Ryanodine Receptor Autoantibodies in Myasthenia Gravis Patients with a Thymoma Ase Mygland, MD,' Ole-Bjarn Tysnes, MD,' Roald Matre, MD,t Pompeo Volpe, MD,S Johan A. Aarli, MD,' and Nils Erik Gilhus, MD'

Sera from patients with myasthenia gravis were examined by Western blot for the presence of antibodies to proteins of the sarcoplasmic reticulum from rabbit skeletal muscle. Fourteen of 30 patients with myasthenia gravis and a thymoma had IgG autoantibodies to the calcium release channel of the sarcoplasmic reticulum (the ryanodine receptor), which plays a crucial role in the mechanism of excitation-contraction coupling in striated muscle. Ryanodine receptor autoantibodies were not detected in any of the 45 sera from patients with myasthenia gravis without a thymoma. Ryanodine receptor autoantibodies may have pathogenetic relevance in thymoma-associated myasthenia gravis. Mygland A, Tysnes 0-B, Matre R, Volpe P, Aarli JA, Gilhus NE. Ryanodine receptor autoantibodies in myasthenia gravis patients with a thymoma. Ann Neurol 1992;32:589-591

Materials and Methods

Sera Sera from 30 patients with MG and a thymoma (lymphoepithelioma), 30 with thymic hyperplasia, and 15 with thymic atrophy were included. Sera were taken from all available patients with a thymoma and thymic atrophy. The sera from those with thymic hyperplasia were randomly selected from our MG pool. The MG diagnosis was based on conventional clinical criteria, a positive finding on the edrophonium test, an increased decrement after repetitive motor nerve stimulation, and the presence of AChR antibodies. Sera were obtained from patients before thymectomy and stored at - 20°C until use. Control sera were collected from 90 healthy blood donors, 10 patients with muscular dystrophy, and 20 patients with autoimmune disease (9 with systemic lupus erythematosus, 9 with rheumatoid arthritis, 2 with polymyositis). Antibodies to the rabbit skeletal muscle ryanodine receptor were raised in guinea pig and purified as previously described f8]. These antibodies have been shown to react specifically with the ryanodine receptor of skeletal muscle SR 191.

Preparation of Sacropkzsmic Reticulum and Ryanodine Receptor Purification Crude SR was prepared from rabbit skeletal muscle by a differential centrifugation technique [lo]. Ryanodine receptor was solubilized from rabbit skeletal muscle SR and purified by density gradient centrifugation, as described by Lai and coauthors (111. The purity of the preparation was verified by staining Western blots with Poinceau red (Fig, lane 1).

The abnormal muscular fatigability in myasthenia gravis (MG) is mainly due to the effect of autoantibodies to the nicotinic acetylcholine receptor (AChR) in motor end-plates El]. Some patients with MG, including the large majority of those with a thymoma, also have autoantibodies to other proteins of the striated muscle fiber [a}. The pathogenetic significance of the latter antibodies is not yet known. There are antibodies to various myofibrillary proteins, that is, titin, myosin, actin, and a-actinin [3-51, and antibodies to the sarcoplasmic reticulum (SR) [6, 71. SR is the intracellular membrane system that regulates the contraction and relaxation of skeletal muscle by the rapid release and reuptake of calcium. This study shows that sera from some MG patients with a thymoma contain autoantibodies to the SR calcium release channel (the so-called ryanodine receptor).

From the *Department of Neurology and the tDepartment of Immunology and Microbiology, University of Bergen, Bergen, Norway, and the $Unit for Muscle Biology and Physiopathology, National Reseatch Council, Institute of General Pathology, University of Padova, Padova, Italy. Received Feb 19, 1992, and in revised form Apr 23. Accepted for publication Apr 23, 1992.

Address correspondence to Dr Mygland, Department of Neurology, Haukeland Hospital, 502 1 Bergen, Norway.

