Acetylcholine Receptor and Muscle-Specific Kinase Autoantibodies

CHAPTER

Acetylcholine Receptor and Muscle-Specific Kinase Autoantibodies

68 Saif Huda and Angela Vincent

Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK

Historical notes Myasthenia gravis (MG) was first hypothesized to be due to antibodies against a muscle “endplate” protein as early as 1960, but it was not until the use of the snake toxin α-bungarotoxin that the antibodies were demonstrated to be against the acetylcholine receptor (AChR) at the neuromuscular junction (NMJ) of skeletal muscles. This stemmed partly from the loss of AChRs at the endplates of patients’ neuromuscular junctions and the induction of a myasthenia-like disease in rabbits by immunization against AChR [1]. The method employed for measuring the antibodies was a radioimmunoprecipitation assay using 125I-α-BuTx to label the AChRs in detergent-solubilized muscle extracts [2]. At the same time, the pathogenicity of the antibodies was shown by passive transfer to mice and by the striking clinical response that occurred when the patients received plasma exchange [3]. In 2001, antibodies to muscle-specific kinase (MuSK) were identified in a proportion of the remaining 10–20% of patients [4]. Other diseases of the NMJ have been discovered using similar experimental and clinical approaches, specifically the Lambert-Eaton syndrome and acquired neuromyotonia. The historical aspects are reviewed in [5].

The autoantigen Definition The nicotinic AChR is a pentameric postsynaptic membrane ion channel. When its ligand, acetylcholine (ACh), is released from the motor nerve terminal it binds to regions on the two α-subunits of the AChR (see Fig. 68.1). This causes a conformational change that opens the central pore and allows cations to flow inward down their electrochemical gradient into the muscle. This results in an endplate potential, which is usually more than sufficient to surpass the threshold necessary for opening of the sodium channels that lead to the muscle action potential. The extent above which the endplate potential exceeds the threshold potential is referred to as the “safety factor” and varies across species, as well as individual muscles. The loss of AChRs in myasthenia gravis reduces the endplate potential so that it may not reach the threshold; this is the main cause of the defective neuromuscular transmission and the characteristic fatigable muscle weakness. Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00068-X Copyright © 2014 Elsevier B.V. All rights reserved.

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CHAPTER 68  Acetylcholine Receptor and Muscle-Specific Kinase Autoantibodies

Structure/origin/sources The adult AChR consists of five different but homologous subunits: α, β, ε, and δ arranged around the central pore in a 2:1:1:1 stochiometry. In fetal muscle the ε-subunit is replaced with a γ-subunit, and the AChRs are expressed throughout the muscle cell surface (see Fig. 68.1). As innervation proceeds, the AChRs become restricted to the region of the neuromuscular junction. In humans, this occurs at around 33 weeks’ gestation, in contrast to rodents where this process occurs after birth. If denervation occurs subsequently, the γ-subunit is once again expressed along the surface of the muscle fiber, although the high density of AChRs at the NMJ persists. Within the thymic medulla there are a population of muscle-like cells called “myoid cells,” which express fetal AChR. They are most common in the fetus and neonate but are also detectable in the adult thymus. Thymic epithelial cells also express individual AChR subunits. The role of thymic AChR in the etiology of MG is controversial, although it is widely agreed that AChR antibody synthesis takes place partly in the thymic germinal centers at least in younger adult female patients [5].

The autoantibody Methods of detection Routine detection of muscle AChR antibodies is still commonly performed with the radioimmunoprecipitation assay (RIA). Initially this used denervated human muscle, but this contains mainly the fetal AChR isoform, limiting the detection of antibodies that are specific to adult isoforms, which are present at the NMJ. Now it uses a mixture of adult and fetal AChRs extracted from engineered human rhabdomyosarcoma cell lines that express both fetal and adult isoforms. The AChRs are labeled with 125I-αBuTx. The RIA is very specific and sensitive, but radioactivity is not suitable for all centers. For these,

(A)

(B) ε

γ α δ

α β

Developing muscle – fetal AChR

α

α δ

β

Neuromuscular junction – adult AChR

FIGURE 68.1 Diagrammatic representation of the acetylcholine receptor (AChR), viewed from above the membrane, showing the five subunits around the central pore. (A) The fetal form contains a γ-subunit and is expressed throughout the muscle during development (and also in cultured muscle cell lines or after denervation). (B) The adult form contains an ε-subunit instead and is concentrated at the neuromuscular junction, clustered under the motor nerve terminal (not shown). Muscle-specific kinase (MuSK) is also present throughout the muscle during development, when it is important for the clustering of AChRs under the motor nerve.

