C-terminally truncation is a prominent post-translational modification of human erythrocyte α-synuclein.

α-Synuclein is a protein related to synucleinopathies with high expression in the central nervous system and erythrocytes which are a major source of peripheral α-synuclein. Recent reports have suggested the presence of α-synuclein within extracellular vesicles derived from erythrocytes, potentially contributing to the pathogenesis of synucleinopathies. While Lewy bodies, intracellular inclusions containing aggregated α-synuclein, are prominently observed within the brain, their occurrence in peripheral neurons implies the dissemination of synucleinopathy pathology throughout the body via the propagation of α-synuclein. In this study, we found erythrocytes and circulating extracellular vesicles obtained from plasma contained α-synuclein, which was separated into four major forms using high-resolution clear native-PAGE and isoelectric focusing. Notably, erythrocyte α-synuclein was classified into full-length and C-terminal truncated forms, with truncation observed between Y133 and Q134 as determined by LC-MS/MS analysis. Our finding revealed that C-terminally truncated α-synuclein, which was previously reported to exist solely within the brain, was also present in erythrocytes and circulating extracellular vesicles obtained from plasma.

Supercomplex formation of mitochondrial respiratory chain complexes in leukocytes from patients with neurodegenerative diseases.

With population aging, cognitive impairments and movement disorders due to neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are increasingly considered as key social issues. Clinically, it has remained challenging to diagnose them before the onset of symptoms because of difficulty to observe the progressive loss of neurons in the brain. Therefore, with exploratory research into biomarkers, a number of candidates have previously been proposed, such as activities of mitochondrial respiratory chain complexes in blood in AD and PD. In this study, we focused on the formation of mitochondrial respiratory chain supercomplexes (SCs) because the formation of SC itself modulates the activity of each complex. Here we investigated the SC formation in leukocytes from patients with AD, PD and DLB. Our results showed that SCs were well formed in AD and PD compared with controls, while poorly formed in DLB. We highlighted that the disruption of the SC formation correlated with the progression of PD and DLB. Taking our findings together, we propose that pronounced SC formation would already have occurred before the onset of AD, PD and DLB and, with the progression of neurodegeneration, the SC formation would gradually be disrupted.

Post-translational modification of lysine residues in erythrocyte α-synuclein.

α-Synuclein is a protein linked to various synuclein-associated diseases ('synucleinopathies'), including Parkinson's disease, dementia with Lewy Bodies and multiple system atrophy, and is highly expressed in the central nervous system and in erythrocytes. Moreover, α-synuclein-containing erythrocyte-derived extracellular vesicles may be involved in the pathogenesis of synucleinopathies and their progression across the blood-brain barrier. Several post-translational modifications of α-synuclein have been reported in brain inclusions, including S129 phosphorylation, but fewer have been found in erythrocytes. In this study, we analysed the post-translational modifications of erythrocyte α-synuclein using liquid chromatography-mass spectrometry. We found that all lysine residues in the α-synuclein protein could be modified by acetylation, glycation, ubiquitination or SUMOylation but that phosphorylation, nitration and acylation were uncommon minor post-translational modifications in erythrocytes. Since the post-translational modification of lysine residues has been implicated in both membrane association and protein clearance, our findings provide new insight into how synucleinopathies may progress and suggest possible therapeutic strategies designed to target α-synuclein.

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.

The in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.