Uncovering the transcriptional regulatory roles of ataxin-2 in neuronal function and neurodegenerative disease

摘要 Aaxin-2(ATXN2)是一种广泛表达的RNA结合蛋白，在真核生物中高度保守 从酵母到人类。猪多聚谷氨酰胺(PolyQ)链的三核苷酸(CAG)重复扩增突变 ATXN2基因与包括脊髓小脑性共济失调2(Sca2)在内的多种神经退行性疾病有关 和肌萎缩侧索硬化症(ALS)，这是一种致命的成人发病疾病，其特征是进行性死亡 大脑和脊髓的运动神经元。活体模型重述了这种与运动神经元退化的联系 和肌萎缩侧索硬化症，因为ataxin-2被报道是TDP-43的有毒修饰物，TDP-43是一种最常见的与 家族性和散发性的肌萎缩侧索硬化。研究已经支持这种有毒的功能获得模型，因为减少 Aaxin-2水平似乎对神经元退化起到保护作用，同时延长 在存在TDP-43过表达的情况下，Atxn2基因缺失小鼠的存活率。同时，内源性神经- Ataxin-2的具体功能以及它在调节ALS发病中的直接作用仍然知之甚少。 该项目旨在揭示ataxin-2在中枢神经系统中的转录组调控作用。 目的是从力学上表征ATXN2突变和运动神经元死亡之间的相互作用。这 该提案寻求使用分子和基因组技术，包括eCLIP-seq、RNA-seq和核糖体图谱， 绘制ataxin-2在小鼠脑和脊髓以及在人类中的物理和功能相互作用组 IPSC-分化的运动神经元。为了解决与运动神经元病的联系，这项提案还旨在 使用CRISPR/Cas9技术通过敲入ALS相关的PolyQ来生成等基因的IPSC模型 将扩增序列插入到ATXN2基因组中。这些细胞系随后将被分化为成熟细胞 运动神经元，以研究神经特有的转录和翻译扰动 重复扩展。这项研究将直接表征ataxin-2在神经元特异性RNA代谢中的作用， 以及识别被多聚Q突变扰乱的特定靶基因。如果成功，这项研究将定义 将ataxin-2与ALS风险联系起来的潜在分子机制，确定了与疾病相关的新途径 潜在地作为有针对性的治疗途径，并提供一个广泛适用的疾病建模策略 在未来的工作中，研究其他重复序列扩展突变的直接遗传影响。

ABSTRACT Ataxin-2 (ATXN2) is a ubiquitously expressed RNA-binding protein (RBP) conserved across eukaryotic species from yeast to human. Trinucleotide (CAG) repeat expansion mutations of the poly-glutamine (polyQ) tract of the ATXN2 gene are associated with several neurodegenerative diseases including spinocerebellar ataxia 2 (SCA2) and amyotrophic lateral sclerosis (ALS), a fatal adult-onset disorder characterized by the progressive death of motor neurons of the brain and spinal cord. In vivo models recapitulate this link to motor neuron degeneration and ALS, as ataxin-2 is reported to be a toxic modifier of TDP-43, a protein most commonly associated with both the familial and sporadic forms of ALS. Studies have supported this toxic gain-of-function model, as reduction of ataxin-2 levels seems to serve a protective role against neuron degeneration while simultaneously prolonging survival in Atxn2-null mice in the presence of TDP-43 overexpression. At the same time, endogenous neural- specific functions of ataxin-2 are still poorly understood, as are its direct role in mediating ALS pathogenesis. This project aims to uncover the transcriptome-wide regulatory role of ataxin-2 in the central nervous system in order to mechanistically characterize the interaction between ATXN2 mutations and motor neuron death. This proposal seeks to use molecular and genomic techniques, including eCLIP-seq, RNA-seq and ribosome profiling, to map the physical and functional “interactome” of ataxin-2 in mouse brain and spinal cord, as well as in human iPSC-differentiated motor neurons. To address the connection to motor neuron disease, this proposal also aims to generate an isogenic iPSC model using CRISPR/Cas9 technologies by knocking-in ALS-associated polyQ expansion sequences into the ATXN2 genomic locus. These cell lines will then be differentiated into mature motor neurons in order to study neural-specific transcriptional and translational perturbations introduced through repeat expansions. This study will directly characterize the role of ataxin-2 in neuron-specific RNA metabolism, as well as identify specific target genes disrupted with polyQ mutations. If successful this research will define the underlying molecular mechanisms linking ataxin-2 to ALS risk, identify novel disease-relevant pathways that potentially serve as targetable avenues for therapy, and provide a broadly applicable disease modeling strategy to investigate the direct genetic influence of other repeat expansion mutations in future work.