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

揭示ataxin-2在神经元功能和神经退行性疾病中的转录调节作用

• 批准号: 9910298
• 负责人: Ryan J Marina
• 金额: $ 3.79万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-02 至 2021-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910298
• 关键词: Address; Adult; Affect; Alleles; Amyotrophic Lateral Sclerosis; Animals; Antisense Oligonucleotides; Binding; Bioinformatics; Biology; Brain; C9ORF72; CAG repeat; CRISPR/Cas technology; Cell Line; Cell model; Cell physiology; Cells; Cessation of life; Disease; Disease model; Etiology; Exhibits; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Human; Immunoprecipitation; In Vitro; Knock-in; Knockout Mice; Length; Link; Maps; Mediating; Metabolism; Modeling; Molecular; Motor Activity; Motor Neuron Disease; Motor Neurons; Mus; Muscular Atrophy; Mutation; Nerve Degeneration; Nervous system structure; Neuraxis; Neurobiology; Neurodegenerative Disorders; Neurons; Onset of illness; Paralysed; Pathogenesis; Pathway interactions; Patients; Physiology; Play; Population; Process; Proteins; RNA; RNA Stability; RNA-Binding Proteins; Recurrence; Repetitive Sequence; Reporting; Research; RiboTag; Ribonucleoproteins; Ribosomal RNA; Risk; Role; SCA2 protein; Spinal Cord; Spinocerebellar Ataxias; Spinocerebellar Degenerations; Techniques; Testing; Time; Tissues; Translational Activation; Translations; Type 2 Spinocerebellar Ataxia; Work; Yeasts; cell type; crosslink; deep sequencing; gain of function; human disease; in vivo; in vivo Model; induced pluripotent stem cell; insight; link protein; motor neuron degeneration; mutant; neuron loss; novel; overexpression; polyglutamine; protein TDP-43; protein function; relating to nervous system; ribosome profiling; stem cell differentiation; stress granule; therapeutic target; tool; transcriptome; transcriptome sequencing

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.

摘要 Ataxin-2（ATXN 2）是一种在真核生物中广泛表达的RNA结合蛋白 从酵母到人类多聚谷氨酰胺（polyQ）序列的三核苷酸（CAG）重复扩增突变 ATXN 2基因与包括脊髓小脑性共济失调2型（SCA 2）在内的多种神经退行性疾病相关 和肌萎缩性侧索硬化症（ALS），一种致命的成人发病性疾病，其特征在于 大脑和脊髓的运动神经元。体内模型概括了这种与运动神经元变性的联系 和ALS，因为据报道共济失调蛋白-2是TDP-43的毒性修饰剂，TDP-43是一种最常见的与这两种疾病相关的蛋白质。 家族性和散发性ALS研究支持这种毒性功能获得模型，因为 共济失调蛋白-2水平似乎对神经元变性起保护作用，同时延长了神经元的功能。 在TDP-43过表达存在下Atxn 2缺失小鼠的存活率。同时，内源性神经- 共济失调蛋白-2的具体功能仍然知之甚少，其在介导ALS发病机制中的直接作用也是如此。 该项目旨在揭示共济失调蛋白-2在中枢神经系统中的转录组调节作用， 为了机械地表征ATXN 2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和核糖体分析， 绘制小鼠脑和脊髓以及人类中共济失调蛋白-2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疾病的联系，该提案还旨在 使用CRISPR/Cas9技术，通过敲入ALS相关的polyQ， 将扩增序列插入ATXN 2基因组基因座。然后这些细胞系将分化成成熟的 运动神经元，以研究神经特异性转录和翻译扰动，通过 重复扩张。这项研究将直接表征共济失调蛋白-2在神经元特异性RNA代谢中的作用， 以及鉴定被polyQ突变破坏的特定靶基因。如果这项研究成功， 将共济失调蛋白-2与ALS风险联系起来的潜在分子机制，确定新的疾病相关途径， 潜在地用作治疗的靶向途径，并提供广泛适用的疾病建模策略 在未来的工作中研究其他重复扩展突变的直接遗传影响。