings show that ethanol induction of Hsc70 requires a functional Sp1-binding site. Additional proximal promoter elements may also play a role in determining whether an Sp1-containing promoter will respond to ethanol.
Animals↗
Gene expression in human alcoholism: microarray analysis of frontal cortex.
BACKGROUND: Changes in brain gene expression are thought to be responsible for the tolerance, dependence, and neurotoxicity produced by chronic alcohol abuse, but there has been no large scale study of gene expression in human alcoholism. METHODS: RNA was extracted from postmortem samples of superior frontal cortex of alcoholics and nonalcoholics. Relative levels of RNA were determined by array techniques. We used both cDNA and oligonucleotide microarrays to provide coverage of a large number of genes and to allow cross-validation for those genes represented on both types of arrays. RESULTS: Expression levels were determined for over 4000 genes and 163 of these were found to differ by 40% or more between alcoholics and nonalcoholics. Analysis of these changes revealed a selective reprogramming of gene expression in this brain region, particularly for myelin-related genes which were down-regulated in the alcoholic samples. In addition, cell cycle genes and several neuronal genes were changed in expression. CONCLUSIONS: These gene expression changes suggest a mechanism for the loss of cerebral white matter in alcoholics as well as alterations that may lead to the neurotoxic actions of ethanol.
Aged↗
Cloning and characterization of the rat and human phosducin-like protein genes: structure, expression and chromosomal localization.
We isolated and characterized the rat gene encoding phosducin-like protein (PhLP), a putative heterotrimeric G protein modulator. The transcription start site was mapped by primer extension. The putative promoter region lacked a TATA sequence but contained a potential initiator element. Two splice variants were identified by RT-PCR of rat brain RNA, potentially generating either the full length or an amino-truncated protein. Only the full-length protein was immunodetected in all mouse tissues surveyed. Comparison of the conceptual translation product of the rat PhLP gene with those from human and Drosophila clones shows a striking conservation in the amino-terminal region of PhLP from these species. This contrasts with the relatively low degree of homology between PhLP and phosducin in this region, suggesting a functional role for this portion of the PhLP protein. Finally, we mapped the human PhLP gene by PCR analysis of somatic cell hybrids and the Stanford G3 radiation hybrid panel. The human PhLP gene (PDCL) is located on chromosome 9, linked to the polymorphic markers D9S1876 and D9S1674 (66-71 cM).
Amino Acid Sequence↗
Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1.
Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.
Adenosine Triphosphatases↗
Interaction of phosducin-like protein with G protein betagamma subunits.
Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gbetagamma signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gbetagamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbetagamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbetagamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbetagamma and PhLP are inhibited by the alpha subunit of Go and Gi3, suggesting that PhLP can bind only free Gbetagamma. Finally, we show that PhLP complexes, at least partially, with Gbetagamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta1gamma2 proteins in COS-7 cells, a PhLP-Gbetagamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbetagamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbetagamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbetagamma-binding domains on PhLP.
Animals↗
Interaction of ethanol with inducers of glucose-regulated stress proteins. Ethanol potentiates inducers of grp78 transcription.
GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.
Animals↗
Effects of alcohol on gene expression in neural cells.
Our studies in the NG108-15 neuroblastoma x glioma cell line previously showed that the molecular chaperonin, Hsc70, is an ethanol-responsive gene (EtRG) regulated at the level of transcription by ethanol. We recently identified two related molecular chaperonins, GRP94 and GRP78, as EtRGs with GRP94 mRNA abundance being induced by ethanol more than three-fold vs. control. Stable transfection studies show that GRP78 transcription is also regulated by ethanol and that ethanol also potentiates GRP78 induction by classical inducing agents such as tunicamycin. Recently, we have found that ethanol induction of Hsc70 may require cis-acting promoter sequences recognized by the DNA-binding protein Sp1. Chronic ethanol exposure does not alter Sp1 DNA-binding activity, thus suggesting a possible ethanol-induced post-translational modification that activates Sp1 function. We predict that the molecular mechanisms underlying ethanol regulation of Hsc70, GRP94 and GRP78 may be similar since they have related functions. GRP94 and GRP78 (GRP94/78) are known to be induced by agents which inhibit glycoprotein processing or deplete endoplasmic reticulum stores of calcium. In turn, induction of GRP78 expression is known to selectively alter the transport of glycoproteins and produce "tolerance" to depletion of sequestered intracellular calcium. The regulation of these genes by ethanol could thus relate to the known effects of ethanol on calcium homeostasis and protein trafficking. The actions of ethanol on chaperonin gene expression may have important mechanistic implications for CNS adaptation to ethanol, particularly if other EtRGs share the same regulatory mechanisms.
