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Tamoxifen improves muscle structure and function of Bin1- and Dnm2-related centronuclear myopathies.Brain : a Journal of Neurology Jul 2023Congenital myopathies define a genetically heterogeneous group of disorders associated with severe muscle weakness, for which no therapies are currently available. Here...
Congenital myopathies define a genetically heterogeneous group of disorders associated with severe muscle weakness, for which no therapies are currently available. Here we investigated the repurposing of tamoxifen in mouse models of mild or severe forms of centronuclear myopathies due to mutations in BIN1 (encoding amphiphysin 2) or DNM2 (encoding dynamin 2), respectively. Exposure to a tamoxifen-enriched diet from 3 weeks of age resulted in significant improvement in muscle contractility without increase in fibre size in both models, underlying an increase in the capacity of the muscle fibres to produce more force. In addition, the histological alterations were fully rescued in the BIN1-centronuclear myopathies mouse model. To assess the mechanism of the rescue, transcriptome analyses and targeted protein studies were performed. Although tamoxifen is known to modulate the transcriptional activity of the oestrogen receptors, correction of the disease transcriptomic signature was marginal on tamoxifen treatment. Conversely, tamoxifen lowered the abnormal increase in dynamin 2 protein level in both centronuclear myopathies models. Of note, it was previously reported that dynamin 2 increase is a main pathological cause of centronuclear myopathies. The Akt/mTOR muscle hypertrophic pathway and protein markers of the ubiquitin-proteasome system (the E3 ubiquitin ligase cullin 3) and autophagy (p62) were increased in both models of centronuclear myopathies. Normalization of dynamin 2 level mainly correlated with the normalization of cullin 3 protein level on tamoxifen treatment, supporting the idea that the ubiquitin-proteasome system is a main target for the tamoxifen effect in the amelioration of these diseases. Overall, our data suggest that tamoxifen antagonizes disease development probably through dynamin 2 level regulation. In conclusion, the beneficial effect of tamoxifen on muscle function supports the suggestion that tamoxifen may serve as a common therapy for several autosomal forms of centronuclear myopathies.
Topics: Animals; Mice; Adaptor Proteins, Signal Transducing; Cullin Proteins; Dynamin II; Muscle, Skeletal; Muscles; Mutation; Myopathies, Structural, Congenital; Nerve Tissue Proteins; Proteasome Endopeptidase Complex; Tumor Suppressor Proteins; Ubiquitins
PubMed: 36562127
DOI: 10.1093/brain/awac489 -
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 -
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 Signalling Jan 2024Mitochondrial dysfunction in pulmonary artery endothelial cells (PAECs) is related to the pathogenesis of pulmonary hypertension (PH). The mitochondrial receptor protein...
Mitochondrial dysfunction in pulmonary artery endothelial cells (PAECs) is related to the pathogenesis of pulmonary hypertension (PH). The mitochondrial receptor protein FUN14 domain containing 1 (FUNDC1) was found to be involved in pulmonary artery smooth muscle cell proliferation in PH. However, its role in PAECs remains unclear. We investigated FUNDC1 expression in the pulmonary artery endothelium in both monocrotaline-induced animal models and TNF-α-stimulated cell models. Additionally, the effect of FUNDC1 on PAECs proliferation and its possible mechanism were also investigated. We observed decreased FUNDC1 protein levels in animals and in vitro in PAECs. FUNDC1 deficiency in PAECs upregulated the expression of the deubiquitination enzyme ubiquitin-specific peptidase 15 (USP15), enhanced dynamin-related protein1 (Drp1)-mediated mitochondrial division, and increased mitochondrial ROS levels via the deubiquitination of Drp1. Additionally, FUNDC1 deficiency increased aerobic glycolysis, the production of ATP and lactic acid, and glucose uptake. FUNDC1 overexpression inhibited PAECs proliferation. Moreover, FUNDC1 overexpression in combination with a mitochondrial division or aerobic glycolysis inhibitor enhanced its inhibitory effect on cell proliferation. Our study findings suggest that FUNDC1 deficiency induced by inflammation can promote PAECs proliferation by regulating mitochondrial dynamics and cell energy metabolism via the USP15/Drp1 pathway.
