nAChR Gene Expression Research Articles

Protective effect of alpha-linoleic acid on Aβ-induced oxidative stress, neuroinflammation, and memory impairment by alteration of α7 nAChR and NMDAR gene expression in the hippocampus of rats

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects many older people around the world. Numerous studies are underway to evaluate the protective effects of natural products in AD. Alpha-linoleic acid (ALA) is an essential unsaturated fatty acid that exhibits neuroprotective outcomes in rat models of ischemic stroke and Parkinson's disease. This research aimed to investigate the effect of ALA on oxidative stress, neuroinflammation, neuronal death, and memory deficit induced by amyloid-beta (Aβ) peptide. After intrahippocampal injection of Aβ1−42, rats received ALA (150 μg/kg, subcutaneously) for 14 consecutive days. ALA decreased the levels of malondialdehyde and nitric oxide, enhanced glutathione content, and increased the activity of catalase in the hippocampus of the rat model of AD. It also reduced the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, nuclear factor-kappa B, and N-methyl-d-aspartate receptor subunits NR2A and NR2B mRNAs in the hippocampus, prevented the neuronal loss in the CA1 region, and enhanced the expression of α7 nicotinic acetylcholine receptor. In addition, ALA allowed Aβ1−42-injected rats to spend less time and distance to reach the hidden platform in the Morris water maze test and to swim longer in the target quadrant. We concluded that ALA reduces the biochemical, molecular, histological, and behavioral changes caused by Aβ1−42 and it may be an effective option for treating AD.

Role of the cutaneous extraneuronal cholinergic system in the pathogenesis of psoriasis: a case-control study.

Although during recent years there have been considerable advances in elucidating the mechanisms of psoriasis pathogenesis, its full understanding is still distant. A cholinergic dysfunction has been proposed in the pathophysiology of some inflammatory and autoimmune diseases, including psoriasis. To determine tissue levels of acetylcholine (ACh) and its muscarinic and nicotinic receptors (mAChR and nAChR) in psoriasis vulgaris lesions in comparison with normal control skin. This case-control study included 30 patients with psoriasis vulgaris and 30 controls. A 4-mm punch skin biopsy was taken from the psoriatic plaques of patients and normal skin of controls. ACh level was measured in tissues using the colorimetric method, while mAChR and nAChR gene expression was determined by real-time PCR. The level of ACh was significantly higher in patients (mean±SD: 5.95±2.69) than in controls (1.12±0.34) (P<0.001). mAChR and nAChR expressions were significantly higher in patients compared with the controls (P<0.001). A significant positive correlation was detected between the expression of nAChR in patients and the duration of psoriasis (r=0.46, P=0.01), and the body mass index of the patients correlated positively with both nAChR (r=0.40, P=0.027) and mAChR expression (r=0.448, P=0.013). Abnormalities in the cutaneous extraneuronal cholinergic system could be involved in the pathogenesis of psoriasis. The high expression of nAChRs in patients with longer disease durations might represent an attempt by the body to regulate the inflammatory cascade in psoriatic lesions. The high expression of mAChR in psoriatic lesions may provide a link between psoriasis and obesity.

Oral exposure to aluminum leads to reduced nicotinic acetylcholine receptor gene expression, severe neurodegeneration and impaired hippocampus dependent learning in mice

