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1.
Eur J Clin Invest ; 50(3): e13204, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31990365

RESUMO

BACKGROUND: The heart is a highly oxidative tissue, thus mitochondria play a major role in maintaining optimal cardiac function. Our previous study established a dietary-induced obese minipig with cardiac fibrosis. The aim of this study was to elucidate the role of mitochondrial dynamics in cardiac fibrosis of obese minipigs. DESIGN: Four-month-old Lee-Sung minipigs were randomly divided into two groups: a control group (C) and an obese group (O) by feeding a control diet or a high-fat diet (HFD) for 6 months. Exposure of H9c2 cardiomyoblasts to palmitate was used to explore the effects of high-fat on induction of myocardial injury in vitro. RESULTS: The O pigs displayed greater heart weight and cardiac collagen accumulation. Obese pigs exhibited a lower antioxidant capacity, ATP concentration, and higher oxidative stress in the left ventricle (LV). The HFD caused downregulation in protein expression of PGC-1α and OPA1, and upregulation of DRP1, FIS1, and PINK1 in the LV of O compared to C pigs. Furthermore, palmitate induced apoptosis and decreased ATP content in H9c2 cells. Palmitate elevated the protein expression of DRP1 and PINK1 in these cells. Inhibition of DRP1 protein expression by siDRP1 in H9c2 cells resulted in enhanced ATP and decreased palmitate-induced apoptosis. CONCLUSIONS: These results suggest that mitochondrial dynamics were linked to the progression of obesity-related cardiac injury. Inhibition of DRP1 after palmitate exposure in H9c2 cells resulted in improved ATP level and decreased apoptosis in vitro suggesting that mitochondrial fission serves a key role in progression of obesity-induced cardiac fibrosis.


Assuntos
Dinaminas/metabolismo , Cardiopatias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Dinaminas/genética , Fibrose/metabolismo , Mitocôndrias Cardíacas/metabolismo , Obesidade , Ratos , Respiração , Suínos , Porco Miniatura
2.
Nat Commun ; 10(1): 4432, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570755

RESUMO

The pathway of homeostatic IgG extravasation is not fully understood, in spite of its importance for the maintenance of host immunity, the management of autoantibody-mediated disorders, and the use of antibody-based biologics. Here we show in a murine model of pemphigus, a prototypic cutaneous autoantibody-mediated disorder, that blood-circulating IgG extravasates into the skin in a time- and dose-dependent manner under homeostatic conditions. This IgG extravasation is unaffected by depletion of Fcγ receptors, but is largely attenuated by specific ablation of dynamin-dependent endocytic vesicle formation in blood endothelial cells (BECs). Among dynamin-dependent endocytic vesicles, IgG co-localizes well with caveolae in cultured BECs. An Abl family tyrosine kinase inhibitor imatinib, which reduces caveolae-mediated endocytosis, impairs IgG extravasation in the skin and attenuates the murine pemphigus manifestations. Our study highlights the kinetics of IgG extravasation in vivo, which might be a clue to understand the pathological mechanism of autoantibody-mediated autoimmune disorders.


Assuntos
Imunoglobulina G/imunologia , Pênfigo/imunologia , Pênfigo/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pele/imunologia , Animais , Autoanticorpos/metabolismo , Doenças Autoimunes/metabolismo , Cavéolas , Modelos Animais de Doenças , Dinaminas/metabolismo , Orelha/patologia , Endocitose , Células Endoteliais/metabolismo , Feminino , Imunoglobulina G/sangue , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Pênfigo/patologia , Pele/patologia , Vesículas Transportadoras/metabolismo
3.
Nat Commun ; 10(1): 4462, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575863

RESUMO

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin's efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.


