Defining The Impaired Proteostasis Network in ALS Patient Motor Neurons

肌萎缩侧索硬化症(ALS)是一种进行性和不可治愈的神经退行性疾病 以上下运动神经元(MN)选择性死亡为特征。绝大多数人 这种疾病本质上是散发性的。然而，相对较小(&lt；12%)但信息量很高的一部分患者 患有家族性疾病，这使得能够识别引起疾病的遗传变异 是他们的状况的基础。这类遗传学研究表明，ALS可由基因突变引起 编码蛋白质的基因涉及多种细胞功能，从RNA处理、囊泡 运输、细胞骨架调节、线粒体功能和蛋白质质量控制途径。不过， ALS患者的共同特点是进行性运动神经变性。这 增加了不同的疾病始发事件可能聚集在一个或多个共同分子中的可能性 小路。具有不同功能的基因突变如何汇聚在MN退行性变中？ 仍然是一个悬而未决的问题。尽管所有肌萎缩侧索硬化症患者都表现出神经病理蛋白 聚集体中，蛋白质动态平衡在引起ALS中的总体作用尚不清楚。如果我们可以 找出一种收敛的机制，它可能提供一个机会来开发一种广泛适用的治疗方法 干预策略。在我们的初步研究中，我们进行了蛋白质降解动力学的全球分析 在突变体SOD1和等基因对照中，来自IPSC系的MNS。有趣的是，我们发现了一些 在SOD1MNS中降解速度较慢的蛋白质。出乎意料的是，这一小组候选人 包括其基因突变导致ALS的蛋白质。在拟议的研究中，我们将使用患者派生的 神经元结合质谱分析来确定蛋白质底物，以及 由于两个最普遍的肌萎缩侧索硬化症基因SOD1和C9orf72的突变而引起的扰动。 首先，我们将确定哪些蛋白质具有降低蛋白质降解动力学的能力。第二，我们将确定 哪些蛋白质改变了合成速率。第三，我们将确定蛋白质组的整体程度 改建。与传统工作流相比，这些方法中的每一种都具有战略优势，并将使我们能够 不仅要确定ALS MN中哪些蛋白质的水平发生了变化，而且还要确定它们的机制 微扰。综上所述，我们提出的目标将揭示 患者神经元中蛋白平衡网络的变化，并可能揭示广泛相关的治疗 肌萎缩侧索硬化症的目标。

Amyotrophic lateral sclerosis (ALS) is a progressive and untreatable neurodegenerative disease that is characterized by the selective death of upper and lower motor neurons (MNs). The overwhelming majority of the disease is sporadic in nature. However a relatively small (<12%) but highly informative fraction of patients suffer from familial forms of disease, which have enabled the identification of causative genetic variants that underlie their condition. Such genetic studies have demonstrated that ALS can be caused by mutations in genes that encode proteins involved in diverse set of cellular functions ranging from RNA processing, vesicle transport, cytoskeletal regulation, mitochondrial function, and protein quality control pathways. Nevertheless, ALS patients are uniformly characterized by a common pattern of progressive motor neurodegeneration. This raises the possibility that different disease initiating events could coalesce in one or more common molecular pathways. How the mutation of genes with dissimilar functions converge on MN degeneration has been and continues to be an outstanding question. Although all ALS patients exhibit neuropathological protein aggregates, the overall contribution of protein homeostasis in causing ALS has remained unclear. If we could identify a convergent mechanism, it may provide an opportunity to develop a broadly applicable therapeutic intervention strategy. In our preliminary studies, we conducted global analysis of protein degradation dynamics in mutant SOD1 and isogenic controls MNs derived from iPSC lines. Interestingly, we identified a number of proteins that are degraded at a slower rate in SOD1 MNs. Unexpectedly, this small panel of candidates included proteins whose genetic mutations cause ALS. In the proposed research we will use patient-derived neurons coupled with mass spectrometry analysis to determine the protein substrates, as well as the nature of the perturbation that arise as a result of mutations in the two most prevalent ALS genes: SOD1 and C9orf72. First, we will determine which proteins have reduced protein degradation dynamics. Second, we will determine which proteins have altered synthesis rates. Third, we will determine the overall degree of proteome-wide remodeling. Each of these approaches has strategic advantages over traditional work-flows and will allow us to determine not only which proteins have altered levels in ALS MNs but also the mechanism responsible for their perturbation. Taken together, our proposed aims will shed light into the cellular mechanisms compromised by changes in the proteostasis network in patient neurons and will likely uncover broadly relevant therapeutic targets for ALS.