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)与包括FTDL和ALS在内的一系列神经退行性疾病有关。不幸的是，其发病机制尚不清楚。有趣的是，虽然正常的TDP-43主要在细胞核内，但其胞浆沉积和核耗竭是疾病状态的特征。这表明，核进口缺陷可能在TDP-43蛋白病变的启动中起着关键作用。在这方面，TdP-43胞质包涵体的形成与核转运蛋白KPNB1(KPNB1)的耗尽之间存在正相关关系。因此，本应用的主要目的是以果蝇为实验模型，确定KPNB1对TDP-43的疾病修饰能力。我们的中心假设是，KPNB1将对TDP-43侮辱起到保护作用。我们的初步数据表明，KPNB1的果蝇同源物Ketel可以改变人TDP-43对果蝇的毒性。例如，Ketel RNAi加剧了TDP-43WT和TDP-43M337V转基因的眼睛表型，而其过表达则有力地抑制了TDP-43M337V的神经毒性。有趣的是，Ketel没有改变TDP-43NLS的眼睛表型，它缺乏核定位信号(NLS)，也没有FUS的毒性，FUS是一种相关的DNA/RNA结合蛋白，其核输入独立于KPNB1/Ketel。值得注意的是，我们还发现病理性的TDP-43诱导NLS标记的报告蛋白的细胞质滞留，这突出了疾病发病的潜在潜在原因。因此，我们计划确定这种核运输损伤的分子基础，并确定靶向核运输机械的治疗潜力。我们的具体目标是：(1)研究Ketel/KPNB1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转基因基因，这些基因具有不同的功能获得和失去功能的Ketel等位基因。然后，我们将评估TDP-43的不解性、裂解、磷酸化和亚细胞分布以及Ketel的潜在封存和/或下调。(2)评价KPNB1对飞行中枢神经系统TDP-43毒性的预防或逆转作用。我们将从羽化后第1天开始在整个中枢系统激活KPNB1，然后在以后的时间点独立激活TDP-43WT和TDP-43M337V。然后，我们将逆转表达方案，首先激活TDP-43结构，然后激活KPNB1，以确定KPNB1是否可以逆转或延缓疾病的进程。这项研究具有创新性和很高的意义，因为这将是第一次在不同的时间模式下对KPNB1和TDP-43进行系统操作。重要的是，我们希望确定KPNB1是否有能力阻止、延迟或预防与TDP-43蛋白病变相关的毒性。

 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.