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1.
Aging (Albany NY) ; 11(22): 10338-10355, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31751314

RESUMO

Glucocerebrosidase (GBA) mutations are the most important genetic risk factor for the development of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase). Loss-of-GCase activity in cellular models has implicated lysosomal and mitochondrial dysfunction in PD disease pathogenesis, although the exact mechanisms remain unclear. We hypothesize that GBA mutations impair mitochondria quality control in a neurosphere model.We have characterized mitochondrial content, mitochondrial function and macroautophagy flux in 3D-neurosphere-model derived from neural crest stem cells containing heterozygous and homozygous N370SGBA mutations, under carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP)- induced mitophagy.Our findings on mitochondrial markers and ATP levels indicate that mitochondrial accumulation occurs in mutant N370SGBA neurospheres under basal conditions, and clearance of depolarised mitochondria is impaired following CCCP-treatment. A significant increase in TFEB-mRNA levels, the master regulator of lysosomal and autophagy genes, may explain an unchanged macroautophagy flux in N370SGBA neurospheres. PGC1α-mRNA levels were also significantly increased following CCCP-treatment in heterozygote, but not homozygote neurospheres, and might contribute to the increased mitochondrial content seen in cells with this genotype, probably as a compensatory mechanism that is absent in homozygous lines.Mitochondrial impairment occurs early in the development of GCase-deficient neurons. Furthermore, impaired turnover of depolarised mitochondria is associated with early mitochondrial dysfunction.In summary, the presence of GBA mutation may be associated with higher levels of mitochondrial content in homozygous lines and lower clearance of damaged mitochondria in our neurosphere model.

2.
BMC Med Res Methodol ; 19(1): 112, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151417

RESUMO

BACKGROUND: From 2005 to 2010, we conducted 2 randomized studies on a journal (Medicina Clínica), where we took manuscripts received for publication and randomly assigned them to either the standard editorial process or to additional processes. Both studies were based on the use of methodological reviewers and reporting guidelines (RG). Those interventions slightly improved the items reported on the Manuscript Quality Assessment Instrument (MQAI), which assesses the quality of the research report. However, masked evaluators were able to guess the allocated group in 62% (56/90) of the papers, thus presenting a risk of detection bias. In this post-hoc study, we analyse whether those interventions that were originally designed for improving the completeness of manuscript reporting may have had an effect on the number of citations, which is the measured outcome that we used. METHODS: Masked to the intervention group, one of us used the Web of Science (WoS) to quantify the number of citations that the participating manuscripts received up December 2016. We calculated the mean citation ratio between intervention arms and then quantified the uncertainty of it by means of the Jackknife method, which avoids assumptions about the distribution shape. RESULTS: Our study included 191 articles (99 and 92, respectively) from the two previous studies, which all together received 1336 citations. In both studies, the groups subjected to additional processes showed higher averages, standard deviations and annual rates. The intervention effect was similar in both studies, with a combined estimate of a 43% (95% CI: 3 to 98%) increase in the number of citations. CONCLUSIONS: We interpret that those effects are driven mainly by introducing into the editorial process a senior methodologist to find missing RG items. Those results are promising, but not definitive due to the exploratory nature of the study and some important caveats such as: the limitations of using the number of citations as a measure of scientific impact; and the fact that our study is based on a single journal. We invite journals to perform their own studies to ascertain whether or not scientific repercussion is increased by adhering to reporting guidelines and further involving statisticians in the editorial process.

3.
Aging (Albany NY) ; 11(11): 3750-3767, 2019 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-31180333

RESUMO

PRKN encodes an E3-ubiquitin-ligase involved in multiple cell processes including mitochondrial homeostasis and autophagy. Previous studies reported alterations of mitochondrial function in fibroblasts from patients with PRKN mutation-associated Parkinson's disease (PRKN-PD) but have been only conducted in glycolytic conditions, potentially masking mitochondrial alterations. Additionally, autophagy flux studies in this cell model are missing.We analyzed mitochondrial function and autophagy in PRKN-PD skin-fibroblasts (n=7) and controls (n=13) in standard (glucose) and mitochondrial-challenging (galactose) conditions.In glucose, PRKN-PD fibroblasts showed preserved mitochondrial bioenergetics with trends to abnormally enhanced mitochondrial respiration that, accompanied by decreased CI, may account for the increased oxidative stress. In galactose, PRKN-PD fibroblasts exhibited decreased basal/maximal respiration vs. controls and reduced mitochondrial CIV and oxidative stress compared to glucose, suggesting an inefficient mitochondrial oxidative capacity to meet an extra metabolic requirement. PRKN-PD fibroblasts presented decreased autophagic flux with reduction of autophagy substrate and autophagosome synthesis in both conditions.The alterations exhibited under neuron-like oxidative environment (galactose), may be relevant to the disease pathogenesis potentially explaining the increased susceptibility of dopaminergic neurons to undergo degeneration. Abnormal PRKN-PD phenotype supports the usefulness of fibroblasts to model disease and the view of PD as a systemic disease where molecular alterations are present in peripheral tissues.

