DIFFERENTIAL MOLECULAR REGULATION OF MITOCHONDRIAL FUSION/FISSION BETWEEN PINK1, PARKIN AND C2-CERAMIDE

Introduction: The role of PINK1 and Parkin in mitophagy is well known, but their role in mitochondrial fusion/fission is controversial [1-3]. Ceramides are toxic lipids that have been associated to mitochondrial alterations, but its role in mitochondrial dynamics has not completely clarify [4, 5] Aim: to analyze mitochondrial dynamics upon silencing of PINK1, Parkin and C2-ceramide treatment. Materials and Methods: CAD cells were silenced for PINK1, Parkin using lentiviral constructs and/or treated with C2-Ceramide (10-25μM) for 6 hours. Mitochondrial morphology was analyzed by confocal microscopy using mitotracker red staining. Protein expression was analyzed by immunoblotting; mitochondrial membrane potential (ΔΨm) analyzed by JC-1 staining and cell viability by flow cytometry. Results: shPINK1, shParkin cells and shcontrol cells treated with C2-ceramide showed fragmented mitochondria. In shPINK1 cells showed mitochondrial fission associated to loss of ΔΨm, DRP1 dephosphorylation, increased OPA1 cleavage and low levels of MNF1; in shParkin cells it was associated to DRP1 dephosphorylation independent of ΔΨm changes; and in C2-ceramide-treated cells it was independent of PINK1/Parkin and associated to increase in FIS1 and OPA1 cleavage, secondary to de-phosphorylation of Akt and ΔΨm loss. Conclusion: mitochondrial fission induced by silencing of PINK1, Parkin and C2-cermide treatment follow different molecular mechanisms.