Modeling DYT1 Dystonia in Patient-derived Neurons

患者源性神经元中 DYT1 肌张力障碍的建模

• 批准号: 10863331
• 负责人: Baojin Ding
• 金额: $ 36.5万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10863331
• 关键词: ATP phosphohydrolase; Address; Animal Model; Animals; Artificial Intelligence; Biochemistry; Biological Assay; Biological Models; Cell Nucleus; Cells; Childhood; Co-Immunoprecipitations; Coupled; Cytoplasm; Deep Brain Stimulation; Diagnosis; Disease; Disease model; Down-Regulation; Dyskinetic syndrome; Dystonia; Electron Microscope; Electrophysiology (science); Etiology; Functional disorder; GAG Gene; Genes; Genome; Goals; Heterozygote; Human; Impairment; In Vitro; Inherited; Intramuscular; LMNB1 gene; Label; Mass Spectrum Analysis; Membrane; Messenger RNA; Modeling; Molecular; Molecular Biology; Morphology; Motor Neurons; Movement Disorders; Muscle Contraction; Mutation; Nervous System; Neurites; Neuromuscular Junction; Neurons; Nuclear; Nuclear Envelope; Nuclear Lamina; Pathogenesis; Pathologic; Pathology; Patients; Posture; Primary Dystonias; Proteins; Proteomics; Recycling; Research; Role; Signal Pathway; Study models; Symptoms; Synaptic Vesicles; System; TOR1A gene; TOR1A protein; Testing; botulinum neurotoxin injection; experimental study; exportin 1 protein; insight; loss of function mutation; neurodevelopment; neuron development; novel; novel strategies; nucleocytoplasmic transport; patch clamp; protein protein interaction; transcriptomics; transmission process

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.

标题 在患者衍生的神经元中建模Dyt1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 使用患者衍生的神经元对Dyt1肌张力纳病的分子发病机理。肌张力障碍是第三常见的 运动障碍和导致肌张力障碍的病理机制在很大程度上是未知的1-5。 当前的疗法在很大程度上是基于症状的，并且仅部分满足工厂6,7。 dyt1肌张力纳米，代表 遗传性原发性肌张力障碍最频繁，最严重的形式为研究提供了一个极好的模型 旨在了解该疾病的发病机理8,9。典型的dyt1 dystonia是由杂合子堵嘴引起的 tor1a基因（ΔE）中的缺失。即使动物模型提供了有关疾病机制的见解，但 存在明显的物种依赖性差异，因为具有相同杂合突变（ΔE）失败的动物 为了显示在人类患者中看到的病理10。此外，对患者神经元的访问有限 阻碍了肌张力障碍研究的进展。在突破中，我们开发了一个新型的细胞系统 用患者特异性神经元建模DYT1肌张力障碍11,12。这些人类神经元保留 杂合Tor1a突变和概括疾病依赖性细胞定义。最出乎意料的 发现核薄片蛋白LMNB1在表达和亚细胞分布下失调。 有趣的是，LMNB1的下调在很大程度上可以改善所有细胞在Dyt1神经元中的定义11。 结果表明，使用人类患者衍生的神经元进行疾病建模的高价值，并表明 核LMNB1的失调可能构成DYT1病理学基础的主要分子机制。如何 失调的LMNB1是否有助于肌张力障碍的发病机理？其他蛋白质和基因可能是什么 被ΔE破坏了吗？这些问题的答案对于理解dyt1肌张力障碍的病理生理学至关重要。 我们假设核LMNB1的失调是导致细胞定义的主要因素，并且 细胞质中错误定位的LMNB1可能会在关键信号通路中捕获因子，并导致宽阔 细胞功能障碍。我们将使用患者衍生的神经元来检验此假设并解决有关的问题 通过三个特定目标。 AIM 1是确定失调的LMNB1如何导致细胞功能障碍 dyt1神经元，包括使用TEM和免疫金标签检查核形态的，以及 鉴定错误定位的LMNB1相互作用蛋白。在AIM 2中，我们将使用 蛋白质组学研究并使用人工的组合检查异常蛋白质蛋白质相互作用 智力（AI）基于分子生物学和生物化学的结构预测和方法。在AIM 3中，我们 将使用转录组研究确定失调的基因，并通过 电生理分析和体外神经肌肉结的结构。成功完成 这些目标将使我们能够对肌张力障碍的发病机理有更深入的了解。