Deciphering the role of Ataxin-2 in amyotrophic lateral sclerosis

摘要： 肌萎缩侧索硬化症（ALS）是一种以进行性脊髓损伤为特征的破坏性神经退行性疾病。 失去自主肌肉控制[1]。我将在吉特勒实验室进行这项研究， ataxin-2基因（ATXN 2）突变是ALS相对常见的遗传风险因素[2]。述突变是 ATXN 2编码区CAG重复序列的中等长度扩增，导致更长的多聚谷氨酰胺 在Ataxin-2蛋白中。野生型Atxn 2转录物的减少延长了存活期，并降低了 在ALS的小鼠模型中，如将该小鼠与共济失调蛋白-2敲除小鼠杂交一样，可以观察到病理学变化[3]。尽管 尽管有这些有希望的结果，但关于野生型Ataxin-2如何导致ALS的知之甚少。RNA缺陷 代谢已成为ALS的中心机制[4-6]。共济失调蛋白-2是mRNA翻译的调节因子， 然而，在其控制下的记录誊本仅在个案基础上确定[7-12]。一是 有兴趣探索Ataxin-2的敲除如何消除翻译缺陷，以及这是否能为运动神经元提供 在转基因TDP-43（TDP-43 tg/tg）ALS小鼠模型中的保护作用。我会利用我在 研究生院进行全基因组和生化翻译测定，但联合收割机这与一套新的 研究复杂组织中mRNA动态的技术。我将执行TRAP-seq，一种技术， 通过纯化与翻译核糖体结合的mRNA来测量单个转录物的翻译水平[13]。 这将使我能够确定TDP-43 tg/tg小鼠运动神经元中具有差异翻译的转录本， 是如何受到共济失调蛋白2基因敲除的影响共济失调蛋白-2是一个不可分割的组成部分， 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 颗粒参与将mRNA转运到细胞的各个部分，以进行适当的转录后调节。 加工[15，16]。在两种培养的外周血中均检测到轴突mRNA定位缺陷。 神经元和来自多个转基因ALS小鼠模型的小鼠胚胎干细胞衍生的运动神经元， 但从未直接从组织中提取，因为该技术以前不可用[17，18]。我会写一本小说 称为APEX-seq的技术来确定mRNA转录物的组成， WT和TDP-43 tg/tg小鼠的外周运动轴突，以及当与Ataxin-2杂交时， 淘汰赛[19，20]。正如我的第二个目标所描述的，我将在人类细胞中进行全基因组siRNA筛选 发现Ataxin-2的调节因子，以阐明上游控制其表达的途径。的 Gitler实验室精通大规模方法来识别疾病修饰物[21-23]。这一目标的目标是 利用我们的研究结果来设计新的治疗策略，并扩大我的培训范围， 筛选这个项目让我有机会扩大我的专业知识在RNA代谢的主题， 神经系统疾病，我计划使我的职业生涯在研究的主题，并破译最有前途的 治疗开发和未来研究的目标。

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.