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E Deneris

Publications and source records attributed to E Deneris.

14 recordsLinked to original sources
PubMed2001

Nicotinic acetylcholine receptor subunit proteins alpha7 and beta4 decrease in the superior cervical ganglion after axotomy.

Synaptic transmission in the superior cervical ganglion (SCG) is mediated by nicotinic acetylcholine receptors (nAChR). After transection of the postganglionic nerves of the SCG in the adult rat, the transcript levels of four of the five nAChR subunits present in the ganglion, alpha3, alpha5, alpha7, and beta4, decrease dramatically. In the present study, the effect of axotomy on nAChR subunit expression was examined at the protein level, focusing on the alpha7 and beta4 subunits. Immunohistochemistry with monoclonal antibody mAb306 (for the alpha7 subunit) and polyclonal antibody 4886 (for the beta4 subunit) showed that immunoreactivities for both alpha7 and beta4 subunits were concentrated in neurons in the intact ganglion. Results from double staining with antibodies to these subunits and to tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the biosynthesis of the sympathetic neurotransmitter norepinephrine, demonstrated that most neurons in the SCG express both the alpha7 and beta4 subunits. Three days after axotomy, the number of immunolabeled neurons and the intensity of the immunostaining per labeled neuron were decreased for both subunits. Decreases in subunit levels were also observed by Western blot analysis. Observing changes in these subunits over time after surgery revealed that, while the protein level of the alpha7 subunit recovered substantially within 2 weeks after the lesion, that of the beta4 subunit stayed low. These data demonstrate that decreases in nicotinic receptor subunits are among the changes in proteins that occur in axotomized sympathetic neurons, and suggest that these decreases may contribute to the depression in ganglionic synaptic transmission observed in axotomized ganglia.

Animals↗
PubMed1998

Differential regulation of levels of nicotinic receptor subunit transcripts in adult sympathetic neurons after axotomy.

Axotomy of adult peripheral neurons produces decreases in the levels of transcripts for a number of proteins involved in synaptic transmission. For example, tyrosine hydroxylase and neuropeptide Y mRNA decrease in axotomized sympathetic neurons in the superior cervical ganglion (SCG). In the present study, the effects of axotomy on the expression of nicotinic receptor subunit transcripts were examined in the SCG and the results were compared to those produced by deafferentation and explantation. Normally, neurons in the SCG express five different nicotinic subunits: alpha3, alpha5, alpha7, beta2, and beta4. Forty-eight hours after axotomy in vivo or explantation, dramatic decreases in these transcripts were seen, except for beta2, which increased. In contrast, deafferentation of the SCG had negligible effects on any of these transcripts. Both leukemia inhibitory factor (LIF) and nerve growth factor (NGF) have been shown to play a role in the decrease in neuropeptide Y mRNA expression after axotomy. In the cases of these nicotinic receptor transcripts, however, similar decreases were seen in wild-type and LIF knockout animals. Furthermore, administration of an antiserum to NGF in intact animals produced no changes in transcript levels. On the other hand, providing exogenous NGF to axotomized SCG in vivo or in explant cultures partially prevented the decreases in the transcripts for alpha3, alpha5, alpha7, and beta4. These data indicate that axotomy produces dramatic decreases in the expression of several nicotinic receptor subunit transcripts, and that the molecular signals underlying these changes differ from those previously shown to mediate the decrease in neuropeptide Y expression.

Animals↗
PubMed1998

Pet-1, a novel ETS domain factor that can activate neuronal nAchR gene transcription.

We report a cDNA clone prepared from adrenal chromaffin-derived PC12 cell RNA that encodes a novel ETS-domain factor, Pet-1. The deduced primary structure of Pet-1 is composed of 340 amino acids and the encoded polypeptide has a predicted molecular mass of 35.4 kD. The pattern of Pet-1 gene expression in the neonatal rat is highly restricted and suggests that Pet-1 functions primarily in the nervous system. Adrenal gland expresses the highest level of Pet-1 among the tissues examined. In situ hybridization indicates that Pet-1 is expressed in the adrenal medulla but not the adrenal cortex. Slightly weaker Pet-1 hybridization is detected in brain and low levels are detectable in intestine and eye. Pet-1 can bind specifically to a PEA3 ETS DNA-binding motif and can modulate transcription of synthetic promoter constructs in a sequence-specific manner. We recently identified a neural cell-type specific enhancer, beta43', within the 3'-untranslated exon of the neuronal nicotinic acetylcholine receptor (nAchR) beta4 subunit gene. Similar to Pet-1, the beta4 gene is also expressed in PC12 cells. The presence of putative ETS-domain binding sites in the beta43' enhancer led us to hypothesize that members of the ets gene family activate neuronal nAchR genes. Cotransfection assays show that Pet-1 can activate reporter gene transcription in a beta43' enhancer-dependent and cell type-dependent manner. Our results lead us to hypothesize that Pet-1 acts as a transcriptional regulator of downstream target genes involved in cholinergic neurotransmission.

