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1.
Arterioscler Thromb Vasc Biol ; 40(3): 583-596, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996026

RESUMO

OBJECTIVE: Inflammatory activation changes the mitochondrial function of macrophages from oxidative phosphorylation to reactive oxygen species production, which may promote necrotic core formation in atherosclerotic lesions. In hypoxic and cancer cells, HIF-1α (hypoxia-inducible factor) promotes oxygen-independent energy production by microRNAs. Therefore, we studied the role of HIF-1α in the regulation of macrophage energy metabolism in the context of atherosclerosis. Approach and Results: Myeloid cell-specific deletion of Hif1a reduced atherosclerosis and necrotic core formation by limiting macrophage necroptosis in apolipoprotein E-deficient mice. In inflammatory bone marrow-derived macrophages, deletion of Hif1a increased oxidative phosphorylation, ATP levels, and the expression of genes encoding mitochondrial proteins and reduced reactive oxygen species production and necroptosis. microRNA expression profiling showed that HIF-1α upregulates miR-210 and downregulates miR-383 levels in lesional macrophages and inflammatory bone marrow-derived macrophages. In contrast to miR-210, which inhibited oxidative phosphorylation and enhanced mitochondrial reactive oxygen species production, miR-383 increased ATP levels and inhibited necroptosis. The effect of miR-210 was due to targeting 2,4-dienoyl-CoA reductase, which is essential in the ß oxidation of unsaturated fatty acids. miR-383 affected the DNA damage repair pathway in bone marrow-derived macrophages by targeting poly(ADP-ribose)-glycohydrolase (Parg), which reduced energy consumption and increased cell survival. Blocking the targeting of Parg by miR-383 prevented the protective effect of Hif1a deletion in macrophages on atherosclerosis and necrotic core formation in mice. CONCLUSIONS: Our findings unveil a new mechanism by which activation of HIF-1α in inflammatory macrophages increases necroptosis through microRNA-mediated ATP depletion, thus increasing atherosclerosis by necrotic core formation.


Assuntos
Aorta/metabolismo , Aterosclerose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Necroptose , Trifosfato de Adenosina/metabolismo , Animais , Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Metabolismo Energético , Regulação da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/genética , Inflamação/patologia , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
2.
Circulation ; 138(18): 2007-2020, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-29748186

RESUMO

BACKGROUND: Alternative macrophage activation, which relies on mitochondrial oxidative metabolism, plays a central role in the resolution of inflammation and prevents atherosclerosis. Moreover, macrophages handle large amounts of cholesterol and triglycerides derived from the engulfed modified lipoproteins during atherosclerosis. Although several microRNAs regulate macrophage polarization, the role of the microRNA-generating enzyme Dicer in macrophage activation during atherosclerosis is unknown. METHODS: To evaluate the role of Dicer in atherosclerosis, Apoe-/- mice with or without macrophage-specific Dicer deletion were fed a high-fat diet for 12 weeks. Anti-argonaute 2 RNA immunoprecipitation chip and RNA deep sequencing combined with microRNA functional screening were performed in the Dicer wild-type and knockout bone marrow-derived macrophages to identify the individual microRNAs and the mRNA targets mediating the phenotypic effects of Dicer. The role of the identified individual microRNA and its target in atherosclerosis was determined by tail vein injection of the target site blockers in atherosclerotic Apoe-/- mice. RESULTS: We show that Dicer deletion in macrophages accelerated atherosclerosis in mice, along with enhanced inflammatory response and increased lipid accumulation in lesional macrophages. In vitro, alternative activation was limited whereas lipid-filled foam cell formation was exacerbated in Dicer-deficient macrophages as a result of impaired mitochondrial fatty acid oxidative metabolism. Rescue of microRNA (miR)-10a, let-7b, and miR-195a expression restored the oxidative metabolism in alternatively activated Dicer-deficient macrophages. Suppression of ligand-dependent nuclear receptor corepressor by miR-10a promoted fatty acid oxidation, which mediated the lipolytic and anti-inflammatory effect of Dicer. miR-10a expression was negatively correlated to the progression of atherosclerosis in humans. Blocking the interaction between ligand-dependent nuclear receptor corepressor and miR-10a by target site blockers aggravated atherosclerosis development in mice. CONCLUSIONS: Dicer plays an atheroprotective role by coordinately regulating the inflammatory response and lipid metabolism in macrophages through enhancing fatty acid-fueled mitochondrial respiration, suggesting that promoting Dicer/miR-10a-dependent metabolic reprogramming in macrophages has potential therapeutic implications to prevent atherosclerosis.


