Trehalose treatment finely tunes the mitophagy pathway in PBMCs of sALS patients

Background. Mitophagy is the pathway responsible of the elimination of damaged mitochondria and its impairment could lead to the accumulation of inefficient mitochondria. Mechanistic and genetic evidences strongly support a critical contribution of mitochondria to ALS pathogenesis, but many questions are still unresolved. Mitochondria alterations can be found in both in ALS animal models and proximal axons of ALS patients, while no data are reported in other patients cells types, like peripheral blood mononuclear cells (PBMCs). Objectives. Aim of this work is to investigate mitophagy in PBMCs of sALS patients and how the pathway can be pharmacologically tuned. Method. Mitochondria morphology and distribution was analyzed in PBMCs of sALS patients and healthy controls by Transmission Electron Microscopy (TEM) and MitoTracker® Red CMXRos. Moreover, we investigated the percentage of healthy mitochondria by cytofluorimetric analysis using Mitotracker and TMRE. Then, mitophagy was also studied by both WB and immunofluorescence analysis. Finally, we tuned mitophagy pathway by treating cells with NH4Cl, rapamycin, and trehalose followed by immunofluorescence and WB analysis. Results. In patients PBMCs, TEM analysis evidenced the presence of morphologically atypical mitochondria, with the presence of an increased number of vacuoles and damaged cristae. We confirmed abnormalities using also MitoTracker. Moreover, we found a decreased number of healthy mitochondria in sALS PBMCs. By WB and immunofluorescence, we found an impairment of mitophagy caused by possible lysosomes dysfunction. After NH4Cl treatment we found a lower increase of LC3 marker in sALS PBMCs, while after rapamycin treatment we found a higher increase of LC3 marker in sALS PBMCs. Finally, after trehalose treatment, we found no modulation of lysosomes level, on the contrary we found a significant decrease in lysosomes levels in sALS PBMCs. Conclusions. In conclusion, our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients relies on an impairment of the mitophagy pathway. In particular, the induction of mTOR-independent autophagy pathway, caused by trehalose treatment, leads to a decrease of lysosomes levels, suggesting a more sensitivity of sALS PBMCs to the effect of trehalose. Such evidences suggest that trehalose can exert protective effects in sALS, representing a potential therapeutic strategy.