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.

肌萎缩性外侧硬化症（ALS）和额颞痴呆（FTD）是致命的神经退行性疾病为此没有实质性的治疗。 ALS和 FTD是C9ORF72基因内的六核苷酸重复膨胀（HRE）突变。转录和随后的HRE序列翻译产生多种有毒的RNA和二肽重复蛋白（DPRS）。苍蝇和酵母模型中的遗传筛选表明，修饰符（增强剂和补充剂）的 C9ALS病理绝大多数在核眼质贩运（NCT）途径中群体群体簇，表明 HRE RNA和/或DPRS会议会议神经毒性通过破坏NCT。但是，我们对我们的理解的关键差距残留的NCT仍存在。例如，有多个NCT途径，每种途径都使用唯一的亚细胞蛋白质中的定位基序被特定的转运蛋白识别。没有以前已经尝试阐明在C9AL中破坏的特定NCT途径或特定的NCT途径负责的有毒HRE产品。调查这些被认为是基础的关键机制在C9ALS中，我们已经产生了旨在询问特定NCT途径的“生物传感器”。这些生物传感器由与独特的核定位和导出信号融合的荧光蛋白组成允许它们被不同的运输蛋白识别。使用交叉方法，我们将共同将每个途径特异性NCT生物传感器转染使用每个HRE产品，以识别NCT途径在C9AL和负责的HRE毒素中破坏。这些实验将在高通量中进行使用永生的细胞和自动图像采集和分析平台（高内含成像）。随后，我们将确定是否在患者中概括了特定NCT途径的扰动衍生的诱导多能干细胞（IPSC）运动神经元，这是一种更相关的细胞模型系统。最后，我们将对C9AL的已知遗传修饰剂进行重点的RNAi屏幕，以识别这些修饰符能够恢复人类细胞中的NCT。研究团队的理想位置可以通过优点具有C9AL的广泛临床知识，治疗性开发方面的专业知识和熟练程度内容成像和IPSC模型系统。此外，初步发现证明了该方法并告知了总体假设，即NCT生物传感器可用于揭示在C9AL中被破坏的特定NCT途径，导致这种破坏的HRE产品，以及在人类细胞模型系统中识别NCT的遗传修饰符。该项目的高通量性质可以适应热筛选，使其独特地定位以加速进度恢复NCT的C9ALS疗法。根据拟议的研究获得的知识将提供对C9AL的病理生物学的批判性洞察力，并导致鉴定相关的治疗靶标。