Abstract
Objective
Munc18 is considered as an intracellular protein that plays an important role in exocytosis of neurotransmitters. Previous studies have demonstrated the presence of autoantibodies against Munc18 in a subgroup of Rasmussen’s encephalitis patients. However, the machinery of Munc18 autoimmunity is still elusive. The present study was aimed to investigate Munc18 release from primary cultured neurons, Munc18 distribution on the outer plasma membrane of neurons, and the neurotoxicity of Munc18 antibody.
Methods
The cerebral cortical neurons from embryonic day 17 Sprague-Dawley rats were prepared and cultured in neurobasal medium. The proteins in culture medium were precipitated with 10% trichloroacetic acid, and analyzed by immunoblotting. The proteins on neuronal surface were biotinylated with EZ-Link-sulfo-NHS-LC-Biotin, and collected with avidin-conjugated agarose beads followed by immunoblotting analysis. For cell surface immunofluorescent staining, the living neurons were labeled with anti-Munc18 antibody at 4 °C. Neuronal injury was assessed by lactate dehydrogenase(LDH) release.
Results
Munc18 was detected in culture medium by immunoblotting analysis. After treatment with 50 μmol/L glutamate for 1 h, Munc18 content in medium was increased. Meanwhile, β-actin and syntaxin1 were not detected in culture medium, and LDH release was not significantly increased. Moreover, glutamate enhanced Munc18 distribution on outer plasma membrane. Living neuron staining also demonstrated the localization of Munc18 on neuronal surface after glutamate treatment, especially at contacting regions between neurons. Glutamate-induced increase of surface Munc18 distribution was suppressed by NMDA receptor antagonist MK801, but not by AMPA receptor antagonist NBQX. Moreover, compared with c-Fos antibody, Munc18 antibody could induce neuronal injury, when culture medium contained the components of serum.
Conclusion
A portion of Munc18 can be released from neurons or distributed on neuronal surface, which can be enhanced by glutamate treatment via activation of NMDA receptors. Besides, Munc18 antibody-induced neuronal injury depends on the serum components.
Keywords: Munc18, neuron, release, cell surface, glutamate
摘要
目的
Munc18 被普遍认为存在于神经元细胞内, 参与神经递质的释放。 虽然已有研究表明在Rasmussen’s脑炎病人中有Munc18 自身抗体的产生, 但对于Munc18 的自身免疫机制目前还不清楚。本研究旨在观察Munc18是否可由神经元释放及是否部分分布于神经元细胞表面, 并对Munc18抗体是否能诱导神经元损伤进行研究。
方法
分离胎龄17 天Sprague-Dawley 胎鼠大脑皮层神经元, 用Neurobasal 培养基培养。培养液中的蛋白经三氯乙酸沉淀后进行免疫印迹检测, 细胞表面蛋白则用EZ-Link-sulfo-NHS-LC-Biotin进行生物素标记, 细胞裂解后用结合有亲和素的琼脂糖珠获取生物素标记的蛋白, 随后进行免疫印迹检测。在4 °C 进行活细胞免疫荧光染色, 观察神经元细胞表面Munc18 的分布。用乳酸脱氢酶(lactate dehydrogenase, LDH)释放指标检测神经元损伤。
结果
用免疫印迹法可在神经元培养的条件培养液中检测到Munc18 蛋白。用谷氨酸(50 μmol/L)处理神经元1 h 后, 培养液中Munc18含量显著增加, 同时培养液中未检测到β-actin 和syntaxin1, LDH 释放也未明显增加。此外, 谷氨酸处理能增强神经元表面的Munc18分布。活细胞免疫荧光染色检测到在谷氨酸处理后, 神经元表面分布有Munc18, 主要位于神经突起与另一神经元接触的部位以及不同神经元的神经末梢交汇部位。谷氨酸诱导的神经元表面Munc18分布增加可被NMDA受体拮抗剂MK801 抑制, 而AMPA受体拮抗剂未有明显的抑制作用。LDH检测显示, 在培养液含有血清成份的情况下, 与对照的c-Fos 抗体相比, Munc18 抗体可明显诱导神经元损伤。
结论
一部分Munc18 可 由神经元释出或在神经细胞表面分布。谷氨酸可通过兴奋NMDA受体促进Munc18 的释放和在神经细胞表面的分布。并且, Munc18 抗体诱导的神经元损伤依赖于血清中的因子。
关键词: Munc18, 神经元, 释放, 细胞表面, 谷氨酸
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