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ENeuro Aug 2023As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2...
As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.
Topics: Animals; Humans; Drosophila; Drosophila Proteins; GTP Phosphohydrolases; Mammals; Microtubules; Mitochondria; Mitochondrial Proteins; Protein Serine-Threonine Kinases; Synapses
PubMed: 37550059
DOI: 10.1523/ENEURO.0409-22.2023 -
Nature Communications Mar 2024NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in...
NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.
Topics: Animals; Mice; Dynamins; Histidine; Hypoxia; Mitophagy; Ubiquitination
PubMed: 38480688
DOI: 10.1038/s41467-024-46385-7 -
European Journal of Pharmacology Jul 2023A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors...
A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors mdivi-1 and dynasore. However, mdivi-1 is capable to inhibit Ba currents through Ca1.2 channels (I), stimulate K1.1 channel currents (I), and modulate pathways key to the maintenance of vessel active tone in a dynamin-independent manner. Using a multidisciplinary approach, the present study demonstrates that dynasore, like mdivi-1, is a bi-functional vasodilator, blocking I and stimulating I in rat tail artery myocytes, as well as promoting relaxation of rat aorta rings pre-contracted by either high K or phenylephrine. Conversely, its analogue dyngo-4a, though inhibiting mitochondrial fission triggered by phenylephrine and stimulating I, did not affect I but potentiated both high K- and phenylephrine-induced contractions. Docking and molecular dynamics simulations identified the molecular basis supporting the different activity of dynasore and dyngo-4a at Ca1.2 and K1.1 channels. Mito-tempol only partially counteracted the effects of dynasore and dyngo-4a on phenylephrine-induced tone. In conclusion, the present data, along with previous observations (Ahmed et al., 2022) rise caution for the use of dynasore, mdivi-1, and dyngo-4a as tools to investigate the role of mitochondrial fission in vascular contraction: to this end, a selective dynamin inhibitor and/or a different experimental approach are needed.
Topics: Rats; Animals; Mitochondrial Dynamics; Dynamins; Niacinamide; Phenylephrine
PubMed: 37179045
DOI: 10.1016/j.ejphar.2023.175786 -
BioRxiv : the Preprint Server For... Aug 2023Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane...
Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane in a GTP hydrolysis-dependent manner. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Phosphorylation of S579 is widely regarded as activating, while S600 phosphorylation is commonly considered inhibiting. However, the direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. In this study, we compare the effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and phosphorylation. The oligomerization state of both phospho-mimetic mutants is shifted toward smaller oligomers. Both phospho-mimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wild-type Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant that lacks GTPase activity, the K38A mutant, stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions, similar to previous results for dynamin-1. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly, and likely requires additional factors for stimulation of mitochondrial fission in cells. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.
PubMed: 37645886
DOI: 10.1101/2023.08.20.554022 -
Shock (Augusta, Ga.) Jul 2023Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of...
Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of ATM in lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) disruption remains unknown. This study aimed to investigate the role and underlying mechanism of ATM in the regulation of the BBB function in sepsis. Methods: We used LPS to induce BBB disruption in vivo and to establish an in vitro model of cerebrovascular endothelial cells. Blood-brain barrier disruption was assessed by measuring Evans blue leakage and expression of vascular permeability regulators. To investigate the role of ATM, its inhibitor AZD1390 and clinically approved doxorubicin, an anthracycline that can activate ATM, were administered as scheduled. To explore the underlying mechanism, protein kinase B (AKT) inhibitor MK-2206 was administered to block the AKT/dynamin-related protein 1 (DRP1) pathway. Results: Lipopolysaccharide challenge induced significant BBB disruption, ATM activation, and mitochondrial translocation. Inhibiting ATM with AZD1390 aggravated BBB permeability as well as the following neuroinflammation and neuronal injury, while activation of ATM by doxorubicin abrogated these defects. Further results obtained in brain microvascular endothelial cells showed that ATM inhibition reduced the phosphorylation of DRP1 at serine (S) 637, promoted excessive mitochondrial fission, and resulted in mitochondrial malfunction. By activating ATM, doxorubicin increased the protein binding between ATM and AKT and promoted the phosphorylated activation of AKT at S473, which could directly phosphorylate DRP1 at S637 to repress excessive mitochondrial fission. Consistently, the protective role of ATM was abolished by the AKT inhibitor MK-2206. Conclusions: Ataxia telangiectasia mutated protects against LPS-induced BBB disruption by regulating mitochondrial homeostasis, at least in part, through the AKT/DRP1 pathway.
