Deciphering the ferroptosis pathways in dorsal root ganglia of Friedreich ataxia models. The role of LKB1/AMPK, KEAP1, and GSK3ß in the impairment of the NRF2 response.

Friedreich ataxia (FA) is a rare neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Frataxin has been related in iron homeostasis, energy metabolism, and oxidative stress. Ferroptosis has recently been shown to be involved in FA cellular degeneration; however, its role in dorsal root ganglion (DRG) sensory neurons, the cells that are affected the most and the earliest, is mostly unknown. In this study, we used primary cultures of frataxin-deficient DRG neurons as well as DRG from the FXNI151F mouse model to study ferroptosis and its regulatory pathways. A lack of frataxin induced upregulation of transferrin receptor 1 and decreased ferritin and mitochondrial iron accumulation, a source of oxidative stress. However, there was impaired activation of NRF2, a key transcription factor involved in the antioxidant response pathway. Decreased total and nuclear NRF2 explains the downregulation of both SLC7A11 (a member of the system Xc, which transports cystine required for glutathione synthesis) and glutathione peroxidase 4, responsible for increased lipid peroxidation, the main markers of ferroptosis. Such dysregulation could be due to the increase in KEAP1 and the activation of GSK3ß, which promote cytosolic localization and degradation of NRF2. Moreover, there was a deficiency in the LKB1/AMPK pathway, which would also impair NRF2 activity. AMPK acts as a positive regulator of NRF2 and it is activated by the upstream kinase LKB1. The levels of LKB1 were reduced when frataxin decreased, in agreement with reduced pAMPK (Thr172), the active form of AMPK. SIRT1, a known activator of LKB1, was also reduced when frataxin decreased. MT-6378, an AMPK activator, restored NRF2 levels, increased GPX4 levels and reduced lipid peroxidation. In conclusion, this study demonstrated that frataxin deficiency in DRG neurons disrupts iron homeostasis and the intricate regulation of molecular pathways affecting NRF2 activation and the cellular response to oxidative stress, leading to ferroptosis.

Skeletal Muscle Involvement in Friedreich Ataxia.

Friedreich Ataxia (FRDA) is an inherited neuromuscular disorder triggered by a deficit of the mitochondrial protein frataxin. At a cellular level, frataxin deficiency results in insufficient iron-sulfur cluster biosynthesis and impaired mitochondrial function and adenosine triphosphate production. The main clinical manifestation is a progressive balance and coordination disorder which depends on the involvement of peripheral and central sensory pathways as well as of the cerebellum. Besides the neurological involvement, FRDA affects also the striated muscles. The most prominent manifestation is a hypertrophic cardiomyopathy, which also represents the major determinant of premature mortality. Moreover, FRDA displays skeletal muscle involvement, which contributes to the weakness and marked fatigue evident throughout the course of the disease. Herein, we review skeletal muscle findings in FRDA generated by functional imaging, histology, as well as multiomics techniques in both disease models and in patients. Altogether, these findings corroborate a disease phenotype in skeletal muscle and support the notion of progressive mitochondrial damage as a driver of disease progression in FRDA. Furthermore, we highlight the relevance of skeletal muscle investigations in the development of biomarkers for early-phase trials and future therapeutic strategies in FRDA.

Ferrostatin-1 specifically targets mitochondrial iron-sulfur clusters and aconitase to improve cardiac function in Sirtuin 3 cardiomyocyte knockout mice.