The autoantibody

577

a fluorescence-immunoprecipitation assay has been described in one report, but the sensitivity and specificity of this assay will require evaluation. A competitive enzyme-linked immunosorbent assay (ELISA) has also been developed, which has much of the sensitivity of the radioimmunoprecipitation assay but has not been extensively validated. Potential problems are that these assays use individual AChRs that are dispersed in solution or on an ELISA plate and may not be entirely in their native conformation. At the NMJ, the AChRs are tightly clustered on the membrane surface, and assays have been developed that rely on binding of antibodies to the surface of cells expressing AChR or MuSK in their native conformation and clustered as they are in vivo [6]. This improves the sensitivity by about 5% but is not yet widely used. The t­ echnique is illustrated in another chapter (see Figure 70.1 of Chapter 70, “Central Nervous System ­Neuronal Surface Antibodies”).

Pathogenic role The pathogenicity of the antibodies was demonstrated early (see above). Plasma exchange, which reduces circulating antibodies, produces marked clinical improvement within a few days; injection of plasma immunoglobulins in mice produces neurophysiological evidence of MG; and AChRs at the neuromuscular junction are inversely correlated with IgG and complement binding to the postsynaptic membrane. In addition, experimental autoimmune MG (EAMG) can be induced in many species by active immunization and exhibits several key features of its human counterpart including serum antibodies to AChR, fatigable weakness, differential involvement of muscle groups, reduction in AChR numbers, and miniature endplate potential amplitudes. There are no spontaneous models of MG in small laboratory animals, but a similar disease associated with AChR antibodies has been reported in dogs and cats. As described above, the passive transfer model using patient immunoglobulin has proven to be an important model despite the limitations of human antibody-animal antigen cross-reactivity. It has helped to clarify pathogenic mechanisms in MG, and similar experiments have now been performed with MuSK antibodies [7].

Factors involved in pathogenicity Three main mechanisms are thought to contribute to antibody pathogenicity. Complement activation results in destruction of the AChR-containing postsynaptic membrane and release of AChR–antibody– complement complexes into the synaptic cleft. Divalent antibody cross-linking of AChRs results in accelerated internalization in a process known as antigenic modulation. Lastly, a small but variable proportion of antibody inhibits ACh binding to the AChRs, causing a pharmacologic blockade of neuromuscular transmission.

Isotypes/subclasses AChR antibodies are typically polyclonal IgG and predominantly of the complement-activating subclasses IgG1 and IgG3. They are of high affinity (around 10–10 M), idiotypically heterogeneous, and variable in antigenic specificity between patients [8]. The antibody response is directed against the AChR in its native configuration. MG sera do not bind in general to peptide epitopes or to AChR subunits after denaturation for Western blotting. In addition, most antibodies induced by denatured AChR subunits do not bind the

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CHAPTER 68  Acetylcholine Receptor and Muscle-Specific Kinase Autoantibodies

intact molecule. However, there is a binding site of antibodies on the two α-subunits called the main immunogenic region (MIR). Monoclonal antibodies to the MIR inhibit a variable but often relatively large proportion of AChR antibodies. Other epitopes include the fetal specific and α-bungarotoxin binding sites.

