Antioxidant defense mechanisms and its dysfunctional regulation in the mitochondrial disease, Friedreich's ataxia.

Redox stress is associated with the pathogenesis of a wide variety of disease states. This can be amplified potentially through redox active iron deposits in oxidatively active organelles such as the mitochondrion. There are a number of disease states, including Friedreich's ataxia (FA) and sideroblastic anemia, where iron metabolism is dysregulated and leads to mitochondrial iron accumulation. Considering FA, which is due to the decreased expression of the mitochondrial protein, frataxin, this iron accumulation does not occur within protective storage proteins such as mitochondrial ferritin. Instead, it forms unbound biomineral aggregates composed of high spin iron(III), phosphorous and sulfur, which probably contributes to the observed redox stress. There is also a dysregulated response to the ensuing redox assault, as the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation. The dysfunctional response of Nrf2 in FA is due to multiple mechanisms including: (1) up-regulation of Keap1 that is involved in Nrf2 degradation; (2) activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3ß (Gsk3ß) signaling; and (3) inhibited nuclear translocation of Nrf2. More recently, increased microRNA (miRNA) 144 expression has been demonstrated to down-regulate Nrf2 in several disease states, including an animal model of FA. Other miRNAs have also demonstrated to be dysregulated upon frataxin depletion in vivo in humans and animal models of FA. Collectively, frataxin depletion results in multiple, complex responses that lead to detrimental redox effects that could contribute to the mechanisms involved in the pathogenesis of FA.

Treatment of dilated cardiomyopathy in a mouse model of Friedreich's ataxia using N-acetylcysteine and identification of alterations in microRNA expression that could be involved in its pathogenesis.

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

During mitosis ZEB1 "switches" from being a chromatin-bound epithelial gene repressor, to become a microtubule-associated protein.

Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.

Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.

Iron is a crucial transition metal for virtually all life. Two major destinations of iron within mammalian cells are the cytosolic iron-storage protein, ferritin, and mitochondria. In mitochondria, iron is utilized in critical anabolic pathways, including: iron-storage in mitochondrial ferritin, heme synthesis, and iron-sulfur cluster (ISC) biogenesis. Although the pathways involved in ISC synthesis in the mitochondria and cytosol have begun to be characterized, many crucial details remain unknown. In this review, we discuss major aspects of the journey of iron from its initial cellular uptake, its modes of trafficking within cells, to an overview of its downstream utilization in the cytoplasm and within mitochondria. The understanding of mitochondrial iron processing and its communication with other organelles/subcellular locations, such as the cytosol, has been elucidated by the analysis of certain diseases e.g., Friedreich's ataxia. Increased knowledge of the molecules and their mechanisms of action in iron processing pathways (e.g., ISC biogenesis) will shape the investigation of iron metabolism in human health and disease.

Fixing frataxin: 'ironing out' the metabolic defect in Friedreich's ataxia.

The metabolically active and redox-active mitochondrion appears to play a major role in the cellular metabolism of the transition metal, iron. Frataxin, a mitochondrial matrix protein, has been identified as playing a key role in the iron metabolism of this organelle due to its iron-binding properties and is known to be essential for iron-sulphur cluster formation. However, the precise function of frataxin remains elusive. The decrease in frataxin expression, as seen in the inherited disorder Friedreich's ataxia, markedly alters cellular and mitochondrial iron metabolism in both the mitochondrion and the cell. The resulting dysregulation of iron trafficking damages affects tissues leading to neuro- and cardiodegeneration. This disease underscores the importance of iron homeostasis in the redox-active environment of the mitochondrion and the molecular players involved. Unravelling the mechanisms of altered iron metabolism in Friedreich's ataxia will help elucidate a biochemical function for frataxin. Consequently, this will enable the development of more effective and rationally designed treatments. This review will focus on the emerging function of frataxin in relation to the observed alterations in mitochondrial iron metabolism in Friedreich's ataxia. Tissue-specific alterations due to frataxin loss will also be discussed, as well as current and emerging therapeutic strategies.