Assuntos
Dinaminas/química , Dinaminas/metabolismo , Endocitose/fisiologia , Domínios e Motivos de Interação entre Proteínas , Animais , Sítios de Ligação , Clatrina/farmacologia , Dinaminas/genética , Endocitose/efeitos dos fármacos , Técnicas de Inativação de Genes , Cinética , Ligantes , Camundongos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Proteômica , Domínios de Homologia de src
4.
Ecotoxicol Environ Saf ; 186: 109749, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31622878

RESUMO

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in various industrial fields. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. The present study was aimed to explore whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. We found that the mitochondrial dysfunction caused by Cr(VI) exposure was characterized by decreased mitochondrial respiratory chain complex (MRCC) I/II activities and levels, collapsed mitochondrial membrane potential (MMP), depleted ATP, and increased reactive oxygen species (ROS) level. Cr(VI) induced abnormal mitochondrial fission/fusion events, the antioxidant Nacetyl-L-cysteine (NAC) restored the abnormal mitochondrial function as well as the fission/fusion dynamics. ROS was the up-stream regulator of extracellular regulated protein kinases (ERK) signaling, and the application of a specific ERK1/2 inhibitor PD98059 confirmed that activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. We also demonstrated that treatment with dynamic-like protein 1 (DLP1)-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. We reached the conclusion that blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes, which may provide the new avenue for developing effective strategies to protect against Cr(VI)-induced hepatotoxicity.


Assuntos
Antioxidantes/farmacologia , Cromo/toxicidade , Dinaminas/farmacologia , Fígado/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Carcinógenos Ambientais , Dinaminas/metabolismo , Flavonoides/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/fisiopatologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais
5.
Eur J Pharmacol ; 861: 172617, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31430457

RESUMO

Mitochondrial dysfunctions contribute to brain injury in ischemic stroke while disturbance of mitochondrial dynamics results in mitochondrial dysfunction. Mitochondrial E3 ubiquitin ligase 1 (Mul1) involves in regulation of mitochondrial fission and fusion. This study aims to explore whether Mul1 contributes to brain injury in ischemic stroke and the underlying mechanisms. First, a rat ischemic stroke model was established by middle cerebral artery occlusion (MCAO), which showed ischemic injuries (increase in neurological deficit score and infarct volume) and upregulation of Mul1 in brain tissues. Next, Mul1 siRNAs were injected intracerebroventricularly to knockdown Mul1 expression, which evidently attenuated brain injuries (decrease in neurological deficit score, infarct volume and caspase-3 activity), restored mitochondrial dynamics and functions (decreases in mitochondrial fission and cytochrome c release while increase in ATP production), and restored protein levels of dynamin-related protein 1 (Drp1, a mitochondrial fission protein) and mitofusin2 (Mfn2, a mitochondrial fusion protein) through suppressing their sumoylation and ubiquitination, respectively. Finally, PC12 cells were cultured under hypoxic condition to mimic the ischemic stroke. Consistently, knockdown of Mul1 significantly reduced hypoxic injuries (decrease in apoptosis and LDH release), restored protein levels of Drp1 and Mfn2, recovered mitochondrial dynamics and functions (decreases in mitochondrial fission, mitochondrial membrane potential, reactive oxygen species production and cytochrome c release while increase in ATP production). Based on these observations, we conclude that upregulation of Mul1 contributes to brain injury in ischemic stroke rats and disturbs mitochondrial dynamics through sumoylation of Drp1 and ubiquitination of Mfn2.


