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This study aimed to comprehensively assess the metabolic, mitochondrial, and inflammatory effects of first-line efavirenz, emtricitabine, and tenofovir disoproxil fumarate (EFV/FTC/TDF) single-tablet regimen (STR) relative to untreated asymptomatic HIV infection. To this end, we analyzed 29 people with HIV (PWH) treated for at least one year with this regimen vs. 33 antiretroviral-naïve PWH. Excellent therapeutic activity was accompanied by significant alterations in metabolic parameters. The treatment group showed increased plasmatic levels of glucose, total cholesterol and its fractions (LDL and HDL), triglycerides, and hepatic enzymes (GGT, ALP); conversely, bilirubin levels (total and indirect fraction) decreased in the treated cohort. Mitochondrial performance was preserved overall and treatment administration even promoted the recovery of mitochondrial DNA (mtDNA) content depleted by the virus, although this was not accompanied by the recovery in some of their encoded proteins (since cytochrome c oxidase II was significantly decreased). Inflammatory profile (TNFα, IL-6), ameliorated after treatment in accordance with viral reduction and the recovery of TNFα levels correlated to mtDNA cell restoration. Thus, although this regimen causes subclinical metabolic alterations, its antiviral and anti-inflammatory properties may be associated with partial improvement in mitochondrial function.
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Alcinos , Fármacos Anti-HIV , Benzoxazinas , Ciclopropanos , DNA Mitocondrial , Emtricitabina , Infecções por HIV , Mitocôndrias , Tenofovir , Humanos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Masculino , Feminino , Adulto , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Benzoxazinas/uso terapêutico , Benzoxazinas/farmacologia , Fármacos Anti-HIV/uso terapêutico , Fármacos Anti-HIV/efeitos adversos , Ciclopropanos/uso terapêutico , Tenofovir/uso terapêutico , Pessoa de Meia-Idade , Emtricitabina/uso terapêutico , DNA Mitocondrial/metabolismo , InflamaçãoRESUMO
Reactive oxygen species (ROS) serve important homeostatic functions but must be constantly neutralized by an adaptive antioxidant response to prevent supraphysiological levels of ROS from causing oxidative damage to cellular components. Here, we report that the cellular plasticity transcription factors ZEB1 and ZEB2 modulate in opposing directions the adaptive antioxidant response to fasting in skeletal muscle. Using transgenic mice in which Zeb1 or Zeb2 were specifically deleted in skeletal myofibers, we show that in fasted mice, the deletion of Zeb1, but not Zeb2, increased ROS production and that the adaptive antioxidant response to fasting essentially requires ZEB1 and is inhibited by ZEB2. ZEB1 expression increased in fasted muscles and protected them from atrophy; conversely, ZEB2 expression in muscles decreased during fasting and exacerbated muscle atrophy. In fasted muscles, ZEB1 reduces mitochondrial damage and increases mitochondrial respiratory activity; meanwhile, ZEB2 did the opposite. Treatment of fasting mice with Zeb1-deficient myofibers with the antioxidant triterpenoid 1[2-cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoro-ethylamide (CDDO-TFEA) completely reversed their altered phenotype to that observed in fasted control mice. These results set ZEB factors as potential therapeutic targets to modulate the adaptive antioxidant response in physiopathological conditions and diseases caused by redox imbalance.
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Antioxidantes , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Animais , Camundongos , Antioxidantes/farmacologia , Jejum , Camundongos Transgênicos , Atrofia Muscular/genética , Espécies Reativas de Oxigênio , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Inclusion body myositis (IBM) is an acquired inflammatory myopathy affecting proximal and distal muscles that leads to weakness in patients over 50. It is diagnosed based on clinical and histological findings in muscle related to inflammation, degeneration, and mitochondria. In relation to IBM, a shortage of validated disease models and a lack of biomarkers and effective treatments constitute an unmet medical need. To overcome these hurdles, we performed an omics analysis of multiple samples from IBM patients (saliva, fibroblasts, urine, plasma, and muscle) to gain insight into the pathophysiology of IBM. Degeneration was evident due to the presence of amyloid ß peptide 1-42 (Aß1-42) in the saliva of the analyzed IBM patients. The presence of metabolic disarrangements in IBM was indicated by an imbalanced organic acid profile in fibroblasts and urine. Specifically, abnormal levels of L-pyroglutamic and orotic acid were supported by the abnormal expression of related metabolites in plasma and urine (glutathione and pyrimidines) and the aberrant expression of upstream gene regulators (L2HGDH, IDH2, OPLAH, and ASL) in muscle. Combined levels of L-pyroglutamic and orotic acid displayed an outstanding biomarker signature in urine with 100% sensitivity and specificity. The confirmation of systemic metabolic disarrangements in IBM and the identification of novel biomarkers reported herein unveil novel insights that require validation in larger cohorts.
