A Novel Genome-Wide Screen to Identify and Characterize Regulators of ALS Disease Modifier Gene Ataxin-2

项目总结/摘要 肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，估计终生风险为1/ 400个人。ALS的临床特征是运动缺陷，病理特征是运动功能的选择性丧失。 脑和脊髓中的神经元，以及TDP的泛素化蛋白质聚集体的沉积， 43.尽管几乎所有（~97%）ALS患者的大脑中都存在TDP-43病理，但遗传学上的 该疾病的基础是高度异质性的，约90%被认为是"散发性的"，或具有 没有已知的遗传原因。潜在原因的可变性使得对这种疾病的治疗 由于缺乏明确的治疗靶点，在过去的十年中，Ataxin-2（ATXN2）已经出现 作为ALS的有希望的治疗靶点，作为TDP-43聚集和毒性的有效遗传修饰剂， TDP-43蛋白质病的多种模型。最令人兴奋的是，使用反义核酸降低ATXN2水平， 在TDP-43过表达的小鼠模型中，寡核苷酸（ASO）导致运动神经元的显著拯救。 损伤和寿命的显著延长。尽管ASO有希望，但使用正交方法来 降低ATXN2水平--例如一种可以靶向其调节因子之一的小分子药物--可能会产生巨大的 在临床上的实际效益。此外，人们对ATXN2通常如何调节知之甚少， 以及它在疾病中的真正作用。为了获得机制的见解以及确定其他治疗靶点，我 开发了一种新的基于FACS（荧光激活细胞分选）的CRISPR/Cas9全基因组敲除 筛选策略这个想法是为了确定ATXN2蛋白水平的抑制剂和增强剂， 敲除后降低ATXN2水平的基因可以作为新的治疗靶点， ALS，而那些在敲除后增加ATXN2水平的人可能会导致ALS的风险增加。 这种疾病筛选产生了大量有希望的命中，其中许多在相同的生物途径中起作用，或 有时编码一个蛋白质复合体的亚基。其中一个例子是溶酶体空泡ATP酶 （v-ATP酶），其中编码几乎每个亚基的基因被发现是ATXN2的显著抑制因子 蛋白质水平的变化除了验证多个疾病相关的初始筛选结果外， 系统-如小鼠原代神经元和人类iPSC衍生的神经元-我将扩展分析， 深入研究v-ATPase如何调节ATXN2蛋白水平的机制。此外，鉴于 有几种FDA批准的小分子药物可以抑制v-ATP酶亚基，我将测试它们的安全性 以及在体内ALS小鼠模型中降低ATXN2水平和挽救疾病表型的功效。如果这 虽然这种方法是成功的，但这种筛选平台和总体目标有许多令人兴奋的可能性。 我相信这种发现方法可以帮助发现许多其他神经退行性疾病的调节因子 基因（例如，FTD和阿尔茨海默病中的tau和Ab，帕金森病中的α-突触核蛋白）， 在不限于ALS的情况下发现新的治疗靶点。

Project Summary / Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with an estimated lifetime risk of 1 in 400 individuals. ALS is clinically characterized by motor deficits, and pathologically by the selective loss of motor neurons in the brain and spinal cord, as well as deposition of ubiquitinated proteinaceous aggregates of TDP- 43. Despite the presence of TDP-43 pathology in nearly all (~97%) brains of ALS patients, the genetic underpinnings of the disease is highly heterogeneous, with ~90% being considered to be ‘sporadic,’ or having no known genetic cause. The variable nature of the underlying causes has made treatment of the disease historically difficult due to a lack of clear therapeutic targets. In the past decade, Ataxin-2 (ATXN2) has emerged as a promising therapeutic target for ALS, as a potent genetic modifier of TDP-43 aggregation and toxicity across multiple models of TDP-43 proteinopathy. Most excitingly, decreasing ATXN2 levels using anti-sense oligonucleotides (ASOs) in a mouse model of TDP-43 overexpression led to a marked rescue of motor impairments and dramatic extension of lifespan. Despite the promise of ASOs, having an orthogonal method to reduce ATXN2 levels—such as a small molecule drug that can target one of its regulators—could have immense practical benefit in the clinical context. Moreover, little remains known on how ATXN2 is normally regulated, as well as its true role in disease. To gain mechanistic insight as well as to identify additional therapeutic targets, I developed a novel FACS (fluorescence activated cell sorting)-based CRISPR/Cas9 genome-wide knockout screening strategy. The idea was to identify suppressors and enhancers of ATXN2 protein levels in a reliable and efficient way; genes that decrease ATXN2 levels upon knockout could serve as novel therapeutic targets for ALS, while those that increase ATXN2 levels upon knockout could potentially contribute to heightened risk for the disease. The screen yielded a multitude of promising hits, with many acting in same biological pathways, or sometimes encoding subunits of one protein complex. One example of this is the lysosomal vacuolar ATPase (v-ATPase), for which genes encoding nearly every subunit were found to be significant suppressors of ATXN2 protein levels in my screens. In addition to validating hits from the initial screens across multiple disease relevant systems—such as in mouse primary neurons and human iPSC-derived neurons—I will expand the analysis to delve deeper into the mechanism of how the v-ATPase is regulating ATXN2 protein levels. Moreover, given that several FDA-approved small molecule drugs are available that inhibit v-ATPase subunits, I will test their safety and efficacy in reducing ATXN2 levels and rescuing disease phenotypes in a mouse model of ALS in vivo. If this approach is successful, there are a multitude of exciting possibilities for this screening platform and overall target discovery approach that I believe could help to uncover regulators of many other neurodegenerative diseases genes (e.g., tau and Ab in FTD and Alzheimer’s Disease, a-synuclein in Parkinson’s Disease) to empower the discovery of novel therapeutic targets in contexts not limited to ALS.