Experimental studies have supported the hypothesis that AMM is the result of the autoimmune response directed against MuSK, by observing weakness and NMJ changes in animals actively 32–34
or passively 35,36
immunized with MuSK. In rabbits and mice repeatedly immunized with MuSK, mild weakness has been observed along with mild electrophysiologic evidence of disordered neuromuscular transmission 32
. In addition, in mice passively injected with human AMM IgG repeatedly over 14 days (total of 0.68 g), mild-moderate weakness occurred in conjunction with reduced MuSK and AChR staining and reduced registration between nerve terminals and endplates at NMJs 35
In contrast, the form of EAMM induced in Lewis rats by a single immunization with 100 ug of xenogeneic N-MuSK 60 is extremely severe with 100% mortality by 27 days and very high anti-MuSK antibody titers (>1: 106
). The animals exhibit marked weight loss and axial muscle wasting, the latter not described in the other models. As the disease progresses, the axial weakness/wasting leads to a striking kyphotic posture and eventually the inability of the forelimbs to lift the chest from the floor of the cage. It is of note that similar posture and gait abnormalities have been observed in adult mice in which MuSK expression was turned off using Cre recombinase-mediated MuSK gene deletion 17
. The reproduction of all the characteristics of AMM in the current form of EAMM supports the hypothesis that the autoimmune response to MuSK in AMM is pathogenically important in this disease, rather than representing an epiphenomenon. Moreover, these observations highlight the potential usefulness of this model for studying the pathogenesis and future treatments of AMM.
It is unclear why the disease in the present study is so much more severe than that induced in the three previous studies involving active immunization. Possible factors include differences in species susceptibility to autoimmunity 37,38
or species differences in sensitivity to the anti-MuSK attack, perhaps related to differences in the safety factor of neuromuscular transmission 37,39
. A third possibility relates to the differences in the antigens used to induce the disease and, hence, the epitope targets of the disease-inducing Abs. The MuSK 60 isoform, used as immunogen in the current study, appears to be an adult form of the protein 24
, which may be an important target of the auto-Abs in EAMM and AMM. For the rabbit and mouse forms of EAMM, the immunogens have been either the fetal isoform or another splicing variant of the protein that is missing not only the 20 amino acid extra domain of MuSK 60 but also the entire third Ig domain 32,33,34
Immunization with a lower dose of N-MuSK 60 resulted in lower titers of anti-N-MuSK 60 Abs (1:105) and less severe disease with a much lower mortality. The correlation between anti-N-MuSK 60 antibody titer and disease severity also supports the hypothesis that EAMM and AMM are the result of the action of the MuSK antibodies. The observation () that the low-dose animals exhibit greater diminution in neuromuscular transmission (at least as measured in a distal extremity muscle), albeit at later times following the immunization, may simply relate to the differential effect observed on axial versus distal muscles in EAMM. However, the observation also raises the possibility that the weakness, weight loss and muscle wasting associated with the rapidly fatal high-dose disease may not solely be the consequence of reduced neuromuscular transmission per se, but rather that other physiologic activities at the NMJ may also play a role.
Within individual EAMM muscles, NMJs exhibit varying degrees of disruption. The architecture of some endplates is altered at the ultrastructural level by hypersegmentation consisting of multiple small axon terminals with marked simplification of post synaptic membranes (, ). The nerve terminals at other NMJs are more severely affected, with complete or partial loss of these structures (, ). In addition, some axons have an abnormal globular appearance and others exhibit local extension beyond the NMJ, or even frank terminal axon sprouting (). In some NMJs, there is misalignment between the presynaptic and postsynaptic portions of these synapses. The latter findings, some of which were also observed in the study of passive transfer of human AMM serum into mice 35
and in one of the three studies of AMM 40
, suggest that there is abnormal “signaling” between nerve terminal and muscle endplate in both directions resulting in failure of maintenance of the mature synapse in these animals 41
In addition, cholinesterase staining of teased muscle bundles from N-MuSK 60-immunized animals reveals segmented and dispersed cholinesterase-stained patches, away from the compact synaptic region (motor point) () reminiscent of newly formed synapses, which raises the possibility that the antibody attack leads first to frank denervation at some NMJs with subsequent and, possibly ongoing, attempts at reinnervation. It is of special note that severe damage to the endplate membrane, as is seen in acute forms of EAMG and MG, shown early on in MG by Drachman and associates42
, produces local cholinesterase spreading but minimal nerve terminal abnormalities and/or denervation/distant reinnervation. Hence, the antibody attack on MuSK appears to have wider ranging effects on the NMJ than is seen with the highly destructive antibody attack on the more abundant endplate AChRs in EAMG/MG.
The mechanism of the prominent axial muscle wasting in EAMM rats, also not seen in EAMG and MG, remains unclear. Both histologic and electrophysiologic studies in human AMM suggest that the muscle wasting is not the result of denervation but rather is the consequence of a myopathic process 40,43–45
. Such observations support a role for MuSK in mediating trophic effects on muscle, perhaps through complex two-way communication between nerve and muscle at the NMJ. On the other hand, focal denervation with accompanying reinnervation, as suggested by some of the morphologic studies described above, might also lead to muscle wasting.
Finally, the observations presented here demonstrate that an immune attack on MuSK can result in weakness, muscle wasting and severe disruption of the architecture of both the postsynaptic and presynaptic portions of the mature NMJ, thereby supporting the hypothesis that, in addition to its role in the developing NMJ, MuSK plays a role in the maintenance and function of the mature structure.