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Journal of Experimental & Clinical... Feb 2022Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is...
BACKGROUND
Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the mechanism underling Drp1 to influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC).
METHODS
Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by fluorescent and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP levels. Xenograft tumor model was performed in BALB/c nude mice to analyze the role of Drp1 on ESCC progression.
RESULTS
We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol thereby inducing cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth.
CONCLUSIONS
Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mtDNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.
Topics: Animals; Autophagy; Cell Line, Tumor; Cell Proliferation; DNA, Mitochondrial; Disease Progression; Dynamins; Female; Humans; Mice; Mice, Knockout; Mice, Nude; Nucleotidyltransferases
PubMed: 35209954
DOI: 10.1186/s13046-022-02262-z -
Molecular Cell Jun 2023Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow...
Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.
Topics: Animals; Mitophagy; Autophagy; Mitochondrial Dynamics; Mitochondrial Proteins; Mitochondria; Dynamins; Lipids; Mammals
PubMed: 37192628
DOI: 10.1016/j.molcel.2023.04.022 -
ALKBH5 attenuates mitochondrial fission and ameliorates liver fibrosis by reducing Drp1 methylation.Pharmacological Research Jan 2023Mitochondrial metabolism plays a pivotal role in various cellular processes and fibrosis. However, the mechanism underlying mitochondrial metabolic function and liver...
Mitochondrial metabolism plays a pivotal role in various cellular processes and fibrosis. However, the mechanism underlying mitochondrial metabolic function and liver fibrosis remains poorly understood. In this study, we determined whether mitochondrial metabolism mediates liver fibrosis using cells, animal models, and clinical samples to elucidate the potential effects and underlying mechanism of mitochondrial metabolism in liver fibrosis. We report that AlkB Homolog 5 (ALKBH5) decreases mitochondrial membrane potential (MMP) and oxygen consumption rate (OCR), suppresses mitochondrial fission and hepatic stellate cell (HSC) proliferation and migration and ameliorates liver fibrosis. Enhancement of mitochondrial fission, an essential event during HSC proliferation and migration, is dependent on decreased ALKBH5 expression. Furthermore, we reveal that low ALKBH5 expression is associated with elevated N6-methyladenosine (mA) mRNA levels. Mechanistically, ALKBH5 mediates mA demethylation in the 3'UTR of Drp1 mRNA and induces its translation in a YTH domain family proteins 1 (YTHDF1)-independent manner. Subsequently, in transforming growth factor-β1 (TGF-β1) induced HSC, Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and increases cell proliferation and migration. Decreased Drp1 expression inhibits mitochondrial fission and suppresses HSC proliferation and migration. Notably, human fibrotic liver and heart tissue exhibited enhanced mitochondrial fission; increased YTHDF1, Drp1, alpha-smooth muscle actin (α-SMA) and collagen I expression; decreased ALKBH5 expression and increased liver fibrosis. Our results highlight a novel mechanism by which ALKBH5 suppresses mitochondrial fission and HSC proliferation and migration by reducing Drp1 methylation in an mA-YTHDF1-dependent manner, which may indicate a demethylation-based approach for liver fibrosis diagnosis and therapy.
Topics: Animals; Humans; Methylation; Mitochondrial Dynamics; Liver Cirrhosis; Dynamins; RNA, Messenger; Hepatic Stellate Cells; AlkB Homolog 5, RNA Demethylase
PubMed: 36566000
DOI: 10.1016/j.phrs.2022.106608 -
Cell Reports Aug 2023Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and...
Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.
Topics: GTP Phosphohydrolases; Phosphoric Monoester Hydrolases; Phosphoglycerate Mutase; Mitochondrial Dynamics; Mitochondrial Proteins; Dynamins
PubMed: 37498743
DOI: 10.1016/j.celrep.2023.112895 -
The EMBO Journal Apr 2022The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and...
The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.
Topics: Apoptosis; Dynamins; Mitochondria; bcl-2-Associated X Protein
PubMed: 35023587
DOI: 10.15252/embj.2021108587 -
Cell Death & Disease Mar 2023The highly widespread and infiltrative nature of glioblastoma multiforme (GBM) makes complete surgical resection hard, causing high recurrence rate and poor patients'...
