Nuclear import receptors as modifiers of TDP-43 phase transition and toxicity in FTD/ALS

该项目的目标是获得核输入受体（NIR）如何预防和逆转RNA结合蛋白TDP-43的细胞质错误定位和不溶性蛋白质聚集体在常见神经退行性疾病发病机制中的积累的详细机制。TDP-43病理学是额颞叶痴呆（FTD）和肌萎缩侧索硬化（ALS）的标志，但也常见于阿尔茨海默病（AD）和其他AD相关痴呆（ADRD），标志着它是治疗开发的高优先级目标。我们的实验室已经发现，1）TDP-43病理学的特征在于TDP-43与FG核孔蛋白（FG-Nups）在细胞质中的共聚集，导致核质转运缺陷，2）NIR可以减少TDP-43和其他具有朊病毒样结构域的致病RNA结合蛋白的异常相变。我们的数据表明，特定的NIR可以逆转FTD和ALS的细胞和动物模型中TDP-43蛋白质病的标志。基于我们的研究结果，我们提出了一种新的非经典的作用，NIR作为有效的分子伴侣，被FG-Nups招募到病理性TDP-43聚集体中，在那里它们可以逆转异常的相变并恢复TDP-43的正常核定位和剪接功能，这表明了一种有希望的新的治疗干预策略。为了验证这一假设，我们的FTD（ADRD）和ALS细胞和动物模型以及结构生物学方法的专家团队将使用尖端的体外和体内方法来详细了解近红外光谱如何恢复溶解度、核定位和正常功能。TDP-43;近红外光谱如何减少神经退行性疾病;近红外功能障碍如何导致人类疾病;以及我们如何利用近红外功能的知识来开发TDP-43蛋白病的未来治疗策略。我们的具体目标是：（i）使用FTD和ALS的高级神经元细胞培养模型和TDP-43液-液相分离（LLPS）和滤过的生物化学表征以及合理的蛋白质工程方法的组合来确定NIR如何在体外恢复适当的TDP-43定位并减少异常相变，以增强NIR的伴侣蛋白活性;和（ii）在TDP-43蛋白质病小鼠模型中采用果蝇、器官型切片培养物和体细胞脑转基因，并阐明ALS和FTD（ADRD）患者脑组织中NIR病理学的性质，以鉴定NIR如何在体内细胞和动物模型中减少TDP-43依赖性神经变性。该项目的成功结果将阐明NIR在发病过程中调节TDP-43相变的作用，以及这种活性如何恢复TDP-43的正常定位和细胞功能。这些知识对于开发新的治疗策略以靶向ALS，FTD和其他破坏性AD相关神经退行性疾病与TDP-43蛋白质病的异常相分离至关重要。

The goal of this project is to gain a detailed mechanistic understanding of how nuclear-import receptors (NIRs) can prevent and reverse the cytoplasmic mislocalization and accumulation of insoluble protein aggregates of the RNA-binding protein TDP-43 in the pathogenesis of common neurodegenerative disorders. TDP-43 pathology is a hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), but is also commonly found in Alzheimer’s disease (AD) and other AD-related dementias (ADRDs), marking it as a high priority target for therapy development. Our labs have discovered that 1) TDP-43 pathology is characterized by the co-aggregation of TDP-43 with FG nucleoporins (FG-Nups) in the cytoplasm, causing nucleocytoplasmic transport defects and 2) NIRs can reduce the aberrant phase transition of TDP-43 and other disease-causing RNA-binding proteins with prion-like domains. Our data demonstrate that specific NIRs can reverse the hallmarks of TDP-43 proteinopathy in cellular and animal models of FTD and ALS. Based on our findings, we propose a novel non-canonical role for NIRs as potent molecular chaperones that are recruited by FG-Nups into pathological TDP-43 aggregates, where they act to to reverse the aberrant phase transition and restore the normal nuclear localization and splicing functions of TDP-43, suggesting a promising new strategy for therapeutic intervention. To test this hypothesis, our team of experts in cellular and animal models of FTD (an ADRD) and ALS, as well as structural biology approaches, will use cutting edge in vitro and in vivo methods to gain a detailed mechanistic understanding of how NIRs restore solubility, nuclear localization and normal function of TDP-43; how NIRs reduce neurodegeneration; how NIR dysfunction contributes to human disease; and how we can use this knowledge of NIR functions to develop future therapeutic strategies for TDP-43 proteinopathies. Our specific aims are: (i) to determine how NIRs restore proper TDP-43 localization and reduce aberrant phase transition in vitro, using a combination of advanced neuronal cell culture models of FTD and ALS and biochemical characterization of TDP-43 liquid-liquid phase separation (LLPS) and fibrillization, and rational protein engineering approaches to potentiate the chaperone activity of NIRs; and (ii) to identify how NIRs reduce TDP-43-dependent neurodegeneration in cellular and animal models in vivo, employing Drosophila, organotypic slice cultures and somatic brain transgenesis in TDP-43 proteinopathy mouse models, and clarifying the nature of NIR pathology in ALS and FTD (an ADRD) patient brain tissue. Successful outcome of this project will clarify the role of NIRs in regulating TDP-43 phase transition during pathogenesis and how this activity can restore normal localization and cellular function of TDP-43. This knowledge will be critical for developing new therapeutic strategies to target aberrant phase separation in ALS, FTD, and other devastating AD-related neurodegenerative disorders with TDP-43 proteinopathy.