Pathogenic Mechanisms of ALS-Linked TDP Gene Mutation

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种常见的神经退行性疾病，目前尚无治愈方法。肌萎缩侧索硬化症无情地发展为瘫痪和死亡，通常发生在确诊后五年内。现有的治疗方法都不能显著延长生命或提高生活质量。ALS研究的最新进展是发现TDP基因产物TDP-43是泛素阳性包涵体的主要蛋白质成分，泛素阳性包涵体是散发性ALS和额颞叶变性(FTLD)的标志。当挫折感困扰TDP-43一段时间时，突破性进展是在ALS中发现了TDP基因突变。了解疾病基因的关键一步是确定致病基因突变的性质。这种致病突变可能导致功能获得、功能丧失或显性的负面影响。在极大的不确定性下，目前的研究表明，TDP基因突变的发病机制可能与功能丧失和功能获得有关。人类形式的致病突变TDP基因的过度表达会在转基因小鼠和大鼠中诱导ALS。然而，转基因研究无法区分突变基因是通过获得功能还是通过显性负面效应导致疾病，因为这两种类型的突变都可以在转基因动物中诱发疾病。因此，需要一个更复杂的模型来确定TDP基因突变的性质。我们已经创造了条件性TDP敲门小鼠，在其中，小鼠的TDP基因被正常或突变的人TDP基因取代，侧翼有loxP位点，允许通过Cre介导的重组来删除TDP敲门基因。我们还建立了来源于TDP敲击小鼠胚胎的神经元培养。利用TDP敲击小鼠和原代神经元作为两个互补的模型系统，我们将明确地确定TDP基因的致病突变是通过功能获得、功能丧失还是显性负面效应导致ALS。这些研究结果将为肌萎缩侧索硬化症的机制研究提供基础。由此产生的敲击小鼠将比目前可用的任何ALS模型更具生理学相关性，将是测试潜在ALS疗法的理想选择。 公共卫生相关性：以条件性TDP敲打小鼠为模型，这项应用将剖析肌萎缩侧索硬化症的致病机制，并将为开发这种致命疾病的治疗方法提供基础。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disease that currently has no cure. ALS inexorably progresses to paralysis and to death that usually occurs within five years of diagnosis. None of the available treatments appreciably prolongs life or improves quality of life. A recent advance in ALS research was the finding that the TDP gene product, TDP-43, is a major protein component of ubiquitin-positive inclusions, which are a hallmark of sporadic ALS and frontotemporal lobe degeneration (FTLD). When frustration haunts TDP-43 for a while, a breakthrough comes up with the discovery of mutation in the TDP gene in ALS. A critical step in understanding a disease gene is determining the nature of the pathogenic gene mutation. Such pathogenic mutations may cause a gain of function, a loss of function, or a dominant negative effect. With great uncertainty, current studies suggest that both loss of function and gain of function possibly contribute to the pathogenesis of the TDP gene mutation. Overexpression of the human form of the pathogenically mutated TDP gene induces ALS in transgenic mice and rats. However, transgenic studies are unable to differentiate whether a mutant gene causes disease by gain of function or by dominant negative effect because both types of mutation can induce disease in transgenic animals. Therefore, a more sophisticated model is required for determining the nature of the TDP gene mutation. We have created conditional TDP knockin mice in which the mouse TDP gene is replaced by the normal or mutant human TDP gene flanked by loxP sites, allowing the TDP knockin genes to be deleted by Cre-mediated recombination. We have also established neuronal cultures derived from TDP knockin mouse embryos. Using TDP knockin mice and primary neurons as two complementary model systems, we will unequivocally determine whether pathogenic mutation of the TDP gene causes ALS by a gain of function, a loss of function, or a dominant negative effect. Findings from these studies will provide a foundation for mechanistic studies of the ALS. The resulting knockin mice will be far more physiologically relevant than any of the ALS models currently available and will be ideal for testing potential ALS therapies. PUBLIC HEALTH RELEVANCE: Using conditional TDP knockin mice as models, this application will dissect pathogenic mechanisms underlying amyotrophic lateral sclerosis and will provide a foundation for development of therapies for this fatal disease.