Aluminum (Al) is known for its neurotoxicity for over a century and is reported to have specifically high toxicity for cholinergic system. The effect of Al on muscarinic acetylcholine receptors is widely reported, but its effect on nicotinic acetylcholine receptors (nAChRs) is less well known. The aim of this study was to determine the effects of Al on hippocampus dependent learning and memory, function and expression of nAChRs in the hippocampus. Al concentration and neurodegeneration were also measured in the hippocampus following Al treatment. The mice were treated with 250 mg/kg AlCl3.6H2O in drinking water for a period of 42 days. Results show that Al treated animals have significantly reduced spatial reference memory as compared to control animals in Morris water maze test. Similarly, Al treated animals showed reduced contextual memory for Pavlovian fear compared to control animals. Al treated animals show higher anxiety in elevated plus maze as compared to control animals. The analysis of nAChR expression via RT-PCR showed reduced expression of α7, α4 and β2 nAChR gene expression in the hippocampus of Al treated animals. High Al accumulation was observed in Al-treated animals (688.14 ± 242.82 μg/g) compared to the control group (115.14 ± 18.18 μg/g) that resulted in severe neurodegeneration in the hippocampus. These results demonstrated that Al exposure caused neurotoxicity in mice hippocampus which is manifested by reduced memory and elevated anxiety. The results were further validated by high Al accumulation in the hippocampus, severe neurodegeneration and reduced expression of nAChRs.

Cortex- and Amygdala-Dependent Learning and Nicotinic Acetylcholine Receptor Gene Expression is Severely Impaired in Mice Orally Treated with AlCl3.

Recent industrialization has increased human exposure to bio-available aluminum (Al). If more Al enters the brain than leaves, Al concentration will rise in the brain leading to neurodegenerative disorders. The aim of the present study was to determine Al concentration, neurodegeneration, and nicotinic acetylcholine receptor (nAChR) gene expression in the cortex and amygdala after oral ingestion of Al salt. The effect of Al on cortex- and amygdala-dependent learning and memory functions was also assessed. Mice were given AlCl3 (250mg/kg) in drinking water for 42days. nAChR gene expression was determined in the cortex and amygdala. The mice were subjected to behavior tests (fear conditioning, fear extinction, and open field), to assess memory deficits. The acquisition of fear memory in the fear conditioning test remained unaffected due to the Al administration. However, fear extinction (which is a new learning) was severely impaired. The behavioral analysis in the open field test showed greater anxiety and less adaptability to the new environment in Al-treated animals. High Al concentration and severe neurodegeneration in the cortex were observed following Al treatment while a slight, non-significant elevation in Al concentration was observed in the amygdala of Al-treated animals. The analysis of nAChR gene expression via RT-PCR showed a significant reduction in expression of α7, α4, and β2 nAChR genes in the cortex of Al-treated animals, while in the amygdala, the level of the α4 nAChR gene remained unaltered. Oral Al ingestion causes neuropathological changes and suppresses expression of nAChR genes that lead to deficits in learning and higher anxiety in Al-treated animals.

Nicotinic cholinergic and dopaminergic receptor mRNA expression in male and female rats with high or low preference for nicotine

ABSTRACTBackground: Nicotine exerts its central actions through nicotinic acetylcholine receptors (nAChRs), which in turn regulate major neurotransmitter systems including dopamine. Nicotinic and dopaminergic systems play significant roles in physiological functions, neuropsychiatric disorders, and addiction. Objectives: To evaluate possible differences in the expression of nAChR subunit and dopamine receptor (DR) mRNAs following voluntary nicotine intake. Methods: Male and female rats (n = 67) were exposed to long-term free-choice oral nicotine (24 hours/day, 6 weeks); rats with maximum and minimum nicotine preference/intake were selected. The mRNA levels of genes encoding α4,β2,α5, and α7 nAChR subunits and DR Drd1and Drd2 subtypes were evaluated in the striatum (STR), prefrontal cortex (PFC), and hippocampus using quantitative real-time polymerase chain reaction in selected rats (n = 30) and their control groups (n = 15). Results: In addition to baseline differences, expression changes were observed in the mRNA levels of evaluated genes in rats exposed to voluntary oral nicotine in a brain region-, sex-, and preference-related manner. Nicotine intake is correlated negatively with Chrnb2, Chrna7 and positively with Drd1 expression. In the cholinergic system, regional differences in Chnrb2 and Chrna5, sex differences in Chrna4 and Chrna5, and nicotine preference effects in the expression of all subunits except α4 were observed. Chrna5 was lower in maximum than in minimum preferring, and in male than female rats, supporting the inhibitory role of the α5 subunit in nicotine dependence. Nicotine increased Drd2 mRNA expression only in minimum preferring female rats in STR and PFC. Conclusion: Modulation of nAChR and DR gene expression by nicotine may have clinical implications and aid drug development. Pharmaceuticals targeting the nicotinic cholinergic and dopaminergic systems might be expected to have differential efficacy that varies with the patient’s sex or smoking status.

