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.

项目概要： 重新编程成人细胞使得有可能将患者特异性神经元从诱导分化的神经元中分化出来。 多能干细胞（iPSC）。这些源自患者的神经元在研究中变得非常宝贵 神经变性的分子机制和潜在治疗靶点的鉴定。肌萎缩 侧索硬化症（ALS）患者来源的脊髓运动神经元已经揭示了突变特异性的见解。 发病机制，但以前的研究几乎只涉及运动神经元内的缺陷， 应激颗粒形成、过度兴奋和减少自噬。然而，ALS的最初病理学， 许多其它运动神经元疾病开始于远端轴突和神经肌肉接头。正常成年轴突 包含数千组不同的mRNA，其蛋白质产物在局部翻译以维持轴突生长。 稳态和健康。然而，轴突mRNA在人运动神经元中的表达， 携带ALS连锁突变的iPSC知之甚少。此外，iPSC衍生的神经肌肉 突触很少被用作询问轴突变性发病机理的系统，部分地， 由于缺乏对不同iPSC形成的神经肌肉突触的可靠和系统的评估， 衍生细胞该应用程序解决的主要问题是： 1)患者和健康对照iPSC之间轴突mRNA的表达是否存在差异？ 衍生的人类脊髓运动神经元 2)不同的致病突变对基因的形成和维持有什么影响？ 人体肌肉纤维神经支配 我们假设ALS引起的突变可能减少了局部翻译的轴突mRNA的丰度， 其蛋白质产物与轴突的健康和功能有关。我们将提取的轴突RNA 我们正在与我们机构的生物信息学小组合作， 探测受影响的轴突通路。我们计划利用一个微流体装置平台， 从轴突/肌纤维的神经元细胞体，使我们能够获得纯的轴突mRNA和创建成熟的 人类神经肌肉突触。我们将测试含有不同突变的人类细胞系： 和C9 orf 72重复扩增，我们将首先检查SOD 1A 4V系和C9 orf 72系， 控件已创建。此外，我们可以获得细胞系，其中疾病的遗传原因具有 通过约翰霍普金斯ALS中心和Answer ALS，我们可以使用 为将来的研究。