RESUMO
Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.
Assuntos
Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Morte Celular , HumanosRESUMO
Juxtaposition between endoplasmic reticulum (ER) and mitochondria is a common structural feature, providing the physical basis for intercommunication during Ca(2+) signalling; yet, the molecular mechanisms controlling this interaction are unknown. Here we show that mitofusin 2, a mitochondrial dynamin-related protein mutated in the inherited motor neuropathy Charcot-Marie-Tooth type IIa, is enriched at the ER-mitochondria interface. Ablation or silencing of mitofusin 2 in mouse embryonic fibroblasts and HeLa cells disrupts ER morphology and loosens ER-mitochondria interactions, thereby reducing the efficiency of mitochondrial Ca(2+) uptake in response to stimuli that generate inositol-1,4,5-trisphosphate. An in vitro assay as well as genetic and biochemical evidences support a model in which mitofusin 2 on the ER bridges the two organelles by engaging in homotypic and heterotypic complexes with mitofusin 1 or 2 on the surface of mitochondria. Thus, mitofusin 2 tethers ER to mitochondria, a juxtaposition required for efficient mitochondrial Ca(2+) uptake.
Assuntos
Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Doença de Charcot-Marie-Tooth/genética , Fibroblastos , GTP Fosfo-Hidrolases/deficiência , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Forma das OrganelasRESUMO
In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities.
Assuntos
Cálcio , Retículo Endoplasmático , GTP Fosfo-Hidrolases , Mitocôndrias , Proteínas Mitocondriais , Animais , Camundongos , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fígado/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Isoformas de Proteínas , Camundongos Knockout , Humanos , Camundongos Endogâmicos C57BL , Células HeLa , Processamento Alternativo , Estresse do Retículo EndoplasmáticoRESUMO
Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , GTP Fosfo-Hidrolases , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp , Ratos , Molécula 1 de Interação EstromalRESUMO
Marburg disease is a fulminant variant of multiple sclerosis (MS), in which the diagnosis may be particularly difficult and with high rates of mortality. We describe the case of a women with a clinical picture, radiographic features, and neuropathological findings consistent with the classical descriptions of Marburg disease. Initially, our patient did not improved with the acute phase treatments but later showed a good response to natalizumab (NTZ) treatment. This report highlights not only the utility of brain biopsy in the accurate diagnosis of this challenging condition but also the potential role of NTZ as an effective therapeutic option.
Assuntos
Esclerose Múltipla , Biópsia , Feminino , Humanos , Imageamento por Ressonância Magnética , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/tratamento farmacológico , Natalizumab/uso terapêuticoRESUMO
INTRODUCTION: Magnetic resonance imaging is recognized as the most important diagnostic test in the diagnosis of multiple sclerosis, differential diagnosis and evaluation of progression/therapeutic response. However, to make optimal use of magnetic resonance imaging in multiple sclerosis, the use of a standard, reproducible and comparable imaging protocol is of uttermost importance. In this context, the Portuguese Society of Neuroradiology and the Group of Studies of Multiple Sclerosis, after a joint discussion, appointed a committee of experts to create recommendations adapted to the national reality on the use of magnetic resonance imaging in multiple sclerosis. This document represents the second part of the first Portuguese consensus recommendations on the use of magnetic resonance imaging in multiple sclerosis in clinical practice. MATERIAL AND METHODS: The Portuguese Society of Neuroradiology and the Group of Studies of Multiple Sclerosis, after discussing the topic in national meetings and after a working group meeting held in Figueira da Foz, May 2017, appointed a committee of experts that have developed several standard protocols on the use of magnetic resonance imaging on multiple sclerosis by consensus. The document obtained was based on the best scientific evidence and expert opinion. Portuguese multiple sclerosis consultants and departments of neuroradiology scrutinized and reviewed the consensus paper; comments and suggestions were considered. Standardized strategies of magnetic resonance imaging referral in clinical practice for diagnosis and follow-up of multiple sclerosis were published in the first part of this paper. RESULTS: We provide magnetic resonance imaging acquisition protocols regarding multiple sclerosis diagnostic and monitoring and the information to be included in the report for application across Portuguese healthcare institutions. CONCLUSION: We hope that these first Portuguese magnetic resonance imaging guidelines will contribute to optimize multiple sclerosis management and improve patient care in Portugal.
