Abstract 110: Role of Beclin1 in Regulating Parkin-mediated Mitophagy

Abstract

Autophagy is a cellular quality control mechanism involved in the selective elimination of damaged organelles and cytotoxic protein aggregates. Beclin1 is a component of the PI3 kinase complex that initiates autophagy in cells. Here, we have investigated the role of Beclin1 in autophagosome formation and Parkin-mediated mitophagy. Using Beclin1-deficient mouse embryonic fibroblasts (MEFs), we found that autophagosomes are still formed in the absence of Beclin1 in response to various treatments known to induce autophagy (FCCP, nutrient deprivation, rapamycin). Autophagic flux is also intact in the Beclin1-/- MEFs, indicating an alternative mechanism of autophagy is induced. Next, we examined whether elimination of mitochondria via autophagy (mitophagy) was intact in Beclin1-/- MEFs. Unexpectedly, we observed that Parkin-mediated mitophagy was significantly reduced in the absence of Beclin1. We confirmed that Parkin was recruited to depolarized mitochondria in both WT and Beclin1-/- MEFs which correlated with increased ubiquitination of mitochondrial proteins. However, high resolution imaging revealed that autophagosomes failed to sequester mitochondria that had been labeled by Parkin. We observed that Parkin was degraded in Beclin1-/- MEF’s, but not WT, after mitochondrial stress. We also found that Beclin1 selectively localized as discrete puncta on Parkin-positive mitochondria suggesting a potential role for Beclin1 in linking mitochondria to autophagosomes. In contrast to our findings in MEFs, characterization of autophagy in mice with cardiac specific deletion of Beclin1 revealed that these hearts had a reduced number of autophagosomes which correlated with a reduction in autophagic flux as indicated by accumulation of cytosolic LC3I and p62. Beclin1-deficient hearts also had significantly reduced Parkin protein levels without changes in mRNA levels, suggesting an impairment in mitophagy. Overall, these findings suggest Beclin1 is required for the proper targeting of mitochondria into autophagosomes and that the adult heart is dependent on Beclin1 to induce formation of autophagosomes.