4.
Hum Mutat ; 40(10): 1700-1712, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31058414

RESUMO

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

5.
J Cell Mol Med ; 23(6): 3962-3973, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941904

RESUMO

Intrauterine growth restriction (IUGR) is an obstetric complication characterised by placental insufficiency and secondary cardiovascular remodelling that can lead to cardiomyopathy in adulthood. Despite its aetiology and potential therapeutics are poorly understood, bioenergetic deficits have been demonstrated in adverse foetal and cardiac development. We aimed to evaluate the role of mitochondria in human pregnancies with IUGR. In a single-site, cross-sectional and observational study, we included placenta and maternal peripheral and neonatal cord blood mononuclear cells (PBMC and CBMC) from 14 IUGR and 22 control pregnancies. The following mitochondrial measurements were assessed: enzymatic activities of mitochondrial respiratory chain (MRC) complexes I, II, IV, I + III and II + III, oxygen consumption (cell and complex I-stimulated respiration), mitochondrial content (citrate synthase [CS] activity and mitochondrial DNA copy number), total ATP levels and lipid peroxidation. Sirtuin3 expression was evaluated as a potential regulator of bioenergetic imbalance. Intrauterine growth restriction placental tissue showed a significant decrease of MRC CI enzymatic activity (P < 0.05) and CI-stimulated oxygen consumption (P < 0.05) accompanied by a significant increase of Sirtuin3/ß-actin protein levels (P < 0.05). Maternal PBMC and neonatal CBMC from IUGR patients presented a not significant decrease in oxygen consumption (cell and CI-stimulated respiration) and MRC enzymatic activities (CII and CIV). Moreover, CS activity was significantly reduced in IUGR new-borns (P < 0.05). Total ATP levels and lipid peroxidation were preserved in all the studied tissues. Altered mitochondrial function of IUGR is especially present at placental and neonatal level, conveying potential targets to modulate obstetric outcome through dietary interventions aimed to regulate Sirtuin3 function.

6.
J Transl Med ; 16(1): 160, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884186

RESUMO

BACKGROUND: Mutations in leucine rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). Mitochondrial and autophagic dysfunction has been described as etiologic factors in different experimental models of PD. We aimed to study the role of mitochondria and autophagy in LRRK2 G2019S -mutation, and its relationship with the presence of PD-symptoms. METHODS: Fibroblasts from six non-manifesting LRRK2 G2019S -carriers (NM-LRRK2 G2019S ) and seven patients with LRRK2 G2019S -associated PD (PD-LRRK2 G2019S ) were compared to eight healthy controls (C). An exhaustive assessment of mitochondrial performance and autophagy was performed after 24-h exposure to standard (glucose) or mitochondrial-challenging environment (galactose), where mitochondrial and autophagy impairment may be heightened. RESULTS: A similar mitochondrial phenotype of NM-LRRK2 G2019S and controls, except for an early mitochondrial depolarization (54.14% increased, p = 0.04), was shown in glucose. In response to galactose, mitochondrial dynamics of NM-LRRK2 G2019S improved (- 17.54% circularity, p = 0.002 and + 42.53% form factor, p = 0.051), probably to maintain ATP levels over controls. A compromised bioenergetic function was suggested in PD-LRRK2 G2019S when compared to controls in glucose media. An inefficient response to galactose and worsened mitochondrial dynamics (- 37.7% mitochondrial elongation, p = 0.053) was shown, leading to increased oxidative stress. Autophagy initiation (SQTSM/P62) was upregulated in NM-LRRK2 G2019S when compared to controls (glucose + 118.4%, p = 0.014; galactose + 114.44%, p = 0.009,) and autophagosome formation increased in glucose media. Despite of elevated SQSTM1/P62 levels of PD-NM G2019S when compared to controls (glucose + 226.14%, p = 0.04; galactose + 78.5%, p = 0.02), autophagosome formation was deficient in PD-LRRK2 G2019S when compared to NM-LRRK2 G2019S (- 71.26%, p = 0.022). CONCLUSIONS: Enhanced mitochondrial performance of NM-LRRK2 G2019S in mitochondrial-challenging conditions and upregulation of autophagy suggests that an exhaustion of mitochondrial bioenergetic and autophagic reserve, may contribute to the development of PD in LRRK2 G2019S mutation carriers.


