Deciphering the role of Ataxin-2 in amyotrophic lateral sclerosis

肌萎缩侧索硬化症(ALS)是一种毁灭性的神经退行性疾病，其特征是进行性丧失自主肌肉控制[1]。吉特勒实验室--我将在那里进行这项研究--发现，ataxin-2基因(ATXN2)突变是ALS相对常见的遗传风险因素[2]。该突变是ATXN2编码区CAG重复序列的中等长度扩张，导致Aaxin-2蛋白中的聚谷氨酰胺区更长。野生型Atxn2转录本的减少延长了ALS小鼠模型的存活率并减少了病理，就像将该小鼠与Aaxin-2基因敲除小鼠杂交一样[3]。尽管有这些有希望的结果，但人们对野生型Aaxin-2在ALS中的作用知之甚少。RNA代谢缺陷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者，然而在其控制下的转录本仅在个案的基础上被识别[7-12]。首先，我有兴趣探索敲除Aaxin-2如何导致翻译缺陷，以及这是否为转基因TDP-43(TDP-43tg/TG)ALS小鼠模型提供运动神经元保护。我将使用我在研究生院期间获得的专业知识进行全基因组和生化翻译分析，但将其与一套研究复杂组织中mRNA动态的新技术结合起来。我将执行TRAP-SEQ，这是一种通过纯化与翻译核糖体结合的mRNA来衡量单个转录本上的翻译水平的技术[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Aaxin-2基因敲除对其的影响。Aaxin-2是特殊信使核糖核蛋白(MRNP)颗粒的组成部分，通过RNA结合与TDP-43相互作用[2，8，14]。MRNP颗粒参与将mRNA运送到细胞的不同部分，以进行适当的转录后处理[15，16]。在培养的外周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中都检测到轴突mRNA定位缺陷，但从来没有直接从组织中检测到，因为这项技术以前没有得到[17，18]。我将使用一种名为APEX-seq的新技术来确定WT和TDP-43TG/TG小鼠中空间限制于外周运动轴突的mRNA转录的组成，以及当与Aaxin-2基因敲除时这种转录产物的组成是如何变化的[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，以发现Aaxin-2的调节因子，它将照亮上游控制其表达的途径。吉特勒实验室精通识别疾病修饰物的大规模方法[21-23]。这一目标的目的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筛查。这个项目让我有机会扩展我在神经疾病的RNA新陈代谢这一主题上的专业知识，这是我计划在我的研究生涯中发展的主题，并破译治疗开发和未来研究的最有前途的目标。

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.