AIMS: Ferroptosis is a form of iron-regulated cell death implicated in ischemic heart disease. Our previous study revealed that Sirtuin 3 (SIRT3) is associated with ferroptosis and cardiac fibrosis. In this study, we tested whether the knockout of SIRT3 in cardiomyocytes (SIRT3cKO) promotes mitochondrial ferroptosis and whether the blockade of ferroptosis would ameliorate mitochondrial dysfunction. METHODS AND RESULTS: Mitochondrial and cytosolic fractions were isolated from the ventricles of mice. Cytosolic and mitochondrial ferroptosis were analyzed by comparison to SIRT3loxp mice. An echocardiography study showed that SIRT3cKO mice developed heart failure as evidenced by a reduction of EF% and FS% compared to SIRT3loxp mice. Comparison of mitochondrial and cytosolic fractions of SIRT3cKO and SIRT3loxp mice revealed that, upon loss of SIRT3, mitochondrial, but not cytosolic, total lysine acetylation was significantly increased. Similarly, acetylated p53 was significantly upregulated only in the mitochondria. These data demonstrate that SIRT3 is the primary mitochondrial deacetylase. Most importantly, loss of SIRT3 resulted in significant reductions of frataxin, aconitase, and glutathione peroxidase 4 (GPX4) in the mitochondria. This was accompanied by a significant increase in levels of mitochondrial 4-hydroxynonenal. Treatment of SIRT3cKO mice with the ferroptosis inhibitor ferrostatin-1 (Fer-1) for 14 days significantly improved preexisting heart failure. Mechanistically, Fer-1 treatment significantly increased GPX4 and aconitase expression/activity, increased mitochondrial iron­sulfur clusters, and improved mitochondrial membrane potential and Complex IV activity. CONCLUSIONS: Inhibition of ferroptosis ameliorated cardiac dysfunction by specifically targeting mitochondrial aconitase and iron­sulfur clusters. Blockade of mitochondrial ferroptosis may be a novel therapeutic target for mitochondrial cardiomyopathies.

The study of iron- and copper-binding proteome of Fusarium oxysporum and its effector candidates.

Fusarium oxysporum f.sp. lycopersici is a phytopathogen which causes vascular wilt disease in tomato plants. The survival tactics of both pathogens and hosts depend on intricate interactions between host plants and pathogenic microbes. Iron-binding proteins (IBPs) and copper-binding proteins (CBPs) play a crucial role in these interactions by participating in enzyme reactions, virulence, metabolism, and transport processes. We employed high-throughput computational tools at the sequence and structural levels to investigate the IBPs and CBPs of F. oxysporum. A total of 124 IBPs and 37 CBPs were identified in the proteome of Fusarium. The ranking of amino acids based on their affinity for binding with iron is Glu > His> Asp > Asn > Cys, and for copper is His > Asp > Cys respectively. The functional annotation, determination of subcellular localization, and Gene Ontology analysis of these putative IBPs and CBPs have unveiled their potential involvement in a diverse array of cellular and biological processes. Three iron-binding glycosyl hydrolase family proteins, along with four CBPs with carbohydrate-binding domains, have been identified as potential effector candidates. These proteins are distinct from the host Solanum lycopersicum proteome. Moreover, they are known to be located extracellularly and function as enzymes that degrade the host cell wall during pathogen-host interactions. The insights gained from this report on the role of metal ions in plant-pathogen interactions can help develop a better understanding of their fundamental biology and control vascular wilt disease in tomato plants.

Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Disulfide Bonds of Thyroid Peroxidase Are Critical Elements for Subcellular Localization, Proteasome-Dependent Degradation, and Enzyme Activity.

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Quantification of human mature frataxin protein expression in nonhuman primate hearts after gene therapy.

Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.

Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1.

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: "GGG" and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

Yeast cells depleted of the frataxin homolog Yfh1 redistribute cellular iron: Studies using Mössbauer spectroscopy and mathematical modeling.

