Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy

Abstract

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pancaspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy.Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and PARKIN are causal genes for autosomal recessive early-onset parkinsonism (1). PINK1 is a unique Ser/Thr kinase localized on the outer membrane of damaged mitochondria, where it is subsequently autophosphorylated, followed by the formation of a larger protein complex that contains a translocase of the outer membrane (TOM) 4 complex (2-4). PINK1localized on damaged mitochondria selectively recruits PAR-KIN (5, 6), and phosphorylates PARKIN to uncover latent ligase activity (7). PINK1 and phosphorylated PARKIN share a cooperative role to modify mitochondrial outer membrane proteins with phospho-ubiquitin chains, and mitochondria decorated by poly-ubiquitin chains are eliminated by selective mitochondrial autophagy (1), thereby maintaining mitochondrial quality. Programmed cell death serves fundamental functions in tissue development and homeostasis and is associated with several human pathologies, including neurodegradation, autoimmune diseases, and cancer (8). Apoptosis, the best studied form of programmed cell death, is characterized by cell shrinkage, blebbing, nuclear fragmentation, and chromatin condensation, and it requires caspase activation (9). Many studies have revealed caspase-independent but genetically regulated forms of cell death that are classified according to their distinct morphologic features and specific inhibitors (10). PARKIN prevents cells from dying in response to proapoptotic stimuli (11,12). The cytoprotective effects of PARKIN are relatively accepted because loss of PARKIN function leads to pro...

PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control

“…In conclusion, consistent with the previous study, PINK1/Parkin induced cell death independently of mitochondrial autophagy. 41 The molecular mechanisms of the PINK1/Parkin-induced cell death need to be elucidated in future studies.…”

Altered expression of IL‐18 binding protein and IL‐18 receptor in basophils and mast cells of asthma patients

“…To detect the expression of IL‐18, IL‐18BP and IL‐18R in sputum mast cells isolated from patients with asthma, dispersed cells were incubated with human Fc receptor blocking solution and a live/dead cell dye (Zombie Green™ Fixable Viability Kit) for 15 minutes before the addition of PE/Cy7‐conjugated anti‐human CD34, PerCP/Cy5.5‐conjugated anti‐human FcεRIα, BV421‐conjugated mouse anti‐human CD117 and APC‐conjugated anti‐human IL‐18Rα antibodies to the tube. Cells were then permeabilized, processed following human blood sample protocols and analysed by flow cytometry.…”

Control of mitochondrial biogenesis and function by the ubiquitin–proteasome system

“…Notably, this cell death pathway requires proteasome activity [91]. This may indicate sensing a rupture of the OM that results from extensive proteasomal digestion [66].…”