Inhibition of apoptotic Bax translocation to the mitochondria is a central function of parkin

Parkinson''s disease (PD) is the second most prevalent neurodegenerative disorder, affecting 1–3% of the population over 65. Mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive PD. The parkin protein possesses potent cell-protective properties and has been mechanistically linked to both the regulation of apoptosis and the turnover of damaged mitochondria. Here, we explored these two functions of parkin and the relative scale of these processes in various cell types. While biochemical analyses and subcellular fractionation were sufficient to observe robust parkin-dependent mitophagy in immortalized cells, higher resolution techniques appear to be required for primary culture systems. These approaches, however, did affirm a critical role for parkin in the regulation of apoptosis in primary cultured neurons and all other cells studied. Our prior work demonstrated that parkin-dependent ubiquitination of endogenous Bax inhibits its mitochondrial translocation and can account for the anti-apoptotic effects of parkin. Having found a central role for parkin in the regulation of apoptosis, we further investigated the parkin-Bax interaction. We observed that the BH3 domain of Bax is critical for its recognition by parkin, and identified two lysines that are crucial for parkin-dependent regulation of Bax translocation. Last, a disease-linked mutation in parkin failed to influence Bax translocation to mitochondria after apoptotic stress. Taken together, our data suggest that regulation of apoptosis by the inhibition of Bax translocation is a prevalent physiological function of parkin regardless of the kind of cell stress, preventing overt cell death and supporting cell viability during mitochondrial injury and repair.Loss-of-function mutations in the ubiquitin E3 ligase parkin are the most common cause of autosomal recessive Parkinson''s disease (PD).¹ Multiple functions have been ascribed to parkin, most notably the inhibition of apoptosis^{2, 3, 4, 5, 6, 7} and the induction of autophagic mitochondrial turnover (mitophagy).^{8, 9} However, the relative scale of these effects mediated by endogenous parkin and whether these processes can occur concomitantly or are mutually exclusive, is not known.Bax is a primary effector of cell death that translocates from the cytosol to the mitochondria upon stress, where it facilitates cytochrome c release and the subsequent caspase cascade.¹⁰ We previously identified Bax as a parkin substrate, and found that the anti-apoptotic effects of parkin can be directly linked to the parkin-dependent ubiquitination of Bax and inhibition of its mitochondrial translocation.³ Recent corroborative evidence showed that primary cultured neurons from parkin knock-out (KO) mice accumulate greater levels of activated Bax at the mitochondria than wild-type (WT) neurons after apoptotic stimulation,¹¹ while a separate report showed the parkin-dependent ubiquitination of Bax during mitophagy.¹²In addition to its anti-apoptotic function, parkin facilitates a depolarization-induced and autophagy-dependent turnover of mitochondria. This process is robustly observed in immortalized cell lines expressing human parkin, where exposure to the mitochondrial depolarizing agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) causes rapid recruitment of parkin from the cytosol to the mitochondrial outer membrane and a coordinated proteasome and autophagosome-mediated turnover of the entire organelle.^{8, 13, 14, 15} Examination of this process in primary neuronal cultures with endogenous parkin expression, however, has been challenging,^{16, 17, 18, 19} and a cooperative role for inhibition of mitochondria-dependent cell death has not been investigated in the context of mitophagy.In this study, we sought further insight into the biological functions of parkin across multiple cell types. Our data showed that whole-cell biochemical techniques were not sufficient to observe the participation of endogenous parkin in mitochondrial turnover but were able to confirm the parkin-dependent regulation of apoptosis. Further examination of the parkin-dependent regulation of apoptosis identified two specific lysines of Bax that are critical for recognition and inhibition of its translocation to the mitochondria by parkin. In addition, the BH3 domain of Bax was critical for its association with parkin. Importantly, we observed parkin-dependent mitophagy and inhibition of apoptotic Bax translocation in the same cell culture systems, suggesting that these two pathways coexist and likely cooperate within neurons. Taken together, our data indicate that the parkin-dependent regulation of Bax is critical for cell survival, irrespective of the nature of cell stress involved.