Expression of Acetylcholine Receptor Isoforms at Extraocular Muscle Endplates

Transcription

1 Expression of Acetylcholine Receptor Isoforms at Extraocular Muscle Endplates Henry J. Kaminski,* Linda L. Kusner* and Christine H. Blocks Purpose. To determine the expression of fetal and adult acetylcholine receptor (AChR) isoforms among extraocular muscle (EOM) en plaque and en grappe endplates. Methods. Antibodies against peptide fragments of the y- and e-subunits of the fetal and adult AChRs and a-bungarotoxin were used in immunofluorescence experiments to stain rat neonatal leg, adult diaphragm, and extraocular rectus muscle endplates. Results. Anti-e antibodies intensely stained diaphragm endplates and weakly stained rare neonatal endplates. Anti-y antibodies stained neonatal, but not diaphragm, endplates. Anti-e antibodies bound to all en plaque and en grappe endplates of extraocular muscle. Anti-7 antibodies bound to global and orbital en grappe endplates. All en plaque endplates of the orbital region and a subset of endplates in the global region stained with anti-y antibodies. Conclusions. All en grappe endplates and certain en plaque endplates of EOM are the only mature endplates that coexpress the adult and fetal AChR isoforms. The expression of both isoforms may be important to determine contractile properties, protein expression regulation, and EOM susceptibility to myasthenia gravis. Invest Ophthalmol Vis Sci. 1996;37: Jlixtraocular muscle (EOM) is the only mature, innervated skeletal muscle to express gene transcripts specific for adult and fetal isoforms of the acetylcholine receptor (AChR). 1 " 3 The adult isoform is composed of two a, and single (3, 8, and e-subunits, whereas the fetal isoform contains a y-subunit substituted for the e-subunit. The subunit substitution confers specific electrophysiological and antigenic properties to the receptor. 4 ' 5 During myogenesis, before any neuronal contact occurs, muscle fibers express fetal AChRs diffusely across their lengths. With innervation, AChRs cluster at the synaptic contact, and, as the endplate matures, fetal AChR concentration decreases and the adult form appears. 4 Denervation leads to reexpression of fetal AChR. The appearance of the isoforms is correlated with expression of transcripts of the y- and e-subunit genes. 4 5 From the Departments of* Neurology and f Pathology, Case Western Reserve University School of Medicine, Cleveland Veterans Affairs Medical Center, University Hospitals of Cleveland, Ohio. Supported l/y National Institutes of Health grant EY and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. Submitted for publication June 20, 1995; revised SejHember 6, 1995; accepted October 4, Projmelaiy interest category: N. Refmnt requests: Henry /. Kaminski, Department of Neurology, University Hospitals of Cleveland, 1U00 Euclid Avenue, Cleveland, OH Extraocular muscle is composed of a heterogenous mixture of muscle fibers, 6 ' 7 but it is unknown if the fetal AChR is expressed by all, or only a subset, of fibers. Extraocular muscle is divided anatomically into a global region, adjacent to the globe, and an orbital region, next to the bony orbit. b The majority of EOM fibers are singly innervated fibers (SIFs) and share a similar endplate morphology with other skeletal muscle because they have a single en plaque neuromuscular junction. Approximately 20% of EOM fibers are innervated at multiple points, multiply innervated fibers (MIFs). Orbital and global MIFs have multiple, small, en grappe endplates at their proximal and distal ends. The orbital MIF also has a single en plaque endplate at its center/' 7 We have hypothesized that MIFs are the site of y-subunit expression for two reasons. 7 ' 8 One, electrophysiological recordings from MIF endplates of snakes demonstrate the presence of AChRs with properties similar to the mammalian adult and fetal isoforms. 9 l0 Two, levator palpebrae superioris, which shares many features with EOM, () does not contain MIFs and does not express y-subunit transcripts. We performed immunocytochemical analysis using antibodies directed toward peptide fragments of the y- and e- subunits to determine the expression of AChR isoforms in EOM. Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 Copyright Association for Research in Vision and Ophthalmology 345