Amino Acid Sequence↗
PubMed1997

beta43': An enhancer displaying neural-restricted activity is located in the 3'-untranslated exon of the rat nicotinic acetylcholine receptor beta4 gene.

Members of a neuronal nicotinic acetylcholine receptor subunit gene cluster ordered beta4, alpha3, alpha5 in the vertebrate genome are expressed in highly restricted patterns in the PNS and CNS. Nothing is known, however, about the regulatory elements that control transcription of these genes in selected neuronal cell populations. We report here a novel enhancer, designated beta43', that is positioned in the beta4 3'-untranslated exon. It is composed of two nearly identical 37 bp direct repeats that are separated by 6 bp. Multimerization of the enhancer upstream of the alpha3 minimal promoter results in synergistic activation. Analysis in different cell types, including three neural lines and primary keratinocytes, shows that beta43' is preferentially active in the neural line PC12, which expresses all members of the cluster. Mobility shift assays reveal a cell-type-specific complex, which forms with the first repeat of the enhancer and PC12 extracts. Complexes co-migrating with the PC12 cell complex are not detected with extracts from other lines, which suggests that PC12 cells contain a differentially expressed factor that may be important for the restricted activity of beta43'. The cell-type-specific activity of the beta43' enhancer suggests that it is important for regulating restricted expression patterns of one or more clustered neuronal acetylcholine receptor genes. Its location within the beta4 gene may be a selective pressure for maintaining tight linkage of clustered neuronal nAchR genes.

Animals↗
PubMed1997

Elements between the protein-coding regions of the adjacent beta 4 and alpha 3 acetylcholine receptor genes direct neuron-specific expression in the central nervous system.

The expression patterns of three clustered neuronal nicotinic acetylcholine receptor (nAchR) subunit genes ordered beta 4, alpha 3, and alpha 5 overlap extensively in the peripheral nervous system (PNS) but only partially in the central nervous system (CNS). We have begun to investigate cell type-specific cis elements regulating these genes by analyzing in both cell culture and transgenic mice, a 2.8-kb fragment (-2732/+47) containing the alpha 3 promoter region, the beta 4/alpha 3 intergenic region, and a portion of the beta 4 3'-untranslated exon. The -2732/+47 fragment is preferentially active in PC12 cells relative to nonneural cell lines. Deletion analysis revealed a cell type-specific positive transcriptional element positioned in the beta 4 3'-untranslated exon. The positive element is likely to be an enhancer and not a second alpha 3 promoter, because no alpha 3 exons are present in this region. Having shown in cell culture that cell-type specific cis elements are positioned between the beta 4 and alpha 3 coding regions, we investigated the activity of -2732/+47 in vivo. Transgenic mice were generated, which carry the lacZ gene fused downstream of -2732/+47. Expression of the lacZ transgene is restricted to neurons of the CNS; no expression was detected in the PNS or in nonneural tissues. LacZ-positive cells were detected virtually exclusively in a subset of CNS nuclei that transcribe the endogenous alpha 3 gene. Some overlap was seen with the beta 4 gene, but nearly none with the alpha 5 gene. Our results demonstrate that cis elements positioned between the alpha 3 and beta 4 coding regions are important for establishing part of the restricted CNS patterns of beta 4, alpha 3, and alpha 5 gene transcription.

Animals↗
PubMed1996

The POU domain of SCIP/Tst-1/Oct-6 is sufficient for activation of an acetylcholine receptor promoter.

In the PC12 neuroendocrine line, the neuronal nicotinic acetylcholine receptor alpha3 gene promoter is activated by SCIP/Tst-1/Oct-6, a POU domain transcription factor proposed to be important for regulating the development of specific neural cell populations. In this study, we have investigated the SCIP polypeptide domains involved in alpha3 promoter activation. The characteristics of activation by a chimeric effector in which the GAL4 DNA binding domain was substituted for the SCIP POU domain were dramatically different from those of wild-type SCIP. At low effector masses, the chimeric polypeptide weakly activated alpha3 in a GAL4 binding-site-dependent manner but then squelched transcription at higher masses. In contrast, wild-type SCIP activation was not modulated by the presence of multimerized SCIP binding sites, and squelching was not observed. Analysis of wild-type SCIP truncations revealed that deletion of the previously characterized SCIP amino-terminal activation domain did not destroy activity of the factor. Surprisingly, a truncation expressing nothing more than the POU domain was nearly as active as wild-type SCIP. Moreover, cotransfection of a GAL4-VP16 effector with an effector expressing just the SCIP POU domain resulted in synergistic activation of the promoter. Synergistic activation did not depend on an Sp1 motif that is the only functional alpha3 cis element outside the transcription start site region. Our results show that the DNA binding domain of a POU factor is capable of transcriptional activation probably through protein-protein interactions with components of the basal transcription complex.

