Toward Understanding Aging Mechanisms of Neuromuscular Junctions

项目摘要 神经肌肉接头（NMJ）是脊髓运动神经元（MN）与肌纤维相遇的独特突触部位， 形成一个功能运动单位（MU），由三个主要组成部分组成，MN的突触前，肌纤维的突触后， 突触和终末许旺细胞（SC）。近年来，越来越多的注意力集中在 “NMJ老化”与肌肉减少症有关，肌肉质量和力量的损失与老化有关。在这里， 我们提出了两个互补的目标，重点是解开与肌肉减少症有关的NMJ衰老机制： <SA-1>确定衰老时最早的肌肉减少症相关分子变化和潜在生物标志物 体内NMJ-我们将首次对在不同时间分离的显微解剖的NMJ进行RNAseq 点从老龄小鼠，使用激光捕获显微切割（LCM）技术。一系列既定的 然后进行生物信息学分析以提取候选分子，然后鉴定它们的细胞 起源.为了在LCM中获得高质量的RNA，我们将应用我们的原始技术，该技术只需要几个 几分钟来观察NMJ。我们将优先验证先前的治疗靶点候选物 与神经肌肉疾病和具有已知配体或其他潜在的生物标志物候选物相关 监测战略。<SA-II>设计一种新的成熟和老化NMJ的体外共培养系统-我们 将采用一个双室系统，重现中枢神经系统（MN）的生理分离 和星形胶质细胞[AST]）和外周（MN轴突、肌管[MT]和SC）神经系统。的 神经胶质细胞（AST和SC）将为MU提供生物学上所需的结构和营养支持， 最小化对培养基补充的需要。这一策略对于一个忠实的后续 NMJ老化的建模，其被认为与构成MU的细胞的功能衰退相关。 此外，我们将用纤维蛋白凝胶支架保护两个隔室中的细胞，以防止细胞脱离 以及由于频繁的MT收缩导致的突触断开。此外，间歇性电刺激 (IES)将提供给MU，以促进其成熟。一旦到期，我们将终止 IES的“肌肉运动效应”并减少营养因子以模拟人类的自然衰老。NMJ成熟度和 将在多个时间点监测老化的分子、形态和功能变化。最后， 所开发的共培养系统将用于筛选和确定分子的性质 在SA-I中识别。<Study impact>我们的新型NMJ-LCM具有尖端的生物信息学分析， 产生宝贵的数据，以揭示病理学，并获得潜在的治疗见解肌肉减少症。在 此外，我们创新的共培养系统可能成为第一个实现完全成熟的体外模型 和随后的NMJ老化，并作为一个强大的工具，机制和翻译研究， 肌肉减少症这项工作也将有助于更广泛的神经肌肉研究领域，从基础 从MU/NMJ的研究，到各种其他神经肌肉疾病的更多翻译工作。

PROJECT SUMMARY Neuromuscular junctions (NMJs), a unique synaptic site where spinal motor neurons (MNs) meet myofibers to form a functional motor unit (MU), consist of three major components, MN’s pre-synapse, myofiber’s post- synapse, and terminal Schwann cells (SCs). Increasing attention has recently been paid to the etiology of “NMJ aging” in relationship to sarcopenia, the loss of muscle mass and strength associated with aging. Here, we propose two complementary aims focusing on unraveling NMJ aging mechanisms in relation to sarcopenia: <SA-1> Identifying the earliest sarcopenia-associated molecular changes and potential biomarkers at aging NMJs in vivo - We will perform, for the first time, RNAseq of micro-dissected NMJs, isolated at different time points from aging mice, using a laser-capture micro-dissection (LCM) technique. A series of established bioinformatics analyses will be then run to extract candidate molecules followed by identification of their cellular origins. To obtain high-quality RNA in the LCM, we will apply our original technique, which requires only a few minutes to visualize the NMJ. We will prioritize the validation of therapeutic target candidates previously associated with neuromuscular diseases and biomarker candidates with known ligands or other potential monitoring strategies. <SA-II> Engineering a novel in vitro co-culture system of mature and aging NMJs - We will employ a double-compartmented system recapitulating the physiological separation of the central (MNs and astrocytes [ASTs]) and peripheral (MN axons, myotubes [MTs] and SCs) nervous systems in vivo. The glial cells (ASTs and SCs) will provide the MU with the biologically required structural and trophic support, minimizing the need for culture media supplementation. This strategy is essential for a faithful subsequent modeling of NMJ aging, which is thought to be associated with functional decline of cells constituting the MUs. In addition, we will protect cells in both compartments with fibrin gel scaffoldings to prevent cell detachment and synaptic disconnection due to frequent MT contraction. Furthermore, intermittent electrical stimulation (IES) will be provided to the MUs to facilitate their maturation. Once maturity is achieved, we will terminate the “muscle exercise effect” of IES and reduce trophic factors to mimic natural aging in humans. NMJ maturity and aging will be monitored at multiple time points for molecular, morphological, and functional changes. Finally, the co-culture system developed will be utilized for screening and determining the properties of the molecules identified in SA-I. <Study impact> Our novel NMJ-LCM with a cutting-edge bioinformatics analysis should generate invaluable data to reveal the pathology and gain potential therapeutic insights into sarcopenia. In addition, our innovative co-culture system could become the first in vitro model to achieve complete maturation and subsequent aging of NMJs and serve as a powerful tool for mechanistic and translational studies of sarcopenia. This work will also contribute to a broader area of neuromuscular research, ranging from basic studies on MUs/NMJs, to more translational works on various other neuromuscular diseases.