Characterization of ganglionic acetylcholine receptor autoantibodies

In myasthenia gravis (MG), autoantibodies bind to the alpha1 subunit and other subunits of the muscle nicotinic acetylcholine receptor (AChR). Autoimmune autonomic ganglionopathy (AAG) is an antibody-mediated neurological disorder caused by antibodies against neuronal AChRs in autonomic ganglia. Subunits of muscle and neuronal AChR are homologous. We examined the specificity of AChR antibodies in patients with MG and AAG. Ganglionic AChR autoantibodies found in AAG patients are specific for AChRs containing the alpha3 subunit. Muscle and ganglionic AChR antibody specificities are distinct. Antibody crossreactivity between AChRs with different alpha subunits is uncommon but can occur.     

Invited Article: Autonomic ganglia: target and novel therapeutic tool

Nicotinic acetylcholine receptors (AChR) are ligand-gated cation channels that are present throughout the nervous system. The muscle AChR mediates transmission at the neuromuscular junction; antibodies against the muscle AChR are the cause of myasthenia gravis. The ganglionic (alpha 3-type) neuronal AChR mediates fast synaptic transmission in sympathetic, parasympathetic, and enteric autonomic ganglia. Impaired cholinergic ganglionic synaptic transmission is one important cause of autonomic failure. Pharmacologic enhancement of ganglionic synaptic transmission may be a novel way to improve autonomic function. Ganglionic AChR antibodies are found in patients with autoimmune autonomic ganglionopathy (AAG). Patients with AAG typically present with rapid onset of severe autonomic failure. Major clinical features include orthostatic hypotension, gastrointestinal dysmotility, anhidrosis, bladder dysfunction, and sicca symptoms. Impaired pupillary light reflex is often seen. Like myasthenia, AAG is an antibody-mediated neurologic disorder. The disease can be reproduced in experimental animals by active immunization or passive antibody transfer. The patient may improve with plasma exchange treatment or other immunomodulatory treatment. Antibodies from patients with AAG inhibit ganglionic AChR currents. Other phenotypes of AAG are now recognized based on the results of antibody testing. These other presentations are generally associated with lower levels of ganglionic AChR antibodies. A chronic progressive form of AAG may resemble pure autonomic failure. Milder forms of dysautonomia, such as postural tachycardia syndrome, are associated with ganglionic AChR in 10-15% of cases. Since ganglionic synaptic transmission is a common pathway for all autonomic traffic, enhancement of autonomic function through inhibition of acetylcholinesterase is a potential specific therapeutic strategy for autonomic disorders. Increasing the strength of ganglionic transmission can ameliorate neurogenic orthostatic hypotension without aggravating supine hypertension. Recent evidence also suggests a potential role for acetylcholinesterase inhibitors in the treatment of postural tachycardia syndrome.     

Antibodies against neuronal nicotinic receptor subtypes in neurological disorders

Patients with myasthenia gravis (MG) have antibodies to the muscle nicotinic acetylcholine receptor (mAChR) which are responsible for their muscle weakness: but some patients with MG and other neuroimmunological disorders have autonomic symptoms. We characterised the neuronal forms of AChRs (nAChRs) into two neuroblastoma cell lines and developed immunoprecipitation assays to test for antibodies to the alpha7- and alpha3-containing nAChR subtypes, present in the autonomic ganglia. We then tested 70 sera samples from MG patients, 38 from subjects with other neurological diseases, and 30 from healthy individuals, for antibodies to these two forms of neuronal AChR subtypes. We used the alpha7 subtype extracted from the human neuroblastoma IMR32 cell line labeled with 125IalphaBungarotoxin (alphaBgtx), and the alpha3-containing subtype extracted from the human neuroblastoma SY5Y cell line labeled with 3H-Epibatidine (Epi). Nine subjects (five MG, one GBS, one CIPD and two LEMS) were positive for the alpha7 subtype; and four for the alpha3-containing subtype (two MG patients, one LEMS and the same GBS patient). None of the MG patients with undetectable levels of antibodies against muscle AChR were positive. The patients with serum antibodies to alpha7 or alpha3-containing neuronal AChRs showed a range of clinical features including autonomic symptoms and thymoma in two MG patients. These results indicate that patients with MG and other immune-mediated disorders can have antibodies to neuronal AChRs, and that these may contribute to the clinical characteristics of the diseases.     