2 346 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 METHODS Antibodies Polyclonal antisera from rabbits designated anti-y 485 and anti-e 360 (a gift from Zach Hall, National Institutes of Health) were produced against peptide fragments of y- and e-achr subunits." These antibodies were shown to bind to rat AChR subunits by immunohistochemistry, enzyme-linked immunoadsorbent assay, and immunoprecipitation. Antibodies were purified by affinity chromatography. Sepharose 4B (Pharmacia, Piscataway, NJ) was prepared according to the manufacturers' instructions, mixed with 1 to 2 /imol of peptide in 0.2 M NaHCO 3 (ph 9.4) to form a slurry, and rotated for 16 hours at 4 C. The slurry was aspirated on a scintered glass filter and washed with five volumes of 0.2 M Na 2 CO B (ph 9.4) and 1 M NaCl alternating with 0.2 M NaC 2 H 3 O 2 (ph 4.5) and 1 M NaCl, followed by one wash in phosphate-buffered saline (PBS)-0.02% sodium azide (ph 7.4). Sepharose was blocked with 0.1 M ethanolamine in PBS-sodium azide for 2 hours at 4 C. The resultant coupled gel was washed in PBS. A 1:10 mixture of the coupled Sepharose and an userum was incubated for 4 hours at 4 C. The entire volume was transferred to a glass wool-stoppered syringe and washed with PBS until the A 28 o of the effluent was less than Antibody was eluted with 0.1 M glycine-hcl (ph 2.5). Fractions were collected in 1.5 M Tris-HCl (ph 8.8), and peak optical density fractions were pooled. Antibody solutions were dialyzed against a 1000-fold volume of PBS-sodium azide for 16 hours at 4 C. Antibody concentration was determined by absorbance at 280 nm. Immunocytochemistry All animal studies were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committees of Case Western Reserve University and the Cleveland Veterans Affairs Medical Center. Adult female Lewis rat rectus EOM and diaphragm, and day 3 newborn leg muscle were sandwiched in liver and frozen in liquid N 2 cooled isobutane. Cholinesterase staining was performed on b-fxm cryostat sections using the method of Karnovsky and Roots 12 to identify innervational regions. Diaphragm and neonatal muscles were cut in cross-section to confirm antibody specificity. Ten rectus EOMs were cut longitudinally, and three rectus muscles were cut in cross-section. The central innervational band, proximal ends, and distal ends of the muscle were sectioned through in their entirety. Sections were air dried and fixed in acetone. Sections were blocked with 5% donkey serum, incubated in 6.5 //g/ml of anti-y 485 or 2.5 //g/ml of anti-e 360 at room temperature for 1 hour, and washed with PBS. Qualitative antibody binding and endplate location were assessed after incubation with anti-rabbit IgG-tetramethyl rhodamine isothiocyanate (TRITC) (Chemicon, Temecula, CA) and biotin labeled a-bungarotoxin (Molecular Probes, Eugene, OR) at 4 /ig/ml for 1 hour at room temperature. After washing in PBS, the sections were treated with streptavidin fluorescein isothiocyanate (FITC; Molecular Probes) at 4 /ig/ml for 30 minutes. Endplates were photographed under rhodamine and fluorescein optics. Fluorescence was not seen in sections incubated with IgG-TRITC alone when excited at wavelengths for FITC. The same was true for streptavidin - FITC, when excited at TRITC wavelengths. RESULTS All diaphragm endplates stained intensely with anti-e antibodies and colocalized with a-bungarotoxin (Fig. 1). The morphology of the endplates was typical of en plaque endplates cut in cross-section. No staining was detected with anti-y antibodies (not shown). Newborn leg muscle endplates stained intensely with antiy antibodies (Fig. 2). Rare neonatal endplates bound anti-e antibodies, but the intensity of staining was punctate and much less than with anti-y antibodies (not shown). Gu and Hall" found anti-e and weak anti-y staining at 9-day and 16-day diaphragm endplates. Endplates of 4-day diaphragms only stained with anti-y. It is likely that the timing of AChR subunit substitution varies