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Neurobiology of Disease Apr 2024Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad... (Review)
Review
Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad clinical phenotype involving the entire nervous system. MFN2, a dynamin-like GTPase protein located on the outer mitochondrial membrane, is well-known for its involvement in mitochondrial fusion. Numerous studies have demonstrated its participation in a network crucial for various other mitochondrial functions, including mitophagy, axonal transport, and its controversial role in endoplasmic reticulum (ER)-mitochondria contacts. Considerable progress has been made in the last three decades in elucidating the disease pathogenesis, aided by the generation of animal and cellular models that have been instrumental in studying disease physiology. A review of the literature reveals that, up to now, no definitive pharmacological treatment for any CMT2A variant has been established; nonetheless, recent years have witnessed substantial progress. Many treatment approaches, especially concerning molecular therapy, such as histone deacetylase inhibitors, peptide therapy to increase mitochondrial fusion, the new therapeutic strategies based on MF1/MF2 balance, and SARM1 inhibitors, are currently in preclinical testing. The literature on gene silencing and gene replacement therapies is still limited, except for a recent study by Rizzo et al.(Rizzo et al., 2023), which recently first achieved encouraging results in in vitro and in vivo models of the disease. The near-future goal for these promising therapies is to progress to the stage of clinical translation.
Topics: Animals; Charcot-Marie-Tooth Disease; Mitochondria; GTP Phosphohydrolases; Phenotype; Mitochondrial Proteins; Mutation
PubMed: 38452947
DOI: 10.1016/j.nbd.2024.106467 -
Life Science Alliance Jan 2024During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar...
During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of (2-12/cell) and (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).
Topics: Animals; Fungal Proteins; Mitochondria; Microsporidia; Saccharomyces cerevisiae; Dynamins; Mammals
PubMed: 37903625
DOI: 10.26508/lsa.202201635 -
Journal of Translational Medicine May 2024Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung diseases, which mainly existed in middle-aged and elderly people. The...
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrotic interstitial lung diseases, which mainly existed in middle-aged and elderly people. The accumulation of reactive oxygen species (ROS) is a common characteristic of IPF. Previous research also shown that lactate levels can be abnormally elevated in IPF patients. Emerging evidence suggested a relationship between lactate and ROS in IPF which needs further elucidation. In this article, we utilized a mouse model of BLM-induced pulmonary fibrosis to detect alterations in ROS levels and other indicators associated with fibrosis. Lactate could induce mitochondrial fragmentation by modulating expression and activity of DRP1 and ERK. Moreover, Increased ROS promoted P65 translocation into nucleus, leading to expression of lung fibrotic markers. Finally, Ulixertinib, Mdivi-1 and Mito-TEMPO, which were inhibitor activity of ERK, DRP1 and mtROS, respectively, could effectively prevented mitochondrial damage and production of ROS and eventually alleviate pulmonary fibrosis. Taken together, these findings suggested that lactate could promote lung fibrosis by increasing mitochondrial fission-derived ROS via ERK/DRP1 signaling, which may provide novel therapeutic solutions for IPF.
Topics: Animals; Reactive Oxygen Species; Mitochondrial Dynamics; Dynamins; Mice, Inbred C57BL; Bleomycin; Signal Transduction; Lactic Acid; Extracellular Signal-Regulated MAP Kinases; Mitochondria; Male; MAP Kinase Signaling System; Pulmonary Fibrosis; Mice; Humans
PubMed: 38773615
DOI: 10.1186/s12967-024-05289-2 -
International Journal of Molecular... Jul 2023Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we...
Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.
Topics: Humans; Angiotensin-Converting Enzyme 2; Cholesterol; Clathrin; COVID-19; Dynamin II; Lipoproteins, LDL; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 37511618
DOI: 10.3390/ijms241411860 -
Journal of Translational Medicine Mar 2024The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation...
Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.
BACKGROUND
The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported.
METHODS
We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway.
RESULTS
CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1β, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1.
CONCLUSION
CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.
