Calcitriol Treatment Is Safe and Increases Frataxin Levels in Friedreich Ataxia Patients.

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.

Mitochondrial impairment, decreased sirtuin activity and protein acetylation in dorsal root ganglia in Friedreich Ataxia models.

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.

Mice harboring the FXN I151F pathological point mutation present decreased frataxin levels, a Friedreich ataxia-like phenotype, and mitochondrial alterations.

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.

Calcitriol increases frataxin levels and restores mitochondrial function in cell models of Friedreich Ataxia.

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.

PPAR gamma agonist leriglitazone improves frataxin-loss impairments in cellular and animal models of Friedreich Ataxia.

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.

Mitochondrial iron and calcium homeostasis in Friedreich ataxia.

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.

Calpain-Inhibitors Protect Frataxin-Deficient Dorsal Root Ganglia Neurons from Loss of Mitochondrial Na+/Ca2+ Exchanger, NCLX, and Apoptosis.

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.

Mitochondrial Localization of the Yeast Forkhead Factor Hcm1.

Hcm1 is a member of the forkhead transcription factor family involved in segregation, spindle pole dynamics, and budding in Saccharomyces cerevisiae. Our group described the role of Hcm1 in mitochondrial biogenesis and stress resistance, and in the cellular adaptation to mitochondrial respiratory metabolism when nutrients decrease. Regulation of Hcm1 activity occurs at the protein level, subcellular localization, and transcriptional activity. Here we report that the amount of protein increased in the G1/S transition phase when the factor accumulated in the nucleus. In the G2/M phases, the Hcm1 amount decreased, and it was translocated outside the nucleus with a network-like localization. Preparation of highly purified mitochondria by a sucrose gradient density demonstrated that Hcm1 colocalized with mitochondrial markers, inducing expression of COX1, a mitochondrial encoded subunit of cytochrome oxidase, in the G2/M phases. Taken together, these results show a new localization of Hcm1 and suggest that it acts as a mitochondrial transcription factor regulating the metabolism of this organelle.

Frataxin-deficient neurons and mice models of Friedreich ataxia are improved by TAT-MTScs-FXN treatment.

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.

Apoptotic cell death and altered calcium homeostasis caused by frataxin depletion in dorsal root ganglia neurons can be prevented by BH4 domain of Bcl-xL protein.

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.

Protein carbonylation: proteomics, specificity and relevance to aging.

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.

Metabolic remodeling in frataxin-deficient yeast is mediated by Cth2 and Adr1.

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.

The FOX transcription factor Hcm1 regulates oxidative metabolism in response to early nutrient limitation in yeast. Role of Snf1 and Tor1/Sch9 kinases.

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.

Memory impairment and hippocampus specific protein oxidation induced by ethanol intake and 3, 4-methylenedioxymethamphetamine (MDMA) in mice.

Ethanol and 3, 4-Methylenedioxymethamphetamine (MDMA) are popular recreational drugs widely abused by adolescents that may induce neurotoxic processes associated with behavioural alterations. Adolescent CD1 mice were subjected to ethanol intake using the drinking in the dark (DID) procedure, acute MDMA or a combination. Considering that both drugs of abuse cause oxidative stress in the brain, protein oxidative damage in different brain areas was analysed 72 h after treatment using a proteomic approach. Damage to specific proteins in treated animals was significant in the hippocampus but not in the prefrontal cortex. The damaged hippocampus proteins were then identified by mass spectrometry, revealing their involvement in energy metabolism, structural function, axonal outgrowth and stability, and neurotransmitter release. Mice treated with MDMA displayed higher oxidative damage than ethanol-treated mice. To determine whether this oxidative damage was affecting hippocampus activity, declarative memory was evaluated at 72 h after treatment using the object recognition assay and the radial arm maze. Although acquisition in the radial arm maze was not impaired by ethanol intake, MDMA treatment impaired long-term memory in both tests. Therefore, oxidative damage to specific proteins observed under MDMA treatment affects important cellular function on the hippocampus that may contribute to declarative memory deficits.

Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation.

BACKGROUND: In the yeast biomass production process, protein carbonylation has severe adverse effects since it diminishes biomass yield and profitability of industrial production plants. However, this significant detriment of yeast performance can be alleviated by increasing thioredoxins levels. Thioredoxins are important antioxidant defenses implicated in many functions in cells, and their primordial functions include scavenging of reactive oxygen species that produce dramatic and irreversible alterations such as protein carbonylation. RESULTS: In this work we have found several proteins specifically protected by yeast Thioredoxin 2 (Trx2p). Bidimensional electrophoresis and carbonylated protein identification from TRX-deficient and TRX-overexpressing cells revealed that glycolysis and fermentation-related proteins are specific targets of Trx2p protection. Indeed, the TRX2 overexpressing strain presented increased activity of the central carbon metabolism enzymes. Interestingly, Trx2p specifically preserved alcohol dehydrogenase I (Adh1p) from carbonylation, decreased oligomer aggregates and increased its enzymatic activity. CONCLUSIONS: The identified proteins suggest that the fermentative capacity detriment observed under industrial conditions in T73 wine commercial strain results from the oxidative carbonylation of specific glycolytic and fermentation enzymes. Indeed, increased thioredoxin levels enhance the performance of key fermentation enzymes such as Adh1p, which consequently increases fermentative capacity.

