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1.
Arterioscler Thromb Vasc Biol ; 40(7): e214-e226, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32493171

RESUMEN

OBJECTIVE: Mitochondria consistently change their morphology in a process regulated by proteins, including Drp1 (dynamin-related protein 1), a protein promoting mitochondrial fission. Drp1 is involved in the mechanisms underlying various cardiovascular diseases, such as myocardial ischemia/reperfusion injury, heart failure, and pulmonary arterial hypertension. However, its role in macrophages, which promote various vascular diseases, is poorly understood. We therefore tested our hypothesis that macrophage Drp1 promotes vascular remodeling after injury. METHOD AND RESULTS: To explore the selective role of macrophage Drp1, we created macrophage-selective Drp1-deficient mice and performed femoral arterial wire injury. In these mice, intimal thickening and negative remodeling were attenuated at 4 weeks after injury when compared with control mice. Deletion of macrophage Drp1 also attenuated the macrophage accumulation and cell proliferation in the injured arteries. Gain- and loss-of-function experiments using cultured macrophages indicated that Drp1 induces the expression of molecules associated with inflammatory macrophages. Morphologically, mitochondrial fission was induced in inflammatory macrophages, whereas mitochondrial fusion was induced in less inflammatory/reparative macrophages. Pharmacological inhibition or knockdown of Drp1 decreased the mitochondrial reactive oxygen species and chemotactic activity in cultured macrophages. Co-culture experiments of macrophages with vascular smooth muscle cells indicated that deletion of macrophage Drp1 suppresses growth and migration of vascular smooth muscle cells induced by macrophage-derived soluble factors. CONCLUSIONS: Macrophage Drp1 accelerates intimal thickening after vascular injury by promoting macrophage-mediated inflammation. Macrophage Drp1 may be a potential therapeutic target of vascular diseases.


Asunto(s)
Dinaminas/metabolismo , Arteria Femoral/metabolismo , Macrófagos Peritoneales/metabolismo , Mitocondrias/metabolismo , Neointima , Remodelación Vascular , Lesiones del Sistema Vascular/metabolismo , Animales , Proliferación Celular , Quimiotaxis , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Dinaminas/deficiencia , Dinaminas/genética , Arteria Femoral/lesiones , Arteria Femoral/patología , Arteria Femoral/fisiopatología , Activación de Macrófagos , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/patología , Dinámicas Mitocondriales , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factores de Tiempo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/patología , Lesiones del Sistema Vascular/fisiopatología
2.
J Cell Mol Med ; 24(2): 1516-1528, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31755231

RESUMEN

Mitochondrial fission and fusion are important for mitochondrial function, and dynamin 1-like protein (DNM1L) is a key regulator of mitochondrial fission. We investigated the effect of mitochondrial fission on mitochondrial function and inflammation in fibroblast-like synoviocytes (FLSs) during rheumatoid arthritis (RA). DNM1L expression was determined in synovial tissues (STs) from RA and non-RA patients. FLSs were isolated from STs and treated with a DNM1L inhibitor (mdivi-1, mitochondrial division inhibitor 1) or transfected with DNM1L-specific siRNA. Mitochondrial morphology, DNM1L expression, cell viability, mitochondrial membrane potential, reactive oxygen species (ROS), apoptosis, inflammatory cytokine expression and autophagy were examined. The impact of mdivi-1 treatment on development and severity of collagen-induced arthritis (CIA) was determined in mice. Up-regulated DNM1L expression was associated with reduced mitochondrial length in STs from patients with RA and increased RA severity. Inhibition of DNM1L in FLSs triggered mitochondrial depolarization, mitochondrial elongation, decreased cell viability, production of ROS, IL-8 and COX-2, and increased apoptosis. DNM1L deficiency inhibited IL-1ß-mediated AKT/IKK activation, NF-κBp65 nuclear translocation and LC3B-related autophagy, but enhanced NFKBIA expression. Treatment of CIA mice with mdivi-1 decreased disease severity by modulating inflammatory cytokine and ROS production. Our major results are that up-regulated DNM1L and mitochondrial fission promoted survival, LC3B-related autophagy and ROS production in FLSs, factors that lead to inflammation by regulating AKT/IKK/NFKBIA/NF-κB signalling. Thus, inhibition of DNM1L may be a new strategy for treatment of RA.


