Human-iPSC derived neuromuscular junctions as a model for neuromuscular diseases.

运动神经元通过脊髓携带来自大脑的电信号，最终产生肌肉 通过神经肌肉接头(NMJ)收缩。涉及启动和维护的机制 中枢神经系统和肌肉之间的正确沟通是令人难以置信的复杂和损害 这种交流是神经肌肉疾病(NMD)的原因，如肌萎缩侧索硬化症 (肌萎缩侧索硬化症)和脊髓和延髓肌萎缩(SBMA)。虽然这些疾病是最常见的 对于NMD，目前还没有治愈或有效的治疗方法。NMD字段确认 在动物模型中发现的大量缓解症状的药物在临床试验中都失败了。连 尽管这突显了开发人性化模型的重要性，但转换研究的一个警告 在IPSC模型中，关注的是单细胞，从而损害成年生物体的复杂系统。在 特别是NMD领域，IPSC的研究主要集中在解决运动神经元表型上， 可以防止它们退化。不幸的是，这种方法已经不够了，因为延长马达 神经元存活并不能保证重新神经支配，也不能保证防止去神经支配。因此，它是 对NMD领域的治疗进步至关重要，干细胞研究不仅需要关注 识别特定于细胞的目标，并在包含两者的正常运行的NMJ系统上测试这些目标 IPSC来源的运动神经元和骨骼肌。为了实现这一目标，我们最近开发了一种2D 功能性人类NMJ系统由IPSC来源的运动神经元和骨骼肌组成。我们的 人类IPSC-NMJ模型对光遗传学有反应，我们能够定量测量NMJ功能 在多电极阵列系统中。因此，在这个R61/R33点火阶段性创新奖系统中，我们 建议利用我们最新开发的NMJ系统来扩展功能和形态评估 (目标1和2)，并通过使用两个NMD分析NMJ特有的功能障碍来验证系统：SBMA和 ALS(目标3和4)。本实验室此前已为SBMA(R01NS121374-01， K01NS116119-01)，并具有丰富的建模经验。此外，我们选择了iPSC 在C9ORF72的第一内含子内，在9号染色体上含有G4C2六核苷酸重复扩增，因为它 是额颞叶痴呆(FTD)和肌萎缩侧索硬化症(ALS)最常见的基因贡献者，占 约占所有这些疾病病例的10%。因此，R61/R33的成功完成将建立和验证 促进NMDS治疗发现的新模型系统。

Motor neurons carry electrical signals from the brain through the spinal cord to ultimately generate muscle contraction via the neuromuscular junction (NMJ). The mechanisms involved in initiating and maintaining proper communication between the central nervous system and muscles are incredibly complex, and damage in this communication is the cause of neuromuscular diseases (NMD), such as Amyotrophic Lateral Sclerosis (ALS) and Spinal and Bulbar Muscular Atrophy (SBMA). While these disorders are among the most common NMDs, there is currently no cure or effective treatment for them. The NMD field acknowledges that a substantial number of drugs found to alleviate symptoms in animal models have failed in clinical trials. Even though this highlights the importance of the development of humanized models, a caveat of converting studies into iPSC models is the focus on single cells in detriment of the complex systems of the adult organism. In the NMD field specifically, iPSC investigations have largely focused on addressing motor neuron phenotypes that would prevent their degeneration. Unfortunately, this approach is no longer sufficient, as prolonging motor neuron survival does not assure re-innervation, nor does it guarantee prevention of denervation. Therefore, it is crucial for therapeutic advancement in the NMD field that stem cell research needs to focus not only on identifying cell-specific targets but also on testing those targets on functional NMJ systems that comprise both iPSC-derived motor neurons and skeletal muscles. To achieve this goal, we have recently developed a 2D functional human NMJ system comprised of both iPSC-derived motor neurons and skeletal muscles. Our human iPSC-NMJ model is responsive to optogenetics and we are able to quantitatively measure NMJ function in a multi-electrode array system. Hence, in this R61/R33 IGNITE Phased Innovation Award System, we propose to leverage our newly developed NMJ system to scale functional and morphological assessment (Aims 1 and 2) and validate the system by assaying NMJ-specific dysfunction using two NMDs: SBMA and ALS (Aims 3 and 4). Our lab has previously established an iPSC model for SBMA (R01NS121374-01, K01NS116119-01) and has extensive experience modeling this disease. Additionally, we selected the iPSCs harboring G4C2 hexanucleotide repeat expansion on chromosome 9 within the first intron of C9ORF72, as it represents is the most common genetic contributor to frontotemporal dementia (FTD) and ALS, accounting for ~10% of all cases of those diseases. Thus, successful completion of this R61/R33 will establish and validate a novel model system to facilitate therapeutic discovery for NMDs.