Modeling DYT1 Dystonia in Patient-derived Neurons

患者源性神经元中 DYT1 肌张力障碍的建模

基本信息

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
项目类别：
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

项目摘要

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.

标题患者源性神经元中 DYT1 肌张力障碍的建模项目概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源性神经元的 DYT1 肌张力障碍的分子发病机制是第三种最常见的肌张力障碍。运动障碍和肌张力障碍的病理机制仍然很大程度上未知 1-5。目前的治疗主要基于症状，并且仅部分令人满意 6,7 代表 DYT1 肌张力障碍。遗传性原发性肌张力障碍最常见和最严重的形式，为研究提供了一个极好的模型目的是了解这种疾病的发病机制 8,9 典型的 DYT1 肌张力障碍是由杂合性 GAG 引起的。尽管动物模型提供了对疾病机制的深入了解，存在显着的物种依赖性差异，因为具有相同杂合突变（ΔE）的动物失败显示人类患者 10 中所见的病理。此外，对患者神经元的访问非常有限阻碍肌张力障碍研究的进展我们取得了突破，开发了一种新型细胞系统。使用患者特异性神经元模拟 DYT1 肌张力障碍 11,12 这些人类神经元保留了杂合 TOR1A 突变和概括疾病依赖性细胞缺陷是最意想不到的。研究结果表明，核纤层蛋白 LMNB1 在表达和亚细胞分布方面失调。 LMNB1 的模糊下调可以在很大程度上改善 DYT1 神经元 11 中的所有细胞缺陷。结果证明了使用人类患者来源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 病理学的主要分子机制。 LMNB1 失调是否会导致肌张力障碍的发病机制？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。我们发现核 LMNB1 的失调是细胞缺陷的主要原因，并且细胞质中错误定位的 LMNB1 可能会捕获关键信号通路中的因子并导致大规模我们将使用患者来源的神经元来检验这一假设并解决相关问题。通过三个具体目标，目标 1 是确定 LMNB1 失调如何导致细胞功能障碍。 DYT1 神经元，包括使用 TEM 和免疫金标记检查核形态，以及鉴定错误定位的 LMNB1 相互作用蛋白在目标 2 中，我们将使用鉴定 ΔE 干扰因子。蛋白质组学研究并使用人工结合检查异常蛋白质-蛋白质相互作用在目标 3 中，我们在分子生物学和生物化学中基于智能 (AI) 的结构预测和方法。将使用转录组研究识别失调基因，并通过以下方式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顺利完成。这些目标将使我们能够更深入地了解肌张力障碍的发病机制。