Mechanistic dissection of novel regulators of TDP-43 aggregation identified in a genome-wide CRISPR-Cas9 knockout screen

项目摘要 RNA结合蛋白（RNA binding proteins，RBP）的错误定位和聚集是肌萎缩性侧索硬化的病理标志 侧索硬化症（ALS）。RBP TAR DNA结合蛋白43（TDP-43）是特别感兴趣的，因为病理性 在近97%的ALS患者中观察到TDP-43的聚集，尽管事实上， TDP-43突变解释了不到5%的疾病发病率。这一观察结果表明， 或其他基因的突变会聚在TDP-43病理学上，然而，仍然不清楚这些基因是如何突变的。 是否会出现聚合物，或者是否可以逆转或防止聚合物。为此，我们开发了脉冲形状 TDP-43的基于分析的聚集报告基因，当与荧光激活细胞分选偶联时， 在单细胞水平上定量聚集。这位记者利用全基因组CRISPR- Cas9敲除筛选以鉴定TDP-43聚集的调节剂。令人欣慰的是，这个屏幕显示了已知的 参与TDP-43调节和病理学的相互作用物和途径。此外，这项工作还揭示了几个 以前未涉及TDP-43生物学或聚集的新蛋白质。对一个热门案件SRRD的初步跟进， 揭示了这种蛋白质在哺乳动物聚集模型中减少TDP-43聚集，并减少TDP-43聚集。 43-在酵母模型中的相关毒性，表明这些基因确实修饰TDP-43聚集。 这项提案的目标是机械地剖析这些新的调节剂如何修饰TDP-43 聚合来使用TDP-43聚集的诱导运动神经元模型、酵母毒性模型和体外 研究，我将评估的相互作用，信号通路，TDP-43的修改模式，为以下四个 基因：胃L5、EIF 2AK 2、XPO 4和SRRD。这项工作不仅将揭示功能的uncharacterized 蛋白质，但剖析其迄今未知的机制，在调节TDP-43聚集。通过更好 了解TDP-43聚集是如何调节的，该领域可以开始回答有关 TDP-43聚集在疾病发病机制和进展中的作用。 通过了解潜在TDP-43聚集的调控网络，我们可以开始 治疗调节导致疾病的途径。

Project Summary The mislocalization and aggregation of RNA-binding proteins (RBP) is a pathological hallmark of Amyotrophic Lateral Sclerosis (ALS). The RBP TAR DNA-binding protein 43 (TDP-43) is of particular interest, as pathological aggregation of TDP-43 is observed in nearly 97% of ALS patients, despite the fact that disease-causing mutations in TDP-43 explain fewer than 5% of disease incidence. This observation suggests that misregulation or mutation of other genes converge on TDP-43 pathology, however it remains poorly understood how the aggregates arise or whether they can be reversed or prevented. To this end, we have developed Pulse-Shape Analysis-based aggregation reporters for the TDP-43 that, when coupled to fluorescence activated cell sorting, quantifies aggregation at the single cell level. This reporter was leveraged against a genome-wide CRISPR- Cas9 knockout screen to identify regulators of TDP-43 aggregation. Reassuringly, this screen revealed known interactors and pathways involved in TDP-43 regulation and pathology. Additionally, this work revealed several novel proteins not previously implicated in TDP-43 biology or aggregation. Initial follow up on one top hit, SRRD, revealed that this protein reduces TDP-43 aggregation in a mammalian aggregation model, and reduces TDP- 43-associated toxicity in a yeast model, indicating that these genes indeed modify TDP-43 aggregation. The goal of this proposal is to mechanistically dissect how these novel regulators modify TDP-43 aggregation. Using an induced motor neuron model of TDP-43 aggregation, yeast toxicity models, and in vitro studies, I will assess the interactors, signaling pathways, and mode of TDP-43 modification for the following four genes: METTL5, EIF2AK2, XPO4, and SRRD. This work will not only reveal the function of uncharacterized proteins, but dissect their heretofore unknown mechanisms in regulating TDP-43 aggregation. Through better understanding of how TDP-43 aggregation is regulated, the field can begin to answer questions regarding the role of TDP-43 aggregation in disease pathogenesis and progression. By understanding the regulatory network of underlying TDP-43 aggregation, we can begin to therapeutically modulate the pathways that contribute to disease.