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 pan-caspase 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.
Highlights
Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and PARKIN are causal genes for autosomal recessive early-onset parkinsonism [1]
The mitochondrial permeability transition (MPT) inhibitors, cyclosporine A and bongkrekic acid, hardly blocked carbonyl cyanide m-chlorophenylhydrazine (CCCP)-induced cell death in HA-PARKIN-expressing cells (Fig. 3J). These results indicated that MPT was not required for PARKIN-mediated cell death, ROS generation transiently occurred in response to mitochondrial depolarization
In HA-PARKIN-expressing cells, PINK1 knockdown significantly suppressed CCCP-induced cell death in the presence of Z-VAD-fmk, whereas PINK1 silencing had no effect on the cell death without the pan-caspase inhibitor (Fig. 4B, right panel) The results indicated that PARKIN-mediated and caspase-insensitive cell death required PINK1, PINK1 down-regulation induced another type of cell death that was not mediated by PARKIN
Summary
PARKIN-dependent Cell Death in Response to Mitochondrial Depolarization—In normal culture conditions with a high glucose concentration, HeLa cell morphology is not significantly altered by treatment with the protonophore CCCP, because cancer cells mainly utilize glycolysis to produce ATP [14]. The MPT inhibitors, cyclosporine A and bongkrekic acid, hardly blocked CCCP-induced cell death in HA-PARKIN-expressing cells (Fig. 3J) These results indicated that MPT was not required for PARKIN-mediated cell death, ROS generation transiently occurred in response to mitochondrial depolarization. Addition of the inhibitors significantly reduced the number of PI-stained cells with PINK1(⌬N34) (Fig. 6A), indicating that impairment of proteasomal activity blocked the PINK1-CA-induced cell death. By vital staining using another membrane-impermeable dye, Zombie Green, proteasome inhibitor-dependent suppression of PINK1-CA-induced cell death was confirmed in PARKIN-expressing HeLa cells (Fig. 6C). This fluorescent dye is amine-reactive and has a different property from PI, which preferentially binds to DNA. Taken together with the requirement for proteasomal activity, these findings suggest that PINK1-CA-induced cell death requires the degradation of mitochondrial outer membrane proteins by proteasomes but not the elimination of mitochondria by autophagy
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