Assuntos
Autofagia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitocôndrias/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Adulto , Idoso , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dinâmica Mitocondrial , Mutação/genética , Doença de Parkinson/epidemiologia , Fenótipo
7.
Neurobiol Aging ; 65: 206-216, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29501959

RESUMO

Mutations in the parkin gene (PRKN) are the most common cause of autosomal-recessive juvenile Parkinson's disease (PD). PRKN encodes an E3 ubiquitin ligase that is involved in multiple regulatory functions including proteasomal-mediated protein turnover, mitochondrial function, mitophagy, and cell survival. However, the precise molecular events mediated by PRKN mutations in PRKN-associated PD (PRKN-PD) remain unknown. To elucidate the cellular impact of parkin mutations, we performed an RNA sequencing study in skin fibroblasts from PRKN-PD patients carrying different PRKN mutations (n = 4) and genetically unrelated healthy subjects (n = 4). We identified 343 differentially expressed genes in PRKN-PD fibroblasts. Gene ontology and canonical pathway analysis revealed enrichment of differentially expressed genes in processes such as cell adhesion, cell growth, and amino acid and folate metabolism among others. Our findings indicate that PRKN mutations are associated with large global gene expression changes as observed in fibroblasts from PRKN-PD patients and support the view of PD as a systemic disease affecting also non-neural peripheral tissues such as the skin.


Assuntos
Fibroblastos , Mutação , Doença de Parkinson/genética , Transcriptoma , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Aminoácidos/metabolismo , Adesão Celular/genética , Processos de Crescimento Celular/genética , Células Cultivadas , Criança , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Ácido Fólico/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA , Pele/citologia , Ubiquitina-Proteína Ligases/fisiologia
8.
J Antimicrob Chemother ; 72(9): 2578-2586, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28859443

RESUMO

Background: HIV infection and HAART trigger genetic and functional mitochondrial alterations leading to cell death and adverse clinical manifestations. Mitochondrial dynamics enable mitochondrial turnover and degradation of damaged mitochondria, which may lead to apoptosis. Objectives: To evaluate markers of mitochondrial dynamics and apoptosis in pregnancies among HIV-infected women on HAART and determine their potential association with obstetric complications. Methods: This controlled, single-site, observational study without intervention included 26 HIV-infected pregnant women on HAART and 18 control pregnancies and their newborns. Maternal PBMCs and neonatal cord blood mononuclear cells (CBMCs) were isolated at the first trimester of gestation and at delivery. The placenta was homogenized at 5% w/v. Mitochondrial dynamics, fusion events [mitofusin 2 (Mfn2)/ß-actin] and fission events [dynamin-related protein 1 (Drp1/ß-actin)] and apoptosis (caspase 3/ß-actin) were assessed by western blot analysis. Results: Obstetric complications were significantly more frequent in pregnancies among HIV-infected women [OR 5.00 (95% CI 1.21-20.70)]. Mfn2/ß-actin levels in PBMCs from controls significantly decreased during pregnancy (202.13 ±â€¯57.45%), whereas cases maintained reduced levels from the first trimester of pregnancy and no differences were observed in CBMCs. Mfn2/ß-actin and Drp1/ß-actin contents significantly decreased in the placenta of cases. Caspase 3/ß-actin levels significantly increased during pregnancy in PBMCs of cases (50.00 ±â€¯7.89%), remaining significantly higher than in controls. No significant differences in caspase 3/ß-actin content of neonatal CBMCs were observed, but there was a slight increased trend in placenta from cases. Conclusions: HIV- and HAART-mediated mitochondrial damage may be enhanced by decreased mitochondrial dynamics and increased apoptosis in maternal and placental compartments but not in the uninfected fetus. However, direct effects on mitochondrial dynamics and implication of apoptosis were not demonstrated in adverse obstetric outcomes.


