RESUMO
BACKGROUND: Calcitriol, the active form of vitamin D (also known as 1,25-dihydroxycholecalciferol), improves the phenotype and increases frataxin levels in cell models of Friedreich ataxia (FRDA). OBJECTIVES: Based on these results, we aimed measuring the effects of a calcitriol dose of 0.25 mcg/24h in the neurological function and frataxin levels when administered to FRDA patients for a year. METHODS: 20 FRDA patients where recluted and 15 patients completed the treatment for a year. Evaluations of neurological function changes (SARA scale, 9-HPT, 8-MWT, PATA test) and quality of life (Barthel Scale and Short Form (36) Health Survey [SF-36] quality of life questionnaire) were performed. Frataxin amounts were measured in isolated platelets obtained from these FRDA patients, from heterozygous FRDA carriers (relatives of the FA patients) and from non-heterozygous sex and age matched controls. RESULTS: Although the patients did not experience any observable neurological improvement, there was a statistically significant increase in frataxin levels from initial values, 5.5 to 7.0 pg/µg after 12 months. Differences in frataxin levels referred to total protein levels were observed among sex- and age-matched controls (18.1 pg/µg), relative controls (10.1 pg/µg), and FRDA patients (5.7 pg/µg). The treatment was well tolerated by most patients, and only some of them experienced minor adverse effects at the beginning of the trial. CONCLUSIONS: Calcitriol dosage used (0.25 mcg/24 h) is safe for FRDA patients, and it increases frataxin levels. We cannot rule out that higher doses administered longer could yield neurological benefits. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Calcitriol , Frataxina , Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Humanos , Ataxia de Friedreich/tratamento farmacológico , Masculino , Feminino , Calcitriol/farmacologia , Calcitriol/administração & dosagem , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Qualidade de Vida , Adolescente , Resultado do TratamentoRESUMO
Friedreich ataxia (FA) is a rare, recessive neuro-cardiodegenerative disease caused by deficiency of the mitochondrial protein frataxin. Mitochondrial dysfunction, a reduction in the activity of iron-sulfur enzymes, iron accumulation, and increased oxidative stress have been described. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in the early stages of this disease. However, its effect on mitochondrial function remains to be elucidated. In the present study, we found that in primary cultures of DRG neurons as well as in DRGs from the FXNI151F mouse model, frataxin deficiency resulted in lower activity and levels of the electron transport complexes, mainly complexes I and II. In addition, altered mitochondrial morphology, indicative of degeneration was observed in DRGs from FXNI151F mice. Moreover, the NAD+/NADH ratio was reduced and sirtuin activity was impaired. We identified alpha tubulin as the major acetylated protein from DRG homogenates whose levels were increased in FXNI151F mice compared to WT mice. In the mitochondria, superoxide dismutase (SOD2), a SirT3 substrate, displayed increased acetylation in frataxin-deficient DRG neurons. Since SOD2 acetylation inactivates the enzyme, and higher levels of mitochondrial superoxide anion were detected, oxidative stress markers were analyzed. Elevated levels of hydroxynonenal bound to proteins and mitochondrial Fe2+ accumulation was detected when frataxin decreased. Honokiol, a SirT3 activator, restores mitochondrial respiration, decreases SOD2 acetylation and reduces mitochondrial superoxide levels. Altogether, these results provide data at the molecular level of the consequences of electron transport chain dysfunction, which starts negative feedback, contributing to neuron lethality. This is especially important in sensory neurons which have greater susceptibility to frataxin deficiency compared to other tissues.