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BACKGROUND: Inclusion body myositis (IBM) is an inflammatory myopathy clinically characterized by proximal and distal muscle weakness, with inflammatory infiltrates, rimmed vacuoles and mitochondrial changes in muscle histopathology. There is scarce knowledge on IBM aetiology, and non-established biomarkers or effective treatments are available, partly due to the lack of validated disease models. METHODS: We have performed transcriptomics and functional validation of IBM muscle pathological hallmarks in fibroblasts from IBM patients (n = 14) and healthy controls (n = 12), paired by age and sex. The results comprise an mRNA-seq, together with functional inflammatory, autophagy, mitochondrial and metabolic changes between patients and controls. RESULTS: Gene expression profile of IBM vs control fibroblasts revealed 778 differentially expressed genes (P-value adj < 0.05) related to inflammation, mitochondria, cell cycle regulation and metabolism. Functionally, an increased inflammatory profile was observed in IBM fibroblasts with higher supernatant cytokine secretion (three-fold increase). Autophagy was reduced considering basal protein mediators (18.4% reduced), time-course autophagosome formation (LC3BII 39% reduced, P-value < 0.05), and autophagosome microscopic evaluation. Mitochondria displayed reduced genetic content (by 33.9%, P-value < 0.05) and function (30.2%-decrease in respiration, 45.6%-decline in enzymatic activity (P-value < 0.001), 14.3%-higher oxidative stress, 135.2%-increased antioxidant defence (P-value < 0.05), 11.6%-reduced mitochondrial membrane potential (P-value < 0.05) and 42.8%-reduced mitochondrial elongation (P-value < 0.05)). In accordance, at the metabolite level, organic acid showed a 1.8-fold change increase, with conserved amino acid profile. Correlating to disease evolution, oxidative stress and inflammation emerge as potential markers of prognosis. CONCLUSIONS: These findings confirm the presence of molecular disturbances in peripheral tissues from IBM patients and prompt patients' derived fibroblasts as a promising disease model, which may eventually be exported to other neuromuscular disorders. We additionally identify new molecular players in IBM associated with disease progression, setting the path to deepen in disease aetiology, in the identification of novel biomarkers or in the standardization of biomimetic platforms to assay new therapeutic strategies for preclinical studies.
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Miosite de Corpos de Inclusão , Miosite , Humanos , Miosite de Corpos de Inclusão/diagnóstico , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Músculos/metabolismo , Inflamação/patologia , Biomarcadores/metabolismoRESUMO
The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.
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Neurodegenerative diseases, such as Parkinson's disease, are heterogeneous disorders with a multifactorial nature involving impaired bioenergetics. Stem-regenerative medicine and bioenergetics have been proposed as promising therapeutic targets in the neurologic field. The rationale of the present study was to assess the potential of human-derived adipose stem cells (hASCs) to transdifferentiate into neuronal-like cells (NhASCs and neurospheres) and explore the hASC bioenergetic profile. hASC neuronal transdifferentiation was performed through neurobasal media and differentiation factor exposure. High resolution respirometry was assessed. Increased MAP-2 neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 28-36 days of differentiation) and increased bIII-tubulin neuronal marker protein expression upon neuronal induction (p<0.05 undifferentiated hASCs vs. 6-28-36 days of differentiation) were found. The bioenergetic profile was detectable through high-resolution respirometry approaches in hASCs but did not lead to differential oxidative capacity rates in healthy or clinically diagnosed PD-hASCs. We confirmed the capability of transdifferentiation to the neuronal-like profile of hASCs derived from the forearms of human subjects and characterized the bioenergetic profile. Suboptimal maximal respiratory capacity trends in PD were found. Neuronal induction leading to positive neuronal protein expression markers is a relevant issue that encourages the suitability of NhASC models in neurodegeneration.
