BackgroundFriedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression.Methodology/Principal FindingsWe thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide.Conclusions/SignificanceThus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress.

Introduction Whether discernible advantages in terms of sensitivity and specificity exist with Acute Kidney Injury Network (AKIN) criteria versus Risk, Injury, Failure, Loss of Kidney Function, End-stage Kidney Disease (RIFLE) criteria is currently unknown. We evaluated the incidence of acute kidney injury and compared the ability of the maximum RIFLE and of the maximum AKIN within intensive care unit hospitalization in predicting inhospital mortality of critically ill patients.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of diseaseassociated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.

Background:MitoNEET is a mammalian iron-sulfur protein with the ability to transfer iron-sulfur (Fe-S) in vitro. Results: MitoNEET conveys Fe-S from the mitochondrion to the cytosol and reactivates cytosolic iron regulatory protein 1 into an Fe-S aconitase. Conclusion: A novel mitoNEET-dependent Fe-S repair pathway affects a key regulator of iron metabolism. Significance: MitoNEET is the first mitochondrial protein found to be involved in mammalian cytosolic Fe-S repair.

BackgroundDespite the considerable progress made in understanding the molecular bases of mitochondrial diseases, no effective treatments have been developed to date. Faithful animal models would be extremely helpful for designing such treatments. We showed previously that the Harlequin mouse phenotype was due to a specific mitochondrial complex I deficiency resulting from the loss of the Apoptosis Inducing Factor (Aif) protein.Methodology/Principal FindingsHere, we conducted a detailed evaluation of the Harlequin mouse phenotype, including the biochemical abnormalities in various tissues. We observed highly variable disease expression considering both severity and time course progression. In each tissue, abnormalities correlated with the residual amount of the respiratory chain complex I 20 kDa subunit, rather than with residual Aif protein. Antioxidant enzyme activities were normal except in skeletal muscle, where they were moderately elevated.Conclusions/SignificanceThus, the Harlequin mouse phenotype appears to result from mitochondrial respiratory chain complex I deficiency. Its features resemble those of human complex I deficiency syndromes. The Harlequin mouse holds promise as a model for developing treatments for complex I deficiency syndromes.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S] ؉ with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the "active state," which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a "dormant form." Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury.MitoNEET (mNT) 5 is an Fe-S protein of the mammalian outer mitochondrial membrane previously identified as a target of the type II diabetes drug pioglitazone (1). This 13-kDa protein is anchored to the outer mitochondrial membrane by its 32-amino acid N terminus with the major part of the protein, including the C-terminal [2Fe-2S] binding domain, located in the cytosol (2). In vivo, the biological activity of mNT has been linked to the regulation of iron/reactive oxygen species homeostasis in vivo (3, 4) to cell proliferation in human breast cancer (5) and to the regulation of lipid and glucose metabolism (4).Crystallographic studies of the soluble form of mNT (mNT ) revealed that the protein dimerizes and accommodates one [2Fe-2S] cluster per monomer coordinated by three cysteines (Cys-72, Cys-74, and Cys-83) and one histidine (His-87) in a CDGSH domain (6 -9). The cluster is redox-active with a midpoint redox potential of roughly 0 mV at pH 7 (10), and its lability depends on its redox state and on the pH (9, 11). mNT is also able to transfer its cluster in vitro to a cyanobacterial (12) and Escherichia coli apoferredoxin or to human ironregulatory protein-1 (IRP-1)/cytosolic aconitase (13). Recently, it has been proposed that mNT plays a specific role in cytosolic Fe-S cluster repair of IRP-1, a key regulator of cellular iron homeostasis in mammalian cells (13).It has been pointed out previously (12) that oxidation of the mNT cluster is necessary to trigger Fe-S ...

Cytochrome c oxidase (COX) deficiency is associated with a wide spectrum of clinical conditions, ranging from early onset devastating encephalomyopathy and cardiomyopathy, to neurological diseases in adulthood and in the elderly. No method of compensating successfully for COX deficiency has been reported so far. In vitro, COX-deficient human cells require additional glucose, pyruvate and uridine for normal growth and are specifically sensitive to oxidative stress. Here, we have tested whether the expression of a mitochondrially targeted, cyanide-resistant, alternative oxidase (AOX) from Ciona intestinalis could alleviate the metabolic abnormalities of COX-deficient human cells either from a patient harbouring a COX15 pathological mutation or rendered deficient by silencing the COX10 gene using shRNA. We demonstrate that the expression of the AOX, well-tolerated by the cells, compensates for both the growth defect and the pronounced oxidant-sensitivity of COX-deficient human cells.