Poly(ADP-ribose)-dependent TDP-43 pathology in oxidative stress (R21)

摘要 额颞叶痴呆(FTD)是一种阿尔茨海默病相关痴呆(ADRD)，涉及核团 出口和细胞质反式激活反应DNA结合蛋白43(TDP-43)。实现以下目标的机制 TDP-43的病理性输出或其在细胞质中的积聚尚不清楚。虽然，研究 关于致病突变的研究表明，核进口中的缺陷可能是导致 随着这些突变改变蛋白质的性质，TDP-43在胞浆中积累。然而，它 TDP-43在很大程度上仍不清楚TDP-43是如何在胞浆中积累的，因为大多数TDP-43依赖于 病理是零星的。TDP-43包含两个RNA识别基序(RRMS)，这是一个核输出信号 (NES)和N-末端的核定位信号(NLS)，并结合聚(ADP-核糖)聚合物(PAR)。 通过嵌入在其NLS中的PAR结合基序。研究表明，PAR抑制剂可以抑制TDP-43 病理，PAR与TDP-43的结合可改变其在溶液中的生物物理特性。出口1 (XPO1)介导NES依赖的蛋白质从细胞核输出。TDP-43是一种43 kDa的蛋白质， 在基础条件下可能不需要XPO1依赖的输出，但我们的数据表明在神经元 应激条件下，TDP-43通过XPO-1依赖的输出使细胞核外移。我们假设在疾病中 如FTD类型的ADRD和相关疾病；PAR在细胞核内结合TDP-43并促进其 与XPO-1的相互作用，从而增强TDP-43从细胞核到胞浆的外流。 这一建议得到了我们的初步数据的支持，即PARP抑制剂BMN673和XPO-1抑制剂KPT- 185抑制PARP后TDP-43在神经细胞内的积聚及其与XPO1的相互作用 激活。在这项计划中，我们计划研究暴露于辐射中的小鼠皮质神经元中TDP-43的变化。 氧化应激。氧化应激是介导蛋白质错误定位的常见病理过程， 包括FTD在内的几乎所有神经疾病中的聚集和细胞死亡。过氧化氢引起的氧化应激 皮质神经元调节TDP-43胞浆的强健定位。因此，很可能是病理生理学 大脑皮层神经元氧化应激模型中识别的机制可以紧密重叠/模拟病理 FTD和其他ADRD中涉及TDP-43变化/错误本地化的机制。 我们将使用生化、细胞生物学和成像技术，并结合邻近连接分析， 和氢/氢交换质谱仪评估依赖于PAR的改变 TDP-43及其与XPO-1的相互作用导致TDP-43从细胞核释放增加 并随后在细胞质中聚集/聚集。这些研究将有助于理解 TDP-43在FTD、ALS和其他ADRD/痴呆相关疾病中积聚的病理生理机制 与TDP-43有关的疾病

Abstract Frontotemporal dementia (FTD) is an Alzheimer-related dementia disease (ADRD) and involves nuclear egress and cytoplasmic Transactivation response DNA binding protein 43 (TDP-43). The mechanisms for pathological export of TDP-43 or its accumulation in the cytoplasm is not clearly defined. Although, studies on disease-causing mutations have revealed that defects in nuclear import may be in part responsible for TDP-43 accumulation in the cytosol as these mutations change the properties of the protein. However, it remains largely unclear how TDP-43 accumulates in the cytosol, as the majority of the TDP-43 dependent pathologies are sporadic. TDP-43 contains two RNA recognition motifs (RRMs), a nuclear export signal (NES), and a nuclear localization signal (NLS) in its N-terminus, and binds poly (ADP-ribose) polymer (PAR) via a PAR-binding motif embedded in its NLS. Studies have shown that PAR inhibitors can inhibit TDP-43 pathology, and binding of PAR to TDP-43 can change its biophysical characteristics in solution. Exportin 1 (XPO1) mediates the NES-dependent export of proteins from the nucleus. TDP-43 is a 43kDa protein that may not require XPO1-dependent export under basal conditions but our data indicates that under neuronal stress conditions TDP-43 egresses the nucleus via XPO-1 dependent export. We hypothesize that in disease conditions like FTD type ADRD and related diseases; PAR binds TDP-43 in the nucleus and facilitates its interaction with XPO-1, which subsequently enhances the egress of TDP-43 from the nucleus to cytosol. This proposal is supported our preliminary data that both PARP inhibitor BMN673 and XPO-1 inhibitor KPT- 185 inhibit the cytosolic accumulation of TDP-43 in neurons and TDP-43 and XPO1 interact following PARP activation. In this proposal, we plan to study the alterations in TDP-43 in mouse cortical neurons exposed to oxidative stress. Oxidative stress is a common pathological process mediating protein mislocalization, aggregation, and cell death in virtually all neurological disorders including FTD. Oxidative stress via H2O2 in cortical neurons mediates a robust TDP-43 cytosolic localization. Therefore, it is likely that pathophysiological mechanisms identified in the oxidative stress model in cortical neurons can closely overlap/mimic pathological mechanisms in FTD and other ADRD involving changes/mislocalization of TDP-43. We will use biochemical, cell biological and imaging techniques in combination with proximity ligation assays, CryoEM, and Hydrogen/Deuterium Exchange mass spectrometry to assess the PAR-dependent alteration in TDP-43 and its interaction with XPO-1 that leads to an increase in the release of TDP-43 from the nucleus and subsequent accumulation/aggregation in the cytoplasm. These studies will help in understanding the pathophysiological mechanisms of TDP-43 accumulation in FTD, ALS, and other ADRD/dementia related diseases involving TDP-43