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Doença de Parkinson , Tecido Adiposo/metabolismo , Diferenciação Celular , Células Cultivadas , Metabolismo Energético , Antebraço , Humanos , Doença de Parkinson/metabolismo , Células-TroncoRESUMO
BACKGROUND: Ganciclovir/valganciclovir is currently indicated during the first 6 months of life in symptomatic children with congenital cytomegalovirus (CMV) infection. However, this treatment may have the potential to induce mitochondrial toxicity due to off-target inhibition of DNA-polymerases. Similar anti-HIV drugs have been associated with mitochondrial toxicity but this has never been explored in CMV. OBJECTIVE: To determine the potential mitochondrial toxicity profile at the genetic, functional and biogenesis level in peripheral blood mononuclear cells from a cohort of newborns and infants with symptomatic congenital CMV infection (treated with valganciclovir, untreated and uninfected controls). DESIGN: Longitudinal, observational and controlled study. SETTING AND PATIENTS: Subjects were recruited at the tertiary referral Hospital Sant Joan de Déu and experiments were conducted at IDIBAPS-Hospital Clínic of Barcelona, Spain. CMV-infected newborns underwent comprehensive monthly clinical follow-up. METHODS: Mitochondrial parameters, audiometry and neurological assessment were measured at baseline, 3-6 and 12 months after inclusion in the study. The Kruskal-Wallis test for k-independent samples and Friedman tests for repeated measurements were applied. RESULTS: Complex IV, citrate synthase enzymatic activities and mtDNA remained preserved in congenital CMV-infected infants treated with valganciclovir compared with controls (p>0.05 in all cases). CONCLUSIONS: No evidence of mitochondrial toxicity was found in infants treated with valganciclovir for congenital CMV.
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Fármacos Anti-HIV , Infecções por Citomegalovirus , Fármacos Anti-HIV/uso terapêutico , Antivirais/efeitos adversos , Criança , Infecções por Citomegalovirus/congênito , Ganciclovir/efeitos adversos , Humanos , Lactente , Recém-Nascido , Leucócitos Mononucleares , Estudos Longitudinais , Valganciclovir/uso terapêuticoRESUMO
Idiopathic Parkinson's disease (iPD) and type 2 diabetes mellitus (T2DM) are chronic, multisystemic, and degenerative diseases associated with aging, with eventual epidemiological co-morbidity and overlap in molecular basis. This study aims to explore if metabolic and mitochondrial alterations underlie the previously reported epidemiologic and clinical co-morbidity from a molecular level. To evaluate the adaptation of iPD to a simulated pre-diabetogenic state, we exposed primary cultured fibroblasts from iPD patients and controls to standard (5 mM) and high (25 mM) glucose concentrations to further characterize metabolic and mitochondrial resilience. iPD fibroblasts showed increased organic and amino acid levels related to mitochondrial metabolism with respect to controls, and these differences were enhanced in high glucose conditions (citric, suberic, and sebacic acids levels increased, as well as alanine, glutamate, aspartate, arginine, and ornithine amino acids; p-values between 0.001 and 0.05). The accumulation of metabolites in iPD fibroblasts was associated with (and probably due to) the concomitant mitochondrial dysfunction observed at enzymatic, oxidative, respiratory, and morphologic level. Metabolic and mitochondrial plasticity of controls was not observed in iPD fibroblasts, which were unable to adapt to different glucose conditions. Impaired metabolism and mitochondrial activity in iPD may limit energy supply for cell survival. Moreover, reduced capacity to adapt to disrupted glucose balance characteristic of T2DM may underlay the co-morbidity between both diseases. Conclusions: Fibroblasts from iPD patients showed mitochondrial impairment, resulting in the accumulation of organic and amino acids related to mitochondrial metabolism, especially when exposed to high glucose. Mitochondrial and metabolic defects down warding cell plasticity to adapt to changing glucose bioavailability may explain the comorbidity between iPD and T2DM.
