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

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种毁灭性的疾病，每年在美国每10万人中约有2人受到影响。最近的发现有可能极大地改变我们对肌萎缩侧索硬化症的病因的理解和确定可能的治疗靶点。肌萎缩侧索硬化症是运动神经元选择性丧失的结果，在肌萎缩侧索硬化症患者中，运动神经元含有胞质蛋白质聚集体。这些聚集体的主要成分是RNA结合蛋白TDP-43，它通常定位在细胞核中，这为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，这提供了这种蛋白与疾病之间的因果联系。在死于其他神经退行性疾病的患者的尸检样本中也发现了TDP-43聚集体，这表明TDP-43病理可能是多种疾病的病原体。了解TDP-43功能障碍如何导致疾病是至关重要的。已经提出了两种可能的机制。首先，TDP-43聚合体具有固有的毒性，会导致运动神经元细胞死亡。第二个模型与第一个模型并不是相互排斥的，即核内正常功能蛋白质的丢失是运动神经元破坏的直接原因。我们正在使用果蝇作为一个模型，来了解TDP-43在神经元中的功能以及异常表达的后果。果蝇TDP-43同源基因的零突变名为TBPH，是幼虫致死的，并显示出幼虫运动缺陷。在所有组织中过度表达三种自然存在的TBPH亚型中的任何一种都是对早期幼虫的致死作用。当表达仅限于运动神经元时，幼虫和成年果蝇都有运动缺陷。在ALS中发现的大部分TDP-43突变位于C-末端结构域，我们的数据表明，TBPH的C-末端结构域是获得最大毒性所必需的。这项应用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与另一个名为瑞士奶酪(SWS)的神经退变基因之间的遗传交互作用，这表明SWS是TDP-43有毒特性所必需的。这种相互作用特别耐人寻味，因为人类SWS同源基因的突变，被称为神经病变靶标酯酶，会导致运动神经元疾病。除了验证我们的假设，即过度表达TDP-43的毒性效应是由于TDP-43和SWS之间的相互作用，我们还建议检查在野生型和SWS突变背景下过度表达TDP-43的运动神经元的解剖和电生理表型。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极大地促进这种理解。 与公共卫生相关：开发ALS治疗方法的第一步是确定直接导致ALS或使其对环境诱因产生ALS易感性的基因/蛋白质。最近的发现将TDP-43与肌萎缩侧索硬化症紧密联系起来，这表明这可能是未来的目标。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。这项应用以果蝇黑腹果蝇为模型，重点了解TDP-43功能障碍导致神经退化的机制，这一方法将极大地加快针对TDP-43的治疗方案的设计过程。

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.