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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 -
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 -
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 -
Metabolism: Clinical and Experimental Sep 2022Mitochondria are essential for myocardial ischemia/reperfusion (I/R) injury. TBC domain family member 15 (TBC1D15) participates in the regulation of mitochondrial...
OBJECTIVE
Mitochondria are essential for myocardial ischemia/reperfusion (I/R) injury. TBC domain family member 15 (TBC1D15) participates in the regulation of mitochondrial homeostasis although its role remains elusive in I/R injury.
METHODS AND MATERIALS
This study examined the role of TBC1D15 in mitochondrial homeostasis under myocardial I/R injury using inducible cardiac-specific TBC1D15 knockin (TBC1D15) and knockout (TBC1D15) mice.
RESULTS
TBC1D15 mRNA/protein levels were downregulated in human ischemic cardiomyopathy samples, mouse I/R hearts and neonatal mouse cardiomyocytes with H/R injury, consistent with scRNA sequencing finding from patients with coronary heart disease. Cardiac-specific knockin of TBC1D15 attenuated whereas cardiac-specific knockout of TBC1D15 overtly aggravated I/R-induced cardiomyocyte apoptosis and cardiac dysfunction. TBC1D15 mice exhibited reduced mitochondrial damage and mitochondrial fragmentation following myocardial I/R injury, while TBC1D15 mice displayed opposite results. TBC1D15 preserved mitochondrial function evidenced by safeguarding MMP and oxygen consumption capacity, antagonizing ROS accumulation and cytochrome C release, which were nullified by TBC1D15 knockdown. Time-lapse confocal microscopy revealed that TBC1D15 activated asymmetrical mitochondrial fission through promoting mitochondria-lysosome contacts untethering in NMCMs under H/R injury, whereas overexpression of TBC1D15 mutants (R400K and ∆231-240) failed to regulate asymmetrical fission and knockdown of TBC1D15 slowed down asymmetrical fission. Moreover, TBC1D15-offered benefits were mitigated by knockdown of Fis1 and Drp1. Mechanistically, TBC1D15 recruited Drp1 to mitochondria-lysosome contact sites via direct interaction with Drp1 through its C terminus (574-624) domain. Interfering with interaction between TBC1D15 and Drp1 abrogated asymmetrical mitochondrial fission and mitochondrial function. Cardiac phenotypes of TBC1D15 mice upon I/R injury were rescued by adenovirus-mediated overexpression of wild-type but not mutants (R400K, ∆231-240 and ∆574-624) TBC1D15.
CONCLUSIONS
TBC1D15 ameliorated I/R injury through a novel modality to preserve mitochondrial homeostasis where mitochondria-lysosome contacts (through the TBC1D15/Fis1/RAB7 cascade) regulate asymmetrical mitochondrial fission (TBC1D15/Drp1 interaction), suggesting promises of targeting TBC1D15 in the management of myocardial I/R injury.
Topics: Animals; Dynamins; GTPase-Activating Proteins; Homeostasis; Humans; Mice; Mitochondria; Mitochondrial Dynamics; Myocardial Reperfusion Injury; Myocytes, Cardiac
PubMed: 35680100
DOI: 10.1016/j.metabol.2022.155239 -
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 -
Cell Metabolism Oct 2018It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related...
It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related GTPases for division (Drp1) and fusion (Opa1) in livers. Mitochondrial stasis rescues liver damage and hypotrophy caused by the single knockout (KO). At the cellular level, mitochondrial stasis re-establishes mitochondrial size and rescues mitophagy defects caused by division deficiency. Using Drp1KO livers, we found that the autophagy adaptor protein p62/sequestosome-1-which is thought to function downstream of ubiquitination-promotes mitochondrial ubiquitination. p62 recruits two subunits of a cullin-RING ubiquitin E3 ligase complex, Keap1 and Rbx1, to mitochondria. Resembling Drp1KO, diet-induced nonalcoholic fatty livers enlarge mitochondria and accumulate mitophagy intermediates. Resembling Drp1Opa1KO, Opa1KO rescues liver damage in this disease model. Our data provide a new concept that mitochondrial stasis leads the spatial dimension of mitochondria to a stationary equilibrium and a new mechanism for mitochondrial ubiquitination in mitophagy.
Topics: Animals; Carrier Proteins; Disease Models, Animal; Dynamins; GTP Phosphohydrolases; Hepatocytes; Kelch-Like ECH-Associated Protein 1; Liver; Mice; Mice, Knockout; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Mitochondrial Size; Mitophagy; Non-alcoholic Fatty Liver Disease; Sequestosome-1 Protein; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 30017357
DOI: 10.1016/j.cmet.2018.06.014 -
Signal Transduction and Targeted Therapy Apr 2022Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the...
Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1 recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1 restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.
Topics: Animals; Dynamins; Mice; Mice, Knockout; Mitochondria; Mitochondrial Dynamics; Phosphorylation; Protein Kinases
PubMed: 35422062
DOI: 10.1038/s41392-022-00933-z -
Cell Proliferation Jun 2021High-mobility group box-1 (HMGB1) and aberrant mitochondrial fission mediated by excessive activation of GTPase dynamin-related protein 1 (Drp1) have been found to be...
OBJECTIVES
High-mobility group box-1 (HMGB1) and aberrant mitochondrial fission mediated by excessive activation of GTPase dynamin-related protein 1 (Drp1) have been found to be elevated in patients with pulmonary arterial hypertension (PAH) and critically implicated in PAH pathogenesis. However, it remains unknown whether Drp1-mediated mitochondrial fission and which downstream targets of mitochondrial fission mediate HMGB1-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration leading to vascular remodelling in PAH. This study aims to address these issues.
METHODS
Primary cultured PASMCs were obtained from male Sprague-Dawley (SD) rats. We detected RNA levels by qRT-PCR, protein levels by Western blotting, cell proliferation by Cell Counting Kit-8 (CCK-8) and EdU incorporation assays, migration by wound healing and transwell assays. SD rats were injected with monocrotaline (MCT) to establish PAH. Hemodynamic parameters were measured by closed-chest right heart catheterization.
RESULTS
HMGB1 increased Drp1 phosphorylation and Drp1-dependent mitochondrial fragmentation through extracellular signal-regulated kinases 1/2 (ERK1/2) signalling activation, and subsequently triggered autophagy activation, which further led to bone morphogenetic protein receptor 2 (BMPR2) lysosomal degradation and inhibitor of DNA binding 1 (Id1) downregulation, and eventually promoted PASMCs proliferation/migration. Inhibition of ERK1/2 cascade, knockdown of Drp1 or suppression of autophagy restored HMGB1-induced reductions of BMPR2 and Id1, and diminished HMGB1-induced PASMCs proliferation/migration. In addition, pharmacological inhibition of HMGB1 by glycyrrhizin, suppression of mitochondrial fission by Mdivi-1 or blockage of autophagy by chloroquine prevented PAH development in MCT-induced rats PAH model.
CONCLUSIONS
HMGB1 promotes PASMCs proliferation/migration and pulmonary vascular remodelling by activating ERK1/2/Drp1/Autophagy/BMPR2/Id1 axis, suggesting that this cascade might be a potential novel target for management of PAH.
Topics: Animals; Autophagy; Cells, Cultured; Dynamins; HMGB1 Protein; MAP Kinase Signaling System; Male; Mitochondria; Mitochondrial Dynamics; Phosphorylation; Pulmonary Arterial Hypertension; Rats, Sprague-Dawley; Rats
PubMed: 33948998
DOI: 10.1111/cpr.13048