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Background: Mitochondrial genome has been used across multiple fields in research, diagnosis, and toxicogenomics. Several compounds damage mitochondrial DNA (mtDNA), including biological and therapeutic agents like the human immunodeficiency virus (HIV) but also its antiretroviral treatment, leading to adverse clinical manifestations. HIV-infected and treated patients may show impaired mitochondrial and metabolic profile, but specific contribution of viral or treatment toxicity remains elusive. The evaluation of HIV consequences without treatment interference has been performed in naïve (non-treated) patients, but assessment of treatment toxicity without viral interference is usually restricted to in vitro assays. Objective: The objective of the present study is to determine whether antiretroviral treatment without HIV interference can lead to mtDNA disturbances. We studied clinical, mitochondrial, and metabolic toxicity in non-infected healthy patients who received HIV post-exposure prophylaxis (PEP) to prevent further infection. We assessed two different PEP regimens according to their composition to ascertain if they were the cause of tolerability issues and derived toxicity. Methods: We analyzed reasons for PEP discontinuation and main secondary effects of treatment withdrawal, mtDNA content from peripheral blood mononuclear cells and metabolic profile, before and after 28 days of PEP, in 23 patients classified depending on PEP composition: one protease inhibitor (PI) plus Zidovudine/Lamivudine (PI plus AZT + 3TC; n = 9) or PI plus Tenofovir/Emtricitabine (PI plus TDF + FTC; n = 14). Results: Zidovudine-containing-regimens showed an increased risk for drug discontinuation (RR = 9.33; 95% CI = 1.34-65.23) due to adverse effects of medication related to gastrointestinal complications. In the absence of metabolic disturbances, 4-week PEP containing PI plus AZT + 3TC led to higher mitochondrial toxicity (-17.9 ± 25.8 decrease in mtDNA/nDNA levels) than PI plus TDF + FTC (which increased by 43.2 ± 24.3 units mtDNA/nDNA; p < 0.05 between groups). MtDNA changes showed a significant and negative correlation with baseline alanine transaminase levels (p < 0.05), suggesting that a proper hepatic function may protect from antiretroviral toxicity. Conclusions: In absence of HIV infection, preventive short antiretroviral treatment can cause secondary effects responsible for treatment discontinuation and subclinical mitochondrial damage, especially pyrimidine analogs such as AZT, which still rank as the alternative option and first choice in certain cohorts for PEP. Forthcoming efforts should be focused on launching new strategies with safer clinical and mitotoxic profile.
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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.
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Autofagia/genética , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Pele/metabolismo , Ubiquitina-Proteína Ligases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mutação , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Doença de Parkinson/genéticaRESUMO
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.
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Retardo do Crescimento Fetal/metabolismo , Coração/fisiopatologia , Leucócitos Mononucleares/metabolismo , Mitocôndrias/metabolismo , Placenta/metabolismo , Sirtuína 3/metabolismo , Adulto , Citrato (si)-Sintase/metabolismo , Estudos Transversais , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Coração/crescimento & desenvolvimento , Humanos , Peroxidação de Lipídeos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Peptídeo Natriurético Encefálico/sangue , Consumo de Oxigênio , Gravidez , Sirtuína 3/genética , Remodelação VentricularRESUMO
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.
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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 , FenótipoRESUMO
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.
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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/fisiologiaRESUMO
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.
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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/virologiaRESUMO
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.
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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/metabolismoRESUMO
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.
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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 OxidativaRESUMO
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.
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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 , EspectrofotometriaRESUMO
OBJECTIVE: The physiopathological mechanisms implicated in hypertensive heart disease are multi-factorial, including myocyte hypertrophy, apoptosis and myocardial remodelling. In this process, some hormonal and local growth factors have a regulatory influence. The aim of this study was to evaluate the potential role of myostatin and insulin-like growth factor-1 (IGF-1) myocardial expression in the development of hypertensive-induced cardiac damage. METHODS: Samples of human myocardium tissue from organ donors were prospectively collected and classified according to the presence of hypertension, alcohol consumption, other causes of myocardial damage and the presence of structural cardiomyopathy (CMP). Myocardial samples were studied by immunohistochemistry and myostatin, and IGF-1 myocardial expression was evaluated in all the different groups of donors. Hypertensive donors were compared to other groups. RESULTS: A total of 66 heart samples from human donors were collected: 33 donors had no previous or present history of hypertension and 33 donors presented defined hypertension. Donors with hypertension presented higher myocyte cell and nuclear hypertrophy and showed similar myostatin myocardial expression as controls, but lower IGF-1 myocardial expression. Myostatin expression was significantly higher in hypertensive donors with CMP compared to non-hypertensive healthy donors. The presence of CMP of diverse origin (alcoholic, valve and coronary) also significantly increased myostatin myocardial expression. CONCLUSION: The presence of hypertension significantly decreases IGF-1 myocardial expression. Myostatin myocardial expression increases in the presence of structural CMP either of hypertensive or other origin. These effects open the possibility of modulating hypertensive-induced cardiac damage.