Introdução: A ressonância magnética é considerada o exame complementar mais importante para o diagnóstico de esclerose múltipla, seus diagnósticos diferenciais e avaliação da sua progressão/resposta terapêutica. No entanto, para um uso ótimo desta ferramenta na esclerose múltipla, é essencial a aplicação de um protocolo de imagem padronizado, reprodutível e comparável. Neste contexto, o Grupo de Estudos de Esclerose Múltipla e a Sociedade Portuguesa de Neurorradiologia, após discussão conjunta, designaram um comité de peritos para a criação de recomendações adaptadas à realidade nacional sobre a utilização da ressonância magnética na esclerose múltipla. Este documento corresponde à segunda parte das primeiras recomendações de consenso portuguesas sobre a utilização da ressonância magnética na esclerose múltipla na prática clínica. Material e Métodos: O Grupo de Estudos de Esclerose Múltipla e a Sociedade Portuguesa de Neurorradiologia após discussão do tema em reuniões de âmbito nacional e de uma reunião do grupo de trabalho que teve lugar na Figueira da Foz em maio de 2017, designaram um comité de peritos que elaboraram por método de consenso protocolos padronizados sobre o uso da ressonância magnética na esclerose múltipla. O documento teve como base a melhor evidência científica e a opinião dos peritos. Posteriormente, o documento foi enviado para escrutínio à maioria dos responsáveis de consulta de esclerose múltipla e dos departamentos de neurorradiologia; tendo sido considerados os seus comentários e sugestões. As estratégias padronizadas de referenciação imagiológica na prática clínica para o diagnóstico e seguimento da esclerose múltipla foram publicadas na primeira parte deste artigo. Resultados: Neste artigo são propostos os protocolos de aquisição de ressonância magnética adequados para o diagnóstico e monitorização da esclerose múltipla, bem como a informação a constar do relatório imagiológico, tendo em vista a sua aplicação nas várias instituições de saúde portuguesas. Conclusão: Os autores esperam que estas primeiras orientações portuguesas sobre a utilização da ressonância magnética na esclerose múltipla na prática clínica contribuam para otimizar a gestão desta patologia e melhorar o tratamento destes doentes em Portugal.
Assuntos
Consenso , Imageamento por Ressonância Magnética , Esclerose Múltipla/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Humanos , Imageamento por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/normas , Portugal , Sociedades Médicas , Medula Espinal/diagnóstico por imagemRESUMO
Mitochondria are central organelles in metabolism, signal transduction, and programmed cell death. To meet their diverse functional demands, their shape is strictly regulated by a growing family of proteins that impinge on fission and fusion of the organelle. Mitochondrial fusion depends on Mitofusin (Mfn) 1 and 2, two integral outer-membrane proteins. Although MFN1 seems primarily involved in the regulation of the docking and fusion of the organelle, mounting evidence is implicating MFN2 in multiple signaling pathways not restricted to the regulation of mitochondrial shape. Here we review data supporting a role for this mitochondria-shaping protein beyond fusion, in regulating mitochondrial metabolism, apoptosis, shape of other organelles, and even progression through cell cycle. In conclusion, MFN2 appears a multifunctional protein whose biologic function is not restricted to the regulation of mitochondrial shape.
Assuntos
Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , GTP Fosfo-Hidrolases , Doenças Genéticas Inatas/fisiopatologia , Humanos , Proteínas de Membrana/química , Proteínas Mitocondriais/química , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Herein, we describe the preformulation study of Ala 125- recombinant human interleukin-2 (rhIL-2A(125)) in solution. This modified form of the natural human IL-2 is obtained by the replacement of cysteine with alanine at position 125. The compatibility of this rhIL-2A(125) with type I borosilicate glass vials showed no significant adsorption at liquid-vial interface. The effect of single excipients on the stability of this lymphokine was evaluated through RP-HPLC, SDS-PAGE and biological activity assay. Polysorbate 80 at high concentrations decreased the stability of rhIL-2A(125) in solution. On the other hand, the use of antioxidants (methionine and EDTA Na(2)) diminished the oxidation rate of the active ingredient. Additionally, a group of amino acids (glutamine, alanine, glycine and histidine) stabilized rhIL-2A(125) in different grades, and glycine at 5 mg mL(-1) allowed for the best stability behaviour. Taken together, these preformulation results can be used to design an adequate liquid vehicle for rhIL-2A(125) to be manufactured for human use.
Assuntos
Interleucina-2/química , Aminoácidos/química , Animais , Antioxidantes/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Embalagem de Medicamentos , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Excipientes , Humanos , Interleucina-2/farmacologia , Cinética , Camundongos , Soluções Farmacêuticas , Polissorbatos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
In this study, we analyzed the effect of Bcl-2 overexpression, an important anti-apoptotic protein, by using two hypothalamic cell lines, GT1-7puro or GT1-7bcl-2. 3-Nitropropionic acid (3-NP) mediated a dose-dependent decrease in cell viability in GT1-7puro cells, as determined by following the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, which was significantly prevented in GT1-7bcl-2 cells. In addition, activation of caspases-2, -3, and -6 induced by 3-NP was prevented by Bcl-2 overexpression. The data suggest that irreversible inhibition of mitochondrial complex II induces apoptotic features of cell death in a process prevented by Bcl-2.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Propionatos/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Hipotálamo/citologia , Hipotálamo/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Neurotoxinas/toxicidade , NitrocompostosRESUMO
Huntington's disease (HD) is a genetic neurodegenerative disorder characterized by striatal neurodegeneration, involving apoptosis. FK506, an inhibitor of calcineurin (or protein phosphatase 3, formerly known as protein phosphatase 2B), has shown neuroprotective effects in several cellular and animal models of HD. In the present study, we show the protective effects of FK506 in two striatal HD models, primary rat striatal neurons treated with 3-nitropropionic acid (3-NP) and immortalized striatal STHdh cells derived from HD knock-in mice expressing normal (STHdh(7/7)) or full-length mutant huntingtin (FL-mHtt) with 111 glutamines (STHdh(111/111)), under basal conditions and after exposure to 3-NP or staurosporine (STS). In rat striatal neurons, FK506 abolished 3-NP-induced increase in caspase-3 activation, DNA fragmentation/condensation and necrosis. Nevertheless, in STHdh(111/111) cells under basal conditions, FK506 did not prevent, in a significant manner, the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, or alter Bax/Bcl-2 ratio, but significantly reverted caspase-3 activation. In STHdh(111/111) cells treated with 0.3mM 3-NP or 25 nM STS, linked to high necrosis, exposure to FK506 exerted no significant effects on caspase-3 activation. However, treatment of STHdh(111/111) cells exposed to 10nM STS with FK506 effectively prevented cell death by apoptosis and moderate necrosis. The results suggest that FK506 may be neuroprotective against apoptosis and necrosis under mild cell death stimulus in the presence of FLmHtt.
