Porcine Circovirus 2 Induction of ROS Is Responsible for Mitophagy in PK-15 Cells via Activation of Drp1 Phosphorylation.
ABSTRACT: Mitochondrial dynamics is essential for the maintenance of cell homeostasis. Previous studies have shown that porcine circovirus 2 (PCV2) infection decreases the mitochondrial membrane potential and causes the elevation of reactive oxygen species (ROS), which may ultimately lead to mitochondrial apoptosis. However, whether PCV2 induce mitophagy remains unknown. Here we show that PCV2-induced mitophagy in PK-15 cells via Drp1 phosphorylation and PINK1/Parkin activation. PCV2 infection enhanced the phosphorylation of Drp1 and its subsequent translocation to mitochondria. PCV2-induced Drp1 phosphorylation could be suppressed by specific CDK1 inhibitor RO-3306, suggesting CDK1 as its possible upstream molecule. PCV2 infection increased the amount of ROS, up-regulated PINK1 expression, and stimulated recruitment of Parkin to mitochondria. N-acetyl-L-cysteine (NAC) markedly decreased PCV2-induced ROS, down-regulated Drp1 phosphorylation, and lessened PINK1 expression and mitochondrial accumulation of Parkin. Inhibition of Drp1 by mitochondrial division inhibitor-1 Mdivi-1 or RNA silencing not only resulted in the reduction of ROS and PINK1, improved mitochondrial mass and mitochondrial membrane potential, and decreased mitochondrial translocation of Parkin, but also led to reduced apoptotic responses. Together, our study shows that ROS induction due to PCV2 infection is responsible for the activation of Drp1 and the subsequent mitophagic and mitochondrial apoptotic responses.
Project description:Corneal endothelium (CE) is a monolayer of mitochondria-rich cells, critical for maintaining corneal transparency compatible with clear vision. Fuchs endothelial corneal dystrophy (FECD) is a heterogeneous, genetically complex disorder, where oxidative stress plays a key role in the rosette formation during the degenerative loss of CE. Increased mitochondrial fragmentation along with excessive mitophagy activation has been detected in FECD; however, the mechanism of aberrant mitochondrial dynamics in CE cell loss is poorly understood. Here, the role of oxidative stress in mitophagy activation in FECD is investigated. Immunoblotting of FECD ex vivo specimens revealed an accumulation of PINK1 and phospho-Parkin (Ser65) along with loss of total Parkin and total Drp1. Similarly, modeling of rosette formation with menadione (MN), led to phospho-Parkin accumulation in fragmented mitochondria resulting in mitophagy-induced mitochondrial clearance, albeit possibly in a PINK1-independent manner. Loss of PINK1, phospho-Drp1, and total Drp1 was prominent after MN-induced oxidative stress, but not after mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone. Moreover, MN-induced mitophagy led to degradation of Parkin along with sequestration of Drp1 and PINK1 that was rescued by mitophagy inhibition. This study shows that in FECD, intracellular oxidative stress induces Parkin-mediated mitochondrial fragmentation where endogenous Drp1 and PINK1 are sequestered and degraded by mitophagy during degenerative loss of post-mitotic cells of ocular tissue.
Project description:Within the mitochondrial matrix, protein aggregation activates the mitochondrial unfolded protein response and PINK1-Parkin-mediated mitophagy to mitigate proteotoxicity. We explore how autophagy eliminates protein aggregates from within mitochondria and the role of mitochondrial fission in mitophagy. We show that PINK1 recruits Parkin onto mitochondrial subdomains after actinonin-induced mitochondrial proteotoxicity and that PINK1 recruits Parkin proximal to focal misfolded aggregates of the mitochondrial-localized mutant ornithine transcarbamylase (ΔOTC). Parkin colocalizes on polarized mitochondria harboring misfolded proteins in foci with ubiquitin, optineurin, and LC3. Although inhibiting Drp1-mediated mitochondrial fission suppresses the segregation of mitochondrial subdomains containing ΔOTC, it does not decrease the rate of ΔOTC clearance. Instead, loss of Drp1 enhances the recruitment of Parkin to fused mitochondrial networks and the rate of mitophagy as well as decreases the selectivity for ΔOTC during mitophagy. These results are consistent with a new model that, instead of promoting mitophagy, fission protects healthy mitochondrial domains from elimination by unchecked PINK1-Parkin activity.
