The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.

The metastasis suppressor, NDRG1, attenuates oncogenic TGF-ß and NF-κB signaling to enhance membrane E-cadherin expression in pancreatic cancer cells.

The metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-ß (TGF-ß) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and ß-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and ß-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/ß and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-ß signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential.

Tumor stressors induce two mechanisms of intracellular P-glycoprotein-mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones.

Multidrug resistance (MDR) is a major obstacle in cancer treatment due to the ability of tumor cells to efflux chemotherapeutics via drug transporters (e.g. P-glycoprotein (Pgp; ABCB1)). Although the mechanism of Pgp-mediated drug efflux is known at the plasma membrane, the functional role of intracellular Pgp is unclear. Moreover, there has been intense focus on the tumor micro-environment as a target for cancer treatment. This investigation aimed to dissect the effects of tumor micro-environmental stress on subcellular Pgp expression, localization, and its role in MDR. These studies demonstrated that tumor micro-environment stressors (i.e. nutrient starvation, low glucose levels, reactive oxygen species, and hypoxia) induce Pgp-mediated drug resistance. This occurred by two mechanisms, where stressors induced 1) rapid Pgp internalization and redistribution via intracellular trafficking (within 1 h) and 2) hypoxia-inducible factor-1α expression after longer incubations (4-24 h), which up-regulated Pgp and was accompanied by lysosomal biogenesis. These two mechanisms increased lysosomal Pgp and facilitated lysosomal accumulation of the Pgp substrate, doxorubicin, resulting in resistance. This was consistent with lysosomal Pgp being capable of transporting substrates into lysosomes. Hence, tumor micro-environmental stressors result in: 1) Pgp redistribution to lysosomes; 2) increased Pgp expression; 3) lysosomal biogenesis; and 4) potentiation of Pgp substrate transport into lysosomes. In contrast to doxorubicin, when stress stimuli increased lysosomal accumulation of the cytotoxic Pgp substrate, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), this resulted in the agent overcoming resistance. Overall, this investigation describes a novel approach to overcoming resistance in the stressful tumor micro-environment.

Pharmacological targeting of mitochondria in cancer stem cells: An ancient organelle at the crossroad of novel anti-cancer therapies.

Mitochondria play vital roles in various cellular processes, ranging from cellular metabolism to signal transduction and cell death regulation. As these properties are critical for cancer growth, the mitochondrion has recently become an attractive target for anti-cancer therapies. In addition, it has come to light that mitochondria are crucially involved in the regulation of stem cell identity, differentiation and fate. A similar role for mitochondria has been also demonstrated in malignant stem-like cells termed cancer stem cells (CSCs), which are implicated in progression and resistance of many tumors. In this review, we summarize different mitochondrial functions reported to promote acquisition and maintenance of CSC phenotype and discuss the rationale for their therapeutic targeting. Particular emphasis is given to therapeutics that act directly through modulation of these mitochondrial functions and have recently emerged as promising anti-CSC drugs in pre-clinical studies. This review highlights the intriguing aspects of mitochondrial biology that may have a crucial role in cancer initiation, progression, and resistance and which might facilitate pharmacological targeting. Indeed, understanding of mitochondrial function in the regulation of CSCs will promote the development of novel CSC-targeted therapeutic strategies, which could significantly improve the long-term survival of cancer patients.

Molecular Alterations in a Mouse Cardiac Model of Friedreich Ataxia: An Impaired Nrf2 Response Mediated via Upregulation of Keap1 and Activation of the Gsk3ß Axis.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a master regulator of the antioxidant response. However, studies in models of Friedreich ataxia, a neurodegenerative and cardiodegenerative disease associated with oxidative stress, reported decreased Nrf2 expression attributable to unknown mechanisms. Using a mouse conditional frataxin knockout (KO) model in the heart and skeletal muscle, we examined the Nrf2 pathway in these tissues. Frataxin KO results in fatal cardiomyopathy, whereas skeletal muscle was asymptomatic. In the KO heart, protein oxidation and a decreased glutathione/oxidized glutathione ratio were observed, but the opposite was found in skeletal muscle. Decreased total and nuclear Nrf2 and increased levels of its inhibitor, Kelch-like ECH-associated protein 1, were evident in the KO heart, but not in skeletal muscle. Moreover, a mechanism involving activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3ß (Gsk3ß) signaling was demonstrated in the KO heart. This process involved the following: i) increased Gsk3ß activation, ii) ß-transducin repeat containing E3 ubiquitin protein ligase nuclear accumulation, and iii) Fyn phosphorylation. A corresponding decrease in Nrf2-DNA-binding activity and a general decrease in Nrf2-target mRNA were observed in KO hearts. Paradoxically, protein levels of some Nrf2 antioxidant targets were significantly increased in KO mice. Collectively, cardiac frataxin deficiency reduces Nrf2 levels via two potential mechanisms: increased levels of cytosolic Kelch-like ECH-associated protein 1 and activation of Gsk3ß signaling, which decreases nuclear Nrf2. These findings are in contrast to the frataxin-deficient skeletal muscle, where Nrf2 was not decreased.