The highly widespread and infiltrative nature of glioblastoma multiforme (GBM) makes complete surgical resection hard, causing high recurrence rate and poor patients' prognosis. However, the mechanism underlying GBM migration and invasion is still unclear. In this study, we investigated the role of a Ras-related protein Rab32 on GBM and uncovered its underlying molecular and subcellular mechanisms that contributed to GBM aggressiveness. The correlation of Rab32 expression with patient prognosis and tumor grade was investigated by public dataset analysis and clinical specimen validation. The effect of Rab32 on migration and invasion of GBM had been evaluated using wound healing assay, cell invasion assay, as well as protein analysis upon Rab32 manipulations. Mitochondrial dynamics of cells upon Rab32 alterations were detected by immunofluorescence staining and western blotting. Both the subcutaneous and intracranial xenograft tumor model were utilized to evaluate the effect of Rab32 on GBM in vivo. The expression level of Rab32 is significantly elevated in the GBM, especially in the most malignant mesenchymal subtype, and is positively correlated with tumor pathological grade and poor prognosis. Knockdown of Rab32 attenuated the capability of GBM's migration and invasion. It also suppressed the expression levels of invasion-related proteins (MMP2 and MMP9) as well as mesenchymal transition markers (N-cadherin, vimentin). Interestingly, Rab32 transported Drp1 to mitochondrial from the cytoplasm and modulated mitochondrial fission in an ERK signaling-dependent manner. Furthermore, silencing of Rab32 in vivo suppressed tumor malignancy via ERK/Drp1 axis. Rab32 regulates ERK/Drp1-dependent mitochondrial fission and causes mesenchymal transition, promoting migration and invasion of GBM. It serves as a novel therapeutic target for GBM, especially for the most malignant mesenchymal subtype. Schematic of Rab32 promotes GBM aggressiveness via regulation of ERK/Drp1-mediated mitochondrial fission. Rab32 transports Drp1 from the cytoplasm to the mitochondria and recruits ERK to activate the ser616 site of Drp1, which in turn mediates mitochondrial fission and promotes mesenchymal transition, migration and invasion of GBM.
Topics: Humans; Glioblastoma; Mitochondrial Dynamics; Signal Transduction; Mitochondria; Cytoplasm; Brain Neoplasms; Cell Line, Tumor; Dynamins; rab GTP-Binding Proteins
PubMed: 36922509
DOI: 10.1038/s41419-023-05721-3 -
Redox Biology Oct 2020Macrophage recruitment and pro-inflammatory differentiation are hallmarks of various diseases, including infection and sepsis. Although studies suggest that mitochondria...
Macrophage recruitment and pro-inflammatory differentiation are hallmarks of various diseases, including infection and sepsis. Although studies suggest that mitochondria may regulate macrophage immune responses, it remains unclear whether mitochondrial mass affects macrophage pro-inflammatory differentiation. Here, we found that lipopolysaccharide (LPS)-activated macrophages possess higher mitochondrial mass than resting cells. Therefore, this study aimed to explore the functional role and molecular mechanisms of increased mitochondrial mass in pro-inflammatory differentiated macrophages. Results show that an increase in the mitochondrial mass of macrophages positively correlates with inflammatory cytokine generation in response to LPS. RNA-seq analysis revealed that LPS promotes signal transducers and activators of transcription 2 (Stat2) and dynamin-related protein 1 (Drp1) expression, which are enriched in positive mitochondrial fission regulation. Meanwhile, knockdown or pharmacological inhibition of Drp1 blunts LPS-induced mitochondrial mass increase and pro-inflammatory differentiation. Moreover, Stat2 boosts Drp1 phosphorylation at serine 616, required for Drp1-mediated mitochondrial fission. LPS also causes Stat2-and Drp1-dependent biogenesis, which contributes to the generation of additional mitochondria. However, these mitochondria are profoundly remodeled, displaying fragmented morphology, loose cristae, reduced Δψm, and metabolic programming. Furthermore, these remodeled mitochondria shift their function from ATP synthesis to reactive oxygen species (ROS) production, which drives NFκB-dependent inflammatory cytokine transcription. Interestingly, an increase in mitochondrial mass with constitutively active phosphomimetic mutant of Drp1 (Drp1) boosted pro-inflammatory response in macrophages without LPS stimulation. In vivo, we also demonstrated that Mdivi-1 administration inhibits LPS-induced macrophage pro-inflammatory differentiation. Importantly, we observed Stat2 phosphorylation and Drp1-dependent mitochondrial mass increase in macrophages isolated from LPS-challenged mice. In conclusion, we comprehensively demonstrate that a Stat2-Drp1 dependent mitochondrial mass increase is necessary for pro-inflammatory differentiation of macrophages. Therefore, targeting the Stat2-Drp1 axis may provide novel therapeutic approaches for treating infection and inflammatory diseases.
Topics: Animals; Cell Differentiation; Dynamins; Macrophages; Mice; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; STAT2 Transcription Factor
PubMed: 33080440
DOI: 10.1016/j.redox.2020.101761 -
Free Radical Biology & Medicine Jan 2022Neuroinflammation following peripheral surgery is a pivotal pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the key site of...
