Glatiramer acetate inhibition of tumor necrosis factor-alpha-induced RANTES expression and release from U-251 MG human astrocytic cells.

Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). RANTES is a beta-family chemokine that manifests chemoattractant activity for T lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the effect of glatiramer acetate on the regulation of RANTES secretion in glial cells is unknown. In the present study, we demonstrate for the first time that treatment of human U-251 MG astrocytic cells with glatiramer acetate blocks tumor necrosis factor-alpha (TNF-alpha)-induced RANTES mRNA and protein in a dose- and time-dependent manner. This effect is attributed to inhibition of transcription and a 40% decrease in transcript stability. Furthermore, our electrophoretic mobility shift assays of nuclear extracts from TNF-alpha-treated cells reveal an increase in DNA-binding activity specific for the nuclear factor-kappa B (NF-kappaB) binding site, in the 5'-flanking promoter region of the human RANTES gene, and that this increase in NF-kappaB binding activity is prevented by pretreatment with glatiramer acetate or the NF-kappaB inhibitors. These findings suggest that glatiramer acetate may exert its therapeutic effect in MS partially through inhibiting NF-kappaB activation and chemokine production.

Induction of RANTES chemokine expression in human astrocytic cells is dependent upon activation of NF-kappaB transcription factor.

RANTES is a C-C (beta)-family chemokine that is implicated in the migration of peripheral blood leukocytes to brain lesions in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Glial cells are active participants in the inflammatory response in the CNS, and they have been shown to respond to and produce a number of cytokines and chemokines in vivo and in vitro. Recently, we have shown inducibility of RANTES gene expression by TNF-alpha in human astrocytic cells. Therefore, the goal of the current study was to investigate the transcription activating factor involved in the process. We found that the induction of RANTES mRNA and protein by TNF-alpha in human astrocytic cells is associated with increased NF-kappaB DNA-binding activity. p65 and p50 were determined to be the components of the activated NF-kappaB transcription factor complex by supershift assay. In addition, the blockade of NF-kappaB activation by three known NF-kappaB inhibitors markedly reduced the TNF-alpha-induced RANTES expression at the mRNA and protein levels. Furthermore, the reduction in NF-kappaB binding activity to the promoter of the human RANTES gene caused by the NF-kappaB inhibitors parallels a decrease in RANTES expression in these cells. Our data suggest that NF-kappaB may mediate the induction of RANTES gene expression, in human glial cells, through its cognate cis-acting element.

The mouse GABA(A) receptor alpha3 subunit gene and promoter.

Gamma-aminobutyric acid (GABA) type A receptors are multisubunit ligand-gated ion channels which mediate inhibition in the brain. The GABA(A) receptor alpha3 subunit gene exhibits extensive variation in its developmental and regional expression, but the detailed mechanisms governing the expression patterns of this gene remain unknown. We have cloned and begun to characterize the murine alpha3 subunit gene Gabra3. All but one of the 10 exons and the intron-exon boundaries have been sequenced; the first intron is in the 5' untranslated region (5'UTR) of the alpha3 mRNA. Rapid amplification of the cDNA 5'-end (5'-RACE) and RNase protection indicated many transcription start sites, with the major site (=+1) corresponding to a 5'UTR of 178 bases. Most sites were in or just downstream of a region of 55 (mouse) and 25 (human) GA repeats in the proximal promoter, as revealed by genome walking of Gabra3 and the human gene GABRA3. No canonical TATA or CAAT boxes or initiator (Inr) sites were found in either promoter, but both contained conserved consensus sites for several transcription factors. Progressive deletion of the mouse promoter produced positive or negative effects on expression of reporter (luciferase) constructs, with the highest observed activity in several types of transiently transfected cells for a construct containing bases -320 to +35. The GA repeats and a much shorter nearby series of four GC repeats, the first three of which are part of a consensus E2F site, appear to contribute significantly to mouse promoter activity. Upstream GA repeats enhanced activity of the SV40 promoter, and the GA repeat sequence bound nuclear proteins from several tissues.

Transcriptional regulation of GABAA receptor gamma2 subunit gene.

