A new human iPSC model of ALS: natural modifiers protecting FUS mutation carriers from the disease

肌萎缩侧索硬化症(ALS)是一种复杂的神经退行性疾病，约10- 15%的家族性病例是由常染色体显性遗传突变引起的。因为家族性和零星的 肌萎缩侧索硬化症临床难以区分，家族性肌萎缩侧索硬化症的研究将有助于了解肌萎缩侧索硬化症的病因 将军。在已鉴定的ALS基因中，有几个编码RNA结合蛋白，包括在肉瘤中融合 (FUS)。FUS在多条RNA代谢途径中发挥作用。突变的FUS蛋白错位到 形成颗粒和包涵体的细胞质，这是ALS的病理特征。我们和其他团体有 研究了不同模型在生理和病理条件下FUS蛋白的表达。令人惊讶的是，我们 最近在一个扩大的肌萎缩侧索硬化症家系中发现了携带肌萎缩侧索硬化症连锁FUS R521G突变的个体，但 如果没有患上肌萎缩侧索硬化症(未受影响的突变携带者，UMCS)，他们可以活到60岁以上。我们的发现 这个扩展的FUS-ALS家系中的不完全外显是此类疾病的第一例。我们假设，尽管 UMCS携带致病的FUS突变，具有防止疾病发展的保护性修饰物。 这些修饰物和潜在机制的确定将指向新的治疗靶点。 患者来源的IPSCs及其分化的运动神经元(MN)促进靶细胞的机制研究 在相关的人类遗传背景下。利用新生成的IPSC行的优势 具有多个UMCs的独特的ALS家系，我们将描述UMCS的细胞和功能表型以及 确定潜在的保护性分子途径。我们提出了两个具体目标。我们将定义 细胞、生化和功能特征保护UMCS免受ALS样表型的发展 目标1中IPSC分化的MN。我们将UMCS、ALS患者和 对照研究不同分化阶段的病理生理功能障碍 免疫细胞化学和电生理学。此外，我们还将比较细胞和功能特征 来自肌萎缩侧索硬化症患者和对照组的IPSC-MNS，这将揭示哪些细胞和生化 功能对于保护UMCS至关重要。在目标2中，我们将破译负责 应用RNA-Seq和全基因组测序分析维持UMCS IPSC-MNS的正常功能。这个 表型数据、转录组数据和基因组数据的整合将提供对基因如何 修饰剂通过分子途径发挥作用，产生保护性表型。 这个MPI R21项目将由辛辛那提儿童医院医疗中心的郭子元博士领导 世卫组织在使用干细胞和IPSCs作为疾病模型方面拥有专业知识，加州大学的朱海宁博士 在肌萎缩侧索硬化症病因学方面有专长的肯塔基州。拟议研究的完成将提供新的见解 转化为自然产生的修饰剂，保护UMCS免受ALS感染。补偿机制 该项目中确定的将为未来开发ALS疗法的研究奠定基础。

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease with approximately 10- 15% familial cases caused by genetic mutations in an autosomal dominant fashion. Since familial and sporadic ALS are clinically indistinguishable, studies of familial ALS will facilitate understanding of ALS etiology in general. Among the ALS genes identified, several encode RNA binding proteins including Fused in Sarcoma (FUS). FUS functions in multiple RNA metabolic pathways. Mutant FUS protein is mis-localized to the cytoplasm where it forms granules and inclusions, a pathological hallmark of ALS. We and other groups have studied the FUS protein under physiological and pathological conditions in various models. Strikingly, we recently identified individuals in an extended ALS kindred who carry the ALS-linked FUS R521G mutation but live well beyond their 60s without developing ALS (Unaffected Mutation Carriers, UMCs). Our discovery of incomplete penetrance in this extended FUS-ALS pedigree is the first of its kind. We hypothesize that, despite carrying a disease-causing FUS mutation, UMCs have protective modifiers preventing disease development. The determination of such modifiers and the underlying mechanisms will point to novel therapeutic targets. Patient-derived iPSCs and their differentiated motor neurons (MNs) facilitate mechanistic studies in target cells in a relevant human genetic background. Taking advantage of newly generated iPSC lines derived from the unique ALS pedigree with multiple UMCs, we will characterize cellular and functional phenotypes of UMCs and determine the underlying protective molecular pathways. We propose two specific aims. We will define the cellular, biochemical and functional features protecting UMCs from developing ALS-like phenotypes using iPSC-differentiated MNs in Aim 1. We will differentiate MNs from iPSC lines of UMCs, ALS patients, and controls to characterize the pathophysiological dysfunctions at different differentiation stages using immunocytochemistry and electrophysiology. In addition, we will compare the cellular and functional features of iPSC-MNs from UMCs with ALS patients and controls, which will reveal which cellular and biochemical features are critical to protecting UMCs. In Aim 2, we will decipher biological pathways responsible for preserving normal functions in UMCs iPSC-MNs using RNA-Seq and whole genome sequencing analysis. The integration of phenotypic, transcriptomic and genomic data will provide in-depth understanding of how genetic modifiers function through molecular pathways to yield protective phenotypes. This MPI R21 project will be led by Dr. Ziyuan Guo at Cincinnati Children's Hospital Medical Center who has expertise on using stem cells and iPSCs as disease models and Dr. Haining Zhu at University of Kentucky who has expertise on ALS etiology. Completion of the proposed studies will provide novel insights into naturally occurring modifiers that protect UMCs from contracting ALS. The compensatory mechanism identified in this project will lay the foundation for future studies to develop future ALS therapies.