Identification of intrinsic and extrinsic regulators of TDP43 splicing function

项目摘要 鉴定TDP43剪接功能的内在和外在调控因子。 痴呆症是一种毁灭性的、无法治愈的神经退行性疾病，对 世界各地的患者、他们的家人和卫生系统。痴呆症的疾病修正疗法有 迫切需要，但由于我们缺乏对弱势群体的了解，它们的发展受到了阻碍 驱动其发病机制的分子网络。RNA结合蛋白TDP43出现功能障碍 许多痴呆的病理特征，包括阿尔茨海默病(AD)，以边缘为主的年龄相关 TDP43脑病(晚期)和额颞叶痴呆(FTD)。TDP43作为蛋白质组的守护者 通过调节隐蔽外显子(CE)、包含提前终止密码子的外显子和 移码突变，存在于数百个前信使核糖核酸中。然而，控制TDP43的监管网络 TDP43功能障碍导致神经变性的机制目前知之甚少 仍然不为人知。为了帮助解决这一关键的知识差距，我们最近开发了一种量化的、 基于荧光的报告，以测量单个活细胞中依赖于TDP43的剪接。综合本文记者 凭借我们在大规模全基因组筛查技术方面的专业知识，我们提出了两种旨在 以全面和公正的方式绘制调控TDP43功能的分子网络。首先，我们 将使用最近开发的深度突变扫描方法来确定所需的TDP43的序列特征 因为它的拼接功能。突变体库将包含50,000多个集中的单点和双点突变 关于TDP43的固有无序结构域(IDR)，因为IDR参与了两个剪接功能 和聚合。为了确定调节TDP43依赖的剪接的反式作用因子和辅助因子，我们将 结合记者和基因组规模的功能丧失CRISPR筛查。来自两个屏幕的最高点击量将 严格验证它们对TDP43依赖的神经元剪接和聚集的影响 小鼠胚胎干细胞。这个项目的成功完成将揭示内在的IDR序列 直接或间接影响TDP43剪接功能的元件和外源性细胞成分， 从而为开发新的治疗策略所需的机制研究提供了基础。

Project Summary Identification of intrinsic and extrinsic regulators of TDP43 splicing function. Dementias are devastating and incurable neurodegenerative diseases that represent an escalating burden on patients, their families and health systems throughout the world. Disease-modifying therapies for dementias are urgently needed, but their development has been hampered by our lack of knowledge about the vulnerable molecular networks that drive their pathogenesis. Dysfunction of the RNA-binding protein TDP43 has emerged pathological signature in many dementias, including Alzheimer’s disease (AD), limbic-predominant age-related TDP43 encephalopathy (LATE) and frontotemporal dementia (FTD). TDP43 functions as a guardian of proteome integrity by regulating the splicing of cryptic exons (CEs), exons that contain premature stop codons and frameshift mutations, present in hundreds of pre-mRNAs. However, the regulatory network that controls TDP43 function is poorly understood and the mechanism by which TDP43 dysfunction leads to neurodegeneration remains unknown. To help address this critical gap in knowledge, we have recently developed a quantitative, fluorescence-based reporter to measure TDP43-dependent splicing in single live cells. Combining this reporter with our expertise in large-scale, genome-wide screening technologies, we propose two screens designed to chart the molecular network that regulates TDP43 function in a comprehensive and unbiased manner. First, we will use recently developed deep mutational scanning methods to identify sequence features of TDP43 required for its splicing function. Mutant libraries will encompass over 50,000 single and double point mutations focused on the intrinsically disordered domain (IDR) of TDP43, since the IDR has been implicated in both splicing function and aggregation. To identify trans-acting factors and co-factors that regulate TDP43-dependent splicing, we will combine the reporter with genome-scale loss-of-function CRISPR screens. The top hits from both screens will be rigorously validated for their effects on TDP43-dependent splicing and aggregation in neurons derived from mouse embryonic stem cells. The successful completion of this project will uncover the intrinsic IDR sequence elements and extrinsic cellular components that directly or indirectly influence the splicing function of TDP43, thereby providing a foundation for the mechanistic studies required to develop new therapeutic strategies.