Role of oligomeric TDP-43 aggregate intermediates in ALS and frontotemporal dementia

这项资助的目的是通过揭示TDP-43聚集的过程， 导致病理学和神经毒性。TDP-43聚集是肌萎缩侧索硬化的病理标志。 硬化症（ALS）和额颞叶痴呆（FTD）病例的一半。此外，TDP-43病变是继发性 大约50%的阿尔茨海默病的病理学。对抗ALS和FTD的主要目标是 通过开发防止或逆转TDP-43的策略来减少TDP-43内含物的积累 聚合来认识到这一点，我们建立了战略方法来研究这种RNA结合的聚集 使用纯化的TDP-43的蛋白质，并确定了以前未知的聚集体中间体。这些是 早期阶段的寡聚体，与研究充分的蛋白质连接的毒性寡聚体物种共享关键特征， 包括Tau、淀粉样蛋白-β和α-突触核蛋白。这些TDP-43聚集体中间体是 能够接种从头细胞内TDP-43聚集，并且它们的形成被ALS连接的 突变。我们最近的研究结果提供了独特的机会，阐明基本机制， TDP-43聚集，确定它们在发病机制中的作用并提供阻断TDP-43病理的模型。目的 1将在细胞聚集模型以及ALS和FTD中确定TDP-43寡聚体的存在。 衍生组织特别是，我们将研究招聘的中间细胞质应激颗粒， 其是富含蛋白质-RNA的体，被认为是病理性聚集的坩埚。这将是 使用我们建立的检测方法和通过开发对中间体特异的抗体来实现 配合物为了确定新发现的TDP-43中间体在聚集和发病机制中的作用， 目标2将定义组装的分子决定因素，并测试疾病相关的条件 上调这一过程。在确定了介导寡聚化的蛋白质区域后，我们将询问是否 聚集和细胞内接种功能在新鉴定的表位被破坏后降低。的 TDP-43聚集体在疾病中作为病理性扩散剂的能力被最近的研究强烈地暗示， FTD衍生的提取物在小鼠脑中播种并传播TDP-43病理学。目标3将测试 单独的TDP-43聚集体是否真的可以在肿瘤细胞中引发从头聚集和病理学传播。 vivo.我们将分析注射早期和晚期TDP-43后TDP-43病理学的传播 在小鼠脑中，来自纯化蛋白质的聚集体。这将确定TDP-43是否聚集， 特别是TDP-43低聚物是成核和神经毒性的主要来源。而且这些 研究我们将确定这种传播是否与增加的神经变性直接相关。与 成功完成这项赠款，我们将：a）确定TDP-43聚合的基本过程; B） 建立体外和体内模型以预防ALS和FTD中神经毒性;和c）提供前所未有的 开发疾病诊断标志物的工具。

The goal of this grant is to elucidate key mechanisms of TDP-43 aggregation by uncovering how this process leads to pathology and neurotoxicity. TDP-43 aggregation is the pathological hallmark of amyotrophic lateral sclerosis (ALS) and half of frontotemporal dementia (FTD) cases. In addition, TDP-43 lesions are a secondary pathology in approximately 50% of Alzheimer's disease. A major goal in combatting ALS and FTD has been to reduce the accumulation of TDP-43 inclusions by developing strategies to prevent or reverse TDP-43 aggregation. Recognizing this, we established strategic methods to study the aggregation of this RNA binding protein using purified TDP-43 and have identified previously unknown aggregate intermediates. These are early stage oligomers that share key characteristics with toxic oligomeric species of well-studied proteins linked to neurodegeneration, including Tau, amyloid-β and α-synuclein. These TDP-43 aggregate intermediates are capable of seeding de novo intracellular TDP-43 aggregation and their formation is accelerated by ALS-linked mutations. Our recent findings provide the unique opportunity to shed light on fundamental mechanisms of TDP-43 aggregation, determine their role in pathogenesis and provide models to block TDP-43 pathology. Aim 1 will establish the presence of TDP-43 oligomers in cellular models of aggregation and in ALS and FTD- derived tissue. In particular, we will examine recruitment of the intermediates to cytoplasmic stress granules, which are protein-RNA rich bodies considered to be crucibles of pathological aggregation. This will be achieved using our established methods of detection and by developing antibodies specific for the intermediate complexes. To establish the role of the newly found TDP-43 intermediates in aggregation and pathogenesis, Aim 2 will define the molecular determinants of assembly and test how disease-associated conditions upregulate this process. After identifying the protein regions mediating oligomerization, we will ask whether aggregation and intracellular seeding function decrease upon disruption of the newly identified epitopes. The ability of TDP-43 aggregates to act as agents of pathological spread in disease is strongly suggested by recent findings that FTD-derived extracts seed and propagate TDP-43 pathology in mouse brain. Aim 3 will test whether TDP-43 aggregates alone can indeed initiate de novo aggregation and propagation of pathology in vivo. We will analyze the spread of TDP-43 pathology upon injection of early and late stage TDP-43 aggregates derived from purified protein in the brain of mice. This will establish whether TDP-43 aggregates, and TDP-43 oligomers in particular, are primary sources of nucleation and neurotoxicity. Furthermore, these studies we will determine whether this propagation directly correlates with increased neurodegeneration. With the successful completion of this grant, we will: a) define fundamental processes in TDP-43 aggregation; b) establish in vitro and in vivo models to forestall neurotoxicity in ALS and FTD; and c) provide unprecedented tools for the development of diagnostic markers of disease.