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 α1 subunit), which encodes muscle-specific AChRs in CRD-Nrg1−/−mice also rescued muscle denervation. Moreover, genetically ablating muscle dihydropyridine receptor (DHPR) β1 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.

施万细胞（Schwann cells）是脊椎动物神经肌肉突触（neuromuscular synapses）的不可或缺的组成部分；当其缺失时，突触前神经末梢会从突触后肌纤维处回缩，进而导致发育中的神经肌肉接头（neuromuscular junction, NMJ）出现肌肉去神经支配与突触丢失。本研究报道了对III型神经调节蛋白1突变小鼠（CRD-Nrg1−/−，该模型缺乏施万细胞）的肌肉去神经支配与神经肌肉突触丢失的挽救策略。我们发现，在CRD-Nrg1−/−小鼠中，通过敲除Snap25（突触体相关蛋白25 kDa，synaptosomal-associated 25 kDa protein）或Chat（胆碱乙酰转移酶，choline acetyltransferase）等特定基因以阻断突触前活动时，肌肉去神经支配与神经肌肉突触丢失的现象可被阻止。进一步研究发现，该效应依赖于突触后乙酰胆碱受体（acetylcholine receptors, AChRs）介导的信号通路：在CRD-Nrg1−/−小鼠中敲除编码肌肉特异性乙酰胆碱受体的Chrna1（乙酰胆碱受体α1亚基，acetylcholine receptor α1 subunit），同样可挽救肌肉去神经支配现象。此外，在CRD-Nrg1−/−小鼠中遗传敲除肌肉二氢吡啶受体（dihydropyridine receptor, DHPR）β1亚基（Cacnb1）或兰尼碱受体1（ryanodine receptor 1, Ryr1），同样可挽救肌肉去神经支配与神经肌肉突触丢失的表型。综上，上述遗传操作所遵循的信号通路为：从突触前到突触后，最终经由DHPR与Ryr1介导的肌肉活动实现。值得注意的是，电生理分析显示，在施万细胞缺陷型的CRD-Nrg1−/−Cacnb1−/−或CRD-Nrg1−/−Ryr1−/−突变小鼠的挽救后神经肌肉接头中，可检测到强烈的突触活动。由此可见，阻断突触活动虽足以维持缺乏施万细胞的神经肌肉接头，但并非维持该接头的必需条件。与之相反，阻断由DHPR与Ryr1介导的肌肉活动，才是维持缺乏施万细胞的神经肌肉接头的充分且必要条件。本研究结果提示，DHPR/Ryr1介导的肌肉活动可能会使发育中的神经肌肉接头不稳定，而施万细胞在发育过程中可通过对抗此种去稳定活性，发挥维持神经肌肉突触的关键作用。