Pathogenic Mechanisms of ALS-Linked TDP Gene Mutation

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种常见的神经退行性疾病，目前无法治愈。ALS无情地发展为瘫痪和死亡，通常发生在诊断后的五年内。现有的治疗方法都不能明显延长生命或改善生活质量。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组分，这是散发性ALS和额颞叶变性（FTLD）的标志。当挫折困扰TDP-43一段时间时，ALS中TDP基因突变的发现带来了突破。了解疾病基因的关键一步是确定致病基因突变的性质。这种致病性突变可能导致功能获得、功能丧失或显性负效应。目前的研究表明，功能丧失和功能获得可能都有助于TDP基因突变的发病机制，但存在很大的不确定性。人类致病突变TDP基因的过度表达可诱导转基因小鼠和大鼠的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效应引起疾病，因为两种类型的突变都可以在转基因动物中诱发疾病。因此，需要更复杂的模型来确定TDP基因突变的性质。我们已经创建了条件TDP敲入小鼠，其中小鼠TDP基因被替换为侧接loxP位点的正常或突变的人TDP基因，允许TDP敲入基因通过Cre介导的重组被删除。我们还建立了TDP敲入小鼠胚胎的神经元培养。使用TDP敲入小鼠和原代神经元作为两个互补的模型系统，我们将明确确定TDP基因的致病性突变是否通过功能的获得、功能的丧失或显性负效应引起ALS。这些研究结果将为ALS的机制研究提供基础。由此产生的基因敲入小鼠将比目前可用的任何ALS模型在生理上更相关，并且将是测试潜在ALS疗法的理想选择。 公共卫生相关性：使用条件TDP敲入小鼠作为模型，该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.