Western blots of the purified ryanodine receptor with myasthenia gravis (MG) sera. Lane 1: Poinceau red staining of transblotted tyanodine receptor (4 pgllane) electrophoresed on a 5 % sodium dodecyl sulfate polyanylamide gel. Lanes 2 to 6: Purified ryanodine receptor stained witb sera from MG patients witb a thym o m . Lanes 7 and 8: Purijied ryanodine receptor reacted with sera from MG patients without a thymoma.

Copyright 0 1992 by the American Neurological Association

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Western Blots Either crude SR or purified ryanodine receptor was electrophoresed on sodium dodecyl sulfate (SDS) polyacrylamide gels as described by Laemmli {12) and transblotted onto nitrocellulose as described by Towbin and colleagues 11 31. After blocking for 60 minutes with 5% dry milk in Trishydrochloric acid-buffered saline (0.15 M sodium chloride, 0.05 M Tris), pH 7.5 (TBS), the blots were immunostained as previously described [6] with MG sera, control sera, or specific ryanodine receptor antibodies. Sere were dilured 1: 50, and peroddase-conjugated rabbit antibodies to guinea pig IgG (Zymed Laboratory, San Francisco, CA) were diluted 1: 500 in TBS containing 0.5% dry milk and 0.05%,Tween 20.

All MG and control sera were tested against crude SR. All 30 sera from MG patients with a thymoma and 15 randomly selected sera from MG patients without a thymoma were tested against the purified ryanodine receptor. Results Fourteen of the 30 sera from MG patients with a thymoma had IgG antibodies to the purified ryanodine receptor (see, e.g., Fig, lanes 2-6), and they immunostained the same single protein in crude SR as did our specific anti-ryanodine receptor antibody (not shown). The positive sera stained both the purified ryanodine receptor and the crude SR preparation with somewhat different degrees of intensity. Sixteen of the 30 sera from MG patients with a thymoma did not react with purified ryanodine receptor or with crude SR. None of the sera from MG patients without a thymoma or control sera stained the purified ryanodine receptor (see, e.g., Fig, lanes 7 and 8), nor did they stain any other SR protein. Discussion This study shows that nearly one half of MG patients with a thymoma have IgG autoantibodies to the ryanodine receptor of skeletal muscle SR. In Western blot experiments, MG sera reacted with the purified ryanodine receptor, and they recognized the same protein in a crude SR preparation as did antibodies with known specificity against the ryanodine receptor. This definitively proves that MG autoantibodies are directed against a single skeletal muscle SR protein that is identical to the ryanodine receptor. The ryanodine receptor of striated muscle SR is a calcium release channel consisting of four homologous subunits, each with a relative molecular mass of about 400 kDa {14]. Several transmembrane segments form the calcium pore. Large cytoplasmic domains project from the SR membrane and come into close apposition with the transverse (T)-tubular sarcolemma invaginations, forming the triad junctions {15]. 'T-tubular depolarization somehow triggers the rapid release of calcium via the ryanodine receptor into the myoplasm, thus producing muscle contraction. The occurrence of ryanodine receptor autoantibodies is strongly related to the presence of a thymoma. 590

Annals of Neurology Vol 32

No 4 October 1992

These antibodies are not detected in MG patients without a thymoma or in patients with degenerative muscular disorders. They are therefore not simply secondary to muscle cell damage. Moreover, these antibodies do not seem to be due to a polyclonal immune activation, since they are absent in patients with other autoinimune disorders. Thus, ryanodine receptor autoantibodies may be relevant in the pathogenesis of thymoma-associated MG. Nearly one half of sera from patients with M C and a thymoma did not react with the ryanodine receptor. It is possible that these sera contained either too low a concentration of ryanodine receptor IgG autoantibodies (i.e, below the detection limit of our Western blots) or ryanodine receptor antibodies of another immunoglobulin class. The majority of thymomas associated with MG are lymphoepitheliomas where neoplastic epithelial cells are surrounded by immature T cells. The neoplastic epithelial cells express proteins that are recognized by antibodies to striated muscle { 161. One possible autoimmune mechanism could be that developing T cells are sensitized to striated muscle antigens in epithelial cells and eventually stimulate B lymphocytes to produce striated muscle antibodies. Further experiments are needed to examine whether T cells from patienrs with a thymoma are sensitized to fragments of the ryanodine receptor, and whether ryanodine receptor (or its fragments) is expressed in thymomas. Dr Mygland is a Research Fellow of the Norwegian Cancer Society.

