Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia

doi:10.1016/0896-6273(88)90180-8

Neuron

Volume 1, Issue 6, August 1988, Pages 495-502

https://doi.org/10.1016/0896-6273(88)90180-8 Get rights and content

Abstract

A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) α3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR α2 or α4 genes. α3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in α3 mRNA levels, implying regulation of neuronal AChR mRNA by cell— cell interactions.

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Cited by (98)

The influence of inhibiting or stimulating the expression of the α3 subunit of the nicotinic receptor in SH-SY5Y cells on levels of amyloid-β peptide and β-secretase
2013, Neurochemistry International
Citation Excerpt :
Examination of nAChRs in living patients by positron emission tomography (PET) indicates that the deficits of these receptors probably reflect an early stage of AD (Nordberg, 2001). The α3 nAChR subunit also may predominate in chick ciliary ganglia (Boyd et al., 1988), superior cervical ganglia (Couturier et al., 1990; Vernallis et al., 1993) and dorsal root ganglion cells (Boyd et al., 1991). Here, we demonstrate that inhibition of the expression of α3 nAChR enhances the amount of Aβ secreted by SH-SY5Y cells.
To examine the effects of the α3 subunit of the nicotinic acetylcholine receptor (nAChR) on the expression of β-secretase and the concomitant level of amyloid-β (Aβ), SH-SY5Y neuroblastoma cells were either transfected with small interference RNAs (siRNAs) specifically targeting this subunit or exposed to nicotine. The levels of α3 nAChR mRNA and protein, as well as the corresponding levels of BACE1 (which cleaves the β-site of APP) and BACE2 (cleaving in the Aβ domain) were determined by real-time PCR and Western blotting, respectively. The levels of Aβ_1–42 in culture media were determined by an Elisa procedure.
In SH-SY5Y cells transfected with siRNA, the levels of α3 nAChR mRNA and protein were reduced by 96% and 88%, respectively; the levels of BACE1 mRNA and protein were significantly enhanced, while those of BACE2 were reduced; and the level of Aβ in the culture medium was elevated. In contrast, when untransfected SH-SY5Y cells were exposed to nicotine, the levels of both α3 nAChR mRNA and protein were enhanced; while the levels of BACE1 mRNA and protein were diminished and the corresponding levels of BACE2 enhanced; and the level of Aβ in the culture medium was attenuated.
These results indicate that the α3 subunit of nAChR inhibits the production of Aβ by reducing the expression of BACE1 and elevating the expression of BACE2, suggesting that this subunit might play an important neuroprotective role in connection with the pathogenesis of AD.
Inhibiting gene expression of α3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease
2008, Neurochemistry International
In order to examine the effects of α3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards α3 nAChR. The expressions of α3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the α-form of secreted amyloid precursor protein (αAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1 μM β-amyloid peptide (Aβ)_1–42, following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of α3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the αAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the α3 nAChR enhanced the toxicity exerted by Aβ. These results indicate that α3 nAChR may improve cleavage of APP by α-secretase, enhance antioxidation and inhibit the toxicity of Aβ, suggesting that the receptor might play an important role in AD.
Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors
2003, Gene Expression Patterns
We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR β3, α2 and α7 subunits expressed in other species. RT-PCR was used to show that the β3 and α2 subunit RNAs were present in zebrafish embryos only 2–5 hours post-fertilization (hpf) while α7 subunit RNA was not detected until 8 hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish β3, α2, and α7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity $[^{3} H]$ -epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC₅₀ values of 28.6 pM and 29.7 nM and in 5 dpf embryos with IC₅₀ values of 28.4 pM and 8.9 nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.
Nicotinic acetylcholine receptor gene expression in developing chick autonomic ganglia
2000, European Journal of Pharmacology
The developmental expression patterns of ten genes encoding nicotinic acetylcholine receptor subunits were analyzed using Northern blots and in situ hybridization in chick peripheral ganglia of neural crest, placodal and dual embryonic origin. The superior cervical and ciliary ganglia were investigated in detail because they accumulated relatively abundant transcripts of the α3, β4, α5 and α7 genes. In the superior cervical ganglion, these four mRNA species had similar developmental time-courses. They appeared at embryonic day 8 (E8), increased steadily until E16 and maintained a rather high plateau level until E18. In the ciliary ganglion, α7 transcripts were already abundant at E6, increased until E10, and considerably decreased thereafter. High-resolution in situ hybridization showed that α7 transcripts were present in all cell types of the E6 ciliary ganglion, whereas they were restricted to large neuronal somas at E16. Transfections with a reporter gene under the control of the α7 promoter demonstrated that a sharp developmental divide occurred at E11-12, after which stage the promoter was activatable in neurons exclusively.
A cysteine-rich isoform of neuregulin controls the level of expression of neuronal nicotinic receptor channels during synaptogenesis
1998, Neuron
We report here that neuregulin (NRG) isoforms with a conserved cysteine-rich domain (CRD) in their N terminus regulate expression of nicotinic acetylcholine receptors (nAChRs) at developing interneuronal synapses and report the isolation of transmembrane NRG isoforms with this CRD within the N-terminal portion. CRD-NRG mRNA and immunoreactive protein are detected early in developing presynaptic (visceral motor) neurons. The levels of expression of CRD-NRG peak prior to the formation of synapses with their postsynaptic partners, the ganglionic sympathetic neurons. Recombinant CRD-NRG mimics the effects of presynaptic input on target neurons. Functional deletion of CRD-NRG from presynaptic neurons abolishes the upregulation of nAChR expression induced by input-derived soluble material. Thus, CRD-NRG appears to be both a necessary and a sufficient signal for the control of neuronal nAChR expression during synaptogenesis.
Multiple Nicotinic Receptor Subtypes and Their Roles on Individual Neurons
1997, Advances in Organ Biology
Nicotinic acetylcholine receptors are widely expressed on neurons in both the central and peripheral nervous systems, but until recently, little was known about their physiological significance. This review first summarizes information from molecular cloning studies and heterologous expression systems examining the properties of neuronal nicotinic receptor gene products. It then considers the distribution and properties of native receptors and reports on the multiplicity of subtypes found in the nervous system. The subunit composition of native receptors is shown to be diverse, ranging from homomeric species to those having as many as four kinds of subunits. Individual neurons are found to express more than a single type of nicotinic receptor, and the subtypes can have distinctive locations on the cell surface. Recent evidence is presented for three kinds of functions performed by neuronal nicotinic receptors: presynaptic modulation of neurotransmitter release, postsynaptic mediation of fast, excitatory cholinergic transmission, and correlative signaling involving activation of second messenger cascades. These functions derive in large part from receptor location, ion selectivity, and ability to elevate intracellular calcium levels.

