Abstract 148: Parkin Mediates Clearance of Dysfunctional Mitochondria via Multiple Pathways

Abstract

The ability to clear damaged mitochondria is critical to prevent unnecessary death. Studies have found that dysfunctional mitochondria are rapidly sequestered by autophagosomes and subsequently delivered to lysosomes for degradation. The E3 ubiquitin ligase Parkin has been identified as an important regulator of mitochondrial autophagy. We have previously shown that Parkin plays an important role in clearing dysfunctional mitochondria via autophagy in the heart after myocardial infarction. In this study, we have discovered that Parkin also induces clearance of mitochondria via an autophagy-independent pathway. We found that Parkin mediated clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Interestingly, mitochondrial clearance in both cell types was dependent on the presence of Parkin, suggesting that Parkin represents a rate-limiting step. Immunofluorescence analysis revealed that FCCP-treatment resulted in activation of the Vps34-Rab5 complex with subsequent sequestration of mitochondria inside Rab5-positive endosomes and LAMP2-positive lysosomes in Atg5-/- MEFs. The presence of mitochondria inside endosomes in Atg5-/- MEFs was confirmed by transmission electron microscopy (TEM). Pharmacological inhibition of the endosomal-lysosomal pathway with 3-methyladenine or Bafilomycin A1 caused a significant increase in FCCP-mediated cell death in Atg5-/- MEFs. Also, although BNIP3 functions as an autophagy receptor on mitochondria by interacting with LC3II on the autophagosome, it induced mitochondrial clearance in Atg5-/- MEFs via activation of the endosomal pathway. Finally, we confirmed that mitochondrial clearance occurs via both the autophagy and endosomal pathways in neonatal cardiomyocytes subjected to simulated ischemia/reperfusion (I/R). TEM analysis revealed the presence of mitochondria inside endosomes rat hearts subjected to ex vivo I/R. These data demonstrate that both autophagy and endosomal pathways contribute to clearance of damaged mitochondria in cells, and represent potential future therapeutic targets to enhance or preserve mitochondrial clearance in patients after MI.