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

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密切相关。它主要定位于细胞核，参与转录和转录后调控的多个步骤。特别是，TDP-43在重要神经发育基因的选择性剪接、稳定性和运输中起着关键作用。TDP-43胞浆异常定位、泛素化、异常磷酸化、裂解和聚集是一系列神经退行性疾病的共同组织病理学特征，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、脑干和脊髓上、下MN的进行性丧失会导致瘫痪，最终导致死亡。在97%的ALS病例中发现异常的TDP-43错误定位，连接了家族性和散发性ALS。鉴于TDP-43的多效性以及TDP-43聚集和/或核耗竭对多种细胞过程的异常影响，建立TDP-43与疾病之间的直接因果联系仍然具有挑战性。TDP-43错误定位影响人类运动神经元的分子机制仍未解决，其在正常运动神经发生中的确切作用也是如此。在这种背景下，我希望了解TDP-43的病理是核功能丧失(例如，异常的前mRNA加工)、细胞质获得毒性功能，还是两者的组合。具体地说，我将回答以下问题：(I)TDP-43如何调控运动神经元的发育；(Ii)这种调控在ALS中如何以及何时出错；(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？我将在帕塔尼实验室产生的健康和ALS捐赠者的HiPSCs MN中探索这些问题，并结合Luscombe小组开发的先进的转录和计算图谱方法。解决这些问题的一个主要障碍是缺乏能够概括人类运动神经元真实ALS表型的体外或体内模型。诱导多能干细胞(HiPSCs)为在人类环境中研究分子致病事件的进展提供了一个前所未有的机会，因此它是探索MN发育和退变中TDP-43RNA靶点的理想模型。我将在HiPSCs来源的MN中进行单核苷酸拆分、紫外光交联和免疫沉淀实验(ICLIP)，以确定健康MN和ALS突变MN分化过程中关键的TDP-43-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介导的替代剪接、基因表达变化和替代多聚腺苷化事件。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变中的一种基本蛋白质TDP-43的理解。我将揭示TDP-43在MN分化中的调控机制(S)，并捕捉其RNA结合靶点的早期扰动，这可能导致ALS病理中的MN退变。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的见解。

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