Assuntos
Isquemia Encefálica/complicações , Encéfalo/patologia , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Hipóxia Celular , Modelos Animais de Doenças , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Células PC12 , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/enzimologia , Sumoilação , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Regulação para Cima
6.
J Recept Signal Transduct Res ; 39(3): 215-225, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31464538

RESUMO

Objective: PTEN has been acknowledged as an anticancer factor in the progression of glioblastoma. Mitochondrial division has been found to be associated with cancer cell death. Objective: The aim of our study is to explore whether PTEN attenuates the development of glioblastoma by modulating mitochondrial division. Materials and methods: PTEN adenovirus was used to overexpress PTEN in U87 cells. Mitochondrial function was detected via western blot and immunofluorescence. Pathway blocker was used to inhibit the Akt activation. Results: The results of our study demonstrated that PTEN overexpression reduced cell viability by increasing cell apoptosis. At the molecular level, PTEN overexpression activated mitochondrial apoptosis by mediating mitochondrial dysfunction. Furthermore, we found that Drp1-related mitochondrial division was required for PTEN-mediated mitochondrial dysfunction and cell death. Finally, we found that PTEN modulated Drp1-related mitochondrial division via the Akt pathway; inactivation of Akt induced cell death, and mitochondrial damage, similar to the results obtained via PTEN overexpression. Conclusions: Taken together, our results clarify that the anticancer mechanism of PTEN in glioblastoma is dependent on the activation of Drp1-related mitochondrial division via Akt pathway modulation. This finding might provide new insight into the tumor-suppressive role played by PTEN in glioblastoma.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Mitocôndrias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Encefálicas/patologia , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Dinaminas/metabolismo , Células HEK293 , Humanos
7.
Oxid Med Cell Longev ; 2019: 1232146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428220

RESUMO

Background: Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. Methods: Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. Results: Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. Conclusions: SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.


Assuntos
Dinaminas/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Substâncias Protetoras/farmacologia , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dinaminas/genética , Coração/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Substâncias Protetoras/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína SUMO-1/antagonistas & inibidores , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Sumoilação/efeitos dos fármacos
8.
Nat Commun ; 10(1): 3906, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467269

RESUMO

Mycobacterium tuberculosis infection remains a major threat to human health worldwide. Drug treatments against tuberculosis (TB) induce expression of several mycobacterial proteins, including IniA, but its structure and function remain poorly understood. Here, we report the structures of Mycobacterium smegmatis IniA in both the nucleotide-free and GTP-bound states. The structures reveal that IniA folds as a bacterial dynamin-like protein (BDLP) with a canonical GTPase domain followed by two helix-bundles (HBs), named Neck and Trunk. The distal end of its Trunk domain exists as a lipid-interacting (LI) loop, which binds to negatively charged lipids for membrane attachment. IniA does not form detectable nucleotide-dependent dimers in solution. However, lipid tethering indicates nucleotide-independent association of IniA on the membrane. IniA also deforms membranes and exhibits GTP-hydrolyzing dependent membrane fission. These results confirm the membrane remodeling activity of BDLP and suggest that IniA mediates TB drug-resistance through fission activity to maintain plasma membrane integrity.


Assuntos
Membrana Celular/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Fusão de Membrana/fisiologia , Mycobacterium smegmatis/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla , Dinaminas/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
9.
Oncol Rep ; 42(3): 1125-1132, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322265

RESUMO

Disturbed mitochondrial dynamics are closely associated with the progression of different types of cancer including hepatocellular carcinoma (HCC). However, the manner in which mitochondrial dynamics are regulated in HCC remains largely unclear. In the present study, via immunofluorescence, real­time PCR and western blot analysis, the effects of dynamin­1­like (DNM1L) on mitochondrial translocation and the mitochondrial permeability transition pore (mPTP) were investigated in HCC cells under hypoxic conditions, and the underlying molecular mechanisms were explored. Our data revealed that hypoxic treatment decreased the mitochondrial membrane potential, elevated generation of reactive oxygen species, and promoted mitochondrial fission in a DNM1L­dependent manner. Instead of changing the levels of DNMlL, hypoxia increased the translocation of DNM1L to mitochondria, leading to excessive mitochondrial fission and decreased the viability of HCC cells. In addition to the effects on mitochondrial dynamics, DNM1L also regulated mPTP opening in HCC. IP analysis revealed that DNM1L interacted with the enzyme hexokinase 2 (HK2), and was involved in its phosphorylation, resulting in HK2 detachment from the mitochondria and consequently mPTP opening.


