Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in PINK1 –Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential mitophagy defects. We aimed to characterise the expression of the PINK1 –Parkin mitophagy machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein expression levels were characterised with reverse transcriptase quantitative PCR and western blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable and PINK1 mRNA levels were remarkably low in control PBMCs. Our findings suggest that the PINK1 –Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable biosample for analysis of mitophagy function in Parkinson disease patients.

Alzheimer’s disease (AD) is a neurodegenerative disorder, accounting for at least two-thirds of dementia cases. A combination of genetic, epigenetic and environmental triggers is widely accepted to be responsible for the onset and development of AD. Accumulating evidence shows that oxidative stress and dysregulation of energy metabolism play an important role in AD pathogenesis, leading to neuronal dysfunction and death. Redox-induced protein modifications have been reported in the brain of AD patients, indicating excessive oxidative damage. Coenzyme A (CoA) is essential for diverse metabolic pathways, regulation of gene expression and biosynthesis of neurotransmitters. Dysregulation of CoA biosynthesis in animal models and inborn mutations in human genes involved in the CoA biosynthetic pathway have been associated with neurodegeneration. Recent studies have uncovered the antioxidant function of CoA, involving covalent protein modification by this cofactor (CoAlation) in cellular response to oxidative or metabolic stress. Protein CoAlation has been shown to both modulate the activity of modified proteins and protect cysteine residues from irreversible overoxidation. In this study, immunohistochemistry analysis with highly specific anti-CoA monoclonal antibody was used to reveal protein CoAlation across numerous neurodegenerative diseases, which appeared particularly frequent in AD. Furthermore, protein CoAlation consistently co-localized with tau-positive neurofibrillary tangles, underpinning one of the key pathological hallmarks of AD. Double immunihistochemical staining with tau and CoA antibodies in AD brain tissue revealed co-localization of the two immunoreactive signals. Further, recombinant 2N3R and 2N4R tau isoforms were found to be CoAlated in vitro and the site of CoAlation mapped by mass spectrometry to conserved cysteine 322, located in the microtubule binding region. We also report the reversible H2O2-induced dimerization of recombinant 2N3R, which is inhibited by CoAlation. Moreover, CoAlation of transiently expressed 2N4R tau was observed in diamide-treated HEK293/Pank1β cells. Taken together, this study demonstrates for the first time extensive anti-CoA immunoreactivity in AD brain samples, which occurs in structures resembling neurofibrillary tangles and neuropil threads. Covalent modification of recombinant tau at cysteine 322 suggests that CoAlation may play an important role in protecting redox-sensitive tau cysteine from irreversible overoxidation and may modulate its acetyltransferase activity and functional interactions.

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14Mutations in the PINK1 and PRKN genes are the most common cause of early-onset familial 15 Parkinson disease. These genes code for the PINK1 and Parkin proteins, respectively, which are 16 involved in the degradation of dysfunctional mitochondria through mitophagy. An early step in 17 PINK1 -Parkin mediated mitophagy is the ubiquitination of the mitofusin proteins MFN1 and -2. The 18 ubiquitination of MFN1 and -2 in patient samples may therefore serve as a biomarker to determine 19 the functional effects of PINK1 and PRKN mutations, and to screen idiopathic patients for potential 20 mitophagy defects. We aimed to characterise the expression of the PINK1 -Parkin mitophagy 21 machinery in peripheral blood mononuclear cells (PBMCs) and assess if these cells could serve as a 22 platform to evaluate mitophagy via analysis of MFN1 and -2 ubiquitination. Mitophagy was induced 23 through mitochondrial depolarisation by treatment with the protonophore CCCP and ubiquitinated 24 MFN proteins were analysed by western blotting. In addition, PINK1 and PRKN mRNA and protein 25 expression levels were characterised with reverse transcriptase quantitative PCR and western 26 blotting, respectively. Whilst CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-27 SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce 28 ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable 29 levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable 30 and PINK1 mRNA levels remarkably low in control PBMCs. Our findings suggest that the PINK1 -31Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable 32 biosample for analysis of mitophagy function in Parkinson disease patients. 33 34