Spatiotemporal analysis of TDP-43 toxicity and endolysosomal turnover mechanisms

肌萎缩性侧索硬化症（ALS）是一种严重的运动神经退行性变， 神经元导致瘫痪，通常导致诊断后2-5年死亡。没有任何疗法 显著提高生活质量或预期寿命。>30个基因的突变与ALS发作有关，尽管 90%以上的ALS病例是散发性的。然而，>95%的ALS病例的统一细胞标志是 核RNA结合蛋白TDP-43（TAR）的细胞质错误定位、积累和聚集 DNA/RNA结合蛋白43）在运动神经元和支持细胞。在其他组织中观察到类似的TDP-43病理学。 神经退行性疾病，包括约50%额颞叶痴呆患者（FTD）的前脑神经元。 TDP-43病理学赋予神经元毒性，但这种毒性的性质仍然存在激烈的争论。损失 核功能（LOF）和细胞质功能获得（GOF）机制已被提出，虽然 分离这些机制和鉴定TDP-43病理学的最早影响仍然难以捉摸。 无论如何，在一些ALS模型中显示出希望的治疗策略正在促进降解。 胞质TDP-43最近，细胞质TDP-43被证明是通过一种新的内溶酶体降解， 途径，当被诱导时，抑制TDP-43毒性。然而，理解这种退化 路径仍然有限。理解上的关键差距包括在ALS相关神经元模型中确定， TDP-43病理学的最早和最疾病相关的影响，并定义内溶酶体TDP-43如何 发生降解。该补助金的目的是：1.）建立一种新的内源性TDP-43报告系统 神经元，其允许通过小分子精确控制TDP-43丰度和细胞定位， 光遗传学方法。使用该系统，改变的TDP-43水平和定位对TDP-43本身的影响， ALS表型和基因表达，重点是RNA丰度和翻译，将进行检查。2.）测试 一种涉及TDP-43泛素化和内体膜内陷的内溶酶体降解模型 在神经元中使用光脉冲标记方法。TDP-43降解机制也将在 使用类似的方法的患者来源的ALS模型。最后，一种新的高通量酵母斑点印迹法将在 用于鉴定TDP-43和融合肉瘤（FUS）丰度的遗传和化学调节剂， 也与ALS和FTD病理学有关。这项赠款是创新的，因为这是一种新的方法， TDP-43表达的时空控制，这保证了TDP 43 LOF和GOF毒性的分离 的影响，并提出了一种手段，以确定调节TDP-43和FUS丰度通过斑点印迹。最后， 机械地定义胞质TDP-43降解的基于内溶酶体的手段有望提供新的基础 深入了解TDP-43和其他底物蛋白质稳定性。总之，TDP-43和FUS是逻辑入口点 用于ALS和FTD的研究。这笔赠款将加强对疾病的了解，并可能导致识别 具有广泛患者适用性的新治疗靶点。

Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which progressive degeneration of motor neurons leads to paralysis, usually resulting in death 2-5 years after diagnosis. No therapies exist that significantly increase quality of life or life expectancy. Mutations in >30 genes are linked to ALS onset, albeit >90% of all ALS cases are sporadic. However, a unifying cellular hallmark in >95% of all ALS cases is the cytoplasmic mis-localization, accumulation, and aggregation of the nuclear RNA binding protein TDP-43 (TAR DNA/RNA binding protein 43) in motor neurons and support cells. Similar TDP-43 pathology is observed in other neurodegenerative diseases, including in forebrain neurons of ~50% of Frontotemporal dementia patients (FTD). TDP-43 pathology confers a toxicity to neurons, but the nature of this toxicity remains fiercely debated. Loss of nuclear function (LOF) and gain of cytoplasmic function (GOF) mechanisms have been proposed, though separating such mechanisms and identifying the earliest impacts of TDP-43 pathology has remained elusive. Regardless, a therapeutic strategy that has shown promise in some ALS models is promoting the degradation of cytoplasmic TDP-43. Recently, cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomal pathway, which, when induced, suppresses TDP-43 toxicity. However, understanding of this degradation pathway remains limited. Key gaps in understanding include determining, in an ALS-relevant neuronal model, the earliest and most disease-relevant impacts of TDP-43 pathology and defining how endolysosomal TDP-43 degradation occurs. The aims of this grant are: 1.) Establish a novel endogenous TDP-43 reporter system in neurons that allows precise control of TDP-43 abundance and cellular localization via small molecule and optogenetic means. Using this system, the impacts of altered TDP-43 levels and localization on TDP-43 itself, ALS phenotypes and gene expression, focusing on RNA abundance and translation, will be examined. 2.) Test an endolysosomal degradation model involving TDP-43 ubiquitination and endosomal membrane invagination in neurons using an optical pulse labelling approach. TDP-43 degradation mechanisms will also be defined in patient-derived ALS models using similar means. Finally, a novel high throughput yeast dot-blot assay will be used to identify genetic and chemical regulators of TDP-43 and Fused in Sarcoma (FUS) abundance, which is also implicated in ALS and FTD pathology. This grant is innovative in that a novel approach to exert spatiotemporal control of TDP-43 expression, which promises separation of TDP43 LOF and GOF toxicity effects, and a means to identify regulators of TDP-43 and FUS abundance via dot blot, are proposed. Finally, mechanistically defining endolysosomal-based means of cytoplasmic TDP-43 degradation promises new basic insight into proteostasis for TDP-43 and other substrates. In summary, TDP-43 and FUS are logical entry points for the study of ALS and FTD. This grant will enhance disease understanding and may lead to identification of new therapeutic targets with broad patient applicability.