The neurodegenerative disease Friedreich's ataxia arises from a deficiency of frataxin, a protein that promotes iron-sulfur cluster (ISC) assembly in mitochondria. Here, primarily using Mössbauer spectroscopy, we investigated the iron content of a yeast strain in which expression of yeast frataxin homolog 1 (Yfh1), oxygenation conditions, iron concentrations, and metabolic modes were varied. We found that aerobic fermenting Yfh1-depleted cells grew slowly and accumulated FeIII nanoparticles, unlike WT cells. Under hypoxic conditions, the same mutant cells grew at rates similar to WT cells, had similar iron content, and were dominated by FeII rather than FeIII nanoparticles. Furthermore, mitochondria from mutant hypoxic cells contained approximately the same levels of ISCs as WT cells, confirming that Yfh1 is not required for ISC assembly. These cells also did not accumulate excessive iron, indicating that iron accumulation into yfh1-deficient mitochondria is stimulated by O2. In addition, in aerobic WT cells, we found that vacuoles stored FeIII, whereas under hypoxic fermenting conditions, vacuolar iron was reduced to FeII. Under respiring conditions, vacuoles of Yfh1-deficient cells contained FeIII, and nanoparticles accumulated only under aerobic conditions. Taken together, these results informed a mathematical model of iron trafficking and regulation in cells that could semiquantitatively simulate the Yfh1-deficiency phenotype. Simulations suggested partially independent regulation in which cellular iron import is regulated by ISC activity in mitochondria, mitochondrial iron import is regulated by a mitochondrial FeII pool, and vacuolar iron import is regulated by cytosolic FeII and mitochondrial ISC activity.

Posttranslational regulation of mitochondrial frataxin and identification of compounds that increase frataxin levels in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a degenerative disease caused by a decrease in the mitochondrial protein frataxin (Fxn), which is involved in iron-sulfur cluster (ISC) synthesis. Diminutions in Fxn result in decreased ISC synthesis, increased mitochondrial iron accumulation, and impaired mitochondrial function. Here, we show that conditions that result in increased mitochondrial reactive oxygen species in yeast or mammalian cell culture give rise to increased turnover of Fxn but not of other ISC synthesis proteins. We demonstrate that the mitochondrial Lon protease is involved in Fxn degradation and that iron export through the mitochondrial metal transporter Mmt1 protects yeast Fxn from degradation. We also determined that when FRDA fibroblasts were grown in media containing elevated iron, mitochondrial reactive oxygen species increased and Fxn decreased compared to WT fibroblasts. Furthermore, we screened a library of FDA-approved compounds and identified 38 compounds that increased yeast Fxn levels, including the azole bifonazole, antiparasitic fipronil, antitumor compound dibenzoylmethane, antihypertensive 4-hydroxychalcone, and a nonspecific anion channel inhibitor 4,4-diisothiocyanostilbene-2,2-sulfonic acid. We show that top hits 4-hydroxychalcone and dibenzoylmethane increased mRNA levels of transcription factor nuclear factor erythroid 2-related factor 2 in FRDA patient-derived fibroblasts, as well as downstream antioxidant targets thioredoxin, glutathione reductase, and superoxide dismutase 2. Taken together, these findings reveal that FRDA progression may be in part due to oxidant-mediated decreases in Fxn and that some approved compounds may be effective in increasing mitochondrial Fxn in FRDA, delaying disease progression.

Modelling Protein Plasticity: The Example of Frataxin and Its Variants.

Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the FXN gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich's ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition.

Heme Oxygenase-1 (HMOX-1) and inhibitor of differentiation proteins (ID1, ID3) are key response mechanisms against iron-overload in pancreatic ß-cells.

Iron overload promotes the generation of reactive oxygen species (ROS). Pancreatic ß-cells can counter oxidative stress through multiple anti-oxidant responses. Herein, RNA-sequencing was used to describe the expression profile of iron regulatory genes in human islets with or without diabetes. Functional experiments including siRNA silencing, qPCR, western blotting, cell viability, ELISA and RNA-sequencing were performed as means of identifying the genetic signature of the protective response following iron overload-induced stress in human islets and INS-1. FTH1 and FTL genes were highly expressed in human islets and INS-1 cells, while hepcidin (HAMP) was low. FXN, DMT1 and FTHL1 genes were differentially expressed in diabetic islets compared to control. Silencing of Hamp in INS-1 cells impaired insulin secretion and influenced the expression of ß-cell key genes. RNA-sequencing analysis in iron overloaded INS-1 cells identified Id1 and Id3 as the top down-regulated genes, while Hmox1 was the top upregulated. Expression of ID1, ID3 and HMOX1 was validated at the protein level in INS-1 cells and human islets. Differentially expressed genes (DEGs) were enriched for TGF-ß, regulating stem cells, ferroptosis, and HIF-1 signaling. Hmox1-silenced cells treated with FAC elevated the expression of Id1 and Id3 expression than untreated cells. Our findings suggest that HMOX1, ID1 and ID3 define the response mechanism against iron-overload-induced stress in ß-cells.