Assuntos
Carcinoma Hepatocelular/patologia , Dinaminas/metabolismo , Hexoquinase/metabolismo , Hipóxia/fisiopatologia , Neoplasias Hepáticas/patologia , Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Dinaminas/genética , Hexoquinase/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas
10.
Chemosphere ; 234: 822-829, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31247492

RESUMO

Mercury is one of the 10 toxic chemicals with major public health concerns. Continuous exposure to low levels of heavy metals including mercury is related to renal injury, especially in children. This study investigated the possible molecular mechanism of inorganic mercury-induced kidney injury. Twenty eight Kunming mice were divided into four groups (n = 7), and treated with 0, 20, 40, 80 mg/L mercuric chloride (HgCl2) in drinking water for 16 weeks respectively. All the HgCl2 exposure mice displayed different degrees of renal injury, which was diagnosed by hematoxylin and eosin stain, biochemical analysis, and ultrastructure examination. The treatment of HgCl2 inhibited the silent information regulator two ortholog 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway and resulted the disorder of mitochondrial dynamics, as evidenced by the increasing expression of dynamin-related protein 1 and decreasing expression of mitofusin 2. Meanwhile, HgCl2 inhibited the nuclear factor erythroid 2-related factor 2 (Nrf2) axis. The abnormality of mitochondrial dynamics and the suppression of Nrf2 axis exacerbated oxidative stress, and then induced cell apoptosis. These findings demonstrated that the disorder of mitochondrial dynamics induced by HgCl2 activated oxidative stress, and further resulted in renal apoptosis through inhibiting the Sirt1/PGC-1α signaling pathway and the Nrf2 axis.


Assuntos
Apoptose , Rim/lesões , Cloreto de Mercúrio/toxicidade , Dinâmica Mitocondrial/efeitos dos fármacos , Estresse Oxidativo , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo
11.
Nat Commun ; 10(1): 2576, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189900

RESUMO

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.


Assuntos
Dinaminas/metabolismo , Mitocôndrias Musculares/patologia , Dinâmica Mitocondrial/fisiologia , Miopatias Mitocondriais/patologia , Músculo Esquelético/patologia , Animais , Cálcio/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Dinaminas/genética , Homeostase/fisiologia , Humanos , Camundongos , Camundongos Knockout , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/mortalidade , Músculo Esquelético/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
12.
Virol J ; 16(1): 80, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196105

RESUMO

BACKGROUND: Rabies virus (RABV), a member of Lyssavirus of Rhabdoviridae family, is a kind of negative-strand RNA virus. The zoonosis caused by RABV leads to high mortality in animals and humans. Though with the extensive investigation, the mechanisms of RABV entry into cells have not been well characterized. METHODS: Chemical inhibitors and RNA interference (RNAi) were used to analysis RABV internalization pathway. The expression level of viral N protein was examined by quantitative PCR and western blot, and the virus infection in the cells was visualized by fluorescence microscopy. RESULTS: We firstly examined the endocytosis pathway of the challenge virus standard (CVS) -11 strain in N2a cells. Chlorpromazine treatment and knockdown of clathrin heavy chain (CHC) significantly reduced viral entry, which proved clathrin was required. Meanwhile neither nystatin nor knocking down caveolin-1 (Cav1) in N2a cells had an effect on CVS-11 infection, suggesting that caveolae was independent for CVS-11 internalization. And when cholesterol of cell membrane was extracted by MßCD, viral infection was strongly impacted. Additionally by using the specific inhibitor dynasore and ammonium chloride, we verified that dynamin and a low-pH environment were crucial for RABV infection, which was confirmed by confocal microscopy. CONCLUSIONS: Our results demonstrated that CVS-11 entered N2a cells through a clathrin-mediated, cholesterol-, pH-, dynamin-required, and caveolae-independent endocytic pathway.