Assuntos
Fármacos Anti-HIV/efeitos adversos , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Apoptose/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Dinâmica Mitocondrial/efeitos dos fármacos , Complicações Infecciosas na Gravidez/tratamento farmacológico , Resultado da Gravidez , Adulto , Fármacos Anti-HIV/uso terapêutico , Caspase 3/genética , Feminino , GTP Fosfo-Hidrolases/genética , Infecções por HIV/virologia , Humanos , Recém-Nascido , Leucócitos Mononucleares , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Placenta/fisiologia , Gravidez , Complicações Infecciosas na Gravidez/virologia
9.
Stem Cells ; 35(7): 1687-1703, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28472853

RESUMO

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.


Assuntos
Ataxia/genética , Deficiência Intelectual/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Debilidade Muscular/genética , Rabdomiólise/genética , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Ataxia/enzimologia , Ataxia/patologia , Sistemas CRISPR-Cas , Diferenciação Celular , Pré-Escolar , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Evolução Fatal , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes/métodos , Expressão Gênica , Genes Letais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Deficiência Intelectual/enzimologia , Deficiência Intelectual/patologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/deficiência , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Debilidade Muscular/enzimologia , Debilidade Muscular/patologia , Cultura Primária de Células , Rabdomiólise/enzimologia , Rabdomiólise/patologia , Ubiquinona/genética
10.
J Transl Med ; 15(1): 29, 2017 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-28183315

RESUMO

BACKGROUND: Metabolic alterations play a role in the development of inflammatory myopathies (IMs). Herein, we have investigated through a multiplex assay whether proteins of energy metabolism could provide biomarkers of IMs. METHODS: A cohort of thirty-two muscle biopsies and forty plasma samples comprising polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (sIBM) and control donors was interrogated with monoclonal antibodies against proteins of energy metabolism using reverse phase protein microarrays (RPPA). RESULTS: When compared to controls the expression of the proteins is not significantly affected in the muscle of PM patients. However, the expression of ß-actin is significantly increased in DM and sIBM in consistence with muscle and fiber regeneration. Concurrently, the expression of some proteins involved in glucose metabolism displayed a significant reduction in muscle of sIBM suggesting a repression of glycolytic metabolism in these patients. In contrasts to these findings, the expression of the glycolytic pyruvate kinase isoform M2 (PKM2) and of the mitochondrial ATPase Inhibitor Factor 1 (IF1) and Hsp60 were significantly augmented in DM when compared to other IMs in accordance with a metabolic shift prone to cancer development. PKM2 alone or in combination with other biomarkers allowed the discrimination of control and IMs with very high (>95%) sensitivity and specificity. Unfortunately, plasma levels of PKM2 were not significantly altered in DM patients to recommend its use as a non-invasive biomarker of the disease. CONCLUSIONS: Expression of proteins of energy metabolism in muscle enabled discrimination of patients with IMs. RPPA identified the glycolysis promoting PKM2 and IF1 proteins as specific biomarkers of dermatomyositis, providing a biochemical link of this IM with oncogenesis.


Assuntos
Carcinogênese/metabolismo , Dermatomiosite/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Piruvato Quinase/metabolismo , Anticorpos/metabolismo , Biomarcadores/metabolismo , Biópsia , Análise por Conglomerados , Metabolismo Energético , Humanos , Inflamação/diagnóstico , Inflamação/patologia , Músculos/metabolismo , Músculos/patologia , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Frações Subcelulares/metabolismo
11.
J Acquir Immune Defic Syndr ; 75(4): e113-e119, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28234688

