Acetylcholine receptor antibodies in a patient with sensory neuropathy.

Antibodies to acetylcholine receptor (AChR) are detected in the vast majority of patients with generalized myasthenia gravis (MG) and are rarely detected in significant titer in other autoimmune diseases. We report a patient with an axonal predominately sensory neuropathy for over 12 years with persistent binding and modulating AChR antibodies as well as striational muscle antibodies with no evidence of MG or any neoplastic disease.

Clinical application of clustered-AChR for the detection of SNMG.

Myasthenia gravis (MG) is an autoantibody-mediated disease of the neuromuscular junction (NMJ). However, accumulating evidence has indicated that MG patients whose serum anti-acetylcholine receptor (AChR) antibodies are not detectable (serumnegative MG; SNMG) in routine assays share similar clinical features with anti-AChR antibody-positive MG patients. We hypothesized that SNMG patients would have low-affinity antibodies to AChRs that would not be detectable using traditional methods but that might be detected by binding to AChR on the cell membrane, particularly if they were clustered at the high density observed at the NMJ. We expressed AChR subunits with the clustering protein rapsyn (an AChR-associated protein at the synapse) in human embryonic kidney (HEK) cells, and we tested the binding of the antibodies using immunofluorescence. With this approach, AChR antibodies to rapsyn-clustered AChR could be detected in the sera from 45.83% (11/24) of SNMG patients, as confirmed with fluorescence-activated cell sorting (FACS). This was the first application in China of cell-based AChR antibody detection. More importantly, this sensitive (and specific) approach could significantly increase the diagnosis rate of SNMG.

Improved neurological outcome by intramuscular injection of human amniotic fluid derived stem cells in a muscle denervation model.

PURPOSE: The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. MATERIALS AND METHODS: Seventy two Sprague-Dawley rats weighing 200-250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. RESULTS: NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. CONCLUSION: Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

Expression of acetylcholine and its receptor in human sympathetic ganglia in primary hyperhidrosis.

BACKGROUND: The pathophysiologic characteristics of primary hyperhidrosis are not well understood and seem to be related to a sympathetic nervous system dysfunction. The resection of thoracic sympathetic chain ganglia is the most effective treatment for hyperhidrosis; however sympathetic ganglia function in normal individuals and in patients with hyperhidrosis is unknown. METHODS: A cross-sectional study, in which 2 groups of 20 subjects were analyzed: the hyperhidrosis group (HYP), comprised of patients with hyperhidrosis who were eligible for thoracic sympathectomy, and the control group (CON) comprised of brain-dead organ donors without a history of hyperhidrosis. For each subject, the following were performed: resection of the third left sympathetic ganglion, measurement of the ganglion's diameter, and immunohistochemical evaluation by quantification of strong and weak expression areas of primary antibodies against acetylcholine and alpha-7 neuronal nicotinic receptor subunit. RESULTS: The presence of a strong alpha-7 subunit expression area was 4.85% in patients with primary hyperhidrosis and 2.34% in controls (p < 0.001), whereas the presence of a weak expression area was 11.48% in the HYP group and 4.59% in the CON group (p < 0.001). Strong acetylcholine expression was found in 4.95% of the total area in the HYP group and in 1.19% in the CON group (p < 0.001), whereas weak expression was found in 18.55% and 6.77% of the HYP and CON groups, respectively (p < 0.001). Furthermore, diameter of the ganglia was 0.71 cm in the HYP group and 0.53 cm in the CON group (p < 0.001). CONCLUSIONS: There is a higher expression of acetylcholine and alpha-7 neuronal nicotinic receptor subunit in the sympathetic ganglia of patients with hyperhidrosis. Furthermore, the diameter of the thoracic sympathetic chain ganglia is larger in such patients.

Myasthenia gravis and autoimmune Addison disease in a patient with thymoma.

The association of thymoma with myasthenia gravis has been well documented. However, the relationship between these two syndromes and Addison disease are very rarely encountered in clinical practice. We report on a 32-year-old man who underwent a resection for thymoma 48 months ago. The diagnosis of Addison disease was made followed by a diagnosis of myasthenia gravis on the basis of a high titer of acetylcholine receptor levels. The treatment of oral prednisolone 7.5 mg/day and oral prostigmine 180 mg/day was initiated. His symptoms and physical signs were improved after this treatment. To our knowledge, this is the fourth reported case of thymoma synchronously associated with myasthenia gravis and Addison disease.