Asunto(s)
Artritis Reumatoide/patología , Dinaminas/antagonistas & inhibidores , Fibroblastos/patología , Inflamación/patología , Dinámicas Mitocondriales , Sinoviocitos/patología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Dinaminas/deficiencia , Dinaminas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos DBA , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/efectos de los fármacos , FN-kappa B/metabolismo , Quinazolinonas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Índice de Severidad de la Enfermedad , Sinoviocitos/efectos de los fármacos , Sinoviocitos/ultraestructura
3.
Cell Tissue Res ; 380(1): 143-153, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31811407

RESUMEN

We recently reported low-density lipoprotein receptor-related protein 6 (LRP6) decreased in dilated cardiomyopathy hearts, and cardiac-specific knockout mice displayed lethal heart failure through activation of dynamin-related protein 1 (Drp1). We also observed lipid accumulation in LRP6 deficiency hearts, but the detailed molecular mechanisms are unclear. Here, we detected fatty acids components in LRP6 deficiency hearts and explored the potential molecular mechanisms. Fatty acid analysis by GC-FID/MS revealed cardiac-specific LRP6 knockout induced the higher level of total fatty acids and some medium-long-chain fatty acids (C16:0, C18:1n9 and C18:2n6) than in control hearts. Carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of mitochondrial ß-oxidation in adult heart, was sharply decreased in LRP6 deficiency hearts, coincident with the activation of Drp1. Drp1 inhibitor greatly improved cardiac dysfunction and attenuated the increase in total fatty acids and fatty acids C16:0, C18:1n9 in LRP6 deficiency hearts. It also greatly inhibited the decrease in the cardiac expression of CPT1b and the transcriptional factors CCCTC-binding factor (CTCF) and c-Myc induced by cardiac-specific LRP6 knockout in mice. C-Myc but not CTCF was identified to regulate CPT1b expression and lipid accumulation in cardiomyocytes in vitro. The present study indicated cardiac-specific LRP6 knockout induced lipid accumulation by Drp1/CPT1b pathway in adult mice, and c-Myc is involved in the process. It suggests that LRP6 regulates fatty acid metabolism in adult heart.


Asunto(s)
Carnitina O-Palmitoiltransferasa/metabolismo , Dinaminas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Dinaminas/deficiencia , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Masculino , Ratones , Ratones Noqueados , Transducción de Señal , Transfección
4.
Biochem Biophys Res Commun ; 500(2): 497-503, 2018 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-29673589

RESUMEN

Stimulator of IFN genes (STING) is essential for the DNA-sensing innate immune pathway. Recently, evidence is emerging that suggests STING also plays important roles in autoimmunity, cancer therapy, and senescence. Although a multitude of post-translational modifications that regulate the STING pathway have been discovered, the cellular events that guide STING translocation remain unclear. Here, we show, paradoxically, that both BAPTA-AM-mediated calcium depletion and ionomycin-induced calcium elevation suppress STING translocation and STING-mediated IFN-ß production. We demonstrate that the mitochondria fission mediator DRP1 is crucial for ionomycin-induced inhibition of IFN-ß production. Furthermore, knockout of DRP1 suppressed ionomycin-induced increases in calcium as well as mitochondrial fragmentation. Collectively, our findings reveal that the induction of STING signaling is contingent on a fine-tuning of intracellular calcium levels.


Asunto(s)
Calcio/metabolismo , Espacio Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Animales , Dinaminas/deficiencia , Dinaminas/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Interferón beta/biosíntesis , Ionomicina/farmacología , Ratones , Células RAW 264.7
5.
Biochim Biophys Acta ; 1857(8): 1267-1276, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26997499