Animals↗
PubMed1991

The nicotinic receptor genes.

The causative factor(s) of Alzheimer's disease (AD) are presently unknown. However, it has been shown that the number as well as the fraction of high- to low-affinity nicotine binding sites is altered in patients suffering from this disease. This finding, along with the identification of seven genes which code for nicotinic receptors expressed in the mammalian brain, has led to the idea that one nicotinic receptor subtype may be specifically altered in AD. The present article reviews how, through a molecular genetic approach, a family of genes coding for nicotinic acetylcholine receptor subtypes was uncovered. Also discussed is the use of in situ hybridization to determine the distribution of expression of the mRNA encoding for each receptor subtype and the patch clamp technique to characterize their biophysical properties. Determination of the promoters of these genes, as well as the properties of the expressed receptor subtypes, may make it possible to design new specific nicotinic receptor subtype drugs that will treat not only the symptoms of AD but the progression of the disease process as well.

Alzheimer Disease↗
PubMed1990

Molecular cloning and functional expression of glutamate receptor subunit genes.

Three closely related genes, GluR1, GluR2, and GluR3, encode receptor subunits for the excitatory neurotransmitter glutamate. The proteins encoded by the individual genes form homomeric ion channels in Xenopus oocytes that are sensitive to glutamatergic agonists such as kainate and quisqualate but not to N-methyl-D-aspartate, indicating that binding sites for kainate and quisqualate exist on single receptor polypeptides. In addition, kainate-evoked conductances are potentiated in oocytes expressing two or more of the cloned receptor subunits. Electrophysiological responses obtained with certain subunit combinations show agonist profiles and current-voltage relations that are similar to those obtained in vivo. Finally, in situ hybridization histochemistry reveals that these genes are transcribed in shared neuroanatomical loci. Thus, as with gamma-aminobutyric acid, glycine, and nicotinic acetylcholine receptors, native kainate-quisqualate-sensitive glutamate receptors form a family of heteromeric proteins.

Amino Acid Sequence↗
PubMed1989

Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat.

Previous studies have revealed the existence of a gene family that encodes a group of neuronal nicotinic acetylcholine receptor (nAChR) subunits. Four members of this family have been characterized thus far; three of these subunits (alpha 2, alpha 3, and alpha 4) are structurally related to the ligand binding subunit expressed in muscle and form functional nAChRs when combined with the beta 2 gene product in Xenopus oocytes. In addition, the alpha 4 gene appears to encode two different products (alpha 4-1 and alpha 4-2) that have been proposed to arise by alternative mRNA splicing. Nine different [35S]-complementary ribonucleic acid (cRNA) probes were used in the present study to map the distribution of these nAChR subunit mRNAs throughout the central nervous system (CNS) of the rat. It was found that the beta 2 gene is expressed in most regions of the CNS, as are the alpha subunit genes as a group. However, each alpha gene is expressed in a unique, although partly overlapping, set of neuronal structures. Alpha 4 is the most widely expressed alpha gene, and the evidence suggests that mRNAs for the alpha 4-1 and alpha 4-2 products are virtually always found in the same regions, in approximately the same ratios (alpha 4-2 greater than alpha 4-1). In addition, there are several examples of cell groups that express beta 2 but none of the alpha subunit mRNAs examined here (particularly in the hypothalamus), as well as all groups that express the converse, thus suggesting that additional neuronal nAChR subunits remain to be characterized. Finally, the extensive expression of multiple alpha subunits in certain regions, particularly for alpha 3 and alpha 4 in the thalamus, suggests that there is microheterogeneity in a small population of cells or that some neurons may express more than one alpha subunit. This problem needs to be examined directly with double labeling methods but raises the possibility that some neuronal nAChRs may be composed of more than one kind of alpha subunit. The wide expression of these receptor genes suggests that nAChRs constitute major excitatory systems in the CNS.

Animals↗
PubMed1987

Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system.

Nicotinic acetylcholine receptors found in the peripheral and central nervous system differ from those found at the neuromuscular junction. Recently we isolated a cDNA clone encoding the alpha subunit of a neuronal acetylcholine receptor expressed in both the peripheral and central nervous system. In this paper we report the isolation of a cDNA encoding the alpha subunit of a second acetylcholine receptor expressed in the central nervous system. Thus it is clear that there is a family of genes coding for proteins with sequence and structural homology to the alpha subunit of the muscle nicotinic acetylcholine receptor. Members of this gene family are expressed in different regions of the central nervous system and, presumably, code for subtypes of the nicotinic acetylcholine receptor.

Amino Acid Sequence↗
PubMed1987

Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family.

A family of genes coding for proteins homologous to the alpha subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha 3, alpha 4, and beta 2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha 3 or alpha 4 protein in combination with the beta 2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha 4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by alpha-bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha 3, alpha 4, and beta 2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha 3, alpha 4, and beta 2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Amino Acid Sequence↗