ABSTRACT

Mitochondrial dysfunction is an early, imminent event in neurodegenerative disorders including Parkinson disease (PD) and Alzheimer disease (AD). The enzymatic pair PINK1 and PRKN/Parkin recognize and transiently label damaged mitochondria with ubiquitin (Ub) phosphorylated at Ser65 (p-S65-Ub) as a signal for degradation via the autophagy-lysosome system (mitophagy). Despite its discovery in cell culture several years ago, robust and quantitative detection of altered mitophagy in vivo has remained challenging. Here we developed a sandwich ELISA targeting p-S65-Ub with the goal to assess mitophagy levels in mouse brain and in human clinical and pathological samples. We characterized five total Ub and four p-S65-Ub antibodies by several techniques and found significant differences in their ability to recognize phosphorylated Ub. The most sensitive antibody pair detected recombinant p-S65-Ub chains in the femtomolar to low picomolar range depending on the poly-Ub chain linkage. Importantly, this ELISA was able to assess very low baseline mitophagy levels in unstressed human cells and in brains from wild-type and prkn knockout mice as well as elevated p-S65-Ub levels in autopsied frontal cortex from AD patients vs. control cases. Moreover, the assay allowed detection of p-S65-Ub in blood plasma and was able to discriminate between PINK1 mutation carriers and controls. In summary, we developed a robust and sensitive tool to measure mitophagy levels in cells, tissue, and body fluids. Our data strongly support the idea that the stress-activated PINK1-PRKN mitophagy pathway is constitutively active in mice and humans under unstimulated, physiological and elevated in diseased, pathological conditions.

Abbreviations: Ab: antibody; AD: Alzheimer disease; AP: alkaline phosphatase; CV: coefficient of variation; ECL: electrochemiluminescence; KO: knockout; LoB: Limit of Blank; LoD: Limit of Detection; LoQ: Limit of Quantification; MSD: meso scale discovery; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated ubiquitin at serine 65; Std.Dev.: standard deviation; Ub: ubiquitin; WT: wild type

摘要

线粒体功能障碍是包括帕金森病 (PD) 和阿尔茨海默病 (AD) 在内的神经退行性疾病的早期迫在眉睫的事件。酶对 PINK1 和 PRKN/Parkin 用 Ser65 (p-S65-Ub) 磷酸化的泛素 (Ub) 识别并瞬时标记受损线粒体，作为通过自噬-溶酶体系统（线粒体自噬）降解的信号。尽管几年前在细胞培养中发现了它，但对体内改变的线粒体自噬进行稳健和定量检测一直充满挑战。在这里，我们开发了一种针对 p-S65-Ub 的夹心 ELISA，目的是评估小鼠大脑和人类临床和病理样本中的线粒体自噬水平。我们通过几种技术对五种总 Ub 和四种 p-S65-Ub 抗体进行了表征，发现它们识别磷酸化 Ub 的能力存在显着差异。最敏感的抗体对检测到飞摩尔到低皮摩尔范围内的重组 p-S65-Ub 链，具体取决于多聚 Ub 链连接。重要的是，这种 ELISA 能够评估未受压的人类细胞和野生型和prkn大脑中非常低的基线线粒体自噬水平AD 患者与对照病例的尸检额叶皮质中 p-S65-Ub 水平升高以及敲除小鼠以及升高的 p-S65-Ub 水平。此外，该测定允许检测血浆中的 p-S65-Ub，并能够区分PINK1突变携带者和对照。总之，我们开发了一种强大而灵敏的工具来测量细胞、组织和体液中的线粒体自噬水平。我们的数据强烈支持这样的观点，即应激激活的 PINK1-PRKN 线粒体自噬途径在小鼠和人类中在未受刺激的、生理的和在患病的病理条件下升高的情况下是组成性活跃的。

缩写：Ab：抗体；AD：阿尔茨海默病；AP：碱性磷酸酶；CV：变异系数；ECL：电化学发光；KO：淘汰赛；LoB：空白极限；LoD：检测限；LoQ：定量限；MSD：中尺度发现；PD：帕金森病；p-S65-PRKN：丝氨酸 65 位磷酸化的 PRKN；p-S65-Ub：丝氨酸 65 位的磷酸化泛素；Std.Dev.：标准差；Ub：泛素；WT：野生型