Frataxin depletion in yeast triggers up-regulation of iron transport systems before affecting iron-sulfur enzyme activities.

The primary function of frataxin, a mitochondrial protein involved in iron homeostasis, remains controversial. Using a yeast model of conditional expression of the frataxin homologue YFH1, we analyzed the primary effects of YFH1 depletion. The main conclusion unambiguously points to the up-regulation of iron transport systems as a primary effect of YFH1 down-regulation. We observed that inactivation of aconitase, an iron-sulfur enzyme, occurs long after the iron uptake system has been activated. Decreased aconitase activity should be considered part of a group of secondary events promoted by iron overloading, which includes decreased superoxide dismutase activity and increased protein carbonyl formation. Impaired manganese uptake, which contributes to superoxide dismutase deficiency, has also been observed in YFH1-deficient cells. This low manganese content can be attributed to the down-regulation of the metal ion transporter Smf2. Low Smf2 levels were not observed in AFT1/YFH1 double mutants, indicating that high iron levels could be responsible for the Smf2 decline. In summary, the results presented here indicate that decreased iron-sulfur enzyme activities in YFH1-deficient cells are the consequence of the oxidative stress conditions suffered by these cells.

The forkhead transcription factor Hcm1 promotes mitochondrial biogenesis and stress resistance in yeast.

In Saccharomyces cerevisiae, the forkhead transcription factor Hcm1 is involved in chromosome segregation, spindle pole dynamics, and budding. We found that Hcm1 interacts with the histone deacetylase Sir2 and shifts from cytoplasm to the nucleus in the G(1)/S phase or in response to oxidative stress stimuli. The nuclear localization of Hcm1 depends on the activity of Sir2 as revealed by activators and inhibitors of the sirtuins and the Δsir2 mutant. Hcm1-overexpressing cells display more mitochondria that can be attributed to increased amounts of Abf2, a protein involved in mitochondrial biogenesis. These cells also show higher rates of oxygen consumption and improved resistance to oxidative stress that would be explained by increased catalase and Sod2 activities and molecular chaperones such as Hsp26, Hsp30, and members of Hsp70 family. Microarray analyses also reveal increased expression of genes involved in mitochondrial energy pathways and those allowing the transition from the exponential to the stationary phase. Taken together, these results describe a new and relevant role of Hcm1 for mitochondrial functions, suggesting that this transcription factor would participate in the adaptation of cells from fermentative to respiratory metabolism.

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation.

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.

Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass.

BACKGROUND: Wine Saccharomyces cerevisiae strains, adapted to anaerobic must fermentations, suffer oxidative stress when they are grown under aerobic conditions for biomass propagation in the industrial process of active dry yeast production. Oxidative metabolism of sugars favors high biomass yields but also causes increased oxidation damage of cell components. The overexpression of the TRX2 gene, coding for a thioredoxin, enhances oxidative stress resistance in a wine yeast strain model. The thioredoxin and also the glutathione/glutaredoxin system constitute the most important defense against oxidation. Trx2p is also involved in the regulation of Yap1p-driven transcriptional response against some reactive oxygen species. RESULTS: Laboratory scale simulations of the industrial active dry biomass production process demonstrate that TRX2 overexpression increases the wine yeast final biomass yield and also its fermentative capacity both after the batch and fed-batch phases. Microvinifications carried out with the modified strain show a fast start phenotype derived from its enhanced fermentative capacity and also increased content of beneficial aroma compounds. The modified strain displays an increased transcriptional response of Yap1p regulated genes and other oxidative stress related genes. Activities of antioxidant enzymes like Sod1p, Sod2p and catalase are also enhanced. Consequently, diminished oxidation of lipids and proteins is observed in the modified strain, which can explain the improved performance of the thioredoxin overexpressing strain. CONCLUSIONS: We report several beneficial effects of overexpressing the thioredoxin gene TRX2 in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in TRX2 overexpressing strain can be of special interest for several industrial applications.

Frataxin-deficient cardiomyocytes present an altered thiol-redox state which targets actin and pyruvate dehydrogenase.

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.