Neurogenic Atrophy‐Induced Nicotinic Acetylcholine Receptor ε to γ Subunit Switching is Impaired in Muscle RING Finger 1 (MuRF1) Null Mice

Skeletal muscle atrophy is caused by a multitude of conditions, including denervation, corticosteroids, immobilization and aging. The nicotinic acetylcholine receptors cluster at the neuromuscular junction (NMJ), which receives incoming signals from neurons and relays the stimulus to skeletal muscle cells causing contraction. The acetylcholine receptor (nAChR) is a multimeric transmembrane protein that consists of two α, one β, and one δ subunit and either the γ subunit expressed during embryonic development or the ε subunit expressed in the adult muscle. Microarray analysis of skeletal muscle tissue isolated from wild-type mice confirmed previous observations that the embryonic nAChRγ subunit is up-regulated in response to denervation, while the adult nAChRε subunit is transiently down-regulated. Interestingly, these changes in nAChRγ and nAChRε expression are altered in MuRF1 null mice in response to neurogenic atrophy. In order to further characterize the transcriptional regulation of the nAChRγ and nAChRε subunit genes, fragments of the promoter regions were cloned, fused to a reporter gene, and transfected into C2C12 cells in combination with MyoD1 or myogenin expression plasmids. MyoD1 and myogenin are well characterized myogenic regulatory factors (MRF) that have previously been shown regulate nAChR gene expression. Ectopic expression of MyoD1 or myogenin had no significant effect on the nAChRγ subunit reporter gene. However, overexpression of MyoD1 or myogenin in combination with the nAChRε reporter gene caused a significant increase in ε subunit reporter activity. Furthermore, when MyoD1 or myogenin were overexpressed in combination with MuRF1, the MRF induction of the nAChRε reporter gene was reversed.

Research tool: Validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches.

There is much interest in α7 nicotinic acetylcholine receptors (nAChRs) in CNS function since they are found throughout peripheral tissues as well as being highly expressed in brain regions implicated in attention, learning and memory. As such, the role of these receptors in many aspects of CNS function and disease is being actively investigated. To date, only one null mouse model (A7KO) is available which is non-conditional and constitutive. Since α7 nAChRs are present on neurons and glia (including astrocytes), as well as being developmentally regulated, there is an unmet need for the technical capability to control α7 nAChR gene expression. Therefore we have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6-Chrna7(LBDEx4007Ehs)) which we refer to as floxed α7 nAChR conditional knockout or α7nAChR(flox). We validated the chosen approach by mating α7nAChR(flox) with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP)-Cre promoter (GFAP-A7KO) to test whether α7nAChR(flox), GFAP-A7KO and appropriate littermate controls performed equally in our standard Rodent In Vivo Assessment Core battery to assess general health, locomotion, emotional and cognitive behaviours. Neither α7nAChR(flox) nor GFAP-A7KO exhibited significant differences from littermate controls in any of the baseline behavioural assessments we conducted, similar to the 'first generation' non-conditional A7KO mice. We also determined that α7 nAChR binding sites were absent on GFAP-positive astrocytes in hippocampal slices obtained from GFAP-A7KO offspring from α7nAChR(flox) and GFAP-Cre crosses. Finally, we validated that Cre recombinase (Cre)-mediated excision led to functional, cell- and tissue-specific loss of α7 nAChRs by demonstrating that choline-induced α7 nAChR currents were present in Cre-negative, but not synapsin promoter-driven Cre-positive, CA1 pyramidal neurons. Additionally, electrophysiological characterization of α7 nAChR-mediated current traces was similar in terms of amplitude and time constants of decay (during desensitization) for the α7nAChR(flox) and wild-type (WT) mice. Thus, we have in vivo and in vitro evidence that the Chrna7 exon 4 targeting strategy does not alter behavioural, cognitive, or electrophysiological properties compared to WT and that Cre-mediated excision is an effective approach to delete α7 nAChR expression in a cell-specific manner.

