Axonal pathogenesis of human iPSC-derived motor neurons

人 iPSC 来源的运动神经元的轴突发病机制

• 批准号: 10604850
• 负责人: Mohamed H Farah
• 金额: $ 45.03万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-19 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10604850
• 关键词: ALS pathology; ALS patients; Address; Adult; Affect; Amyotrophic Lateral Sclerosis; Autophagocytosis; Axon; Back; Bioinformatics; C9ORF72; Cell Line; Cells; Cessation of life; Coculture Techniques; Data; Degenerative Disorder; Disease; Disease Progression; Distal; Drug Screening; Evaluation; Future; Gene Expression; Genetic; Health; Homeostasis; Human; Human Cell Line; Induced pluripotent stem cell derived neurons; Institution; Investigation; Link; Maintenance; Messenger RNA; Microfluidic Microchips; Molecular; Morphology; Motor; Motor Neuron Disease; Motor Neurons; Muscle Fibers; Muscle denervation procedure; Muscular Atrophy; Mutation; Nerve Degeneration; Neuromuscular Junction; Neurons; Pathogenesis; Pathology; Pathway interactions; Patients; Physiological; Proteins; Proteomics; Quality of life; RNA; Regulation; Research; Rodent; Sampling; Signal Transduction; Site; Spinal; Survival Rate; Synapses; System; Testing; Therapeutic; Time; Translating; amyotrophic lateral sclerosis therapy; axonal degeneration; disease-causing mutation; induced pluripotent stem cell; insight; mRNA Expression; mutant; nerve supply; neuromuscular; neuronal cell body; sporadic amyotrophic lateral sclerosis; stress granule; superoxide dismutase 1; synaptogenesis; therapeutic target; therapeutically effective; transcriptome sequencing

Project Summary: Reprogramming adult cells has made it possible to differentiate patient-specific neurons from induced pluripotent stem cells (iPSCs). These patient-derived neurons have become invaluable in the investigation of molecular mechanisms of neurodegeneration and identification of potential therapeutic targets. Amyotrophic Lateral Sclerosis (ALS) patient-derived spinal motor neurons have revealed insights into mutation-specific pathogenesis, but previous studies have almost exclusively addressed deficits within motor neurons such as stress granule formation, hyperexcitability, and reduced autophagy. However, the initial pathology of ALS, and many other motor neuron diseases, begins at the distal axon and neuromuscular junction. Normal adult axons contain thousands of diverse sets of mRNAs whose protein products are locally translated to maintain axonal homeostasis and health. However, the expression of axonal mRNA in human motor neurons derived from iPSCs harboring ALS-linked mutations are poorly understood. Additionally, iPSC-derived neuromuscular synapses are rarely utilized as a system to interrogate the pathogenesis of axon degeneration, in part, because of a lack of robust and systematic evaluations of neuromuscular synapses formed by different iPSC- derived cells. The main questions that this application addresses are: 1) Are there differences in the expression of axonal mRNA between patient and healthy control iPSC- derived human spinal motor neurons? 2) What are the effects of distinct disease-causing mutations on the formation and maintenance of human muscle fiber innervations? We hypothesize that ALS-causing mutations may reduce the abundance of locally translated axonal mRNA whose protein products are involved in axonal health and function. We will subject axonal RNA extracted from control and mutant samples to RNAseq, and we are collaborating with bioinformatic group at our institution to probe for effected axonal pathways. We plan to take advantage of a microfluidic device platform that separate neuronal cell bodies from axons/muscle fibers, permitting us to obtain pure axonal mRNA and create mature human-derived neuromuscular synapses. We will test human cell lines containing distinct mutations: SOD1 and C9orf72 repeat expansions, and we will first examine SOD1A4V lines and C9orf72 lines for which isogenic controls have been created. In addition, we have access to cell lines in which the genetic cause of disease has not been determined (sporadic ALS) through the Johns Hopkins ALS center and Answer ALS that we can use for future studies.

项目摘要： 重编程成人细胞已使区分患者特异性神经元与诱导的 多能干细胞（IPSC）。这些患者衍生的神经元在研究中变得无价 神经退行性的分子机制和潜在治疗靶标的鉴定。肌萎缩 横向硬化症（ALS）患者衍生的脊柱运动神经元揭示了对突变特异性的见解 发病机理，但以前的研究几乎完全解决了运动神经元（例如 应力颗粒的形成，过度兴奋和自噬降低。但是，ALS的最初病理和 许多其他运动神经元疾病始于远端轴突和神经肌肉连接。正常的成年轴突 包含成千上万种不同的mRNA，其蛋白质产物被局部翻译以保持轴突 稳态和健康。然而，源自人类运动神经元中的轴突mRNA的表达 具有与ALS连接突变的IPSC了解不足。另外，IPSC衍生的神经肌肉 突触很少用作系统来审问轴突变性的发病机理，部分 由于缺乏对不同ipsc-形成的神经肌肉突触的强大和系统评估 衍生细胞。该申请解决的主要问题是： 1）患者和健康对照IPSC之间的轴突mRNA表达是否存在差异 衍生的人脊柱运动神经元？ 2）独特的致病突变对形成和维持的影响 人类肌肉纤维神经？ 我们假设引起ALS的突变可能会减少局部翻译的轴突mRNA的丰度 其蛋白质产物参与轴突健康和功能。我们将从中提取的轴突RNA 控制和突变样本到RNASEQ，我们正在与机构的生物信息集团合作 探测有效的轴突途径。我们计划利用单独的微流体设备平台 来自轴突/肌肉纤维的神经元细胞体，使我们能够获得纯轴突mRNA并创建成熟 人源性神经肌肉突触。我们将测试包含不同突变的人类细胞系：SOD1 和C9ORF72重复扩展，我们将首先检查SOD1A4V线和C9ORF72线路的等源性。 控件已创建。此外，我们可以使用疾病遗传原因的细胞系 未通过约翰·霍普金斯ALS中心确定（零星的ALS），并回答我们可以使用的ALS 用于未来的研究。