among muscle groups, e-subunit gene transcripts are found in the triceps surae of 2- day-old rats 13 ; therefore, some level of adult AChR expression could be expected in the neonatal leg muscle used in the current study. No cross-reactivity between the antibodies used in this investigation was observed by us or Gu and Hall.'' However, it is possible that these antibodies may cross-react with unidentified endplate antigens that colocalize with the AChR isoforms. En grappe endplates were concentrated at the proximal and distal ends of the EOM. All en grappe endplates identified by a-bungarotoxin stained with anti-e and anti-y antibodies indicating that the adult and fetal AChR isoforms were coexpressed at these endplates (Figs. 3, 4). En grappe endplates were either strung along the fiber (Figs. 3, 4) or clustered in a single region of a fiber (Fig. 4). Such endplate distributions have been observed by us and others. 0 ' 8 ' 14 ' 15 * Extraocular muscle cut in cross-section demonstrated that global and orbital MIFs were stained by both antibodies. All en plaque EOM endplates of the global and orbital regions were stained by anti-e antibodies and colocalized with a-bungarotoxin (Fig. 5). Anti-y anti-

3 Receptors at Ocular Muscle Endplates sodium channels despite the fact that they are only one sodium channel gene transcript. However, given the use of polyclonal antibodies in this investigation, a similar occurrence in EOM fibers is unlikely. DISCUSSION Results of experiments with neonatal leg muscle and adult diaphragm support previous investigations demonstrating that mammals undergo a AChR substitution during myogenesis. Gu and Hall," using the same antibodies, found anti- staining in 9-day-old and older diaphragms, whereas anti-y staining was observed only in developing muscle. In cow and rat, gene transcripts of the y-subunit are present at birth and disappear soon afterward, whereas e-subunit gene transcript levels increase Oocyte expression studies demonstrated that these subunits' gene transcripts correlate with the electrophysiological properties of the AChR isoforms. 18 En grappe endplates of MIFs bound anti-e and anti-y antibodies, indicating the coexpression of adult and fetal AChR isoforms.^' 815 This finding is consistent FIGURE 1. Diaphragm muscle fibers cut in cross-section and incubated with biotin-labeled a-bungarotoxin (A) and antie 360 (B). Binding was detected using fluorescein isothiocyanate streptavidin (A) and TRITC secondary antibodies (B). The two center fibers have typical en plaque endplates of twitch muscle fibers. All diaphragm endplates stained with anti-e 360, indicating the presence of the adult acetylcholine receptor isoform. Scale bar = 25 ^m. TRITC = tetramethyl rhodamine isothiocyanate. bodies stained all en plaque endplates of the orbital region, and we conclude that the MIF and SIF of the orbital region express both AChR isoforms at their en plaque endplates. The most striking feature of the anti-y-stained endplates of the global region was their location at the periphery of the innervational band (Fig. 6). No anti-y-stained endplates were found in the central area of the innervational region. Identification of the global fibers with anti-y-stained endplates could not be performed using the current methods and awaits colocalization of AChR isoforms with myosin heavy chain expression to allow fiber typing. The microenvironment of these anti-y-stained endplates is such that the adult isoform possibly binds anti-y antibodies, and this has been supported by another study." 1 This demonstrates differential immunohistochemical staining patterns of fast and slow twitch muscle fibers stained with monoclonal antibodies to FIGURE 2. Neonatal leg muscle fibers cut in cross-section and incubated with biotin-labeled a-bungarotoxin (A) and antiy 485 (B). Binding was detected using fluorescein isothiocyanate streptavidin (A) and TRITC secondary antibodies (B). En plaque endplates are seen on severalfibers.all endplates stained with anti-y 485 antibody, indicating the expression of the fetal acetylcholine receptor. Scale bar = 25 /xm. TRITC = tetramethyl rhodamine isothiocyanate.