The Expression and Pharmacological Characterization of Nicotinic Acetylcholine Receptor Subunits in HBE16 Airway Epithelial Cells

This study characterizes the expression and the biological effects of the nicotinic acetylcholine receptor (nAChR) on human airway epithelial cells. Cultured HBE16 airway epithelial cells were incubated with either nicotine or cigarette smoke extract (CSE). The nAChR gene and protein expression in cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and western blot. The protein expression of the nAChR subunits, α1, α5, and α7, were evaluated by immunohistochemistry. Cells were subsequently transfected with α1-, α5-, and α7-specific siRNAs, and the effects of nicotine on the production of the pro-inflammatory factors, TNF-α, IL-8, and IL-6 in transfected cells were analyzed using an enzyme-linked immunosorbent assay and real-time PCR. We detected α1, α5, α7, and β2 subunits in untreated HBE16 cells, and their expression was elevated after nicotinic incubation. Importantly, the most significant increase in expression was observed in the α5 and α7 subunits. However, CSE did not cause a significant enhancement in the expression of these genes and proteins. Cells pretreated with nicotine prior to lipopolysaccharide (LPS) stimulation exhibited a lower production of TNF-α, IL-8, and IL-6 compared to LPS-treated (only) cells. Cells that were transfected with α7 siRNA and subsequently incubated with nicotine and LPS, exhibited a higher expression of TNF-α, IL-8, and IL-6 compared with non-transfected cells or α1 and α5 siRNA-transfected cells. In α1- and α5-siRNA-transfected cells, the expression of TNF-α, IL-8, and IL-6 showed no significant difference compared with non-transfected cells. Therefore, we concluded that α1, α5, α7, and β2 nAChR subunits are highly expressed in human bronchial epithelial cells (HBE16) after nicotinic incubation and that the α7 subunit is involved in the nicotine-induced inhibitory effect on the production of inflammatory factors. Moreover, α1, α5, and β2 subunits did not play an important role in this process.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

SK2 channels are required for function and long-term survival of efferent synapses on mammalian outer hair cells

Cochlear hair cells use SK2 currents to shape responses to cholinergic efferent feedback from the brain. Using SK2 −/− mice, we demonstrate that, in addition to their previously defined role in modulating hair cell membrane potentials, SK2 channels are necessary for long-term survival of olivocochlear fibers and synapses. Loss of the SK2 gene also results in loss of electrically driven olivocochlear effects in vivo, and down regulation of ryanodine receptors involved in calcium-induced calcium release, the main inducer of nAChR evoked SK2 activity. Generation of double-null mice lacking both the α10 nAChR gene, loss of which results in hypertrophied olivocochlear terminals, and the SK2 gene, recapitulates the SK2 −/− synaptic phenotype and gene expression, and also leads to down regulation of α9 nAChR gene expression. The data suggest a hierarchy of activity necessary to maintain early olivocochlear synapses at their targets, with SK2 serving an epistatic, upstream, role to the nAChRs.

Myogenin-dependent nAChR clustering in aneural myotubes

During development of the neuromuscular junction, nerve-derived agrin and the cell substrate laminin stimulate postsynaptic nAChR clustering. This clustering is dependent on activation of the tyrosine kinase, MuSK, which signals receptor clustering via a rapsyn-dependent mechanism. Myogenin is a muscle-specific transcription factor that controls myoblast differentiation and nAChR gene expression. Here, we used RNA interference to investigate if myogenin is also necessary for nAChR clustering. We find that myogenin expression is essential for robust nAChR clustering and cannot be compensated by the muscle regulatory factors MyoD, myf5, and MRF4. In addition, we show that clustering cannot be rescued in myogenin-depleted myotubes by simply overexpressing the essential clustering molecules MuSK, rapsyn, and nAChRs. These data suggest that myogenin controls the expression of molecules crucial to nAChR clustering in addition to its role in regulating nAChR gene expression.