RESUMEN

During apoptosis mitochondria undergo cristae remodeling and fragmentation, but how the latter relates to outer membrane permeabilization and downstream caspase activation is unclear. Here we show that the mitochondrial fission protein Dynamin Related Protein (Drp) 1 participates in cytochrome c release by selected intrinsic death stimuli. While Bax, Bak double deficient (DKO) and Apaf1(-/-) mouse embryonic fibroblasts (MEFs) were less susceptible to apoptosis by Bcl-2 family member BID, H(2)O(2), staurosporine and thapsigargin, Drp1(-/-) MEFs were protected only from BID and H(2)O(2). Resistance to cell death of Drp1(-/-) and DKO MEFs correlated with blunted cytochrome c release, whereas mitochondrial fragmentation occurred in all cell lines in response to all tested stimuli, indicating that other mechanisms accounted for the reduced cytochrome c release. Indeed, cristae remodeling was reduced in Drp1(-/-) cells, potentially explaining their resistance to apoptosis. Our results indicate that caspase-independent mitochondrial fission and Drp1-dependent cristae remodeling amplify apoptosis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.


Asunto(s)
Apoptosis/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Dinaminas/genética , Fibroblastos/metabolismo , Dinámicas Mitocondriales/genética , Animales , Apoptosis/efectos de los fármacos , Factor Apoptótico 1 Activador de Proteasas/deficiencia , Factor Apoptótico 1 Activador de Proteasas/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular , Citocromos c/metabolismo , Dinaminas/deficiencia , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Noqueados , Dinámicas Mitocondriales/efectos de los fármacos , Estrés Oxidativo , Transducción de Señal , Estaurosporina/farmacología , Tapsigargina/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética
6.
Biochim Biophys Acta ; 1857(8): 1307-1312, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26992930

RESUMEN

Parkin is familiar to many because of its link to Parkinson's disease, and to others because of its well-characterized role as a central factor mediating selective mitophagy of damaged mitochondria for mitochondrial quality control. The genetic connection between Parkin and Parkinson's disease derives from clinical gene-association studies, whereas our mechanistic understanding of Parkin functioning in mitophagy is based almost entirely on work performed in cultured cells. Surprisingly, experimental evidence linking the disease and the presumed mechanism derives almost entirely from fruit flies; germline Parkin deficient mice do not develop Parkinson's disease phenotypes. Moreover, genetic manipulation of Parkin signaling in mouse hearts does not support a central role for Parkin in homeostatic mitochondrial quality control in this mitochondria-rich and -dependent organ. Here, I provide an overview of data suggesting that (in mouse hearts at least) Parkin functions more as a stress-induced and developmentally-programmed facilitator of cardiomyocyte mitochondrial turnover. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016.


Asunto(s)
Dinaminas/genética , Mitocondrias Cardíacas/metabolismo , Infarto del Miocardio/genética , Miocardio/metabolismo , Proteínas Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dinaminas/deficiencia , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/patología , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Mitofagia/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteínas Quinasas/deficiencia , Transducción de Señal , Ubiquitina-Proteína Ligasas/deficiencia
7.
J Am Soc Nephrol ; 27(9): 2733-47, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26825530

RESUMEN

Mitochondrial fission has been linked to the pathogenesis of diabetic nephropathy (DN). However, how mitochondrial fission affects progression of DN in vivo is unknown. Here, we report the effect of conditional podocyte-specific deletion of dynamin-related protein 1 (Drp1), an essential component of mitochondrial fission, on the pathogenesis and progression of DN. Inducible podocyte-specific deletion of Drp1 in diabetic mice decreased albuminuria and improved mesangial matrix expansion and podocyte morphology. Ultrastructure analysis revealed a significant increase in fragmented mitochondria in the podocytes of wild-type diabetic mice but a marked improvement in mitochondrial structure in Drp1-null podocytes of diabetic mice. When isolated from diabetic mice and cultured in high glucose, Drp1-null podocytes had more elongated mitochondria and better mitochondrial fitness associated with enhanced oxygen consumption and ATP production than wild-type podocytes. Furthermore, administration of a pharmacologic inhibitor of Drp1, Mdivi1, significantly blunted mitochondrial fission and rescued key pathologic features of DN in mice. Taken together, these results provide novel correlations between mitochondrial morphology and the progression of DN and point to Drp1 as a potential therapeutic target in DN.