Clinical utility Disease association Patients with MG can be stratified into groups based on presence or absence of generalized symptoms, age of onset, thymus pathology, and antibody status (Table 68.1). The etiology of the disease in these different subgroups is discussed elsewhere [7]. The prevalence is approximately 100 patients per million population, but the incidence in older patients is increasing, only in part due to heightened awareness of the condition. About 15–25% of patients present with a purely ocular confined muscle fatigability. Approximately 85% of patients with generalized MG will have antibodies to the AChR. About 5–8% will have antibodies to MuSK. Of the remaining patients, approximately 50% will have antibodies to clustered AChR . Thymoma occurrence is relatively uncommon (about 15%) and is almost always associated with antibodies to the AChR as well as striated muscle antigens. The levels of AChR antibodies vary greatly in MG patients and there is no clear correlation with disease severity between individuals, whereas within an individual serial titers usually correlate well with the clinical course of the disease. A reduction of more than 50% of the initial titer can often be associated with a marked clinical improvement. This means that even in clinical remission the antibodies can be positive. Though highly specific for MG, AChR antibodies can be found occasionally in other conditions and are usually associated with an increased risk of developing MG; these include polymyositis, primary biliary cirrhosis, penicillamine-treated rheumatoid arthritis, and thymoma without evidence of MG.

Fetal development and neonatal myasthenia gravis Although now rather rare, there are children born to MG mothers who have a transient neonatal form of the disease, presenting with poor sucking and generalized hypotonia within the first 3 days. The condition responds well to anticholinesterase inhibitors and generally remits within 1 to 2 months as the antibody levels decrease. Rarer still, a form of arthrogryposis multiplex congenital (AMC) may occur. Restriction of movement in utero results in joint contractures and other abnormalities [8]. The condition has been reported in successive pregnancies of asymptomatic mothers, apparently caused by antibodies that bind selectively to the fetal form of the AChR. In 179 mothers with children with AMC, maternal antibodies to AChR or MuSK were found in 10 patients. Three of the mothers with AChR antibodies and two with MuSK antibodies were asymptomatic [8]; thus lack of maternal weakness should not prevent testing the mother’s serum for AChR antibodies, which are often relatively specific for the fetal receptor but can be detected using the conventional RIA, which contains both forms (see above). Intense immunotherapy early during the second trimester may prevent AMC in subsequent pregnancies.

Muscle-specific kinase MuSK is a 110-kDa membrane protein that has an integral role in both fetal muscle during development and the mature neuromuscular junction. MuSK is a receptor tyrosine kinase with a large

Low density lipoprotein receptor-related protein 4

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Table 68.1  Different Forms of Myasthenia Gravis

Subtype

Proportion

Age of Onset (years)

Ocular MG Earlyonset MG

15–25%

15–25

3:2

20–25%

< 41

1:3

Lateonset MG

30–40%

> 40

1.5:1

MuSK MG 5–8%

22–52

1:3

SNMG

5–10

1:2

5–10%

Sex M:F

Clinical Features Ptosis, ­opthalmoplegia Ptosis, opthalmoplegia, generalized weakness Ptosis, opthalmoplegia, generalized weakness Oculo-fasciobulbar weakness Ptosis, opthalmoplegia, generalized weakness

AChR Abx

MuSK Abx

Thymic Abnormality

≈ 50%

Rare

≈ 85%

Absent

Mild hyperplasia (30%) Hyperplastic (> 80%)

≈ 60%

Absent

Normal or atrophied

Absent

100%

Normal or hyperplasia Mild hyperplasia

≈ 50% clustered Absent -AChR ≈ 2–50% -LRP4

AChR Abx: acetylcholine receptor antibodies; MG: myasthenia gravis; MuSK Abx: Muscle-specific kinase antibodies, SNMG: seronegative myasthenia gravis, LRP4: low density lipoprotein receptor-related protein 4.

extracellular domain, a short transmembrane, and typical intracellular kinase domain. During development, a growth factor agrin is released from the nerve and binds to a coreceptor called LRP4 (low density lipoprotein receptor-related protein 4), which then interacts with MuSK. Signaling to the inside of the cell by MuSK results in clustering of the AChRs and formation of the NMJ. MuSK is found mainly at the NMJ in mature muscle. The protein can be expressed, purified, and radiolabeled for use in immunoprecipitation assays [4]. A cell-based method, however, is more sensitive. MuSK antibodies have been shown to be pathogenic by passive transfer and active immunization experiments [9]. They are mainly IgG4 in humans and may block the binding of LRP4 to MuSK, thereby inhibiting the clustering mechanism and leading to loss of AChRs [6]. There is strong geographical variation, with prevalence being less than 5% in some countries and as high as 40% in others. Patients are typically but not exclusively female and under 30 years of age. The disease has preponderance for the fascio-bulbar and respiratory muscles. Relapses are often severe when they involve respiratory failure. Facial and tongue muscle atrophy may also occur. In contrast to AChR-MG, thymus pathology is uncommon, and as such the role for thymectomy is less clear [8]. Hypersensitivity to cholinesterase inhibitors can be seen, and often steroids and additional immunosuppression are required.

