Effects of virus infection and interferon-y on the development of the neuromuscular junction in vitro

Sammanfattning: Normal development of the nervous system depends upon complex interactions between cells and their local environment. These interactions may be disturbed by toxic and infectious agents. It is well known that such agents can cause malformations, due to interference with neural tube formation or neuronal proliferation and migration. However, little is known about potential effects on neuronal differentiation, and in particular on synapse formation. Neuromuscular junctions have served as models in studies of synaptic structure and function and as such they have been used in this thesis. The thesis examines if mumps virus andIFN-y can affect the interactions between nerve and muscle cells in culture, with particular reference to the myotubes capacity to aggregate nicotinic acetylcholine receptors (AChRs), which is an early event during the development of the neuromuscular junctions. Fluorescence-conjugated a-bungarotoxin (FlTC-a-Btx), that binds specifically to the AChR was used in order to study changes of receptor distribution during development. The localization of various virus proteins and skeletal muscle specific cytoskeleton proteins was studied with immunohistochemical techniques. Myoblast proliferation was studied using a thymidine analog as a marker of cell proliferation. Different nerve fiber populations coming to the myotubes were visualized with immunofluorescence of synaptophysin and a novel cholinergic-specific plasma-membrane antigen. The specificity of this novel cholinergic antigen, designated asChol-2, was recognized by its antisera ability to sensitize the cholinergic subpopulation of rat-brain synaptosomes and to lyse them by the complement system. From the presented studies, it was shown that the RW-strain of mumps virus can selectively infect skeletal muscle cells during the myoblast stage of development; and that such infected myoblasts can fuse to form virus antigen containing myotubes. The formation of AChR aggregates in these infected myotubes was inhibited when they were co-cultured with spinal cord cells. Similar results were obtained by exposure of the cultures to lymphocyte derived IFN-y, as well as to a neuronal IFN-y like molecule isolated from sensory ganglia. This isolated molecule had a molecular weight distinct from that of Iymphocyte-derived IFN-y, but shared a number of bioactivities, namely antiviral effects, increase of skeletal muscle cell proliferation and induction of MHC antigens expression. A notable effect was also that the molecule, like lymphocyte-derived IFN-y, induced nitric oxide (NO)production in cultures from the nervous system. NO has been implied to play a role in the modulation of synapse formation, but no effects on AChR aggregation was observed in the present studies. In conclusion, this thesis demonstrates that a non-lytic infection with a common paramyxovirus and a key regulative cytokine can inhibit AChR aggregation and thus disturb the formation of synapses. This may be of significance for our understanding of the pathogenic mechanisms underlying disturbances of the nervous system which may evolve as a result of infections during infancy.