CaM kinase II-dependent phosphorylation of myogenin contributes to activity-dependent suppression of nAChR gene expression in developing rat myotubes

During development of the neuromuscular junction (NMJ), extrajunctional expression of genes, whose products are destined for the synapse, is suppressed by muscle activity. One of the best-studied examples of activity-dependent gene regulation in muscle are those encoding nicotinic acetylcholine receptor (nAChR) subunits. We recently showed that nAChR gene expression is inhibited by calcium/calmodulin-dependent protein kinase II (CaMKII) and CaMKII inhibitors block activity-dependent suppression of these genes. Here we report results investigating the mechanism by which CaMKII suppresses nAChR gene expression. We show that the muscle helix–loop–helix transcription factor, myogenin, is necessary for activity-dependent control of nAChR gene expression in cultured rat myotubes and is a substrate for CaMKII both in vitro and in vivo. CaMKII phosphorylation of myogenin is induced by muscle activity and this phosphorylation influences DNA binding and transactivation. Thus we have identified a signaling mechanism by which muscle activity controls nAChR gene expression in developing muscle.

Protein Kinase C and Calcium/Calmodulin-activated Protein Kinase II (CaMK II) Suppress Nicotinic Acetylcholine Receptor Gene Expression in Mammalian Muscle: A SPECIFIC ROLE FOR CaMK II IN ACTIVITY-DEPENDENT GENE EXPRESSION

Nicotinic acetylcholine receptor (nAChR) gene expression is regulated by both muscle activity and increased intracellular calcium. This regulation is an important developmental event that rids receptors from the extrajunctional region of the developing muscle fiber. In avian muscle, it has been proposed that muscle activity suppresses nAChR gene expression via calcium-activated protein kinase C (PKC)-dependent phosphorylation of the myogenic transcription factor, myogenin. Here, we examined the role that PKC and other kinases play in mediating calcium- and activity-dependent suppression of nAChR genes in rat primary myotubes. We found that although activated PKC could regulate nAChR promoter activity and transiently suppressed both nAChR and myogenin gene expression, it did not appear to be required for calcium- or activity-dependent control of nAChR gene expression in mammalian muscle. Neither depletion of PKC from myotubes nor specific pharmacological inhibition of PKC blocked the suppression of nAChR gene expression produced by calcium or muscle depolarization. In contrast, we provide evidence that calcium/calmodulin-activated protein kinase II participates in mediating the effects of muscle depolarization on nAChR and myogenin gene expression.

CaM Kinase II-dependent Suppression of Nicotinic Acetylcholine Receptor δ-Subunit Promoter Activity

Nerve-induced muscle activity suppresses nicotinic acetylcholine receptor (nAChR) gene expression by increasing intracellular calcium levels. This suppression is mediated by nAChR promoter sequences harboring at least 1 E-box (CANNTG) that bind myogenic helix-loop-helix transcription factors. How muscle depolarization or increased calcium mediates changes in nAChR promoter activity is not well understood. In chick muscle, protein kinase C (PKC) activation is necessary for activity-dependent nAChR gene suppression. Similar effects of PKC activation have not been found in mammalian skeletal muscle. Therefore, we used rat primary muscle cultures to screen for other calcium-regulated enzymatic activities that may mediate the effects of muscle activity and calcium on nAChR promoter activity. We report here that calcium/calmodulin-dependent protein kinase II (CaM kinase II) can specifically suppress nAChR promoter activity in mammalian muscle. This regulation was mediated by a single E-box sequence residing in the previously characterized nAChR delta-subunit genes 47-base pair activity-dependent enhancer. In vitro protein/DNA interaction studies suggest that CaM kinase II inhibits binding of the myogenic factor, myogenin, to the delta-promoter 47-base pair activity-dependent enhancer. CaM kinase activity is increased in active muscle and inhibition of this enzymatic activity results in increased nAChR delta-promoter activity. Therefore, CaM kinase II may represent a previously unappreciated activity that participates in coupling muscle depolarization to nAChR gene expression.

