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Original Contributions |

Acute Severe Animal Model of Anti–Muscle-Specific Kinase Myasthenia:  Combined Postsynaptic and Presynaptic Changes

David P. Richman, MD; Kayoko Nishi, PhD; Stuart W. Morell, MS; Jolene Mi Chang, PhD; Michael J. Ferns, PhD; Robert L. Wollmann, MD, PhD; Ricardo A. Maselli, MD; Joachim Schnier, PhD; Mark A. Agius, MD
Arch Neurol. 2012;69(4):453-460. doi:10.1001/archneurol.2011.2200.
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Objectives To determine the pathogenesis of anti–muscle-specific kinase (MuSK) myasthenia, a newly described severe form of myasthenia gravis associated with MuSK antibodies characterized by focal muscle weakness and wasting and absence of acetylcholine receptor antibodies, and to determine whether antibodies to MuSK, a crucial protein in the formation of the neuromuscular junction (NMJ) during development, can induce disease in the mature NMJ.

Design, Setting, and Participants Lewis rats were immunized with a single injection of a newly discovered splicing variant of MuSK, MuSK 60, which has been demonstrated to be expressed primarily in the mature NMJ. Animals were assessed clinically, serologically, and by repetitive stimulation of the median nerve. Muscle tissue was examined immunohistochemically and by electron microscopy.

Results Animals immunized with 100 μg of MuSK 60 developed severe progressive weakness starting at day 16, with 100% mortality by day 27. The weakness was associated with high MuSK antibody titers, weight loss, axial muscle wasting, and decrementing compound muscle action potentials. Light and electron microscopy demonstrated fragmented NMJs with varying degrees of postsynaptic muscle end plate destruction along with abnormal nerve terminals, lack of registration between end plates and nerve terminals, local axon sprouting, and extrajunctional dispersion of cholinesterase activity.

Conclusions These findings support the role of MuSK antibodies in the human disease, demonstrate the role of MuSK not only in the development of the NMJ but also in the maintenance of the mature synapse, and demonstrate involvement of this disease in both presynaptic and postsynaptic components of the NMJ.

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Figure 1. Purification of the N-terminal extracellular domain of muscle-specific kinase 60 (N–MuSK 60). The culture medium from COS7 cells transfected with N–MuSK 60 vector (M60) or empty vector (V) was applied to a nickel column and eluted with buffer containing imidazole. A, Western blot demonstrated a strong immunoreacting band in the original culture medium, the first effluent fraction (E1), and the second effluent fraction (E2). B, Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The immunoreacting band in E2 corresponds to a single Coomassie-stained band.

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Figure 2. Clinical course. A, Rats immunized with 100 μg of the N-terminal extracellular domain of muscle-specific kinase 60 (N–MuSK 60) developed more severe weakness than those immunized with 50 μg. A clinical score of 0 indicates normal; 1, weak grip; 2, abnormal gait; 3, walking only a few steps at a time with waddle and kyphosis; 4, inability to stand; and 5, moribund. B, Mean weight of these animals, demonstrating more severe weight loss in rats immunized with 100 μg of N–MuSK 60. Error bars indicate SEM.

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Figure 3. Immunodot blot of serum samples at day 27. A, Serum samples diluted from 1:104 through 1:106 from rat immunized with 100 μg of the N-terminal extracellular domain of muscle-specific kinase 60 (N–MuSK 60) (titer >1:106) and one immunized with 50 μg (titer of 1:105) were blotted against 0.5 μg of affinity-purified mouse N–MuSK 60 or 0.5 μg of bovine serum albumin (BSA) as an antigen control. B, Serum samples from 3 adjuvant control animals diluted 1:500 showed no reaction.

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Figure 4. Clinical findings at day 25. A, Rat immunized with 100 μg of the N-terminal extracellular domain of muscle-specific kinase 60 (right) had significant weight loss, flank and neck muscle wasting, extremity weakness, kyphotic posture, and ruffled, ungroomed fur, whereas the adjuvant control (left) was healthy. B, Lateral view of the same immunized rat.

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Figure 5. Frozen sections of diaphragm muscle obtained at day 27 following immunization, stained with α-bungarotoxin to label acetylcholine receptor (AChR) (red) and with antisynapsin plus antineurofilament antibodies to label presynaptic nerve terminals and axons (green). A, Adjuvant control animals. B-D, Animals immunized with 100 μg of the N-terminal extracellular domain of muscle-specific kinase 60 (N–MuSK 60) and demonstrating increasingly severe disruptions of the neuromuscular junctions. Scale bar = 20 μm.

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Figure 6. Cholinesterase-stained muscle bundle. Teased gastrocnemius bundle from rat immunized with 100 μg of the N-terminal extracellular domain of muscle-specific kinase 60 at 27 days earlier and stained for cholinesterase activity demonstrated patchy staining beyond the end plate region along the entire muscle fiber (A) and, at higher magnification, punctate staining within and adjacent to end plate regions (B).

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Figure 7. Electron micrographs of animals immunized with the N-terminal extracellular domain of muscle-specific kinase 60. A, Electron micrograph of neuromuscular junctions from gastrocnemius of immunized rat (same muscle bundle as in Figure 6) demonstrating hypersegmented neuromuscular junctions. B, At higher magnification, the postsynaptic membranes of these neuromuscular junctions are markedly simplified with sparse synaptic folds (Table 3).

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