Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin-decorated mitochondria are selectively recruiting autophagy receptors, which are required to terminate the organelle via autophagy. In this work, we show a previously uncharacterized molecular pathway that correlates the activation of the Ca²⁺-dependent phosphatase Calcineurin to Parkin translocation and Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.

Mitochondrial homeostasis relies on a tight balance between mitochondrial biogenesis and degradation. Although mitophagy is one of the main pathways involved in the clearance of damaged or old mitochondria, its coordination with mitochondrial biogenesis is poorly characterized. Here, by unbiased approaches including last-generation liquid chromatography coupled to mass spectrometry and transcriptomics, we identify the protein phosphatase PP2A-B55α/PPP2R2A as a Parkin-dependent regulator of mitochondrial number. Upon mitochondrial damage, PP2A-B55α determines the amplitude of mitophagy induction and execution by regulating both early and late mitophagy events. A few minutes after the insult, ULK1 is released from the inhibitory regulation of PP2A-B55α, whereas 2 to 4 hours later, PP2A-B55α promotes the nuclear translocation of TFEB, the master regulator of autophagy and lysosome genes, to support mitophagy execution. Moreover, PP2A-B55α controls a transcriptional program of mitochondrial biogenesis by stabilizing the Parkin substrate and PGC-1α inhibitor PARIS. PP2A-B55α targeting rescues neurodegenerative phenotypes in a fly model of Parkinson’s disease, thus suggesting potential therapeutic application.

Stress-induced mitophagy, a tightly regulated process that targets dysfunctional mitochondria for autophagy-dependent degradation, mainly relies on two proteins, PINK1 and Parkin, which genes are mutated in some forms of familiar Parkinson’s Disease (PD). Upon mitochondrial damage, the protein kinase PINK1 accumulates on the organelle surface where it controls the recruitment of the E3-ubiquitin ligase Parkin. On mitochondria, Parkin ubiquitinates a subset of mitochondrial-resident proteins located on the outer mitochondrial membrane, leading to the recruitment of downstream cytosolic autophagic adaptors and subsequent autophagosome formation. Importantly, PINK1/Parkin-independent mitophagy pathways also exist that can be counteracted by specific deubiquitinating enzymes (DUBs). Down-regulation of these specific DUBs can presumably enhance basal mitophagy and be beneficial in models in which the accumulation of defective mitochondria is implicated. Among these DUBs, USP8 is an interesting target because of its role in the endosomal pathway and autophagy and its beneficial effects, when inhibited, in models of neurodegeneration. Based on this, we evaluated autophagy and mitophagy levels when USP8 activity is altered. We used genetic approaches in D. melanogaster to measure autophagy and mitophagy in vivo and complementary in vitro approaches to investigate the molecular pathway that regulates mitophagy via USP8. We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy. These results suggest the existence of a yet uncharacterized mitophagic pathway that is inhibited by USP8.