Novel Thiosemicarbazones Inhibit Lysine-Rich Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1) Coisolated (LYRIC) and the LYRIC-Induced Epithelial-Mesenchymal Transition via Upregulation of N-Myc Downstream-Regulated Gene 1 (NDRG1).

Tumor necrosis factor α (TNFα) plays a vital role in cancer progression as it is associated with inflammation and promotion of cancer angiogenesis and metastasis. The effects of TNFα are mediated by its downstream target, the oncogene lysine-rich CEACAM1 coisolated protein (LYRIC, also known as metadherin or astrocyte elevated gene-1). LYRIC plays an important role in activating the nuclear factor-ĸB (NF-κB) signaling pathway, which controls multiple cellular processes, including proliferation, apoptosis, migration, etc. In contrast, the metastasis suppressor N-myc downstream regulated gene 1 (NDRG1) has the opposite effect on the NF-κB pathway, being able to inhibit NF-κB activation and reduce angiogenesis, proliferation, migration, and cancer cell invasion. These potent anticancer properties make NDRG1 an ideal therapeutic target. Indeed, a novel class of thiosemicarbazone anticancer agents that target this molecule has been developed; the lead agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, has recently entered clinical trials for advanced and resistant cancers. To further elucidate the interaction between NDRG1 and oncogenic signaling, this study for the first time assessed the effects of NDRG1 on the tumorigenic properties of TNFα and its downstream target, LYRIC. We have demonstrated that NDRG1 inhibits the TNFα-mediated epithelial-to-mesenchymal transition. Further, NDRG1 also potently inhibited LYRIC expression, with a negative feedback loop existing between these two molecules. Examining the mechanism involved, we demonstrated that NDRG1 inhibited phosphatidylinositol 3-kinase/AKT signaling, leading to reduced levels of the LYRIC transcriptional activator, c-Myc. Finally, we demonstrated that novel thiosemicarbazones that upregulate NDRG1 also inhibit LYRIC expression, further highlighting their marked potential for cancer treatment.

Roads to melanoma: Key pathways and emerging players in melanoma progression and oncogenic signaling.

Melanoma has markedly increased worldwide during the past several decades in the Caucasian population and is responsible for 80% of skin cancer deaths. Considering that metastatic melanoma is almost completely resistant to most current therapies and is linked with a poor patient prognosis, it is crucial to further investigate potential molecular targets. Major cell-autonomous drivers in the pathogenesis of this disease include the classical MAPK (i.e., RAS-RAF-MEK-ERK), WNT, and PI3K signaling pathways. These pathways play a major role in defining the progression of melanoma, and some have been the subject of recent pharmacological strategies to treat this belligerent disease. This review describes the latest advances in the understanding of melanoma progression and the major molecular pathways involved. In addition, we discuss the roles of emerging molecular players that are involved in melanoma pathogenesis, including the functional role of the melanoma tumor antigen, p97/MFI2 (melanotransferrin).

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics.

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.

Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway.

Frataxin and the molecular mechanism of mitochondrial iron-loading in Friedreich's ataxia.

