Expression Of Acetylcholine Receptor Subunits Research Articles

Neuregulin-1/ErbB4 upregulates acetylcholine receptors via Akt/mTOR/p70S6K: a study in a rat model of obstetric brachial plexus palsy and in vitro.

In obstetric brachial plexus palsy (OBPP), the operative time window for nerve reconstruction of the intrinsic muscles of the hand (IMH) is much shorter than that of biceps. The reason is that the atrophy of IMH becomes irreversible more quickly than that of biceps. A previous study confirmed that the motor endplates of denervated intrinsic muscles of the forepaw (IMF) were destabilized, while those of denervated biceps remained intact. However, the specific molecular mechanism of regulating the self-repair of motor endplates is still unknown. In this study, we use a rat model of OBPP with right C5-C6 rupture plus C7-C8-T1 avulsion and left side as a control. Bilateral IMF and biceps are harvested at 5 weeks postinjury to assess relative protein and mRNA expression. We also use L6 skeletal myoblasts to verify the effects of signaling pathways regulating acetylcholine receptor (AChR) protein synthesis in vitro. The results show that in the OBPP rat model, the protein and mRNA expression levels of NRG-1/ErbB4 and phosphorylation of Akt/mTOR/p70S6K are lower in denervated IMF than in denervated biceps. In L6 myoblasts stimulated with NRG-1, overexpression and knockdown of ErbB4 lead to upregulation and downregulation of AChR subunit protein synthesis and Akt/mTOR/p70S6K phosphorylation, respectively. Inhibition of mTOR abolishes protein synthesis of AChR subunits elevated by NRG-1/ErbB4. Our findings suggest that in the OBPP rat model, lower expression of AChR subunits in the motor endplates of denervated IMF is associated with downregulation of NRG-1/ErbB4 and phosphorylation of Akt/mTOR/p70S6K. NRG-1/ErbB4 can promote protein synthesis of the AChR subunits in L6 myoblasts via phosphorylation of Akt/mTOR/p70S6K.

Mice lacking α4 nicotinic acetylcholine receptors are protected against alcohol-associated liver injury.

Chronic heavy alcohol consumption is a major risk factor for the development of liver steatosis, fibrosis, and cirrhosis, but the mechanisms by which alcohol causes liver damage remain incompletely elucidated. This group has reported that α4 nicotinic acetylcholine receptors (α4 nAChRs) act as sensors for alcohol in lung cells. This study tested the hypothesis that α4 nAChRs mediate the effects of alcohol in the liver. Expression of acetylcholine receptor subunits in mouse liver was determined by RNA sequencing (RNA-seq). α4 nAChR knockout (α4 KO) mice were generated in C57BL/6J mice by introducing a mutation encoding an early stop codon in exon 4 of Chrna4, the gene encoding the α4 subunit of the nAChR. The presence of the inactivating mutation was established by polymerase chain reaction and genomic sequencing, and the lack of α4 nAChR function was confirmed in primary fibroblasts isolated from the α4 KO mice. Wild-type (WT) and α4 KO mice were fed the Lieber-DeCarli diet (with 36% of calories from alcohol) or pair fed an isocaloric maltose-dextrin control diet for a 6-week period that included a ramping up phase of increasing dietary alcohol. Chrna4 was the most abundantly expressed nAChR subunit gene in mouse livers. After 6 weeks of alcohol exposure, WT mice had elevated serum transaminases and their livers showed increased fat accumulation, decreased Sirt1 protein levels, and accumulation of markers of oxidative stress and inflammation including Cyp2E1, Nos2, Sod1, Slc7a11, TNFα, and PAI1. All these responses to alcohol were either absent or significantly attenuated in α4 KO animals. Together, these observations support the conclusion that activation of α4 nAChRs by alcohol or one of its metabolites is one of the initial events promoting the accumulation of excess fat and expression of inflammatory mediators. Thus, α4 nAChRs may represent viable targets for intervention in chronic alcohol-related liver disease.

Acetylcholine receptor subunit expression in Huntington's disease mouse muscle

Huntington's disease (HD) causes neurological impairments, as well as muscle dysfunction, including smaller neuromuscular junctions (NMJs). This study assessed the expression levels of the subunits of the nicotinic acetylcholine receptor (nAChR) in muscles of the R6/2 mouse model of HD. Based on our previous findings of reduced NMJ size in R6/2 mice, it was hypothesized that muscles from R6/2 mice would also show an altered expression pattern of nAChR subunits compared to wild-type (WT) mice. Therefore, the mRNA levels of nAChR subunits were quantified in R6/2 and WT mouse muscles using qRT-PCR. Denervated muscles from WT mice served as positive controls for alterations in nAChR expression. Although some changes in nAChR subunit expression occurred in R6/2 muscles, the expression levels closely resembled WT. However, the expression of nAChR subunit-ε (Chrne) was significantly decreased in R6/2 muscles relative to WT. This study demonstrates that only minor changes in nAChR subunit expression occurs in R6/2 mouse muscles and that reduction in Chrne expression may be related to a reduction in NMJ size in R6/mice.

