RNA-based regulation of TDP-43 nuclear export in ALS/FTD

TAR DNA结合蛋白-43(TDP-43)是一种必需的DNA/RNA结合蛋白，在RNA中起重要作用 加工性能和稳定性。尽管在定位上以核为主，但超过95%的肌萎缩侧索硬化 硬化症(ALS)患者和多达一半的额颞叶痴呆(FTD)患者显示TDP-43的错误定位 进入细胞质。大量证据表明TDP-43错误定位与神经退行性变的发病机制有关 在ALS/FTD中，已在部分家族性ALS病例中发现TDP-43突变，进一步支持 TDP-43基因中断在疾病中的主要作用。然而，ALS/FTD中TDP-43核清除的原因 仍然不为人知。一个主要障碍是对TDP-43核出口机制缺乏了解。 直到最近，TDP-43还被认为是核出口受体Exportin-1(XPO1)的货物。然而， 最近的多项研究表明，XPO1不与TDP-43结合或介导其输出。替代出口 机制可能包括从核的被动扩散，或与RNA的主动共输出，例如通过 Nxf1/TREX mRNA输出复合体。我们的初步数据显示，TDP-43的核出口是ATP-和RNA- 取决于，但可能需要孔上游的能量，以实现TDP-43的核内迁移，而不是 穿过核孔复合体(NPC)的易位。事实上，渗透性细胞测试表明，一旦 TDP-43从核RNA中移位，很可能扩散到核外。此外，对Nxf1生长素的研究- 诱导性降解(AID)细胞株提示，TDP-43的输出不需要大量的mRNA途径。基座 根据这些数据，我们假设TDP-43的依赖于ATP的核拴系，最有可能与核RNA， 关键决定了它扩散到细胞质的速度。在建议的研究中，我们会进行 排列CRISPRi和CRISPRa筛选靶向文库以确定能量依赖机制 TDP-43的核拴系。HITS将在核运输和光漂白分析中得到验证，此外 在ALS/FTD患者组织中进行评估。然后我们将把高含量显微镜和核质相结合 转运分析，包括新开发的通透性细胞输出分析，以确认 TDP-43在出口过程中跨核孔易位。更多的艾滋病细胞株和基因敲除研究 将被用来明确排除信使核糖核酸输出途径的作用。最后，我们将测试治疗 在我们的初步研究中确定的促进TDP-43核滞留的新方法的有效性。 测试将包括TDP-43突变体果蝇(与Daniela Zarnescu实验室合作)、TDP-43突变体 INeurons和小鼠坐骨神经切断，以确定我们是否可以促进TDP-43在体内的核滞留， 以及这一策略是否会减轻TDP-43蛋白病的分子和神经病理特征。 这些研究将使对TDP-43的机制和调控有一个精确的机制理解 核出口，以及促进TDP-43核保留的候选战略的临床前测试。

TAR DNA-binding protein-43 (TDP-43) is an essential DNA/RNA-binding protein that plays a major role in RNA processing and stability. Although predominantly nuclear in localization, over 95% of amyotrophic lateral sclerosis (ALS) cases and up to half of frontotemporal dementia (FTD) cases show mislocalization of TDP-43 to the cytoplasm. Substantial evidence links TDP-43 mislocalization to the pathogenesis of neurodegeneration in ALS/FTD, and TDP-43 mutations have been identified in subset of familial ALS cases, further supporting a primary role for TDP-43 disruption in disease. However, the cause of TDP-43 nuclear clearance in ALS/FTD remains unknown. A major barrier has been lack of understanding of the mechanism of TDP-43 nuclear export. Until recently, TDP-43 was thought to be a cargo of the nuclear export receptor, exportin-1 (XPO1). However, multiple recent studies demonstrate that XPO1 does not bind TDP-43 or mediate its export. Alternative export mechanisms may include passive diffusion from the nucleus, or active co-export with RNA, such as via the NXF1/TREX mRNA export complex. Our preliminary data show that TDP-43 nuclear export is ATP- and RNA- dependent, but the energy may be required upstream of the pore, for TDP-43 intranuclear mobility, rather than translocation across the nuclear pore complex (NPC). Indeed, a permeabilized cell assay suggests that, once displaced from nuclear RNA, TDP-43 likely diffuses out of the nucleus. Moreover, studies in an NXF1 auxin- inducible degron (AID) cell line suggest that the bulk mRNA pathway is not required for TDP-43 export. Based on these data, we hypothesize that ATP-dependent nuclear tethering of TDP-43, most likely to nuclear RNA, critically dictates the rate at which it diffuses into the cytoplasm. In the proposed studies, we will perform an arrayed CRISPRi and CRISPRa screen of a targeted library to determine the mechanism of energy-dependent nuclear tethering of TDP-43. Hits will be validated in nuclear transport and photobleaching assays, and also evaluated in ALS/FTD patient tissue. We will then combine high content microscopy with nucleocytoplasmic transport assays, including a newly developed permeabilized cell export assay, to confirm the mechanism of TDP-43 translocation across the nuclear pore during export. Additional AID cell lines and knockdown studies will be used to definitively exclude a role for the mRNA export pathway. Finally, we will test the therapeutic efficacy of a novel approach to promote TDP-43 nuclear retention, identified during our preliminary studies. Testing will include TDP-43 mutant Drosophila (in collaboration with the Daniela Zarnescu lab), TDP-43 mutant iNeurons, and mouse sciatic nerve axotomy, to determine if we can promote TDP-43 nuclear retention in vivo, and whether this strategy will attenuate molecular and neuropathologic features of TDP-43 proteinopathy. These studies will enable a precise mechanistic understanding of the mechanism and regulation of TDP-43 nuclear export, and advance preclinical testing of a candidate strategy to promote TDP-43 nuclear retention.