Spatiotemporal analysis of TDP-43 toxicity and endolysosomal turnover mechanisms

肌萎缩侧索硬化症(ALS)是一种进行性运动变性的破坏性疾病 神经元会导致瘫痪，通常会在确诊后2-5年内死亡。不存在任何治疗方法 显著提高生活质量或预期寿命。-gt；30基因突变与肌萎缩侧索硬化症发病有关，尽管 90%的肌萎缩侧索硬化症病例是散发性的。然而，95%的肌萎缩侧索硬化症病例的一个统一的细胞标志是 核RNA结合蛋白TDP-43(TAR)在细胞质中的错误定位、聚集和聚集 DNA/RNA结合蛋白43)在运动神经元和支持细胞中表达。在其他组织中观察到类似的TDP-43病理 神经退行性疾病，包括约50%的额颞叶痴呆患者(FTD)的前脑神经元。 TDP-43病理对神经元具有毒性，但这种毒性的性质仍存在激烈的争论。损失 虽然已经提出了核功能(LOF)和细胞质功能增益(GOF)的机制 分离这些机制并确定TDP-43病理的最早影响仍然难以捉摸。 无论如何，一种在一些ALS模型中显示出希望的治疗策略是促进退化 细胞质中的TDP-43。最近，细胞质中的tdp-43被发现通过一种新的内切酶降解。 途径，当被诱导时，抑制TDP-43的毒性。然而，对这种退化的理解 途径仍然有限。理解上的关键差距包括，在与ALS相关的神经元模型中， TDP-43病理的最早和最与疾病相关的影响以及内切酶如何定义TDP-43 退化就会发生。这笔赠款的目的是：1.)建立新型内源性TDP-43报告系统 神经元，允许精确控制TDP-43的丰度和通过小分子和 光遗传意味着。使用该系统，改变TDP-43水平和定位对TDP-43本身的影响， ALS的表型和基因表达，重点是RNA的丰度和翻译，将被检查。2.)测试 包含TDP-43泛素化和内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 使用类似方法的患者衍生的ALS模型。最后，一种新的高通量酵母斑点杂交方法将被应用于 用于识别TDP-43和融合在肉瘤(FUS)丰度中的遗传和化学调节因子，这是 也与肌萎缩侧索硬化症和FTD病理有关。这笔赠款是创新的，因为它是一种新的方法来发挥 TDP-43表达的时空调控，保证了TDP43 LOF和GOF毒性的分离 效应，并提出了一种通过斑点杂交识别TDP-43和FUS丰度调节因子的方法。最后， 机械定义基于内溶酶体的细胞质TDP-43降解方式有望获得新的基础 深入了解TDP-43和其他底物的蛋白质平衡。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 新的治疗靶点具有广泛的患者适用性。

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.