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侧索硬化症（ALS）背后的病理机制。最近，在散发性ALS患者的运动神经元中的包涵体中鉴定出蛋白质TDP-43，并且已经发现TDP-43的突变与ALS的家族性病例的子集有关。拟议的研究旨在确定TDP-43在神经元中聚集的后果，并确定潜在毒性TDP-43聚集体可以从受影响细胞中清除的机制。该提议的一个关键方面是开发黑腹果蝇中TDP-43毒性的模型。TDP-43将在果蝇的神经元中表达，并将分析与TDP-43毒性相关的表型。遗传技术，包括无偏筛选，然后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理解，或成为未来的治疗靶点。此外，Ubiquilin，最近确定的TDP-43相互作用蛋白的作用，将使用该模型进行探索。泛素与未折叠蛋白质反应密切相关，并且认为这种蛋白质在神经元中具有保护功能。这一假设将直接在体内使用果蝇进行测试。该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，并且对于正常神经元的稳态是至关重要的。药理学、显微镜和生物化学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。 公共卫生相关性：如果自噬确实被发现对毒性TDP-43聚集体的降解很重要，那么增强自噬的药物可能值得进一步探索作为潜在的治疗方法。重要的是，ALS和相关疾病具有高发病率、死亡率和社会经济成本。作为拟议研究的结果，对疾病机制的新见解对于理解和潜在治疗这种毁灭性疾病至关重要。 注：下文以基本上未经编辑的形式提供了个别审查者的评论。这些评论是在评审会议之前编写的，在会议讨论之后可能没有更新或修订。因此，它们可能无法充分反映小组讨论结束时单个评审员的最终意见或小组的最终多数意见。以上讨论的摘要和总结总结了小组讨论的最终结果。

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. PUBLIC HEALTH RELEVANCE: If autophagy is indeed found to be important for the degradation of toxic TDP-43 aggregates, drugs that enhance autophagy may warrant further exploration as potential therapies. Importantly, ALS and related diseases have high morbidity, mortality, and socioeconomic cost. New insights into disease mechanisms as a result of the proposed research will be critical for understanding and potentially treating this devastating disease. NOTE: The critiques of individual reviewers are provided below in an essentially unedited form. These critiques were prepared prior to the review meeting and may not have been updated or revised subsequent to the discussion at the meeting. Therefore, they may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. The Resume and Summary of Discussion above summarizes the final outcome of the group discussion.