Determining the pathogenesis of DYT1 dystonia in reprogrammed human neurons

标题 确定重新编程的人类神经元肌张力障碍的发病机制 项目总结 这个项目的总体目标是通过重新编程人类神经元来确定肌张力障碍的发病机制。 来自病人的成纤维细胞。肌张力障碍是第三种最常见的运动障碍，其特征是持续性或 间歇性肌肉收缩导致不正常的动作、姿势或两者兼而有之。病态的 肌张力障碍的机制仍不清楚，也没有有效的治疗方法来治愈这种疾病。这个 早发性DYT1肌张力障碍也属于神经发育障碍，是最常见的 和严重形式的肌张力障碍，为了解这种疾病的发病机制提供了一个很好的例子。 然而，接触患者神经元的途径有限，以及缺乏体外人类神经元系统，这极大地阻碍了这一进程 肌张力障碍的研究进展。令人兴奋的是，使用慢病毒传递的转录因子，我有 从DYT1患者和健康对照组的成纤维细胞中成功地培养出了人类神经元 策略：1)直接转化和2)诱导基于多能干细胞(IPSCs)的重编程和 差异化。这些与疾病相关的人类神经元的产生为此奠定了坚实的基础 拟开展的研究。 典型的DYT1肌张力障碍是由蛋白质Torsin A(ΔE)的功能缺失突变引起的，Torsin A是一种膜- 嵌入式ATPase。Torsin A在轴突延伸和突触小泡回收中的作用强调了 扭蛋白A在神经元发育和功能中的关键作用。另外，现在积累的证据 表明扭转蛋白A在核膜(NE)也起着关键作用。在果蝇中，mRNA需要扭转蛋白 通过不依赖于核孔复合体的机制(NE-萌芽)输出。在细胞层面上，一 DYT1肌张力障碍小鼠的病理特征是神经元NE形态异常，尤其是在 脊髓，提示下运动神经元可能是DYT1中受影响最严重的神经元类型 肌张力障碍。在小鼠中发生的核膜被破坏是否也发生在人类DYT1神经元中？这些是如何做到的 异常导致人类肌张力障碍综合征？在本项目中，我们将解决这些相关问题 直接在与疾病相关的人类神经元中提出问题。在我们的初步研究中，我们发现核子 DYT1神经元的包膜形态在光镜和电子显微镜下都有明显的破坏 神经突起生长也明显慢于对照组。我假设这些反常现象 在DYT1，去甲肾上腺素损害核质运输。在这个项目中，我们将系统地测量 核质转运中的mRNA输出和蛋白质核转运，并鉴定 失调因子，如错误定位的mRNAs。这项研究的预期结果将提供 对肌张力障碍病理的新见解，并可能导致治疗干预的分子靶点。

Title Determining the pathogenesis of dystonia in reprogrammed human neurons PROJECT SUMMARY The overall goal of this project is to determine the pathogenesis of dystonia via reprogramming human neurons from patient fibroblasts. Dystonia is the third most common movement disorder characterized by sustained or intermittent muscle contractions causing abnormal movements, postures, or both. The pathological mechanisms of dystonia remain largely unknown and there is no effective treatment to cure this disease. The early-onset DYT1 dystonia also belongs to neurodevelopmental disorders and represents the most frequent and severe form of dystonia, providing an excellent example to understand the pathogenesis of this disease. However, the limited access to patient neurons and the lack of in vitro human neuron systems greatly impede the progress of dystonia research. Excitingly, using lentiviral delivery of transcription factors, I have successfully generated human neurons from fibroblasts of DYT1 patients and healthy controls via two strategies: 1) direct conversion and 2) induced pluripotent stem cells (iPSCs)-based reprogramming and differentiation. The generation of these disease-relevant human neurons laid a solid foundation for this proposed research. Typically, DYT1 dystonia is caused by a loss-of-function mutation in protein torsin A (ΔE), a membrane- embedded ATPase. The effects of torsin A on neurite extension and synaptic vesicle recycling underscore the critical roles of torsin A in neuronal development and function. Additionally, accumulating evidence now indicates that torsin A also plays critical roles at the nuclear envelope (NE). In flies, torsin is required for mRNA exporting via a nuclear pore complex-independent mechanism (NE-budding). At the cellular level, one pathological hallmark in DYT1 dystonia mice is abnormal neuronal NE morphology, particularly severe in the spinal cord, suggesting that lower motor neurons could be the most severely affected neuron type in DYT1 dystonia. Do disrupted nuclear envelopes occurring in mice also occur in human DYT1 neurons? How do these abnormalities contribute to the human dystonia syndrome? In this project, we will address these pertinent questions directly in disease-relevant human neurons. In our preliminary studies, we found that the nuclear envelope morphology of DYT1 neurons was obviously disrupted at both light and electron microscopy levels and also the neurite outgrowth was significantly slower than that of controls. I hypothesize that the abnormities in DYT1 NE impair nucleocytoplasmic transport. In this project, we will systematically measure the nucleocytoplasmic transport of both mRNA exporting and protein nuclear transport, and to identify dysregulated factors, such as mis-localized mRNAs. Expected results emanating from this study will provide novel insights into dystonia pathology and potentially lead to molecular targets for therapeutic interventions.