Deciphering the role of Ataxin-2 in amyotrophic lateral sclerosis

解读 Ataxin-2 在肌萎缩侧索硬化症中的作用

• 批准号: 10395882
• 负责人: Caitlin Marie Rodriguez
• 金额: $ 3.43万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2022-04-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10395882
• 关键词: Deciphering; role; Ataxin; amyotrophic; lateral

Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of voluntary muscle control[1]. The Gitler lab—where I will be conducting this research—discovered that a mutation in the ataxin-2 gene (ATXN2) is a relatively common genetic risk factor for ALS[2]. The mutation is an intermediate-length expansion of a CAG repeat in the ATXN2 coding region, leading to longer polyglutamine tracts in the Ataxin-2 protein. Reduction of the wild-type Atxn2 transcript extended survival and reduced pathology in a mouse model of ALS, as did crossing this mouse with the Ataxin-2 knockout mouse[3]. Despite these promising results, little is known about how wild-type Ataxin-2 contributes to ALS. Defects in RNA metabolism has emerged as a central mechanism in ALS[4-6]. Ataxin-2 is a regulator of mRNA translation, however transcripts under its control have only been identified on a case-by-case basis[7-12]. First, I am interested in exploring how knockout of Ataxin-2 elicits deficits in translation, and if this affords motor neurons protection in the transgenic TDP-43 (TDP-43tg/tg) ALS mouse model. I will use the expertise I gained during graduate school to perform genome-wide and biochemical translation assays, but combine this with a new set of techniques for investigating mRNA dynamics in complex tissue. I will perform TRAP-seq, a technique for gauging the level of translation on individual transcripts by purifying mRNA bound to translating ribosomes[13]. This will allow me to determine transcripts with differential translation in TDP-43tg/tg mouse motor neurons, and how that is affected by the Ataxin-2 knockout. Ataxin-2 is an integral component of specialized messenger ribonucleoprotein (mRNP) granules, and interacts with TDP-43 through RNA association[2, 8, 14]. mRNP granules are involved in the transport of mRNA to various parts of the cell for proper posttranscriptional processing[15, 16]. Deficits in axonal mRNA localization have been detected in both cultured peripheral neurons and mouse embryonic stem cell-derived motor neurons from multiple transgenic ALS mouse models, but never directly from tissue as the technology was not previously available[17, 18]. I will employ a novel technique called APEX-seq to determine the composition of mRNA transcripts spatially constricted to peripheral motor axons in WT and TDP-43tg/tg mice, and how this changes when crossed to the Ataxin-2 knockout[19, 20]. As described in my second aim, I will perform a genome-wide siRNA screen in human cells to discover regulators of Ataxin-2 that will illuminate pathways that work upstream to control its expression. The Gitler lab is proficient in large-scale approaches to identifying disease modifiers[21-23]. The goal of this aim is to harness our results to devise novel therapeutic strategies and to expand my training to include genome-wide screening. This project allows me the opportunity to expand my expertise in the topic of RNA metabolism in neurological disease, the topic I plan to make my career in researching, and to decipher the most promising targets for therapeutic development and future study.

抽象的： 肌萎缩性侧索硬化症（ALS）是一种毁灭性的神经退行性疾病，其特征是进行性疾病 自愿控制的丧失[1]。吉特勒实验室（我将在哪里进行这项研究）发现 ataxin-2基因中的突变（ATXN2）是ALS的相对常见的遗传危险因素[2]。突变是 CAG在ATXN2编码区重复的中间长度扩展，导致多谷氨酰胺 ataxin-2蛋白质中的区域。野生型ATXN2转录本的降低延长存活率并降低 ALS的小鼠模型中的病理，将小鼠与ataxin-2基因敲除小鼠越过[3]。尽管 这些希望的结果，关于野生型ataxin-2对ALS的贡献知之甚少。 RNA中的缺陷 代谢已成为ALS中的一种核心机制[4-6]。 ataxin-2是mRNA翻译的调节剂， 但是，仅逐案[7-12]才鉴定出其控制下的转录本。首先，我是 有兴趣探索ataxin-2的敲除如何在翻译中定义，并且是否提供了运动神经元 转基因TDP-43（TDP-43TG/TG）ALS鼠标模型中的保护。我将使用我在 研究全基因组和生化翻译分析的研究生院，但将其与新集合结合起来 用于研究复杂组织中mRNA动力学的技术。我将执行Trap-Seq，这是一种技术 通过净化与翻译核糖体的mRNA纯化mRNA来评估单个转录本上的翻译水平[13]。 这将使我能够在TDP-43TG/TG小鼠运动神经元中确定具有差分翻译的成绩单，并且 这是如何受ataxin-2敲除的影响。 Ataxin-2是专用Messenger不可或缺的组成部分 核糖核蛋白（MRNP）颗粒，并通过RNA缔合与TDP-43相互作用[2，8，14]。 mrnp 颗粒参与mRNA到细胞的各个部分的运输，以进行适当的转录后 处理[15，16]。在两个培养的周围都发现了轴突mRNA定位的缺陷 来自多个转基因ALS小鼠模型的神经元和小鼠胚胎干细胞衍生的运动神经元， 但是，由于该技术以前无法使用，因此永远不会直接来自组织[17，18]。我将雇用一本小说 称为Apex-Seq的技术，以确定mRNA转录的组成在空间上限制为 WT和TDP-43TG/TG小鼠中的外围运动轴突，以及交叉到ataxin-2时的变化 淘汰赛[19，20]。如我的第二个目标所述，我将在人类细胞中执行全基因组siRNA筛选 发现Ataxin-2的调节剂将照亮在上游控制其表达的途径。 吉特勒实验室（Gitler Lab）精通鉴定疾病修饰剂的大规模方法[21-23]。这个目标的目的是 为了利用我们的结果制定新颖的治疗策略并扩大我的培训以包括全基因组 筛选。这个项目使我有机会扩大我在RNA代谢主题中的专业知识 神经疾病，我打算从事研究职业的话题，并破译最有前途的 治疗发展和未来研究的目标。