Modeling DYT1 Dystonia in Patient-derived Neurons

标题 在患者源性神经元中建模DYT 1肌张力障碍 项目摘要 该项目的总体目标是开发用于肌张力障碍研究的新型细胞系统，并确定 DYT 1肌张力障碍的分子发病机制，使用患者衍生的神经元。肌张力障碍是第三常见 运动障碍和导致肌张力障碍的病理机制在很大程度上仍然是未知的1-5。 目前的治疗方法主要是基于维生素A的，只有部分令人满意6，7。DYT 1肌张力障碍，代表 遗传性原发性肌张力障碍的最常见和最严重的形式，提供了一个很好的研究模型， 目的是了解这种疾病的发病机制8，9.典型的DYT 1肌张力障碍是由杂合子GAG引起的 TOR 1A基因缺失（ΔE）。尽管动物模型提供了对疾病机制的见解， 存在显著的种属依赖性差异，因为具有相同杂合突变（ΔE）的动物 以显示在人类患者中观察到的病理学10.此外，患者神经元的有限获取极大地 阻碍了肌张力障碍研究的进展。在一项突破中，我们开发了一种新的细胞系统， 用患者特异性神经元模拟DYT 1肌张力障碍11，12.这些人类神经元保留了 杂合TOR 1A突变和重演疾病依赖性细胞缺陷。最意想不到的 发现核纤层蛋白LMNB 1在表达和亚细胞分布方面失调。 有趣的是，LMNB 1的下调可以在很大程度上改善DYT 1神经元中的所有细胞缺陷11。这些 结果证明了使用源自人类患者的神经元的疾病建模的高价值，并表明， 核LMNB 1的失调可能构成DYT 1病理学的主要分子机制。如何 LMNB 1失调是否与肌张力障碍的发病机制有关？还有哪些蛋白质和基因 被ΔE破坏？这些问题的答案对于理解DYT 1肌张力障碍的病理生理学至关重要。 我们假设核LMNB 1的调节异常是细胞缺陷的主要原因，并且 LMNB 1在细胞质中的错误定位可以捕获关键信号通路中的因子，并导致细胞内的细胞毒性。 细胞功能障碍我们将使用患者源性神经元来验证这一假设，并解决相关问题 三个具体目标。目的1是确定LMNB 1的失调如何导致细胞功能障碍， DYT 1神经元，包括使用TEM和免疫金标记的核形态学检查，以及 错误定位的LMNB 1相互作用蛋白的鉴定。在目标2中，我们将使用 蛋白质组学研究和检查异常蛋白质-蛋白质相互作用的结合使用人工 分子生物学和生物化学中基于智能（AI）的结构预测和方法。在目标3中， 将使用转录组学研究鉴定失调基因，并通过以下方法检查功能改变： 电生理学分析和体外神经肌肉接头形成测定。圆满完成 这些目的将使我们对肌张力障碍的发病机制有更深入的了解。

TITLE Modeling DYT1 Dystonia in Patient-derived Neurons PROJECT SUMMARY The overall goal of this project is to develop novel cellular systems for dystonia research and determine the molecular pathogenesis of DYT1 dystonia using patient-derived neurons. Dystonia is the third most common movement disorder and the pathological mechanisms responsible for dystonia remain largely unknown 1-5. Current therapies are largely symptom-based and only partially satisfactory 6,7. DYT1 dystonia, which represents the most frequent and severe form of hereditary primary dystonia, provides an excellent model for studies that aim to understand the pathogenesis of this disease 8,9. Typical DYT1 dystonia is caused by a heterozygous GAG deletion in the TOR1A gene (ΔE). Even though animal models provide insights into disease mechanisms, significant species-dependent differences exist because animals with identical heterozygous mutation (ΔE) fail to show the pathology seen in human patients 10. In addition, the limited access to patient neurons greatly impedes the progress of research in dystonia. In a breakthrough, we have developed a novel cellular system for modeling DYT1 dystonia with patient-specific neurons 11,12. These human neurons retain the heterozygous TOR1A mutation and recapitulate disease-dependent cellular deficits. The most unexpected finding is that nuclear lamina protein LMNB1 was dysregulated at expression and subcellular distribution. Interestingly, downregulation of LMNB1 can largely ameliorate all the cellular deficits in DYT1 neurons 11. These results demonstrate the high value of disease modeling using human patient-derived neurons and indicate that dysregulation of nuclear LMNB1 may constitute a major molecular mechanism underlying DYT1 pathology. How does dysregulated LMNB1 contribute to the pathogenesis of dystonia? What other proteins and genes could be disrupted by ΔE? Answers to these questions are critical in understanding the pathophysiology of DYT1 dystonia. We hypothesize that the dysregulation of nuclear LMNB1 is the major contributor to the cellular deficits, and the mislocalized LMNB1 in the cytoplasm could trap factors in critical signaling pathways and lead to widescale cellular dysfunction. We will use patient-derived neurons to test this hypothesis and address pertaining questions via three specific aims. Aim 1 is to determine how dysregulated LMNB1 contributes to the cellular dysfunction in DYT1 neurons, including examination of nuclear morphology using TEM and immunogold labeling, and identification of mislocalized LMNB1-interacting proteins. In Aim 2, we will identify ΔE-disrupted factors using proteomic studies and examine the abnormal protein-protein interactions using the combination of Artificial Intelligence (AI)-based structural prediction and approaches in molecular biology and biochemistry. In Aim 3, we will identify dysregulated genes using transcriptomic study and examine the functional alterations via electrophysiology analysis and in vitro neuromuscular junction formation assay. The successful completion of these Aims will allow us to gain a more in-depth understanding of the pathogenesis of dystonia.