RESUMO

OBJECTIVE: To determine whether mitochondrial, oxidative, and apoptotic abnormalities in placenta derived from HIV and combined antiretroviral therapy (cART) containing zidovudine (AZT) could be associated with adverse perinatal outcome. DESIGN: Cross-sectional, controlled, observational study. METHODS: We studied obstetric results and mitochondrial, oxidative, and apoptotic state in placenta of 24 treated HIV-infected and 32 -uninfected pregnant women. We measured mitochondrial DNA (mtDNA) content by quantitative reverse transcriptase-polymerase chain reaction (mtND2/n18SrRNA), oxidative stress by the spectrophotometric quantification of lipid peroxidation and apoptosis by Western blot analysis of active caspase-3 respect to ß-actin content and analysis of the terminal deoxynucleotidyl transferase dUTP nick end labeling. RESULTS: Global adverse perinatal outcome (defined as preterm delivery or/and small newborns for gestational age) was significantly increased in HIV pregnancies [or 6.7 (1.3-33.2); P < 0.05]. mtDNA content in HIV-infected women was significantly depleted (39.20% ± 2.78%) with respect to controls (0.59 ± 0.03 vs. 0.97 ± 0.07; P < 0.001). A significant 29.50% ± 9.14% increase in oxidative stress was found in placentas of HIV-infected women (23.23 ± 1.64 vs. 17.94 ± 1.03; P < 0.01). A trend toward 41.18% ± 29.41% increased apoptosis active caspase-3/ß-actin was found in HIV patients (0.48 ± 0.10 vs. 0.34 ± 0.05; P = not significant), confirmed by transferase dUTP nick end labeling assay. Adverse perinatal outcome did not correlate mitochondrial, oxidative, or apoptotic findings. CONCLUSIONS: Placentas of HIV-infected pregnant women under AZT cART showed evidence of mtDNA depletion, increased oxidative stress levels, and apoptosis suggestive of secondary mitochondrial failure, potential base of associated adverse perinatal outcome. Despite the fact that further demonstration of causality would need new approaches and bigger sample sizes, AZT-sparing cART should be considered in the context of pregnancy.


Assuntos
Fármacos Anti-HIV/efeitos adversos , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Apoptose/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Troca Materno-Fetal/efeitos dos fármacos , Placenta/efeitos dos fármacos , Complicações Infecciosas na Gravidez/tratamento farmacológico , Zidovudina/efeitos adversos , Adulto , Fármacos Anti-HIV/uso terapêutico , Estudos de Casos e Controles , Contraindicações , Estudos Transversais , Feminino , Sangue Fetal/efeitos dos fármacos , Infecções por HIV/sangue , Infecções por HIV/complicações , Humanos , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Estresse Oxidativo , Gravidez , Complicações Infecciosas na Gravidez/sangue , Resultado da Gravidez , Efeitos Tardios da Exposição Pré-Natal , Espanha/epidemiologia , Zidovudina/uso terapêutico
12.
J Cell Mol Med ; 21(1): 26-34, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27577111

RESUMO

To assess the impact of HIV-infection and highly active anti-retroviral treatment in mitochondria and apoptotic activation of caspases during pregnancy and their association with adverse perinatal outcome. Changes of mitochondrial parameters and apoptotic caspase activation in maternal peripheral blood mononuclear cells were compared at first trimester of pregnancy and delivery in 27 HIV-infected and -treated pregnant women versus 24 uninfected pregnant controls. We correlated immunovirological, therapeutic and perinatal outcome with experimental findings: mitochondrial DNA (mtDNA) content, mitochondrial protein synthesis, mitochondrial function and apoptotic caspase activation. The HIV pregnancies showed increased adverse perinatal outcome (OR: 4.81 [1.14-20.16]; P < 0.05) and decreased mtDNA content (42.66 ± 5.94%, P < 0.01) compared to controls, even higher in naïve participants. This depletion caused a correlated decrease in mitochondrial protein synthesis (12.82 ± 5.73%, P < 0.01) and function (20.50 ± 10.14%, P < 0.001), not observed in controls. Along pregnancy, apoptotic caspase-3 activation increased 63.64 ± 45.45% in controls (P < 0.001) and 100.00 ± 47.37% in HIV-pregnancies (P < 0.001), in correlation with longer exposure to nucleoside analogues. HIV-infected women showed increased obstetric problems and declined genetic and functional mitochondrial parameters during pregnancy, especially those firstly exposed to anti-retrovirals. The apoptotic activation of caspases along pregnancy is emphasized in HIV pregnancies promoted by nucleoside analogues. However, we could not demonstrate direct mitochondrial or apoptotic implication in adverse obstetric outcome probably because of the reduced sample size.


Assuntos
Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Complicações Infecciosas na Gravidez/induzido quimicamente , Adulto , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , DNA Mitocondrial/genética , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Gravidez
13.
J Cell Mol Med ; 21(2): 402-409, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27758070

RESUMO

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or to study HIV reservoirs.


