Increased expression of rapsyn in muscles prevents acetylcholine receptor loss in experimental autoimmune myasthenia gravis

Abstract

Myasthenia gravis is usually caused by autoantibodies to the acetylcholine receptor (AChR). The AChR is clustered and anchored in the postsynaptic membrane of the neuromuscular junction (NMJ) by a cytoplasmic protein called rapsyn. We previously showed that resistance to experimental autoimmune myasthenia gravis (EAMG) in aged rats correlates with increased rapsyn concentration at the NMJ. It is possible, therefore, that endogenous rapsyn expression may be an important determinant of AChR loss and neuromuscular transmission failure in the human disease, and that upregulation of rapsyn expression could be used therapeutically. To examine first a potential therapeutic application of rapsyn upregulation, we induced acute EAMG in young rats by passive transfer of AChR antibody, mAb 35, and used in vivo electroporation to over-express rapsyn unilaterally in one tibialis anterior. We looked at the compound muscle action potentials (CMAPs) in the tibialis anterior, at rapsyn and AChR expression by quantitative radioimmunoassay and immunofluorescence, and at the morphology of the NMJs, comparing the electroporated and untreated muscles, as well as the control and EAMG rats. In control rats, transfected muscle fibres had extrasynaptic rapsyn aggregates, as well as slightly increased rapsyn and AChR concentrations at the NMJ. In EAMG rats, despite deposits of the membrane attack complex, the rapsyn-overexpressing muscles showed no decrement in the CMAPs, no loss of AChR, and the majority had normal postsynaptic folds, whereas endplates of untreated muscles showed typical AChR loss and morphological damage. These data suggest not only that increasing rapsyn expression could be a potential treatment for selected muscles of myasthenia gravis patients, but also lend support to the hypothesis that individual differences in innate rapsyn expression could be a factor in determining disease severity.

Immunosuppression of experimental autoimmune myasthenia gravis by mycophenolate mofetil

“…Anti-rat AChR antibody titer measurements were carried out at 4°C by a double-antibody radioimmunoassay (RIA) as described previously (Losen et al, 2005;Martinez-Martinez et al, 2007). Briefly, antibodies directed against rat AChRs were measured using a crude extract of denervated rat muscle (approximately 5 nmol/L AChR), labeled with an excess of 125 I-α-bungarotoxin (IM109, 6.12 TBq/mmol, GE Healthcare, Amersham Place, UK), and incubated overnight with 5 µL rat test serum.…”

“…Left tibialis anterior muscles were frozen in isopentane cooled with liquid nitrogen to perform immunohistochemical staining as described previously (Losen et al, 2005;MartinezMartinez et al, 2007) with the following modifications: 10 µm cryosections were incubated with mouse anti-rapsyn mAb 1234 (1:500 in PBS with 2% bovine serum albumin (PBSA), Sigma, USA), rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500 in PBSA, Sigma, USA), and Alexa 594-conjugated α-bungarotoxin (1:300 in PBSA, Molecular Probes, USA) for 1 h. Subsequently, sections were incubated for 45 min with the appropriate secondary antibodies: biotinylated goat antimouse IgG (1:400 in PBSA; Jackson Immunoresearch, USA) and Alexa 350-conjugated goat anti-rabbit (1:100 in PBSA; Molecular Probes, USA). Finally, the biotinylated secondary antibody was stained for 30 min with Alexa 488-conjugated streptavidin (1:2000 in PBSA; Molecular Probes, USA).…”

“…Extraction was performed on a reciprocal shaker for 1 h at 4°C, followed by centrifugation at 22,100 g for 30 min at 4°C. AChR concentrations were then measured by RIA using an excess of 125 I-α-bungarotoxin (IM209, 74 TBq/mmol, GE healthcare, Amersham Place, UK) as described previously (Losen et al, 2005). Total muscle membrane AChR concentration was calculated per g of fresh muscle and thus expressed in fmol/g.…”

“…Decrement of CMAP during repetitive nerve stimulation was measured during concurrently continuous curare (D-tubocurarine chloride, ICN Biomedicals, Illkirch France) infusions in left tibialis anterior muscles of rats 8 weeks after immunization using the EMG system Viking IV (Nicolet Biomedicals Inc., Madison, USA) as previously described (Losen et al, 2005;Martinez-Martinez et al, 2007;Seybold et al, 1976) with the following modifications: curare infusions (Terfusion syringe pump, model STC-521, Terumo, Japan; 1 mL/h, 0.33 µg curare/ min) were proceeded after intubation and catheterization of the tail vene. To detect a decrementing response, series of 8 supramaximal stimuli were given to the nervus tibialis at 3 Hz with stimulus duration of 0.2 ms and an interval of 2 min.…”

“…To detect a decrementing response, series of 8 supramaximal stimuli were given to the nervus tibialis at 3 Hz with stimulus duration of 0.2 ms and an interval of 2 min. The signal was considered positive when both amplitude and area of the fourth negative peak of the CMAP showed a decrease of at least 10% when compared to the first response (Losen et al, 2005;Martinez-Martinez et al, 2007). To demonstrate reproducibility, a minimum of three recordings were made with an interval of at least 30 s between the repetitive stimulations.…”