Molecular Details of the Frataxin-Scaffold Interaction during Mitochondrial Fe-S Cluster Assembly.

Iron-sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe-S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron-sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe-2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe-S cluster assembly.

Generation of transgene-free iPSC lines from three patients with Friedreich's ataxia (FRDA) carrying GAA triplet expansions in the first intron of FXN gene.

Friedreich's ataxia (FRDA) is a rare neurodegenerative disorder which is caused by triplet repeat expansion (GAA) in the first intron of FXN gene. In this present study, we generated induced pluripotent stem cells (iPSC) lines from fibroblasts of three unrelated FRDA patients using integration-free episomal vectors. All iPSC lines express the pluripotency markers such as OCT4 and SSEA4, display normal karyotypes and can differentiate into all three germ layers via in vivo teratoma formation assay.

Drp1-dependent peptide reverse mitochondrial fragmentation, a homeostatic response in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive, neurodegenerative disease characterized by the deficiency of the iron-sulfur cluster assembly protein frataxin. Loss of this protein impairs mitochondrial function. Mitochondria alter their morphology in response to various stresses; however, such alterations to morphology may be homeostatic or maladaptive depending upon the tissue and disease state. Numerous neurodegenerative diseases exhibit excessive mitochondrial fragmentation, and reversing this phenotype improves bioenergetics for diseases in which mitochondrial dysfunction is a secondary feature of the disease. This paper demonstrates that frataxin deficiency causes excessive mitochondrial fragmentation that is dependent upon Drp1 activity in Friedreich ataxia cellular models. Drp1 inhibition by the small peptide TAT-P110 reverses mitochondrial fragmentation but also decreases ATP levels in frataxin-knockdown fibroblasts and FRDA patient fibroblasts, suggesting that fragmentation may provide a homeostatic pathway for maintaining cellular ATP levels. The cardiolipin-stabilizing compound SS-31 similarly reverses fragmentation through a Drp1-dependent mechanism, but it does not affect ATP levels. The combination of TAT-P110 and SS-31 does not affect FRDA patient fibroblasts differently from SS-31 alone, suggesting that the two drugs act through the same pathway but differ in their ability to alter mitochondrial homeostasis. In approaching potential therapeutic strategies for FRDA, an important criterion for compounds that improve bioenergetics should be to do so without impairing the homeostatic response of mitochondrial fragmentation.

The Regulator OmpR in Yersinia enterocolitica Participates in Iron Homeostasis by Modulating Fur Level and Affecting the Expression of Genes Involved in Iron Uptake.

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.

DNA repair pathways are altered in neural cell models of frataxin deficiency.

