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Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples.

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.

SUBMITTER: Watzlawik JO

PROVIDER: S-EPMC8496550 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sciatic nerve chronic constriction injury (CCI) in rodents produces nerve demyelination via proteolysis of myelin basic protein (MBP), the major component of myelin sheath. Proteolysis releases the cryptic MBP epitope, a demyelination marker, which is hidden in the native MBP fold. It has never been established if the proteolytic release of this cryptic MBP autoantigen stimulates the post-injury increase in the respective circulating autoantibodies. To measure these autoantibodies, we developed the ELISA that employed the cryptic 84-104 MBP sequence (MBP84-104) as bait. This allowed us, for the first time, to quantify the circulating anti-MBP84-104 autoantibodies in rat serum post-CCI. The circulating IgM (but not IgG) autoantibodies were detectable as soon as day 7 post-CCI. The IgM autoantibody level continually increased between days 7 and 28 post-injury. Using the rat serum samples, we established that the ELISA intra-assay (precision) and inter-assay (repeatability) variability parameters were 2.87% and 4.58%, respectively. We also demonstrated the ELISA specificity by recording the autoantibodies to the liberated MBP84-104 epitope alone, but not to intact MBP in which the 84-104 region is hidden. Because the 84-104 sequence is conserved among mammals, we tested if the ELISA was applicable to detect demyelination and quantify the respective autoantibodies in humans. Our limited pilot study that involved 16 female multiple sclerosis and fibromyalgia syndrome patients demonstrated that the ELISA was efficient in measuring both the circulating IgG- and IgM-type autoantibodies in patients exhibiting demyelination. We believe that the ELISA measurements of the circulating autoantibodies against the pathogenic MBP84-104 peptide may facilitate the identification of demyelination in both experimental and clinical settings. In clinic, these measurements may assist neurologists to recognize patients with painful neuropathy and demyelinating diseases, and as a result, to personalize their treatment regimens.

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Sensitive ELISA-based detection method for the mitophagy marker p-S65-Ub in human cells, autopsy brain, and blood samples.

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