Animals↗
Art in hospitals: does it work? A survey of evaluation of arts projects in the NHS.
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Art↗
Ethanol-responsive genes in neural cells include the 78-kilodalton glucose-regulated protein (GRP78) and 94-kilodalton glucose-regulated protein (GRP94) molecular chaperones.
Previously we found that ethanol increases expression of the constitutive 70-kDa heat shock protein (Hsc70) in NG108-15 neuroblastoma x glioma cells. We suggested that known ethanol actions on cellular protein trafficking may relate to Hsc70 induction because Hsc70 functions as a molecular chaperone. Here we use a subtractive hybridization protocol to isolate ethanol-responsive genes (EtRGs). Northern blot hybridization verified ethanol-induced increases in mRNA abundance for five cDNA clones isolated from ethanol-treated NG108-15 neuroblastoma x glioma cells. DNA sequence analysis identified one EtRG as 94-kDa glucose-regulated protein (GRP94), a member of the "glucose-responsive" subgroup of stress proteins. Other identified EtRGs included an insulin-induced growth-response protein gene and an intracisternal A-type particle gene. Sequence analysis of the remaining two EtRGs showed no homology in DNA sequence databases. All EtRGs showed wide tissue expression, except SL64, which was not detected in Northern blot analyses of adult mouse or rat tissues. Ethanol also increased mRNA abundance for 78-kDa glucose-regulated protein (GRP78), a molecular chaperone known to function in glycoprotein trafficking and usually coordinately regulated with GRP94. However, ethanol induced GRP94 more than GRP78, a pattern distinct from those of other inducers of these genes. All EtRGs, including GRP94 and GRP78, showed similar ethanol concentration-dependent increases in mRNA abundance. In contrast, thapsigargin and other inducers of glucose-responsive proteins increased GRP94 and GRP78 mRNA levels without altering expression of other EtRGs. Our studies demonstrate that several molecular chaperones constitute a subset of EtRGs. Ethanol appears to regulate these EtRGs by a unique mechanism, rather than one shared by classical inducers of stress proteins.
Carrier Proteins↗
Phosducin-like protein: an ethanol-responsive potential modulator of guanine nucleotide-binding protein function.
Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein beta gamma subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern hybridization. We have identified four forms of PhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus. Treatment of NG108-15 cells for 24 hr with concentrations of ethanol seen in actively drinking alcoholics (25-100 mM) causes up to a 3-fold increase in PhLP mRNA levels. Induction of PhLP by ethanol could account for at least some of the widespread alterations in signal transduction and G-protein function that are known to occur with chronic exposure to ethanol.
Amino Acid Sequence↗
Ethanol-responsive gene expression in neural cell cultures.
We have studied the molecular mechanisms underlying neuronal adaptation to chronic ethanol exposure. NG108-15 neuroblastoma cells were used to perform a detailed analysis of ethanol-induced changes in neuronal gene expression. High resolution, quantitative two-dimensional (2-D) gel electrophoresis of in vitro translation products showed both dose-dependent increases and decreases in specific mRNA abundance following treatment with ethanol at concentrations seen in actively drinking alcoholics (50-200 mM). Dose response curves for representative members of the increasing or decreasing response groups had very similar profiles, suggesting that similar mechanisms may regulate members of a response group. Some mRNAs that increased with ethanol treatment appeared identical to species induced by heat shock while other mRNAs were only induced by ethanol. We conclude that chronic ethanol exposure can produce specific coordinate changes in expression of neuronal mRNAs, including some members of the stress protein response. However, the overall pattern of ethanol-responsive gene expression is distinct from the classical heat shock subgroup of stress proteins response. Changes in gene expression and specifically, mechanisms regulating a subset of stress protein expression, could be an important aspect of neuronal adaptation to chronic ethanol seen in alcoholics.
Dose-Response Relationship, Drug↗
Ethanol increases tyrosine hydroxylase gene expression in N1E-115 neuroblastoma cells.
