The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

Receptor-mediated mitophagy is a crucial process involved in mitochondria quality control. AMBRA1 is a mitophagy receptor for the selective removal of damaged mitochondria in mammalian cells. A critical unresolved issue is how AMBRA1-mediated mitophagy is controlled in response to cellular stress. Here, we investigated the role of BCL2-family proteins on AMBRA1-dependent mitophagy and showed that MCL1 delays AMBRA1-dependent mitophagy. Indeed, MCL1 overexpression is sufficient to inhibit recruitment to mitochondria of the E3 Ubiquitin ligase HUWE1, a crucial dynamic partner of AMBRA1, upon AMBRA1-mediated mitophagy induction. In addition, we found that during mitophagy induced by AMBRA1, MCL1 levels decreased but were sustained by inhibition of the GSK-3β kinase, which delayed AMBRA1-mediated mitophagy. Also, we showed that MCL1 was phosphorylated by GSK-3β at a conserved GSK-3 phosphorylation site (S159) during AMBRA1-mediated mitophagy and that this event was accompanied by HUWE1dependent MCL1 degradation. Altogether, our results demonstrate that MCL1 stability is regulated by the kinase GSK-3β and the E3 ubiquitin ligase HUWE1 in regulating AMBRA1-mediated mitophagy. Our work thus defines MCL1 as an upstream stress-sensitive protein, functional in AMBRA1-mediated mitophagy.

Therapeutic strategies are needed to protect dopaminergic neurons in Parkinson’s disease (PD) patients. Oxidative stress caused by dopamine may play an important role in PD pathogenesis. Selective autophagy of mitochondria (mitophagy), mainly regulated by PINK1 and PARKIN, plays an important role in the maintenance of cell homeostasis. Mutations in those genes cause accumulation of damaged mitochondria, leading to nigral degeneration and early-onset PD. AMBRA1ActA is a fusion protein specifically expressed at the mitochondria, and whose expression has been shown to induce a powerful mitophagy in mammalian cells. Most importantly, the pro-autophagy factor AMBRA1 is sufficient to restore mitophagy in fibroblasts of PD patients carrying PINK1 and PARKIN mutations. In this study, we investigated the potential neuroprotective effect of AMBRA1-induced mitophagy against 6-hydroxydopamine (6-OHDA)- and rotenone-induced cell death in human neuroblastoma SH-SY5Y cells. We demonstrated that AMBRA1ActA overexpression was sufficient to induce mitochondrial clearance in SH-SY5Y cells. We found that apoptosis induced by 6-OHDA and rotenone was reversed by AMBRA1-induced mitophagy. Finally, transfection of SH-SY5Y cells with a vector encoding AMBRA1ActA significantly reduced 6-OHDA and rotenone-induced generation of reactive oxygen species (ROS). Altogether, our results indicate that AMBRA1ActA is able to induce mitophagy in SH-SY5Y cells in order to suppress oxidative stress and apoptosis induced by both 6-OHDA and rotenone. These results strongly suggest that AMBRA1 may have promising neuroprotective properties with an important role in limiting ROS-induced dopaminergic cell death, and the utmost potential to prevent PD or other neurodegenerative diseases associated with mitochondrial oxidative stress.