Robust and sensitive ELISA detection of total and activated PRKN
- 1Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
- 2Mayo Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA
- 3Department of Neurology, Mayo Clinic, Jacksonville, FL, USA
Abstract
Parkinson disease (PD) is closely linked to disruptions in mitochondrial quality control, a process regulated by the ubiquitin kinase PINK1 and the E3 ubiquitin ligase PRKN/parkin. Upon mitochondrial damage, PINK1 phosphorylates ubiquitin, which in turn recruits and activates PRKN. Full activation of PRKN is mediated by PINK1-dependent phosphorylation of PRKN at serine 65, which leads to widespread ubiquitination of mitochondrial substrates and amplifies the mitophagy response. Disruption of this pathway results in mitochondrial accumulation, oxidative stress, and neuronal death, all key mechanisms of PD pathogenesis. Genetic studies have shown biallelic loss-of-function mutations in PRKN are the most common cause of early-onset PD. Although the role of haploinsufficiency remains under investigation, PRKN protein becomes insoluble and inactive with aging or post-translational modifications, indicating that functional protein levels are a key determinant of disease risk. Reliable quantification of total and activated PRKN in samples has not been feasible, limiting research and clinical assessment. To address this, we developed and validated knockout (KO)-verified sandwich ELISA assays that quantify both total PRKN and PINK1-phosphorylated p-S65-PRKN. These assays provide absolute quantification of PRKN, improving functional diagnosis, and patient stratification in PD. Application of these methods established the concentration of PRKN in cells and in brain and revealed significant effects of a common genetic PRKN variant, further highlighting the importance of determining functional PRKN protein levels. The developed immunoassays complement previously established PINK1 and p-S65-Ub measurements, enhancing mechanistic insight into mitophagy and enabling effective monitoring of PD therapies and other neurodegenerative diseases.
Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; ELISA: enzyme-linked immunosorbent assay; IBR: In-between-RING; iPSC: induced pluripotent stem cell; KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MSD: Meso Scale Discovery; PD: Parkinson disease; p-S65-PRKN: serine 65 phosphorylated PRKN; p-S65-Ub: serine 65 phosphorylated ubiquitin; REP: repressor element of PRKN; RING: really interesting new gene; Ub: ubiquitin; UBL: ubiquitin-like domain; VCL: vinculin; WT: wild-type
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BibTeX
@article{watzlawik2026robust,
title = {{Robust and sensitive ELISA detection of total and activated PRKN}},
author = {Jens O. Watzlawik and Bernardo A. Bustillos and Claudia Rodriguez Martinez and Dennis W. Dickson and Zbigniew K. Wszolek and Owen A. Ross and Wolfdieter Springer and Fabienne C. Fiesel},
journal = {Autophagy},
year = {2026},
month = jul,
doi = {10.1080/15548627.2026.2694658},
url = {https://doi.org/10.1080/15548627.2026.2694658},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Robust and sensitive ELISA detection of total and activated PRKN
AU - Jens O. Watzlawik
AU - Bernardo A. Bustillos
AU - Claudia Rodriguez Martinez
AU - Dennis W. Dickson
AU - Zbigniew K. Wszolek
AU - Owen A. Ross
AU - Wolfdieter Springer
AU - Fabienne C. Fiesel
PY - 2026
DA - 2026/07/03
JO - Autophagy
DO - 10.1080/15548627.2026.2694658
UR - https://doi.org/10.1080/15548627.2026.2694658
AB - Parkinson disease (PD) is closely linked to disruptions in mitochondrial quality control, a process regulated by the ubiquitin kinase PINK1 and the E3 ubiquitin ligase PRKN/parkin. Upon mitochondrial damage, PINK1 phosphorylates ubiquitin, which in turn recruits and activates PRKN. Full activation of PRKN is mediated by PINK1-dependent phosphorylation of PRKN at serine 65, which leads to widespread ubiquitination of mitochondrial substrates and amplifies the mitophagy response. Disruption of this pathway results in mitochondrial accumulation, oxidative stress, and neuronal death, all key mechanisms of PD pathogenesis. Genetic studies have shown biallelic loss-of-function mutations in PRKN are the most common cause of early-onset PD. Although the role of haploinsufficiency remains under investigation, PRKN protein becomes insoluble and inactive with aging or post-translational modifications, indicating that functional protein levels are a key determinant of disease risk. Reliable quantification of total and activated PRKN in samples has not been feasible, limiting research and clinical assessment. To address this, we developed and validated knockout (KO)-verified sandwich ELISA assays that quantify both total PRKN and PINK1-phosphorylated p-S65-PRKN. These assays provide absolute quantification of PRKN, improving functional diagnosis, and patient stratification in PD. Application of these methods established the concentration of PRKN in cells and in brain and revealed significant effects of a common genetic PRKN variant, further highlighting the importance of determining functional PRKN protein levels. The developed immunoassays complement previously established PINK1 and p-S65-Ub measurements, enhancing mechanistic insight into mitophagy and enabling effective monitoring of PD therapies and other neurodegenerative diseases. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; ELISA: enzyme-linked immunosorbent assay; IBR: In-between-RING; iPSC: induced pluripotent stem cell; KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MSD: Meso Scale Discovery; PD: Parkinson disease; p-S65-PRKN: serine 65 phosphorylated PRKN; p-S65-Ub: serine 65 phosphorylated ubiquitin; REP: repressor element of PRKN; RING: really interesting new gene; Ub: ubiquitin; UBL: ubiquitin-like domain; VCL: vinculin; WT: wild-type
SN - 1554-8627
SN - 1554-8635
ER -