Myasthenia, thymoma, presynaptic antibodies, and a continuum of neuromuscular hyperexcitability.

BACKGROUND: Autoantibodies specific for the nicotinic acetylcholine receptor (AChR) of skeletal muscle impair neuromuscular transmission in myasthenia gravis (MG). Autoantibodies specific for alpha3 neuronal AChRs or voltage-gated potassium channels have been reported in patients with Isaacs syndrome, an acquired disorder of continuous muscle fiber activity characterized by neuromyotonia. OBJECTIVE: To report the neuromuscular autoantibody profiles of three patients with a syndrome of MG and neuromuscular hyperexcitability. RESULTS: All three patients reported here had clinical and electrophysiologic evidence of MG and neuromuscular hyperexcitability. None had neuromyotonia. Thymoma was proven in two patients and suspected in the third. One had MG and thymoma and subsequently developed cramp-fasciculation syndrome; MG and rippling muscle syndrome appeared simultaneously in the other two. All patients had muscle and neuronal AChR binding antibodies and striational antibodies. Only one had antibodies reactive with alpha-dendrotoxin-complexed potassium channels. CONCLUSIONS: The coexistence of cramp-fasciculation syndrome and acquired rippling muscle syndrome with MG, thymoma, and neuronal AChR autoantibodies suggests that there is a continuum of autoimmune neuromuscular hyperexcitability disorders related pathogenically to Isaacs syndrome. Manifestations of neuromuscular hyperexcitability may be altered and less apparent in the context of MG because of the coexisting defect of neuromuscular transmission.     

Neuromuscular junction autoimmune disease: muscle specific kinase antibodies and treatments for myasthenia gravis

PURPOSE OF REVIEW: Some of the 20% of myasthenia gravis patients who do not have antibodies to acetylcholine receptors (AChRs) have antibodies to muscle specific kinase (MuSK), but a full understanding of their frequency, the associated clinical phenotype and the mechanisms of action of the antibodies has not yet been achieved. Moreover, some patients do not respond well to conventional corticosteroid therapy. Here we review recent clinical and experimental studies on MuSK antibody associated myasthenia gravis, and summarize the results of newer treatments for myasthenia gravis. RECENT FINDINGS: MuSK antibodies are found in a variable proportion of AChR antibody negative myasthenia gravis patients who are often, but not exclusively, young adult females, with bulbar, neck, or respiratory muscle weakness. The thymus histology is normal or only very mildly abnormal. Surprisingly, limb or intercostal muscle biopsies exhibit no reduction in AChR numbers or complement deposition. However, patients without AChR or MuSK antibodies appear to be similar to those with AChR antibodies and may have low-affinity AChR antibodies. A variety of treatments, often intended to enable corticosteroid doses to be reduced, have been used in all types of myasthenia gravis with some success, but they have not been subjected to randomized clinical trials. SUMMARY: MuSK antibodies define a form of myasthenia gravis that can be difficult to diagnose, can be life threatening and may require additional treatments. An improved AChR antibody assay may be helpful in patients without AChR or MuSK antibodies. Clinical trials of drugs in other neuroimmunological diseases may help to guide the treatment of myasthenia gravis.     

Autonomic ganglia, acetylcholine receptor antibodies, and autoimmune ganglionopathy