Assuntos
Colesterol/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitose , Vírus da Raiva/fisiologia , Internalização do Vírus , Linhagem Celular , Clorpromazina/farmacologia , Concentração de Íons de Hidrogênio , Proteínas do Nucleocapsídeo/genética , Interferência de RNA , Vírus da Raiva/efeitos dos fármacos
13.
Mol Biol Cell ; 30(16): 2037-2052, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31166831

RESUMO

The epithelial junctional complex, composed of tight junctions, adherens junctions, desmosomes, and an associated actomyosin cytoskeleton, forms the apical junctional ring (AJR), which must maintain its continuity in the face of external mechanical forces that accompany normal physiological functions. The AJR of umbrella cells, which line the luminal surface of the bladder, expands during bladder filling and contracts upon voiding; however, the mechanisms that drive these events are unknown. Using native umbrella cells as a model, we observed that the umbrella cell's AJR assumed a nonsarcomeric organization in which filamentous actin and ACTN4 formed unbroken continuous rings, while nonmuscle myosin II (NMMII) formed linear tracts along the actin ring. Expansion of the umbrella cell AJR required formin-dependent actin assembly, but was independent of NMMII ATPase function. AJR expansion also required membrane traffic, RAB13-dependent exocytosis, specifically, but not trafficking events regulated by RAB8A or RAB11A. In contrast, the voiding-induced contraction of the AJR depended on NMMII and actin dynamics, RHOA, and dynamin-dependent endocytosis. Taken together, our studies indicate that a mechanism by which the umbrella cells retain continuity during cyclical changes in volume is the expansion and contraction of their AJR, processes regulated by the actomyosin cytoskeleton and membrane trafficking events.


Assuntos
Polaridade Celular , Bexiga Urinária/citologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Junções Aderentes/metabolismo , Animais , Dinaminas/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Miosina Tipo II/metabolismo , Ratos Sprague-Dawley , Sarcômeros/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
14.
Cells ; 8(6)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208084

RESUMO

The maintenance of muscle mass and its ability to function relies on a bioenergetic efficient mitochondrial network. This network is highly impacted by fusion and fission events. We have recently shown that the acute deletion of the fusion protein Opa1 induces muscle atrophy, systemic inflammatory response, precocious epithelial senescence, and premature death that are caused by muscle-dependent secretion of FGF21. However, both fusion and fission machinery are suppressed in aging sarcopenia, cancer cachexia, and chemotherapy-induced muscle wasting. We generated inducible muscle-specific Opa1 and Drp1 double-knockout mice to address the physiological relevance of the concomitant impairment of fusion and fission machinery in skeletal muscle. Here we show that acute ablation of Opa1 and Drp1 in adult muscle causes the accumulation of abnormal and dysfunctional mitochondria, as well as the inhibition of autophagy and mitophagy pathways. This ultimately results in ER stress, muscle loss, and the reduction of force generation. However, the simultaneous inhibition of the fission protein Drp1 when Opa1 is absent alleviates FGF21 induction, oxidative stress, denervation, and inflammation rescuing the lethal phenotype of Opa1 knockout mice, despite the presence of any muscle weakness. Thus, the simultaneous inhibition of fusion and fission processes mitigates the detrimental effects of unbalanced mitochondrial fusion and prevents the secretion of pro-senescence factors.


