Data_Sheet_1_IRE1α-XBP1 Affects the Mitochondrial Function of Aβ25–35-Treated SH-SY5Y Cells by Regulating Mitochondria-Associated Endoplasmic Reticulum Membranes.PDF

Background: Neurotoxicity induced by the amyloid beta (Aβ) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism.
Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aβ25–35, with or without 4μ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays.
Results: Aβ-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4μ8c, reduced Aβ-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aβ treatment in the presence of 4μ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aβ-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aβ treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)–mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis.
Conclusion: These findings demonstrate that Aβ peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aβ-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

背景：淀粉样β（amyloid beta, Aβ）肽诱导的神经毒性是阿尔茨海默病（Alzheimer's disease, AD）最核心的病理机制之一。适应性IRE1α-XBP1通路的激活参与AD的发病进程，使其成为AD治疗的潜在靶点。然而，IRE1α-XBP1通路在AD中的作用机制仍未阐明。因此，本研究在体外AD细胞模型中探究了IRE1α-XBP1轴的调控作用，并对其潜在分子机制进行了深入探索。方法：本研究采用人神经母细胞瘤细胞系SH-SY5Y。将细胞用Aβ₂₅₋₃₅处理，同时设置是否添加IRE1α抑制剂4μ8c的实验组与对照组。收集细胞后，通过蛋白质印迹（Western blotting）、实时定量聚合酶链反应（quantitative real-time PCR）、电子显微镜、荧光显微镜、钙成像及其他生化实验手段进行检测分析。结果：经Aβ处理的SH-SY5Y细胞中，XBP1s与p-IRE1α的表达水平显著升高。3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）实验与钙成像分析结果显示，IRE1α抑制剂4μ8c可有效减轻Aβ诱导的细胞毒性。在添加4μ8c的条件下，Aβ处理后的细胞ATP水平回升、线粒体膜电位得以恢复，线粒体活性氧生成量显著降低，这表明抑制IRE1α-XBP1轴可有效缓解SH-SY5Y细胞中Aβ诱导的线粒体功能障碍。此外，Aβ处理可上调IP3R、Grp75与vdac1的表达并增强其相互作用，进而导致内质网（endoplasmic reticulum, ER）与线粒体的连接增加、线粒体相关内质网膜（mitochondria-associated ER-membranes, MAMs）功能异常以及线粒体功能障碍。上述病理缺陷均可通过抑制IRE1α-XBP1轴得到挽救。结论：本研究结果证实，Aβ肽可诱导IRE1α-XBP1轴激活，该轴可能通过靶向线粒体相关内质网膜，加重SH-SY5Y细胞的细胞毒性与线粒体损伤。抑制IRE1α-XBP1轴可对SH-SY5Y细胞起到抗Aβ诱导损伤的保护作用，因此有望成为阿尔茨海默病治疗的全新策略。