Nicotinic acetylcholine receptors (AChR) are ligand-gated cation channels that are present throughout the nervous system. The ganglionic (alpha3-type) neuronal AChR mediates fast synaptic transmission in sympathetic, parasympathetic and enteric autonomic ganglia. Autonomic ganglia are an important site of neural integration and regulation of autonomic reflexes. Impaired cholinergic ganglionic synaptic transmission is one important cause of autonomic failure. Ganglionic AChR antibodies are found in many patients with autoimmune autonomic ganglionopathy (AAG). These antibodies recognize the alpha3 subunit of the ganglionic AChR, and thus do not bind non-specifically to other nicotinic AChR. Patients with high levels of ganglionic AChR antibodies typically present with rapid onset of severe autonomic failure, with orthostatic hypotension, gastrointestinal dysmotility, anhidrosis, bladder dysfunction and sicca symptoms. Impaired pupillary light reflex is often seen. Like myasthenia gravis, AAG is an antibody-mediated neurological disorder. Antibodies from patients with AAG inhibit ganglionic AChR currents and impair transmission in autonomic ganglia. An animal model of AAG in the rabbit recapitulates the important clinical features of the human disease and provides additional evidence that AAG is an antibody-mediated disorder caused by impairment of synaptic transmission in autonomic ganglia.     

Acetylcholine receptors and myasthenia

Much progress has been made in the 26 years since initial studies of the first purified acetylcholine receptors (AChRs) led to the discovery that an antibody-mediated autoimmune response to AChRs causes the muscular weakness and fatigability characteristic of myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG). Now, the structure of muscle AChRs is much better known. Monoclonal antibodies to muscle AChRs, developed as model autoantibodies for studies of EAMG, were used for initial purifications of neuronal AChRs, and now many homologous subunits of neuronal nicotinic AChRs have been cloned. There is a basic understanding of the pathological mechanisms by which autoantibodies to AChRs impair neuromuscular transmission. Immunodiagnostic assays for MG are used routinely. Nonspecific approaches to immunosuppressive therapy have been refined. However, fundamental mysteries remain regarding what initiates and sustains the autoimmune response to muscle AChRs and how to specifically suppress this autoimmune response using a practical therapy. Many rare congenital myasthenic syndromes have been elegantly shown to result from mutations in muscle AChRs. These studies have provided insights into AChR structure and function as well as into the pathological mechanisms of these diseases. Evidence has been found for autoimmune responses even to some central nervous system neurotransmitter receptors, but only one neuronal AChR has so far been implicated in an autoimmune disease. Thus far, only two neuronal AChR mutations have been found to be associated with a rare form of epilepsy, but many more neuronal AChR mutations will probably be found to be associated with disease in the years ahead.     

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.     

Inhibition of acetylcholine receptor function by seronegative myasthenia gravis non-IgG factor correlates with desensitisation

15% of myasthenia gravis (MG) patients do not have antibodies against the acetylcholine receptor (AChR). Some of these "seronegative" MG patients have antibodies against muscle specific kinase (MuSK), and many have a non-IgG factor that acutely inhibits AChR function in a muscle-like cell line, CN21. Here we show, using mainly one plasma negative for both AChR and MuSK antibodies, that the inhibitory effect of the non-IgG fraction correlates well with the desensitisation caused by 100 microM nicotine, and is found also when AChRs are expressed in a non-muscle cell line (HEK). Moreover, a similar effect was seen with M3C7-a monoclonal antibody against human AChR. The results suggest that, rather than acting indirectly as previously proposed, the SNMG factor may bind directly to an allosteric site that induces or enhances AChR desensitisation.     

Myasthenia gravis, thymoma, intestinal pseudo-obstruction, and neuronal nicotinic acetylcholine receptor antibody.

Intestinal pseudo-obstruction occurs rarely in patients with myasthenia gravis (MG) and thymoma. The etiology of the intestinal pseudo-obstruction remains to be elucidated, although an autoimmune mechanism is postulated. We present the first report of neuronal nicotinic acetylcholine receptor (AChR)-specific antibody in a patient with seropositive MG, malignant thymoma, and intestinal pseudo-obstruction. This finding provides evidence that intestinal pseudo-obstruction associated with thymoma and possibly other neoplasms may be related to antibodies against the neuronal nicotinic receptors at autonomic ganglia.     