The mitochondrion is a major site for the metabolism of the transition metal, iron, which is necessary for metabolic processes critical for cell vitality. The enigmatic mitochondrial protein, frataxin, is known to play a significant role in both cellular and mitochondrial iron metabolism due to its iron-binding properties and its involvement in iron-sulfur cluster (ISC) and heme synthesis. The inherited neuro- and cardio-degenerative disease, Friedreich's ataxia (FA), is caused by the deficient expression of frataxin that leads to deleterious alterations in iron metabolism. These changes lead to the accumulation of inorganic iron aggregates in the mitochondrial matrix that are presumed to play a key role in the oxidative damage and subsequent degenerative features of this disease. Furthermore, the concurrent dys-regulation of cellular antioxidant defense, which coincides with frataxin deficiency, exacerbates oxidative stress. Hence, the pathogenesis of FA underscores the importance of the integrated homeostasis of cellular iron metabolism and the cytoplasmic and mitochondrial redox environments. This review focuses on describing the pathogenesis of the disease, the molecular mechanisms involved in mitochondrial iron-loading and the dys-regulation of cellular antioxidant defense due to frataxin deficiency. In turn, current and emerging therapeutic strategies are also discussed.

Letter to the Editor: "Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques".

n/a.

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Potent antimycobacterial activity of the pyridoxal isonicotinoyl hydrazone analog 2-pyridylcarboxaldehyde isonicotinoyl hydrazone: a lipophilic transport vehicle for isonicotinic acid hydrazide.

The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

Hepcidin, show some self-control! How the hormone of iron metabolism regulates its own expression.

Does the hormone of iron metabolism, hepcidin, exhibit 'self-control'? Hepcidin is a small, disulfide-rich peptide synthesized by the liver, which plays a keystone role in regulating systemic iron metabolism in mammals. Hepcidin acts by binding and triggering the lysosomal degradation of the cellular iron exporter ferroportin. Ultimately, decreased ferroportin leads to decreased plasma iron levels. Although various modulators of HAMP (the hepcidin antimicrobial peptide gene) expression are known, no auto-regulatory pathway has been described. In their paper published in the Biochemical Journal in April 2013, Pandur et al. identify an auto-regulatory pathway in which prohepcidin regulates HAMP expression. The authors observe that prohepcidin can bind to the inflammation-regulated STAT3 (signal transducer and activator of transcription 3)-binding site in the HAMP promoter to negatively regulate HAMP expression. Furthermore, the authors find that the prohepcidin-binding partner, α-1 antitrypsin, inhibits prohepcidin's ability to decrease HAMP activity. This is significant as α-1 antitrypsin, similar to hepcidin, is an acute-phase reactant that is up-regulated by inflammation. In conclusion, the discovery of a hepcidin auto-regulatory pathway, first, supports the emerging notion that hepcidin regulation is exquisitely fine-tuned through a process of combinatorial control; and secondly, suggests that hepcidin may play a hand in its own deregulation in diseases of iron metabolism that involve aberrant cytokine signalling (e.g. the anaemia of inflammation).

Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol.

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

Mitochondrial DNA copy number in adults with and without Type 1 diabetes.

AIMS: Mitochondrial damage is implicated in diabetes pathogenesis and complications. Mitochondrial DNA copy number (mtDNA-cn) in human Type 1 diabetes (T1D) studies are lacking. We related mtDNA-cn in T1D and non-diabetic adults (CON) with diabetes complications and risk factors. METHODS: Cross-sectional study: 178 T1D, 132 non-diabetic controls. Associations of whole blood mtDNA-cn (qPCR) with complications, inflammation, and C-peptide. RESULTS: mtDNA-cn (median (LQ, UQ)) was lower in: T1D vs. CON (271 (189, 348) vs. 320 (264, 410); p < 0.0001); T1D with vs. without kidney disease (238 (180, 309) vs. 294 (198, 364); p = 0.02); and insulin injection vs. pump-users (251 (180, 340) vs. 322 (263, 406); p = 0.008). Significant univariate correlates of mtDNA-cn: T1D: (positive) HDL-C; (negative) fasting glucose, white cell count (WCC), sVCAM-1, sICAM-1; CON: (negative) WHR (waist-hip-ratio). Detectable C-peptide in T1D increased with lowest-highest mtDNA-cn tertiles (54%, 69%, 79%, p = 0.02). Independent determinants of mtDNA-cn: T1D: (positive) HDL-C; (negative) age, sICAM-1; AUROC 0.71; CON: WCC (negative), never smoking, (positive) female, pulse pressure; AUROC 0.74. CONCLUSIONS: mtDNA-cn is lower in T1D vs. CON, and in T1D kidney disease. In T1D, mtDNA-cn correlates inversely with age and inflammation, and positively with HDL-C, detectable C-peptide and pump use. Further clinical and basic science studies are merited.