Catecholamines appear to be involved in behavioral responses to acute and chronic ethanol consumption. Since tyrosine hydroxylase (TH) is the rate-limiting enzyme for catecholamine biosynthesis and is regulated by second messenger systems known to be modulated by ethanol, we studied ethanol-induced changes in TH gene expression. In the N1E-115 neural cell line, Northern and Western blot analyses showed that treatment with 25-200 mM ethanol for 3 days caused a dose-dependent increase in TH mRNA and protein levels. N1E-115 cells were also stably transfected with pTH5'CAT, a plasmid containing 773 base pairs of the TH promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene. Subclones expressing pTH5'CAT showed ethanol-induced increases in CAT activity, suggesting that ethanol modulates TH gene transcription. Furthermore, simultaneous treatment of transfected cells with 100 mM ethanol and 1 nM to 1 microM prostaglandin E1 increased prostaglandin E1-mediated stimulation of TH-promoter activity. Similarly, simultaneous treatment of transfected cells with 100 mM ethanol and either 10 mM (-)-N6-(R-phenylisopropyl)adenosine or 0.5 mM 8-bromo-cAMP also resulted in increased TH-promoter activity compared to treatment with these agents without ethanol. These results suggest that ethanol treatment of N1E-115 cells has a prominent effect on both basal and cAMP-regulated TH expression. Ethanol-induced changes in TH expression may be a critical molecular event in adaptation of the central nervous system to ethanol.
8-Bromo Cyclic Adenosine Monophosphate↗
Mechanisms of neuronal adaptation to ethanol. Ethanol induces Hsc70 gene transcription in NG108-15 neuroblastoma x glioma cells.
The mechanisms underlying neuronal adaptation to ethanol are poorly understood but appear to involve alterations in cellular membrane structure and/or function. Using a two-dimensional gel analysis, we have recently identified Hsc70 as an ethanol-responsive gene (Miles, M.F. (1989) Neurology 39, (Suppl. 1), 425). Hsc70 is a constitutive member of the 70-kDa stress protein family which plays an important role in protein trafficking and coated vesicle processing. Thus, modulation of Hsc70 by ethanol could produce widespread changes in cellular membrane functioning. Here, we report a detailed study on the regulation of Hsc70 by ethanol in NG108-15 neuroblastoma x glioma cells. Northern and Western blot analyses showed that ethanol concentrations observed in actively drinking alcoholics caused an induction of Hsc70 mRNA and protein. Increases in Hsc70 mRNA were seen as early as 4 h after exposure to ethanol. In comparison with ethanol, propanol and butanol caused proportionally greater increases in Hsc70 mRNA. This is consistent with known anesthetic and intoxicating potencies of these aliphatic alcohols and suggested that lipophilicity, rather than an osmotic effect, was critical for ethanol induction of Hsc70. Induction of Hsc70 mRNA by ethanol resulted, at least in part, from increased Hsc70 gene transcription as determined by nuclear runoff studies. Stable transfection analysis revealed an ethanol-responsive cis-acting element in the proximal 2500 base pairs of the Hsc70 promoter. Regulation of Hsc70 by 50-200 mM ethanol appeared to be a specific change in expression of an ethanol-responsive gene rather than a typical stress protein response since no induction of the highly inducible stress protein, Hsp70, was seen at these ethanol concentrations. These results suggest that ethanol-induced changes in Hsc70 transcription may be important for neuronal adaptation to ethanol and the development of tolerance and dependence in alcoholics.
Blotting, Northern↗
Up regulation of calbindin-D28K mRNA in the rat hippocampus following focal stimulation of the perforant path.
Calbindin-D28K is a constitutive Ca2(+)-binding protein expressed in hippocampal neurons that are resistant to various forms of excitotoxic injury. However, the local factors controlling calbindin-D28K expression within the central nervous system are unknown. We report that neuronal excitation via the perforant path leads to an increased expression of calbindin-D28K mRNA within dentate granule cells. This response is related specifically to stimulation that induces prolonged periods of bursting afterdischarges and precedes cellular injury. The up regulation of calbindin-D28K mRNA occurs during the type of neuronal activation associated with elevated cytosolic Ca2+ and suggests that the maintenance of Ca2+ homeostasis includes a system of feedback control at the level of gene expression.
Animals↗
The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity.
We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.
Animals↗