Low density lipoprotein receptor-related protein 4 The identity of LRP4 as the functional muscle receptor for agrin is a relatively recent discovery. Up until this point, it was generically referred to as myotube-associated specificity component (MASC). Like MuSK it has an intracellular, transmembrane, and extracellular domain, the former of which is dispensable to functions subsequently described. LRP4 has bidirectional functionality. Postsynpatically it forms a complex with MuSK and mediates agrin-stimulated MuSK activation, which is important for clustering of AChR. It is also acts as a

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CHAPTER 68  Acetylcholine Receptor and Muscle-Specific Kinase Autoantibodies

retrograde signal in the early steps of presynaptic differentiation at neuromuscular synapses. Early neonatal lethality is seen in mice with LRP4 mutations, with severe neuromuscular synapse formation defects. Variable and generally low numbers of MG patients without detectable antibodies to MuSK or AChR have recently been shown to have antibodies to LRP4 by both cell-based and ELISA methods (2–50%). It is likely that the antibodies inhibit the LRP4–agrin interaction [11]. Our knowledge of the clinical phenotype, given the antigen’s recent discovery, is still evolving.

Take-home messages • T  he NMJ and AChR antibodies that result in MG represent the paradigm of autoimmune disease. • Antibodies to postsynaptic receptors such as AChR and MuSK result in defective neuromuscular transmission leading to the clinical hallmark of MG – fatigable muscle weakness • Methods of detecting antibodies to these and other antigens are evolving, improving our ­pathophysiologic characterization of this disease.  

References [1]  Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science 1973;180:871–2. [2]  Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD. Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates and diagnostic value. Neurology 1976;26:1054–9. [3]  Tokya KV, Drachman DB, Pestronk A, Kao I. Myasthenia gravis: passive transfer from man to mouse. ­Science 1975;190:397–9. [4]  Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A. Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat Med 2001;7:365–8. [5]  Vincent A. Unravelling the pathogenesis of myasthenia gravis. Nat Rev Immunol 2002;2:797–804. [6]  Leite MI, Jacob S, Viegas S, Cossins J, Clover L, Morgan PB, et al. IgG1 antibodies to acetylcholine receptors in ‘seronegative’ myasthenia gravis. Brain 2008;131:1940–52. [7]  Vincent A, Wilcox N, Hill M, Curnow J, Maclennan C, Beeson D. Determinant spreading and immune responses to acetylcholine receptors in myasthenia gravis. Immunol Rev 1998;164:157–68. [8]  Dalton P, Clover L, Wallerstein R, Stewart H, Genzel-Boroviczeny O, Dean A, et al. Fetal arthrogryposis and maternal serum antibodies. Neuromuscul Disord 2006;16:481–91. [9]  Klooster R, Plomp JJ, Huijbers MG, Niks EH, Straasheijm KR, Detmers FJ, et al. Muscle-specific kinase myasthenia gravis IgG4 autoantibodies cause severe neuromuscular junction dysfunction in mice. Brain 2012;135:1081–101. [10] Leite MI, Strobel P, Jones M, Micklem K, Moritz R, Gold R, et al. Fewer thymic changes in MuSK antibodypositive than in MuSK antibody negative MG. Ann Neurol 2005;57:444–8. [11] Zhang B, Tzartos JS, Belimezi M, Ragheb S, Bealmear B, Lewis RA, et al. Autoantibodies to lipoproteinrelated protein 4 in patients with double seronegative myasthenia gravis. Arch Neurol 2012;69:445–51.