Topics: Animals; Mice; Anti-Inflammatory Agents; Caffeic Acids; Inflammation; Methylprednisolone; Mitochondrial Diseases; Molecular Docking Simulation; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phenylethyl Alcohol; Reactive Oxygen Species; Signal Transduction; Sirtuin 1; Spinal Cord; Spinal Cord Injuries; Dynamins
PubMed: 38528569
DOI: 10.1186/s12967-024-05089-8 -
CNS Neuroscience & Therapeutics Apr 2024Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in...
OBJECTIVES
Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia -reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO-1ylation and deSUMO-2/3ylation of dynamin-related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics.
METHODS
Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co-immunoprecipitation (Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot.
RESULTS
In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH-SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression.
CONCLUSIONS
This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO-1ylation of Drp1, augment the SUMO-2/3ylation of Drp1, and thereby exert a protective effect against CIRI.
Topics: Humans; Animals; Rats; Mitochondrial Dynamics; Neuroblastoma; Reactive Oxygen Species; Adenosine Triphosphate; Dynamins; Cysteine Endopeptidases; Pyrazines; Saponins; Triterpenes
PubMed: 38615367
DOI: 10.1111/cns.14725 -
Renal Failure Dec 2023Lupus nephritis (LN) is a potentially fatal autoimmune disease. The purpose of this study was to find potential key molecular markers of LN to aid in the early diagnosis...
Revealing common differential mRNAs, signaling pathways, and immune cells in blood, glomeruli, and tubulointerstitium of lupus nephritis patients based on transcriptomic data.
Lupus nephritis (LN) is a potentially fatal autoimmune disease. The purpose of this study was to find potential key molecular markers of LN to aid in the early diagnosis and management of the disease. Datasets GSE99967_blood, GSE32591_glomeruli, and GSE32591_tubulointerstitium were included in this study. Differentially expressed mRNAs (DEmRNAs) were identified between the normal control and LN groups using the limma package in R. Common DEmRNAs in the three datasets were taken. Subsequently, functional enrichment analysis, immune correlation analysis, receiver operating characteristic (ROC) curve analysis and real-time polymerase chain reaction (RT-PCR) verification were performed. In this study, 11 common DEmRNAs were obtained and all of them were up-regulated. In protein-protein interaction (PPI) networks, we found that MX dynamin like GTPase 1 (MX1) and radical S-adenosyl methionine domain containing 2 (RSAD2) had the highest interaction score (0.997). Functional enrichment analysis revealed that MX1 and RSAD2 were enriched in influenza A and hepatitis C signaling pathways. The area under the curve (AUC) values of interferon-induced protein 44 (IFI44) and MX1 in GSE32591_glomeruli and GSE32591_tubulointerstitium datasets are 1, which is worthy of further study on their diagnostic value and molecular mechanism. The xCell analysis showed abnormal distribution of granulocyte-macrophage progenitor (GMP) cells in blood, glomeruli, and tubulointerstitium. Pearson's correlation analysis found that GMP cells were significantly correlated with lactotransferrin (LTF) and cell cycle. Identification of common DEmRNAs and key pathways in the blood, glomeruli, and tubulointerstitium of patients with LN provides potential research directions for exploring the molecular mechanisms of the disease.
Topics: Humans; Lupus Nephritis; Transcriptome; RNA, Messenger; Gene Expression Profiling; Signal Transduction
PubMed: 37334926
DOI: 10.1080/0886022X.2023.2215344 -
Frontiers in Cellular and Infection... 2023Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles,...
Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs . Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.
Topics: Mice; Animals; Muromegalovirus; Endothelial Cells; Neuropilin-1; Liver; Dynamins
PubMed: 38029264
DOI: 10.3389/fcimb.2023.1249894 -
RSC Medicinal Chemistry Aug 2023We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC 45 μM) and...
We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC 45 μM) and transferrin (Tfn) endocytosis in U2OS cells (IC 205 μM). Synthesis gave a new class of GTP-competitive dynamin inhibitors, the Sulfonadyns™. The introduction of a terminal cinnamyl moiety greatly enhanced dynI inhibition. Rigid diamine or amide links between the dansyl and cinnamyl moieties were detrimental to dynI inhibition. Compounds with inhibition of dynI activity <10 μM were tested in-cell for inhibition of CME. These data unveiled a number of compounds, analogues 33 (()--(6-{[(3-(4-bromophenyl)-2-propen-1-yl]amino}hexyl)-5-isoquinolinesulfonamide)) and 47 (()--(3-{[3-(4-bromophenyl)-2-propen-1-yl]amino}propyl)-1-naphthalenesulfonamide)isomers that showed dyn IC <4 μM, IC <30 μM and IC from 12-265 μM. Both analogues (33 and 47) are at least 10 times more potent that the initial lead, dansylcadaverine (1). Enzyme kinetics revealed these sulfonamide analogues as being GTP competitive inhibitors of dynI. Sulfonadyn-47, the most potent SVE inhibitor observed (IC = 12.3 μM), significantly increased seizure threshold in a 6 Hz mouse psychomotor seizure test at 30 ( = 0.003) and 100 mg kg ip ( < 0.0001), with similar anti-seizure efficacy to the established anti-seizure medication, sodium valproate (400 mg kg). The Sulfonadyn™ class of drugs target dynamin and show promise as novel leads for future anti-seizure medications.
PubMed: 37593570
DOI: 10.1039/d2md00371f -
Redox Biology Jul 2024Inflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1...
Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α signaling by phenothiazine in hypothermic and normothermic neuroprotection after acute ischemic stroke.
BACKGROUND
Inflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1 (RIPK1) activated dynamin-related protein 1 (DRP1) in a NLRPyrin domain containing 3 (NLRP3) inflammasome-dependent fashion and Hypoxia-Inducible Factor (HIF)-1α play key roles in the process. This study determined how phenothiazine drugs (chlorpromazine and promethazine (C + P)) with the hypothermic and normothermic modality impacts the RIPK1/RIPK3-DRP1 and HIF-1α pathways in providing neuroprotection.
METHODS
A total of 150 adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. 8 mg/kg of C + P was administered at onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation and cytochrome c-apoptosis were assessed. Apoptotic cell death, infiltration of neutrophils and macrophages, and mitochondrial function were evaluated. Interaction between RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. In SH-SY5Y cells subjected to oxygen/glucose deprivation (OGD), the normothermic effect of C + P on inflammation and apoptosis were examined.
RESULTS
C + P significantly reduced infarct volumes, mitochondrial dysfunction (ATP and ROS concentration, citrate synthase and ATPase activity), inflammation and apoptosis with and without induced hypothermia. Overexpression of RIPK1, RIPK3, DRP-1, NLRP3-inflammasome and cytochrome c-apoptosis were all significantly reduced by C + P at 33 °C and the RIPK1 inhibitor (Nec1s), suggesting hypothermic effect of C + P via RIPK1/RIPK3-DRP1pathway. When body temperature was maintained at 37 °C, C + P and HIF-1α inhibitor (YC-1) reduced HIF-1α expression, leading to reduction in mitochondrial dysfunction, NLRP3 inflammasome and cytochrome c-apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced in vitro, indicating a normothermic effect of C + P via HIF-1α.
CONCLUSION
Hypothermic and normothermic neuroprotection of C + P involve different pathways. The normothermic effect was mediated by HIF-1α, while hypothermic effect was via RIPK1/RIPK3-DRP1 signaling. This provides a theoretical basis for future precise exploration of hypothermic and normothermic neuroprotection.
Topics: Animals; Receptor-Interacting Protein Serine-Threonine Kinases; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Ischemic Stroke; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Signal Transduction; Inflammasomes; Dynamins; Rats, Sprague-Dawley; Phenothiazines; Inflammation; Neuroprotection; Humans; Disease Models, Animal; Hypothermia, Induced
PubMed: 38692093
DOI: 10.1016/j.redox.2024.103169