Molecular mechanisms mediating synapse-specific gene expression during development of the neuromuscular junction.

Adult skeletal muscle locally expresses nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction by selective induction of their subunit-encoding genes (alpha beta epsilon delta) in endplate-associated myonuclei. Neuregulin/ARIA is a nerve-derived factor that is thought to be largely responsible for this local gene induction. Neuregulin/ARIA activates a Ras/MAP kinase signalling cascade, which ultimately induces nAChR epsilon-subunit gene expression via a 15 bp sequence that harbors a core Ets transcription factor binding site (GGA). Interestingly, this same sequence also appears to participate in extrajunctional repression of the epsilon-subunit gene. Muscle Ets 2 overexpression induces nAChR epsilon-subunit gene promoter activity, whereas a dominant/negative Ets blocks neuregulin-dependent induction. These results suggest that Ets transcription factors play a role in mediating synapse-specific and neuregulin-mediated motor neuron control of nAChR gene expression.

Role for calcium from the sarcoplasmic reticulum in coupling muscle activity to nicotinic acetylcholine receptor gene expression in rat.

Neurally evoked muscle electrical activity suppresses nicotinic acetylcholine receptor (nAChR) gene expression in extrajunctional domains of adult muscle fibers. It has been proposed that this regulation is mediated by calcium influx through voltage-dependent L-type calcium channels but bypasses the sarcoplasmic reticulum in chick and mouse C2C12 cells. Here we report that in rat muscle calcium influx through L-type calcium channels preferentially reduced nAChR epsilon-subunit RNA via a post-transcriptional mechanism. In contrast, calcium release from the sarcoplasmic reticulum (SR) suppressed nAChR subunit RNA levels as a result of decreasing nAChR subunit promoter activity. Finally, we show that this decreased promoter activity is mediated through the same DNA sequences that control activity-dependent gene expression. Therefore, we propose that in rat muscle, calcium release from the SR participates in coupling muscle depolarization to nAChR gene expression.

Axon–target interactions maintain synaptic gene expression in retinae transplanted to intracranial regions of the rat

In the present study we examined the effects of optic axon–CNS target interactions on gene expression in the rat retina. These studies took advantage of a transplantation paradigm that allowed us to assay gene expression in retinae transplanted to different intracranial locations in the neonatal rat that either promoted (dorsal midbrain) or precluded (cerebral cortex) the formation of retino-collicular connections. Using in situ hybridization experiments, we observed that transplantation to the dorsal midbrain resulted in a relatively normal pattern of nicotinic acetylcholine receptor (nAChR) β-3 subunit and glutamate receptor 3 (GluR3) gene expression. In contrast, retinae transplanted to the cerebral cortex (which did not result in normal retino-collicular interactions) showed a dramatic reduction in nAChR β-3 subunit and GluR3 gene expression. These results agree with those obtained in the adult goldfish retina, where it has been demonstrated that an optic nerve–optic tectum interaction is responsible for the re-induction nAChR and NMDA receptor gene expression during optic nerve regeneration. Taken together, these results support the hypothesis that proper axon–target interactions are required for maintenance of nAChR and glutamate receptor gene expression in the mature vertebrate retina.

Protein-tyrosine phosphatases specifically regulate muscle adult-type nicotinic acetylcholine receptor gene expression.