Assuntos
Ataxia de Friedreich , Sirtuína 3 , Sirtuínas , Camundongos , Animais , Sirtuína 3/metabolismo , Gânglios Espinais/metabolismo , Sirtuínas/metabolismo , Acetilação , Proteínas de Ligação ao Ferro/genética , Frataxina , Mitocôndrias/metabolismo , Superóxido Dismutase/metabolismo , Ferro/metabolismoRESUMO
Friedreich's ataxia is a neurodegenerative disease caused by mutations in the frataxin gene. Frataxin homologues, including bacterial CyaY proteins, can be found in most species and play a fundamental role in mitochondrial iron homeostasis, either promoting iron assembly into metaloproteins or contributing to iron detoxification. While several lines of evidence suggest that eukaryotic frataxins are more effective than bacterial ones in iron detoxification, the residues involved in this gain of function are unknown. In this work, we analyze conservation of amino acid sequence and protein structure among frataxins and CyaY proteins to identify four highly conserved residue clusters and group them into potential functional clusters. Clusters 1, 2, and 4 are present in eukaryotic frataxins and bacterial CyaY proteins. Cluster 3, containing two serines, a tyrosine, and a glutamate, is only present in eukaryotic frataxins and on CyaY proteins from the Rickettsia genus. Residues from cluster 3 are blocking a small cavity of about 40 Å present in E. coli's CyaY. The function of this cluster is unknown, but we hypothesize that its tyrosine may contribute to prevent formation of reactive oxygen species during iron detoxification. This cluster provides an example of gain of function during evolution in a protein involved in iron homeostasis, as our results suggests that Cluster 3 was present in the endosymbiont ancestor of mitochondria and was conserved in eukaryotic frataxins.
Assuntos
Proteínas de Ligação ao Ferro , Doenças Neurodegenerativas , Rickettsia , Humanos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Eucariotos/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ferro/metabolismo , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Rickettsia/metabolismo , Tirosina/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , FrataxinaRESUMO
Friedreich Ataxia (FA) is a rare neuro-cardiodegenerative disease caused by mutations in the frataxin (FXN) gene. The most prevalent mutation is a GAA expansion in the first intron of the gene causing decreased frataxin expression. Some patients present the GAA expansion in one allele and a missense mutation in the other allele. One of these mutations, FXNI154F, was reported to result in decreased content of mature frataxin and increased presence of an insoluble intermediate proteoform in cellular models. By introducing this mutation into the murine Fxn gene (I151F, equivalent to human I154F) we have now analyzed the consequences of this pathological point mutation in vivo. We have observed that FXNI151F homozygous mice present low frataxin levels in all tissues, with no evidence of insoluble proteoforms. Moreover, they display neurological deficits resembling those observed in FA patients. Biochemical analysis of heart, cerebrum and cerebellum have revealed decreased content of components from OXPHOS complexes I and II, decreased aconitase activity, and alterations in antioxidant defenses. These mitochondrial alterations are more marked in the nervous system than in heart, precede the appearance of neurological symptoms, and are similar to those observed in other FA models. We conclude that the primary pathological mechanism underlying the I151F mutation is frataxin deficiency, like in patients carrying GAA expansions. Therefore, patients carrying the I154F mutation would benefit from frataxin replacement therapies. Furthermore, our results also show that the FXNI151F mouse is an excellent tool for analyzing tissue-specific consequences of frataxin deficiency and for testing new therapies.
Assuntos
Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Mutação Puntual , Alelos , Animais , Comportamento Animal , Biomarcadores/metabolismo , Códon , Modelos Animais de Doenças , Feminino , Ataxia de Friedreich/fisiopatologia , Células HEK293 , Humanos , Íntrons , Proteínas de Ligação ao Ferro/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Mitocondriais/fisiopatologia , Mutação , Mutação de Sentido Incorreto , Fenótipo , Proteômica , Aumento de Peso , FrataxinaRESUMO
Friedreich Ataxia is a neuro-cardiodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. Many evidences indicate that frataxin deficiency causes an unbalance of iron homeostasis. Nevertheless, in the last decade many results also highlighted the importance of calcium unbalance in the deleterious downstream effects caused by frataxin deficiency. In this review, the role of these two metals has been gathered to give a whole view of how iron and calcium dyshomeostasys impacts on cellular functions and, as a result, which strategies can be followed to find an effective therapy for the disease.