Assuntos
Envelhecimento/patologia , GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Animais , Autofagia , Dinaminas/deficiência , Dinaminas/metabolismo , Estresse do Retículo Endoplasmático , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/deficiência , Camundongos Knockout , Mitocôndrias/patologia , Debilidade Muscular/complicações , Debilidade Muscular/patologia , Atrofia Muscular/complicações , Atrofia Muscular/patologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo
15.
J Diabetes Res ; 2019: 8463125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098384

RESUMO

Mitochondrial fission and fusion are dependent on cellular nutritional states, and maintaining this dynamics is critical for the health of cells. Starvation triggers mitochondrial fusion to maintain bioenergetic efficiency, but during nutrient overloads (as with hyperglycemic conditions), fragmenting mitochondria is a way to store nutrients to avoid waste of energy. In addition to ATP production, mitochondria play an important role in buffering intracellular calcium (Ca2+). We found that in cultured 661W cells, a photoreceptor-derived cell line, hyperglycemic conditions triggered an increase of the expression of dynamin-related protein 1 (DRP1), a protein marker of mitochondrial fission, and a decrease of mitofusin 2 (MFN2), a protein for mitochondrial fusion. Further, these hyperglycemic cells also had decreased mitochondrial Ca2+ but increased cytosolic Ca2+. Treating these hyperglycemic cells with melatonin, a multifaceted antioxidant, averted hyperglycemia-altered mitochondrial fission-and-fusion dynamics and mitochondrial Ca2+ levels. To mimic how people most commonly take melatonin supplements, we gave melatonin to streptozotocin- (STZ-) induced type 1 diabetic mice by daily oral gavage and determined the effects of melatonin on diabetic eyes. We found that melatonin was not able to reverse the STZ-induced systemic hyperglycemic condition, but it prevented STZ-induced damage to the neural retina and retinal microvasculature. The beneficial effects of melatonin in the neural retina in part were through alleviating STZ-caused changes in mitochondrial dynamics and Ca2+ buffering.


Assuntos
Retinopatia Diabética/metabolismo , Dinaminas/metabolismo , Melatonina/farmacologia , Dinâmica Mitocondrial/efeitos dos fármacos , Retina/patologia , Trifosfato de Adenosina/metabolismo , Angiografia , Animais , Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Eletrorretinografia , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Neoplasias da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
PLoS Pathog ; 15(4): e1007512, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947298

RESUMO

The single mitochondrion of apicomplexan protozoa is thought to be critical for all stages of the life cycle, and is a validated drug target against these important human and veterinary parasites. In contrast to other eukaryotes, replication of the mitochondrion is tightly linked to the cell cycle. A key step in mitochondrial segregation is the fission event, which in many eukaryotes occurs by the action of dynamins constricting the outer membrane of the mitochondria from the cytosolic face. To date, none of the components of the apicomplexan fission machinery have been identified and validated. We identify here a highly divergent, dynamin-related protein (TgDrpC), conserved in apicomplexans as essential for mitochondrial biogenesis and potentially for fission in Toxoplasma gondii. We show that TgDrpC is found adjacent to the mitochondrion, and is localised both at its periphery and at its basal part, where fission is expected to occur. We demonstrate that depletion or dominant negative expression of TgDrpC results in interconnected mitochondria and ultimately in drastic changes in mitochondrial morphology, as well as in parasite death. Intriguingly, we find that the canonical adaptor TgFis1 is not required for mitochondrial fission. The identification of an Apicomplexa-specific enzyme required for mitochondrial biogenesis and essential for parasite growth highlights parasite adaptation. This work paves the way for future drug development targeting TgDrpC, and for the analysis of additional partners involved in this crucial step of apicomplexan multiplication.


Assuntos
Dinaminas/metabolismo , Fibroblastos/metabolismo , Dinâmica Mitocondrial , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/metabolismo , Células Cultivadas , Dinaminas/genética , Fibroblastos/citologia , Fibroblastos/parasitologia , Humanos , Proteínas de Protozoários/genética , Toxoplasmose/genética , Toxoplasmose/parasitologia
17.
Neurotox Res ; 36(1): 27-38, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30993548

