Deciphering the role of Ataxin-2 in amyotrophic lateral sclerosis

摘要： 肌萎缩侧索硬化症(ALS)是一种以进行性为特征的破坏性神经退行性疾病 失去自主肌肉控制[1]。吉特勒实验室--我将在那里进行这项研究--发现一个 Ataxin-2基因突变(ATXN2)是ALS相对常见的遗传风险因素。这种突变是一种 ATXN2编码区CAG重复序列的中等长度扩展，导致更长的聚谷氨酰胺 Aaxin-2蛋白中的区域。野生型Atxn2转录本的减少延长了生存时间并降低了 ALS小鼠模型的病理学，就像将该小鼠与Aaxin-2基因敲除小鼠杂交一样[3]。尽管 这些有希望的结果，对野生型Aaxin-2在ALS中的作用知之甚少。RNA中的缺陷 代谢已成为肌萎缩侧索硬化症的中心机制[4-6]。Aaxin-2是信使核糖核酸翻译的调节因子， 然而，在其控制下的文字记录只是在个案的基础上确定的[7-12]。首先，我是 有兴趣研究敲除Aaxin-2如何导致翻译缺陷，以及这是否为运动神经元提供 转基因TDP-43(TDP-43TG/TG)ALS小鼠模型的保护作用我会用我在 研究生院进行全基因组和生化翻译分析，但将其与一套新的 研究复杂组织中信使核糖核酸动力学的技术。我将执行Trap-Seq，这是一种 通过提纯与翻译核糖体结合的mRNA来衡量单个转录本上的翻译水平[13]。 这将使我能够确定TDP-43TG/TG小鼠运动神经元中差异翻译的转录本，以及 这是如何受到Aaxin-2基因敲除的影响的。Aaxin-2是专门化信使的组成部分 核糖核蛋白(MRNP)颗粒，并通过RNA结合与TDP-43相互作用[2，8，14]。MRNP 颗粒参与将信使核糖核酸运输到细胞的不同部分，以进行适当的转录后转录。 正在处理[15，16]。在两种培养的外周血中都检测到轴突mRNA定位缺陷。 来自多个转基因ALS小鼠模型的神经元和小鼠胚胎干细胞来源的运动神经元， 但从未直接从组织中获得，因为这项技术以前是不可用的[17，18]。我将用一本小说 一种被称为APEX-SEQ的技术，用于确定空间限制为 WT和TDP-43TG/TG小鼠的外周运动轴突起，以及当与Aaxin-2交叉时这种变化是如何变化的 淘汰赛[19，20]。正如我在第二个目标中所描述的，我将在人类细胞中进行全基因组siRNA筛选 发现Aaxin-2的调节子，它将照亮上游控制其表达的途径。这个 吉特勒实验室精通识别疾病修饰物的大规模方法[21-23]。这一目标的目标是 利用我们的结果来设计新的治疗策略，并将我的培训扩展到包括全基因组 放映。这个项目让我有机会扩展我在RNA新陈代谢方面的专业知识 神经疾病，这是我计划以研究为职业的主题，并破译最有希望的 治疗发展和未来研究的目标。

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.