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的痴呆症（ADRD）， 出口和细胞质反式激活反应DNA结合蛋白43（TDP-43）。的机制 TDP-43的病理性输出或其在细胞质中的积累并不清楚。虽然，研究 关于致病突变的研究表明，核输入的缺陷可能是导致 由于这些突变改变了蛋白质的性质，因此TDP-43在细胞质中积累。但 TDP-43如何在胞质溶胶中积累仍然很不清楚，因为大多数依赖TDP-43的 病理是偶发的。TDP-43含有两个RNA识别基序（RRM），即核输出信号 (NES)和核定位信号（NLS）在其N-末端，并结合聚（ADP-核糖）聚合物（PAR） 通过其NLS中嵌入的PAR结合基序。研究表明，PAR抑制剂可以抑制TDP-43 PAR与TDP-43的结合可以改变其在溶液中的生物物理特性。输出1 （XPO 1）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依赖XPO 1的输出，但我们的数据表明，在神经元条件下， 应激条件下TDP-43通过XPO-1依赖性输出而从细胞核中排出。我们假设在疾病中 如FTD型ADRD和相关疾病; PAR在细胞核中结合TDP-43， 与XPO-1的相互作用，其随后增强TDP-43从细胞核到胞质溶胶的流出。 这一提议得到了我们的初步数据的支持，即PARP抑制剂BMN 673和XPO-1抑制剂KPT-1都是PARP抑制剂。 185抑制TDP-43在神经元中的胞质蓄积，并且TDP-43和XPO 1在PARP后相互作用 activation.在这个提议中，我们计划研究暴露于以下物质的小鼠皮层神经元中TDP-43的改变： 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， 在几乎所有神经系统疾病（包括FTD）中的聚集和细胞死亡。通过H2 O2的氧化应激， 皮质神经元介导了稳健的TDP-43胞质定位。因此，很可能病理生理学 在皮质神经元中的氧化应激模型中鉴定的机制可以紧密重叠/模拟病理学 涉及TDP-43变化/错误定位的FTD和其他ADRD机制。 我们将使用生物化学，细胞生物学和成像技术与邻近连接试验相结合， CryoEM和氢/氘交换质谱，以评估PAR依赖性改变， TDP-43及其与XPO-1的相互作用导致TDP-43从细胞核释放增加 以及随后在细胞质中的积累/聚集。这些研究将有助于了解 FTD、ALS和其他ADRD/痴呆相关疾病中TDP-43蓄积的病理生理学机制 TDP-43相关疾病

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