Barik Arnab, Lu Yisheng, Sathyamurthy Anupama, Bowman Andrew, Shen Chengyong, Li Lei, Xiong Wen-cheng, Mei Lin
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912.
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912, Department of Neurology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912, and Charlie Norwood Veterans Administration Medical Center, Augusta, Georgia 30904.
J Neurosci. 2014 Oct 15;34(42):13892-905. doi: 10.1523/JNEUROSCI.1733-14.2014.
The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.
神经肌肉接头（NMJ）是运动神经元与骨骼肌纤维之间的突触，对肌肉收缩的控制至关重要。其形成需要神经元聚集蛋白，该蛋白通过与低密度脂蛋白受体相关蛋白4（LRP4）结合来刺激肌肉特异性激酶（MuSK）发挥作用。在先天性肌无力综合征患者中已鉴定出聚集蛋白、MuSK和LRP4的突变，重症肌无力患者会产生针对聚集蛋白、LRP4和MuSK的抗体。然而，由于这三个基因中的任何一个基因的无效突变在围产期都是致死性的，因此聚集蛋白信号通路对神经肌肉接头维持是否至关重要仍不清楚。在本研究中，我们构建了诱导型敲除（imKO）小鼠，这是一种突变品系，其LRP4基因可通过强力霉素（Dox）处理在肌肉中被删除。在成年肌肉中敲除LRP4基因能够研究其在神经肌肉接头维持中的作用。我们证明，对30日龄小鼠进行Dox处理会降低肌肉力量和复合肌肉动作电位。乙酰胆碱受体（AChR）簇变得碎片化，连接褶皱和突触小泡减少。微小终板电位的幅度和频率降低，表明神经肌肉传递受损，并提供了成年LRP4缺乏的细胞机制。我们发现，LRP4基因敲除导致突触聚集蛋白和90 kDa片段丢失，这发生在其他突触前和突触后成分之前，表明LRP4可能调节突触聚集蛋白的稳定性。这些观察结果表明，LRP4对于维持神经肌肉接头的结构和功能完整性至关重要，并且仅成年期肌肉LRP4的缺失就足以导致肌无力症状。