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Mechanisms of impaired mitochondrial homeostasis and NAD+ metabolism in a model of mitochondrial heart disease exhibiting redox active iron accumulation.

Due to the high redox activity of the mitochondrion, this organelle can suffer oxidative stress. To manage energy demands while minimizing redox stress, mitochondrial homeostasis is maintained by the dynamic processes of mitochondrial biogenesis, mitochondrial network dynamics (fusion/fission), and mitochondrial clearance by mitophagy. Friedreich's ataxia (FA) is a mitochondrial disease resulting in a fatal hypertrophic cardiomyopathy due to the deficiency of the mitochondrial protein, frataxin. Our previous studies identified defective mitochondrial iron metabolism and oxidative stress potentiating cardiac pathology in FA. However, how these factors alter mitochondrial homeostasis remains uncharacterized in FA cardiomyopathy. This investigation examined the muscle creatine kinase conditional frataxin knockout mouse, which closely mimics FA cardiomyopathy, to dissect the mechanisms of dysfunctional mitochondrial homeostasis. Dysfunction of key mitochondrial homeostatic mechanisms were elucidated in the knockout hearts relative to wild-type littermates, namely: (1) mitochondrial proliferation with condensed cristae; (2) impaired NAD+ metabolism due to perturbations in Sirt1 activity and NAD+ salvage; (3) increased mitochondrial biogenesis, fusion and fission; and (4) mitochondrial accumulation of Pink1/Parkin with increased autophagic/mitophagic flux. Immunohistochemistry of FA patients' heart confirmed significantly enhanced expression of markers of mitochondrial biogenesis, fusion/fission and autophagy. These novel findings demonstrate cardiac frataxin-deficiency results in significant changes to metabolic mechanisms critical for mitochondrial homeostasis. This mechanistic dissection provides critical insight, offering the potential for maintaining mitochondrial homeostasis in FA and potentially other cardio-degenerative diseases by implementing innovative treatments targeting mitochondrial homeostasis and NAD+ metabolism.

Novel multifunctional iron chelators of the aroyl nicotinoyl hydrazone class that markedly enhance cellular NAD+ /NADH ratios.

BACKGROUND AND PURPOSE: Alzheimer's disease (AD) is a multifactorial condition leading to cognitive decline and represents a major global health challenge in ageing populations. The lack of effective AD therapeutics led us to develop multifunctional nicotinoyl hydrazones to target several pathological characteristics of AD. EXPERIMENTAL APPROACH: We synthesised 20 novel multifunctional agents based on the nicotinoyl hydrazone scaffold, which acts as a metal chelator and a lipophilic delivery vehicle, donating a NAD+ precursor to cells, to target metal dyshomeostasis, oxidative stress, ß-amyloid (Aß) aggregation, and a decrease in the NAD+ /NADH ratio. KEY RESULTS: The most promising compound, 6-methoxysalicylaldehyde nicotinoyl hydrazone (SNH6), demonstrated low cytotoxicity, potent iron (Fe)-chelation efficacy, significant inhibition of copper-mediated Aß aggregation, oxidative stress alleviation, effective donation of NAD+ to NAD-dependent metabolic processes (PARP and sirtuin activity) and enhanced cellular NAD+ /NADH ratios, as well as significantly increased median Caenorhabditis elegans lifespan (to 1.46-fold of the control); partly decreased BACE1 expression, resulting in significantly lower soluble amyloid precursor protein-ß (sAPPß) and Aß1-40 levels; and favourable blood-brain barrier-permeation properties. Structure-activity relationships demonstrated that the ability of these nicotinoyl hydrazones to increase NAD+ was dependent on the electron-withdrawing or electron-donating substituents on the aldehyde- or ketone-derived moiety. Aldehyde-derived hydrazones containing the ONO donor set and electron-donating groups were required for NAD+ donation and low cytotoxicity. CONCLUSIONS AND IMPLICATIONS: The nicotinoyl hydrazones, particularly SNH6, have the potential to act as multifunctional therapeutic agents and delivery vehicles for NAD+ precursors for AD treatment.

Acireductone dioxygenase 1 (ADI1) is regulated by cellular iron by a mechanism involving the iron chaperone, PCBP1, with PCBP2 acting as a potential co-chaperone.

The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.