Mapping TDP-43 RNA binding partners in motor neuron differentiation and ALS pathology

绘制运动神经元分化和 ALS 病理学中 TDP-43 RNA 结合伴侣的图谱

• 批准号: 2250125
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2250125
• 关键词: Mapping; TDP; 43; RNA; binding

TAR DNA Binding Protein 43 kDa (TDP-43) is an RNA/DNA-binding protein that is fundamentally implicated in the development and disease of the nervous system. It is predominantly localized in the nucleus, where it is implicated in multiple steps of transcriptional and post-transcriptional regulation. In particular, TDP-43 has a key role in alternative splicing, stability and transport of essential neurodevelopmental genes. Aberrant cytoplasmic mislocalization, ubiquitination, abnormal phosphorylation, cleavage and aggregation of TDP-43 are common histopathological hallmarks of a spectrum of neurodegenerative disorders, collectively known as TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS). ALS is an age-related, incurable disease characterized by selective motor neuron (MN) degeneration. The progressive loss of upper and lower MNs in the brain, brainstem and spinal cord, cause paralysis and ultimately death. Aberrant TDP-43 mislocalization is found in 97% of all ALS cases, bridging both familial and sporadic forms of ALS. Given its pleiotropic nature and the multiple cellular processes that are aberrantly affected by TDP-43 aggregation and/or nuclear depletion, establishing direct causal links between TDP-43 and disease remains challenging. The molecular mechanisms by which TDP-43 mislocalization affects human motor neurons remains unresolved, as do its precise roles in normal motor neurogenesis. Against this background, I wish to understand whether TDP-43 pathology is the result of a nuclear loss of function (e.g aberrant pre-mRNA processing), a cytoplasmic gain of toxic function, or a combination of the two. Specifically, I will address the following questions: (i) how does TDP-43 regulate motor neuron development; (ii) how and when does this regulation go awry in ALS (iii) and what target genes are affected; (iv) and how does TDP-43 post-translational modification influence its RNA-binding targets and splicing function? I will explore these questions in hiPSCs derived MNs from healthy and ALS donors generated in the Patani lab, combined with advanced transcriptomic and computational profiling approaches developed in the Luscombe group.A major obstacle in addressing these questions is the lack of in vitro or in vivo models that recapitulate authentic ALS phenotypes in human motor neurons. Induced pluripotent stem cells (hiPSCs) provide an unprecedented opportunity to investigate the progression of molecular pathogenic events in a human setting that is conventionally inaccessible and therefore represents an ideal model to explore TDP-43 RNA targets in MN development and degeneration.I will perform individual-nucleotide resolution UV-crosslinking and immunoprecipitation experiments (iCLIP) in the hiPSCs derived MNs to identify key TDP-43-RNA interactions over the course of MN differentiation in healthy and ALS-mutant MNs, and investigate conformation specific changes in TDP-43 binding targets. I will then computationally integrate iCLIP and RNA-seq data to uncover TDP-43-mediated alternative splicing, gene expression changes and alternative polyadenylation events. The analysis will reveal the link between binding sites and specific functional outcomes, and will guide downstream molecular investigations. This work will expand our understanding of a fundamental protein in neurodegeneration, TDP-43. I will uncover the regulatory mechanism(s) of TDP-43 in MN differentiation and to capture early perturbations in its RNA binding targets, which may cause MN degeneration in ALS pathology. Ultimately, this work may provide key insight into molecular mechanisms and new therapeutic targets in ALS

TAR DNA结合蛋白43 kDa（TDP-43）是一种RNA/DNA结合蛋白，与神经系统的发育和疾病有根本性的关系。它主要位于细胞核中，在那里它涉及转录和转录后调节的多个步骤。特别是，TDP-43在选择性剪接、稳定性和运输必需的神经发育基因中具有关键作用。TDP-43的异常细胞质错误定位、泛素化、异常磷酸化、切割和聚集是一系列神经退行性疾病的常见组织病理学标志，统称为TDP-43蛋白病，包括肌萎缩侧索硬化症（ALS）。ALS是一种与年龄相关的不治之症，其特征是选择性运动神经元（MN）变性。大脑、脑干和脊髓中上部和下部MN的逐渐丧失导致瘫痪并最终死亡。异常TDP-43错误定位在97%的ALS病例中发现，桥接了家族性和散发性ALS。鉴于其多效性和受TDP-43聚集和/或核耗竭异常影响的多种细胞过程，建立TDP-43和疾病之间的直接因果关系仍然具有挑战性。TDP-43错误定位影响人类运动神经元的分子机制仍然没有得到解决，其在正常运动神经发生中的确切作用也是如此。在此背景下，我希望了解TDP-43病理学是否是核功能丧失（例如异常前mRNA加工）、细胞质获得毒性功能或两者结合的结果。具体来说，我将解决以下问题：（i）TDP-43如何调节运动神经元的发育;（ii）如何以及何时这种调节在ALS中出错;（iii）哪些靶基因受到影响;（iv）TDP-43翻译后修饰如何影响其RNA结合靶点和剪接功能？我将在Patani实验室产生的来自健康和ALS供体的hiPSC衍生的MN中探索这些问题，结合Luscombe组开发的先进转录组学和计算分析方法。解决这些问题的主要障碍是缺乏体外或体内模型来重现人类运动神经元中真实的ALS表型。诱导性多能干细胞（hiPSC）为研究人类环境中分子致病事件的进展提供了前所未有的机会，这是传统上无法获得的，因此代表了探索MN发育和变性中TDP-43 RNA靶点的理想模型。在健康和ALS突变MN的MN分化过程中的RNA相互作用，并研究TDP-43结合靶点的构象特异性变化。然后，我将计算整合iCLIP和RNA-seq数据，以揭示TDP-43介导的选择性剪接，基因表达变化和选择性聚腺苷酸化事件。该分析将揭示结合位点和特定功能结果之间的联系，并将指导下游分子研究。这项工作将扩大我们对神经退行性疾病中一种基本蛋白质TDP-43的理解。我将揭示TDP-43在MN分化中的调节机制，并捕获其RNA结合靶点的早期扰动，这可能导致ALS病理学中的MN变性。最终，这项工作可能为ALS的分子机制和新的治疗靶点提供关键见解