Assuntos
Cálcio/metabolismo , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ferro/metabolismo , Mitocôndrias/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/fisiopatologia , Homeostase , Humanos , Quelantes de Ferro/farmacologia , Proteínas de Ligação ao Ferro/química , FrataxinaRESUMO
Calpains are calcium-dependent proteases activated in apoptotic cell death and neurodegeneration. Friedreich Ataxia is a neurodegenerative rare disease caused by frataxin deficiency, a mitochondrial protein. Dorsal root ganglion (DRG) sensory neurons are among the cellular types most affected in this disease. We have previously demonstrated that frataxin-deficient DRGs show calpain activation, alteration in calcium levels and decreased content of the Na+/Ca2+ exchanger (NCLX). This transporter is involved in mitochondrial calcium efflux. In this study, we have performed a time-course analysis of several parameters altered in a frataxin-deficient DRGs. These include decline of NCLX levels, calcium accumulation, mitochondrial depolarization, α-fodrin fragmentation and apoptotic cell death. Furthermore, we have analysed the effect of the calpain inhibitors MDL28170 and Calpeptin on these parameters. We have observed that these inhibitors increase NCLX levels, protect sensory neurons from neurite degeneration and calcium accumulation, and restore mitochondrial membrane potential. In addition, calpain 1 reduction alleviated neurodegeneration in frataxin-deficient DRG neurons. These results strengthen the hypothesis of a central role for calcium homeostasis and calpains in frataxin-deficient dorsal root ganglia neurons.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Calpaína/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Gânglios Espinais/citologia , Proteínas de Ligação ao Ferro/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos , FrataxinaRESUMO
Friedreich ataxia (FA) is a neurodegenerative disease caused by the deficiency of frataxin, a mitochondrial protein. In primary cultures of dorsal root ganglia neurons, we showed that frataxin depletion resulted in decreased levels of the mitochondrial calcium exchanger NCLX, neurite degeneration and apoptotic cell death. Here, we describe that frataxin-deficient dorsal root ganglia neurons display low levels of ferredoxin 1 (FDX1), a mitochondrial Fe/S cluster-containing protein that interacts with frataxin and, interestingly, is essential for the synthesis of calcitriol, the active form of vitamin D. We provide data that calcitriol supplementation, used at nanomolar concentrations, is able to reverse the molecular and cellular markers altered in DRG neurons. Calcitriol is able to recover both FDX1 and NCLX levels and restores mitochondrial membrane potential indicating an overall mitochondrial function improvement. Accordingly, reduction in apoptotic markers and neurite degeneration was observed and, as a result, cell survival was also recovered. All these beneficial effects would be explained by the finding that calcitriol is able to increase the mature frataxin levels in both, frataxin-deficient DRG neurons and cardiomyocytes; remarkably, this increase also occurs in lymphoblastoid cell lines derived from FA patients. In conclusion, these results provide molecular bases to consider calcitriol for an easy and affordable therapeutic approach for FA patients.
Assuntos
Calcitriol/farmacologia , Ferredoxinas/metabolismo , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Apoptose/efeitos dos fármacos , Calcitriol/biossíntese , Calcitriol/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Vitamina D/metabolismo , FrataxinaRESUMO
Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing ironsulfur clusters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid ß-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.
Assuntos
Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/efeitos dos fármacos , Gotículas Lipídicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Ataxia de Friedreich/patologia , Ataxia de Friedreich/fisiopatologia , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neuritos/efeitos dos fármacos , Neuritos/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , FrataxinaRESUMO
Friedreich ataxia (FA) is a cardioneurodegenerative disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Previously, we set up a cardiac cellular model of FA based on neonatal rat cardiac myocytes (NRVM) and lentivirus-mediated frataxin RNA interference. These frataxin-deficient NRVMs presented lipid droplet accumulation, mitochondrial swelling and signs of oxidative stress. Therefore, we decided to explore the presence of protein thiol modifications in this model. With this purpose, reduced glutathione (GSH) levels were measured and the presence of glutathionylated proteins was analyzed. We observed decreased GSH content and increased presence of glutahionylated actin in frataxin-deficient NRVMs. Moreover, the presence of oxidized cysteine residues was investigated using the thiol-reactive fluorescent probe iodoacetamide-Bodipy and 2D-gel electrophoresis. With this approach, we identified two proteins with altered redox status in frataxin-deficient NRVMs: electron transfer flavoprotein-ubiquinone oxidoreductase and dihydrolipoyl dehydrogenase (DLDH). As DLDH is involved in protein-bound lipoic acid redox cycling, we analyzed the redox state of this cofactor and we observed that lipoic acid from pyruvate dehydrogenase was more oxidized in frataxin-deficient cells. Also, by targeted proteomics, we observed a decreased content on the PDH A1 subunit from pyruvate dehydrogenase. Finally, we analyzed the consequences of supplementing frataxin-deficient NRVMs with the PDH cofactors thiamine and lipoic acid, the PDH activator dichloroacetate and the antioxidants N-acetyl cysteine and Tiron. Both dichloroacetate and Tiron were able to partially prevent lipid droplet accumulation in these cells. Overall, these results indicate that frataxin-deficient NRVMs present an altered thiol-redox state which could contribute to the cardiac pathology.