References Lindstrom J. lmmunobiology of myasthenia gravis, experimental autoimmune myasthenia gravis, and Laniberr-Eaton syndrome. Annu Rev Immunol 1985;3:109-13 1 Strauss AJL, Seegal BC, Hsu KC, er al. Irnmunofluorescence demonstration of a muscle binding, complement-fixing serum globulin fraction in myasthenia gravis. Proc Soc Exp B i d Med 1960;105:184- 191 Aarli JA, Stefansson K, Marron LSG, Wollmann RL. Patients with myastheniagravis and thymoma have in their sera IgG autoantibodies against titin. Clin Exp Immunol 1990;82:284-288 Williams CL, Lennon VA. Thymic B-lymphocyte clones from patients with myasthenia gravis secrete monoclonal srriarional autoantibodies reacting with myosin, u-actinin or acrin. J Exp Med 1986;164: 1043- 1059 Ohm M, Ohta K, Iroh N, er al. Anti-skeletal muscle antibodies in the sera from myasthenic patients with thymorna: idenrification of anti-myosin, actomyosin, actin, and cu-actinin antibodies by a solid-phase radioimmunoassay and a Western blot1ing analysis. Clin Shim Acta 1990;187:255-264 Mygland A, Tysnes 0 - B , Aarli JA, et al. Myasrhenia gravis patients with a thymoma have antibodies against a high m&cular weight protein in sarcoplasmic reticulum. J Neuroimmunc.~l 199257 :1-7 Flood PR, Bjugn R, Gilhus NE, ct al. The ultrastructural localization of antigens for skeletal muscle antibodies in myasthenii gravis. Ann N Y Acad Sci 1987;505:332-734 Volpe P, Bravin M, Zorzato F, Margreth F. Isolation of terniinal cisrernae of frog skeletal muscle. J I3iol Chern 1988;263: 990 1-9907

9. Zorzato F, Chu A, Volpe P. Antibodies to junctional sarcoplas10.

11.

12 13.

14.

15.

16.

mic reticulum proteins: probes for the Ca” release channel. Biochem J 1989;261:863-870 de Meis L, Hasselbach W. Acetyl phosphate as substrate for Ca” uptake in skeletal muscle microsomes. J Biol Chem 1971;246:4759-4763 Lai FA, Erickson HP, Rousseau E, et al. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature 1988;331:315-3 19 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970,227:680-685 Towbin H , Stahelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350-1354 Lai FA, Meissner G. Structure of the calcium release channel of skeletal muscle sacroplasmic reticulum and its regulation by calcium. Adv Exp Med Biol 1990;269:73-77 Zorzato F, Fujii J, Otsu K, et al. Molecular cloning of cDNA encoding human and rabbit forms of the Ca” release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 1990;265:2244-2256 Gilhus NE, Aarli JA, Christensson B, Matre R. Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasrhenia gravis patients. J Neuroimmunol 1984;7:55-64

a-Tocopherol Levels in Brain Are Not Altered in Parkmson’s Disease D. T. Dexter, PhD,’ R. J. Ward, PhD,? F. R. Wells, MA(Oxon),f S. E. Daniel, MRCPath,S A. J. Lees, FRCP,f T. J. Peters, DSc,? P. Jenner, DSc,’ and C. D. Marsden, FRSf

a-Tocopherol (vitamin E) levels in normal brain were lower in the cerebellum than in the cerebral cortex or basal ganglia. There was no difference in a-tocopherol levels in the cerebellum, basal ganglia, or cerebral cortex between control subjects and patients with Parkinson’s disease. DT, Ward RJ, Wells FR, Daniel SE, Lees AJ, Peters TJ, Jenner P, Marsden CD. a-Tocopherol levels in brain are not altered in Parkinson’s disease. Ann Neurol 1992;32:591-593