Nicotinic acetylcholine receptor subunit expression in the gastrocnemius and in the orbicularis oris before and after facial nerve injury in rats

ABSTRACTObjectives: To observe the expression of nicotinic acetylcholine receptor (AChR) subunits in normal orbicularis oris and gastrocnemius muscles and to explore the relationships between the expression of AChR subunits and the severity of facial nerve injury.Methods: Gene and protein expression of AChR subunits in the orbicularis oris and gastrocnemius muscles of male Sprague–Dawley rats was measured by reverse transcription polymerase chain reaction and western blotting, respectively, 1–90 days after graded facial nerve injury.Results: Expression of ε-AChR in the normal orbicularis oris was significantly higher than that in the gastrocnemius, whereas no γ subunit expression was observed. Expression of α, β, δ, ε, and γ subunits was upregulated in the orbicularis oris and was positively correlated with the degree of facial nerve injury.Discussion: We demonstrated the higher expression of the AChR subunits in the orbicularis oris, compared to gastrocnemius muscles. The differences in expression of these subunits between muscles innervated by the facial nerve and somatic nerves and the correlation of AChR subunit expression with the degree of facial nerve injury yield insights into the sensitivity to muscle relaxants during intraoperative facial nerve monitoring.

Differences in pharmacodynamic responses to rocuronium in normal or injured orbicularis oris are associated with expression of acetylcholine receptor subunits

Previous research has indicated that differences in sensitivities to muscle relaxants exist between facial nerve- and somatic nerve-innervated muscles. Here, we report that the 50% inhibitory concentration (IC50) values for rocuronium were significantly larger in the normal orbicularis oris than those in the gastrocnemius. Increased IC50 values and reduced twitch tension were observed after facial nerve injury. The normal orbicularis oris had a smaller muscle fiber cross-sectional area (CSA) and a larger ratio of endplate surface area (ESA) to muscle fiber CSA (ESA/CSA), but no difference was found in the density of nicotinic acetylcholine receptor (nAChR) subunits on endplates between normal orbicularis oris and gastrocnemius. Expression of the nAChR α1, β1, δ, ε, and γ subunits increased significantly on the postsynaptic membranes of endplates and extra-junctional muscle membranes after facial nerve injury. Our results suggest that facial nerve-innervated muscle was less sensitive than somatic nerve-innervated muscle, and the mechanisms underlying this result may be related to muscle fiber CSA and the ESA/CSA ratio, but not to the density of nAChR subunits on endplates. Facial nerve injury caused the resistance to neuromuscular blockers and reduced twitch tension, which was related to qualitative, quantitative, and locational changes in nAChR subunits.

Disruption of Basal Lamina Components in Neuromotor Synapses of Children with Spastic Quadriplegic Cerebral Palsy

Cerebral palsy (CP) is a static encephalopathy occurring when a lesion to the developing brain results in disordered movement and posture. Patients present with sometimes overlapping spastic, athetoid/dyskinetic, and ataxic symptoms. Spastic CP, which is characterized by stiff muscles, weakness, and poor motor control, accounts for ∼80% of cases. The detailed mechanisms leading to disordered movement in spastic CP are not completely understood, but clinical experience and recent studies suggest involvement of peripheral motor synapses. For example, it is recognized that CP patients have altered sensitivities to drugs that target neuromuscular junctions (NMJs), and protein localization studies suggest that NMJ microanatomy is disrupted in CP. Since CP originates during maturation, we hypothesized that NMJ disruption in spastic CP is associated with retention of an immature neuromotor phenotype later in life. Scoliosis patients with spastic CP or idiopathic disease were enrolled in a prospective, partially-blinded study to evaluate NMJ organization and neuromotor maturation. The localization of synaptic acetylcholine esterase (AChE) relative to postsynaptic acetylcholine receptor (AChR), synaptic laminin β2, and presynaptic vesicle protein 2 (SV2) appeared mismatched in the CP samples; whereas, no significant disruption was found between AChR and SV2. These data suggest that pre- and postsynaptic NMJ components in CP children were appropriately distributed even though AChE and laminin β2 within the synaptic basal lamina appeared disrupted. Follow up electron microscopy indicated that NMJs from CP patients appeared generally mature and similar to controls with some differences present, including deeper postsynaptic folds and reduced presynaptic mitochondria. Analysis of maturational markers, including myosin, syntrophin, myogenin, and AChR subunit expression, and telomere lengths, all indicated similar levels of motor maturation in the two groups. Thus, NMJ disruption in CP was found to principally involve components of the synaptic basal lamina and subtle ultra-structural modifications but appeared unrelated to neuromotor maturational status.