Assuntos
Ataxia de Friedreich , Miócitos Cardíacos , Actinas/metabolismo , Animais , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro , Miócitos Cardíacos/metabolismo , Oxirredução , Estresse Oxidativo , Oxirredutases/metabolismo , Piruvatos/metabolismo , Ratos , Compostos de Sulfidrila/metabolismo , FrataxinaRESUMO
Friedreich ataxia is a neurodegenerative disease with an autosomal recessive inheritance. In most patients, the disease is caused by the presence of trinucleotide GAA expansions in the first intron of the frataxin gene. These expansions cause the decreased expression of this mitochondrial protein. Many evidences indicate that frataxin deficiency causes the deregulation of cellular iron homeostasis. In this review, we will discuss several hypotheses proposed for frataxin function, their caveats, and how they could provide an explanation for the deregulation of iron homeostasis found in frataxin-deficient cells. We will also focus on the potential mechanisms causing cellular dysfunction in Friedreich Ataxia and on the potential use of the iron chelator deferiprone as a therapeutic agent for this disease.
RESUMO
Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.
Assuntos
Proteínas de Ligação ao Ferro/metabolismo , Óxido Nítrico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Mutagênese , Plasmídeos/genética , Plasmídeos/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/genética , Transcriptoma , FrataxinaRESUMO
Friedreich ataxia (FA) is a rare disease caused by deficiency of frataxin, a mitochondrial protein. As there is no cure available for this disease, many strategies have been developed to reduce the deleterious effects of such deficiency. One of these approaches is based on delivering frataxin to the tissues by coupling the protein to trans-activator of transcription (TAT) peptides, which enables cell membranes crossing. In this study, we tested the efficiency of TAT-MTScs-FXN fusion protein to decrease neurodegeneration markers on frataxin-depleted neurons obtained from dorsal root ganglia (DRG), one of the most affected tissues. In mice models of the disease, we tested the ability of TAT-MTScs-FXN to penetrate the mitochondria and its effect on lifespan. In DRG neurons, treatment with TAT-MTScs-FXN increased cell survival, decreased neurite degeneration and reduced apoptotic markers, such as α-fodrin cleavage and caspase 9 activation. Also, we show that heat-shock protein 60 (HSP60), a molecular chaperone targeted to mitochondria, suffered an impaired processing in frataxin-deficient neurons that was relieved by TAT-MTScs-FXN addition. In mice models of the disease, administration of TAT-MTScs-FXN was able to reach muscle mitochondria, restore the activity of the succinate dehydrogenase and produce a significant lifespan increase. These results support the use of TAT-MTScs-FXN as a treatment for Friedreich ataxia.
Assuntos
Ataxia de Friedreich/patologia , Ataxia de Friedreich/terapia , Proteínas de Ligação ao Ferro/metabolismo , Neurônios/patologia , Sinais Direcionadores de Proteínas , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Animais , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Chaperonina 60/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/patologia , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Mitocôndrias/metabolismo , Músculos/metabolismo , Degeneração Neural/patologia , Neuritos/metabolismo , Ratos , Análise de Sobrevida , FrataxinaRESUMO
Calcium is utilised by cells in signalling and in regulating ATP production; it also contributes to cell survival and, when concentrations are unbalanced, triggers pathways for cell death. Mitochondria contribute to calcium buffering, meaning that mitochondrial calcium uptake and release is intimately related to cytosolic calcium concentrations. This review focuses on the proteins contributing to mitochondrial calcium homoeostasis, the roles of the mitochondrial permeability transition pore (MPTP) and mitochondrial calcium-activated proteins, and their relevance in neurodegenerative pathologies. It also covers alterations to calcium homoeostasis in Friedreich ataxia (FA).