In substantia nigra in patients with Parkinson’s disease there is evidence for increased lipid peroxidation El], From the “Parlunson’s Disease Society Experimental Research Laboratories, Pharmacology Group, Biomedical Sciences Division, King’s College London, $Department of Clinical Biochemistry, King’s College School of Medicine and Dentistry, and $Parkinson’s Disease Society Brain Bank, University Department of Clinical Neurology, Institute of Neurology, National Hospital, London, UK. Received Jan 27, 1992, and in revised form May 5. Accepted for publication May 5 , 1992. Address correspondence to Prof Jenner, Pharmacology Group, Biomedical Sciences Division, King’s College London, Manresa Road, London SW3 6LX, UK.

decreased reduced glutathione levels {Z), increased mitochondrial superoxide dismutase activity [ 3 ] , altered iron metabolism {4-61, and impaired mitochondrial function 171. Such findings support the concept of oxidative stress as a component of nigral degeneration and suggest that antioxidants may be valuable in treating patients with Parkinson’s disease. In an open trial, newly diagnosed patients with Parkinson’s disease treated with high doses of vitamins E and C extended the period before introducing L-dopa therapy by 2.5 years [8}. Vitamin C levels are reported to be normal in the brain in patients with Parkinson’s disease {2). We now report on a-tocopherol levels in patients with this disorder.

Methods Brain tissue from patients dying with a clinical diagnosis of Parkinson’s disease (n = 12), and from control subjects dying of nonneurological disorders (n = 12), was obtained from the Parkinson’s Disease Society Brain Bank, Institute of Neurology, London. Details of the patients’ characteristics and postmortem parameters are presented in the Table. At autopsy, brains were removed and divided midsagittally. One-half of the brain was neuropathologically examined, whereas the other half of the brain was immediately frozen and subsequently dissected [I}.

Characteristics of Patient Gronps and Details of Postmortem Parameters

Control Subjects (n = 12) Age Sex (FIM) Age at onset of Parkinson’s disease (yr) Duration of Parkinson’s disease (yr) L-Dopa dosage (mglday) at time of death“ Cell loss and presence of Lewy bodies in midbrain Caudate dopamine concentration (pg/gm wet weight of tissue) Time between death and body refrigeration (hr) Time between death and autopsy (hr) Freezer storage time at - 70°C (yr)

72.8 t 3.4 517

... ...

... No 4.3

%

0.9

Patients with Parkinson’s Disease (n = 12) 78.8 k 1.7 319 68.2 -+ 1.7 (58-7 5) 10.9 2 1.7 (5-24) 522.2 ? 111.8 (150-1,000) Yes 1.0 2 0.2b

3.2 t 0.3 2.3 i 0.3b 14.6 t 2.0 2.4 ir 0.2

11.8 2 2.2 2.0

2

0.3

*

Data are mean 1 SEM. Numbers in parentheses show the range of values. Caudate dopamine levels were measured by standard highperformance liquid chromatography with electrochemical detection technique. ”With

a peripheral decarboxylase inhibitor.

hp < 0.05, compared with control subjects (Student’s t test).

Copyright 0 1992 by the American Neurological Association

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Ryanodine receptor autoantibodies in myasthenia gravis patients with a thymoma.

Sera from patients with myasthenia gravis were examined by Western blot for the presence of antibodies to proteins of the sarcoplasmic reticulum from ...
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