Asunto(s)
Nefropatías Diabéticas/etiología , Dinaminas/deficiencia , Dinámicas Mitocondriales , Animales , Nefropatías Diabéticas/prevención & control , Progresión de la Enfermedad , Dinaminas/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Podocitos
8.
Proc Natl Acad Sci U S A ; 110(29): 11863-8, 2013 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-23821750

RESUMEN

Mammalian cells typically contain thousands of copies of mitochondrial DNA assembled into hundreds of nucleoids. Here we analyzed the dynamic features of nucleoids in terms of mitochondrial membrane dynamics involving balanced fusion and fission. In mitochondrial fission GTPase dynamin-related protein (Drp1)-deficient cells, nucleoids were enlarged by their clustering within hyperfused mitochondria. In normal cells, mitochondrial fission often occurred adjacent to nucleoids, since localization of Mff and Drp1 is dependent on the nucleoids. Thus, mitochondrial fission adjacent to nucleoids should prevent their clustering by maintaining small and fragmented nucleoids. The enhanced clustering of nucleoids resulted in the formation of highly stacked cristae structures in enlarged bulb-like mitochondria (mito-bulbs). Enclosure of proapoptotic factor cytochrome c, but not of Smac/DIABLO, into the highly stacked cristae suppressed its release from mitochondria under apoptotic stimuli. In the absence of nucleoids, Drp1 deficiency failed to form mito-bulbs and to protect against apoptosis. Thus, mitochondrial dynamics by fission and fusion play a critical role in controlling mitochondrial nucleoid structures, contributing to cristae reformation and the proapoptotic status of mitochondria.


Asunto(s)
Citocromos c/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/fisiología , Apoptosis/efectos de los fármacos , Benzotiazoles , Diaminas , Dinaminas/deficiencia , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Compuestos Orgánicos , Consumo de Oxígeno , Quinolinas , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de Tiempo
9.
J Neurosci ; 34(43): 14304-17, 2014 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-25339743

RESUMEN

Disruptions in mitochondrial dynamics may contribute to the selective degeneration of dopamine (DA) neurons in Parkinson's disease (PD). However, little is known about the normal functions of mitochondrial dynamics in these neurons, especially in axons where degeneration begins, and this makes it difficult to understand the disease process. To study one aspect of mitochondrial dynamics-mitochondrial fission-in mouse DA neurons, we deleted the central fission protein dynamin-related protein 1 (Drp1). Drp1 loss rapidly eliminates the DA terminals in the caudate-putamen and causes cell bodies in the midbrain to degenerate and lose α-synuclein. Without Drp1, mitochondrial mass dramatically decreases, especially in axons, where the mitochondrial movement becomes uncoordinated. However, in the ventral tegmental area (VTA), a subset of midbrain DA neurons characterized by small hyperpolarization-activated cation currents (Ih) is spared, despite near complete loss of their axonal mitochondria. Drp1 is thus critical for targeting mitochondria to the nerve terminal, and a disruption in mitochondrial fission can contribute to the preferential death of nigrostriatal DA neurons.


Asunto(s)
Axones/metabolismo , Neuronas Dopaminérgicas/metabolismo , Dinaminas/deficiencia , Mesencéfalo/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Animales , Axones/patología , Neuronas Dopaminérgicas/patología , Dinaminas/genética , Femenino , Masculino , Potenciales de la Membrana/fisiología , Mesencéfalo/patología , Ratones , Ratones Noqueados , Mitocondrias/patología , Técnicas de Cultivo de Órganos
10.
Biochem J ; 461(1): 137-46, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24758576

RESUMEN

Oxidative-stress-induced necrosis is considered to be one of the main pathological mediators in various neurological disorders, such as brain ischaemia. However, little is known about the mechanism by which cells modulate necrosis in response to oxidative stress. In the present study, we showed that Drp1 (dynamin-related protein 1), a primary mitochondrial fission protein, stabilizes the well-known stress gene p53 and is required for p53 translocation to the mitochondria under conditions of oxidative stress. We found that Drp1 binding to p53 induced mitochondria-related necrosis. In contrast, inhibition of Drp1 hyperactivation by Drp1 siRNA reduced necrotic cell death in cell cultures exposed to oxidative stress. Most significantly, we demonstrated that inhibition of Drp1 by the Drp1 peptide inhibitor P110, which was developed recently by our group, abolished p53 association with the mitochondria and reduced brain infarction in rats subjected to brain ischaemia/reperfusion injury. Taken together, these findings reveal a novel mechanism of Drp1 hyperactivation in the induction of mitochondrial damage and subsequent cell death. We propose that a Drp1 inhibitor such as P110 is a possible therapeutic agent for diseases in which hyperactivated Drp1 contributes to the pathology.