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner.

Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential.Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy.However, its precise function at the synapse is unknown. Here we show that two mutations in the alpha subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine(Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ).These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a pump activity-independent, allele-specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy.

Myotonic dystrophy type 1 coexisting with myasthenia gravis and thymoma.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multisystemic disorder that may rarely be associated with benign and malignant neoplasms. Cases of both thymoma and myasthenia gravis in association with DM1 are extremely rare. A literature review revealed only three prior reports. We present a 51-year-old man with a family history of DM1 and fluctuating diplopia and ptosis, who was found to have acetylcholine receptor-binding antibodies, thymoma, and a clinical presentation compatible with ocular myasthenia gravis as well as positive genetic testing for DM1. Needle electromyographic (EMG) study demonstrated diffuse runs of myotonic discharges in multiple muscles, consistent with the diagnosis of DM1. Single-fiber EMG showed both increased jitter and blocking. Due to somatic instability, which has been shown previously in DM1, the myotonin protein kinase (DMPK) gene appears to act as a tumor suppressor. Therefore, abnormal CTG repeat expansions in the gene could lead to the development of thymoma and myasthenia gravis.

Rhabdomyosarcoma lysis by T cells expressing a human autoantibody-based chimeric receptor targeting the fetal acetylcholine receptor.

Rhabdomyosarcomas are the most frequent malignant soft tissue tumors of childhood; however, because current multimodality treatments fail to improve the poor survival rate of children with metastatic rhabdomyosarcoma, new treatments are required. We previously identified the gamma-subunit of the fetal acetylcholine receptor (fAChR) as a specific cell surface target in rhabdomyosarcoma. Here, we engineered human T lymphocytes to express chimeric receptors composed of the antigen-binding domain of a human anti-fAChR antibody joined to the signaling domain of the human T-cell receptor zeta-chain. The interaction of fAChRzeta-transduced T cells with fAChR-positive rhabdomyosarcoma cell lines, but not with fAChR-negative control cells, induced T-cell activation characterized by strong secretion of IFN-gamma and delayed lysis of tumor cells. Importantly, we found that in six of six rhabdomyosarcoma patients, chemotherapy increased fAChR expression on residual tumor cells in vivo. Our observations suggest that these fully human chimeric fAChRzeta-transduced T cells, which should be well tolerated by the patient, have potential use in vivo both as a primary treatment for rhabdomyosarcoma and as a complementary approach to eradicate residual tumor cells after chemotherapy.

Succinylcholine-induced hyperkalemia in acquired pathologic states: etiologic factors and molecular mechanisms.

Lethal hyperkalemic response to succinylcholine continues to be reported, but the molecular mechanisms for the hyperkalemia have not been completely elucidated. In the normal innervated mature muscle, the acetylcholine receptors (AChRs) are located only in the junctional area. In certain pathologic states, including upper or lower motor denervation, chemical denervation by muscle relaxants, drugs, or toxins, immobilization, infection, direct muscle trauma, muscle tumor, or muscle inflammation, and/or burn injury, there is up-regulation (increase) of AChRs spreading throughout the muscle membrane, with the additional expression of two new isoforms of AChRs. The depolarization of these AChRs that are spread throughout the muscle membrane by succinylcholine and its metabolites leads to potassium efflux from the muscle, leading to hyperkalemia. The nicotinic (neuronal) alpha7 acetylcholine receptors, recently described to be expressed in muscle also, can be depolarized not only by acetylcholine and succinylcholine but also by choline, persistently, and possibly play a critical role in the hyperkalemic response to succinylcholine in patients with up-regulated AChRs.

Neuromuscular synapse formation in mice lacking motor neuron- and skeletal muscle-derived Neuregulin-1.