4 348 Investigative Ophthalmology & Visual Science, Februaiy 1996, Vol. 37, No. 2 endplates has properties similar to the global MIF. 19 Fetal AChR has a longer open time and is more resistant to desensitization than the adult isoform. 5 ' 18 A long open time provides a longer depolarization and tends to produce a more uniform depolarization along the global MIF and the distal portions of the orbital MIF. 7 Resistance to desensitization would allow the fiber to respond better to repeated or prolonged stimulation. Because EOM fibers are subject to high neuronal firing frequencies and because some fibers are under constant stimulation, the presence of the fetal AChR is particularly advantageous. 78 Our results indirecdy support electrophysiological data that demonstrate two channel types at the synapses of MIFs. Snake and frog MIF endplates have AChRs with open channel times and conductances FIGURE 3. Longitudinal section of extraocular muscle (COM) fiber incubated with biotin-labed a-bungarotoxin (A) and anti-c 360 (B). Binding was detected using fluorescein isothiocyanate streptavidin (A) and TRITC secondary antibodies (B). (large arrow) Myelinated nerve innervates the EOM fiber, (small arrows) Individual en grappe endplates are strung along the surface of the muscle fiber. En grappe endplates are much smaller than en plaque endplates. Antie 360 staining indicates the presence of the adult acetylcholine receptor at these endplates. Scale bar = 25 /zm. TRITC = tetramethyl rhodamine isothiocyanate. with our previous investigations and those of Horton et al 1 " 3 demonstrating gene transcripts of the fetal and adult AChR isoforms in EOM. The function of both AChR isoforms at the en grappe endplates is unclear. Global MIFs are similar to tonic fibers in that they develop tonic contractions and relax and contract more slowly than twitch fibers, but they do not generate action potentials. Orbital MIFs share properties of twitch and tonic fibers 19 and more closely resemble intermediate-type fibers of reptilian and amphibian muscle. 7 ' 10 The central portion in the area of the en plaque endplate is twitch-like, generates action potentials, and develops sustained contractions to continued depolarization, whereas the area of the en grappe FIGURE 4. Longitudinal section of extraocular muscle fiber (EOM) incubated with biotin-labeled a-bungarotoxin (A) and anti-y 485 (B). Binding was detected using fluorescein isothiocyanate streptavidin (A) and TRITC secondary antibodies (B). (large arrow) cluster of en grappe endplates. The nerve to the endplates can be seen crossing among the endplates. Global and orbital en grappe endplates demonstrated anti-y 485 immunoreactivity, which indicates the presence of the fetal acerylcholine receptor at these endplates. Other fluorescent markings represent nonspecific staining caused by the large amounts of connective tissue in EOM. Scale bar 25 /Am. TRITC = tetramethyl rhodamine isothiocyanate.

5 Receptors at Ocular Muscle Endplates 349 McLoon et al 22 raised the possibility that active regeneration of EOM fibers occurs in the adult animal. Perhaps the fibers expressing fetal AChR are regenerating SIFs. However, it is unlikely that all orbital SIFs are regenerating fibers. Fetal AChR would prolong endplate depolarization, perhaps beyond the refractory period for action potential generation, and would lead to repetitive action potentials after a single nerve stimulus. 5 Such an occurrence may have some functional benefit for certain eye movements. Innervation plays a significant role in the control of AChR subunit expression, which is regulated primarily at a transcriptional level, 4 ' l8l2s and electrical stimulation is thought to be the major influence. Neuromuscular blockade leads to an increase of AChR subunit mrnas, whereas electrical stimulation suppresses AChR synthesis. Cultured myotubes respond in a similar fashion.' 