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

这项资助的目标是通过揭示TDP-43聚集的关键机制来揭示这一过程 会导致病理和神经毒性。TDP-43聚集是肌萎缩侧索硬化症的病理标志 硬化症(ALS)和一半的额颞部痴呆(FTD)病例。此外，TDP-43的损害是继发性的 约50%的阿尔茨海默病患者的病理改变。抗击ALS和FTD的一个主要目标是 通过制定防止或逆转TDP-43的战略，减少TDP-43包裹体的积累 聚合。认识到这一点，我们建立了战略性的方法来研究这种RNA结合的聚集 使用纯化的TDP-43的蛋白质，并鉴定了以前未知的聚集中间体。这些是 早期寡聚体与研究良好的连接蛋白的有毒寡聚体具有共同的关键特征 神经退行性变，包括牛磺酸、淀粉样蛋白-β和α-突触核蛋白。这些TDP-43聚合中间体是 能够诱导TDP-43的细胞内聚集，ALS连锁可加速其形成 突变。我们最近的发现提供了一个独特的机会来阐明 TDP-43聚集，确定其在发病机制中的作用，并为阻断TDP-43的病理机制提供模型。目标 1将确定TDP-43寡聚体在聚集的细胞模型以及ALS和FTD中的存在。 衍生组织。特别是，我们将研究中间体向细胞质应激颗粒的募集， 它们是富含蛋白质-RNA的小体，被认为是病理性聚集的熔炉。这将是 使用我们已建立的检测方法和开发针对中间体的特异性抗体来实现 复合体。为了确定新发现的TDP-43中间体在聚集和发病中的作用， 目标2将定义组装的分子决定因素，并测试疾病相关条件如何 提升这一过程。在确定了介导寡聚的蛋白质区域后，我们将询问是否 一旦新识别的表位被破坏，聚集和细胞内播种功能就会降低。这个 TDP-43聚集体在疾病中作为病理性传播因子的能力最近被强烈地提示 FTD提取物在小鼠脑内的提取、种子和TDP-43病理的传播。AIM 3将测试 TDP-43聚集体是否真的能启动病理的从头聚集和传播 活着。我们将分析注射早期和晚期TDP-43后TDP-43的病理扩散情况 从小鼠大脑中纯化的蛋白质中衍生的聚集体。这将确定TDP-43是否聚合， 尤其是TDP-43低聚物，是成核和神经毒性的主要来源。此外，这些 通过研究，我们将确定这种传播是否与神经退行性变的增加直接相关。使用 为了成功完成这笔赠款，我们将：a)确定TDP-43汇总的基本程序；b) 建立体外和体内模型以预防ALS和FTD的神经毒性；以及c)提供前所未有的 开发疾病诊断标记物的工具。

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.