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.

肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）是致命的神经退行性疾病 ALS 和 ALS 的最常见的已知遗传原因尚不存在。 FTD 是 C9ORF72 基因内的六核苷酸重复扩展 (HRE) 突变。 HRE 序列的后续翻译产生多个有毒 RNA 和二肽重复蛋白（DPR）在果蝇和酵母模型中的遗传筛选揭示了修饰子（增强子和抑制子）。 C9ALS 病理学绝大多数聚集在核细胞质运输 (NCT) 途径中，表明 HRE RNA 和/或 DPR 通过破坏 NCT 来赋予神经毒性。然而，我们对 NCT 的理解存在重大差距。例如，有多种 NCT 途径，每种途径都利用独特的亚细胞。蛋白质货物内的定位基序可以被特定的转运蛋白识别。已经尝试阐明在 C9ALS 中被破坏的特定 NCT 途径，也没有阐明特定的 NCT 途径。有毒的 HRE 产品是为了研究这些被认为是潜在的关键机制。为了解决 C9ALS 中的神经毒性，我们开发了“生物传感器”，旨在询问特定的 NCT 通路。这些生物传感器由融合到独特核定位和输出信号的荧光蛋白组成使它们能够被不同的转运蛋白识别，我们将共同使用交叉方法。用每种 HRE 产品转染每个途径特异性 NCT 生物传感器，以识别以下 NCT 途径： C9ALS 和相关的 HRE 毒素中被破坏。这些实验将以高通量进行。使用永生化细胞和自动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身上重现衍生的诱导多能干细胞（iPSC）运动神经元，一种与疾病更相关的细胞模型系统。最后，我们将对 C9ALS 的已知遗传修饰物进行重点 RNAi 筛选，以识别那些能够恢复人体细胞中的 NCT，该研究小组非常有能力通过以下方式开展这些研究。凭借丰富的 C9ALS 临床知识、治疗开发方面的专业知识以及高水平的熟练程度此外，初步结果证明了内容成像和 iPSC 模型系统的可行性。该方法并提供了 NCT 生物传感器可用于揭示的总体假设 C9ALS 中被破坏的特定 NCT 途径，导致这种破坏的 HRE 产品，以及识别人类细胞模型系统中 NCT 的遗传修饰剂该项目的高通量性质。可以适用于治疗筛选，使其具有独特的优势，可以加速实现这一目标的进展预计将提供恢复 NCT 的 C9ALS 疗法。对 C9ALS 病理学的重要洞察并导致相关治疗靶点的确定。