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.

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.

Deciphering molecular consequences of the proprotein convertase 1/3 inhibition in macrophages for application in anti-tumour immunotherapy.

During tumour development, macrophages are recruited to the tumour site and orientated towards an anti-inflammatory phenotype. Due to their immunosuppressive function, tumour associated macrophages (TAMs) are recognized as major components in tumour progression. Changing these macrophages to a pro-inflammatory phenotype is thus extensively studied as a potential means for developing novel anti-tumour therapy. In this context, we found that the Proprotein convertase 1/3 (PC1/3) is a relevant target. Proteomic analysis reveals that PC1/3 knockdown (KD) macrophages present all the characteristic of activated pro-inflammatory macrophages. Moreover, in PC1/3 KD macrophages, TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and anti-tumour factors. To develop an efficient anti-tumour immunotherapy, we may (i) target TAMs directly inside the tumour site for PC1/3 inhibition and TLR activation and used them as "Trojan macrophages" or (ii) directly take advantage of PC1/3 inhibited macrophages and use them as "drone macrophages" by activating them "at distance" with a TLR ligand. Therefore, PC1/3 inhibited macrophages constitute an innovative cell therapy to treat tumours efficiently.