Project description:Mitophagy plays a key role in cleaning damaged and depolarized mitochondria to maintain cellular homeostasis and viability. Although it was originally found in neurodegenerative diseases, mitophagy is reported to play an important role in acute kidney injury. PINK1 and Parkin are key molecules in mitophagy pathway. Here, we used PINK1 knockout rats to examine the role of PINK1/Parkin-mediated mitophagy in cisplatin nephrotoxicity. After cisplatin treatment, PINK1 knockout rats showed lower plasma creatinine and less tubular damage when compared with wild-type rats. Meanwhile, mitophagy indicated by autophagosome formation and LC3B-II accumulation was also attenuated in PINK1 knockout rats. Renal expression of PINK1 and Parkin were down-regulated while BNIP3L was up-regulated by cisplatin treatment, indicating a major role of BNIP3/BNIP3L pathway in cisplatin-induced mitophagy. Transmission electron microscopy showed that PINK1 deficiency inhibited cisplatin-induced mitochondrial fragmentation indicating an involvement of mitochondrial fusion and fission. Renal expression of mitochondrial dynamics related proteins including Fis1, Drp1, Mfn1, Mfn2, and Opa1 were checked by real-time PCR and western blots. The results showed PINK1 deficiency distinctly prevented cisplatin-induced up-regulation of DRP1. Finally, PINK1 deficiency alleviated cisplatin-induced tubular apoptosis indicated by TUNEL assay as well as the expression of caspase3 and cleaved caspase3. Together, these results suggested PINK1 deficiency ameliorated cisplatin-induced acute kidney injury in rats, possibly via inhibiting DRP1-mediated mitochondrial fission and excessive mitophagy.
Project description:Mitochondrial fission is essential for the degradation of damaged mitochondria. It is currently unknown how the dynamin-related protein 1 (DRP1)-associated fission machinery is selectively targeted to segregate damaged mitochondria. We show that PTEN-induced putative kinase (PINK1) serves as a pro-fission signal, independently of Parkin. Normally, the scaffold protein AKAP1 recruits protein kinase A (PKA) to the outer mitochondrial membrane to phospho-inhibit DRP1. We reveal that after damage, PINK1 triggers PKA displacement from A-kinase anchoring protein 1. By ejecting PKA, PINK1 ensures the requisite fission of damaged mitochondria for organelle degradation. We propose that PINK1 functions as a master mitophagy regulator by activating Parkin and DRP1 in response to damage. We confirm that PINK1 mutations causing Parkinson disease interfere with the orchestration of selective fission and mitophagy by PINK1.
Project description:PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.
Project description:PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.
Project description:Mitophagy is a crucial process in controlling mitochondrial biogenesis. Balancing mitophagy and mitochondrial functions is required for maintaining cellular homeostasis. In this study, we found that Gerontoxanthone I (GeX1) and Macluraxanthone (McX), xanthone derivatives isolated from Garcinia bracteata C. Y. Wu ex Y. H. Li, induced Parkin puncta accumulation and promoted mitophagy. GeX1 and McX treatment induced the degradation of mitophagy-related proteins such as Tom20 and Tim23. GeX1 and McX directly stabilized PTEN-induced putative kinase 1 (PINK1) on the outer membrane of the mitochondria, and then recruited Parkin to mitochondria. This significantly induced phosphorylation and ubiquitination of Parkin, suggesting that GeX1 and McX mediate mitophagy through the PINK1-Parkin pathway. Transfecting ParkinS65A or pretreated MG132 abolished the induction effects of GeX1 and McX on mitophagy. Furthermore, GeX1 and McX treatment decreased cell death and the level of ROS in an ischemia/reperfusion (IR) injury model in H9c2 cells compared to a control group. Taken together, our data suggested that GeX1 and McX induce PINK1-Parkin-mediated mitophagy and attenuate myocardial IR injury in vitro.
Project description:Parkinson's disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (??m), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism - an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ??m. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.
Project description:The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.
Project description:Contrast-induced acute kidney injury (CI-AKI) occurs in more than 30% of patients after intravenous iodinated contrast media and causes serious complications, including renal failure and mortality. Recent research has demonstrated that routine antioxidant and alkaline therapy failed to show benefits in CI-AKI patients with high risk for renal complications. Mitophagy is a mechanism of selective autophagy, which controls mitochondrial quality and mitochondrial reactive oxygen species (ROS) through degradation of damaged mitochondria. The role of mitophagy and its regulation of apoptosis in CI-AKI are poorly understood. In this study, we demonstrated that mitophagy was induced in renal tubular epithelial cells (RTECs) during CI-AKI, both in vivo and in vitro. Meanwhile, contrast media-induced mitophagy was abolished when silencing PINK1 or PARK2 (Parkin), indicating a dominant role of the PINK1-Parkin pathway in mitophagy. Moreover, mitochondrial damage, mitochondrial ROS, RTEC apoptosis, and renal injury under contrast exposure were more severe in PINK1- or PARK2-deficient cells and mice than in wild-type groups. Functionally, PINK1-Parkin-mediated mitophagy prevented RTEC apoptosis and tissue damage in CI-AKI through reducing mitochondrial ROS and subsequent NLRP3 inflammasome activation. These results demonstrated that PINK1-Parkin-mediated mitophagy played a protective role in CI-AKI by reducing NLRP3 inflammasome activation.