Asunto(s)
Dinaminas/fisiología , Potencial de la Membrana Mitocondrial , Estrés Oxidativo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Infarto Encefálico/genética , Infarto Encefálico/patología , Infarto Encefálico/prevención & control , Muerte Celular/genética , Células Cultivadas , Dinaminas/antagonistas & inhibidores , Dinaminas/deficiencia , Humanos , Células MCF-7 , Masculino , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Noqueados , Necrosis , Estrés Oxidativo/genética , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética
11.
BMC Microbiol ; 12: 298, 2012 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-23249255

RESUMEN

BACKGROUND: In eukaryotic cells, dynamin and flotillin are involved in processes such as endocytosis and lipid raft formation, respectively. Dynamin is a GTPase that exerts motor-like activity during the pinching off of vesicles, while flotillins are coiled coil rich membrane proteins with no known enzymatic activity. Bacteria also possess orthologs of both classes of proteins, but their function has been unclear. RESULTS: We show that deletion of the single dynA or floT genes lead to no phenotype or a mild defect in septum formation in the case of the dynA gene, while dynA floT double mutant cells were highly elongated and irregularly shaped, although the MreB cytoskeleton appeared to be normal. DynA colocalizes with FtsZ, and the dynA deletion strain shows aberrant FtsZ rings in a subpopulation of cells. The mild division defect of the dynA deletion is exacerbated by an additional deletion in ezrA, which affects FtsZ ring formation, and also by the deletion of a late division gene (divIB), indicating that DynA affects several steps in cell division. DynA and mreB deletions generated a synthetic defect in cell shape maintenance, showing that MreB and DynA play non-epistatic functions in cell shape maintenance. TIRF microscopy revealed that FloT forms many dynamic membrane assemblies that frequently colocalize with the division septum. The deletion of dynA did not change the pattern of localization of FloT, and vice versa, showing that the two proteins play non redundant roles in a variety of cellular processes. Expression of dynamin or flotillin T in eukaryotic S2 cells revealed that both proteins assemble at the cell membrane. While FloT formed patch structures, DynA built up tubulated structures extending away from the cells. CONCLUSIONS: Bacillus subtilis dynamin ortholog DynA plays a role during cell division and in cell shape maintenance. It shows a genetic link with flotillin T, with both proteins playing non-redundant functions at the cell membrane, where they assemble even in the absence of any bacterial cofactor.


Asunto(s)
Bacillus subtilis/citología , Bacillus subtilis/crecimiento & desarrollo , División Celular , Dinaminas/metabolismo , Proteínas de la Membrana/metabolismo , Bacillus subtilis/genética , Membrana Celular/metabolismo , Dinaminas/deficiencia , Eliminación de Gen , Proteínas de la Membrana/deficiencia , Unión Proteica , Multimerización de Proteína
12.
Cell Microbiol ; 12(12): 1809-20, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20690924

RESUMEN

Clostridium botulinum C2 toxin is an ADP-ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft-dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant-negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA-mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin-independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho-mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin-dependent mechanisms and we provide evidence for involvement of clathrin and Rho.


Asunto(s)
Toxinas Botulínicas/metabolismo , Clatrina/metabolismo , Clostridium botulinum/patogenicidad , Dinaminas/metabolismo , Endocitosis , Proteínas de Unión al GTP rho/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/antagonistas & inhibidores , Factores de Ribosilacion-ADP/metabolismo , Dinaminas/deficiencia , Eliminación de Gen , Silenciador del Gen , Células HeLa , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo
13.
Commun Biol ; 4(1): 894, 2021 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-34290349

