Using Drosophila as a model to understand TDP-43 function in ALS

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种毁灭性的疾病，在美国每年影响约2/100，000人。最近的发现有可能极大地改变我们对ALS病因的理解，并确定可能的治疗靶点。ALS是由运动神经元的选择性丧失引起的，在ALS患者中，运动神经元含有蛋白质的细胞质聚集体。这些聚集体的主要成分是RNA结合蛋白TDP-43，它通常位于细胞核中，这一发现为ALS的研究开辟了许多新的方向。在ALS患者中也发现了TDP-43的大量突变，提供了这种蛋白质与疾病之间的因果关系。TDP-43聚集体也已在死于其他神经变性疾病（例如额颞痴呆、阿尔茨海默病和帕金森病）的患者的死后样品中鉴定，表明TDP-43病理学可能是多种疾病中的病原体。了解TDP-43功能障碍如何导致疾病至关重要。提出了两种可能的机制。第一个是TDP-43聚集体具有固有毒性，并导致运动神经元细胞死亡。第二个模型与第一个模型并不相互排斥，它认为细胞核中正常功能蛋白质的丢失是运动神经元破坏的直接原因。我们正在使用果蝇，果蝇，作为一个模型，以了解TDP- 43在神经元中的功能和异常表达的后果。TDP-43的果蝇直向同源物中的突变，命名为TBPH，是蛹致死的，并显示幼虫运动缺陷。TBPH的三种天然存在的异构体中的任何一种在所有组织中的过表达都是早期幼虫致死的。当表达仅限于运动神经元时，幼虫和成虫都有运动缺陷。ALS中TDP-43突变的大部分在C-末端结构域中发现，我们的数据表明TBPH的C-末端结构域是最大毒性所必需的。本申请的主要目标是确定这些缺陷背后的细胞和分子机制。我们已经确定了TBPH/TDP-43和另一个神经变性基因，名为瑞士奶酪（sws），这表明sws是TDP-43的毒性所需的遗传相互作用。这种相互作用特别有趣，因为sws的人类直向同源物（称为神经病靶向酯酶）的突变会导致运动神经元疾病。除了测试我们的假设，过度表达的TDP-43的毒性作用，从TDP-43和sws之间的相互作用的结果，我们还建议检查运动神经元的解剖和电生理表型，过度表达TDP-43在野生型和sws突变体的背景。任何依赖于靶向TDP-43功能障碍的治疗ALS的治疗方法都需要了解TDP-43毒性作用的分子机制。使用果蝇中可用的强大遗传工具将大大加速这种理解。 公共卫生相关性：开发ALS治疗方法的第一步是鉴定直接导致ALS或赋予ALS易感性以响应环境触发因素的基因/蛋白质。最近发现TDP-43与ALS密切相关，表明这是未来可能的目标。任何依赖于靶向TDP-43功能障碍的治疗ALS的治疗方法都需要了解TDP-43毒性作用的分子机制。该应用程序的重点是使用果蝇Drosophila melanogaster作为模型来理解TDP-43功能障碍导致神经变性的机制，这种方法将大大加快设计靶向TDP-43的治疗方法的过程。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating disease affecting about 2 in 100,000 people in the USA each year. Recent discoveries have the potential to dramatically change our understanding of the causes of ALS and identification of possible targets for treatment. ALS results from the selective loss of motor neurons, which in ALS patients contain cytoplasmic aggregates of proteins. Identification that a major constituent of these aggregates is the RNA-binding protein TDP-43, which is normally localized in the nucleus, has opened up many new directions in ALS research. A large number of mutations in TDP-43 have also been identified in ALS patients providing a causative link between this protein and the disease. TDP-43 aggregates have also been identified in post-mortem samples of patients who have died of other neurodegenerative diseases such as fronto-temporal dementias, Alzheimer's and Parkinson's diseases, suggesting that TDP-43 pathology might be a causative agent in a wide variety of diseases. It is critical to understand how TDP-43 dysfunction leads to disease. Two possible mechanisms have been proposed. The first is that the TDP-43 aggregates are inherently toxic and lead to motor neuron cell death. The second model, which is not mutually exclusive with the first, is that the loss of normally functioning protein in the nucleus is the proximate cause of motor neuron disruption. We are using the fruit fly, Drosophila melanogaster, as a model to understand the function of TDP- 43 in neurons and the consequences of aberrant expression. Null mutations in the Drosophila orthologue of TDP-43, named TBPH, are pupal lethal and show larval motor defects. Over-expression of any of three naturally occurring isoforms of TBPH in all tissues is early larval lethal. When expression is restricted to motor neurons, both larvae and adult flies have motor deficits. The bulk of the TDP-43 mutations in ALS are found in the C-terminal domain and our data shows that the C-terminal domain of TBPH is necessary for maximum toxicity. The primary goals of this application are to determine the cellular and molecular mechanisms underlying these defects. We have identified a genetic interaction between TBPH/TDP-43 and another neurodegeneration gene, named swiss cheese (sws), which suggests that sws is required for the toxic properties of TDP-43. This interaction is particularly intriguing because mutations in the human orthologue of sws, named neuropathy target esterase, lead to motor neuron disease. In addition to testing our hypothesis that the toxic effects of over-expressed TDP-43 result from an interaction between TDP-43 and sws we also propose to examine the anatomical and electrophysiological phenotypes of motor neurons that over-express TDP-43 in wild-type and sws mutant backgrounds. Any therapeutic approaches to treat ALS that rely on targeting TDP-43 dysfunction require an understanding of the molecular mechanisms that underlie the toxic effects of TDP-43. Using the powerful genetic tools available in Drosophila will greatly accelerate this understanding. PUBLIC HEALTH RELEVANCE: The first step in developing therapies for ALS is to identify the genes/proteins that either directly cause ALS or confer susceptibility to develop ALS in response to environmental triggers. Recent discoveries that strongly link TDP-43 to ALS suggest that this is a possible future target. Any therapeutic approaches to treat ALS that rely on targeting TDP-43 dysfunction require an understanding of the molecular mechanisms that underlie the toxic effects of TDP-43. This application focuses on understanding the mechanisms that lead to neurodegeneration by TDP-43 dysfunction using the fruit fly, Drosophila melanogaster as a model, an approach that will greatly speed up the process of designing therapies targeting TDP-43.