1 Innervation in other muscles suppresses y-subunit gene expression. Our findings indicate that AChR y-subunit expression in EOM must be influenced by factors other than innervation and electrical stimulation or that the nature of the electri- FIGURE 5. Longitudinal section of extraocular muscle fiber (EOM) incubated with biotin-labeled a-bungarotoxin (A) and anti-e 360 (B). Binding was detected using fluorescein isothiocyanate streptavidin (A) and TR1TC secondary antibodies (B). An en plaque endplate is seen. All EOM en plaque endplates demonstrated anti-e 360 immunoreactivity, which indicates the expression of the adult acetylcholine receptor. Scale bar = 25 ^m. TRITC = tetramethyl rhodamine isothiocyanate. similar to the mammalian adult and fetal isoforms. 9 ' 10^0 A subunit substitution has not been identified in snake to account for two populations of channels, but we have identified gene transcripts of an e- subunit in snake (unpublished observations, 1995) that make this possibility likely. Orbital SIF and some global SIF endplates expressed adult and fetal AChRs. It is unlikely that these fibers were denervated partially and that they expressed the fetal isoform because such endplates would be expected in diaphragm. Using electrophysiological recording, Brehm and Kullberg 21 found that 3% of channels at flexor digitorum brevis endplates had fetal properties. Such levels would have been too low to detect by immunocytochemistry." Therefore, the en plaque endplates, which stained with anti-y AChR antibodies, express the fetal AChR in high concentrations. The possible function of the fetal AChR in the SIFs is not as easily considered as in the MIFs. FlGURE 6. Extraocular muscle global region cut in cross-section incubated with biotin-labeled a-bungarotoxin (A) and anti-y 485 (B). Binding was detected using fluorescein isotriiocyanate streptavidin (A) and TRITC secondary antibodies (B). (large arrows) En plaque endplates bind both a- bungarotoxin and anti-y 485, indicating the expression of the fetal acetylcholine receptor, (small arroxvs) Endplates without anti-y 485 immunoreactivity. Scale bar = 50 /^m. TRITC = tetramethyl rhodamine isothiocyanate.

6 350 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2 cal stimulus may be important in y-subunit gene regulation. Numerous proteins, in addition to the AChR, are expressed differentially or exclusively at or in the region of the neuromuscular junction. 4 Sodium channels, dystrophin, and N-CAM are concentrated near the junctional folds. In orbital MIF, myosin heavy chain expression is associated with innervation pattern. 24 The fast myosin heavy chain isoform is localized to the region of the en plaque endplate, whereas the area of en grappe endplates expresses embryonic myosin heavy chain. In avian muscle, the cell adhesion molecule, N-CAM, is localized to MIFs. 25 How the nerve mediates this influence is unclear, but both electrical stimulation and trophic factors have a role. 4 During myogenesis, the fetal AChR allows spontaneous contractions to occur in response to miniature endplate currents, and it appears to be critical for normal neuromuscular development. 26 The presence of the fetal AChR or the ratio of fetal-to-adult AChRs may influence muscle protein expression by the nature of endplate depolarization. Expression of the fetal AChR may be relevant to the preponderance of EOM involvement by myasthenia gravis. Most people with myasthenia have ocular manifestations, and 10% of them have restricted ocular muscle involvement. Oda 27 found ocular myasthenic sera that were directed exclusively toward MIF endplates; however, the identity of the antigen bound by these sera was not determined. Given the results of the current investigation, these antibodies probably are directed toward the fetal AChR. People with myasthenia do have antibodies against the fetal AChR, but such antibodies have not been correlated with ocular manifestations. 28 The frequency of ptosis among people with myasthenia cannot be explained by fetal AChR expression because the levator palpebrae does not express the y-subunit. 3 Several reasons may account for the preferential involvement of EOM, 8 and the presence of the fetal AChR isoform offers a target for differential, immune-mediated attack of EOM by myasthenia gravis. Key Words acetylcholine receptor, extraocular muscle, immunocytochemistry, myasthenia gravis Acknowledgments The authors thank Zach Hall for the gift of anti- and antiy antisera, Joyce Pressly for technical assistance, and Robert L. Ruff for helpful discussion. References 1. Kaminski HJ, Fenstermaker R, Ruff RL. Adult extraocular and intercostal muscle express the gamma-subunit of fetal AChR. BiophysJ. 