Huang Lushan, Ma Liyi, Zhao Qiaowen, Zhu Qichen, She Guangwei, Mu Lixuan, Shi Wensheng
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2025 Jan 14;97(1):194-202. doi: 10.1021/acs.analchem.4c03646. Epub 2024 Dec 26.
pH and peroxynitrite (ONOO) are two critical biomarkers to unveil the corresponding status of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are closely related to Alzheimer's disease (AD). Simultaneously monitoring pH and ONOO fluctuations in the ER and mitochondria during AD progression is pivotal for clarifying the interplay between the disorders of the two organelles and revealing AD pathogenesis. Herein, we designed and synthesized a dual-channel fluorescent probe (DCFP) to visualize pH and ONOO in the ER and mitochondria. DCFP possessed excellent sensitivity and selectivity to pH and ONOO without spectral crosstalk and was utilized in monitoring the two analytes within AD model cells and larval zebrafish. Importantly, DCFP could preferentially target mitochondria in normal cells and be enriched in the ER after mitochondrial depolarization. With the aid of DCFP, the slower acidification rate of the ER than that of mitochondria induced by Aβ oligomers (AβOs) was first identified, which could be ascribed to the relief of the AβOs-triggered ER stress through the Ca migration from the ER to mitochondria. Moreover, continuous exposure to AβOs led to mitochondrial Ca overload, accelerating the acidification and ONOO overproduction within mitochondria. As a result, intracellular oxidative stress levels were elevated, further exacerbating ER stress and aggravating ER acidification in turn. The advanced understanding of the potential interplay between the ER and mitochondria in this work may offer new insights and methodologies for studying AD pathogenesis. The DCFP developed in this work could also be employed to study other diseases related to ER stress and mitochondrial dysfunction.
pH值和过氧亚硝酸根（ONOO）是揭示内质网（ER）应激和线粒体功能障碍相应状态的两个关键生物标志物，这两者与阿尔茨海默病（AD）密切相关。在AD进展过程中同时监测内质网和线粒体中pH值和ONOO的波动，对于阐明这两个细胞器紊乱之间的相互作用以及揭示AD发病机制至关重要。在此，我们设计并合成了一种双通道荧光探针（DCFP），用于可视化内质网和线粒体中的pH值和ONOO。DCFP对pH值和ONOO具有出色的灵敏度和选择性，且无光谱串扰，并被用于监测AD模型细胞和斑马鱼幼体中的这两种分析物。重要的是，DCFP在正常细胞中可优先靶向线粒体，而在线粒体去极化后会在内质网中富集。借助DCFP，首次发现由Aβ寡聚体（AβOs）诱导的内质网酸化速率比线粒体慢，这可归因于通过Ca从内质网迁移到线粒体而缓解了AβOs触发的内质网应激。此外，持续暴露于AβOs会导致线粒体Ca过载，加速线粒体内的酸化和ONOO过量产生。结果，细胞内氧化应激水平升高，进而进一步加剧内质网应激并加重内质网酸化。这项工作中对内质网和线粒体之间潜在相互作用的深入理解可能为研究AD发病机制提供新的见解和方法。这项工作中开发的DCFP也可用于研究其他与内质网应激和线粒体功能障碍相关的疾病。
