ALS-associated TDP-43 Aggregation: A Drosophila Model and A Role for Autophagy

描述（由申请人提供）：本提案的长期目标是了解肌萎缩性侧索硬化症（ALS）背后的病理机制。最近，在散发性肌萎缩侧索硬化患者的运动神经元包裹体中发现了蛋白TDP-43，并且发现TDP-43的突变与一部分家族性肌萎缩侧索硬化病例有关。拟议的研究旨在确定神经元中TDP-43聚集的后果，并确定从受影响细胞中清除潜在毒性TDP-43聚集的机制。该建议的一个关键方面是建立TDP-43对果蝇的毒性模型。TDP-43将在果蝇神经元中表达，并分析与TDP-43毒性相关的表型。基因技术，包括无偏见筛选，将用于确定消除这些表型的因素。在这些检测中发现的基因可能导致对疾病机制的新认识或成为未来治疗的目标。此外，将使用该模型探索最近发现的与tdp -43相互作用的蛋白Ubiquilin的作用。泛素与未折叠蛋白反应密切相关，被认为该蛋白在神经元中具有保护功能。这一假设将直接在果蝇体内进行验证。本提案的另一个目标是确定自噬在TDP-43内含物降解中的作用。自噬是细胞质成分的大量降解，包括错误折叠和聚集的蛋白质，对正常的神经元稳态至关重要。药理学、显微镜和生化技术将用于探索自噬参与TDP-43聚集体清除的假设。此外，上述果蝇模型也将用于在体内解决这个问题。

DESCRIPTION (provided by applicant): The long term objective of this proposal is to understand the pathologic mechanisms behind the disease amyotrophic lateral sclerosis (ALS). Recently, the protein TDP-43 was identified in inclusions in motor neurons of patients with sporadic ALS, and mutations in TDP-43 have been found to be responsible for a subset of familial cases of ALS. The proposed research seeks to identify the consequences of TDP-43 aggregation in neurons and identify mechanisms by which potentially toxic TDP-43 aggregates can be cleared from affected cells. A key aspect of this proposal is to develop a model of TDP-43 toxicity in Drosophila melanogaster. TDP-43 will be expressed in the neurons of flies, and phenotypes associated with TDP-43 toxicity will be analyzed. Genetic techniques, including unbiased screens, will then be used to identify factors that abrogate these phenotypes. Genes identified in these assays could lead to new understanding of disease mechanisms or become future targets of therapy. In addition, the role of Ubiquilin, a recently identified TDP-43-interacting protein, will be explored using this model. Ubiquilin is intimately involved in the unfolded protein response, and it is thought that this protein has a protective function in neurons. This hypothesis will be directly tested in vivo using Drosophila. Another goal of this proposal is to determine the role of autophagy in the degradation of TDP-43 inclusions. Autophagy is the bulk degradation of cytoplasmic components, including misfolded and aggregated proteins, and is critical for normal neuronal homeostasis. Pharmacologic, microscopic, and biochemical techniques will be used to explore the hypothesis that autophagy is involved in TDP-43 aggregate clearance. In addition, the Drosophila model described above will also be used to address this question in vivo.