Development and validation of a sensitive sandwich ELISA against human PINK1.

Abstract

The ubiquitin kinase and ligase PINK1 and PRKN together label damaged mitochondria for their elimination in lysosomes by selective autophagy (mitophagy). This cytoprotective quality control pathway is genetically linked to familial Parkinson disease but is also altered during aging and in other neurodegenerative disorders. However, the molecular mechanisms of these mitophagy changes remain uncertain. In healthy mitochondria, PINK1 protein is continuously imported, cleaved, and degraded, but swiftly accumulates on damaged mitochondria, where it triggers the activation of the mitophagy pathway by phosphorylating its substrates ubiquitin and PRKN. Levels of PINK1 protein can therefore be used as a proxy for mitochondrial damage and mitophagy initiation. However, validated methodologies to sensitively detect and quantify PINK1 protein are currently not available. Here, we describe the development and thorough validation of a novel immunoassay to measure human PINK1 on the Meso Scale Discovery platform. The final assay showed excellent linearity, parallelism, and sensitivity. Even in the absence of mitochondrial stress (i.e. at basal conditions), when PINK1 protein is usually not detectable by immunoblotting, significant differences were obtained when comparing samples from patient fibroblasts or differentiated neurons with and without PINK1 expression. Of note, PINK1 protein levels were found increased in human postmortem brain with normal aging, but not in brains with Alzheimer disease, suggesting that indeed different molecular mechanisms are at play. In summary, we have developed a novel sensitive PINK1 immunoassay that will complement other efforts to decipher the roles and biomarker potential of the PINK1-PRKN mitophagy pathway in the physiological and pathological context. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; ELISA: enzyme-linked immunosorbent assay; iPSC: induced pluripotent stem cell; KO: knockout; LLOQ: lower limit of quantification; MSD: Meso Scale Discovery; PD: Parkinson disease; p-S65-Ub: serine-65 phosphorylated ubiquitin; Ub: ubiquitin; ULOQ: upper limit of quantification; WT: wild-type.