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Neuron

ArticleExpression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia

Abstract

Purification of biologically active globin messenger RNA by chromatography over oligothymidylic acid-cellulose

Influences on the expression of acetylcholine receptors on rat nodose neurones in cell culture

J. Physiol.

Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor asubunit

Nature

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

Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells

J. Physiol.

A method for isolation of intact, translationally active ribonucleic acid

DNA

Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation

J. Physiol.

Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects

J. Neurosci.

Factors affecting the expression of acetylcholine receptors on rat sensory neurones in culture

J. Physiol.

Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes

Dev. Biol.

Temporal patterns of neurogenesis in avian cranial sensory and autonomic ganglia

Am. J. Ant.

Primary structure and expression of β2: a novel subunit of neuronal nicotinic acetylcholine receptors

Neuron

Lack of nicotinic supersensitivity in frog sympathetic neurones following denervation

J. Physiol.

Cell division in the ciliary ganglion of quail embryos in situ and after back-transplantation into the neural crest migration pathways of chick embryos

Dev. Biol.

Affinity alkylation labels two subunits of the reduced acetylcholine receptor from mammalian muscle

Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor α-subunit cDNAs

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

Cell

Identification of a nicotinic acetylcholine receptor on neurons using an α-neurotoxin that blocks receptor function

J. Neurosci.

Affinity labeling of neuronal acetylcholine receptor subunits with an α-neurotoxin that blocks receptor function

J. Neurosci.

Article
Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia