Modifiers of Age of Onset in ALS Studied Using Patient iPS-derived Motor Neurons

描述(由申请人提供)：使用患者iPS衍生运动神经元进行的ALS发病年龄修饰物研究肌萎缩侧索硬化症(ALS)是一种一致致命的与年龄相关的神经退行性疾病，破坏运动神经元。虽然所有的肌萎缩侧索硬化症患者都是成年人，但发病年龄可能相差很大，大约60岁。在ALS中，决定衰老和神经退变相互作用的确切因素尚不清楚。我对发表的ALS患者运动神经元的表达谱进行了重新分析，发现了一组基因的表达水平与症状开始时的年龄高度相关。这些基因是ALS疾病严重程度的潜在修饰者。这项工作的总体目标是使用来自患者特异性诱导多能干细胞(iPS细胞)的运动神经元来验证这些候选的疾病发病修饰物，并在iPS来源的运动神经元中发现新的ALS依赖的基因表达变化。直到最近，还不可能从ALS患者身上研究活的、症状前的人类运动神经元。然而，我们已经从ALS患者和对照中获得了多个iPS系，并将它们分化为患者特有的运动神经元(IPSMN)。这项工作的第一个目标将是表征和改进我开发的从分化过程中产生的混合培养物中纯化iPSMN的系统。为了达到这个目的，iPS来源的运动神经元将被一种编码运动神经元特异性报告基因的慢病毒感染。然后，病毒标记的细胞将被表征，以揭示报告策略的任何运动神经元亚型偏见。在此之后，我将尝试通过分离共表达病毒报告基因和我在先前工作中鉴定的运动神经元丰富的表面标记的细胞，来改进通过这项技术实现的运动神经元的大量丰富。[然后，我将使用这一浓缩策略来测试这样一个假设，即先前确定的候选发病修改基因将在iPSMN中显示与ALS患者捐赠者的疾病发病相关的表达模式，而不是与对照组的年龄相关。任何阳性结果都表明这些候选修饰物的表达是运动神经元的一个稳定特征，并可能在决定ALS的发病中发挥作用。最后，我将确定ALS患者来源的iPSMN中任何有效的发病依赖表达模式的SOD1依赖性。这最后一组实验将有助于确定是否有任何经过验证的候选基因是真正的发病修饰基因或疾病的早期标记。这一目标有可能定义第一个与肌萎缩侧索硬化症相关的iPSMN差异。了解这些差异可能反过来导致改善ALS预后的先验措施和更相关的治疗目标。]] 与公共卫生相关：该项目使用从ALS(Lou Gehrig病)患者身上产生的干细胞，试图了解是什么原因导致一些患者在生命早期患上这种致命疾病，而另一些患者直到老年才表现出症状。最终，这项研究可能会导致ALS的新疗法。

DESCRIPTION (provided by applicant): Modifiers of Age of Onset in ALS Studied Using Patient iPS-derived Motor Neurons Amyotrophic lateral sclerosis (ALS) is a uniformly fatal age-associated neurodegenerative disease that destroys motor neurons. Although all ALS patients are adults, age of onset can vary widely, with a range of about 60 years. The precise factors that determine the interaction of aging and neurodegeneration in ALS are unknown. I performed a re-analysis of published expression profiles from ALS-patient motoneurons and uncovered a group of genes expressed at levels highly correlated with age at symptom onset. These genes are potential modifiers of ALS disease severity. The overall goal of this work is to use motoneurons derived from patient-specific induced pluripotent stem cells (iPS cells) to validate these candidate disease onset- modifiers and to discover new ALS-dependent gene expression changes in iPS-derived motoneurons. Until recently, it was not possible to study living, pre-symptomatic human motoneurons from ALS patients. However, we have derived multiple iPS lines from ALS patients and controls and differentiated them into patient-specific motor neurons (iPSMNs). The first aim of this work will be to characterize and improve upon a system that I have developed to purify iPSMNs from the mixed cultures produced during differentiation. In this aim, iPS-derived motoneurons will be infected with a lentivirus that encodes a motoneuron-specific reporter. Virally labeled cells will then be characterized to reveal any motoneuron-subtype biases of the reporter strategy. Following this, I will attempt to improve upon the already substantial enrichment of motoneurons achieved through this technique by isolating cells that co-express the viral reporter and motoneuron-enriched surface markers that I have identified in previous work. [[I will then use this enrichment strategy to test the hypothesis that the candidate onset-modifying genes identified earlier will show expression patterns in iPSMNs that correlate with disease onset in ALS-patient donors, but not the age of controls. Any positive results would indicate that expression of these candidate- modifiers is a stable trait of motoneurons and may play a role in determining ALS onset. Finally, I will determine the SOD1 dependence of any validated onset-dependent expression patterns in ALS patient-derived iPSMNs. This final set of experiments will help to determine whether any validated candidate genes are true onset-modifiers or very early markers of disease. This aim has the potential to define the first ALS-related differences in iPSMNs. Understanding these differences may, in turn, lead to improved a priori measures of ALS prognosis and more relevant therapeutic targets.]] PUBLIC HEALTH RELEVANCE: This project uses stem cells generated from patients with ALS (Lou Gehrig's disease) in an attempt to understand what causes some patients to fall ill with this fatal disease early in life, while others do not show symptoms until old-age. Eventually, this research could lead to new treatments for ALS.