Low-dose hexavalent chromium induces mitophagy in rat liver via the AMPK-related PINK1/Parkin signaling pathway
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ABSTRACT:
SUBMITTER: Li N
PROVIDER: S-EPMC11296300 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:MitoNEET, a mitochondrial outer membrane protein containing the Asn-Glu-Glu-Thr (NEET) sequence, controls the formation of intermitochondrial junctions and confers autophagy resistance. Moreover, mitoNEET as a mitochondrial substrate undergoes ubiquitination by activated Parkin during the initiation of mitophagy. Therefore, mitoNEET is linked to the regulation of autophagy and mitophagy. Mitophagy is the selective removal of the damaged or unnecessary mitochondria, which is crucial to sustaining mitochondrial quality control. In numerous human diseases, the accumulation of damaged mitochondria by impaired mitophagy has been observed. However, the therapeutic strategy targeting of mitoNEET as a mitophagy-enhancing mediator requires further research. Herein, we confirmed that mitophagy is indeed activated by mitoNEET inhibition. CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which leads to mitochondrial depolarization, induces mitochondrial dysfunction and superoxide production. This, in turn, contributes to the induction of mitophagy; mitoNEET protein levels were initially increased before an increase in LC3-Ⅱ protein following CCCP treatment. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) promoted accumulation of Pink1 and Parkin, which are mitophagy-associated proteins, and activation of mitochondria-lysosome crosstalk, in comparison to CCCP alone. Inhibition of mitoNEET using NL-1, or mitoNEET shRNA transfected into RAW264.7 cells, abrogated CCCP-induced ROS and mitochondrial cell death; additionally, it activated the expression of PGC-1α and SOD2, regulators of oxidative metabolism. In particular, the increase in PGC-1α, which is a major regulator of mitochondrial biogenesis, promotes mitochondrial quality control. These results indicated that mitoNEET is a potential therapeutic target in numerous human diseases to enhance mitophagy and protect cells by maintaining a network of healthy mitochondria. [BMB Reports 2022; 55(7): 354-359].
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Project description:Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
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Project description:Gastric cancer (GC) is one of the most common tumors worldwide, and cisplatin is a standard chemotherapeutic reagent for GC treatment. However, chemoresistance is an inherent challenge which limits its application and effectiveness in clinic. This study aims to investigate the mechanism of metformin-induced cisplatin resistance in GC. Intriguingly, the upregulation of mitophagy markers, mitochondrial fission, autophagy and mitophagosome were observed in SGC-7901/DDP cells compared to those in the SGC-7901 cells. Treatment with metformin significantly increased mitochondrial fission and mitophagy in both AGS and SGC-7901 cells, resulting in decreased ATP production, which unexpectedly protected GC cells against the cytotoxicity of cisplatin. In contrast, application of Chloroquine and 3-methyladenine, two inhibitors of autophagy, significantly alleviated the protective effect of metformin on SGC-7901 and AGS cells against cytotoxicity of cisplatin. Moreover, metformin also stimulated the phosphorylation of AMPK (Thr172) and increased the expression of mitophagy markers including Parkin and PINK1 in the AMPK signaling-dependent manner. Consistently, the cell viability and cell apoptosis assay showed that metformin-induced cisplatin resistance was prevented by knockdown of AMPKα1. Taken together, all data in this study indicate that metformin induced AMPK activation and PINK1/Parkin dependent mitophagy, which may contribute to the progression of cisplatin resistance in GC.
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Project description:NaAsO2-induced liver damage leads to autophagy, which plays an important role in cell quality control. Mitophagy plays an important role in hepatocyte damage, and PINK1 and Parkin constitute an important pathway in mitophagy. PINK1 selectively degrades abnormal mitochondria, and Parkin can recognize damaged mitochondria. However, the mechanism underlying the involvement of PINK1/Parkin in NaAsO2-induced mitophagy is unclear. Transfection plasmids containing dsRNA were used to interfere with the expression of Parkin in the following groups: the empty plasmid group was established by add the empty plasmid only, the PINK1-knockdown (KD) group was established by adding 5 μg of PINK1 dsRNA and then by adding 10 mM NaAsO2, and the Parkin-KD group was established by adding 5 μg of Parkin dsRNA and then by adding 10 mM NaAsO2. The expression of PINK1 and Parkin in autophagy was detected by western blotting and immunofluorescence staining. The ultrastructures of autophagosomes and mitochondria were observed by transmission electron microscopy. The successful KD of PINK1 and Parkin aggravated the NaAsO2-induced damage to mitophagy. The degeneration of mitochondrial vacuoles and the appearance of autophagosomes were detected in the NaAsO2, NaAsO2 + PINK1-KD and NaAsO2 + Parkin-KD groups. NaAsO2 can induce mitophagy in rat hepatocytes, and the silencing of PINK1 and Parkin can aggravate mitochondrial damage during this process. This study explored the mechanism of NaAsO2-induced mitophagy in BRL-3A cells after PINK1 and Parkin gene silencing.