RESUMO
Friedreich ataxia is a genetic disease caused by the deficiency of frataxin, a mitochondrial protein. Frataxin deficiency impacts in the cell physiology at several levels. One of them is oxidative stress with consequences in terms of protein dysfunctions and metabolic alterations. Among others, alterations in lipid metabolism have been observed in several models of the disease. In this review we summarize the current knowledge of the molecular basis of the disease, the relevance of oxidative stress and the therapeutic strategies based on reduction of mitochondrial reactive oxygen species production. Finally, we will focus the interest in alterations of lipid metabolism as a consequence of mitochondrial dysfunction and describe the therapeutic approaches based on targeting lipid metabolism.
Assuntos
Ataxia de Friedreich/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Modelos Animais de Doenças , HumanosRESUMO
Friedreich ataxia (FRDA) is a hereditary disease caused by deficient frataxin expression. This mitochondrial protein has been related to iron homeostasis, energy metabolism, and oxidative stress. Patients with FRDA experience neurologic alterations and cardiomyopathy, which is the leading cause of death. The specific effects of frataxin depletion on cardiomyocytes are poorly understood because no appropriate cardiac cellular model is available to researchers. To address this research need, we present a model based on primary cultures of neonatal rat ventricular myocytes (NRVMs) and short-hairpin RNA interference. Using this approach, frataxin was reduced down to 5 to 30% of control protein levels after 7 days of transduction. At this stage the activity and amount of the iron-sulfur protein aconitase, in vitro activities of several OXPHOS components, levels of iron-regulated mRNAs, and the ATP/ADP ratio were comparable to controls. However, NRVMs exhibited markers of oxidative stress and a disorganized mitochondrial network with enlarged mitochondria. Lipids, the main energy source of heart cells, also underwent a clear metabolic change, indicated by the increased presence of lipid droplets and induction of medium-chain acyl-CoA dehydrogenase. These results indicate that mitochondria and lipid metabolism are primary targets of frataxin deficiency in NRVMs. Therefore, they contribute to the understanding of cardiac-specific mechanisms occurring in FRDA and give clues for the design of cardiac-specific treatment strategies for FRDA.
Assuntos
Cardiomiopatias/patologia , Proteínas de Ligação ao Ferro/genética , Metabolismo dos Lipídeos/genética , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/metabolismo , Aconitato Hidratase/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Ataxia de Friedreich/patologia , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , Estresse Oxidativo/fisiologia , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , FrataxinaRESUMO
Detection and quantification of protein carbonyls present in biological samples has become a popular, albeit indirect, method to determine the existence of oxidative stress. Moreover, the rise of proteomics has allowed the identification of the specific proteins targeted by protein carbonylation. This review discusses these methodologies and proteomic strategies and then focuses on the relationship between protein carbonylation and aging and the parameters that may explain the increased sensitivity of certain proteins to protein carbonylation.
Assuntos
Envelhecimento , Carbonilação Proteica , Proteínas/metabolismo , Proteômica/métodos , Animais , Humanos , Estresse Oxidativo , Proteínas/análiseRESUMO
Friedreich ataxia (FRDA) is a neurodegenerative disease characterized by a decreased expression of the mitochondrial protein frataxin. Major neurological symptoms of the disease are due to degeneration of dorsal root ganglion (DRG) sensory neurons. In this study we have explored the neurodegenerative events occurring by frataxin depletion on primary cultures of neurons obtained from rat DRGs. Reduction of 80% of frataxin levels in these cells was achieved by transduction with lentivirus containing shRNA silencing sequences. Frataxin depletion caused mitochondrial membrane potential decrease, neurite degeneration and apoptotic cell death. A marked increase of free intracellular Ca(2+) levels and alteration in Ca(2+)-mediated signaling pathways was also observed, thus suggesting that altered calcium homeostasis can play a pivotal role in neurodegeneration caused by frataxin deficiency. These deleterious effects were reverted by the addition of a cell-penetrant TAT peptide coupled to the BH4, the anti-apoptotic domain of Bcl-x(L). Treatment of cultured frataxin-depleted neurons with TAT-BH4 was able to restore the free intracellular Ca(2+) levels and protect the neurons from degeneration. These observations open the possibility of new therapies of FRDA based on modulating the Ca(2+) signaling and prevent apoptotic process to protect DRG neurons from neurodegeneration.
