Toward Understanding Aging Mechanisms of Neuromuscular Junctions

项目总结 神经肌肉接头(NMJ)，一个独特的突触位置，脊髓运动神经元(MN)在这里与肌纤维相遇 形成一个功能运动单位(MU)，由MN的突触前、肌纤维的后突触和肌纤维的后突触三个主要成分组成。 突触和终末雪旺细胞(SCs)。最近，人们越来越多地关注该病的病因。 “NMJ老化”与骨质疏松症有关，也就是与衰老相关的肌肉质量和力量的丧失。这里, 我们提出了两个互补的目标，重点是解开与骨质疏松症相关的NMJ衰老机制： &lt；SA-1&gt；确定最早的与骨质疏松症相关的分子变化和衰老时潜在的生物标志物 NMJ在活体中-我们将首次对不同时间分离的显微解剖NMJ进行RNASEQ 使用激光捕获显微解剖(LCM)技术从衰老小鼠身上提取斑点。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，这只需要几个 几分钟就能想象出NMJ。我们将优先验证之前的治疗目标候选 与神经肌肉疾病和具有已知配体或其他潜力的生物标记物候选有关 监控策略。&lt；SA-II&GT；设计一种新型的成熟和老化NMJ-WE体外共培养体系 将采用双隔室系统，概括中央(MNS)的生理分离 和周围神经系统(MN轴突、肌管[MTS]和SCs)。这个 胶质细胞(AST和SCs)将为MU提供生物学上所需的结构和营养支持， 最大限度地减少对培养基补充的需求。这个策略对于一个忠实的后来者来说是必不可少的 NMJ衰老的模型，这被认为与组成MU的细胞的功能衰退有关。 此外，我们将用纤维蛋白凝胶支架保护两个隔室中的细胞，以防止细胞分离。 以及由于MT频繁收缩而导致的突触断开。此外，间歇性电刺激 将向MU提供(IES)，以促进其成熟。一旦达到成熟，我们将终止 IES的“肌肉锻炼效应”和减少营养因子，以模拟人类的自然衰老。NMJ成熟度和 将在多个时间点监测衰老，以了解分子、形态和功能的变化。最后， 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。&lt；研究Impact&gt；我们具有尖端生物信息学分析的新型NMJ-LCM应该 生成宝贵的数据来揭示病理并获得对石棺减少症的潜在治疗见解。在……里面 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 以及随后NMJ的老化，并作为机械和翻译研究的有力工具 石棺减少症。这项工作还将有助于神经肌肉研究的更广泛领域，从基础 对MUS/NMJ的研究，到其他各种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.