Friedreich's ataxia (FRDA) is a hereditary and predominantly neurodegenerative disease caused by a deficiency of the protein frataxin (FXN). As part of the overall efforts to understand the molecular basis of neurodegeneration in FRDA, a new human neural cell line with doxycycline-induced FXN knockdown was established. This cell line, hereafter referred to as iFKD-SY, is derived from the human neuroblastoma SH-SY5Y and retains the ability to differentiate into mature neuron-like cells. In both proliferating and differentiated iFKD-SY cells, the induction of FXN deficiency is accompanied by increases in oxidative stress and DNA damage, reduced aconitase enzyme activity, higher levels of p53 and p21, activation of caspase-3, and subsequent apoptosis. More interestingly, FXN-deficient iFKD-SY cells exhibit an important transcriptional deregulation in many of the genes implicated in DNA repair pathways. The levels of some crucial proteins involved in DNA repair appear notably diminished. Furthermore, similar changes are found in two additional neural cell models of FXN deficit: primary cultures of FXN-deficient mouse neurons and human olfactory mucosa stem cells obtained from biopsies of FRDA patients. These results suggest that the deficiency of FXN leads to a down-regulation of DNA repair pathways that synergizes with oxidative stress to provoke DNA damage, which may be involved in the pathogenesis of FRDA. Thus, a failure in DNA repair may be considered a shared common molecular mechanism contributing to neurodegeneration in a number of hereditary ataxias including FRDA.

Thyroid peroxidase in human endometrium and placenta: a potential target for anti-TPO antibodies.

Autoimmune thyroid disease is the most common endocrine disorder during pregnancy. Thyroid autoantibodies (TAs) have been suggested to serve a role in implantation failure and spontaneous abortion. Until now, there are no data on the potential interaction of TAs with human reproductive organs. Here, we set out for the first time to test this hypothesis by studying the expression of thyroid peroxidase (TPO) at gene and protein level in human reproductive organs. Endometrial samples were taken from normal women, and placenta tissues were collected after full-term caesarian section. Expression of TPO messenger RNA (mRNA) was investigated by qRT-PCR. In addition, polyclonal anti-TPO antibodies were produced and the expression of TPO protein in mentioned tissues was evaluated by immunohistochemistry and Western blot analysis. The reactivity of anti-TPO antibody in human embryos was evaluated by immunofluorescent staining. For the first time, our study showed that TPO is expressed at gene and protein levels in endometrium and placenta. TPO expression was mainly localized to glandular and luminal epithelial cells in the endometrium. In placenta, the syncytiotrophoblasts and invasive trophoblast cells were the main cell types that expressed TPO protein. Specific band of approximately 110 kDa was observed in all endometrial and placental tissues by Western blot analysis. However, no expression of TPO protein was observed in human embryo. TPO expression in endometrium and placenta may explain higher frequency of abortion and infertility in patients with thyroid autoimmunity.

Association of Thyroid Peroxidase Gene Polymorphisms and Serum Anti- TPO Levels in Egyptian Patients with Autoimmune Hypothyroidism.

BACKGROUND: Thyroid peroxidase (TPO) gene mutation leads to a change in enzyme built structure resulting in the anti-TPO autoantibodies production that may cause thyroid destruction. AIM: To evaluate the association of three single nucleotide polymorphisms (SNPs) of the TPO gene and anti-TPO levels in Egyptian patients with autoimmune hypothyroidism and correlate them with the disease severity. METHODS: Two hundred patients with newly discovered autoimmune hypothyroidism were included in the study (100 with subclinical hypothyroidism and 100 of them with overt hypothyroidism) and 100 healthy individuals as a control group were genotyped by PCR-REFLP. RESULTS: The TT genotype of rs2071400 C/T and the T allele were significantly more frequent in patients with subclinical hypothyroidism and overt hypothyroidism than in the control group. But there were no significant differences in the TT genotype and T allele between subclinical and overt hypothyroidism patients. As regards TPO rs732609 A/C polymorphism, the CC genotype of rs732609 A/C and the C allele were significantly increased in patients with subclinical hypothyroidism and overt hypothyroidism than in controls. There was a significant difference in the CC genotype and C allele between subclinical and overt hypothyroidism patients. Concerning TPO rs1126797 C/T polymorphism, there were no significant differences of genotype or allele frequencies between patients groups and control group. CONCLUSION: We found an association of rs2071400 C/T and rs732609A/C polymorphisms with autoimmune hypothyroidism and correlated anti-TPO levels with different genotypes in hypothyroid patients. Also, we found an association of rs732609A/C polymorphism with the disease severity.