Assuntos
Apoptose/genética , Gânglios Espinais/citologia , Proteínas de Ligação ao Ferro/genética , Células Receptoras Sensoriais/citologia , Proteína bcl-X/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Células Cultivadas , Ataxia de Friedreich/genética , Produtos do Gene tat/genética , Homeostase , Proteínas de Ligação ao Ferro/biossíntese , Proteínas de Ligação ao Ferro/metabolismo , Potencial da Membrana Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Neuritos/patologia , Doenças Neurodegenerativas , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Interferente Pequeno , Ratos , FrataxinaRESUMO
Frataxin is a mitochondrial protein involved in iron metabolism whose deficiency in humans causes Friedreich ataxia. We performed transcriptomic and proteomic analyses of conditional Yeast Frataxin Homologue (Yfh1) mutants (tetO7-YFH1) to investigate metabolic remodeling upon Yfh1 depletion. These studies revealed that Yfh1 depletion leads to downregulation of many glucose-repressed genes. Most of them were Adr1 targets, a key transcription factor required for growth in non-fermentable carbon sources. Using a GFP-tagged Adr1, we observed that Yfh1 depletion promotes the export of Adr1 from the nucleus to the cytosol without affecting its protein levels. This effect was also observed upon H2O2 treatment, but not by iron overload/starvation, indicating the presence of a regulatory pathway involved in Adr1 export and inactivation upon stress conditions. We also observed that CTH2, a gene involved in the mRNA degradation of several iron-containing enzymes, was induced upon Yfh1 depletion. Accordingly, decreased levels of aconitase and succinate dehydrogenase were observed. Nevertheless, their levels were maintained in a Δcth2 mutant even in the absence of Yfh1. From these results we can conclude that, in addition to altering iron homeostasis, frataxin depletion involves drastic metabolic remodeling governed by Adr1 and Cth2 that finally leads to downregulation of iron-sulfur proteins and other proteins involved in respiratory metabolism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Tristetraprolina/metabolismo , Aconitato Hidratase/metabolismo , Álcool Desidrogenase/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Doxiciclina/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Glutarredoxinas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , FrataxinaRESUMO
Within Saccharomyces cerevisiae, Hcm1is a member of the forkhead transcription factor family with a role in chromosome organization. Our group recently described its involvement in mitochondrial biogenesis and stress resistance, and reports here that Hcm1 played a role in adaptation to respiratory metabolism when glucose or nitrogen was decreased. Regulation of Hcm1 activity occurs in at least three ways: i) protein quantity, ii) subcellular localization, and iii) transcriptional activity. Transcriptional activity was measured using a reporter gene fused to a promoter that contains a binding site for Hcm1. We also analyzed the levels of several genes whose expression is known to be regulated by Hcm1 levels and the role of the main kinases known to respond to nutrients. Lack of sucrose-nonfermenting (Snf1) kinase increases cytoplasmic localization of Hcm1, whereas Δtor1 cells showed a mild increase in nuclear Hcm1. In vitro experiments showed that Snf1 clearly phosphorylates Hcm1 while Sch9 exerts a milder phosphorylation. Although in vitroTor1 does not directly phosphorylate Hcm1, in vivo rapamycin treatment increases nuclear Hcm1. We conclude that Hcm1 participates in the adaptation of cells from fermentation to respiratory metabolism during nutrient scarcity. According to our hypothesis, when nutrient levels decrease, Snf1 phosphorylates Hcm1. This results in a shift from the cytoplasm to the nucleus and increased transcriptional activity of genes involved in respiration, use of alternative energy sources, NAD synthesis and oxidative stress resistance.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Alimentos , Fatores de Transcrição Forkhead/genética , Glucose/genética , Glucose/metabolismo , Nitrogênio/metabolismo , Estresse Oxidativo/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sacarose/metabolismo , Transcrição GênicaRESUMO
Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.