We have cloned the promoter regions of the genes for the mouse and human gamma2 subunits of the type A receptors for gamma-aminobutyric acid (GABA). For the mouse, the two major transcription start sites were at +1 (by definition) and +43, as established by rapid amplification of cDNA ends (RACE) and primer extension. This numbering places the start methionine at +297. There was no TATA or CCAAT box. Both mouse and human sequences have a candidate neuron-restrictive silencer element (NRSE) site in the first intron (+956 in mouse). We made assorted mouse-based promoter/reporter (luciferase) constructs starting from a core extending from -331 to +136, varying sizes at both ends, and including and excluding the putative NRSE and more proximal sequences. These were tested by transient transfection in several neuron-like and non-neuronal cell lines. Both proximal and distal downstream elements appeared to help direct expression to neuron-like cells, the NRSE in the intron, by repression in non-neurons, and a 24-bp portion of the 5' untranslated region starting at +113 (named GPE1) by preferentially promoting expression in neuron-like cells. Cotransfected human NRSF (transcription factor for NRSE) reduced reporter expression in neuron-like cells for constructs containing the NRSE in two locations. In gel mobility shift assays, the mouse gamma2 NRSE and a consensus NRSE both bound in vitro translated NRSF very similarly, and the NRSF gave the same major shifted band with the mouse gamma2 NRSE as was observed with nuclear extracts.

GABAA receptor subunit mRNAs in rat superior cervical ganglia.

gamma-Aminobutyric acid (GABA), one of the most important neurotransmitters in the brain, is also found in the periphery. GABAA receptors are chloride channels opened by GABA whose presence in the rat superior cervical ganglion has been indicated by functional and binding measurements. We describe the first molecular data on the possible subunit composition of these receptors, detecting mRNAs for 12 subunits (alpha1-5, beta1-3, gamma1-3, delta) by reverse-transcriptase polymerase chain reaction. Preliminary quantitation gave highest levels (in descending order) for the gamma2 (both short and long forms), beta3, gamma3, and alpha1 subunits.

The alpha5 subunit of the murine type A GABA receptor.

GABA[A] receptors in the brain convert binding of GABA (gamma-aminobutyric acid) to inhibition by chloride currents. Several important classes of drugs, including benzodiazepines and alcohol, modulate these receptors, which have also been implicated in epilepsy. We describe the alpha5 subunit of GABAA receptors in mice, comparing inbred DBA/2J mice, prone to juvenile audiogenic seizures, with seizure resistant C57BL/6J mice. We find no sequence differences between the strains, although there are several interesting amino acid differences from the rat. We also compare the expression of the alpha5 subunit in whole brains of DBA/2J mice to that in C57BL/6J mice at 21 days, the peak of the former's seizure susceptibility, again finding no significant difference. We further describe the pattern of expression of alpha5 mRNA during mouse brain development, with a peak at 3 days after birth, and among five brain regions in the adult mouse, with the highest levels in the hippocampus. Finally, we present preliminary evidence for rare alternative splicing of this subunit's message, in the N-terminal extracellular domain, to give a form not translatable into a functional protein.

The GABA(A) receptor gamma1-subunit in seizure prone (DBA/2) and resistant (C57BL/6) mice.

Gamma-aminobutyric acid (GABA)A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the gamma1-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the gamma1-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse gamma1-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the gamma1-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit's mRNA in whole brain by Northern blots at 3 weeks of age.

A synthetic standard for competitive RT-PCR quantitation of 13 GABA receptor type A subunit mRNAs in rats and mice.

We describe a synthetic 769-bp DNA internal standard, GABARQuant 1, for measuring mRNAs of 13 GABA(A) receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR). When it is transcribed into cRNA, added in known amounts to target mRNAs in extracts from rat or mouse tissue. competitively reverse transcribed into cDNA, and amplified by the polymerase chain reaction (PCR), the relative intensities of the amplified, stained target and standard DNA bands enable measurement of small amounts of mRNAs for GABA(A) receptor subunits alpha1-6, beta1-3, gamma1-3 and delta and the three cellular markers beta-actin, light neurofilament protein, and glutamine synthetase. For the subunits, most standard products (263-504 bp) differ in size from target products (398-564 bp) by 10-20%. Primer pairs span at least one intron, to prevent interference by genomic DNA, and at least one rat versus mouse restriction fragment length polymorphism (RFLP), to enable rat products to be distinguished from mouse products.

GABA(A) receptor alpha4 subunit in DBA/2J and C57BL/6J mice.

GABA(A) receptors are chloride channels in the brain activated by binding of gamma-aminobutyric acid (GABA). Several important classes of drugs, including alcohol and certain antiepileptic drugs, modulate the actions of GABA. We report the sequence and expression of alpha4 subunits of GABA(A) receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ in susceptibility to seizures and to behavioral effects of alcohol. We find no differences between the two strains in cDNA sequence, or in levels of alpha4 mRNA in whole brains of the two strains at 21 days of age, when DBA/2J are most susceptible to audiogenic seizures. We also describe the pattern of developmental expression and brain regional distribution of this subunit in mice, finding the highest developmental expression at about 14 days of age in whole brains, and the highest regional levels in hippocampus and basal forebrain (including thalamus) in adults.

Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs.

Tritiated haloperidol and tritiated dopamine label postsynaptic dopamine receptors in mammalian brain. Clinical potencies of butyrophenones, phenothiazines, and related drugs correlate closely with their ability to inhibit tritiated haloperidol binding. These binding methods provide a simple in vitro means for evaluating new drugs as potential antischizophrenic agents.

GABAA receptor beta 1, beta 2, and beta 3 subunits: comparisons in DBA/2J and C57BL/6J mice.

GABAA receptors link binding of GABA (gamma-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the beta 1, beta 2 and beta 3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the beta 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse beta 1 and beta 2 subunits for internal alternative splicing, detecting none.

A PCR shortcut to oocyte expression.

We describe a new method, RNA amplification with oocyte expression, for efficient expression of proteins in the Xenopus oocyte after PCR amplification of cDNA coding regions, using as examples mouse GABAA receptor alpha 1 and beta 2 subunits. RNA is reverse-transcribed and the cDNAs are amplified using 5' primers containing a T7 RNA polymerase promoter and a consensus ribosome binding site and 3' primers giving a short poly(A) tail. This is followed by direct in vitro transcription of the PCR products and injection of the resulting mRNAs into Xenopus oocytes. The method gave abundant GABA-gated chloride channels in the oocyte membrane, as measured by recording agonist-induced currents. It promises to be a rapid route to expression of cloned proteins in oocytes, without requiring actual clones, and is free of possible variations in efficiency from untranslated regions.

Thyrotropin-releasing hormone gene expression and receptors are differentially modified in limbic foci by seizures.

Previous studies using two seizure paradigms, electroconvulsive shock and kindling, suggested potential sites of endogenous thyrotropin-releasing hormone (TRH) action in specific epileptogenic areas. We studied TRH gene expression and TRH receptors in rat limbic areas using the kindling model of epilepsy. Immunoassayable TRH increased 4- to 20-fold over control levels in specific subregions of the hippocampus 24 hours after a single stage 5 seizure. Concurrently, TRH receptor binding was significantly reduced in hippocampal (23-39%) and amygdaloid (21-22%) membranes. Dramatic temporal and spatial changes in prepro-TRH messenger RNA were visualized by in situ hybridization histochemistry in the hippocampal dentate gyrus, the piriform cortex, and the amygdala. Peak hybridization occurred 6 and 12 hours postictally in these loci and returned toward basal levels by 24 hours. These results are consistent with the hypothesis that TRH may have an important role in the pathophysiology epilepsy by modulating excitatory processes.

Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.

The alpha 1, alpha 2, and alpha 3 subunits of GABAA receptors: comparison in seizure-prone and -resistant mice and during development.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the alpha 1, alpha 2, and alpha 3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the alpha 1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.

Differential expression of two forms of GABAA receptor gamma 2-subunit in mice.

The gamma-aminobutyric acid type A (GABAA) receptor gamma 2-subunit is important for benzodiazepine action. In previous studies, two forms of gamma 2-subunit have been found which are generated by RNA alternative splicing (gamma 2L and gamma 2S) and differ by the presence or absence of an 8-amino acid insertion in the major intracellular loop between proposed transmembrane domains M3 and M4. We have used the RNase protection assay to study regulation of expression of the two forms of gamma 2-subunit in mouse brain, as judged from mRNA levels. We find that gamma 2L and gamma 2S are differentially expressed in brain regions and that gamma 2S is expressed at a fairly constant level during brain development while gamma 2L increases dramatically with maturation. Their differentially regulated expression suggests further that gamma 2L and gamma 2S form receptors with important functional differences.

Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit.

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Characterization of recombinant GABAA receptors produced in transfected cells from murine alpha 1, beta 1, and gamma 2 subunit cDNAs.

In order to explore the structural basis of GABAA receptor function, we have expressed murine alpha 1, beta 1, and gamma 2 subunit cDNAs by transient transfection of human 293 cells. Expression of GABAA receptors was measured by ligand binding assay and by electrophysiological analysis. As in other species, expression of the alpha 1 and beta 1 subunits produced a receptor that was insensitive to modulation by benzodiazepines as measured by electrophysiological analysis; however, a small number of flunitrazepam binding sites were detectable. The coexpression of the gamma 2 subunit was found to be essential for this modulation, and also resulted in a dramatic (14-fold) increase in the number of binding sites for flunitrazepam. On the coexpression of all 3 subunit cDNAs, a receptor was produced that exhibited a similar number of binding sites for flunitrazepam and muscimol.

Generation of two forms of the gamma-aminobutyric acidA receptor gamma 2-subunit in mice by alternative splicing.

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.