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    • br Introduction Myasthenia gravis MG

    2023-05-29


    Introduction Myasthenia gravis (MG) is an autoimmune disease characterised by failure of transmission at the neuromuscular junction (NMJ). In 80–85% of myasthenia gravis patients, the disease is mediated by antibodies to the nicotinic KPT-335 receptor (AChR; (Lindstrom et al., 1976). Many investigations, both in vitro and in passive transfer models, have shown that these antibodies reduce the number of functional AChRs at the postsynaptic membrane by increasing AChR degradation (Drachman et al., 1978), inducing complement-mediated damage to the postsynaptic membrane (Engel et al., 1977), and sometimes by blocking AChR function (Burges et al., 1990). In 2001, IgG antibodies to muscle specific kinase, MuSK, were identified in 70% of the patients without AChR antibodies (Hoch et al., 2001), and have since been detected in varying proportions of patients from Europe, the USA, Japan and Taiwan (reviewed by Vincent and Leite, 2005). MuSK plays an essential role in the agrin-induced clustering of AChRs at the NMJ during development, and preliminary studies showed that MuSK-MG IgG preparations inhibited the agrin-induced clustering of AChRs in the mouse myoblast cell line, C2C12 (Hoch et al., 2001). Moreover, immunoglobulin preparations (IgG) from AChR-antibody negative MG patients, since identified as positive for MuSK antibodies, transferred electrophysiological defects to mice (Mossman et al., 1986, Burges et al., 1994). But when motor endplates were studied in biopsied muscles, Shiraishi et al. (2005) reported no loss of AChR numbers or evidence of immune-complex deposition in MuSK-MG patients, suggesting that the MuSK antibodies do not act by fixing complement and do not reduce AChR numbers overall. It is unclear, therefore, how MuSK antibodies cause a transmission defect in mature muscle, and some authors have questioned their pathogenicity (Selcen et al., 2004). Moreover, the target(s) and mechanism(s) of action of those antibodies in AChR/MuSK antibody negative MG patients (SNMG) are also unexplained.
    Materials and methods Sera or plasma samples were obtained from our archives. MG had been diagnosed on the basis of clinical features, neurophysiological investigations and the presence of AChR or MuSK antibodies when present. Sera were retested to confirm their antibody status. We initially used sera from a total of 13 AChR-MG sera, 12 MuSK-MG, 9 SNMG sera and 8 healthy control sera but for the main experiments, only sera from MuSK-MG 1, 2 and 3 and SNMG 1 and 3 were used (see Table 2).
    Results
    Discussion MuSK antibodies have, to date, only been found in patients with myasthenia gravis and only in those patients without AChR antibodies, except in one report (Ohta et al., 2004), which was subsequently corrected. The manner by which these antibodies reduce neuromuscular transmission is not known, but a reasonable hypothesis is that they reduce the number or stability of AChRs. We used two in vitro cell lines to investigate the effects of MuSK antibodies on AChR numbers, distribution and mRNA expression. We found a small reduction in AChR numbers in TE671 cells but unexpectedly no change in C2C12 myotubes when incubated in the presence of MuSK-MG sera over varying time periods and at different concentrations. Moreover, the effect on agrin-induced AChR clusters in C2C12 myotubes, previously reported (Hoch et al., 2001), appears to relate as much to coalescence of small clusters to form larger ones, as to dispersion of clusters. Overall there was little effect of the MuSK-MG sera on AChR or MuSK expression, whereas SNMG sera marginally increased AChR γ subunit and MuSK expression. Thus these experiments, which are the first to quantify AChRs in C2C12 cells treated with MuSK-MG antibodies, do not demonstrate a potentially pathogenic effect on AChR or MuSK expression. MuSK is crucial for neuromuscular development and MuSK deficient mice have profound NMJ defects as do agrin-deficient mice (DeChiara et al., 1996, Gautam et al., 1996). Myotubes derived from these mice failed to undergo agrin-induced AChR clustering (Glass et al., 1996). AChR cluster formation is dependent on rapsyn (Frail et al., 1988, Froehner, 1991) and mice that are null for rapsyn fail to form AChR clusters during embryological formation (Gautam et al., 1995). However, the importance of MuSK in the maintenance of the mature NMJ has not been clear until recently when Kong et al. (2004) showed that inhibition of MuSK synthesis in rodent muscle, by injection of siRNA, leads to a slow but dramatic dispersion of AChRs and disruption of the NMJ. The mechanisms by which MuSK antibodies alter neuromuscular transmission, however, are not yet clear and it has been suggested that these antibodies may not be pathogenic (Selcen et al., 2004).