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

项目摘要/摘要 肌萎缩侧索硬化症(ALS)是一种致命的神经退行性疾病，估计终身风险为1 400个人。肌萎缩侧索硬化症的临床特征是运动障碍，病理上表现为选择性运动丧失。 大脑和脊髓中的神经元，以及泛素化的TDP蛋白聚集体的沉积。 43.尽管ALS患者几乎所有(~97%)的脑中都存在TDP-43病理，但基因 这种疾病的基础是高度不同的，大约90%被认为是散发性的，或有 没有已知的遗传原因。潜在原因的多变性质使得对这种疾病的治疗 由于缺乏明确的治疗目标，这在历史上是困难的。在过去的十年中，出现了Aaxin-2(ATXN2) 作为ALS的一个有前途的治疗靶点，作为TDP-43聚集和毒性的有效遗传修饰物 多种TDP-43蛋白病模型。最令人兴奋的是，使用反义技术降低ATXN2水平 寡核苷酸(ASO)在TDP-43过度表达的小鼠模型中对运动的明显拯救作用 损伤和寿命的显著延长。尽管有ASOS的承诺，但有一种正交方法来 降低ATXN水平--例如一种可以针对其调节器之一的小分子药物--可能会产生巨大的影响 在临床环境中的实际益处。此外，关于ATXN2通常是如何调控的，目前还知之甚少，因为 以及它在疾病中的真正作用。为了获得机械性的洞察力并确定其他治疗靶点，我 开发了一种新的基于荧光激活细胞分类(FACS)的CRISPR/Cas9全基因组敲除 筛选策略。我们的想法是在一个可靠的 在基因敲除后降低ATXN2水平的基因可以作为新的治疗靶点 而那些在基因敲除时增加ATXN2水平的基因可能会增加患ALS的风险 这种疾病。这部银幕产生了许多有希望的热门作品，其中许多都是通过相同的生物途径发挥作用，或者 有时编码一种蛋白质复合体的亚基。溶酶体空泡ATPase就是其中的一个例子 (v-ATPase)，编码几乎每个亚基的基因都被发现是ATXN2的显著抑制者 我屏幕上的蛋白质水平。除了验证来自多个相关疾病的初始屏幕的命中之外 系统--如小鼠原代神经元和人类IPSC来源的神经元--I将把分析范围扩大到 深入研究v-ATPase如何调节ATXN2蛋白水平的机制。此外，鉴于 几种FDA批准的小分子药物可以抑制v-ATPase亚基，我将测试它们的安全性 并在体内降低ATXN2水平和挽救ALS小鼠模型的疾病表型方面的有效性。如果这个 方法是成功的，这个筛查平台和总体目标有很多令人兴奋的可能性 发现方法，我相信这可能有助于发现许多其他神经退行性疾病的调节者 基因(如FTD和阿尔茨海默病中的tau和Ab，帕金森病中的a-突触核蛋白)增强 在不限于肌萎缩侧索硬化症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.