Topics: Humans; Blood-Brain Barrier; Proto-Oncogene Proteins c-akt; Lipopolysaccharides; Ataxia Telangiectasia; Endothelial Cells; Phosphorylation; Homeostasis; Dynamins; Doxorubicin; Mitochondrial Dynamics
PubMed: 37141173
DOI: 10.1097/SHK.0000000000002139 -
Molecular Metabolism Nov 2023Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported a strong correlation between enhanced Drp1 activity and...
OBJECTIVE
Dynamin-related protein 1 (Drp1) is the key regulator of mitochondrial fission. We and others have reported a strong correlation between enhanced Drp1 activity and impaired skeletal muscle insulin sensitivity. This study aimed to determine whether Drp1 directly regulates skeletal muscle insulin sensitivity and whole-body glucose homeostasis.
METHODS
We employed tamoxifen-inducible skeletal muscle-specific heterozygous Drp1 knockout mice (mDrp1). Male mDrp1 and wildtype (WT) mice were fed with either a high-fat diet (HFD) or low-fat diet (LFD) for four weeks, followed by tamoxifen injections for five consecutive days, and remained on their respective diet for another four weeks. In addition, we used primary human skeletal muscle cells (HSkMC) from lean, insulin-sensitive, and severely obese, insulin-resistant humans and transfected the cells with either a Drp1 shRNA (shDrp1) or scramble shRNA construct. Skeletal muscle and whole-body insulin sensitivity, skeletal muscle insulin signaling, mitochondrial network morphology, respiration, and HO production were measured.
RESULTS
Partial deletion of the Drp1 gene in skeletal muscle led to improved whole-body glucose tolerance and insulin sensitivity (P < 0.05) in diet-induced obese, insulin-resistant mice but not in lean mice. Analyses of mitochondrial structure and function revealed that the partial deletion of the Drp1 gene restored mitochondrial dynamics, improved mitochondrial morphology, and reduced mitochondrial Complex I- and II-derived HO (P < 0.05) under the condition of diet-induced obesity. In addition, partial deletion of Drp1 in skeletal muscle resulted in elevated circulating FGF21 (P < 0.05) and in a trend towards increase of FGF21 expression in skeletal muscle tissue (P = 0.095). In primary myotubes derived from severely obese, insulin-resistant humans, ShRNA-induced-knockdown of Drp1 resulted in enhanced insulin signaling, insulin-stimulated glucose uptake and reduced cellular reactive oxygen species (ROS) content compared to the shScramble-treated myotubes from the same donors (P < 0.05).
CONCLUSION
These data demonstrate that partial loss of skeletal muscle-specific Drp1 expression is sufficient to improve whole-body glucose homeostasis and insulin sensitivity under obese, insulin-resistant conditions, which may be, at least in part, due to reduced mitochondrial HO production. In addition, our findings revealed divergent effects of Drp1 on whole-body metabolism under lean healthy or obese insulin-resistant conditions in mice.
Topics: Animals; Humans; Male; Mice; Diet, High-Fat; Dynamins; Glucose; Hydrogen Peroxide; Insulin; Insulin Resistance; Mice, Obese; Muscle, Skeletal; Obesity; RNA, Small Interfering; Tamoxifen
PubMed: 37690520
DOI: 10.1016/j.molmet.2023.101802 -
Journal of Neurochemistry Feb 2024The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults,...
The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.
Topics: Animals; Humans; HEK293 Cells; Protein Transport; Endosomes; Endocytosis; Astrocytes; Aquaporin 4; Mammals
PubMed: 38102893
DOI: 10.1111/jnc.16029 -
Cellular & Molecular Biology Letters Mar 2024Acute kidney injury (AKI) is a common clinical disorder with complex etiology and poor prognosis, and currently lacks specific and effective treatment options....
BACKGROUND
Acute kidney injury (AKI) is a common clinical disorder with complex etiology and poor prognosis, and currently lacks specific and effective treatment options. Mitochondrial dynamics dysfunction is a prominent feature in AKI, and modulation of mitochondrial morphology may serve as a potential therapeutic approach for AKI.
METHODS
We induced ischemia-reperfusion injury (IRI) in mice (bilateral) and Bama pigs (unilateral) by occluding the renal arteries. ATP depletion and recovery (ATP-DR) was performed on proximal renal tubular cells to simulate in vitro IRI. Renal function was evaluated using creatinine and urea nitrogen levels, while renal structural damage was assessed through histopathological staining. The role of Drp1 was investigated using immunoblotting, immunohistochemistry, immunofluorescence, and immunoprecipitation techniques. Mitochondrial morphology was evaluated using confocal microscopy.