The localization of acetylcholine receptors (AChRs) to the vertebrate neuromuscular junction is mediated, in part, through selective transcription of AChR subunit genes in myofiber subsynaptic nuclei. Agrin and the muscle-specific receptor tyrosine kinase, MuSK, have critical roles in synapse-specific transcription, because AChR genes are expressed uniformly in mice lacking either agrin or MuSK. Several lines of evidence suggest that agrin and MuSK stimulate synapse-specific transcription indirectly by regulating the distribution of other cell surface ligands, which stimulate a pathway for synapse-specific gene expression. This putative secondary signal for directing AChR gene expression to synapses is not known, but Neuregulin-1 (Nrg-1), primarily based on its presence at synapses and its ability to induce AChR gene expression in vitro, has been considered a good candidate. To study the role of Nrg-1 at neuromuscular synapses, we inactivated nrg-1 in motor neurons, skeletal muscle, or both cell types, using mice that express Cre recombinase selectively in developing motor neurons or in developing skeletal myofibers. We find that AChRs are clustered at synapses and that synapse-specific transcription is normal in mice lacking Nrg-1 in motor neurons, myofibers, or both cell types. These data indicate that Nrg-1 is dispensable for clustering AChRs and activating AChR genes in subsynaptic nuclei during development and suggest that these aspects of postsynaptic differentiation are dependent on Agrin/MuSK signaling without a requirement for a secondary signal.

Effects of cytokines on acetylcholine receptor expression: implications for myasthenia gravis.

Myasthenia gravis is an autoimmune disease associated with thymic pathologies, including hyperplasia. In this study, we investigated the processes that may lead to thymic overexpression of the triggering Ag, the acetylcholine receptor (AChR). Using microarray technology, we found that IFN-regulated genes are more highly expressed in these pathological thymic tissues compared with age- and sex-matched normal thymus controls. Therefore, we investigated whether proinflammatory cytokines could locally modify AChR expression in myoid and thymic epithelial cells. We found that AChR transcripts are up-regulated by IFN-gamma, and even more so by IFN-gamma and TNF-alpha, as assessed by real-time RT-PCR, with the alpha-AChR subunit being the most sensitive to this regulation. The expression of AChR protein was increased at the cytoplasmic level in thymic epithelial cells and at the membrane in myoid cells. To examine whether IFN-gamma could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-gamma gene knock-out mice, and found a significant decrease in AChR transcript levels in the thymus but not in the muscle, compared with wild-type mice. However, up-regulation of AChR protein expression was found in the muscles of animals with myasthenic symptoms treated with TNF-alpha. Altogether, these results indicate that proinflammatory cytokines influence the expression of AChR in vitro and in vivo. Because proinflammatory cytokine activity is evidenced in the thymus of myasthenia gravis patients, it could influence AChR expression and thereby contribute to the initiation of the autoimmune anti-AChR response.

Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta.

The juvenile hormones (JHs) regulate a diverse array of insect developmental and reproductive processes. One molecular target of JH action is its transporter, hemolymph JH binding protein (hJHBP); in the larva of the tobacco hornworm, Manduca sexta, low doses of JH can immediately increase hJHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to affect embryonic development and pupation. This study examines the egg form of JHBP and its gene expression during embryogenesis of M. sexta, as well as the phenotypic effect JH treatment has on embryos and on JHBP gene expression. We here demonstrate that the preponderance of JHBP found in the egg is maternally derived and that the embryonic gene and protein appear identical to those found in the larva. Expression of the JHBP gene begins in both the embryo itself and extra-embryonic tissues 15 h after fertilization, long before emergence of a functional fat body and circulatory system. Topical application of low JH doses to early embryos resulted in larval abnormalities while high doses of the hormone induced embryonic mortality. These effects are not mediated through regulation of the JHBP gene, since embryonic expression appears invariant in response to JH challenge. The toxicity of JH is tightly correlated with the concentration of unbound hormone.

A new model linking intrathymic acetylcholine receptor expression and the pathogenesis of myasthenia gravis.

The thymus is thought to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, its role remains a mystery. The studies described represent our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine receptors (nAChRs) is involved in the immunopathogenesis of MG. We review our work characterizing the expression of the alpha subunit of nAChR (nAChRalpha) in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Regulation of MuSK expression by a novel signaling pathway.

MuSK is a receptor tyrosine kinase essential for neuromuscular junction formation. Expression of the MuSK gene is tightly regulated during development and at the neuromuscular junction. However, little is known about molecular mechanisms regulating its gene expression. Here we report a characterization of the promoter of the mouse MuSK gene. The transcription of MuSK starts at multiple sites with a major site 51 nt upstream of the translation start site. We have identified an E-box-like cis-element that is both required and sufficient for differentiation-dependent transcription. Interestingly, the promoter activity of the MuSK gene did not respond to neuregulin, a factor believed to mediate the synapse-specific transcription of acetylcholine receptor subunit genes. Rather, MuSK expression is increased in muscle cells stimulated with Wnt or at conditions when the Wnt signaling was activated. These results suggest a novel mechanism for the MuSK synapse-specific expression.

