Role of calcium channels in presynaptic differentiation

The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle, and it represents one of the foremost models for the study of synaptogenesis. The development of the neuromuscular synapse requires a series of reciprocal interactions between the presynaptic motor neuron and postsynaptic muscles cells. Very recently we reported that calcium influx and release in skeletal muscle cooperatively regulate the organization and innervation of acetylcholine receptors (AChR) during NMJ formation (Kaplan et al., Cell Reports 23, 3891-3904, 2018). Unexpectedly, we further detected that in mouse mutants lacking the skeletal muscle calcium channel (CaV1.1) motor axons failed to stop and differentiate nerve terminals at postsynaptic AChR clusters. This observation suggests that CaV1.1 regulates the expression or distribution of factors involved in directing the motor nerve to prospective synaptic sites and the differentiation of functional nerve terminals. Therefore, in the proposed project we will apply several genetic calcium channel mouse models and double-mutants thereof, as well as a nerve-muscle co-culture system to examine the importance of the postsynaptic CaV1.1 channel in regulating the guidance and differentiation of the presynaptic motor nerve terminal and to elucidate the underlying signaling mechanism. Moreover, we will identify the muscle-derived retrograde signals regulated by CaV1.1 and attempt to rescue the presynaptic defects in CaV1.1-/- mice by generating double-mutant mice overexpressing or deficient in the putative synaptogenic factor. The expected results will elucidate an important trans-synaptic signaling mechanism involved in the coordinated development of pre- and postsynaptic structures of the NMJ, and it will assign a hitherto unnoticed function in synapse formation to the skeletal muscle calcium channel CaV1.1. Because the NMJ is central in the pathology of several detrimental neurological diseases, including Lampert Eaton syndrome, myasthenia gravis, and ALS, and trophic factors involved in the differentiation and stabilization of synapses represent therapeutic targets, unravelling the signaling pathway regulating such factors will advance our understanding of the disease mechanisms and possibly contribute to modern therapeutically strategies.

THE ROLE OF CaV1.1 CALCIUM CHANNELS IN PRESYNAPTIC DIFFERENTIATION DURING NEUROMUSCULAR JUNCTION DEVELOPMENT. The neuromuscular junction (NMJ) is the synapse between motor neurons and skeletal muscle, and it represents one of the foremost models for the study of synaptogenesis. The development of the neuromuscular synapse requires a series of reciprocal interactions between the presynaptic motor neuron and postsynaptic muscles cells. Recently we reported that calcium influx and release in skeletal muscle cooperatively regulate the organization and innervation of acetylcholine receptors (AChR) during NMJ formation (Kaplan et al., Cell Reports 23, 3891-3904, 2018). Unexpectedly, we further detected that in mouse mutants lacking the skeletal muscle calcium channel (CaV1.1) motor axons failed to stop and differentiate nerve terminals at postsynaptic AChR clusters. This observation suggested that CaV1.1 regulates the expression or distribution of factors involved in directing the motor nerve to prospective synaptic sites and the differentiation of functional nerve terminals. In this project, first we thoroughly characterized the effects of lacking CaV1.1-derived calcium signals on different aspects of nerve guidance and presynaptic differentiation in a CaV1.1-null mouse model (Kaplan and Flucher, Scientific Reports 9:18450, 2019). Recognizing the striking similarity between the presynaptic phenotype of CaV1.1-null mutant mice and mouse models of the signal transduction protein -catenin, we investigated the relationship of both signaling mechanisms in double-mutant mice unraveling their counteractive and cooperative actions on pre- and postsynaptic differentiation during neuromuscular junction formation (Kaplan and Flucher, iScience 25:104025, 2022). Finally, we performed the first spatial genomics analysis of developing mouse diaphragm muscle of normal and mutant mice, in order to identify candidate genes for muscle-to-nerve signaling involved in the CaV1.1-dependent regulation of presynaptic differentiation (publication in progress). Together, these results elucidated an important trans-synaptic signaling mechanism involved in the coordinated development of pre- and postsynaptic structures of the NMJ, and thus assigned a hitherto unnoticed function in synapse formation to the skeletal muscle calcium channel CaV1.1. Because the NMJ is central in the pathology of several detrimental neurological diseases, including Lampert Eaton syndrome, myasthenia gravis, and ALS, and trophic factors involved in the differentiation and stabilization of synapses represent therapeutic targets, unravelling the signaling pathway regulating such factors advanced our understanding of the disease mechanisms and might possibly contribute to modern therapeutically strategies.