运动神经元ev传递的microrna和wnt在促进AChR聚集中的协同作用。

IF 8.2 2区 生物学 Q1 CELL BIOLOGY
Rachele Agostini, Paola Ceccaroli, Emanuela Polidori, Manuela Ferracin, Ilaria Pace, Serena Maggio, Andrea Cioccoloni, Michela Battistelli, Giulia Matacchione, Matilde Sbriscia, Fabiola Olivieri, Fabrizia Cesca, Vilberto Stocchi, Michele Guescini
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引用次数: 0

摘要

背景:神经肌肉连接(NMJ)的建立是通过运动神经元和肌肉纤维之间复杂的交流事件发生的;然而,导致NMJ形成的分子机制尚未完全阐明。细胞外囊泡(EVs)在NMJ形成过程中介导运动神经元和肌纤维相互作用的重要性尚不清楚;本研究探讨了运动神经元来源的电动汽车在NMJ形成的早期阶段的作用。方法:以NSC-34细胞为运动神经元模型;在神经突发育过程中,使用专门用于分离大型和小型电动汽车的系列超离心方案分离电动汽车。通过纳米颗粒跟踪法对分离的ev进行定量,并通过Western Blot和TEM分析对其进行表征。通过小rna测序鉴定EV亚群的microRNA (miRNA)载货,并对预测的miRNA下游靶标进行了研究。结果:NGS分析了NSC-34衍生EV携带的小rna,共鉴定出245个EV特异性mirna,其中大部分在NSC-34细胞和EV的神经突拉伸过程中上调。靶标预测分析证明了这些mirna如何协同靶向Wnt信号通路。此外,我们发现nsc -34衍生的ev携带Wnt蛋白,包括Wnt11、Wnt4和Wnt3a。由于一些研究表明wnt相关信号网络在NMJ形成中的作用,我们研究了NSC-34 EV在NMJ形成中的潜在作用,并通过α-bungarotoxin免疫荧光染色显示,EV给肌管增加了乙酰胆碱受体(AChR)簇的形成。此外,用nsc -34来源的ev处理肌管导致GSK3β和JNK磷酸化,随后是β-catenin核易位,这表明神经元来源的ev可以通过Wnt通路激活诱导AChR聚集。结论:这些数据表明,分化的运动神经元释放的ev携带多模态信号、mirna和wnt,可以刺激肌管AChR聚集,这是NMJ形成的基本准备阶段。这些新数据表明,在生理和病理条件下,特别是神经退行性疾病下,ev可能在NMJ的建立和功能中发挥作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Synergic action of MicroRNAs and Wnts delivered by motor neuron EVs in promoting AChR clustering.

Background: The neuromuscular junction (NMJ) establishment occurs through complex communication events between motor neurons and muscle fibers; however, the molecular mechanisms leading to NMJ formation have yet to be fully elucidated. Little is known about the significance of extracellular vesicles (EVs) in mediating the interaction between motor neurons and muscle fiber in the NMJ establishment; this study investigates the role of motor neuron-derived EVs during the earliest stages of NMJ formation.

Methods: NSC-34 cells have been used as a model of motor neurons; EVs have been isolated during neurite development using a serial ultracentrifugation protocol specifically adjusted to isolate large and small EVs. Isolated EVs were quantified through Nanoparticles Tracking Assay and characterized by Western Blot and TEM analyses. The microRNA (miRNA) cargo of EV subpopulations was identified by small-RNA sequencing and the predicted miRNA downstream targets were investigated.

Results: NGS analysis of small RNAs carried by NSC-34-derived EVs identified a total of 245 EV specific miRNAs, most of which are up-regulated in NSC-34 cells and EVs during neurite stretching. Target prediction analysis evidenced how these miRNAs synergically target the Wnt signaling pathway. Moreover, we found that NSC-34-derived EVs carry Wnt proteins, including Wnt11, Wnt4 and Wnt3a. Since several studies suggested a role for the Wnt-associated signaling network in NMJ formation, we investigated the potential role of NSC-34 EVs in NMJ development and demonstrated that EV administration to myotubes increases acetylcholine receptor (AChR) cluster formation, as revealed by immunofluorescence staining with α-bungarotoxin. Moreover, myotube treatment with NSC-34-derived EVs led to GSK3β and JNK phosphorylation, followed by β-catenin nuclear translocation, suggesting that neuron-derived EVs can induce AChR clustering through Wnt pathway activation.

Conclusion: These data demonstrate that EVs released from differentiated motor neurons carry multimodal signals, miRNAs, and Wnts, which can stimulate AChR clustering in myotubes, a fundamental preparatory stage for NMJ formation. These new data highlight that EVs may play a role in the NMJ establishment and function under physiological and pathological conditions, particularly neurodegenerative diseases.

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来源期刊
CiteScore
11.00
自引率
0.00%
发文量
180
期刊介绍: Cell Communication and Signaling (CCS) is a peer-reviewed, open-access scientific journal that focuses on cellular signaling pathways in both normal and pathological conditions. It publishes original research, reviews, and commentaries, welcoming studies that utilize molecular, morphological, biochemical, structural, and cell biology approaches. CCS also encourages interdisciplinary work and innovative models, including in silico, in vitro, and in vivo approaches, to facilitate investigations of cell signaling pathways, networks, and behavior. Starting from January 2019, CCS is proud to announce its affiliation with the International Cell Death Society. The journal now encourages submissions covering all aspects of cell death, including apoptotic and non-apoptotic mechanisms, cell death in model systems, autophagy, clearance of dying cells, and the immunological and pathological consequences of dying cells in the tissue microenvironment.
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