Assuntos
Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Doença de Huntington/patologia , Imunossupressores/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , Tacrolimo/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Morte Celular , Linhagem Celular , Citosol/metabolismo , Fragmentação do DNA , Humanos , Proteína Huntingtina , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Necrose , Proteínas do Tecido Nervoso/genética , Nitrocompostos/toxicidade , Proteínas Nucleares/genética , Propionatos/toxicidade , Estaurosporina/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Communication between endoplasmic reticulum (ER) and mitochondria is crucial for Ca(2+) homeostasis, lipid biosynthesis and therefore for the regulation of mitochondrial metabolism and apoptosis. The mitochondrial GTPase mitofusin (MFN) 2 is enriched in mitochondria associated membranes (MAM) and localizes also on the ER, where it interacts with mitofusins on mitochondria to form interorganellar bridges. MFN2 also binds and inhibits the proto-oncogene Ras that controls proliferation, cell cycle and morphology. Mutants of MFN2 lacking the Ras-binding domain fail to tether the two organelles, raising the question of whether signaling cascades downstream of Ras can influence its ability to juxtapose ER and mitochondria. Here we show that extracellular regulated kinase (ERK) 1 is hyperactivated in cells lacking MFN2. However, genetic or pharmacological manipulation of the Ras-MAPK-ERK cascade does not influence the morphology of ER and mitochondria or their tethering. Thus, sustained Ras signaling is not the mechanism through which loss of MFN2 affects organelle shape and juxtaposition, solidifying a direct role for MFN2 in these processes.
Assuntos
Retículo Endoplasmático/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/ultraestrutura , Proteínas ras/metabolismo , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Deleção de Genes , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Ligação Proteica , Transdução de SinaisRESUMO
Mitochondria amplify activation of caspases during apoptosis by releasing cytochrome c and other cofactors. This is accompanied by fragmentation of the organelle and remodeling of the cristae. Here we provide evidence that Optic Atrophy 1 (OPA1), a profusion dynamin-related protein of the inner mitochondrial membrane mutated in dominant optic atrophy, protects from apoptosis by preventing cytochrome c release independently from mitochondrial fusion. OPA1 does not interfere with activation of the mitochondrial "gatekeepers" BAX and BAK, but it controls the shape of mitochondrial cristae, keeping their junctions tight during apoptosis. Tightness of cristae junctions correlates with oligomerization of two forms of OPA1, a soluble, intermembrane space and an integral inner membrane one. The proapoptotic BCL-2 family member BID, which widens cristae junctions, also disrupts OPA1 oligomers. Thus, OPA1 has genetically and molecularly distinct functions in mitochondrial fusion and in cristae remodeling during apoptosis.
Assuntos
Apoptose/genética , GTP Fosfo-Hidrolases/metabolismo , Fusão de Membrana/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/fisiologia , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The regulated equilibrium between mitochondrial fusion and fission is essential to maintain integrity of the organelle. Mechanisms of mitochondrial fusion are largely uncharacterized in mammalian cells. It is unclear whether OPA1, a dynamin-related protein of the inner membrane mutated in autosomal dominant optic atrophy, participates in fusion or fission. OPA1 promoted the formation of a branched network of elongated mitochondria, requiring the integrity of both its GTPase and C-terminal coiled-coil domain. Stable reduction of OPA1 levels by RNA interference resulted in small, fragmented, and scattered mitochondria. Levels of OPA1 did not affect mitochondrial docking, but they correlated with the extent of fusion as measured by polyethylene glycol mitochondrial fusion assays. A genetic analysis proved that OPA1 was unable to tubulate and fuse mitochondria lacking the outer membrane mitofusin 1 but not mitofusin 2. Our data show that OPA1 functionally requires mitofusin 1 to regulate mitochondrial fusion and reveal a specific functional difference between mitofusin 1 and 2.