Role of Ca2+ channels in AChR pre-patterning / neuromuscular junction development

The role of calcium channels in acetylcholine receptor pre-patterning during neuromuscular junction development The neuromuscular junction (NMJ) is the synapse between the motor neuron and muscle cells. Due to its experimental accessibility and the availability of unique molecular tools and animal models, the NMJ served as the prime model for research on synapse formation, and in particular to study the mechanisms that govern the accumulation of acetylcholine receptors (AChR) at the crests of the postsynaptic folds. Today it is generally accepted that both muscle-intrinsic and nerve-derived factors cooperate in the aggregation and stabilization of AChR in the postsynaptic membrane. Nevertheless the signaling mechanisms regulating these processes are still incompletely understood. AChR pre- patterning is the initial step in the formation of the NMJ by which muscle-autonomous mechanisms cause the aggregation of AChR clusters in the central zone of the muscle fiber and thus determine the territory accessible for innervation by the motor neuron. Recently an essential role of L-type calcium currents (LTCC) in this process was reported. Moreover, LTCC have been implicated in the signaling mechanisms regulating the subsequent stabilization of synaptic AChRs as well as the destabilization of extrasynaptic AChRs. Our laboratory recently discovered a high-conductance embryonic splice variant of the voltage- gated calcium channel (CaV1.1e), which likely is the source of the LTCC that regulate the aggregation and stabilization AChR. In this research project we will take advantage of several available genetic mouse models (classical and newly generated) for calcium channels to test the importance of CaV1.1e in NMJ formation and to elucidate the mechanisms by which calcium signals contribute to AChR aggregation and stabilization. We hypothesize thatif LTCC are essential for AChR pre-patterning or other calcium-regulated steps of NMJ formationthese processes should fail in mice lacking CaV1.1 or expressing a non-conducting CaV1.1 channel. Furthermore, mice lacking the ryanodine receptor and double mutants will establish the relative importance of calcium influx and release. Finally, hybrids of calcium channel mutants and mice lacking either the motor neurons or the AChR- aggregating factor agrin will allow us to scrutinize the specific roles of the nerve and the muscle cell in the calcium-mediated signaling mechanisms regulating AChR clustering in the postsynaptic membrane. Thus, studying AChR aggregation in these mouse models is expected to elucidate the importance of calcium signaling in the development of the NMJ, to determine the underlying calcium source, and possibly to assign a function in synapse formation to the newly found CaV1.1e calcium channel splice variant.

The role of calcium channels in acetylcholine receptor pre-patterning during neuromuscular junction development Synapses are molecularly highly specialized site of communication between nerves and their target cells. Their formation during early development requires the reciprocal interaction between the presynaptic nerve terminal and the postsynaptic target cell to establish specific contacts and to differentiate a functional synapse The neuromuscular junction (NMJ) is the synapse between the motor neuron and the muscle fiber, by which the nervous system controls contractile activity of the muscle. In the adult, each muscle fiber receives input from a motor neuron at a single NMJ located in the center of the muscle fiber. Interestingly, the muscle cell dictates where the synapse is formed by arranging the postsynaptic receptors for the neurotransmitter acetylcholine (AChR) in a narrow band in the center of the muscle prior to the nerves arrival. In this project we studied the underlying mechanisms; in particular the role of activity-dependent calcium signals in AChR patterning during early synapse formation. Using a range of genetic mouse models with altered calcium channels we found that muscle calcium signaling is the key to correct AChR patterning. In the absence of activity-dependent calcium signals AChR clusters failed to be aggregated in the center of the muscle and the incoming nerve processes showed unrestricted growth and over-branching. Importantly either influx of calcium through a voltage-activated channel in the plasma membrane or the activity-dependent release of calcium from intracellular stores, were sufficient to restore proper AChR patterning and innervation. Our results show a delicate dependence of this important initial step in NMJ formation on the magnitude of the calcium signal. Unexpectedly, our results further demonstrate that the growth pattern of the motor nerve, its recognition of the postsynaptic AChR clusters, and the differentiation of functional nerve terminals also is controlled by the muscle cell in a calcium-dependent manner. Together these results demonstrate that the same signaling mechanism that in adults regulates muscle contraction, during early development is used for the proper formation of its synapses and that voltage-gated calcium channels are of central importance for this essential developmental process.