1991; 59:444a. 2. Horton RM, Manfredi AA, Conti-Tronconi BM. The 'embryonic' gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle. Neurology. 1993; 43: Kaminski HJ, Kusner LL, Nash KV, Ruff RL. The y- subunit of the acetylcholine receptor is not expressed in the levator palpebrae superioris. Neurology. 1995;45: Hall ZW, Sanes JR. Synaptic structure and development: The neuromuscular junction. Cell. 1993; 72: Kaminski HJ, Ruff RL. Insights into possible skeletal muscle nicotinic acetylcholine receptor (AChR) changes in some congenital myasthenias from physiological studies, point mutations, subunit substitutions of the AChR. Ann NY Acad Sci. 1993;681: Spencer R, Porter J. Structural organization of the extraocular muscles. In: Buttner-Ennever J, ed. Revies of Oculomotor Research. Vol. 2. New York: Elsevier; 1988: Ruff RL, Kaminski HJ, Maas E, Spiegel P. Ocular muscles: Physiology and structure-function correlations. Bull Soc Beige Ophtalmol. 1989;237: Kaminski HJ, Maas E, Spiegel P, Ruff RL. Why are eye muscles frequently involved by myasthenia gravis? Neurology. 1990; 40: Dionne VE. Two types of nicotinic acetylcholine receptor channels at slow fibre end-plates of the garter snake. / Physiol. 1989; 409: Ruff R, Spiegel P. Ca sensitivity and AChR currents of twitch and tonic snake muscle fibers. Am J Physiol. 1990; (Cell Physiol 28):C911-C Gu Y, Hall ZW. Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle. Neuron. 1988; 1: Karnovsky J, Roots L. A direct coloring tricholine method for cholinesterase. / Histochem Cytochem. 1964; 12: Witzemann V, Barg B, Criado M, Stein E, Sakmann B. Developmental regulation of five subunits specific mrnas encoding acetylcholine receptor subtypes in rat muscle. FEBS Lett. 1989; 242: Chiarandini D, Davidowitz J. Structure and function of extraocular muscle. Curr Top Eye Res. 1979; 1: Oda K. Motor innervation and acetylcholine receptor distribution of human extraocular muscle fibers. / NeurolSci. 1986; 74: Cohen SA, Barchi RL. Localization of epitopes for antibodies that differentially label sodium channels in skeletal muscle surface and T-tubular membranes. / MembrBiol. 1992; 128: Takai T, Noda M, Mishina M, et al. Cloning, sequencing and expression of cdna for a novel subunit of acetylcholine receptor from calf muscle. Nature. 1985;315: Mishna M, Takai T, Imoto K, et al. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 1986; 321: JacobyJ, Chiarandini D, Stefani E. Electrical proper-

7 Receptors at Ocular Muscle Endplates 351 ties and innervation of fibers in the orbital layer of rat extraocular muscles. J Neurophysiol. 1989;61: Henderson LP, Brehm P. The single-channel basis for the slow kinetics of synaptic currents in vertebrate slow muscle fibers. Neuron. 1989;2: Brehm P, Kullberg R. Acetylcholine receptor channel on adult mouse skeletal muscle are functionally identical in synaptic and nonsynaptic membrane. Proc Natl Acad Sci USA. 1987; 84: McLoon L, Dodge N, Wirtschafter J. Mature rabbit extraocular muscles contain activated satellite cells. Invest Ophthalmol Vis Sci. 1995;36:S Witzemann V, Barg B, Nishikawa Y, Sakmann B, Numa S. Differential regulation of muscle acetylcholine receptor y- and e-subunit mrnas. FEBS Lett. 1987; 223: Jacoby J, Ko K, Weiss C, Rushbrook J. Systematic variation in myosin expression along extraocular muscle fibers of the adult rat. / Muscle Res Cell Motil. 1990; 11: Bleisch W, Scharff C, Nottebohm F. Neural cell adhesion moleculae (N-CAM) is elevated in adult avian slow muscle fibers with multiple terminals. Proc Natl Acad Sci USA. 1989;86: Jaramillo F, Vicini S, Schuetze SM. Embryonic acetylcholine receptors guarantee spontaneous contractions in rat developing muscle. Nature. 1988; 335: Oda K. Myasthenia gravis: Antibodies to endplates of human extraocular muscle. Ann NY Acad Sci. 1987; 505: Tzartos S, Seybold M, Lindstrom J. Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci. 1982;79: