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1.
Nat Cell Biol ; 23(10): 1073-1084, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616024

RESUMO

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.


Assuntos
Neoplasias da Mama/patologia , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Transporte Biológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Clatrina/genética , Dinaminas/genética , Feminino , Humanos , Integrina beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas rab de Ligação ao GTP/genética
2.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639129

RESUMO

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that finally leads to demyelination. Demyelinating optic neuritis is a frequent symptom in MS. Recent studies also revealed synapse dysfunctions in MS patients and MS mouse models. We previously reported alterations of photoreceptor ribbon synapses in the experimental auto-immune encephalomyelitis (EAE) mouse model of MS. In the present study, we found that the previously observed decreased imunosignals of photoreceptor ribbons in early EAE resulted from a decrease in synaptic ribbon size, whereas the number/density of ribbons in photoreceptor synapses remained unchanged. Smaller photoreceptor ribbons are associated with fewer docked and ribbon-associated vesicles. At a functional level, depolarization-evoked exocytosis as monitored by optical recording was diminished even as early as on day 7 after EAE induction. Moreover compensatory, post-depolarization endocytosis was decreased. Decreased post-depolarization endocytosis in early EAE correlated with diminished synaptic enrichment of dynamin3. In contrast, basal endocytosis in photoreceptor synapses of resting non-depolarized retinal slices was increased in early EAE. Increased basal endocytosis correlated with increased de-phosphorylation of dynamin1. Thus, multiple endocytic pathways in photoreceptor synapse are differentially affected in early EAE and likely contribute to the observed synapse pathology in early EAE.


Assuntos
Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Endocitose , Exocitose , Esclerose Múltipla/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Sinapses/patologia , Animais , Dinaminas/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/etiologia , Esclerose Múltipla/metabolismo , Fosforilação , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/patologia
3.
Nat Commun ; 12(1): 5305, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489447

RESUMO

Mitochondrial dysfunction is a common hallmark of neurological disorders, and reducing mitochondrial damage is considered a promising neuroprotective therapeutic strategy. Here, we used high-throughput small molecule screening to identify CHIR99021 as a potent enhancer of mitochondrial function. CHIR99021 improved mitochondrial phenotypes and enhanced cell viability in several models of Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Notably, CHIR99201 treatment reduced HD-associated neuropathology and behavioral defects in HD mice and improved mitochondrial function and cell survival in HD patient-derived neurons. Independent of its known inhibitory activity against glycogen synthase kinase 3 (GSK3), CHIR99021 treatment in HD models suppressed the proteasomal degradation of calpastatin (CAST), and subsequently inhibited calpain activation, a well-established effector of neural death, and Drp1, a driver of mitochondrial fragmentation. Our results established CAST-Drp1 as a druggable signaling axis in HD pathogenesis and highlighted CHIR99021 as a mitochondrial function enhancer and a potential lead for developing HD therapies.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Dinaminas/genética , Doença de Huntington/genética , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dinaminas/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Injeções Intraperitoneais , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais
4.
Biochem Biophys Res Commun ; 577: 80-88, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34509082

RESUMO

Atherosclerosis still remains the leading cause of morbidity and mortality worldwide, and deeper understanding of target signaling that protect from the atherosclerosis progression may provide novel therapeutic strategies. CDGSH iron-sulfur domain-containing protein 1 (CISD1) is a protein localized on the outer membrane of mitochondria, and plays key roles in regulating cell death and oxidative stress. However, its potential on atherosclerosis development and the underlying mechanisms are largely unknown. Here, in our study, we found markedly decreased CISD1 expression in lipid-laden THP1 macrophages. Notably, lentivirus (LV)-mediated CISD1 over-expression remarkably ameliorated lipid deposition in macrophages stimulated by ox-LDL. Furthermore, cellular total ROS and mitochondrial ROS generation, and impairment of mitochondrial membrane potential (MMP) were highly induced by ox-LDL in THP1 cells, while being considerably reversed upon CISD1 over-expression. Inflammatory response caused by ox-LDL was also significantly restrained in macrophages with CISD1 over-expression. Mechanistically, we found that CISD1 could interact with dynamin-related protein 1 (Drp1). Intriguingly, CISD1-improved mitochondrial dysfunction and inflammation in ox-LDL-treated macrophages were strongly abolished by Drp1 over-expression, indicating that Drp1 suppression might be necessary for CISD1 to perform its protective effects in vitro. In high fat diet (HFD)-fed apolipoprotein E-deficient (ApoE-/-) mice, tail vein injection of lentiviral vector expressing CISD1 remarkably decreased atherosclerotic lesion area, serum LDL cholesterol levels and triglyceride contents. Inflammatory response, cellular total and mitochondrial ROS production, and Drp1 expression levels in aorta tissues were also dramatically ameliorated in HFD-fed ApoE-/- mice, contributing to the inhibition of atherosclerosis in vivo. Therefore, improving CISD1 expression may be a novel therapeutic strategy for atherosclerosis treatment.


Assuntos
Aterosclerose/metabolismo , Dinaminas/metabolismo , Inflamação/metabolismo , Metabolismo dos Lipídeos , Proteínas Mitocondriais/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Western Blotting , Dieta Hiperlipídica/efeitos adversos , Humanos , Inflamação/genética , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Substâncias Protetoras/metabolismo , Ligação Proteica , Células THP-1
5.
Elife ; 102021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34545812

RESUMO

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Dinaminas/metabolismo , Dinâmica Mitocondrial , Células-Tronco/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Ciclina E/genética , Ciclina E/metabolismo , Dinaminas/genética , Células HaCaT , Humanos , Queratina-15/genética , Queratina-15/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Fosforilação , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
6.
J Biol Chem ; 297(4): 101196, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34529976

RESUMO

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.


Assuntos
Sinalização do Cálcio , Dinaminas/metabolismo , Mitocôndrias Musculares/metabolismo , Dinâmica Mitocondrial , Linhagem Celular , Dinaminas/genética , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias Musculares/genética , Oxirredução
7.
Exp Cell Res ; 407(2): 112828, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34508745

RESUMO

BACKGROUND: Particulate matter≤ 2.5 µm (PM2.5) is a type of environmental agent associated with air pollution, which induces hepatic fibrosis. However, the function and mechanism of PM2.5 on hepatic stellate cell (HSC) proliferation and fibrosis remain largely unknown. METHODS: Human HSC line (LX-2) and murine HSCs were exposed to various doses of PM2.5. microRNA (miR)-411 expression was detected via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation, fibrosis, mitochondrial dynamics dysfunction and mitophagy were determined via cell counting kit-8 (CCK-8), qRT-PCR, Western blotting and immunofluorescence. RESULTS: PM2.5 facilitated HSC proliferation and fibrosis via increasing the levels of ACTA2, Collagen 1, TIMP1 and TGF-ß1. PM2.5 reduced miR-411 expression, and contributed to mitochondrial dynamics dysfunction via increasing Drp1 and decreasing OPA1, TOM20 and PGC-1α levels. PM2.5 promoted mitophagy by upregulating the levels of Beclin-1, LC3II/I, PINK1 and Parkin. miR-411 overexpression or autophagy blockage using 3-methyladenine (3-MA) relieved PM2.5-mediated cell proliferation and fibrosis-associated factor expression in HSCs. Drp1 was targeted by miR-411. miR-411 mitigated PM2.5-induced mitophagy via targeting Drp1. Drp1 overexpression abolished the inhibitory role of miR-411 in cell proliferation and fibrosis-associated factor levels in HSCs. CONCLUSION: PM2.5 induced HSC activation and fibrosis via promoting Drp1-mediated mitophagy by decreasing miR-411, thereby causing liver fibrosis.


Assuntos
Dinaminas/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/patologia , MicroRNAs/genética , Dinâmica Mitocondrial , Mitofagia , Material Particulado/efeitos adversos , Animais , Autofagia , Proliferação de Células , Dinaminas/genética , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Camundongos , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
8.
Nat Commun ; 12(1): 4990, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404808

RESUMO

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Cálcio/metabolismo , Citoplasma , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Membranas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo
9.
Nanoscale ; 13(28): 12356-12369, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34254625

RESUMO

Previous studies have revealed that the liver is the main target organ of deposition for engineered nanoparticles. The hepatotoxicity of silver nanoparticles (AgNPs), the widely used antimicrobial nanoparticles, has been of great interest. However, little is known about the regulatory mechanism of the mitochondria in AgNP-induced hepatotoxicity. In the present study, we found that AgNPs, rather than silver ions, induced mitochondrial dynamics disorders, oxidative stress, and mitochondria-dependent hepatocyte apoptosis in mice. Using human hepatocellular carcinoma (HepG2) cells, we confirmed that the interaction between dynamin-related protein 1 (DRP1)-dependent mitochondrial fission and oxidative stress promoted mitochondrial damage and mitochondria-dependent apoptosis induced by AgNPs, as determined by the elimination of DRP1 or addition of N-acetylcysteine (NAC). Interestingly, the crosstalk between DRP1-dependent mitochondrial fission and oxidative stress also activated mitophagy and autophagy flux blocking. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene silencing contributed to the aggravation of mitochondrial damage, oxidative stress, and apoptosis. These results revealed that the interplay between mitochondrial fission and oxidative stress induced mitophagy defects and triggered AgNP-induced mitochondria-dependent apoptosis in liver cells both in vivo and in vitro. Our findings provide a perspective for the mechanism of hepatotoxicity induced by exposure to metal NPs.


Assuntos
Nanopartículas Metálicas , Dinâmica Mitocondrial , Animais , Apoptose , Dinaminas/metabolismo , Hepatócitos/metabolismo , Nanopartículas Metálicas/toxicidade , Camundongos , Estresse Oxidativo , Prata/toxicidade
10.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281194

RESUMO

Cockayne syndrome group A (CS-A) is a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. Cells derived from CS-A patients present as pathological hallmarks excessive oxidative stress, mitochondrial fragmentation and apoptosis associated with hyperactivation of the mitochondrial fission dynamin related protein 1 (DRP1). In this study, by using human cell models we further investigated the interplay between DRP1 and CSA and we determined whether pharmacological or genetic inhibition of DRP1 affects disease progression. Both reactive oxygen and nitrogen species are in excess in CS-A cells and when the mitochondrial translocation of DRP1 is inhibited a reduction of these species is observed together with a recovery of mitochondrial integrity and a significant decrease of apoptosis. This study indicates that the CSA-driven modulation of DRP1 pathway is key to control mitochondrial homeostasis and apoptosis and suggests DRP1 as a potential target in the treatment of CS patients.


Assuntos
Síndrome de Cockayne/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Síndrome de Cockayne/fisiopatologia , Progressão da Doença , Dinaminas/genética , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Estresse Oxidativo , Quinazolinonas/metabolismo , Quinazolinonas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
11.
Cancer Sci ; 112(10): 4013-4025, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34252226

RESUMO

Although the role of bromodomain-containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP-activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin-related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c-Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.


Assuntos
Apoptose/fisiologia , Azepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Melanoma/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Respiração Celular/efeitos dos fármacos , Dinaminas/efeitos dos fármacos , Dinaminas/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo
12.
Cryobiology ; 102: 42-55, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34331901

RESUMO

Hypothermia is a valuable clinical tool in mitigating against the consequences of ischemia in surgery, stroke, cardiac arrest and organ preservation. Protection is afforded principally by a reduction of metabolism, manifesting as reduced rates of oxygen uptake, preservation of ATP levels, and a curtailing of ischemic calcium overload. The effects of non-ischemic hypothermic stress are relatively unknown. We sought to investigate the effects of clinically mild-to-severe hypothermia on mitochondrial morphology, oxygen consumption and protein expression in normoxic hearts and cardiac cells. Normoxic perfusion of rat hearts at 28-32 °C was associated with inhibition of mitochondrial fission, evidenced by a reduced abundance of the active phosphorylated form of the fission receptor Drp1 (pDrp1S616). Abundance of the same residue was reduced in H9c2 cells subjected to hypothermic culture (25-32 °C), in addition to a reduced abundance of the Drp1 receptor MFF. Hypothermia-treated H9c2 cardiomyocytes exhibited elongated mitochondria and depressed rates of mitochondrial-associated oxygen consumption, which persisted upon rewarming. Hypothermia also promoted a reduction in mRNA expression of the capsaicin receptor TRPV1 in H9c2 cells. When normothermic H9c2 cells were transfected with TRPV1 siRNA we observed reduced pDrp1S616 and MFF abundance, elongated mitochondria, and reduced rates of mitochondrial-associated oxygen consumption, mimicking the effects of hypothermic culture. In conclusion hypothermia promoted elongation of cardiac mitochondria via reduced pDrp1S616 abundance which was also associated with suppression of cellular oxygen consumption. Silencing of TRPV1 in H9c2 cardiomyocytes reproduced the morphological and respirometric phenotype of hypothermia. This report demonstrates a novel mechanism of cold-induced inhibition of mitochondrial fission.


Assuntos
Dinaminas , Hipotermia , Animais , Criopreservação/métodos , Dinaminas/genética , Dinaminas/metabolismo , Hipotermia/metabolismo , Mitocôndrias , Miócitos Cardíacos/metabolismo , Ratos
13.
Cells ; 10(6)2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204906

RESUMO

High glucose (HG)-induced Drp1 overexpression contributes to mitochondrial dysfunction and promotes apoptosis in retinal endothelial cells. However, it is unknown whether inhibiting Drp1 overexpression protects against the development of retinal vascular cell loss in diabetes. To investigate whether reduced Drp1 level is protective against diabetes-induced retinal vascular lesions, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Drp1+/- mice, and STZ-induced diabetic Drp1+/- mice were examined after 16 weeks of diabetes. Western Blot analysis indicated a significant increase in Drp1 expression in the diabetic retinas compared to those of WT mice; retinas of diabetic Drp1+/- mice showed reduced Drp1 level compared to those of diabetic mice. A significant increase in the number of acellular capillaries (AC) and pericyte loss (PL) was observed in the retinas of diabetic mice compared to those of the WT control mice. Importantly, a significant decrease in the number of AC and PL was observed in retinas of diabetic Drp1+/- mice compared to those of diabetic mice concomitant with increased expression of pro-apoptotic genes, Bax, cleaved PARP, and increased cleaved caspase-3 activity. Preventing diabetes-induced Drp1 overexpression may have protective effects against the development of vascular lesions, characteristic of diabetic retinopathy.


Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Dinaminas , Vasos Retinianos/metabolismo , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/prevenção & controle , Dinaminas/genética , Dinaminas/metabolismo , Feminino , Masculino , Camundongos , Camundongos Mutantes
14.
J Physiol ; 599(17): 4045-4063, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34269418

RESUMO

KEY POINTS: The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in the muscle ageing process remains unclear. Here we report that both Drp1 knockdown and Drp1 overexpression late in life in mice is detrimental to skeletal muscle function and mitochondrial health. Drp1 knockdown in 18-month-old mice resulted in severe skeletal muscle atrophy, mitochondrial dysfunction, muscle degeneration/regeneration, oxidative stress and impaired autophagy. Overexpressing Drp1 in 18-month-old mice resulted in mild skeletal muscle atrophy and decreased mitochondrial quality. Our data indicate that silencing or overexpressing Drp1 late in life is detrimental to skeletal muscle integrity. We conclude that modulating Drp1 expression is unlikely to be a viable approach to counter the muscle ageing process. ABSTRACT: Sarcopenia, the ageing-related loss of skeletal muscle mass and function, is a debilitating process negatively impacting the quality of life of afflicted individuals. Although the mechanisms underlying sarcopenia are still only partly understood, impairments in mitochondrial dynamics, and specifically mitochondrial fission, have been proposed as an underlying mechanism. Importantly, conflicting data exist in the field and both excessive and insufficient mitochondrial fission were proposed to contribute to sarcopenia. In Drosophila melanogaster, enhancing mitochondrial fission in midlife through overexpression of dynamin-1-like protein (Drp1) extended lifespan and attenuated several key hallmarks of muscle ageing. Whether a similar outcome of Drp1 overexpression is observed in mammalian muscles remains unknown. In this study, we investigated the impact of knocking down and overexpressing Drp1 protein for 4 months in skeletal muscles of late middle-aged (18 months) mice using intra-muscular injections of adeno-associated viruses expressing shRNA targeting Drp1 or full Drp1 cDNA. We report that knocking down Drp1 expression late in life triggers severe muscle atrophy, mitochondrial dysfunctions, degeneration/regeneration, oxidative stress and impaired autophagy. Drp1 overexpression late in life triggered mild muscle atrophy and decreased mitochondrial quality. Taken altogether, our results indicate that both overexpression and silencing of Drp1 in late middle-aged mice negatively impact skeletal muscle mass and mitochondrial health. These data suggest that Drp1 content must remain within a narrow physiological range to preserve muscle and mitochondrial integrity during ageing. Altering Drp1 expression is therefore unlikely to be a viable target to counter sarcopenia.


Assuntos
Drosophila melanogaster , Dinâmica Mitocondrial , Animais , Proteínas do Citoesqueleto/metabolismo , Drosophila melanogaster/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Proteínas de Ligação ao GTP , Camundongos , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Qualidade de Vida
15.
Cells ; 10(5)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068960

RESUMO

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.


Assuntos
Dinaminas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miocárdio/metabolismo , Tretinoína/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial
16.
Biochem Biophys Res Commun ; 559: 113-120, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33940381

RESUMO

Retinal damage caused by blue light has become an important public health concern. Mitochondria have been found to play a key role in light-induced retinal cell death. In this study, we aimed to clarify the molecular mechanism involved in mitochondrion-related retinal cell damage caused by blue light, the major component of light-emitting diodes (LEDs). Our results show that blue light (450 nm, 300lux)-induced R28 cell death is caspase independent and can be attenuated by necrostatin-1. Apoptosis-inducing factor (AIF) cleavage and translocation to the nucleus are involved in the cell death progress. Blue light exposure causes mitochondrial fragmentation, which is mediated by phosphorylation at dynamin-related protein 1 (Drp1) Ser616 site, but it does not alter the protein levels of fission or fusion machinery. Knocking down Drp1 or treatment with Drp1 inhibitor Mdivi-1 protects R28 cells from blue light. Overproduction of reactive oxygen species (ROS) is induced by blue light. The ROS scavenger Trolox decreases Drp1 Ser616 phosphorylation level and mitochondrial fragmentation upon blue light exposure. Moreover, Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 blocks Drp1 phosphorylation and rescues mitochondrial fragmentation and AIF-mediated cell death caused by blue light. In conclusion, our data suggest that the CaMKII-Drp1 pathway plays a major role in blue light-induced AIF-mediated retinal cell damage.


Assuntos
Fator de Indução de Apoptose/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dinaminas/metabolismo , Necroptose , Retina/citologia , Animais , Linhagem Celular , Fosforilação , Ratos , Retina/metabolismo
17.
Biomed Pharmacother ; 140: 111689, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34004510

RESUMO

Vascular smooth muscle cell (VSMC) phenotypic switch plays an essential role in the pathogenesis of hypertension. Mitochondrial dynamics, such as mitochondrial fission, can also contribute to VSMC phenotypic switch. Whether mitochondrial fission act as a novel target for anti-hypertensive drug development remains unknown. In the present study, we confirmed that angiotensin II (AngII) rapidly and continuously induced mitochondrial fission in VSMCs. We also detected the phosphorylation status of dynamin-related protein-1 (Drp1), a key protein involved in mitochondrial fission, at Ser616 site; and observed Drp1 mitochondrial translocation in VSMCs or arteries of AngII-induced hypertensive mice. The Drp1 inhibitor mitochondrial division inhibitor-1 (Mdivi-1) dramatically reversed AngII-induced Drp1 phosphorylation, mitochondrial fission, and reactive oxidative species generation. Treatment with Mdivi-1 (20 mg/kg/every other day) significantly attenuated AngII-induced hypertension (22 mmHg), arterial remodeling, and cardiac hypertrophy, in part by preventing VSMC phenotypic switch. In addition, Mdivi-1 treatment was not associated with liver or renal functional injury. Collectively, these results indicate that Mdivi-1 inhibited mitochondrial fission, recovered mitochondrial activity, and prevented AngII-induced VSMC phenotypic switch, resulting in reduced hypertension.


Assuntos
Angiotensina II/farmacologia , Hipertensão/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Quinazolinonas/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Células Cultivadas , Dinaminas/metabolismo , Hipertensão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
18.
Cell Prolif ; 54(6): e13048, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33948998

RESUMO

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.


Assuntos
Autofagia , Dinaminas/metabolismo , Proteína HMGB1/metabolismo , Sistema de Sinalização das MAP Quinases , Dinâmica Mitocondrial , Hipertensão Arterial Pulmonar/metabolismo , Animais , Células Cultivadas , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação , Hipertensão Arterial Pulmonar/patologia , Ratos Sprague-Dawley
19.
Cell Death Dis ; 12(5): 442, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947842

RESUMO

Despite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia-reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dinaminas/metabolismo , Fígado/irrigação sanguínea , Mitocôndrias Hepáticas/metabolismo , Traumatismo por Reperfusão/metabolismo , Adenosina/metabolismo , Animais , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/patologia , Traumatismo por Reperfusão/patologia
20.
Cell Death Dis ; 12(5): 445, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953167

RESUMO

Dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction is associated with synaptic injury in the diabetic brain. However, the dysfunctional mitochondria by Drp1 deletion in the diabetic brain are poorly understood. Here, we investigated the effects of neuron-specific Drp1 deletion on synaptic damage and mitophagy in the hippocampus of a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. HFD/STZ-induced diabetic mice exhibited metabolic disturbances and synaptic damages. Floxed Drp1 mice were crossed with Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre mice, to generate neuron-specific Drp1 knockout (Drp1cKO) mice, which showed marked mitochondrial swelling and dendritic spine loss in hippocampal neurons. In particular, diabetic Drp1cKO mice exhibited an increase in dendritic spine loss and higher levels of oxidative stress and neuroinflammation compared with diabetic wild-type (WT) mice. Diabetic WT mice generally displayed increased Drp1-induced small mitochondrial morphology in hippocampal neurons, but large mitochondria were prominently observed in diabetic Drp1cKO mice. The levels of microtubule-associated protein 1 light-chain 3 and lysosomal-associated membrane protein 1 proteins were significantly increased in the hippocampus of diabetic Drp1cKO mice compared with diabetic WT mice. The inhibition of Drp1 adversely promotes synaptic injury and neurodegeneration in the diabetic brain. The findings suggest that the exploratory mechanisms behind Drp1-mediated mitochondrial dysfunction could provide a possible therapeutic target for diabetic brain complications.


Assuntos
Dinaminas/metabolismo , Hipocampo/metabolismo , Dinâmica Mitocondrial/imunologia , Animais , Camundongos
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