Assuntos
Apoptose , HIV-1/fisiologia , Linfócitos/virologia , Mitocôndrias/metabolismo , Modelos Biológicos , Monócitos/virologia , Linhagem Celular , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Linfócitos/metabolismo , Monócitos/metabolismo , Subunidades Proteicas/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo
15.
Clin Sci (Lond) ; 130(19): 1741-51, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27413019

RESUMO

Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in elderly people. Mitochondrial abnormalities at the histological level are present in these patients. We hypothesize that mitochondrial dysfunction may play a role in disease aetiology. We took the following measurements of muscle and peripheral blood mononuclear cells (PBMCs) from 30 sIBM patients and 38 age- and gender-paired controls: mitochondrial DNA (mtDNA) deletions, amount of mtDNA and mtRNA, mitochondrial protein synthesis, mitochondrial respiratory chain (MRC) complex I and IV enzymatic activity, mitochondrial mass, oxidative stress and mitochondrial dynamics (mitofusin 2 and optic atrophy 1 levels). Depletion of mtDNA was present in muscle from sIBM patients and PBMCs showed deregulated expression of mitochondrial proteins in oxidative phosphorylation. MRC complex IV/citrate synthase activity was significantly decreased in both tissues and mitochondrial dynamics were affected in muscle. Depletion of mtDNA was significantly more severe in patients with mtDNA deletions, which also presented deregulation of mitochondrial fusion proteins. Imbalance in mitochondrial dynamics in muscle was associated with increased mitochondrial genetic disturbances (both depletion and deletions), demonstrating that proper mitochondrial turnover is essential for mitochondrial homoeostasis and muscle function in these patients.


Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Idoso , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fosforilação Oxidativa
17.
Curr Drug Metab ; 17(7): 648-62, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27000075

RESUMO

BACKGROUND: Certain therapeutic drugs used in medical practice may trigger mitochondrial toxicity leading to a wide range of clinical symptoms including deafness, neuropathy, myopathy, hyperlactatemia, lactic acidosis, pancreatitis and lipodystrophy, among others, which could even compromise the life of the patient. OBJECTIVES: The aim of this work is to review the potential mitochondrial toxicity derived from drugs used in health care, including anesthetics, antiepileptics, neuroleptics, antidepressants, antivirals, antibiotics, antifungals, antimalarics, antineoplastics, antidiabetics, hypolipemiants, antiarrhythmics, anti-inflammatories and nitric oxide. METHODS: We herein have reviewed data from experimental and clinical studies to document the molecular mitochondrial basis, potential biomarkers and putative clinical symptoms associated to secondary effects of drugs. RESULTS: One hundred and forty-five articles were selected and the information was organized by means of the primary target to which pharmacologic drugs were directed. Adverse toxic events were classified depending on the mitochondrial offtarget effect and whether they had been demonstrated in the experimental or clinical setting. CONCLUSIONS: Since treatment of acquired mitochondriopathies remains supportive and therapeutic interventions cannot be avoided, information of molecular and clinical consequences of toxic exposure becomes fundamental to assess riskbenefit imbalance of treatment prescription. Additionally, there is a crucial need to develop less mitochondrial toxic compounds, novel biomarkers to follow up mitochondrial toxicity (or implement those already proposed) and new approaches to prevent or revert unintended mitochondrial damage.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Mitocôndrias/efeitos dos fármacos , Doenças Mitocondriais/induzido quimicamente , Animais , Humanos , Doenças Mitocondriais/tratamento farmacológico
18.
Am J Pathol ; 186(3): 517-23, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26784526

RESUMO

Niemann-Pick C disease is a neurovisceral disorder caused by mutations in the NPC gene that result in systemic accumulation of intracellular cholesterol. Although neurodegeneration defines the disease's severity, in most patients it is preceded by hepatic complications such as cholestatic jaundice or hepatomegaly. To analyze the contribution of the hepatic disease in Niemann-Pick C disease progression and to evaluate the degree of primary and secondary hepatic damage, we generated a transgenic mouse with liver-selective expression of NPC1 from embryonic stages. Hepatic NPC1 re-expression did not ameliorate the onset and progression of neurodegeneration of the NPC1-null animal. However, the mice showed reduced hepatomegalia and dramatic, although not complete, reduction of hepatic cholesterol and serum bile salts, bilirubin, and transaminase levels. Therefore, hepatic primary and secondary cholesterol deposition and damage occur simultaneously during Niemann-Pick C disease progression.