Identification of nicotinic acetylcholine receptor subunit expression in early avian embryos

Most previous studies on nicotine used relatively high concentrations of nicotine, administered later in development. Using chick embryos as our model system, we have found a single low dose of nicotine (100 nM) administered during early development significantly retarded embryo growth, reduced heart rate, and increased the incidence of defects four days later. It is often assumed that nicotine's teratogenic effects are mediated by nicotinic acetylcholine receptors (nAChRs), whose expression at very early stages of embryonic development has not been thoroughly investigated. The objective was to identify which nAChR subunits are expressed during early avian embryogenesis. We performed RT‐PCR expression analysis for eleven nAChR subunits, including alpha1‐alpha7, alpha9, alpha10, beta3, and beta4, at 24‐, 48‐, 72‐, and 96‐hrs of development. Surprisingly, we find nAChR expression is widespread during early development, which suggests the teratogenic effects of nicotine were mediated by the early activation of nAChRs.Nebraska NIH‐INBRE Program; Nebraska Health and Human Services (LB595); Ferlic Undergraduate Summer Research Program, Creighton University College of Arts and SciencesGrant Funding Source: none current

Retinoic acid and nerve growth factor induce differential regulation of nicotinic acetylcholine receptor subunit expression in SN56 cells

Retinoic acid (RA) and nerve growth factor (NGF) have multiple functions in the regulation of neuronal development. In the present study, we characterized the expression of different nicotinic acetylcholine receptor (nAChR) subtypes in the cholinergic SN56 cell line and investigated the roles of RA and NGF in the expression of choline acetyltransferase (ChAT) and different nAChR subtypes. The nAChR agonist [(3)H]epibatidine was bound to two sites, with apparent affinities of 13 and 380 pM. RT-PCR analysis revealed expression of alpha3, alpha4, alpha5, alpha7, beta2, and beta4 nAChR subunits. RA treatment induced morphological changes, and the mRNA level of ChAT was maximally elevated after 4 days of exposure. The density of [(3)H]epibatidine binding sites and the mRNA and protein level of the alpha3 and beta2 nAChR subunits were also increased by RA-induced differentiation. RA down-regulated the mRNA and protein level of the alpha4 nAChR subunit, whereas no significant change was observed in the mRNA and protein level of the alpha7 nAChR subunit. NGF treatment increased the mRNA and protein level of the alpha3 and beta2 nAChR subunits. No morphological effects of NGF were observed, and the mRNA level of ChAT and mRNA and protein level of the alpha4 and alpha7 nAChR subunits were not significantly altered. Validation was performed with real-time RT-PCR. The present results show that RA and NGF have different effects on the expression of ChAT and the morphology and the expression pattern of different nAChR subunits in cholinergic SN56 cells.

Gene Expression of Acetylcholine Receptor Subunits and Myogenic Regulatory Factors Following Peripheral Nerve Injury

Peripheral nerve lesions cause impairment to both sensory and motor function. Motor functional recovery correlates with the gene expression of specific nicotinic acetylcholine receptor (nAChR) subunits and myogenic regulatory factors (MRF) which stabilize the neuromuscular junction. Sensory functional recovery correlates with the gene expression of Growth Associated Protein-43 (GAP-43) in the dorsal root ganglion (DRG), which affects axon growth. Understanding the gene expression of nAChR, MRF, and GAP-43 is critical in predicting motor and sensory recovery following nerve injury and repair. The goals of this study were to document the gene expression of nAChr, MRF, and GAP-43 following nerve injury.

Nicotine-induced Ca 2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line

We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M 3 mAChRs on lymphocytes increases the intracellular free Ca 2+ concentration ([Ca 2+] i) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca 2+] i in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca 2+-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca 2+, nicotine (30 μM) evoked rapid, transient increases in [Ca 2+] i. This effect was concentration-dependently inhibited by the α7 nAChR subunit antagonists, α-bungarotoxin (0.01-10 μM) and methyllycaconitine (0.01-10 mM), suggesting that the α7 nAChR subunit mediates Ca 2+ signaling in T-lymphocytes. Nicotine (0.01–10 μM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the α6 and α7 subunits was down-regulated within 1 week, while expression of the α3 and α5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine — and therefore likely smoking — affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca 2+ signaling in lymphocytes.

Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of α and β subunits with specific structural, functional and pharmacological properties. In this study the distribution of α3, α4, α7, β2 and β4 nAChR subunits in the human hippocampus was investigated using immunohistochemistry. Most pyramidal neurons, pre-alpha cells of the entorhinal cortex and dentate granule cells were immunoreactive for all subunits. Small islands of α7 immunoreactive cells were present in the outer presubiculum. α4 and β2, and α3, α4 and β2 immunoreactive fibre tracts were present in the stratum radiatum and subiculum, respectively, suggesting nAChRs may play a role in modulating inputs to the hippocampus via Schaffer collaterals and along the perforant pathway. Some astrocytes were immunoreactive for α3, α7 and β4 subunits. Immunoreactivity to all subunits was noted in association with blood vessels. These results indicate the involvement of multiple nAChR subtypes in the modulation of both neuronal and non-neuronal functions in the human hippocampus.

Variation in inter-animal susceptibility to noise damage is associated with alpha 9 acetylcholine receptor subunit expression level.

Large intersubject variabilities in acoustic injury are known to occur in both humans and animals; however, the mechanisms underlying such differences are poorly understood. The olivocochlear efferent system has been hypothesized to play a significant role in protecting the cochlea from noise overexposure. In this study, we demonstrate that a newly developed test for determining average efferent system strength can predict intersubject variations in acoustic injury. In addition, the intersubject variability in cochlear expression of the alpha9 subunit of the nicotinic acetylcholine receptor was found to be proportional to an animals average efferent strength. Therefore, the inter-animal variability in the alpha9-containing acetylcholine receptor expression may be one mechanism contributing to the inter-animal variability in acoustic injury.

Mice Lacking α-Calcitonin Gene-Related Peptide Exhibit Normal Cardiovascular Regulation and Neuromuscular Development

α-Calcitonin gene-related peptide (αCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration, nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated αCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking αCGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in αCGRP-deficient animals. These results suggest that αCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Monoclonal antibodies raised against human acetylcholine receptor bind to all five subunits of the fetal isoform

The human muscle acetylcholine receptor (AChR) is an oligomeric membrane protein consisting of (α1) 2,β,δ,ε subunits in the adult form and (α1) 2,β,γ,δ in the fetal form. The adult AChR is the target for autoantibodies in myasthenia gravis (MG), and antibodies that block the function of fetal AChR can cross the placenta and paralyse the developing baby causing joint contractures. Monoclonal antibodies (mAbs) raised against purified AChR were characterised previously in terms of binding to five regions, three of which appeared to partially overlap, but the subunit localisation of the regions was not clearly established and they were assumed to be mainly on the immunodominant α subunits. We have studied binding of the mAbs to AChR subunit extracellular fragments expressed in E. coli, and to AChRs derived from TE671 cells and from fibroblast cell lines expressing human/ Torpedo and Torpedo/mouse hybrid receptors. Using a combination of Western blotting and immunoprecipitation experiments, we demonstrate the subunit specificity of each mAb. The results confirm our previous observations but importantly show that only two of the regions are on the α subunit, the three others being on the β, γ and δ subunits of human AChR. Thus these mAbs should be useful in studies of AChR subunit expression in normal and diseased tissue, and to define further the binding sites of antibodies in MG patients.

Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.

The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG.

Innervation and target tissue interactions differentially regulate acetylcholine receptor subunit mRNA levels in developing neurons in situ.

Neurons engage in two distinct types of cell-cell interactions: they receive innervation and establish synapses on target tissues. Regulatory events that influence synapse formation and function on developing neurons are largely undefined. We show here that nicotinic acetylcholine receptor (AChR) subunit transcript levels are differentially regulated by innervation and target tissue interactions in developing chick ciliary ganglion neurons in situ. Using ganglia that have developed in the absence of pre- or postganglionic tissues and quantitative RT-PCR, we demonstrate that alpha 3 and beta 4 transcript levels are increased by innervation and target tissue interactions. In contrast, alpha 5 transcript levels are increased by innervation, but target tissues have little effect. Whole-cell ACh-induced currents, used to estimate the number of functional AChRs, change in correlation with alpha 3 and beta 4, but not alpha 5, transcript levels. A model is proposed in which the changes in AChR subunit expression regulate levels of synaptic activity, which is a critical determinant of synapse stabilization and elimination, and neuronal cell death.

Control of acetylcholinesterase gene expression during myogenesis

Acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor show enhanced expression upon differentiation of C2–C12 myoblasts into myotubes. Northern blots and RNAase protection show substantial increases in the mRNAs encoding both proteins. In contrast to transcriptional control of gene expression of acetylcholine receptor subunits during differentiation, Ache mRNA levels increase through stabilization of the transcript. The transcriptional rate remains unchanged during Ache mRNA accumulation, whereas transcription rate increase for the receptor subunits. Inhibition of protein synthesis produces a dramatic superinduction of Ache mRNA in undifferentiated cells without affecting transcription rates, suggesting the presence of labile proteins that uniquely destabilize the mRNA. Hence, Ache mRNA in myoblasts shows rapid turnover, and upon differentiation, its level is increased by slowing the rate of mRNA degradation.