Acetylcholine receptors in human thymic myoid cells in situ: an immunohistological study

Myoid cells were studied by double immunofluorescence in sections of thymus from 47 patients with myasthenia gravis and 15 control subjects, using polyclonal sheep anti-troponin T and monoclonal antibodies to troponin I, striated muscle myosin, and acetylcholine receptor (AChR). The myoid cells were rare and located mainly in the medulla, and most were clearly positive for AChR; labeling was similar with four individual monoclonal antibodies specific for extrajunctional AChR and five that also recognize endplate AChR. They were mostly keratin-positive and consistently HLA-DR-negative. In the myasthenia gravis samples, the myoid cells were similar but largely confined to medullary epithelial areas; AChR labeling was slightly weaker, but otherwise they did not differ noticeably from those of control subjects. A preliminary finding was of even rarer AChR-positive/HLA-DR-positive antigen-presenting (possibly) cells seen in 9 of 9 myasthenia gravis samples and in none of 9 control samples. Although myoid-cell AChR appears antigenically similar to extrajunctional muscle AChR, and must therefore express the epitopes that myasthenics' antibodies recognize, these cells do not appear to be foci of immunological stimulation in myasthenia gravis.     

Interaction of myasthenic immunoglobulins and cholinergic agonists on acetylcholine receptors of rat myotubes

Acetylcholine receptor (AChR) antibodies from patients with myasthenia gravis (MG) impair neuromuscular transmission by altering the number of AChRs at the skeletal muscle motor endplate. Cholinergic agonists similarly impair transmission by altering the number and affinity of AChRs. The frequent clinical resistance ot anticholinesterase medication in patients with MG has raised the question of possible synergistic effects of these agents on regulation of AChRs and neuromuscular transmission. To investigate this question, rat myotube cultures were incubated with MG globulin and carbamylcholine (CMC) for 20 hours. The number of AChRs was assayed by 125I-alpha-bungarotoxin binding. Incubation of cultures with both MG globulins and CMC consistently produced greater reductions of AChRs than incubation with either substance alone. Cholinergic antagonists blocked the CMC effect but not the MG globulin effect. A muscarinic antagonist had no effect. The effects of short-term incubation with these substances on the affinity of AChR were assessed by the rate of 125I-alpha-bungarotoxin-AChR complex formation. CMC desensitized AChRs, but MG globulins did not alter the affinity of AChRs. Results with this in vitro model suggest that chronic anticholinesterase therapy in the presence of MG AChR antibodies may aggravate failure of neuromuscular transmission in MG.     

Titin and ryanodine receptor epitopes are expressed in cortical thymoma along with costimulatory molecules

Cortical-type thymomas are associated with myasthenia gravis (MG) in 50% of the cases. MG is caused by antibodies against the acetylcholine receptors (AChR), but additional non-AChR muscle autoantibodies such as those against titin and ryanodine receptor (RyR) are found in up to 95% of MG patients with thymoma. To elucidate the induction of non-AChR autoantibodies in thymoma-associated MG, we studied cortical-type thymomas from seven thymoma MG patients, and sera from six of them. All six had titin antibodies, and four had RyR antibodies. Titin and RyR epitopes were co-expressed along with LFA3 and B7 (BB1) costimulatory molecules on thymoma antigen-presenting cells (APC) in all thymomas. In normal thymus, the staining by anti-titin, anti-RyR, anti-LFA3, and anti-BB1 antibodies was weak and occurred exclusively in the medulla and perivascularly. Our results indicate a primary autosensitization against titin and RyR antigens inside the thymoma. In MG-associated thymoma, the mechanisms involved in the initial autosensitization against titin and RyR are probably similar to those implicated in the autosensitization against AChR. In all cases, there is an overexpression of muscle-like epitopes and costimulatory molecules indicating that the T-cell autoimmunization is actively promoted by the pathogenic microenvironment inside the thymoma.     

Ion channel and striational antibodies define a continuum of autoimmune neuromuscular hyperexcitability

Neuromuscular hyperexcitability is a characteristic of Isaacs' syndrome. Autoantibodies specific for voltage-gated potassium channels (VGKC) or ganglionic nicotinic acetylcholine receptors (AChR) are markers of this disorder. To determine the frequency of these ion channel antibodies and of related neuron- and muscle-specific antibodies in patients with acquired neuromuscular hyperexcitability, we tested serum specimens from 77 affected patients (35 neuromyotonia, 32 cramp-fasciculation syndrome, 5 rippling muscle syndrome, and 5 focal neuromuscular hyperexcitability) and 85 control subjects. Among study patients, 14% had coexisting myasthenia gravis, and 16% had an associated neoplasm. We found that 35% had VGKC antibodies, 12% ganglionic AChR antibodies, 16% muscle AChR antibodies, and 10% striational antibodies. Overall, 55% had serological evidence of neurological autoimmunity compared to 2% of control subjects. Patients with neuromyotonia were more frequently seropositive (71%) than patients with cramp-fasciculation syndrome (31%). We conclude that acquired neuromuscular hyperexcitability consists of a continuum of clinical disorders with a common autoimmune pathogenesis.     

Seropositive myasthenia and autoimmune autonomic ganglionopathy: cross reactivity or subclinical disease?

Autoimmune autonomic ganglionopathy (AAG) and myasthenia gravis (MG) are both autoimmune channelopathies mediated by antibodies directed against nicotinic acetylcholine receptors. While both diseases target acetylcholine receptors, skeletal muscle and ganglionic receptor subtypes have key immunologic and genetic distinctions, and reports of patients with both AAG and MG are rare. We report a patient with antibody-confirmed AAG and elevated levels of ACh binding antibodies that did not meet clinical or electrodiagnostic criteria for MG. We presume that his skeletal muscle nAChR seropositivity was a false positive, perhaps due to the cross reactivity of the patient's ganglionic nAChR antibodies with skeletal nAChR subtypes.     

Idiopathic autonomic neuropathy: comparison of cases seropositive and seronegative for ganglionic acetylcholine receptor antibody

BACKGROUND: The clinical characteristics of autoimmune autonomic neuropathy are only partially defined. More than 50% of patients with high levels of ganglionic acetylcholine receptor (AChR) autoantibodies have a combination of sicca complex (marked dry eyes and dry mouth), abnormal pupillary light response, upper gastrointestinal tract symptoms, and neurogenic bladder. OBJECTIVE: To compare patients with idiopathic autonomic neuropathy who were seropositive (n = 19) and seronegative (n = 87) for ganglionic AChR antibodies. DESIGN: Retrospective review of autonomic programmatic database. SETTING: Autonomic Disorders Program Project at Mayo Clinic College of Medicine, Rochester, Minn. PATIENTS: We evaluated a cohort of 87 patients with idiopathic autonomic neuropathy who had undergone full autonomic testing and neurological evaluation and who had a complete panel of paraneoplastic and ganglionic AChR antibodies. We compared patients seropositive (n = 19) and seronegative (n = 87) for ganglionic AChR antibodies. RESULTS: The seropositive group had a significant overrepresentation of abnormal pupillary responses (12/18 [67%] vs 12/87 [14%]; P<.001), sicca complex (9/15 [60%] vs 11/47 [23%]; P =.01), and lower gastrointestinal tract dysautonomia (16/19 [84%] vs 48/85 [56%]; P =.02). A subacute mode of onset was more common in the seropositive group (12/19 [63%] vs 23/84 [27%]; P =.004). Results of quantitative autonomic function tests differed significantly in the 2 groups only in the cardiovagal domain. Because subacute onset was overrepresented in the seropositive group, we analyzed the data separately, controlling for temporal profile (ie, the relationship between antibody status and symptoms while controlling for rate of onset). The relationships between antibody status and clinical profile (eg, presence of sicca complex, pupillary abnormalities, and lower gastrointestinal tract symptoms) generally remained significant regardless of onset rate, indicating that the associations are not due to temporal profile. CONCLUSIONS: These observations support the concept that ganglionic AChR antibodies are diagnostically and pathophysiologically important. Patients with orthostatic hypotension and prominent cholinergic dysautonomia are most likely to be seropositive for ganglionic AChR antibody.     