RESUMO

Alterations in the basal ganglia circuitry are critical events in the pathophysiology of Parkinson's disease (PD). We earlier compared MPTP-susceptible C57BL/6J and MPTP-resistant CD-1 mice to understand the differential prevalence of PD in different ethnic populations like Caucasians and Asian-Indians. The MPTP-resistant CD-1 mice had 33% more nigral neurons and lost only 15-17% of them following MPTP administration. In addition to other cytomorphological features, their basal ganglia neurons had higher calcium-buffering protein levels. During disease pathogenesis as well as in MPTP-induced parkinsonian models, the loss of nigral neurons is associated with reduction in mitochondrial complex-1. Under these conditions, mitochondria respond by undergoing fusion or fission. 17ß-hydroxysteroid type 10, i.e., hydroxysteroid dehydrogenase10 (HSD10) and dynamin-related peptide1 (Drp1) are proteins involved in mitochondrial hyperfusion and fission, respectively. Each plays an important role in mitochondrial structure and homeostasis. Their role in determining susceptibility to the neurotoxin MPTP in basal ganglia is however unclear. We studied their expression using immunohistochemistry and Western blotting in the dorsolateral striatum, ventral tegmental area, and substantia nigra pars compacta (SNpc) of C57BL/6J and CD-1 mice. In the SNpc, which exhibits more neuron loss following MPTP, C57BL/6J had higher baseline Drp1 levels; suggesting persistence of fission under normal conditions. Whereas, HSD10 levels increased in CD-1 following MPTP administration. This suggests mitochondrial hyperfusion, as an attempt towards neuroprotection. Thus, the baseline differences in HSD10 and DRP1 levels as well as their contrasting MPTP-responses may be critical determinants of the magnitude of neuronal loss/survival. Similar differences may determine the variable susceptibility to PD in humans.


Assuntos
Corpo Estriado/metabolismo , Neurônios Dopaminérgicos/metabolismo , Dinaminas/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Transtornos Parkinsonianos/metabolismo , Parte Compacta da Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Masculino , Camundongos Endogâmicos C57BL
18.
Cell Stress Chaperones ; 24(3): 595-608, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30993599

RESUMO

Irisin plays a protective effect in acute and chronic myocardial damage, but its role in septic cardiomyopathy is unclear. The aim of our study was to explore the in vivo and in vitro effects of irisin using an LPS-induced septic cardiomyopathy model. Our results demonstrated that irisin treatment attenuated LPS-mediated cardiomyocyte death and myocardial dysfunction. At the molecular level, LPS application was associated with mitochondrial oxidative injury, cardiomyocyte ATP depletion and caspase-related apoptosis activation. In contrast, the irisin treatment sustained mitochondrial function by inhibiting DRP1-related mitochondrial fission and the reactivation of mitochondrial fission impaired the protective action of irisin on inflammation-attacked mitochondria and cardiomyocytes. Additionally, we found that irisin modulated DRP1-related mitochondrial fission through the JNK-LATS2 signaling pathway. JNK activation and/or LATS2 overexpression abolished the beneficial effects of irisin on LPS-mediated mitochondrial stress and cardiomyocyte death. Altogether, our results illustrate that LPS-mediated activation of DRP1-related mitochondrial fission through the JNK-LATS2 pathway participates in the pathogenesis of septic cardiomyopathy. Irisin could be used in the future as an effective therapy for sepsis-induced myocardial depression because it corrects DRP1-related mitochondrial fission and normalizes the JNK-LATS2 signaling pathway.


Assuntos
Cardiomiopatias/tratamento farmacológico , Dinaminas/metabolismo , Fibronectinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Cardiomiopatias/induzido quimicamente , Lipopolissacarídeos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Miócitos Cardíacos/patologia
19.
Phytomedicine ; 59: 152922, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981186