RESULTS
Renal IRI induced significant mitochondrial fragmentation, accompanied by Dynamin-related protein 1 (Drp1) translocation to the mitochondria and Drp1 phosphorylation at Ser616 in the early stages (30 min after reperfusion), when there was no apparent structural damage to the kidney. The use of the Drp1 inhibitor P110 significantly improved kidney function and structural damage. P110 reduced Drp1 mitochondrial translocation, disrupted the interaction between Drp1 and Fis1, without affecting the binding of Drp1 to other mitochondrial receptors such as MFF and Mid51. High-dose administration had no apparent toxic side effects. Furthermore, ATP-DR induced mitochondrial fission in renal tubular cells, accompanied by a decrease in mitochondrial membrane potential and an increase in the translocation of the pro-apoptotic protein Bax. This process facilitated the release of dsDNA, triggering the activation of the cGAS-STING pathway and promoting inflammation. P110 attenuated mitochondrial fission, suppressed Bax mitochondrial translocation, prevented dsDNA release, and reduced the activation of the cGAS-STING pathway. Furthermore, these protective effects of P110 were also observed renal IRI model in the Bama pig and folic acid-induced nephropathy in mice.
CONCLUSIONS
Dysfunction of mitochondrial dynamics mediated by Drp1 contributes to renal IRI. The specific inhibitor of Drp1, P110, demonstrated protective effects in both in vivo and in vitro models of AKI.
Topics: Animals; Mice; Swine; bcl-2-Associated X Protein; Acute Kidney Injury; Dynamins; Nucleotidyltransferases; Adenosine Triphosphate
PubMed: 38439028
DOI: 10.1186/s11658-024-00553-1 -
CNS Neuroscience & Therapeutics Mar 2024As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive...
The cold-inducible RNA-binding protein-Thioredoxin 1 pathway ameliorates mitochondrial dysfunction and mitochondrial dynamin-related protein 1 level in the hippocampus of aged mice with perioperative neurocognitive dysfunction.
BACKGROUND
As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive dysfunction (PND). Dynamin-related protein 1 (Drp1) could mediate mitochondrial fission and play important roles in mitochondrial dynamic homeostasis and mitochondria function. The Drp1 may be involved in PND development. The cold-inducible RNA-binding protein (Cirbp) could bind to the 3'-UTR of the thioredoxin 1 (Trx1) mRNA, control oxidative stress, and improve mitochondrial function. In this study, we hypothesized that the Cirbp-Trx1 pathway could ameliorate mitochondrial dysfunction and Drp1 levels in PND mice.
METHODS
Differentially expressed genes were screened using the Gene Expression Omnibus (GEO) database GSE95426 and validated using PCR. Eighteen-month-old C57BL/6 mice were subjected to tibial fracture surgery to generate a PND model. Cirbp was upregulated by hippocampal stereotaxic injections of over-Cirbp plasmid according to the manufacturer's instructions for the in vivo DNA transfection reagent. Cirbp expression was measured using western blot (WB) and immunofluorescence (IF). The Morris water maze (MWM) was used to assess cognitive function. After behavioral testing, the hippocampal tissue was extracted to examine changes in mitochondrial Drp1, mitochondrial function, neuroinflammation, and oxidative stress.
RESULTS
Differential gene screening showed that Cirbp expression was significantly downregulated (fold change >1.5, p = 0.003272) in the PND model. In this study, we also found that Cirbp protein levels were downregulated, accompanied by an impairment of cognition, a decrease in superoxide dismutase (SOD) activity, and an increase in malondialdehyde (MDA) content, mitochondrial Drp1 levels, neuroinflammation, and apoptosis. Cirbp overexpression increased Trx1 protein levels and reversed the damage. However, this protective effect was abolished by PX-12 treatment with a Trx1 inhibitor.
CONCLUSIONS
The Cirbp-Trx1 pathway may regulate mitochondrial dysfunction and mitochondrial Drp1 expression in the hippocampus of PND mice to ameliorate cognitive dysfunction.
Topics: Animals; Mice; Cognitive Dysfunction; Dynamins; Hippocampus; Mice, Inbred C57BL; Mitochondria; Mitochondrial Diseases; Mitochondrial Dynamics; Neuroinflammatory Diseases; RNA-Binding Proteins; Thioredoxins
PubMed: 37641878
DOI: 10.1111/cns.14433 -
Ecotoxicology and Environmental Safety Mar 2024Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to...
Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.
Topics: Adenosine Triphosphate; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 9; Dynamins; Mitophagy; Nanoparticles; Protein Kinases; Reactive Oxygen Species; Silicon Dioxide; Superoxide Dismutase; Ubiquitin-Protein Ligases; Animals; Mice; Cell Line, Tumor
PubMed: 38325272
DOI: 10.1016/j.ecoenv.2024.116050