| S-EPMC8882795 | biostudies-literature
Project description:Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.
| S-EPMC5739729 | biostudies-literature
Project description:Dysregulation of the PINK1/Parkin-mediated mitophagy is essential to Parkinson's disease. Although important progress has been made in previous researches, the biochemical reagents that induce global and significant mitochondrial damage may still hinder deeper insights into the mechanisms of mitophagy. The origin of PINK1/Parkin pathway activation in mitophagy remains elusive. In this study, we develop an optical method, ultra-precise laser stimulation (UPLaS) that delivers a precise and noninvasive stimulation onto a submicron region in a single mitochondrial tubular structure. UPLaS excites localized mitochondrial Ca2+ (mitoCa2+) oscillations with tiny perturbation to mitochondrial membrane potential (MMP) or mitochondrial reactive oxygen species. The UPLaS-induced mitoCa2+ oscillations can directly induce PINK1 accumulation and Parkin recruitment on mitochondria. The Parkin recruitment by UPLaS requires PINK1. Our results provide a precise and noninvasive technology for research on mitophagy, which stimulates target mitochondria with little damage, and reveal mitoCa2+ oscillation directly initiates the PINK1-Parkin pathway for mitophagy without MMP depolarization.
| S-EPMC8214625 | biostudies-literature
Project description:In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation-induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.
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Project description:PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson's disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.
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Project description:Hexavalent chromium, Cr(VI), is an environmental toxicant and is associated with hepatotoxicity. However, very little is known about the intracellular antioxidant defense mechanism against Cr(VI)-induced cytotoxicity in hepatocytes. In the present study, we cultured human liver (HepG2) cells in the absence or presence of Cr(VI) and determined its effect on cellular oxidative stress, mitochondrial damage, apoptosis and the expression of the transcription factor Nrf2 and the Nrf2-dependent antioxidant enzymes. Cr(VI) intoxication at a dose of 0, 3.125, 6.25, 12.5, 25, or 50 μM for 24 h exhibited a dose dependent cytotoxic effects in hepatocytes. Besides, Cr(VI) induced oxidative stress and subsequent mitochondrial damage. Cr(VI) also induced caspase 3-dependent apoptosis in HepG2 cells. In addition, Cr(VI) induced the translocation of Nrf2 into the nucleus and up-regulated the expression of Nrf2-dependent antioxidant enzymes, including SOD2, GCLC, and HO1. Our present experimental data support the notion that Cr(VI) caused mitochondrial damage, apoptosis, oxidative stress, and subsequently lead to a strong induction of HO1, GCLC and SOD2 via the Nrf-2 signaling pathway in hepatocytes.
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Project description:Mitophagy is a selective form of autophagy involving the removal of damaged mitochondria via the autophagy-lysosome pathway. PINK1-Parkin-mediated mitophagy is one of the most important mechanisms in cardiovascular disease, cerebral ischemia-reperfusion (I/R) injury, and neurodegenerative diseases. In this study we conducted an image-based screening in YFP-Parkin HeLa cells to discover new mitophagy regulators from natural xanthone compounds. We found that garciesculenxanthone B (GeB), a new xanthone compound from Garcinia esculenta, induced the formation of YFP-Parkin puncta, a well known mitophagy marker. Furthermore, treatment with GeB dose-dependently promoted the degradation of mitochondrial proteins Tom20, Tim23, and MFN1 in YFP-Parkin HeLa cells and SH-SY5Y cells. We revealed that GeB stabilized PINK1 and triggered Parkin translocation to the impaired mitochondria to induce mitophagy, and these effects were abolished by knockdown of PINK1. Finally, in vivo experiments demonstrated that GeB partially rescued ischemia-reperfusion-induced brain injury in mice. Taken together, our findings demonstrate that the natural compound GeB can promote the PINK1-Parkin-mediated mitophagy pathway, which may be implicated in protection against I/R brain injury.
| S-EPMC8026581 | biostudies-literature