RESUMO

BACKGROUND: Inflammation is a major contributor to stroke pathology, making it a promising strategy for intervention. Microglia, the resident macrophages in the brain, play essential roles in both the generation and resolution of neuroinflammation. In particular, mitochondrial homeostasis is critical for microglial function and its dysregulation is involved in the pathogenesis of ischemic stroke. Atractylenolide III (A III), a sesquiterpene lactone found in Atractylodes macrocephala Koidz, has been shown to have an inhibitory effect on inflammation. However, its effect specifically on neuroinflammation and microglial mitochondrial homeostasis following stroke remains elusive. HYPOTHESIS: We hypothesized that A III protects against brain ischemia through inhibition of neuroinflammation mediated by JAK2/STAT3/Drp1-dependent mitochondrial fission. METHODS: The neuroprotective and anti-neuroinflammatory effects of A III were investigated in vivo in mice with transient occlusion to the middle cerebral artery (MCAO) and in vitro in oxygen glucose deprivation-reoxygenation (OGDR)-stimulated primary microglia from mice. RESULTS: A III and AG490, an inhibitor of JAK2, treatment reduced brain infarct size, restored cerebral blood flow (CBF), ameliorated brain edema and improved neurological deficits in MCAO mice. Furthermore, A III and AG490 inhibited mRNA and protein expressions of proinflammatory (IL-1ß, TNF-α, and IL-6) and anti-inflammatory cytokines in both MCAO mice and OGDR-stimulated primary microglia. The JAK2/STAT3 pathway was effectively suppressed by A III, similar to the effect of AG490 treatment. In addition, A III and AG490 treatments significantly decreased Drp1 phosphorylation, translocation and mitochondrial fission in primary microglia stimulated with OGDR for 24 h. CONCLUSION: Our study demonstrated that A III was able to reduce complications associated with ischemia through inhibiting neuroinflammation, which was mediated in part by JAK2/STAT3-dependent mitochondrial fission in microglia.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Dinaminas/metabolismo , Inflamação/tratamento farmacológico , Janus Quinase 2/metabolismo , Lactonas/farmacologia , Fator de Transcrição STAT3/metabolismo , Sesquiterpenos/farmacologia , Animais , Isquemia Encefálica/patologia , Citocinas/metabolismo , Dinaminas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Interleucina-1beta/metabolismo , Janus Quinase 2/genética , Masculino , Camundongos , Microglia/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo
20.
Cell Mol Life Sci ; 76(10): 1967-1985, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30840087

RESUMO

Mitochondria are essential components of eukaryotic cells, carrying out critical physiological processes that include energy production and calcium buffering. Consequently, mitochondrial dysfunction is associated with a range of human diseases. Fundamental to their function is the ability to transition through fission and fusion states, which is regulated by several GTPases. Here, we have developed new methods for the non-subjective quantification of mitochondrial morphology in muscle and neuronal cells of Caenorhabditis elegans. Using these techniques, we uncover surprising tissue-specific differences in mitochondrial morphology when fusion or fission proteins are absent. From ultrastructural analysis, we reveal a novel role for the fusion protein FZO-1/mitofusin 2 in regulating the structure of the inner mitochondrial membrane. Moreover, we have determined the influence of the individual mitochondrial fission (DRP-1/DRP1) and fusion (FZO-1/mitofusin 1,2; EAT-3/OPA1) proteins on animal behaviour and lifespan. We show that loss of these mitochondrial fusion or fission regulators induced age-dependent and progressive deficits in animal movement, as well as in muscle and neuronal function. Our results reveal that disruption of fusion induces more profound defects than lack of fission on animal behaviour and tissue function, and imply that while fusion is required throughout life, fission is more important later in life likely to combat ageing-associated stressors. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology, with no correlation found between morphological changes and behavioural defects. Surprisingly, we find that disruption of either mitochondrial fission or fusion significantly reduces median lifespan, but maximal lifespan is unchanged, demonstrating that mitochondrial dynamics play an important role in limiting variance in longevity across isogenic populations. Overall, our study provides important new insights into the central role of mitochondrial dynamics in maintaining organismal health.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Longevidade/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Mutação , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Estimativa de Kaplan-Meier , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura
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