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Elucidation of specific nucleocytoplasmic trafficking pathways that are disrupted in C9ORF72 ALS

阐明 C9ORF72 ALS 中被破坏的特定核细胞质运输途径

基本信息

批准号：
9544330
负责人：
Zane Zeier
金额：
$ 19.19万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9544330
关键词：
Affect Amyotrophic Lateral Sclerosis Biogenesis Biological Models Biosensor C9ORF72 Carrier Proteins Cell Nucleus Cell model Cells Clinical Cytoplasm DNA Sequence Alteration Defect Dipeptides Disease Enhancers Ensure Exportins Family Frontotemporal Dementia Gene-Modified Genes Genetic Genetic Screening Genetic Transcription Green Fluorescent Proteins Human Image Importins Karyopherins Knowledge Lead Microscopy Molecular Abnormality Motor Neurons Mutation Nature Neurodegenerative Disorders Nuclear Nuclear Export Nuclear Import Nuclear Pore Other Genetics Pathology Pathway interactions Patients Pharmacology Phenotype Positioning Attribute Protein Import Proteins RNA RNA Interference RNA interference screen RNA-Binding Proteins Reporter Research Role Severities Signal Transduction Specificity Testing Therapeutic Toxin Translations Weight Yeast Model System c9FTD/ALS cell immortalization design effective therapy experimental study exportin 1 protein fly gain of function human model immortalized cell induced pluripotent stem cell insight intersectionality neurotoxicity novel therapeutics nucleocytoplasmic transport overexpression premature protein transport screening small hairpin RNA therapeutic development therapeutic target trafficking

项目摘要

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases for which no substantially effective treatments exist. The most common known genetic cause of both ALS and FTD is a hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene. Transcription and subsequent translation of the HRE sequence produces multiple toxic RNAs and dipeptide repeat proteins (DPRs). Genetic screens in fly and yeast models have revealed that modifiers (enhancers and suppressors) of C9ALS pathology overwhelmingly cluster within nucleocytoplasmic trafficking (NCT) pathways, suggesting that HRE RNAs and/or DPRs confer neurotoxicity by disrupting NCT. However, critical gaps in our understanding of disrupted NCT remain. For example, there are multiple NCT pathways, each utilizing unique sub-cellular localization motifs within protein cargos that are recognized by specific transport proteins. No previous attempts have been made to elucidate the specific NCT pathways that are disrupted in C9ALS nor the specific toxic HRE product(s) that are responsible. To investigate these critical mechanisms thought to underlie neurotoxicity in C9ALS, we have generated “biosensors” designed to interrogate specific NCT pathways. These biosensors are composed of fluorescent proteins fused to unique nuclear localization and export signals allowing them to be recognized by different transport proteins. Using an intersectional approach, we will co- transfect each pathway-specific NCT biosensor with each HRE product to identify NCT pathways that are disrupted in C9ALS and the responsible HRE toxin(s). These experiments will be performed in high-throughput using immortalized cells and an automated image acquisition and analysis platform (high-content imaging). Subsequently, we will determine whether the perturbation of specific NCT pathways is recapitulated in patient- derived induced pluripotent stem cell (iPSC) motor neurons, a more disease relevant cellular model system. Finally, we will carry out a focused RNAi screen of the known genetic modifiers of C9ALS to identify those capable of restoring NCT in human cells. The research team is ideally positioned to carry out these studies by virtue of a vast clinical knowledge of C9ALS, expertise in therapeutic development and proficiency in both high content imaging and iPSC model systems. Furthermore, preliminary findings demonstrate both the feasibility of the approach and have informed the overarching hypothesis that NCT biosensors can be used to reveal specific NCT pathways that are disrupted in C9ALS, the HRE products that cause this disruption, and to identify genetic modifiers of NCT in human cellular model systems. The high-throughput nature of the project could be adapted for therapeutic screening, making it uniquely positioned to accelerate progress toward C9ALS therapies that restore NCT. Knowledge gained under the proposed studies is expected to provide critical insight into the pathobiology of C9ALS and lead to the identification of relevant therapeutic targets.

肌萎缩侧索硬化（ALS）和额颞叶痴呆（FTD）是致命的神经退行性疾病对于这些疾病没有实质上有效的治疗方法。最常见的已知遗传原因的ALS和 FTD是C9 ORF 72基因内的六核苷酸重复扩增（HRE）突变。转录和 HRE序列的后续翻译产生多种毒性RNA和二肽重复蛋白（DPR）。在果蝇和酵母模型中进行的遗传筛选已经揭示， C9 ALS病理学绝大多数集中在核质运输（NCT）途径内，表明 HRE RNA和/或DPR通过破坏NCT赋予神经毒性。然而，我们对这些问题的理解存在重大差距，被打乱的NCT仍然存在。例如，存在多种NCT途径，每种途径利用独特的亚细胞表达。被特定转运蛋白识别的蛋白质货物内的定位基序。还从未有一位已经尝试阐明在C9 ALS中被破坏的特异性NCT途径，有毒的HRE产品负责。为了研究这些被认为是在C9 ALS的神经毒性中，我们已经产生了设计用于询问特定NCT途径的“生物传感器”。这些生物传感器是由荧光蛋白融合到独特的核定位和输出信号使它们能够被不同的转运蛋白识别。通过一种交叉的方法，我们将共同- 用每种HRE产品检测每种途径特异性NCT生物传感器，以鉴定在C9 ALS和负责的HRE毒素中被破坏。这些实验将在高通量使用永生化细胞和自动化图像采集和分析平台（高内涵成像）。随后，我们将确定特定NCT通路的扰动是否在患者中重现- 衍生的诱导多能干细胞（iPSC）运动神经元，一种更疾病相关的细胞模型系统。最后，我们将对C9 ALS的已知遗传修饰剂进行集中的RNAi筛选，以鉴定那些能够在人类细胞中恢复NCT。该研究小组是开展这些研究的理想场所，凭借C9 ALS的丰富临床知识，治疗开发方面的专业知识和对两种高水平内容成像和iPSC模型系统。此外，初步研究结果表明，该方法并告知了NCT生物传感器可用于揭示在C9 ALS中被破坏的特定NCT途径，引起这种破坏的HRE产物，以及在人细胞模型系统中鉴定NCT遗传修饰剂。项目的高吞吐量性质可以适用于治疗筛选，使其具有独特的地位，以加速进展， C9 ALS疗法可以恢复NCT。通过拟议的研究获得的知识预计将提供对C9 ALS的病理生物学的重要见解，并导致相关治疗靶点的鉴定。