RESUMEN

Mitochondrial fusion and fission, which are strongly related to normal mitochondrial function, are referred to as mitochondrial dynamics. Mitochondrial fusion defects in the liver cause a non-alcoholic steatohepatitis-like phenotype and liver cancer. However, whether mitochondrial fission defect directly impair liver function and stimulate liver disease progression, too, is unclear. Dynamin-related protein 1 (DRP1) is a key factor controlling mitochondrial fission. We hypothesized that DRP1 defects are a causal factor directly involved in liver disease development and stimulate liver disease progression. Drp1 defects directly promoted endoplasmic reticulum (ER) stress, hepatocyte death, and subsequently induced infiltration of inflammatory macrophages. Drp1 deletion increased the expression of numerous genes involved in the immune response and DNA damage in Drp1LiKO mouse primary hepatocytes. We administered lipopolysaccharide (LPS) to liver-specific Drp1-knockout (Drp1LiKO) mice and observed an increased inflammatory cytokine expression in the liver and serum caused by exaggerated ER stress and enhanced inflammasome activation. This study indicates that Drp1 defect-induced mitochondrial dynamics dysfunction directly regulates the fate and function of hepatocytes and enhances LPS-induced acute liver injury in vivo.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Dinaminas/deficiencia , Inflamación/inmunología , Lipopolisacáridos/farmacología , Animales , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL
14.
Cells ; 9(2)2020 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-32012656

RESUMEN

Mitochondria are involved in many cellular processes and their main role is cellular energy production. They constantly undergo fission and fusion, and these counteracting processes are under strict balance. The cytosolic dynamin-related protein 1, Drp1, or dynamin-1-like protein (DNM1L) mediates mitochondrial and peroxisomal division. Defects in the DNM1L gene result in a complex neurodevelopmental disorder with heterogeneous symptoms affecting multiple organ systems. Currently there is no curative treatment available for this condition. We have previously described a patient with a de novo heterozygous c.1084G>A (p.G362S) DNM1L mutation and studied the effects of a small molecule, bezafibrate, on mitochondrial functions in this patient's fibroblasts compared to controls. Bezafibrate normalized growth on glucose-free medium, as well as ATP production and oxygen consumption. It improved mitochondrial morphology in the patient's fibroblasts, although causing a mild increase in ROS production at the same time. A human foreskin fibroblast cell line overexpressing the p.G362S mutation showed aberrant mitochondrial morphology, which normalized in the presence of bezafibrate. Further studies would be needed to show the consistency of the response to bezafibrate, possibly using fibroblasts from patients with different mutations in DNM1L, and this treatment should be confirmed in clinical trials. However, taking into account the favorable effects in our study, we suggest that bezafibrate could be offered as a treatment option for patients with certain DNM1L mutations.


Asunto(s)
Bezafibrato/farmacología , Dinaminas/deficiencia , Dinámicas Mitocondriales/efectos de los fármacos , Adenosina Trifosfato/biosíntesis , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dinaminas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mutación/genética , Consumo de Oxígeno/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo
15.
Elife ; 82019 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-31603426

RESUMEN

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.


Asunto(s)
Encéfalo/metabolismo , Dendritas/metabolismo , Dinaminas/metabolismo , Endocitosis , Dinámicas Mitocondriales , Sinapsis/metabolismo , Animales , Dinaminas/deficiencia , Ratones , Ratones Noqueados , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/metabolismo
16.
Aging Cell ; 18(3): e12912, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30767411

RESUMEN

Abnormal mitochondrial dynamics contributes to mitochondrial dysfunction in Alzheimer's disease (AD), yet the underlying mechanism remains elusive. In the current study, we reported that DLP1, the key mitochondrial fission GTPase, is a substrate of calpain which produced specific N-terminal DLP1 cleavage fragments. In addition, various AD-related insults such as exposure to glutamate, soluble amyloid-ß oligomers, or reagents inducing tau hyperphosphorylation (i.e., okadaic acid) led to calpain-dependent cleavage of DLP1 in primary cortical neurons. DLP1 cleavage fragments were found in cortical neurons of CRND8 APP transgenic mice which can be inhibited by calpeptin, a potent small molecule inhibitor of calpain. Importantly, these N-terminal DLP1 fragments were also present in the human brains, and the levels of both full-length and N-terminal fragments of DLP1 and the full-length and calpain-specific cleavage product of spectrin were significantly reduced in AD brains along with significantly increased calpain. These results suggest that calpain-dependent cleavage is at least one of the posttranscriptional mechanisms that contribute to the dysregulation of mitochondrial dynamics in AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Calpaína/metabolismo , Dinaminas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Dinaminas/deficiencia , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos
17.
Cells ; 8(6)2019 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-31208084

RESUMEN

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.


