Axonal pathogenesis of human iPSC-derived motor neurons

项目总结： 重新编程成体细胞使区分患者特有的神经元和诱导的神经元成为可能 多能干细胞(IPSCs)。这些患者来源的神经元在研究人类免疫缺陷方面变得非常有价值。 神经退行性变的分子机制和潜在治疗靶点的确定。肌萎缩性 侧索硬化症(ALS)患者来源的脊髓运动神经元揭示了对突变特异性的见解 发病机制，但以前的研究几乎只涉及运动神经元内的缺陷，如 应激颗粒形成，过度兴奋，减少自噬。然而，ALS的最初病理，以及 许多其他运动神经元疾病始于远端轴突和神经肌肉交界处。正常成人轴突 包含数千组不同的mRNAs，它们的蛋白质产物被局部翻译以维持轴突 动态平衡与健康然而，轴突mRNA在人运动神经元中的表达源于 携带肌萎缩侧索硬化症相关突变的ipscs目前知之甚少。此外，IPSC衍生的神经肌肉 突触很少被用来作为一个系统来询问轴突变性的发病机制，在某种程度上， 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- 派生的细胞。此应用程序解决的主要问题包括： 1)IPSC患者和健康对照组的轴突mRNA表达有无差异？ 人类脊髓运动神经元的来源？ 2)不同的致病突变对人类免疫缺陷病毒的形成和维持有什么影响 人体肌肉纤维的神经支配？ 我们推测引起肌萎缩侧索硬化症的突变可能会降低局部翻译的轴突mRNA的丰度。 其蛋白质产品与轴突的健康和功能有关。我们将用从 对照和突变样本到RNAseq，我们正在与我们机构的生物信息学小组合作 探查受影响的轴突通路。我们计划利用一种微流控设备平台 来自轴突/肌肉纤维的神经元胞体，使我们能够获得纯净的轴突mRNA并创造成熟的 人类衍生的神经肌肉突触。我们将测试含有不同突变的人类细胞系：SOD1 和C9orf72重复扩增，我们将首先检查SOD1A4V株和C9orf72株的等位基因 已创建控件。此外，我们还可以接触到致病基因具有 没有通过约翰·霍普金斯肌萎缩侧索硬化症中心确定(散发性肌萎缩侧索硬化)，并回答我们可以使用的肌萎缩侧索硬化症 以备将来研究之用。

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.