Assuntos
Aterosclerose/patologia , Macrófagos/metabolismo , Ribonuclease III/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Antagomirs/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Células da Medula Óssea/citologia , Dieta Hiperlipídica , Ácidos Graxos/química , Feminino , Humanos , Macrófagos/citologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Correpressor 2 de Receptor Nuclear/química , Correpressor 2 de Receptor Nuclear/metabolismo , Estresse Oxidativo , Ribonuclease III/genética
3.
EMBO J ; 31(14): 3038-62, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22735187

RESUMO

Neurons are critically dependent on mitochondrial integrity based on specific morphological, biochemical, and physiological features. They are characterized by high rates of metabolic activity and need to respond promptly to activity-dependent fluctuations in bioenergetic demand. The dimensions and polarity of neurons require efficient transport of mitochondria to hot spots of energy consumption, such as presynaptic and postsynaptic sites. Moreover, the postmitotic state of neurons in combination with their exposure to intrinsic and extrinsic neuronal stress factors call for a high fidelity of mitochondrial quality control systems. Consequently, it is not surprising that mitochondrial alterations can promote neuronal dysfunction and degeneration. In particular, mitochondrial dysfunction has long been implicated in the etiopathogenesis of Parkinson's disease (PD), based on the observation that mitochondrial toxins can cause parkinsonism in humans and animal models. Substantial progress towards understanding the role of mitochondria in the disease process has been made by the identification and characterization of genes causing familial variants of PD. Studies on the function and dysfunction of these genes revealed that various aspects of mitochondrial biology appear to be affected in PD, comprising mitochondrial biogenesis, bioenergetics, dynamics, transport, and quality control.


Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Animais , Humanos , Mitocôndrias/patologia , Neurônios/patologia , Doença de Parkinson/patologia
4.
Hum Mol Genet ; 22(14): 2829-41, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23525905

RESUMO

PTEN-induced kinase 1 (PINK1) is a serine/threonine kinase that is localized to mitochondria. It protects cells from oxidative stress by suppressing mitochondrial cytochrome c release, thereby preventing cell death. Mutations in Pink1 cause early-onset Parkinson's disease (PD). Consistently, mitochondrial function is impaired in Pink1-linked PD patients and model systems. Previously, in vitro analysis implied that the protective effects of PINK1 depend on phosphorylation of the downstream factor, TNF receptor-associated protein 1 (TRAP1). Furthermore, TRAP1 has been shown to mitigate α-Synuclein-induced toxicity, linking α-Synuclein directly to mitochondrial dysfunction. These data suggest that TRAP1 seems to mediate protective effects on mitochondrial function in pathways that are affected in PD. Here we investigated the potential of TRAP1 to rescue dysfunction induced by either PINK1 or Parkin deficiency in vivo and in vitro. We show that overexpression of human TRAP1 is able to mitigate Pink1 but not parkin loss-of-function phenotypes in Drosophila. In addition, detrimental effects observed after RNAi-mediated silencing of complex I subunits were rescued by TRAP1 in Drosophila. Moreover, TRAP1 was able to rescue mitochondrial fragmentation and dysfunction upon siRNA-induced silencing of Pink1 but not parkin in human neuronal SH-SY5Y cells. Thus, our data suggest a functional role of TRAP1 in maintaining mitochondrial integrity downstream of PINK1 and complex I deficits but parallel to or upstream of Parkin.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Doença de Parkinson/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Feminino , Técnicas de Inativação de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Masculino , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
EMBO J ; 29(20): 3571-89, 2010 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-20842103

RESUMO

Aggregation of α-synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA-mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin Δ1-79 or by DJ-1 C106A.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fusão de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/ultraestrutura , Proteínas Oncogênicas/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1 , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/genética
6.
EMBO Mol Med ; 13(8): e14323, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34151532

