Abstract 223: Parkin is Critical for the Clearance of Damaged Mitochondria via an Autophagy-independent Pathway

Abstract

Functional mitochondria are essential for highly metabolic organs such as the heart. When mitochondria are damaged they can release pro-death factors and reactive oxygen species which in turn can result in cell death. The E3 ubiquitin ligase Parkin plays an important role in clearing damaged mitochondria via the autophagy pathway to protect cells against unnecessary cell death. Interestingly, we have found that Parkin can mediate clearance of damaged mitochondria via an autophagy-independent pathway. In fact, Parkin promotes clearance of depolarized mitochondria at the same rate in both wild-type (WT) and autophagy deficient Atg5-/- mouse embryonic fibroblasts (MEFs) in response to the mitochondrial uncoupler FCCP. We also found that Parkin-mediated ubiquitination is critical for this process as disease associated mutants of Parkin were incapable of inducing mitochondrial clearance in Atg5-/- MEFs. Upon further investigation, we observed a significant increase in the number of Rab5+ early- and Rab7+ late endosomes in both WT and Atg5-/- MEFs after depolarization of mitochondria with FCCP or valinomycin, indicating activation of the endosomal-lysosomal degradation pathway. We did not observe activation of the endosomal pathway after exposure to actinomycin D, an inhibitor RNA synthesis and activator of apoptosis, confirming that mitochondrial damage specifically activates the endosomal degradation pathway. We also observed activation of the endosomal pathway in neonatal myocytes in response to FCCP treatment or after exposure to simulated ischemia/reperfusion (sI/R). Overexpression of the dominant negative Rab5S34N significantly enhanced sI/R-mediated cell death, suggesting that this is a protective pathway activated by cells in response to stress. Moreover, Beclin1 is well known to regulate activation of autophagy. Here, we found that knockdown of Beclin1 inhibited both the number of Rab5+ early endosomes and their colocalization with mitochondria in response to either FCCP or sI/R in myocytes, suggesting that Beclin1 is a critical upstream regulator of the endosomal degradation pathway. Thus, our data suggest that Parkin mediates clearance of damaged mitochondria via both the autophagy and endosomal pathways in cells.