Innervation of skeletal muscles results in expression of adult-type nicotinic acetylcholine receptors (alpha 2 beta epsilon delta) beneath the neuromuscular junction. This local expression is largely a result of selective induction of adult-type nicotinic acetylcholine receptor (nAChR) genes in endplate-associated myonuclei. The molecular mechanism by which the nerve induces gene expression in these nuclei is not known. We have shown previously that ionophore-induced calcium influx across the plasma membrane preferentially decreases expression from the adult-type specific nAChR epsilon-subunit gene (Walke, W., Staple, J., Adams, L., Gnegy, M., Chahine, K., and Goldman, D. (1994) J. Biol. Chem. 269, 19447-19456). Here we provide evidence that the genes encoding adult-type nAChRs are specifically regulated by protein-tyrosine phosphatase activity. Orthovanadate, a specific protein-tyrosine phosphatase inhibitor, caused increased expression of the epsilon-subunit gene in rat primary myotubes and was able to completely block the suppressive effects of increased calcium influx on epsilon-subunit RNA expression. Overexpression of protein-tyrosine phosphatases selectively decreased expression from the adult-type nAChR genes with no effect on the embryonic-type specific gamma-subunit gene. These results demonstrate that protein-tyrosine phosphatases regulate mammalian adult-type nAChR gene expression and suggest a mechanism by which muscle innervation selectively regulates gene expression in endplate-associated myonuclei.

Calcium-dependent regulation of rat and chick muscle nicotinic acetylcholine receptor (nAChR) gene expression.

Muscle depolarization leads to decreased expression of nicotinic acetylcholine receptor (nAChR) genes in extrajunctional regions of the muscle fiber with little effect on their expression at the neuromuscular junction (NMJ). Depolarization-dependent decreases in nAChR gene expression have been linked to a cAMP-dependent signaling system in rat (Chahine, K. G., Baracchini, E., and Goldman, D. (1993) J. Biol. Chem. 2893-2898), and a calcium-dependent protein kinase C (PKC) signaling system in chick (Klarsfeld, A., Laufer, R., Fontaine, B., Devillers-Thiery, A., Bubreuil, C., and Changeux, J. P. (1989) Neuron 2, 1229-1236). We report here on experiments investigating the role of calcium and PKC in regulating rat muscle nAChR gene expression. These studies indicate that depolarization-dependent regulation of rat muscle nAChR gene expression is independent of PKC activity. However, these genes are regulated by a calcium-dependent signal transduction system. Calcium influx across the plasma membrane decreases nAChR gene expression in inactive rat myotubes. Surprisingly, this influx of extracellular calcium is most effective at reducing nAChR epsilon-subunit gene expression. We also provide evidence that a similar signal transduction system is capable of regulating nAChR gene expression in chick muscle. Based on these data we propose that calcium, in addition to mediating depolarization-dependent regulation of nAChR expression, may also participate in restricting their expression to the neuromuscular junctions of adult muscle fibers.

Cues intrinsic to the retina induce nAChR gene expression during development.

Recent studies of optic nerve regeneration in goldfish have indicated that the optic tectum plays an important role in modulating the induction of nicotinic acetylcholine receptor (nAChR) gene expression in regenerating retinal ganglion cells (Heiber, Agranoff, and Goldman, 1992, J. Neurochem. 58:1009-1015). These observations suggest that induction of these genes is regulated by brain target regions. The appearance of nAChR mRNA in the developing rat retina coincides with a time when ganglion cells are sending axons to their brain targets (Hoover and Goldman, 1992, Exp. Eye Res. 54:561-571). Might a mechanism similar to that seen during goldfish optic nerve regeneration also mediate induction of nAChR gene expression during development of the mammalian retina? This possibility was tested by either transplanting embryonic rat retina to different brain regions, or explanting it to organ culture and assaying for nAChR gene expression. These studies showed that induction of the nAChR genes in developing rat retina is independent of the environment in which the retina develops. These results indicate that either the retinal microenvironment or a signal intrinsic to the retinal ganglion cell is responsible for this induction.