Asunto(s)
Envejecimiento/patología , GTP Fosfohidrolasas/metabolismo , Dinámicas Mitocondriales , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Animales , Autofagia , Dinaminas/deficiencia , Dinaminas/metabolismo , Estrés del Retículo Endoplásmico , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , GTP Fosfohidrolasas/deficiencia , Ratones Noqueados , Mitocondrias/patología , Mitofagia , Debilidad Muscular/complicaciones , Debilidad Muscular/patología , Atrofia Muscular/complicaciones , Atrofia Muscular/patología , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo
18.
Neurochem Int ; 117: 77-81, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-28527629

RESUMEN

Purkinje neurons, one of the largest neurons in the brain, are critical for controlling body movements, and the dysfunction and degeneration of these cells cause ataxia. Purkinje neurons require a very efficient energy supply from mitochondria because of their large size and extensive dendritic arbors. We have previously shown that mitochondrial division mediated by dynamin-related protein 1 (Drp1) is critical for the development and survival of Purkinje neurons. Drp1 deficiency has been associated with one of the major types of ataxia: autosomal recessive spastic ataxia of Charlevoix Saguenay. Using post-mitotic Purkinje neuron-specific Drp1 knockout (KO) in mice, we investigated the molecular mechanisms that mediate the progressive degeneration of Drp1-KO Purkinje neurons in vivo. In these Purkinje neurons, p62/sequestosome-1, a multi-functional adaptor protein that balances apoptotic cell death and cell survival, was recruited to large mitochondria resulting from unopposed fusion in the absence of mitochondrial division. To test the role of p62 in Drp1-deficient neurodegeneration, we created mice lacking both Drp1 and p62 and found that the additional loss of p62 significantly extended the survival of Purkinje neurons lacking Drp1. These results provide insights into the neurodegenerative mechanisms of mitochondrial ataxia and a critical foundation for therapeutic interventions for this disease.


Asunto(s)
Dinaminas/deficiencia , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/prevención & control , Células de Purkinje/metabolismo , Proteína Sequestosoma-1/deficiencia , Animales , Dinaminas/genética , Técnicas de Inactivación de Genes/métodos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Degeneración Nerviosa/patología , Células de Purkinje/patología , Proteína Sequestosoma-1/genética
19.
J Comp Neurol ; 525(11): 2535-2548, 2017 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-28324645

RESUMEN

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation.


Asunto(s)
Dinaminas/deficiencia , Imagenología Tridimensional/métodos , Microscopía Electrónica de Rastreo/métodos , Dinámicas Mitocondriales/fisiología , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Animales , Dinaminas/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos
20.
FEBS Lett ; 590(20): 3544-3558, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27664110

RESUMEN

Mitochondria continually fuse and divide to dynamically adapt to changes in metabolism and stress. Mitochondrial dynamics are also required for mitochondrial DNA (mtDNA) integrity; however, the underlying reason is not known. In this study, we examined the link between mitochondrial fusion and mtDNA maintenance in Schizosaccharomyces pombe, which cannot survive without mtDNA, by screening for suppressors of the lethality induced by loss of the dynamin-related large GTPase Msp1p. Our findings reveal that inactivation of Msp1p induces a ROS-dependent nuclear mutator phenotype that affects mitochondrial fission genes involved in suppressing mitochondrial fragmentation and mtDNA depletion. This indicates that mitochondrial fusion is crucial for maintaining the integrity of both mitochondrial and nuclear genetic information. Furthermore, our study suggests that the primary roles of Msp1p are to organize mitochondrial membranes, thus making them competent for fusion, and maintain the integrity of mtDNA.


Asunto(s)
Dinaminas/deficiencia , GTP Fosfohidrolasas/deficiencia , Mitocondrias/fisiología , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/enzimología , ADN Mitocondrial/metabolismo , Regulación Fúngica de la Expresión Génica , Dinámicas Mitocondriales , Fenotipo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
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