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter-reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

Autoreactive T cells to the P3A+ isoform of AChR alpha subunit in myasthenia gravis.

In vitro T cell proliferative response to an alternative splicing variant of acetylcholine receptor alpha subunit (AChR alpha) with the P3A exon-encoded region was examined in peripheral blood samples from 28 myasthenia gravis (MG) patients and 14 healthy donors using recombinant fragments and synthetic peptides. T cells responsive to the P3A region-specific sequences were detected in five MG patients, all of whom were late-onset disease with thymoma, but in none of healthy donors. These autoreactive T cells may be involved in the pathogenic process in a subset of MG patients.

Synthesis of acetylcholine by lung cancer.

The role of autocrine growth factors in the stimulation of lung cancer growth is well established. Nicotine is an agonist for acetylcholine receptors and stimulates lung cancer growth. This suggests that if lung cancers synthesize acetylcholine (ACh), then ACh may be an autocrine growth factor for lung cancer. Analysis of normal lung demonstrated that the cells of origin of lung cancers express the proteins necessary for non-neuronal ACh storage and synthesis. Analysis of mRNA from squamous cell lung carcinoma, small cell lung carcinoma (SCLC) and adenocarcinoma showed synthesis of choline acetyltransferase (ChAT) and nicotinic receptors. Immunohistochemical analysis of a retrospective series of SCLC and adenocarcinomas showed that more than 50% of the lung cancers screened expressed ChAT and nicotinic receptors. To study the effect of endogenous ACh synthesis on growth, SCLC cell lines were studied. SCLC cell lines were found to express ChAT mRNA and to secrete ACh into the medium as measured by HPLC separation and enzymatically-coupled electrochemical detection. The SCLC cell line NCI-H82 synthesized highest levels of ACh. Showing that the endogenously synthesized ACh interacted with its receptors to stimulate cell growth, addition of muscarinic and nicotinic antagonists slowed H82 cell proliferation. These findings demonstrate that lung cancer cell lines synthesize and secrete ACh to act as an autocrine growth factor. The existence of a cholinergic autocrine loop in lung cancer provides a basis for understanding the effects of nicotine in cigarette smoke on lung cancer growth and provides a new pathway to investigate for potential therapeutic approaches to lung cancer.

Neuromuscular junctions in cerebral palsy: presence of extrajunctional acetylcholine receptors.

BACKGROUND: Cerebral palsy (CP) is the most prevalent neurologic disease in children. A primary deficit in CP is neuromuscular dysfunction; however, neuromuscular junctions in children with CP have not been studied. Evidence exists that up-regulation of acetylcholine receptors (AChRs) may be present in children with CP, and the current study was undertaken to examine this possibility. METHODS: Thirty-nine children with spastic CP and 25 neurologically normal children were enrolled in the study. Paraspinal muscles underwent biopsy during scheduled spinal fusion surgery. Two sets of assessments were performed on the biopsy specimens: (1) reverse-transcription polymerase chain reaction and Western blotting to evaluate the expression of the gamma subunit of the AChR; and (2) histologic evaluation using a double-stain technique for AChR and acetylcholinesterase, wherein acetylcholinesterase staining defined the limits of the neuromuscular junction, and AChR staining that appeared outside of these limits indicated an abnormal distribution of AChRs. RESULTS: Reverse-transcription polymerase chain reaction and Western blot analyses showed that neither the CP nor non-CP samples had detectable gamma-AChR subunit. Histologic analysis indicated that 11 of 39 children with CP and none of 20 children with idiopathic scoliosis scored positive for the presence of AChR outside of the neuromuscular junction (P = 0.0085). CONCLUSION: A subset of children with CP have an abnormal distribution of AChR relative to the acetylcholinesterase found at the neuromuscular junction. The altered distribution of AChR in CP was not associated with a detectable presence of the gamma-AChR subunit, suggesting that the nonjunctional AChRs in CP does not contain the gamma subunit.

Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle.

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.