Targeting nuclear transport dysfunction in TDP-43 proteinopathies

 描述（由申请人提供）：TAR DNA结合蛋白43（TDP-43）与一系列神经退行性疾病（包括FTDL和ALS）相关。不幸的是，其发病机制的基础是知之甚少。有趣的是，虽然正常的TDP-43主要在细胞核中，但其胞质沉积和核消耗表征了疾病状态。这表明，核输入的缺陷可能在TDP-43蛋白病的起始中起关键作用。在这方面，已经记录了TDP-43细胞质内含物的形成与核转运蛋白Karyopherin β（KPNB 1）的消耗之间的正相关性。因此，本申请的主要目的是使用果蝇作为实验模型来确定KPNB 1针对TDP-43的疾病修饰能力。我们的中心假设是KPNB 1将对TDP-43损伤发挥保护活性。这得到了我们的初步数据的支持，这些数据显示Ketel（KPNB 1的果蝇同源物）改变了果蝇中的人类TDP-43毒性。例如，Ketel RNAi加剧了TDP-43 WT和TDP-43 M337 V转基因的眼表型，而其过表达强烈抑制了TDP-43 M337 V的神经毒性。有趣的是，Ketel不改变TDP-43 NLS的眼表型，其缺乏核定位信号（NLS），也不改变FUS的毒性，FUS是一种相关的DNA/RNA结合蛋白，其核输入不依赖于KPNB 1/Ketel。引人注目的是，我们还发现病理性TDP-43诱导NLS标记的报告蛋白的细胞质保留，这突出了疾病发病机制的潜在原因。因此，我们计划确定这种核转运障碍的分子基础，并确定靶向核转运机制的治疗潜力。我们的具体目标是：（1）表征Ketel/KPNB 1和TDP-43毒性之间的相互作用。我们将首先共表达一组野生型和突变型TDP-43转基因与各种获得和丧失功能的Ketel等位基因。然后，我们将评估TDP-43的不溶性，切割，磷酸化和亚细胞分布以及Ketel的潜在螯合和/或下调的概况。(2)评估KPNB 1在果蝇CNS中预防或逆转TDP-43毒性的能力。我们将在羽化后第1天开始激活整个CNS中的KPNB 1，随后在稍后的时间点独立激活TDP-43 WT和TDP-43 M337 V。然后，我们将逆转表达方案，首先激活TDP-43构建体，然后激活KPNB 1，以确定KPNB 1是否可以逆转或延迟疾病的进程。该研究具有创新性和高度重要性，因为这将是KPNB 1和TDP-43在不同时间模式下的首次系统操作。重要的是，我们希望确定KPNB 1是否有能力阻止，延迟或预防与TDP-43蛋白病相关的毒性。

 DESCRIPTION (provided by applicant): TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative disorders that include FTDL and ALS. Unfortunately, the mechanisms underlying its pathogenesis are poorly understood. Interestingly, although normal TDP-43 is primarily in the nucleus, its cytosolic deposition and nuclear depletion characterize the disease state. This suggests that defects in nuclear import may play a pivotal role in the initiation of TDP-43 proteinopathies. In this regard, a positive correlation between formation of TDP-43 cytoplasmic inclusions and depletion of the nuclear transporter Karyopherin β (KPNB1) has been documented. Thus, the major goal of this application is to determine the disease-modifying ability of KPNB1 against TDP-43 using Drosophila as experimental model. Our central hypothesis is that KPNB1 will exert a protective activity against TDP-43 insults. This is supported by our preliminary data showing that Ketel, the Drosophila homologue of KPNB1, modifies human TDP-43 toxicity in flies. For instance, Ketel RNAi exacerbates the eye phenotypes of TDP-43WT and TDP-43M337V transgenes, while its overexpression robustly suppresses the neurotoxicity of TDP-43M337V. Interestingly, Ketel does not modify the eye phenotype of TDP-43NLS, which lacks a nuclear localization signal (NLS), neither the toxicity of FUS, a related DNA/RNA binding protein whose nuclear import is independent of KPNB1/Ketel. Strikingly, we also found that pathological TDP-43 induces cytoplasmic retention of NLS-tagged reporter proteins, which highlights a potential underlying cause of disease pathogenesis. Therefore, we plan to determine the molecular basis of this nuclear transport impairment and to define the therapeutic potential of targeting the nuclear transport machinery. Our specific aims are: (1) Characterize the interplay between Ketel/KPNB1 and TDP-43 toxicity. We will first co-express a group of wild type and mutant TDP-43 transgenes with various gain- and loss-of-function Ketel alleles. Then, we will assess the profiles of TDP-43 insolubility, cleavage, phosphorylation and subcellular distribution as well as the potential sequestration and/or down-regulation of Ketel. (2) Assess the ability of KPNB1 to prevent or reverse TDP-43 toxicity in the fly CNS. We will activate KPNB1 in the entire CNS starting at day 1 post-eclosion followed by independent activation of TDP-43WT and TDP-43M337V at later time points. Then, we will reverse the expression scheme, activating TDP-43 constructs first followed by KPNB1 to determine if KPNB1 can reverse or delay the course of the disease. The proposed research is innovative and highly significant because this will be the first systematic manipulation of KPNB1 and TDP-43 under different temporal patterns. Importantly, we expect to define whether KPNB1 has the ability to stop, delay, or prevent the toxicity associated with TDP-43 proteinopathies.