Topics: Animals; Pulmonary Artery; Tumor Necrosis Factor-alpha; Endothelial Cells; Mitochondrial Dynamics; Dynamins; Cell Proliferation; Hypertension, Pulmonary; Mitochondrial Proteins
PubMed: 37871666
DOI: 10.1016/j.cellsig.2023.110939 -
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 -
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 -
Neuroscience Dec 2023Spinal cord injuries (SCIs) often result in limited prospects for recovery and a high incidence of disability. Melatonin (Mel), a hormone, is acknowledged for its...
Spinal cord injuries (SCIs) often result in limited prospects for recovery and a high incidence of disability. Melatonin (Mel), a hormone, is acknowledged for its neuroprotective attributes. Mel was examined in this study to discover if it alleviates SCIs via the sirtuin1/dynamin-related protein1 (SIRT1/Drp1) signaling pathway. SCIs were simulated in mice by inducing cord contusion at the T9-T10 vertebrae and causing inflammation in primary spinal neurons using lipopolysaccharide (LPS). The findings of our study demonstrated that Mel treatment effectively promoted neuromotor recovery through multiple mechanisms, including the reduction of neuronal death, suppression of astrocyte and microglia activation, and attenuation of neuroinflammation. Moreover, Mel therapy significantly upregulated the expression of SIRT1 in both spinal cord tissues and spinal neurons of mice. Additionally, Mel exhibited the potential to mitigate neuronal mitochondrial dysfunction by modulating the levels of Drp1 and TOMM20, thereby addressing the underlying factors contributing to this dysfunction. Furthermore, when SIRT1 was downregulated, it reversed the positive effects of Mel. Overall, our present study suggests that Mel has the capacity to modulate the SIRT1/Drp1 pathway, thereby ameliorating mitochondrial dysfunction, attenuating inflammation and apoptosis, and enhancing neural function subsequent to SCIs.
Topics: Rats; Mice; Animals; Melatonin; Rats, Sprague-Dawley; Sirtuin 1; Signal Transduction; Spinal Cord; Neurons; Spinal Cord Injuries; Apoptosis; Inflammation; Dynamins
PubMed: 37865165
DOI: 10.1016/j.neuroscience.2023.10.010 -
Cell Biochemistry and Biophysics Mar 2024Drp1 (Dynamin-Related Protein 1) is a cytoplasmic GTPase protein encoded by the DNM1L gene that influences mitochondrial dynamics by mediating mitochondrial fission... (Review)
Review
Drp1 (Dynamin-Related Protein 1) is a cytoplasmic GTPase protein encoded by the DNM1L gene that influences mitochondrial dynamics by mediating mitochondrial fission processes. Drp1 has been demonstrated to play an important role in a variety of life activities such as cell survival, proliferation, migration, and death. Drp1 has been shown to play different physiological roles under different physiological conditions, such as normal and inflammation. Recently studies have revealed that Drp1 plays a critical role in the occurrence, development, and aggravation of a series of diseases, thereby it serves as a potential therapeutic target for them. In this paper, we review the structure and biological properties of Drp1, summarize the biological processes that occur in the inflammatory response to Drp1, discuss its role in various cancers triggered by the mitochondrial pathway and investigate effective methods for targeting Drp1 in cancer treatment. We also synthesized the phenomena of Drp1 involving in the triggering of other diseases. The results discussed herein contribute to our deeper understanding of mitochondrial kinetic pathway-induced diseases and their therapeutic applications. It is critical for advancing the understanding of the mechanisms of Drp1-induced mitochondrial diseases and preventive therapies.
PubMed: 38438751
DOI: 10.1007/s12013-024-01245-5