Mitophagy dysfunction in PBMCs of sporadic ALS patients

Background: The presence of dysfunctional mitochondria is widely accepted as a central hallmark in amyotrophic lateral sclerosis (ALS). Although many questions are still unresolved, mechanistic and genetic evidence strongly support a critical contribution of mitochondria to ALS pathogenesis. Mitochondria alterations can be readily found in tissues from patients and animal models, while no data are reported in peripheral blood mononuclear cells (PBMCs). Objectives: Aim of this work is to study mitochondrial dynamism, caspase-dependent apoptosis and mitophagy in sporadic ALS (sALS) PBMCs used as peripheral cellular model of the disease (Cereda et al, 2013; Bordoni et al., 2019). Methods: We analyzed mitochondria morphology in PBMCs of 32 sALS patients and 32 healthy controls by both Transmission Electron Microscopy (TEM) and MitoTracker® Red CMXRos. We performed cytofluorimetry analysis to evaluate the percentage of damaged mitochondria by staining with Mitotracker and TMRE. We evaluated proteins involved in 32 mitochondrial dynamism and in mitochondria- dependent apoptosis. We evaluated by WB LC3-II/LC3-I ratio and PINK1 protein level for mitophagy. We also analyzed accumulation of damaged mitochondria by the evaluation of localization of PINK1 and LC3 by immunofluorescence microscopy. Finally, we tested the effect of trehalose, which induces autophagy by damaging lysosomes. Results: In patients PBMCs, TEM analysis evidenced the presence of morphologically atypical mitochondria. Moreover, we found an increase in mitochondrial area in sALS PBMCs. We then found that the total number of mitochondria is the same, but in sALS PBMCs the percentage of damaged mitochondria is higher respect to healthy control. We did not find any significant change in proteins involved in mitochondrial dynamism and in caspase-dependent apoptosis We further found the clustering of mitochondria in sALS PBMCs. Thus, we investigated mitophagy pathway, finding significant increase in LC3-II/LC3-I ratio (p<0.05) and in PINK1 levels (p<0.05) in PBMCs of sALS patients. These data suggested us an accumulation of damaged mitochondria, confirmed by the co-localization of PINK1 and LC3 in PBMCs of sALS patients by immunofluorescence microscopy. Finally, treahlose treatment was demonstrated to resolve mitophagy impairment on sALS PBMCs. Discussion: Our data suggest that the presence of morphologically altered mitochondria in PBMCs of sALS patients does not rely on an impairment of mitochondrial dynamism or caspase-dependent apoptosis. On the other hand, we found an involvement of mitophagy pathway. Thus, we hypothesized that this impairment could lead to an accumulation of damaged mitochondria, resulting in an inefficient turnover. Finally, trehalose represents an intriguing approach as potential treatments for ALS.