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Hypertension (Dallas, Tex. : 1979) Jul 2020Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited....
Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited. Mitochondrial dysfunction is frequently associated with mitochondrial fission/fragmentation mediated by the GTPase Drp1 (dynamin-related protein 1). Nuclear factor (NF)-κB, a master regulator of inflammation, is implicated in endothelial dysfunction and resultant complications. Here, we explore a causal relationship between mitochondrial fission and NF-κB activation in endothelial inflammatory responses. In cultured endothelial cells, TNF-α (tumor necrosis factor-α) or lipopolysaccharide induces mitochondrial fragmentation. Inhibition of Drp1 activity or expression suppresses mitochondrial fission, NF-κB activation, vascular cell adhesion molecule-1 induction, and leukocyte adhesion induced by these proinflammatory factors. Moreover, attenuations of inflammatory leukocyte adhesion were observed in Drp1 heterodeficient mice as well as endothelial Drp1 silenced mice. Intriguingly, inhibition of the canonical NF-κB signaling suppresses endothelial mitochondrial fission. Mechanistically, NF-κB p65/RelA seems to mediate inflammatory mitochondrial fission in endothelial cells. In addition, the classical anti-inflammatory drug, salicylate, seems to maintain mitochondrial fission/fusion balance against TNF-α via inhibition of NF-κB. In conclusion, our results suggest a previously unknown mechanism whereby the canonical NF-κB cascade and a mitochondrial fission pathway interdependently regulate endothelial inflammation.
Topics: 3T3 Cells; Animals; Aorta; Cell Adhesion; Cells, Cultured; Dynamins; Endothelial Cells; Endothelium, Vascular; Leukocytes, Mononuclear; Membrane Proteins; Mice; Mitochondrial Dynamics; Mitochondrial Proteins; Mutation, Missense; NF-kappa B; Phosphorylation; Phosphoserine; Protein Processing, Post-Translational; Proteome; RNA Interference; RNA, Small Interfering; Rats; Sodium Salicylate; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vasculitis
PubMed: 32389075
DOI: 10.1161/HYPERTENSIONAHA.120.14686 -
Methods in Molecular Biology (Clifton,... 2022This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds....
This protocol describes the chemical synthesis of the dynamin inhibitors Dynole 34-2 and Acrylo-Dyn 2-30, and their chemical scaffold matched partner inactive compounds. The chosen active and inactive paired compounds represent potent dynamin inhibitors and very closely related dynamin-inactive compounds, with the synthesis of three of the four compounds readily possible via a common intermediate. Combined with the assay data provided, this allows the interrogation of dynamin in vitro and potentially in vivo.
Topics: Cyanoacrylates; Dynamins; Endocytosis; Indoles
PubMed: 35099803
DOI: 10.1007/978-1-0716-1916-2_17 -
The FEBS Journal Jun 2022Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The...
Nek4 is a serine/threonine kinase which has been implicated in primary cilia stabilization, DNA damage response, autophagy and epithelial-to-mesenchymal transition. The role of Nek4 in cancer cell survival and chemotherapy resistance has also been shown. However, the precise mechanisms by which Nek4 operates remain to be elucidated. Here, we show that Nek4 overexpression activates mitochondrial respiration coupled to ATP production, which is paralleled by increased mitochondrial membrane potential, and resistance to mitochondrial DNA damage. Congruently, Nek4 depletion reduced mitochondrial respiration and mtDNA integrity. Nek4 deficiency caused mitochondrial elongation, probably via reduced activity of the fission protein DRP1. In Nek4 overexpressing cells, the increase in mitochondrial fission was concomitant to enhanced phosphorylation of DRP1 and Erk1/2 proteins, and the effects on mitochondrial respiration were abolished in the presence of a DRP1 inhibitor. This study shows Nek4 as a novel regulator of mitochondrial function that may explain the joint appearance of high mitochondrial respiration and mitochondrial fragmentation.
Topics: DNA, Mitochondrial; Dynamins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Phosphorylation; Respiration
PubMed: 34986513
DOI: 10.1111/febs.16343 -
Proceedings of the National Academy of... Aug 2021Pancreatic β cells operate with a high rate of membrane recycling for insulin secretion, yet endocytosis in these cells is not fully understood. We investigate this...