Neuroinflammation following peripheral surgery is a pivotal pathogenic mechanism of postoperative cognitive dysfunction (POCD). However, the key site of inflammation-mediated neural damage remains unclear. Impaired mitochondrial function is a vital feature of degenerated neurons. Dynamin-related protein 1 (DRP1), a crucial regulator of mitochondrial dynamics, has been shown to play an essential role in synapse formation. Here, we designed experiments to assess whether Drp1-regulated mitochondrial dynamics and function are involved in the pathological processes of POCD and elucidate its relationship with neuroinflammation. Aged mice were subjected to experimental laparotomy under isoflurane anesthesia. Primary neurons and SH-SY5Y cells were exposed to tumor necrosis factor (TNF). We found an increase in Drp1 activation as well as mitochondrial fragmentation both in the hippocampus of mice after surgery and primary neurons after TNF exposure. Pretreatment with Mdivi-1, a Drp1 specific inhibitor, reduced this mitochondrial fragmentation. Drp1 knockdown with small interfering RNA blocked TNF-induced mitochondrial fragmentation in SH-SY5Y cells. However, the application of Mdivi-1 exhibited a negative impact on mitochondrial function and neurite growth in primary neurons. Calcineurin activity was increased in primary neurons after TNF exposure and contributed to the Drp1 activation. The calcineurin inhibitor FK506 exhibited a Drp1-independent function that mitigated mitochondrial dysfunction. Finally, we found that FK506 pretreatment ameliorated the neurite growth in neurons treated with TNF and the learning ability of mice after surgery. Overall, our research indicated a crucial role of mitochondrial function in the pathological processes of POCD, and neuronal metabolic modulation may represent a novel and important target for POCD.
Topics: Animals; Dynamins; Hippocampus; Mice; Mitochondria; Mitochondrial Dynamics; Neuroinflammatory Diseases; Postoperative Cognitive Complications
PubMed: 34875338
DOI: 10.1016/j.freeradbiomed.2021.12.004 -
Redox Biology Jul 2023Silver nanoparticles (AgNPs) have widely used in industrial and medical applications for their excellent antibacterial activities. AgNPs can penetrate into the brain and...
Silver nanoparticles (AgNPs) have widely used in industrial and medical applications for their excellent antibacterial activities. AgNPs can penetrate into the brain and cause neuronal death, but limited evidence focused on toxic effects and mechanic study in hippocampal neuron. This study aimed to investigate the molecular mechanisms of mitochondrial damage and apoptosis in mouse hippocampal HT22 cells and further to explore role of reactive oxygen species (ROS) and GTPase dynamin-related protein 1 (Drp1) in AgNPs-induced neurotoxicity. Our results showed that acute exposure to AgNPs at low doses (2-8 μg/mL) increased ROS generation, decreased mitochondrial membrane potential (MMP) and ATP synthesis in HT22 cells. In addition, AgNPs promoted mitochondrial fragmentation and mitochondria-dependent apoptosis via excessive mitochondrial fission/fusion by 8 μg/mL AgNPs treatment for 24 h. The mechanism was involved in increased protein expression of Drp1, mitochondrial fission protein 1 (Fis1), mitofusin 1/2 (Mfn1/2) and inhibited optic atrophy 1 (OPA1), and mainly mediated by phosphorylation of Drp1 Ser616. The AgNPs-induced mitochondrial impairment and apoptosis was mainly due to their particle-specific effect rather than silver ions release. Furthermore Drp1-mediated mitochondrial fission contributed to mitochondria-dependent apoptosis induced by AgNPs, all aforementioned changes were significantly rescued by N-acetyl-l-cysteine (NAC) and Mdivi-1 except for OPA1 protein expression. Hence, our results provide a novel neurotoxic mechanism to AgNPs-induced neurotoxicity and revealed that the mechanism of mitochondria-dependent apoptosis in HT22 cells was mediated by excessive activation of ROS-Drp1-mitochondrial fission axis. These findings can deepen current evidences on neurotoxicological evaluation of AgNPs and aid in guiding their proper applications in different areas, especially in biomedical use.
Topics: Mice; Animals; Reactive Oxygen Species; Silver; Metal Nanoparticles; Dynamins; Apoptosis; Mitochondria; Hippocampus; Mitochondrial Dynamics
PubMed: 37187014
DOI: 10.1016/j.redox.2023.102739 -
Autophagy Nov 2019The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an system in which mitophagy is slowed...
The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an system in which mitophagy is slowed by inhibiting mitochondrial division through knockout of , a dynamin related GTPase that mediates mitochondrial division. Using this system, we revealed that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/parkin, during mitophagy. Here, we tested the role of PINK1, a mitochondrial protein kinase that activates mitophagy by phosphorylating ubiquitin, in mitochondrial ubiquitination by knocking out in -knockout liver. We found mitochondrial ubiquitination did not decrease in the absence of PINK1; instead, PINK1 was required for the degradation of MFN1 (mitofusin 1) and MFN2, two homologous outer membrane proteins that mediate mitochondrial fusion in -knockout hepatocytes. These data suggest that mitochondrial ubiquitination is promoted by SQSTM1 independently of PINK1 and PRKN during mitophagy. PINK1 and PRKN appear to control the balance between mitochondrial division and fusion . DNM1L/DRP1: dynamin 1-like; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN1/2: mitofusin 1/2; OPA1: OPA1, mitochondrial dynamin like GTPase; PDH: pyruvate dehydrogenase E1; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase.
Topics: Animals; Dynamins; GTP Phosphohydrolases; Hepatocytes; Mice; Mitochondria; Mitochondrial Dynamics; Mitophagy; Protein Kinases; Sequestosome-1 Protein; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31339428
DOI: 10.1080/15548627.2019.1643185