Assuntos
Cardiopatias/metabolismo , Hipertensão/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Miocárdio/metabolismo , Miostatina/metabolismo , Adulto , Idoso , Alcoolismo/metabolismo , Apoptose , Estudos de Casos e Controles , Feminino , Expressão Gênica , Cardiopatias/etiologia , Humanos , Hipertensão/complicações , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Doadores de TecidosRESUMO
BACKGROUND: Mitochondrial consequences from foetal exposure to HIV infection and antiretrovirals could be further investigated. OBJECTIVE: The main objective of this study was to evaluate maternofoetal mitochondrial disturbances in HIV infection and antiretroviral administration in human pregnancies as the aetiopathogenic basis of suboptimal perinatal-clinical features. DESIGN: Cross-sectional, prospective, observational, exploratory and controlled study. METHODS: Clinical/epidemiological data of 35 HIV-infected pregnant women and 17 controls were collected. Mitochondrial DNA (mtDNA) and RNA (mtRNA) content (real time-PCR), enzymatic activities and content (spectrophotometry) were measured in leucocytes. Genetic-functional, maternofoetal and molecular-clinical correlations were assessed. RESULTS: Birth weight was lower in infants from HIV-infected mothers compared with controls. MtDNA values were slightly decreased in HIV cases, although not reaching statistical significance. MtRNA values were lower in HIV-infected mothers. Similarly, binary complex II+III enzymatic activity decreased to 50% in both HIV-infected mothers (44.45â±â3.77%) and their infants (48.79â±â3.41%) (Pâ=â0.001 and Pâ<â0.001). Global CI+III+IV enzymatic activity was lower in HIV-infected mothers and infants (90.43â±â2.39% and 51.16â±â9.30%) (Pâ<â0.005 and Pâ<â0.05). MtDNA content correlated with function in mothers and infants. Maternofoetal parameters correlated at genetic and functional levels. CONCLUSION: HAART toxicity caused mitochondrial damage in HIV-infected pregnant women and their newborns, being present at a genetic and functional level with a maternofoetal correlation.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Infecções por HIV/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Complicações Infecciosas na Gravidez/virologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Estudos Transversais , DNA Mitocondrial/genética , Feminino , Infecções por HIV/transmissão , Humanos , Lactente , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Leucócitos Mononucleares/citologia , Masculino , Mitocôndrias/genética , Gravidez , Estudos Prospectivos , RNA/genética , RNA MitocondrialRESUMO
Mitochondrial toxicity in perinatally human immunodeficiency virus (HIV)-infected pediatric patients has been scarcely investigated. Limited data are available about HIV or antiretroviral (ARV)-mediated mitochondrial damage in this population group, specifically, regarding oxygen consumption and apoptosis approach. We aimed to elucidate whether a given mitochondrial DNA depletion is reflected at downstream levels, to gain insight on the pathology of HIV and highly active antiretroviral therapy (HAART) in perinatally HIV-infected pediatric patients. We studied 10 healthy control participants and 20 perinatally HIV-infected pediatric patients (10 under ARV treatment and 10 off treatment). We determined mitochondrial mass, subunits II and IV of complex IV, global and specific mitochondrial enzymatic and oxidative activities, and apoptosis from peripheral blood mononuclear cells. Global oxygen consumption was significantly compromised in HIV-infected untreated patients, compared to the control group (0.76 ± 0.01 versus 1.59 ± 0.15; P = 0.014). Apoptosis showed a trend to increase in untreated patients as well. The overall complex (C) CI-III-IV activity of the mitochondrial respiratory chain (MRC) was significantly decreased in HIV-infected treated patients with respect to the control group (1.52 ± 0.38 versus 6.38 ± 1.53; P = 0.02). No statistically significant differences were found between untreated and HAART-treated patients. These findings suggest the pathogenic role of both HIV and HAART in mitochondrial dysfunction in vertical infection. The abnormalities in mitochondrial genome may be downstream reflected through a global alteration of the MRC. Mitochondrial impairment associated with HIV and HAART was generalized, rather than localized, in this series of perinatally HIV-infected patients.