RESUMO

Very-low-carbohydrate diet triggers the endogenous production of ketone bodies as alternative energy substrates. There are as yet unproven assumptions that ketone bodies positively affect human immunity. We have investigated this topic in an in vitro model using primary human T cells and in an immuno-nutritional intervention study enrolling healthy volunteers. We show that ketone bodies profoundly impact human T-cell responses. CD4+ , CD8+ , and regulatory T-cell capacity were markedly enhanced, and T memory cell formation was augmented. RNAseq and functional metabolic analyses revealed a fundamental immunometabolic reprogramming in response to ketones favoring mitochondrial oxidative metabolism. This confers superior respiratory reserve, cellular energy supply, and reactive oxygen species signaling. Our data suggest a very-low-carbohydrate diet as a clinical tool to improve human T-cell immunity. Rethinking the value of nutrition and dietary interventions in modern medicine is required.


Assuntos
Dieta com Restrição de Carboidratos , Corpos Cetônicos , Humanos , Cetonas , Mitocôndrias , Espécies Reativas de Oxigênio
7.
J Biol Chem ; 284(34): 22938-51, 2009 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-19546216

RESUMO

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.


Assuntos
Proteínas de Drosophila/fisiologia , Mitocôndrias/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genética
8.
J Neurosci ; 27(45): 12413-8, 2007 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-17989306

RESUMO

Degeneration of dopaminergic neurons in the substantia nigra is characteristic for Parkinson's disease (PD), the second most common neurodegenerative disorder. Mitochondrial dysfunction is believed to contribute to the etiology of PD. Although most cases are sporadic, recent evidence points to a number of genes involved in familial variants of PD. Among them, a loss-of-function of phosphatase and tensin homolog-induced kinase 1 (PINK1; PARK6) is associated with rare cases of autosomal recessive parkinsonism. In HeLa cells, RNA interference-mediated downregulation of PINK1 results in abnormal mitochondrial morphology and altered membrane potential. Morphological changes of mitochondria can be rescued by expression of wild-type PINK1 but not by PD-associated PINK1 mutants. Moreover, primary cells derived from patients with two different PINK1 mutants showed a similar defect in mitochondrial morphology. Human parkin but not PD-associated mutants could rescue mitochondrial pathology in human cells like wild-type PINK1. Our results may therefore suggest that PINK1 deficiency in humans results in mitochondrial abnormalities associated with cellular stress, a pathological phenotype, which can be ameliorated by enhanced expression of parkin.


Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/patologia , Proteínas Quinases/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Células Cultivadas , Células HeLa , Humanos , Mitocôndrias/genética , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Fenótipo , Proteínas Quinases/deficiência , Proteínas Quinases/genética
9.
EMBO Mol Med ; 10(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29789341

RESUMO

CHCHD10 mutations are linked to amyotrophic lateral sclerosis, but their mode of action is unclear. In a 29-year-old patient with rapid disease progression, we discovered a novel mutation (Q108P) in a conserved residue within the coiled-coil-helix-coiled-coil-helix (CHCH) domain. The aggressive clinical phenotype prompted us to probe its pathogenicity. Unlike the wild-type protein, mitochondrial import of CHCHD10 Q108P was blocked nearly completely resulting in diffuse cytoplasmic localization and reduced stability. Other CHCHD10 variants reported in patients showed impaired mitochondrial import (C122R) or clustering within mitochondria (especially G66V and E127K) often associated with reduced expression. Truncation experiments suggest mitochondrial import of CHCHD10 is mediated by the CHCH domain rather than the proposed N-terminal mitochondrial targeting signal. Knockdown of Mia40, which introduces disulfide bonds into CHCH domain proteins, blocked mitochondrial import of CHCHD10. Overexpression of Mia40 rescued mitochondrial import of CHCHD10 Q108P by enhancing disulfide-bond formation. Since reduction in CHCHD10 inhibits respiration, mutations in its CHCH domain may cause aggressive disease by impairing mitochondrial import. Our data suggest Mia40 upregulation as a potential therapeutic salvage pathway.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Mitocondriais/genética , Adulto , Respiração Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Estudos de Associação Genética , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Interferência de RNA
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