Functional characterization of MuSK, receptor tyrosine kinase required for the formation and the maintenance of nerve-muscle synapses : "in vivo" and "in vitro" approaches

The developing neuromuscular junction (NMJ) serves as one of the best model systems for studying synapse formation since changes in shape, size, and molecular composition can be followed with high spatial and temporal resolution. Formation of the NMJ depends on coordinated interactions between nerve terminals and muscle fibres [1] and requires reciprocal signals from both cells to efficiently regulate all the events taking place during its development. This includes synapse-specific gene expression, generation of action potentials and stabilization events leading to the formation of a sophisticated apparatus which ensures that the muscle fibre is provided with trophic factors as well as electrical stimuli. The receptor tyrosine kinase MuSK and its natural ligand, a neuron-specific isoform of the extracellular matrix molecule agrin, are considered to play a fundamental role in the formation and maintenance of the NMJ. In cultured myotubes, MuSK is activated by neural agrin, and this causes its phosphorylation and results in the formation of AChRs clusters on the cell surface [2-5]. The present study discusses different approaches to understand better the mechanisms of how the NMJ is formed and maintained. In the first project, we addressed the question of MuSK – neural agrin interaction and the necessity for an additional component of the agrin receptor complex. We generated transgenic mice overexpressing MuSK or neural mini-agrin as well as both proteins throughout the entire muscle fibre. We found evidence that in muscle cells MuSK is sufficient to respond to neural agrin with no necessity of any additional co-receptor protein. We also show that Dok-7, a MuSK adaptor protein, limits the formation of ectopic postsynaptic like structures in innervated muscle. From this, we conclude that it is very likely that in muscle cells MuSK serves as a functional receptor for neural agrin. The second project refers to the regulation of the NMJ formation. We found that signal transduction downstream of agrin involves the mitogen-activated protein kinase (MAPK) pathway, particularly ERK1/2 and JNK. It involves MuSK signaling, requires Dok7 and is ErbB-independent. We also show that MAPK phosphatase-1, MKP-1, plays a crucial regulatory role in formation of the nerve-muscle connection. Results of the third project describe that a miniaturized form of agrin is able to fully rescue perinatal death of agrin-deficient mice, and that this function does not depend on local deposition of agrin at synapses. Moreover, we show that acetylcholine together with neural agrin stabilizes the postsynaptic structures at the NMJ. The function of agrin in CNS was our main interest in the fourth project. Using
agrin-deficient mice with a transgenic reconstitution of the expression of neural agrin by motor neurons, we found that in the brain, agrin is localized to the excitatory synapses. Lack of agrin resulted in a strong reduction of synaptic structures in the cerebral cortex coinciding with the attenuation of the frequency of miniature postsynaptic currents. Additionally we found that muscle specific kinase MuSK is also expressed in the brain, thus possibly involved in the formation of the nerve-nerve connections. Finally, we show that agrin function involves MAP kinase signaling.