Assuntos
Colesterol/metabolismo , Modelos Animais de Doenças , Hepatopatias/complicações , Fígado/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Animais , Ácidos e Sais Biliares/sangue , Bilirrubina/sangue , Colesterol/análise , Progressão da Doença , Células-Tronco Embrionárias , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/patologia , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Knockout , Doença de Niemann-Pick Tipo C/complicações , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Proteínas/metabolismo , Transaminases/sangue
19.
Addict Biol ; 21(1): 159-70, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25186090

RESUMO

In utero exposure of fetuses to tobacco is associated with reduced birth weight. We hypothesized that this may be due to the toxic effect of carbon monoxide (CO) from tobacco, which has previously been described to damage mitochondria in non-pregnant adult smokers. Maternal peripheral blood mononuclear cells (PBMCs), newborn cord blood mononuclear cells (CBMCs) and placenta were collected from 30 smoking pregnant women and their newborns and classified as moderate and severe smoking groups, and compared to a cohort of 21 non-smoking controls. A biomarker for tobacco consumption (cotinine) was assessed by ELISA (enzyme-linked immunosorbent assay). The following parameters were measured in all tissues: mitochondrial chain complex IV [cytochrome c oxidase (COX)] activity by spectrophotometry, mitochondrial DNA levels by reverse transcription polymerase chain reaction, oxidative stress by spectrophotometric lipid peroxide quantification, mitochondrial mass through citrate synthase spectrophotometric activity and apoptosis by Western blot parallelly confirmed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) assay in placenta. Newborns from smoking pregnant women presented reduced birth weight by 10.75 percent. Materno-fetal mitochondrial and apoptotic PBMC and CBMC parameters showed altered and correlated values regarding COX activity, mitochondrial DNA, oxidative stress and apoptosis. Placenta partially compensated this dysfunction by increasing mitochondrial number; even so ratios of oxidative stress and apoptosis were increased. A CO-induced mitotoxic and apoptotic fingerprint is present in smoking pregnant women and their newborn, with a lack of filtering effect from the placenta. Tobacco consumption correlated with a reduction in birth weight and mitochondrial and apoptotic impairment, suggesting that both could be the cause of the reduced birth weight in smoking pregnant women.


Assuntos
Apoptose , Peso ao Nascer/fisiologia , DNA Mitocondrial/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Placenta/metabolismo , Fumar/metabolismo , Adulto , Western Blotting , Monóxido de Carbono , Estudos de Casos e Controles , Cotinina/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Recém-Nascido de Baixo Peso , Recém-Nascido , Leucócitos Mononucleares/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrofotometria
20.
Mol Med ; 21(1): 817-823, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26552061

RESUMO

Sporadic inclusion body myositis (sIBM) is a rare disease that is difficult to diagnose. Muscle biopsy provides three prominent pathological findings: inflammation, mitochondrial abnormalities and fibber degeneration, represented by the accumulation of protein depots constituted by ß-amyloid peptide, among others. We aim to perform a screening in plasma of circulating molecules related to the putative etiopathogenesis of sIBM to determine potential surrogate biomarkers for diagnosis. Plasma from 21 sIBM patients and 20 age- and gender-paired healthy controls were collected and stored at -80°C. An additional population of patients with non-sIBM inflammatory myopathies was also included (nine patients with dermatomyositis and five with polymyositis). Circulating levels of inflammatory cytokines (interleukin [IL]-6 and tumor necrosis factor [TNF]-α), mitochondrial-related molecules (free plasmatic mitochondrial DNA [mtDNA], fibroblast growth factor-21 [FGF-21] and coenzyme-Q10 [CoQ]) and amyloidogenic-related molecules (beta-secretase-1 [BACE-1], presenilin-1 [PS-1], and soluble Aß precursor protein [sAPPß]) were assessed with magnetic bead-based assays, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA) and high-pressure liquid chromatography (HPLC). Despite remarkable trends toward altered plasmatic expression of inflammatory and mitochondrial molecules (increased IL-6, TNF-α, circulating mtDNA and FGF-21 levels and decreased content in CoQ), only amyloidogenic degenerative markers including BACE-1, PS-1 and sAPPß levels were significantly increased in plasma from sIBM patients compared with controls and other patients with non-sIBM inflammatory myopathies (p < 0.05). Inflammatory, mitochondrial and amyloidogenic degeneration markers are altered in plasma of sIBM patients confirming their etiopathological implication in the disease. Sensitivity and specificity analysis show that BACE-1, PS-1 and sAPPß represent a good predictive noninvasive tool for the diagnosis of sIBM, especially in distinguishing this disease from polymyositis.

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