Acetylcholine receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis

Muscle-specific tyrosine kinase (MuSK) antibodies are found in some patients with "seronegative" myasthenia gravis (MG), but how they cause myasthenic symptoms is not clear. We visualized acetylcholine receptors (AChRs) and complement component 3 (C3) in muscle biopsies from 10 Japanese MG patients with MuSK antibodies, compared with 42 with AChR antibodies. The AChR density was not significantly decreased in MuSK antibody (Ab)-positive end-plates compared with AChR antibody-positive end-plates, and C3 was detected in only two of eight MuSK Ab-positive patients. MuSK antibodies do not appear to cause substantial AChR loss, complement deposition, or morphological damage. Effects on MuSK function need to be explored.     

Anti-LRP4 autoantibodies in AChR- and MuSK-antibody-negative myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder characterized by a defect in synaptic transmission at the neuromuscular junction causing fluctuating muscle weakness with a decremental response to repetitive nerve stimulation or altered jitter in single-fiber electromyography (EMG). Approximately 80% of all myasthenia gravis patients have autoantibodies against the nicotinic acetylcholine receptor in their serum. Autoantibodies against the tyrosine kinase muscle-specific kinase (MuSK) are responsible for 5–10% of all myasthenia gravis cases. The autoimmune target in the remaining cases is unknown. Recently, low-density lipoprotein receptor-related protein 4 (LRP4) has been identified as the agrin receptor. LRP4 interacts with agrin, and the binding of agrin activates MuSK, which leads to the formation of most if not all postsynaptic specializations, including aggregates containing acetylcholine receptors (AChRs) in the junctional plasma membrane. In the present study we tested if autoantibodies against LRP4 are detectable in patients with myasthenia gravis. To this end we analyzed 13 sera from patients with generalized myasthenia gravis but without antibodies against AChR or MuSK. The results showed that 12 out of 13 antisera from double-seronegative MG patients bound to proteins concentrated at the neuromuscular junction of adult mouse skeletal muscle and that approximately 50% of the tested sera specifically bound to HEK293 cells transfected with human LRP4. Moreover, 4 out of these 13 sera inhibited agrin-induced aggregation of AChRs in cultured myotubes by more than 50%, suggesting a pathogenic role regarding the dysfunction of the neuromuscular endplate. These results indicate that LRP4 is a novel target for autoantibodies and is a diagnostic marker in seronegative MG patients.     

T cells in myasthenia gravis specific for embryonic acetylcholine receptor.

In myasthenia gravis (MG) there is an autoimmune response against muscle acetylcholine receptor (AChR). Embryonic and adult muscles express different AChRs; embryonic AChR contains a gamma subunit, instead of the homologous epsilon subunit that contributes to form adult AChR. We report propagation from the blood of MG patients of T helper (TH) cell lines specific for human embryonic AChR, by cycles of stimulation with a pool of synthetic peptides corresponding to the complete sequence of the gamma subunit (gamma pool). The TH lines strongly recognized AChR from embryonic mammalian muscle, and reacted less or not at all with adult muscle AChR. The existence of TH cells specific for embryonic AChR strongly suggests that the primary anti-AChR sensitization in MG occurs in a tissue other than the innervated skeletal muscle. This may be within the thymus, which expresses an AChR similar or identical to embryonic muscle AChR.     

Morphologic and immunologic studies in experimental autoimmune myasthenia gravis and myasthenia gravis.

An indirect immunoperoxidase technique was used to study by light microscopy the binding of serum from experimental autoimmune myasthenia gravis (EAMG) rabbits to junctionally and extrajunctionally located acetylcholine receptors (AChRs) in human and rat muscles. Binding was restricted to junctional AChR. Alpha bungarotoxin (a-BGT) partially blocked the binding of EAMG serum, while myasthenia gravis serum, carbamylcholine, decamethonium, and tubocurarine did not. A radioimmunoassay showed significant binding of antibodies in EAMG sera to 125l AChR. This binding was not inhibited by a-BGT, nor by carbamylcholine, decamethonium, or tubocurarine. Sera from 10 myasthenia gravis patients did not contain antibodies binding to the 125l AChR. We suggest that EAMG in rabbits induced by Torpedo AChR differs serologically from myasthenia gravis in patients, probably owing to antigenic differences between Torpedo and human AChR, and that antigenic differences also exist between junctional and extrajunctional receptors.