FUS Gain-of-Function Mechanisms in Animal and Cellular Models of ALS

肌萎缩性侧索硬化症（ALS）是一种进行性神经退行性疾病，其中优选的肌萎缩性侧索硬化是一种神经退行性疾病。 运动神经元（MN）的丧失导致瘫痪和死亡。虽然ALS在很大程度上是一种散发性疾病，但研究 一直专注于遗传形式的疾病，因为临床和病理学证据表明， 致病机制基因FUS的突变导致一些最具侵略性的早发性形式的 人症在最近的一项研究中，我们的实验室在小鼠疾病模型中证明，突变的FUS导致运动神经元 不是通过功能丧失，而是通过不涉及过量FUS活性的毒性功能获得而引起的变性。 FUS是许多RNA结合蛋白之一-包括TDP-43和hnRNP A1 -其已经被因果地 与ALS有关我们最近的工作--连同来自几个实验室的相关研究--已经导致了一种疾病模型 其中FUS和相关蛋白质的低复杂性（LC）“朊病毒样”结构域驱动其相变为 不可逆的神经毒性组装我们的数据表明FUS中ALS相关突变增加了 蛋白质形成这些有毒组件的自然趋势，这些组件捕获并隔离其他核糖核蛋白 颗粒组分，包括参与翻译控制的蛋白质。在这个项目中，我们将探索 FUS在一系列转基因和基因敲入突变小鼠中的毒性机制， FUS-ALS表型的关键方面。此外，使用运动神经元和星形胶质细胞的体外研究表明， 这些小鼠模型将用于研究细胞自主和非自主机制， 疾病 在目标1中，我们将使用条件性敲入小鼠模型在MN或星形胶质细胞中表达突变型FUS， 或更广泛地在神经系统中探索受调节的突变FUS表达对MN存活的影响 和功能，以及可能导致FUS-ALS中MN变性的基因表达变化。我们还将 分析在FUS的LC结构域中具有导致ALS的突变的小鼠，以测试该关键结构域在FUS中的作用。 疾病在目标2中，我们将使用微流体分离MN轴突，并测试FUS依赖性缺陷在MN轴突中的作用。 轴突蛋白质合成有助于MN变性，我们将继续我们的发现，hnRNP U选择性 通过探索这种RNA结合蛋白在疾病中的功能作用， 进展最后，在目标3中，我们将应用单细胞RNA测序和拓扑数据的组合 对源自我们的FUS敲入突变小鼠的体外分化MN的混合分布的分析。这 体内和体外实验系统的复杂整合，结合我们的综合 计算和分析的方法将使我们能够阐明疾病的途径， MN的亚群，并确定用于治疗FUS-ALS和相关疾病的潜在治疗靶点。 运动神经元疾病的症状

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS cause some of the most aggressive early-onset forms of ALS. In a recent study, our lab demonstrated in a mouse model of disease that mutant FUS causes motor neuron degeneration not by a loss-of-function, by a toxic gain-of-function that does not involve an excess of FUS activity. FUS is one of a number of RNA binding proteins – including TDP-43 and hnRNP A1 – that have been causally related to ALS. Our recent work – together with related studies from several labs – has led to a disease model in which the low complexity (LC) “prion-like” domain of FUS and related proteins drives its phase transition to an irreversible, neurotoxic assembly. Our data demonstrates that ALS-related mutations in FUS increase the natural tendency of the protein to form these toxic assemblies, which trap and sequester other ribonucleoprotein granule components, including proteins involved in translational control. In this project we will explore the mechanisms of FUS toxicity in a series of transgenic and knock-in mutant mice with which we have modelled key aspect of the FUS-ALS phenotype. In addition, in vitro studies using motor neurons and astrocytes derived from these mouse models will be used to investigate cell autonomous and non-autonomous mechanisms of disease. In Aim 1, we will use a conditional knock-in mouse model to express mutant FUS in MNs or astrocytes, or more broadly in the nervous system to explore the effects of regulated mutant FUS expression on MN survival and function, and on gene expression changes that may underlie MN degeneration in FUS-ALS. We will also analyze mice with ALS-causing mutations in the LC domain of FUS to test the role of this critical domain in the disease. In Aim 2, we will use microfluidics to isolate MN axons and test the idea that FUS-dependent defects in axonal protein synthesis contribute to MN degeneration, and we will pursue our finding that hnRNP U selectively interacts with ALS-mutant FUS by exploring in vivo the functional role of this RNA binding protein in disease progression. Finally in Aim 3, we will apply a combination of single-cell RNA sequencing and topological data analysis to a mixed distribution of in vitro differentiated MNs derived from our FUS knock-in mutant mice. This sophisticated integration of in vivo and in vitro experimental systems, combined with our integrative computational and analytical approach will allow us to elucidate pathways of disease in vulnerable subpopulations of MNs and to identify potential therapeutic targets for the treatment of FUS-ALS and related forms of motor neuron disease.