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疗的神经退行性疾病， 其特征在于上运动神经元和下运动神经元（MN）的选择性死亡。绝大多数 这种疾病是散发性的。然而，相对较小（<12%）但信息量较高的患者比例 患有家族性疾病，这使得能够鉴定致病性遗传变异， 他们的情况。这些遗传学研究表明，ALS可能是由以下基因突变引起的： 编码蛋白质的基因参与多种细胞功能，包括RNA加工、囊泡 转运、细胞骨架调节、线粒体功能和蛋白质质量控制途径。然而，尽管如此， ALS患者的共同特征是进行性运动神经变性的共同模式。这 不同的疾病引发事件可能在一个或多个共同的分子中合并， 途径。具有不同功能的基因突变如何会聚在MN变性上已经被研究， 仍然是个悬而未决的问题。尽管所有ALS患者都表现出神经病理蛋白 尽管蛋白质聚集体的存在，但蛋白质稳态在引起ALS中的总体贡献仍不清楚。如果我们能 确定一个会聚机制，它可以提供一个机会，以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，我们对蛋白质降解动力学进行了全局分析 在突变体SOD1和源自iPSC系的同基因对照MN中。有趣的是，我们发现了一些 在SOD1 MN中以较慢速率降解的蛋白质。出乎意料的是，这一小组候选人 包括基因突变导致ALS的蛋白质。在拟议的研究中，我们将使用患者来源的 神经元与质谱分析相结合，以确定蛋白质底物，以及 由于两个最常见的ALS基因突变而引起的扰动：SOD1和C9orf72。 首先，我们将确定哪些蛋白质具有降低的蛋白质降解动力学。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 重塑这些方法中的每一种都比传统的工作流程具有战略优势， 不仅可以确定哪些蛋白质在ALS MN中的水平发生了改变，而且还可以确定导致其改变的机制。 扰动总之，我们提出的目标将揭示细胞机制受到损害， 患者神经元中蛋白质稳态网络的变化，并可能揭示广泛相关的治疗方法。 ALS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.