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Toward Understanding Aging Mechanisms of Neuromuscular Junctions

理解神经肌肉接头的老化机制

基本信息

批准号：
9353712
负责人：
Diane Berengere Re
金额：
$ 20万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-30 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9353712
关键词：
Affect Aging Anoikis Area Astrocytes Attention Axon Bioinformatics Biological Biological Markers Cells Coculture Techniques Culture Media Data Denervation Development Disease Elderly Electric Stimulation Engineering Ensure Etiology Event Exercise Fibrin Functional disorder Future Gel Gene Expression Profiling Genetic Transcription Health Human Impairment In Vitro Industrialization Interneurons Investigation Ligands Longevity Maintenance Masks Minor Modeling Molecular Monitor Morphology Motor Motor Neurons Mus Muscle Muscle Contraction Muscle Fibers Muscle Weakness Muscular Atrophy Nervous system structure Neuroglia Neuromuscular Diseases Neuromuscular Junction Neuromuscular research Neurons Pathologic Processes Pathology Patients Peripheral Pharmaceutical Preparations Physiological Population Process Production Property Proteomics RNA Role Running Schwann Cells Series Site Skeletal Muscle Societies Spinal Supplementation Synapses System Techniques Testing Therapeutic Time Transcript Validation Work aged base candidate marker functional decline in vitro Model in vivo innovation insight laser capture microdissection middle age muscle form muscle strength neuromuscular neuron loss neuronal cell body novel novel therapeutics potential biomarker pre-clinical presynaptic prevent sarcopenia scaffold screening therapeutic target tool transcriptome transcriptome sequencing translational study

项目摘要

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.

项目概要神经肌肉接头 (NMJ) 是脊髓运动神经元 (MN) 与肌纤维相遇的独特突触位点形成一个功能性运动单元（MU），由三个主要部分组成，MN的前突触，肌纤维的后突触突触和末端雪旺细胞 (SC)。近年来，该病的病因学越来越受到人们的关注。 “NMJ 衰老”与肌少症有关，即与衰老相关的肌肉质量和力量的损失。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相关的 NMJ 衰老机制： <SA-1> 识别衰老过程中最早的肌少症相关分子变化和潜在生物标志物体内 NMJ - 我们将首次对不同时间分离的显微解剖 NMJ 进行 RNA 测序使用激光捕获显微解剖（LCM）技术从衰老小鼠身上提取点。一系列既定的然后将进行生物信息学分析以提取候选分子，然后识别其细胞起源。 To obtain high-quality RNA in the LCM, we will apply our original technique, which requires only a few 分钟可视化 NMJ。我们将优先考虑先前候选治疗靶点的验证与神经肌肉疾病和具有已知配体或其他潜在的候选生物标志物相关监测策略。 <SA-II> 设计成熟和老化 NMJ 的新型体外共培养系统 - 我们将采用双室系统来概括中枢（MNs）的生理分离和星形胶质细胞 [AST]）和体内外周（MN 轴突、肌管 [MT] 和 SC）神经系统。这胶质细胞（AST 和 SC）将为 MU 提供生物学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。这一策略对于忠实的后续行动至关重要 NMJ 衰老模型，被认为与构成 MU 的细胞功能衰退有关。此外，我们将用纤维蛋白凝胶支架保护两个隔室中的细胞，以防止细胞脱离 MT 频繁收缩导致突触断开。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“肌肉锻炼效果”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衰老的分子、形态和功能变化。最后，开发的共培养系统将用于筛选和确定分子的特性在 SA-I 中确定。 <研究影响> 我们的新型 NMJ-LCM 具有尖端的生物信息学分析应该生成宝贵的数据来揭示病理学并获得对肌肉减少症的潜在治疗见解。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，并作为机制和转化研究的强大工具肌肉减少症。这项工作还将有助于更广泛的神经肌肉研究领域，从基础对 MU/NMJ 的研究，以及对各种其他神经肌肉疾病的更多转化工作。