Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction
Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction
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DOI：
10.1016/j.neuropharm.2021.108637
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发表时间：
2021-06-19
影响因子：
4.7
通讯作者：
Ohno, Kinji
中科院分区：
文献类型：
--
作者：
Inoue, Taro;Ohkawara, Bisei;Ohno, Kinji
重症肌无力、先天性肌无力综合征和运动神经元疾病中乙酰胆碱受体（AChR）聚集减少损害神经肌肉接头（NMJ）的信号传递。尽管增强乙酰胆碱受体在NMJ的聚集是治疗这些疾病的一种有前途的策略，但目前还没有药物可用于这种增强。我们以前报道过，唑尼沙胺（ZNS），一种抗癫痫和抗帕金森病的药物，可增强初级脊髓运动神经元（SMNs）的轴突伸长。由于神经发芽发生，以弥补人类疾病中AChR簇的损失，我们研究了ZNS对NMJ AChR簇的影响。为此，我们建立了一个简单而快速的共培养系统，使用C2 C12肌管和NSC 34运动神经元可重复地在体外制造NMJ。ZNS在1-20 μ M增强AChR簇的形成剂量依赖性在共培养的C2 C12肌管，但不是在agrin处理的单一培养的C2 C12肌管。我们观察到赋予对ZNS的响应性的分子不分泌到共培养基中。我们发现，10 μ M ZNS上调神经调节蛋白-1（Nrg 1）的表达在共培养的细胞，但不是在单一培养的C2 C12肌管或单一培养的NSC 34运动神经元。根据这一观察，Nrg 1/ErbB信号通路的抑制抵消了10 μ M ZNS对体外NMJ中AChR聚集增强的作用。虽然在共培养的细胞中，10 μ M ZNS不能诱导聚集蛋白，但抗聚集蛋白抗体减弱了ZNS介导的AChR聚集增强。我们的结论是，ZNS增强聚集蛋白依赖AChR的集群通过上调Nrg 1/ErbB信号通路的存在下，NMJ。
Decreased acetylcholine receptor (AChR) clustering compromises signal transmission at the neuromuscular junction (NMJ) in myasthenia gravis, congenital myasthenic syndromes, and motor neuron diseases. Although the enhancement of AChR clustering at the NMJ is a promising therapeutic strategy for these maladies, no drug is currently available for this enhancement. We previously reported that zonisamide (ZNS), an anti-epileptic and anti-Parkinson's disease drug, enhances neurite elongation of the primary spinal motor neurons (SMNs). As nerve sprouting occurs to compensate for the loss of AChR clusters in human diseases, we examined the effects of ZNS on AChR clustering at the NMJ. To this end, we established a simple and quick co-culture system to reproducibly make in vitro NMJs using C2C12 myotubes and NSC34 motor neurons. ZNS at 1-20 mu M enhanced the formation of AChR clusters dose-dependently in co-cultured C2C12 myotubes but not in agrin-treated single cultured C2C12 myotubes. We observed that molecules that conferred responsiveness to ZNS were not secreted into the co-culture medium. We found that 10 mu M ZNS upregulated the expression of neuregulin-1 (Nrg1) in co-cultured cells but not in single cultured C2C12 myotubes or single cultured NSC34 motor neurons. In accordance with this observation, inhibition of the Nrg1/ErbB signaling pathways nullified the effect of 10 mu M ZNS on the enhancement of AChR clustering in in vitro NMJs. Although agrin was not induced by 10 mu M ZNS in co-cultured cells, anti-agrin antibody attenuated ZNS-mediated enhancement of AChR clustering. We conclude that ZNS enhances agrin-dependent AChR-clustering by upregulating the Nrg1/ErbB signaling pathways in the presence of NMJs.