Socrates J. Tzartos

Production and assay of antibodies to acetylcholine receptors.

Publisher Summary This chapter describes the production and assay of antibodies to acetylcholine receptors. All the basic methods necessary to assay and purify acetylcholine receptors (AChRs) and AChR subunits for use as immunogens to produce antisera and monoclonal antibodies (mAbs) to AChRs and to induce experimental autoimmune myasthenia gravis are discussed in the chapter. AChR is routinely identified and quantitated, by the binding of radioactively labeled krait or cobra venom toxins, which are competitive inhibitors of acetylcholine binding. An easy approach to study the specificity of antibodies to intact AChR is to test their reaction, with 123 I-toxin-labeled AChR, from various species by radioimmunoassay. The competitive binding to native AChR method uses mAbs to AChR that have been mapped, by their ability to react, with denatured subunits to help map the subunit specificity of antibodies, that react only, with native AChR, and to distinguish between mAbs to various antigenic determinants on the same subunit. It is demonstrated that the antigenicity of the native AChR molecule is dominated, by a small region, on the α-subunit.

Muscle and neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand‐gated ion‐channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (α1–10, β1–4, γ, δ, and ε). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non‐neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimers and Parkinsons diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high‐resolution X‐ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high‐resolution X‐ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype‐specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.

α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer's Disease

Abstract : Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimers disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age‐matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for α3, α4, and α7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to α3, α4, and α7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and α4‐like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the α4 subunit) were reduced in AD cases compared with control subjects (p <0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p <0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic α4‐like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p <0.05). There were no significant changes in α3‐ or α7‐like immunoreactivity associated with AD or tobacco use. The selective involvement of α4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.

Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases

Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimers disease or Parkinsons disease are more selective. In Alzheimers disease, in the cortex there is a selective loss of the alpha4 (but not alpha3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not alpha-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Abeta1-42, but not related to plaque or tangle densities. In contrast, alpha-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinsons disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of alpha4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased alpha4 immunoreactivity in the cortex and lower densities of amyloid-beta plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the alpha4 subunit in beta-amyloidosis but not in nigro-striatal dopaminergic degeneration.

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor

Summary: The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti‐AChR antibody response is polyclonal. However, a small extracellular region of the AChR a‐subunit, the main immunogenic region (MIR), seems to be a major target for anti‐AChR antibodies. A major loop containing overlapping epitopes for several anti‐MIR monoclonal antibodies (mAbs) lies within residues α67–76 at the extreme synaptic end of each a‐subunit; however, anti‐MIR mAbs are functionally and structurally quite heterogeneous. Anti‐MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplas‐mic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine‐binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti‐AChR antibodies cross‐reactive with noti‐AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR, especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in‐depth understanding, and for possible specific immunotherapy, of MG.

Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor

Experimental autoimmune myasthenia gravis (EAMG) was passively transferred to rats by injecting monoclonal antibodies (mAbs) directed at the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR). The MIR is located on the extracellular part of the AChR alpha-subunit. All four mAbs directed at the MIR which were tested were very efficient in inducing EAMG: within 2 days the rats became moribund or very weak and their muscle AChR content decreased to about 50% of normal. These mAbs are of two different IgG subclasses (IgG1 and IgG2a) and derived from rats immunized with AChR from either fish electric organs or mammalian muscles. One mAb directed at the extracellular side of the beta-subunit did not cause AChR loss or induce symptoms of EAMG. mAbs to the cytoplasmic side were, as expected, ineffective.

Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity.

Seventy-five monoclonal antibodies (mAbs) to sodium dodecyl sulfate-denatured Torpedo californica (66 mAbs) and intact fetal calf (9 mAbs) acetylcholine receptor (AChR) were produced. These mAbs were characterized for subunit, region and species specificity, for Ig class and subclass, for protein A binding and for antigen-crosslinking capacity. Fourteen were identified as anti-alpha, 35 were anti-beta, 8 were anti-gamma and 15 were anti-delta. None of the 11 anti-alpha derived from denatured AChR bound to the main immunogenic region (MIR) as judged by antibody competition assays. This contrasts with previous results using mAbs against native AChr, the majority of which bind to the MIR. Thirty-eight mAbs crossreacted with some or all of the tested AChRs from fish electric organs and mammalian muscles in addition to the immunogen. Eight anti-alpha, anti-beta and 1 anti-delta mAbs showed good to excellent autoantibody activity. Analysis by sucrose gradient centrifugation of some AChR-mAb complexes revealed that some mAbs form intermolecular and others form intramolecular crosslinkings of the AChR. The described mAbs have proven valuable tools in AChR and myasthenia gravis research.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

Epidemiology of seropositive myasthenia gravis in Greece

OBJECTIVES To study the epidemiological characteristics of myasthenia gravis in Greece. METHODS A population based study was carried out of seropositive myasthenia gravis in Greece for the period from 1 January 1983 to 30 June 1997; 843 patients were studied. RESULTS The average annual incidence for the period 1992–7, for which the database is complete, was 7.40/million population/year (women 7.14; men 7.66). On 1 July 1997, there were 740 prevalent cases. The point prevalence rate was 70.63/million (women 81.58; men 59.39). The average overall annual mortality rate in the patients was 0.67/million population (women 0.53; men 0.82), and the mortality rate attributed to myasthenia gravis was 0.43/million population (women 0.41; men 0.45). The average age at onset was 46.50 years (women 40.16; men 54.46), and the mean age of the prevalent patients was 52.58 (women 47.65; men 59.48). The women:men incidence ratio was 1:1.04, and the prevalence ratio was 1.41:1. It is predicted that the prevalence and women: men prevalence ratio would increase if the patient list included all patients with a date of onset before 1983. CONCLUSIONS The largest epidemiological study ever performed on myasthenia gravis is presented. The most important epidemiological indexes are provided.

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-γ, and even more so by IFN-γ and TNF-α, as assessed by real-time RT-PCR, with the α-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-γ could influence AChR expression in vivo, we analyzed AChR transcripts in IFN-γ 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-α. 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.