Blocking skeletal muscle DHPRs/Ryr1 prevents neuromuscular synapse loss in mutant mice deficient in type III Neuregulin 1 (CRD-Nrg1)

Schwann cells are integral components of vertebrate neuromuscular synapses; in their absence, pre-synaptic nerve terminals withdraw from post-synaptic muscles, leading to muscle denervation and synapse loss at the developing neuromuscular junction (NMJ). Here, we report a rescue of muscle denervation and neuromuscular synapses loss in type III Neuregulin 1 mutant mice (CRD-Nrg1(-/-)), which lack Schwann cells. We found that muscle denervation and neuromuscular synapse loss were prevented in CRD-Nrg1(-/-)mice when presynaptic activity was blocked by ablating a specific gene, such as Snap25 (synaptosomal-associated 25 kDa protein) or Chat (choline acetyltransferase). Further, these effects were mediated by a pathway that requires postsynaptic acetylcholine receptors (AChRs), because ablating Chrna1 (acetylcholine receptor 1 subunit), which encodes muscle-specific AChRs in CRD-Nrg1(-/-)mice also rescued muscle denervation. Moreover, genetically ablating muscle dihydropyridine receptor (DHPR) 1 subunit (Cacnb1) or ryanodine receptor 1 (Ryr1) also rescued muscle denervation and neuromuscular synapse loss in CRD-Nrg1(-/-)mice. Thus, these genetic manipulations follow a pathway-from presynaptic to postsynaptic, and, ultimately to muscle activity mediated by DHPRs and Ryr1. Importantly, electrophysiological analyses reveal robust synaptic activity in the rescued, Schwann-cell deficient NMJs in CRD-Nrg1(-/-)Cacnb1(-/-)or CRD-Nrg1(-/-)Ryr1(-/-)mutant mice. Thus, a blockade of synaptic activity, although sufficient, is not necessary to preserve NMJs that lack Schwann cells. Instead, a blockade of muscle activity mediated by DHRPs and Ryr1 is both necessary and sufficient for preserving NMJs that lack Schwann cells. These findings suggest that muscle activity mediated by DHPRs/Ryr1 may destabilize developing NMJs and that Schwann cells play crucial roles in counteracting such a destabilizing activity to preserve neuromuscular synapses during development. Author summary Nerve cells (neurons) communicate with each other through specialized focal connections called synapses. Synapses are commonly assembled into a tripartite structure composed of pre- and post-synaptic neurons, and a glial cell that is closely associated with both pre- and post-synaptic components. At the vertebrate neuromuscular synapse (the neuromuscular junction, NMJ), the tripartite structure is composed of a nerve terminal, a muscle fiber and a glial cell called the terminal Schwann cell. Previous studies have shown that Schwann cells are vital to the NMJ-without Schwann cells, the NMJ fails to form. Here, we report unexpected findings that bipartite NMJs can be established in vivo, in the absence of Schwann cells. These results were accomplished by ablating specific genes in mice, ultimately resulting in a total blockade of muscle activity. Thus, Schwann cells become dispensable for the NMJ if muscle activity is blocked. These findings suggest that muscle activity normally destabilizes the NMJ during synapse formation and that Schwann cells play crucial roles in NMJ formation by opposing this muscle-derived destabilizing activity in order to preserve the developing neuromuscular synapses.