Pancreatic β cells operate with a high rate of membrane recycling for insulin secretion, yet endocytosis in these cells is not fully understood. We investigate this process in mature mouse β cells by genetically deleting dynamin GTPase, the membrane fission machinery essential for clathrin-mediated endocytosis. Unexpectedly, the mice lacking all three dynamin genes (, , ) in their β cells are viable, and their β cells still contain numerous insulin granules. Endocytosis in these β cells is severely impaired, resulting in abnormal endocytic intermediates on the plasma membrane. Although insulin granules are abundant, their release upon glucose stimulation is blunted in both the first and second phases, leading to hyperglycemia and glucose intolerance in mice. Dynamin triple deletion impairs insulin granule exocytosis and decreases intracellular Ca responses and granule docking. The docking defect is correlated with reduced expression of Munc13-1 and RIM1 and reorganization of cortical F-actin in β cells. Collectively, these findings uncover the role of dynamin in dense-core vesicle endocytosis and secretory capacity. Insulin secretion deficiency in the absence of dynamin-mediated endocytosis highlights the risk of impaired membrane trafficking in endocrine failure and diabetes pathogenesis.
Topics: Animals; Blood Glucose; Calcium Signaling; Dense Core Vesicles; Dynamin II; Dynamins; Endocytosis; Female; GTP-Binding Proteins; Hyperglycemia; Insulin Secretion; Insulin-Secreting Cells; Male; Mice, Knockout; Mice, Transgenic; Nerve Tissue Proteins; Mice
PubMed: 34362840
DOI: 10.1073/pnas.2021764118 -
Cells Aug 2019The purpose of this article is to highlight the role of dynamin-related protein 1 (Drp1) in abnormal mitochondrial dynamics, mitochondrial fragmentation,... (Review)
Review
The purpose of this article is to highlight the role of dynamin-related protein 1 (Drp1) in abnormal mitochondrial dynamics, mitochondrial fragmentation, autophagy/mitophagy, and neuronal damage in Alzheimer's disease (AD) and other neurological diseases, including Parkinson's, Huntington's, amyotrophic lateral sclerosis, multiple sclerosis, diabetes, and obesity. Dynamin-related protein 1 is one of the evolutionarily highly conserved large family of GTPase proteins. Drp1 is critical for mitochondrial division, size, shape, and distribution throughout the neuron, from cell body to axons, dendrites, and nerve terminals. Several decades of intense research from several groups revealed that Drp1 is enriched at neuronal terminals and involved in synapse formation and synaptic sprouting. Different phosphorylated forms of Drp1 acts as both increased fragmentation and/or increased fusion of mitochondria. Increased levels of Drp1 were found in diseased states and caused excessive fragmentation of mitochondria, leading to mitochondrial dysfunction and neuronal damage. In the last two decades, several Drp1 inhibitors have been developed, including Mdivi-1, Dynasore, P110, and DDQ and their beneficial effects tested using cell cultures and mouse models of neurodegenerative diseases. Recent research using genetic crossing studies revealed that a partial reduction of Drp1 is protective against mutant protein(s)-induced mitochondrial and synaptic toxicities. Based on findings from cell cultures, mouse models and postmortem brains of AD and other neurodegenerative disease, we cautiously conclude that reduced Drp1 is a promising therapeutic target for AD and other neurological diseases.
Topics: Animals; Dynamins; Humans; Mice; Mitochondria; Neurodegenerative Diseases
PubMed: 31450774
DOI: 10.3390/cells8090961 -
Biochemical and Biophysical Research... Sep 2021Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of...
Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cells, Cultured; Dynamin II; Humans; Mice; Podosomes
PubMed: 34325130
DOI: 10.1016/j.bbrc.2021.07.041 -
ChemMedChem Dec 2022The Bis-T series of compounds comprise some of the most potent inhibitors of dynamin GTPase activity yet reported, e. g.,...
The Bis-T series of compounds comprise some of the most potent inhibitors of dynamin GTPase activity yet reported, e. g., (2E,2'E)-N,N'-(propane-1,3-diyl)bis(2-cyano-3-(3,4-dihydroxyphenyl)acrylamide) (2), Bis-T-22. The catechol moieties are believed to limit cell permeability, rendering these compounds largely inactive in cells. To solve this problem, a prodrug strategy was envisaged and eight ester analogues were synthesised. The shortest and bulkiest esters (acetate and butyl/tert-butyl) were found to be insoluble under physiological conditions, whilst the remaining five were soluble and stable under these conditions. These five were analysed for plasma stability and half-lives ranged from ∼2.3 min (propionic ester 4), increasing with size and bulk, to greater than 24 hr (dimethyl carbamate 10). Similar profiles where observed with the rate of formation of Bis-T-22 with half-lives ranging from ∼25 mins (propionic ester 4). Propionic ester 4 was chosen to undergo further testing and was found to inhibit endocytosis in a dose-dependent manner with IC ∼8 μM, suggesting this compound is able to effectively cross the cell membrane where it is rapidly hydrolysed to the desired Bis-T-22 parent compound.
Topics: Prodrugs; Dynamins; Esters; Endocytosis
PubMed: 36351775
DOI: 10.1002/cmdc.202200400 -
BMB Reports Dec 2023C-reactive protein (CRP) is an inflammatory marker and risk factor for atherosclerosis and cardiovascular diseases. However, the mechanism through which CRP induces...
C-reactive protein (CRP) is an inflammatory marker and risk factor for atherosclerosis and cardiovascular diseases. However, the mechanism through which CRP induces myocardial damage remains unclear. This study aimed to determine how CRP damages cardiomyocytes via the change of mitochondrial dynamics and whether survivin, an anti-apoptotic protein, exerts a cardioprotective effect in this process. We treated H9c2 cardiomyocytes with CRP and found increased intracellular ROS production and shortened mitochondrial length. CRP treatment phosphorylated ERK1/2 and promoted increased expression, phosphorylation, and translocation of DRP1, a mitochondrial fission-related protein, from the cytoplasm to the mitochondria. The expression of mitophagy proteins PINK1 and PARK2 was also increased by CRP. YAP, a transcriptional regulator of PINK1 and PARK2, was also increased by CRP. Knockdown of YAP prevented CRP-induced increases in DRP1, PINK1, and PARK2. Furthermore, CRP-induced changes in the expression of DRP1 and increases in YAP, PINK1, and PARK2 were inhibited by ERK1/2 inhibition, suggesting that ERK1/2 signaling is involved in CRP-induced mitochondrial fission. We treated H9c2 cardiomyocytes with a recombinant TAT-survivin protein before CRP treatment, which reduced CRP-induced ROS accumulation and reduced mitochondrial fission. CRP-induced activation of ERK1/2 and increases in the expression and activity of YAP and its downstream mitochondrial proteins were inhibited by TAT-survivin. This study shows that mitochondrial fission occurs during CRPinduced cardiomyocyte damage and that the ERK1/2-YAP axis is involved in this process, and identifies that survivin alters these mechanisms to prevent CRP-induced mitochondrial damage. [BMB Reports 2023; 56(12): 663-668].
Topics: Myocytes, Cardiac; Dynamins; Survivin; Mitochondrial Dynamics; C-Reactive Protein; MAP Kinase Signaling System; Reactive Oxygen Species; Protein Kinases
PubMed: 37817437
DOI: 10.5483/BMBRep.2023-0127 -
Nature Communications Aug 2023Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene...
Dynamin-1 is a large GTPase with an obligatory role in synaptic vesicle endocytosis at mammalian nerve terminals. Heterozygous missense mutations in the dynamin-1 gene (DNM1) cause a novel form of epileptic encephalopathy, with pathogenic mutations clustering within regions required for its essential GTPase activity. We reveal the most prevalent pathogenic DNM1 mutation, R237W, disrupts dynamin-1 enzyme activity and endocytosis when overexpressed in central neurons. To determine how this mutation impacted cell, circuit and behavioural function, we generated a mouse carrying the R237W mutation. Neurons from heterozygous mice display dysfunctional endocytosis, in addition to altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes are corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates endocytosis. Here, we demonstrate a credible link between dysfunctional endocytosis and epileptic encephalopathy, and importantly reveal that synaptic vesicle recycling may be a viable therapeutic target for monogenic intractable epilepsies.
Topics: Animals; Mice; Dynamin I; Seizures; Disease Models, Animal; Drug Resistant Epilepsy; Biological Transport; Mammals
PubMed: 37648685
DOI: 10.1038/s41467-023-41035-w -
Trends in Cell Biology Apr 2021Mitochondria are dynamic organelles that undergo cycles of fission and fusion events depending on cellular requirements. During mitochondrial division, the GTPase... (Review)
Review
Mitochondria are dynamic organelles that undergo cycles of fission and fusion events depending on cellular requirements. During mitochondrial division, the GTPase dynamin-related protein-1 is recruited to endoplasmic reticulum (ER)-induced mitochondrial constriction sites where it drives fission. However, the events required to complete scission of mitochondrial membranes are not well understood. Here, we emphasize the recently described roles for Golgi-derived phosphatidylinositol 4-phosphate (PI4P)-containing vesicles in the last steps of mitochondrial division. We then propose how trans-Golgi network vesicles at mitochondria-ER contact sites and PI4P generation could mechanistically execute mitochondrial division, by recruiting PI4P effectors and/or the actin nucleation machinery. Finally, we speculate on mechanisms to explain why such a complex dance of different organelles is required to facilitate the remodelling of mitochondrial membranes.
Topics: Dynamins; Endoplasmic Reticulum; GTP Phosphohydrolases; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membranes; Mitochondrial Proteins
PubMed: 33446409
DOI: 10.1016/j.tcb.2020.12.005